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9.12C: RNA Oncogenic Viruses - Biology

9.12C: RNA Oncogenic Viruses - Biology


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An estimated 15% of all human cancers worldwide may be attributed to viruses.

Learning Objectives

  • Classify the viruses with oncogenic properties

Key Points

  • Both DNA and RNA viruses have been shown to be capable of causing cancer in humans.
  • Human T lymphotrophic virus type 1 and hepatitis C viruses are the two RNA viruses that contribute to human cancers.
  • Hepatitis C virus is an enveloped RNA virus capable of causing acute and chronic hepatitis in humans by infecting liver cells. It is estimated 3% of the world’s population are carriers. Chronic infection with hepatitis C virus results in cirrhosis, which in turn can lead to liver cancer.

Key Terms

  • oncogenic: Tending to cause the formation of tumors.
  • hepatocellular: Of or pertaining to the cells of the liver

There are two classes of cancer viruses: DNA and RNA viruses. Several viruses have been linked to certain types of cancer in humans. These viruses have varying ways of reproduction and represent several different virus families. Specifically, RNA viruses have RNA as their genetic material and can be either single-stranded RNA (ssRNA) or double-stranded (dsRNA). RNA viruses are classified based on the Baltimore classification system and do not take into account viruses with DNA intermediates in their life cycle. Viruses which contain RNA for their genetic material but do include DNA intermediates in their life cycle are called “retroviruses. ” There are numerous RNA oncogenic viruses that have been linked to various cancer types. These various oncogenic viruses include:

1. Human T lymphotrophic virus type 1 (HTLV-I), a retrovirus, has been linked to T-cell leukemia. 2. The hepatitis C virus has been linked to liver cancer in people with chronic infections.

2. Hepatitis viruses includes hepatitis B and hepatitis C have been linked to hepatocellular carcinoma.

3. Human papillomaviruses (HPV) have been linked to cancer of the cervix, anus, penis, vagina/vulva, and some cancers of the head and neck.

4. Kaposi’s sarcoma-associated herpesvirus (HHV-8) has been linked to Kaposi’s sarcoma and primary effusion lymphoma.

5. Epstein-Barr virus (EBV) has been linked to Burkitt’s lymphoma, Hodgkin’s lymphoma, post-transplantation lymphoproliferative disease, and nasopharyngeal carcinoma.

RNA Retroviruses

Retroviruses are different from DNA tumor viruses in that their genome is RNA, but they are similar to many DNA tumor viruses in that the genome is integrated into host genome. Since RNA makes up the genome of the mature virus particle, it must be copied to DNA prior to integration into the host cell chromosome. This lifestyle goes against the central dogma of molecular biology in which that DNA is copied into RNA. The outer envelope comes from the host cell plasma membrane. Coat proteins (surface antigens) are encoded by env (envelope) gene and are glycosylated. One primary gene product is made, but this is cleaved so that there are more than one surface glycoprotein in the mature virus (cleavage is by host enzyme in the Golgi apparatus). The primary protein (before cleavage) is made on ribosomes attached to the endoplasmic reticulum and is a transmembrane (type 1) protein. Inside the membrane is an icosahedral capsid containing proteins encoded by the gag gene (group-specific AntiGen). Gag-encoded proteins also coat the genomic RNA. Again, there is one primary gene product. This is cleaved by a virally-encoded protease (from the pol gene). There are two molecules of genomic RNA per virus particle with a 5′ cap and a 3′ poly A sequence. Thus, the virus is diploid. The RNA is plus sense (same sense as mRNA). About 10 copies of reverse transcriptase are present within the mature virus, these are encoded by the pol gene. Pol gene codes for several functions (again, as with gag and env, a polyprotein is made that is then cut up).


An Oncogenic Virus Promotes Cell Survival and Cellular Transformation by Suppressing Glycolysis

Aerobic glycolysis is essential for supporting the fast growth of a variety of cancers. However, its role in the survival of cancer cells under stress conditions is unclear. We have previously reported an efficient model of gammaherpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV)-induced cellular transformation of rat primary mesenchymal stem cells. KSHV-transformed cells efficiently induce tumors in nude mice with pathological features reminiscent of Kaposi’s sarcoma tumors. Here, we report that KSHV promotes cell survival and cellular transformation by suppressing aerobic glycolysis and oxidative phosphorylation under nutrient stress. Specifically, KSHV microRNAs and vFLIP suppress glycolysis by activating the NF-κB pathway to downregulate glucose transporters GLUT1 and GLUT3. While overexpression of the transporters rescues the glycolytic activity, it induces apoptosis and reduces colony formation efficiency in softagar under glucose deprivation. Mechanistically, GLUT1 and GLUT3 inhibit constitutive activation of the AKT and NF-κB pro-survival pathways. Strikingly, GLUT1 and GLUT3 are significantly downregulated in KSHV-infected cells in human KS tumors. Furthermore, we have detected reduced levels of aerobic glycolysis in several KSHV-infected primary effusion lymphoma cell lines compared to a Burkitt’s lymphoma cell line BJAB, and KSHV infection of BJAB cells reduced aerobic glycolysis. These results reveal a novel mechanism by which an oncogenic virus regulates a key metabolic pathway to adapt to stress in tumor microenvironment, and illustrate the importance of fine-tuning the metabolic pathways for sustaining the proliferation and survival of cancer cells, particularly under stress conditions.


Luc Montagnier

I was born on August 18, 1932 in Chabris, a “bourg”, larger than a village but smaller than a town, located in Berry south of the Loire Valley. This was – and still is – a region of agriculture with some renowned products such as welsh rabbit, goat cheeses and white asparagus. It was the place where my mother had grown up but, in fact, I never lived there.

On my father’s side, his parents came from Auvergne, a province in the centre of France, made of rich plains and old volcanoes, the latter probably being at the origin of my family name: Montagnier, the man living in mountains.

In his youth, my father had caught a terrible disease: streptococcal arthritis, ending in irreversible lesions in the aortic valves. He was therefore declared unfit for military service and had to find a sedentary job: he became an accountant and excelled in this profession, which implied, at that time, mainly hand-written work. He started working in the Poitiers area and then moved a little farther north to Châtellerault, a small city between Tours and Poitiers.

As an only child, I was cherished by my mother, a housewife, but two events dominated this pre-war period, of which I keep a vivid memory:

I was badly injured by a high speed car while crossing a main road: multiple wounds of which I keep some visible scars. After two days in a coma, I emerged as if I was born again, at the age of 5 (Figure 1).

Figure 1. Luc Montagnier at the age of 5.

… and two years later came the declaration of war in 1939, while the whole family was harvesting grapes in the vineyards of my mother’s brother. I still remember the images in a newspaper of Warsaw ruins after a bombing by German planes.

And then, in 1940, came the “real” war: the German invasion, my parents and I leaving their house (close to a risky railway station), fleeing on the roads in a little car, and finally more exposed to German bombing during this “exodus” than if we had stayed home.

The first year of German occupation was terrible, in that we had no food reserves and most of the time we were starving. I was a rather puny boy and during the four years of the war did not gain a gram! The “ersatz” did not stimulate my appetite, when I was dreaming of chocolate and oranges! My father had chronic enterocolitis and, worse, my grandfather (his father) was diagnosed with rectal cancer. He died in 1947 after terrible suffering and each time I visited him, I could see the inexorable progression of the disease. This affected me so much that it is probably one reason why I decided later to study medicine and to start research on cancer.

In June 1944, our house (so close to the railway) was partly destroyed − this time by an Allied bombing. I keep a mixed feeling of this year of the liberation of France. It was a great relief but I could not forget also the vision of so many dead people, civilians and soldiers, and the images of skinny deportees released from concentration camps. I will hate wars and their atrocities for the rest of my life.

At high school I did well, being usually ahead of my classmates. This is when I became curious about scientific knowledge, having left behind my religious Catholic belief.

Following the example of my father, who was tinkering in his leisure days with electric batteries, I set up a chemistry laboratory in the cellar of the new house which was requisitioned to accommodate us. There, I enthusiastically produced hydrogen gas, sweet-smelling aldehydes and nitro compounds (not nitro-glycerine!) that had the unfortunate habit of blowing up in my face.

I was delighted to read – in popularised books – the impressive progress of physics, especially atomic physics. Being good in physics and chemistry – but not as good in maths – I decided not to prepare to compete for the “Grandes Ecoles” but instead to register both at the School of Medicine and the Faculty of Sciences in Poitiers. My goal was in fact to start a research carrier in human biology, but there was no such specialty in Poitiers, either in Medicine or in Sciences. Since both the Faculty and School were within walking distance, I could spend the morning at the hospital and the afternoon attending courses in botany, zoology and geology, which were the main disciplines of the degree course in Sciences.

Fortunately the new Professor of Botany, Pierre Gavaudan, was a very atypical professor in that his scientific interests went far beyond the classification of plants. In fact, I owe him for having opened me a large window on what was the beginning of a new Biology, the DNA double helix, the in vitro synthesis of proteins by ribosomes and the structure of viruses.

At the same time, I was installing at home a device combining a time-lapse movie camera and a microscope, thanks to a gift by my father. This allowed me to do my first research work. I was studying a phenomenon known since 1908 as the phototaxy of chloroplasts: the property of some algae living at the surface of ponds to orient their large unique chloroplast according to the intensity of light if the light was too intense, the chloroplast turned inside the tubular cell to present its edge. In dark or weaker light, the chloroplast, a flat plate, exposed its larger surface. The phenomenon took a few minutes, which could be analysed by time-lapse cinematography. Using different glass filters, I could show that it was not the wavelength absorbed by the chlorophyll (red light) which regulated the orientation of the chloroplasts but indirectly some yellowish pigments absorbing the blue light. I was very proud, at the age of 21, to defend this work as a small thesis at the Faculty of Sciences of Poitiers. I was asked by my mentor, Pierre Gavaudan, to do research also on a literature-based subject: the L-forms of bacteria. This allowed me to make my first incursion – not the last – into the world of filtering bacteria. I could only find the references on this controversial subject at the library of the Institut Pasteur in Paris. This was indeed the time when I left Poitiers for Paris, where I was able to complete my medical studies as well as explore some aspects of biology closer to human beings, particularly neurophysiology, virology and oncology.

Having been hired as an assistant at the Sorbonne at the age of 23, I started learning old-fashioned technologies derived from Alexis Carrel‘s work on chick embryo heart cultures, as well as that of human cell lines in monolayers. Although my research was not productive at all, I keep from this period a solid expertise of Pasteurian technologies for working in perfectly sterile conditions without the use of antibiotics.

In 1957, the first description of infectious viral RNA from the tobacco mosaic virus by Fraenkel-Conrat and Gierer and Schramm determined my vocation: to become a virologist using the modern approach of molecular biology.

I started with the foot and mouth virus and then, in Kingsley Sanders’ laboratory at Carshalton near London, I was proud to identify for the first time an infectious double-stranded RNA from cells infected with the murine encephalomyocarditis virus, a small single-stranded RNA virus. This demonstrated for the first time that RNA could replicate like DNA by making a base-paired complementary strand.

In order to perfect my knowledge of oncogenic viruses, I moved from Carshalton to Glasgow where a new Institute of Virology had been recently inaugurated, headed by a remarkable virologist, Michael Stocker, and where many high-ranking visitors, among them Renato Dulbecco, were spending sabbatical years.

Working on a small oncogenic DNA virus, polyoma, I could show there, with I. Macpherson, a new property of transformed cells, that of growing in soft agar. Using this technique, it was easy to detect the transforming capacity of polyoma virus and its DNA. We showed that naked DNA alone carried all the oncogenic potential of the virus. This now looks pretty obvious, but it was not so at that time.

Back to France at the Institut Curie, I extended this finding to a number of cancer cells, transformed or not by oncogenic RNA or DNA viruses. However, this property allowed me to distinguish some in vitro steps in the process of transformation, which were correlated with some modifications of the plasma membrane and of the carbohydrate layer surrounding it.

A great mystery remained at that time: that of the replication of the oncogenic RNA viruses, now known as retroviruses. Howard Temin (Figure 2) had proposed the hypothesis of a DNA intermediate, but other possibilities could be considered. I myself tried to find a double-stranded RNA specific of the Rous sarcoma virus, a virus able to infect and transform chick embryo cells. I indeed isolated double-stranded RNA sequences, but they were of cellular origin and existed at the same level in non-infected cells! With Louise Harel, I later showed that this RNA was partly coming from repetitious sequences of DNA. In retrospect, it could at least in part represent the recently identified interfering RNAs involved in the negative control of messenger RNA translation.

Figure 2. Receiving an award plate of the American Society of Pathology from Howard Temin’s hands in 1985.

In 1969–70, the isolation of an RNA-polymerase associated with the viral particles of the vesicular stomatitis virus led to the idea that perhaps a key enzyme was also associated with the oncogenic RNA viruses. Indeed, Howard Temin and Mizutani, and independently David Baltimore, discovered in 1970 a specific enzyme associated with Rous sarcoma virus (RSV), the reverse transcriptase (RT), capable of reversely transcribing the viral RNA into DNA.

At about the same time, Hill and Hillova in Villejuif, France, demonstrated that the DNA extracted from RSV transformed cells was infectious and carry the genetic information of the viral RNA, confirming that the enzyme was working faithfully in infected cells.

I myself, with P. Vigier, confirmed and extended this discovery by showing that the infectious DNA was associated with the chromosomal DNA of the cells, showing integration of the proviral DNA, as earlier postulated by Temin.

Work on the chicken RSV was extended to similar viruses in mammals, so that many researchers at that time believed that RT activity was a new, highly sensitive tool for detecting similar viruses in human leukaemia and cancer. This was stimulated by the generously funded virus-cancer program launched by America’s National Institutes of Health. Unfortunately, the hunt for human retroviruses was basically unsuccessful but led to important basic work on the molecular biology of animal retroviruses.

In 1972, I was asked by Jacques Monod, then head of the Institut Pasteur, to create a research unit in the newly created Department of Virology of the Institute. I accepted, and this new laboratory allowed me to develop new avenues of research within the general theme of Viral Oncology, the ultimate goal remaining the detection of viruses involved in human cancers.

Thus, I became interested in the mechanism of action of interferon and its role in its expression of retroviruses. I came into this field after having demonstrated the biological activity of interferon messenger RNA in collaboration with two world-renowned experts in the field, Edward and Jacqueline De Maeyer.

From 1973 on, Ara Hovanessian and his co-workers joined my unit and brought a new dimension: the complex biochemical mechanism sustaining the antiviral activity of this remarkable group of cellular proteins.

In 1975, two other researchers joined my unit and brought their expertise on murine retroviruses: J. C. Chermann and his collaborator, Françoise Barré-Sinoussi (Figure 3). The latter mastered particularly the detection of retroviruses by their RT activity. I convinced them to participate in a joint study inside the unit to look again for retroviruses in human cancers. We started in 1977 with blood samples coming from different Paris hospitals and biopsy specimens.

Figure 3. HIV discoverers in the park of the Institut Pasteur Annex in Garches, near Paris, during a break of a � guards meeting” in 1987. From left to right: Jonas Salk, Jean- Claude Gluckman, Jean-Claude Chermann, Luc Montagnier, Robert Gallo, Françoise Barré-Sinoussi, Willy Rozenbaum, Charles Mérieux.

Two advances made in other laboratories boosted this search:

In Villejuif, France, Ion Gresser had prepared a potent antiserum neutralising any molecule of alpha endogenous interferon produced by individual cells. This interferon, we realised, was produced by mouse cells induced to express some of their endogenous retroviruses. Its blockade by the antiserum increased by up to 50 times the production of endogenous retroviruses in the culture medium. We could conclude that, despite the fact that endogenous retroviruses have been integrated in the genome of vertebrates for millions of years, their expression is still controlled by the interferon system, like that of exogenous viruses.

At about the same period, the discovery by Denis Morgan and Frank Ruscetti in Dr. Gallo’s laboratory of a growth factor allowing the in vitro multiplication of human T lymphocytes (TCGF, then named interleukin 2, Il2) made it possible to propagate T lymphocytes in sustained cultures.

We knew at that time that some retroviruses involved in mouse mammary tumour formation (MMTV) could not only be expressed in the tumour cells but also in the circulating lymphocytes.

Taking advantage of these two advances, we started a search for retroviruses in human cancers. Using anti-interferon serum and Il2, we focused particularly on the T lymphocyte cultures from breast cancer patients.

Indeed, in 1980, we were able to detect a DNA sequence close to that MMTV, not only in the cells of an inflammatory breast cancer (from a North African woman), but also in her cultured T lymphocytes. A second patient showed similar results.

Unfortunately, the molecular tools we had at that time could not tell us whether we were dealing with endogenous retroviral sequences or with an exogenous virus. Nowadays, having access to more powerful technologies, I am planning to reinitiate these studies.

But in 1983, the same approach, the use of anti-interferon serum, and the use of long term cultures of T lymphocytes greatly facilitated the isolation of HIV.

My involvement in AIDS began in 1982, when the information circulated that a transmissible agent – possibly a virus – could be at the origin of this new mysterious disease. At that time there were only a few cases in France, but they attracted the interest of a group of young clinicians and immunologists. They were looking for virologists, especially retro-virologists, as a likely hypothesis was that HTLV – the only human retrovirus known so far, recently described by R. C. Gallo – could be involved. Retrovirus causing leukaemia in rodents often also causes a wasting syndrome, which could be the result of secondary immune depression. This was also the case of patients suffering from leukaemia induced by HTLV.

A member of the working group, Françoise Brun-Vézinet, was a former student of the virology course that I was then directing. She called me up to organise the search for the putative retrovirus from a patient presenting with an early sign of the disease, lymphodenopathy. The patient was a young gay man who had been travelling to the USA and who was consulting Dr. Willy Rozenbaum – one of the leaders of the working group – for a swollen lymph node in the neck.

The reasoning was that if we were to find a virus at this early stage of the disease, it could be more a cause than a consequence of the immune depression.

Another incentive to start this research was a request from the producers of hepatitis B virus vaccine in the industrial subsidiary of the Institut Pasteur. They were using plasmas from American blood donors and were concerned by the risk of transmission of the AIDS agent through their procedure of viral antigen purification.

The lymph node biopsy arrived on January 3, 1983, a date which I remember well because it was also the first day of the virology course at the Institut Pasteur, which I had to introduce. I could only dissect the small hard piece at the end of the day. I dissociated the lymphocytes with a Dounce glass homogeniser and started their stimulation in culture with a bacterial mitogen, Protein A, known as an activator of B and T lymphocytes, since I did not know which fraction of lymphocytes could produce the putative virus. Three days later, I added the T cell growth factor I had obtained from a colleague working in the laboratory of Jean Dausset.

The T cells grew well. As previously established in a protocol for the search of retrovirus in human cancers, it was decided with my associates, Françoise Barré-Sinoussi and Jean-Claude Chermann, to measure the RT activity in the culture medium every 3 days. On day 15, Françoise showed me a hint of positivity (incorporation of radioactive thymidine in polymeric DNA), which was confirmed the following week.

We had evidence of a retrovirus, but this was just the beginning of a series of questions:
• Was it close to HTLV or not?
• Was it a passenger virus or, on the contrary, the real cause of the disease?

In order to answer these basic questions, we had to characterise the virus biochemically and immunologically, and to do that, we needed to propagate it in sufficient amounts. Fortunately, the virus could be easily propagated on activated T lymphocytes from adult blood donors. No cytopathic effect was observed with this first isolate, but unlike HTLV infected cultures, no transformed immortalised cell lines could emerge from the cultures, which always died after 3–4 weeks as do normal lymphocytes.

By contrast, subsequent isolates I made from culture of lymphocytes of sick patients with AIDS were cytopathic for T lymphocytes culture and – we discovered later – could be cultivated in larger amounts in tumour cell lines derived from leukaemia.

Shortly after the virus isolation, my co-workers and I were able to show that it was not immunologically related to HTLV, and in electron microscopy, it was very different from HTLV viral particles. In fact, as soon as June 1983, I noticed the quasi-identity of our virus with the published electron microscopy pictures of the visna virus in sheep, the infectious anaemia virus in horses and the bovine lymphocytic virus: it was a retrolentivirus, a sub-family of viruses causing long-lasting disease in animals without immunodeficiency.

This indicated clearly that we were dealing with a virus very different from HTLV, and my task was now to organise a team of researchers to accumulate evidence that this new virus was indeed the cause of AIDS.

It was an exciting period, since every Saturday morning when we had a meeting in my office, new data were brought by my associates favouring the causative role of the virus. The viral isolates were called LAV, for Lymphadenopathy Associated Virus, when it was isolated from patients displaying swollen lymph nodes, a frequent sign of the early phase of infection. The isolates made from patients with full-blown AIDS were called Immuno Deficiency Associated Viruses (IDAV). The latter generally grew better in T lymphocyte culture and induced the formation of large syncitia, resulting from the fusion between several infected cells. Some of them – we found out later – could also multiply in continuous cell lines of B or T cell origin. The latter property greatly facilitated the mass production of the virus for commercial use.

By September 1983, I was able to make a synthesised presentation of all our data favouring a causal link between the virus and the disease at a meeting on the HTLV organised by L. Gross and R. Gallo at Cold Spring Harbor.

This presentation was received with scepticism by a small audience (it was a late night session) and the HTLV theory still prevailed. Mentally, most attendants were not prepared to accept the idea of a second family of retroviruses (lentiretroviruses) existing in humans and causing immune deficiency, and having no counterpart in animals!

This situation is not infrequent in science, since new discoveries often raise controversy. The only problem is that it was a matter of life and death for blood transfused people and haemophiliacs, since a serologic blood test using our virus antigen was already working at laboratory scale but awaited industrial and commercial development.

This came in 1985, after two other teams of researchers, first that of Dr. Gallo at the NIH in early 1984 and that of Jay Levy in San Francisco, confirmed and extended our findings. In particular, Dr. Gallo and his associates gave more strength to the correlation between the virus and the disease, improved the detection of the antibody response and were able to grow several viral strains, including ours, in T cell lines of cancer origin. Meanwhile, my co-workers showed the tropism of the virus for CD4T cells and identified the CD4 surface molecule as the main receptor to the virus.

The rest of the story is described in the next chapter. I would just like to illustrate how I discovered what I believe are two important phenomena for explaining the destruction of the immune system induced by HIV.

During the latent phase of the infection, no virus is found in the blood. It is mostly localised in lymphocytes of lymphatic tissues and yet, we found that most of the lymphocytes present in the blood are sick! In 1987, a young visitor from Sweden, Jan Alberts, came to my lab. He wanted to cultivate human lymphocytes in a serum-free synthetic medium and to learn some technologies about HIV culture. The surprise came when we compared the viability in his medium of lymphocytes from healthy donors and those from HIV infected patients, even in their early asymptomatic stage of infection. While the former could survive several days without dying, the majority (more than 50%) of the latter died very quickly. Addition of interleukin 2 partially prevented their death.

When we used normal culture medium supplemented with foetal calf serum, the same difference was observed, although the survival time of the lymphocytes from infected patients was longer.

It did not take very long before three of my collaborators found the reason for such deaths: apoptosis. This is an active process by which the cell “decides” to die in a clean way, without releasing too many toxic compounds into the medium.

It is a physiological way of preventing abnormal proliferation of activated lymphocyte clones, but here the phenomenon was enormous and bore not only on the main cellular target of HIV infection, CD4+ T-lymphocytes, but also on cells which were not infectable by the virus, such as CD8+ T-lymphocytes, B-lymphocytes, monocytes, natural killer cells … Clearly, it was a general phenomenon, the culture simply revealing a predisposition to apoptosis of the majority of circulating blood cells, although most of them were not infected. Indeed, my collaborator Marie-Lise Gougeon found a very good relation between in vitro apoptosis and the in vivo observed drop of CD4 T cells in patients.

We have spent a lot of time trying to find the origin of this massive apoptosis, without finding a completely satisfactory explanation: the most likely is the intensive oxidative stress existing in patients since the beginning of their infection. This is also a finding I am very proud of: although oxidative stress has been – and still is – completely overlooked by AIDS researchers, it is likely to aggravate the wrong activation of the immune system at the origin of its decline and also it triggers inflammation through the production of cytokines.

Of course, the next question arises: what are the factors causing oxidative stress: viral proteins, fragments of viral DNA, co-infection with mycoplasmas? Even after 25 years, we still do not know the complete answer. But the phenomenon does exist and needs to be treated, while most AIDS clinicians do not care about it at all!

The treatment by combined antiretroviral therapy has, without doubt, changed the prognosis of this lethal disease, from a death sentence to an almost “normal” life. However, the virus is still there, ready to multiply when the treatment is interrupted, and not all HIV infected patients in the developing world have access to it. And the epidemics still kill 2–3 million people each year. It is thus absolutely necessary to resolve these problems. Basic research, as well as clinical research, has to be continued.

In addition, I realised in the 1990s that research should not only be localised in the wealthy laboratories of the developed countries, but also in southern countries where a lot of patients were suffering from AIDS and many other diseases like tuberculosis and malaria.

Too many examples showed that collaboration between northern and southern research laboratories is unequal, the south providing serum samples to be analysed in the north. This “safari” concept is wrong. There are now many young researchers trained in northern laboratories who would like to return to their own countries, but are prevented from doing so because laboratories and adequate structures are missing. Moreover, one has to be in the regions where disease proliferates to realise how complex the reality is.

This is why I joined with the former Director General of UNESCO, Federico Mayor, in initiating a foundation aimed at creating centres for research and prevention in African countries. Although the task was difficult, this concept was met with enthusiasm from colleagues and medical doctors and also found the support of governments, particularly in Côte d’Ivoire and Cameroon.

I wish that based on these pilot experiments, a whole network of similar centres could cover all the countries of the developing world where the populations are badly hit by epidemics.

Another lesson I drew from my AIDS experience was the weakening effect of oxidative stress on the immune system and its pro-inflammatory role in many chronic diseases, such as Parkinson’s, Alzheimer’s and rheumatoid arthritis: a likely consequence of chronic infections? Or both consequence and cause? There are many questions, which can be resolved only by hard work and innovative thinking. I hope to be able to continue both.

From Les Prix Nobel. The Nobel Prizes 2008, Editor Karl Grandin, [Nobel Foundation], Stockholm, 2009

This autobiography/biography was written at the time of the award and later published in the book series Les Prix Nobel/ Nobel Lectures/The Nobel Prizes. The information is sometimes updated with an addendum submitted by the Laureate.

Copyright © The Nobel Foundation 2008

To cite this section
MLA style: Luc Montagnier – Biographical. NobelPrize.org. Nobel Prize Outreach AB 2021. Wed. 30 Jun 2021. <https://www.nobelprize.org/prizes/medicine/2008/montagnier/biographical/>

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Nobel Prizes 2020

Twelve laureates were awarded a Nobel Prize in 2020, for achievements that have conferred the greatest benefit to humankind.

Their work and discoveries range from the formation of black holes and genetic scissors to efforts to combat hunger and develop new auction formats.


Biologists Uncover History of Ancient Retroviruses as Far Back as 33 Million Years Ago

A group of scientists at Boston College, Chestnut Hill, has reconstructed the natural history of a specific retrovirus lineage — ERV-Fc — that disseminated widely between 33 and 15 million years ago (Oligocene and early Miocene).

Retrovirus particles budding from rhesus macaque placenta cells. Image credit: Dorothy Feldman, via aacrjournals.org.

Retroviruses are abundant in nature and include human immunodeficiency viruses (HIV-1 and HIV-2), human T-cell leukemia viruses (HTLV-1 and -2), and the well-studied oncogenic retroviruses of mice and other model organisms, among many others.

The team’s findings, reported online on March 8, 2016, in the journal eLife, show that an ancient group of retroviruses known as ERV-Fc infected the ancestors of at least 28 mammal species — including carnivores, rodents and primates — between 15 million and 33 million years ago.

The distribution of ERV-Fc retroviruses among these ancient mammals suggests the viruses spread to every continent except Antarctica and Australia, and that they jumped from one species to another more than 20 times.

The study also places the origins of ERV-Fc at least as far back as the beginning of Oligocene.

“Unfortunately, viruses do not leave fossils behind, meaning we know very little about how they originate and evolve,” said team member Prof. Welkin Johnson, from the Boston College’s Biology Department.

“Over the course of millions of years, however, viral genetic sequences accumulate in the DNA genomes of living organisms, including humans, and can serve as molecular ‘fossils’ for exploring the natural history of viruses and their hosts.”

Using such ‘fossil’ remnants, Prof. Johnson and co-authors sought to uncover the natural history of ERV-Fc.

They were especially curious to know where and when these pathogens were found in the ancient world, which species they infected, and how they adapted to their mammalian hosts.

To do this, they first performed an exhaustive search of mammalian genome sequence databases for ERV-Fc loci and then compared the recovered sequences.

For each genome with sufficient ERV-Fc sequence, they reconstructed the sequences of proteins representing the virus that colonized the ancestors of that particular species.

These sequences were then used to infer the natural history and evolutionary relationships of ERV-Fc-related viruses.

“Mammalian genomes contain hundreds of thousands of ancient viral fossils similar to ERV-Fc,” the scientists said.

“Future work could study these to improve our understanding of when and why new viruses emerge and how long-term contact with viruses affects the evolution of their host organisms.”

William E. Diehl et al. 2016. Tracking interspecies transmission and long-term evolution of an ancient retrovirus using the genomes of modern mammals. eLife 5: e12704 doi: 10.7554/eLife.12704


RNA-binding protein IGF2BP3 targeting of oncogenic transcripts promotes hematopoietic progenitor proliferation

1 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.

2 Department of Molecular, Cellular and Integrative Physiology, UCLA, Los Angeles, California, USA.

3 Department of Molecular, Cellular and Developmental Biology, UCSC, Santa Cruz, California, USA.

4 Bioo Scientific Corporation, Austin, Texas, USA.

5 Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA.

6 Department of Women’s and Children’s Health SDB, University of Padova, Padova, Italy.

7 Jonsson Comprehensive Cancer Center (JCCC) and

8 Broad Stem Cell Research Center, UCLA, Los Angeles, California, USA.

Address correspondence to: Dinesh S. Rao, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 650 Charles E Young Drive, 12-272 Factor, Los Angeles, California 90095, USA. Phone: 310.825.1675 E-mail: [email protected]

Authorship note: J. Kumar Palanichamy, T.M. Tran, and J.M. Howard contributed equally to this work.

Find articles by Palanichamy, J. in: JCI | PubMed | Google Scholar

1 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.

2 Department of Molecular, Cellular and Integrative Physiology, UCLA, Los Angeles, California, USA.

3 Department of Molecular, Cellular and Developmental Biology, UCSC, Santa Cruz, California, USA.

4 Bioo Scientific Corporation, Austin, Texas, USA.

5 Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA.

6 Department of Women’s and Children’s Health SDB, University of Padova, Padova, Italy.

7 Jonsson Comprehensive Cancer Center (JCCC) and

8 Broad Stem Cell Research Center, UCLA, Los Angeles, California, USA.

Address correspondence to: Dinesh S. Rao, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 650 Charles E Young Drive, 12-272 Factor, Los Angeles, California 90095, USA. Phone: 310.825.1675 E-mail: [email protected]

Authorship note: J. Kumar Palanichamy, T.M. Tran, and J.M. Howard contributed equally to this work.

1 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.

2 Department of Molecular, Cellular and Integrative Physiology, UCLA, Los Angeles, California, USA.

3 Department of Molecular, Cellular and Developmental Biology, UCSC, Santa Cruz, California, USA.

4 Bioo Scientific Corporation, Austin, Texas, USA.

5 Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA.

6 Department of Women’s and Children’s Health SDB, University of Padova, Padova, Italy.

7 Jonsson Comprehensive Cancer Center (JCCC) and

8 Broad Stem Cell Research Center, UCLA, Los Angeles, California, USA.

Address correspondence to: Dinesh S. Rao, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 650 Charles E Young Drive, 12-272 Factor, Los Angeles, California 90095, USA. Phone: 310.825.1675 E-mail: [email protected]

Authorship note: J. Kumar Palanichamy, T.M. Tran, and J.M. Howard contributed equally to this work.

1 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.

2 Department of Molecular, Cellular and Integrative Physiology, UCLA, Los Angeles, California, USA.

3 Department of Molecular, Cellular and Developmental Biology, UCSC, Santa Cruz, California, USA.

4 Bioo Scientific Corporation, Austin, Texas, USA.

5 Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA.

6 Department of Women’s and Children’s Health SDB, University of Padova, Padova, Italy.

7 Jonsson Comprehensive Cancer Center (JCCC) and

8 Broad Stem Cell Research Center, UCLA, Los Angeles, California, USA.

Address correspondence to: Dinesh S. Rao, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 650 Charles E Young Drive, 12-272 Factor, Los Angeles, California 90095, USA. Phone: 310.825.1675 E-mail: [email protected]

Authorship note: J. Kumar Palanichamy, T.M. Tran, and J.M. Howard contributed equally to this work.

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1 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.

2 Department of Molecular, Cellular and Integrative Physiology, UCLA, Los Angeles, California, USA.

3 Department of Molecular, Cellular and Developmental Biology, UCSC, Santa Cruz, California, USA.

4 Bioo Scientific Corporation, Austin, Texas, USA.

5 Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA.

6 Department of Women’s and Children’s Health SDB, University of Padova, Padova, Italy.

7 Jonsson Comprehensive Cancer Center (JCCC) and

8 Broad Stem Cell Research Center, UCLA, Los Angeles, California, USA.

Address correspondence to: Dinesh S. Rao, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 650 Charles E Young Drive, 12-272 Factor, Los Angeles, California 90095, USA. Phone: 310.825.1675 E-mail: [email protected]

Authorship note: J. Kumar Palanichamy, T.M. Tran, and J.M. Howard contributed equally to this work.

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1 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.

2 Department of Molecular, Cellular and Integrative Physiology, UCLA, Los Angeles, California, USA.

3 Department of Molecular, Cellular and Developmental Biology, UCSC, Santa Cruz, California, USA.

4 Bioo Scientific Corporation, Austin, Texas, USA.

5 Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA.

6 Department of Women’s and Children’s Health SDB, University of Padova, Padova, Italy.

7 Jonsson Comprehensive Cancer Center (JCCC) and

8 Broad Stem Cell Research Center, UCLA, Los Angeles, California, USA.

Address correspondence to: Dinesh S. Rao, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 650 Charles E Young Drive, 12-272 Factor, Los Angeles, California 90095, USA. Phone: 310.825.1675 E-mail: [email protected]

Authorship note: J. Kumar Palanichamy, T.M. Tran, and J.M. Howard contributed equally to this work.

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1 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.

2 Department of Molecular, Cellular and Integrative Physiology, UCLA, Los Angeles, California, USA.

3 Department of Molecular, Cellular and Developmental Biology, UCSC, Santa Cruz, California, USA.

4 Bioo Scientific Corporation, Austin, Texas, USA.

5 Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA.

6 Department of Women’s and Children’s Health SDB, University of Padova, Padova, Italy.

7 Jonsson Comprehensive Cancer Center (JCCC) and

8 Broad Stem Cell Research Center, UCLA, Los Angeles, California, USA.

Address correspondence to: Dinesh S. Rao, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 650 Charles E Young Drive, 12-272 Factor, Los Angeles, California 90095, USA. Phone: 310.825.1675 E-mail: [email protected]

Authorship note: J. Kumar Palanichamy, T.M. Tran, and J.M. Howard contributed equally to this work.

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1 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.

2 Department of Molecular, Cellular and Integrative Physiology, UCLA, Los Angeles, California, USA.

3 Department of Molecular, Cellular and Developmental Biology, UCSC, Santa Cruz, California, USA.

4 Bioo Scientific Corporation, Austin, Texas, USA.

5 Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA.

6 Department of Women’s and Children’s Health SDB, University of Padova, Padova, Italy.

7 Jonsson Comprehensive Cancer Center (JCCC) and

8 Broad Stem Cell Research Center, UCLA, Los Angeles, California, USA.

Address correspondence to: Dinesh S. Rao, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 650 Charles E Young Drive, 12-272 Factor, Los Angeles, California 90095, USA. Phone: 310.825.1675 E-mail: [email protected].

Authorship note: J. Kumar Palanichamy, T.M. Tran, and J.M. Howard contributed equally to this work.

1 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.

2 Department of Molecular, Cellular and Integrative Physiology, UCLA, Los Angeles, California, USA.

3 Department of Molecular, Cellular and Developmental Biology, UCSC, Santa Cruz, California, USA.

4 Bioo Scientific Corporation, Austin, Texas, USA.

5 Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA.

6 Department of Women’s and Children’s Health SDB, University of Padova, Padova, Italy.

7 Jonsson Comprehensive Cancer Center (JCCC) and

8 Broad Stem Cell Research Center, UCLA, Los Angeles, California, USA.

Address correspondence to: Dinesh S. Rao, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 650 Charles E Young Drive, 12-272 Factor, Los Angeles, California 90095, USA. Phone: 310.825.1675 E-mail: [email protected]

Authorship note: J. Kumar Palanichamy, T.M. Tran, and J.M. Howard contributed equally to this work.

1 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.

2 Department of Molecular, Cellular and Integrative Physiology, UCLA, Los Angeles, California, USA.

3 Department of Molecular, Cellular and Developmental Biology, UCSC, Santa Cruz, California, USA.

4 Bioo Scientific Corporation, Austin, Texas, USA.

5 Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA.

6 Department of Women’s and Children’s Health SDB, University of Padova, Padova, Italy.

7 Jonsson Comprehensive Cancer Center (JCCC) and

8 Broad Stem Cell Research Center, UCLA, Los Angeles, California, USA.

Address correspondence to: Dinesh S. Rao, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 650 Charles E Young Drive, 12-272 Factor, Los Angeles, California 90095, USA. Phone: 310.825.1675 E-mail: [email protected]

Authorship note: J. Kumar Palanichamy, T.M. Tran, and J.M. Howard contributed equally to this work.

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1 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.

2 Department of Molecular, Cellular and Integrative Physiology, UCLA, Los Angeles, California, USA.

3 Department of Molecular, Cellular and Developmental Biology, UCSC, Santa Cruz, California, USA.

4 Bioo Scientific Corporation, Austin, Texas, USA.

5 Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA.

6 Department of Women’s and Children’s Health SDB, University of Padova, Padova, Italy.

7 Jonsson Comprehensive Cancer Center (JCCC) and

8 Broad Stem Cell Research Center, UCLA, Los Angeles, California, USA.

Address correspondence to: Dinesh S. Rao, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 650 Charles E Young Drive, 12-272 Factor, Los Angeles, California 90095, USA. Phone: 310.825.1675 E-mail: [email protected]

Authorship note: J. Kumar Palanichamy, T.M. Tran, and J.M. Howard contributed equally to this work.

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1 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.

2 Department of Molecular, Cellular and Integrative Physiology, UCLA, Los Angeles, California, USA.

3 Department of Molecular, Cellular and Developmental Biology, UCSC, Santa Cruz, California, USA.

4 Bioo Scientific Corporation, Austin, Texas, USA.

5 Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA.

6 Department of Women’s and Children’s Health SDB, University of Padova, Padova, Italy.

7 Jonsson Comprehensive Cancer Center (JCCC) and

8 Broad Stem Cell Research Center, UCLA, Los Angeles, California, USA.

Address correspondence to: Dinesh S. Rao, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 650 Charles E Young Drive, 12-272 Factor, Los Angeles, California 90095, USA. Phone: 310.825.1675 E-mail: [email protected]

Authorship note: J. Kumar Palanichamy, T.M. Tran, and J.M. Howard contributed equally to this work.

Find articles by Sanford, J. in: JCI | PubMed | Google Scholar

1 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.

2 Department of Molecular, Cellular and Integrative Physiology, UCLA, Los Angeles, California, USA.

3 Department of Molecular, Cellular and Developmental Biology, UCSC, Santa Cruz, California, USA.

4 Bioo Scientific Corporation, Austin, Texas, USA.

5 Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA.

6 Department of Women’s and Children’s Health SDB, University of Padova, Padova, Italy.

7 Jonsson Comprehensive Cancer Center (JCCC) and

8 Broad Stem Cell Research Center, UCLA, Los Angeles, California, USA.

Address correspondence to: Dinesh S. Rao, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, 650 Charles E Young Drive, 12-272 Factor, Los Angeles, California 90095, USA. Phone: 310.825.1675 E-mail: [email protected]

Authorship note: J. Kumar Palanichamy, T.M. Tran, and J.M. Howard contributed equally to this work.

Posttranscriptional control of gene expression is important for defining both normal and pathological cellular phenotypes. In vitro, RNA-binding proteins (RBPs) have recently been shown to play important roles in posttranscriptional regulation however, the contribution of RBPs to cell specification is not well understood. Here, we determined that the RBP insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is specifically overexpressed in mixed lineage leukemia–rearranged (MLL-rearranged) B-acute lymphoblastic leukemia (B-ALL), which constitutes a subtype of this malignancy associated with poor prognosis and high risk of relapse. IGF2BP3 was required for the survival of B-ALL cell lines, as knockdown led to decreased proliferation and increased apoptosis. Enforced expression of IGF2BP3 provided murine BM cells with a strong survival advantage, led to proliferation of hematopoietic stem and progenitor cells, and skewed hematopoietic development to the B cell/myeloid lineage. Cross-link immunoprecipitation and high throughput sequencing uncovered the IGF2BP3-regulated transcriptome, which includes oncogenes MYC and CDK6 as direct targets. IGF2BP3 regulated transcripts via targeting elements within 3′ untranslated regions (3′UTR), and enforced IGF2BP3 expression in mice resulted in enhanced expression of Myc and Cdk6 in BM. Together, our data suggest that IGF2BP3-mediated targeting of oncogenic transcripts may represent a critical pathogenetic mechanism in MLL-rearranged B-ALL and support IGF2BP3 and its cognate RNA-binding partners as potential therapeutic targets in this disease.

Oncogenesis in early B cell progenitors results in B cell acute lymphoblastic leukemia (B-ALL), the most prevalent hematological neoplasm in children and young adults ( 1 ). The majority of B-ALL cases exhibit genetic alterations, including recurring chromosomal rearrangements, which contribute to the heterogeneity of the observed clinical behavior ( 2 ). Specifically, B-ALL with chromosomal rearrangements of the mixed lineage leukemia (MLL) gene accounts for 5%–6% of all B-ALL cases and is associated with poor prognosis and risk of early relapse after treatment ( 3 ). MLL, which encodes a H3K4 methyltransferase, plays a critical role in the transcriptional dysregulation that occurs during leukemogenesis ( 3 , 4 ). Previously demonstrated targets of MLL include genes critical in cell survival and proliferation, such as BCL2, MYC, and CDK6 ( 5 – 7 ). Additionally, MLL is known to regulate hematopoiesis, and its expression correlates with the maintenance of hematopoietic stem cell (HSC) self-renewal and differentiation ( 8 , 9 ). In line with such a role in normal HSC function, MLL fusion proteins induce HOXA9 and MEIS1, generating leukemia that displays stem cell–like properties ( 10 – 12 ). These findings demonstrate an intimate connection between the dysregulation of gene expression and malignant transformation, and they highlight the importance of investigating key players in the regulation of gene expression.

Simplistically, gene expression may be regulated at the transcriptional and posttranscriptional levels. Recent work has revealed the complexity of the latter mechanism, which not only includes sequences intrinsic to the regulated mRNA but also other factors such as miRs, RNA-binding proteins (RBPs), and noncoding RNA ( 13 ). A complex interplay between the protein coding mRNA and the 3′ untranslated region (3′UTR) targeting miRs and RBPs has been reported ( 14 ). However, the role of gene expression regulation by RBPs in the malignant transformation of B cells is not understood. In an effort to identify critical RBP-mediated regulation in B-ALL, we began by examining a high throughput dataset generated in our laboratory, identifying the insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) as one of the top dysregulated genes in MLL-translocated B-ALL. IGF2BP3 belongs to a family of mRNA-binding proteins that consists of 3 structurally and functionally related paralogs (IGF2BP1, IGF2BP2, and IGF2BP3) that influence the cytoplasmic fate of mRNAs through localization, stability, and translation ( 15 , 16 ). IGF2BP3 is an oncofetal protein with high expression during embryogenesis, low expression in adult tissues, and reexpression in malignant tissues. In epithelial cancer, IGF2BP3 expression is associated with a range of neoplastic phenotypes ( 17 – 20 ). However, many of these studies have been largely correlative, and a bona fide functional role of IGF2BP3, or any RBP, in B cell oncogenesis has not been established.

In this study, we sought to delineate the function of IGF2BP3 in B cell leukemogenesis. We overexpressed IGF2BP3 in the BM of lethally irradiated mice and found that it plays a critical role in the proliferation of hematopoietic stem and progenitor cells, recapitulating some features of MLL-rearranged B-ALL. IGF2BP3 provided BM progenitors with a competitive survival advantage and increased their proliferation. We also found that IGF2BP3 was essential for the survival of B-ALL cell lines. We used individual nucleotide resolution cross-linking immunoprecipitation (iCLIP) to capture the in situ specificity of protein-RNA interactions and to reveal the positional context of protein binding sites across the transcriptome. In total, we identified IGF2BP3 binding sites in several hundred transcripts in 2 B-ALL cell lines. IGF2BP3 cross-linking sites are strongly enriched in the 3′UTRs of target transcripts. Of the many IGF2BP3 target transcripts, we demonstrated IGF2BP3-mediated enhancement of the expression of oncogenic targets CDK6 and MYC in B-ALL cells and hematopoietic progenitor cells in vivo. Deletion of the RNA-binding domains of IGF2BP3 abrogated target mRNA binding as well as the hematopoietic stem and progenitor expansion. Together, our studies suggest that IGF2BP3-mediated upregulation of oncogenic targets represents a key pathogenetic mechanism operant in MLL-rearranged B-ALL.

IGF2BP3 is differentially expressed in MLL-rearranged B-ALL. We have previously described a microarray experiment performed on patient B-ALL samples ( 21 ). Following correction for multiple-hypotheses testing, we performed unsupervised hierarchical clustering with significantly differentially expressed protein-coding genes (adjusted P ≤ 0.01). This generated a list of RBPs differentially expressed between the 3 cytogenetic subtypes of B-ALL used in our microarray experiments (ETV-RUNX1, E2A-PBX, and MLL-rearranged). In the list of RBPs whose expression was highest in MLL-rearranged leukemia, IGF2BP3 was among the top candidates (Figure 1A). MLL-rearranged leukemias show a stem-cell signature with high expression of stemness-associated genes like HOXA9, MEIS1, and CD44 ( 11 , 22 ). Concordant with this, we observed that HOXA9, MEIS1A, CDK6, and MYC — putative targets of the oncogenic MLL fusion protein — were significantly overexpressed in the MLL-rearranged group when compared with the other 2 subsets (Supplemental Figure 1, A–D). By performing quantitative PCR (qPCR) on a large cohort of B-ALL patient–derived BMs, we confirmed that IGF2BP3 and CD44 were highly expressed in the MLL group (total n = 134) (Figure 1, B and C). Additionally, IGF2BP3 expression was significantly higher in all B-ALL samples when compared with CD19 + B cells isolated from healthy donors (Figure 1B). To examine the dependence of IGF2BP3 on MLL-mediated effects on gene expression, we utilized I-BET151, a bromodomain and extra terminal (BET) domain inhibitor that has recently been shown to inhibit MLL-dependent gene expression ( 23 ). Treatment of RS411 — an MLL-AF4–expressing human B-ALL cell line — with I-BET151 caused a dose-dependent decrease in the expression of MYC, CDK6, and IGF2BP3 (Figure 1D). It also caused cell cycle arrest in the G1-S phase (Figure 1, E and F). These experiments confirm the overexpression of IGF2BP3 in B-ALL, with the highest expression seen in MLL-rearranged B-ALL. In line with IGF2BP3 being downstream of MLL fusion proteins, a fall in IGF2BP3 mRNA levels, along with a fall in other MLL-AF4 target levels, is seen after BET inhibition.

IGF2BP3 is overexpressed in MLL-translocated B-ALL. (A) Heatmap from the microarray data showing differentially expressed RBPs between B-ALL. IGF2BP3 is highly expressed in MLL-rearranged B-ALL. (B and C) qPCR-based confirmation of overexpression of IGF2BP3 (B) and its previously defined target, CD44 (C), in MLL-rearranged B-ALL (total n = 134 one-way ANOVA followed by Bonferroni’s multiple comparisons test **P < 0.01, ***P < 0.001, ****P < 0.0001). (DF) Treatment of RS411 cell line with increasing doses of I-BET151. (D) qPCR of MYC, CDK6, and IGF2BP3 levels in RS411 cells shows a significant decrease in all 3 mRNA levels (t test MYC, P = 0.04, P = 0.06 IGF2BP3, P < 0.0001, P = 0.1 CDK6, P = 0.005, P = 0.004 1 μM and 2 μM, respectively). (E and F) Cell cycle analysis by propidium iodide staining after I-BET151 treatment of RS411 cells shows G1 arrest secondary to CDK6 inhibition. Experiments were conducted 3× for validation. qPCR assays were normalized to actin (B and C) and RNA Pol II (D). Data represent mean ±SD. See also Supplemental Figure 1.

IGF2BP3 loss of function causes apoptosis in B-ALL cells. Given the oncogenic expression pattern of IG2BP3 in human B-ALL, we proceeded to examine its expression in 4 different B-ALL cell lines, including 697 (E2A-PBX translocated), RS411, REH (ETV-RUNX1 translocated), and NALM6 (Figure 2A). To examine the effects of IGF2BP3 knockdown, we used a lentiviral vector expressing 2 different miR formatted siRNA sequences to transduce RS411 cells (Figure 2B). Both siRNAs caused decreased IGF2BP3 expression by qPCR (Figure 2C). Propidium iodide staining showed an increase in the apoptotic sub-G1 fraction, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay showed a significant reduction in cell proliferation with IGF2BP3 knockdown, confirming the dependence of B-ALL cell lines on IGF2BP3 for survival (Figure 2, D and E). Deletion of the IGF2BP3 locus using the CRISPR-Cas9 system was also undertaken in the RS411 cell line. We utilized the LentiCRISPR system ( 24 ) with 2 different guide strands, Cr1 and Cr2, to target the IGF2BP3 locus for deletion. CRISPR-mediated deletion was confirmed by a T7 endonuclease assay (Supplemental Figure 2, D and E) with complete abrogation of IGF2BP3 protein with the guide RNA Cr2, whereas residual protein was detected with Cr1 (Figure 2F). Cr2-mediated deletion resulted in reduced cell proliferation by MTS assay, increased sub-G1 staining, and increased annexin V positivity (Figure 2, G–I). To confirm these findings, we also targeted IGF2BP3 for knockdown in NALM6 cells using a lentiviral siRNA expression system (Supplemental Figure 2A). Reduced IGF2BP3 mRNA levels were observed with both siRNAs, with si2 giving a stronger reduction in cell proliferation, as seen by the MTS assay (Supplemental Figure 2, B and C). Together, these findings highlight the importance of IGF2BP3 in maintaining cell survival and proliferation in B-ALL.

IGF2BP3 knockdown leads to disruptions of cell growth and increased apoptosis. (A) IGF2BP3 expression in human B-ALL cell lines. (B) Schematic of lentiviral vector used for IGF2BP3 knockdown. (C) IGF2BP3 knockdown, measured by qPCR shown in RS411 cell line (t test ***P = 0.0005). (D) Cell cycle analysis with propidium iodide staining. (E) MTS assay showing significantly reduced cell proliferation with IGF2BP3 knockdown. (F) Western blot showing IGF2BP3 expression after CRISPR-Cas9–mediated targeting using the Cr1 or Cr2 constructs. Cr1-mediated targeting results in some residual protein. β-Actin is used as a loading control. (G) MTS assay showing significantly reduced cell proliferation after Cr2 targeting (t test **P ≤ 0.01 for all marked comparisons). (H) Cell cycle analysis by propidium iodide staining showing increased cell death (sub-G1 peak) in Cr2-expressing cells. (I) Increased annexin V staining in Cr2-targeted cells with IGF2BP3 KO. I3, IGF2BP3. Experiments were conducted 3× for validation. Data represent mean ±SD. See also Supplemental Figure 2. UbC, ubiquitin C promoter Puro, puromycin LC, lentiCRISPR control.

Enforced expression of IGF2BP3 leads to high levels of engraftment and increased leukocytes. To directly assess the role of IGF2BP3 in the hematopoietic system, we undertook an in vivo experiment to examine the effects of enforced expression. We initially cloned the human or mouse coding sequence of IGF2BP3 into MIG, a murine stem cell virus–based (MSCV-based) retroviral vector (Figure 3A), and confirmed the functionality of the vector in expressing both IGF2BP3 and the GFP marker (Figure 3, B–D, and data not shown). A peripheral bleed of these mice at 4 weeks showed a significant increase in GFP + cells that was sustained over time in mice with enforced expression of human and mouse IGF2BP3, as measured by the congenic CD45.2 versus CD45.1 FACS markers (Figure 3, E and F). Moreover, significantly increased GFP + leukocyte cells were found, confirming increased hematopoietic output attributable to IGF2BP3 expression (Figure 3G). These changes were restricted to the B cell and myeloid cell counts in the peripheral blood following complete engraftment (Figure 3, H and I). There was no difference in the number of T cells in the periphery (Figure 3J). Interestingly, the number of platelets and red blood cells were significantly lower with IGF2BP3-enforced expression (Figure 3, K and L). Together these findings suggest that IGF2BP3 promotes overall hematopoietic output from the BM and skews BM development toward the B cell/myeloid lineage and away from T-cells, erythroid cells, and megakaryocytes. These findings, notably the preferential increase in B cells and myeloid cells, are interesting in light of the fact that MLL rearrangements are found not only in B-ALL, but also in acute myeloid leukemia and mixed lineage acute leukemia, most commonly expressing both B cell and myeloid markers.

Enforced expression of IGF2BP3 leads to enhanced engraftment and skewing toward B cell/myeloid development. (A) Schematic of the bicistronic vector used for enforced expression of IGF2BP3. (B) Western blot showing overexpression of IGF2BP3 in the murine pre–B cell line, 7OZ/3, and the human embryonic kidney cell line, 293T. (C) qPCR showing overexpression in 7OZ/3 at the mRNA level (t test **P = 0.0013). (D) FACS analysis of PB from mice 6 weeks after BMT showing successful engraftment and transduction (GFP + ). (E) FACS of PB done at 4 weeks after BMT, showing CD45.2 and GFP positivity (one-way ANOVA followed by Bonferroni’s test ****P < 0.0001). (F) Quantitation of GFP expression in the PB between 4 and 16 weeks after transplant shows that the effect is marked and sustained. (G) PB leukocyte counts at 16 weeks show increased leukocytes (one-way ANOVA with Bonferroni’s test ***P < 0.001). (H and I) Significantly higher numbers of B220 + cells (H) and CD11b + cells (I) in PB (one-way ANOVA with Bonferroni’s test ***P < 0.001). (J) FACS-based enumeration of T cells shows no significant change in circulating T cells. (K and L) Enumeration of RBCs and platelets by CBC show significant reductions (one-way ANOVA with Bonferroni’s test ***P < 0.001, ****P < 0.0001). n = 8 for all 3 groups. PB, peripheral blood BMT, BM transplantation hI3, human IGF2BP3 mI3, murine IGF2BP3 CBC, complete blood count LTR, long terminal repeat IRES, internal ribosome entry site. Three separate BMT experiments were completed for validation. Data represent mean ±SD.

Enforced expression of IGF2BP3 leads to increased progenitors in the BM with higher rates of proliferation. To further characterize these hematopoietic changes, IGF2BP3-overexpressing mice were sacrificed and hematopoietic organs were collected for analysis at 6 months after transplant. The percentage of GFP + cells was significantly higher in the IGF2BP3-overexpressing BM, similar to the peripheral blood (Supplemental Figure 3, A and B). The overall proportion of myeloid and B cells in the BM were similar between control and IGF2BP3-expressing mice (Supplemental Figure 3, C and D). qPCR from the RNA collected from the mouse BM confirmed human and mouse IGF2BP3 overexpression (Supplemental Figure 3, E and F). These changes led us to query whether there were changes in hematopoietic progenitors in the BM. Indeed, enforced expression of IGF2BP3 led to an increase in the fraction of HSCs, lymphoid-primed multipotent progenitors (LMPPs), and common lymphoid progenitors (CLPs) (Figure 4, A–C). We followed the developmental pathway of B cells by following the schema created by Hardy et al. ( 25 ). Among the Hardy fractions, we observed a significant increase in the number of cells in fractions A and B with no significant differences observed in developmentally subsequent stages (Supplemental Figure 3, H, I, and K). Hence, overexpression of IGF2BP3 led to an increase in immature hematopoietic fractions starting at the level of the HSC and on to the pro–B cell stage. To analyze the proliferation rate of the various progenitor cells in the BM, we performed intracellular staining with Ki67 in conjunction with progenitor cell stains. Ki67 was significantly higher in Lin – Sca1 + c-Kit + (LSK) population and the LMPPs in IGF2BP3-overexpressing BM (Figure 4, D and E). The CLPs did not show a significant difference in Ki67 expression (Supplemental Figure 3, G and J). These findings imply an increase in the proliferation rate of the early progenitors (HSCs and LMPPs), secondary to increased IGF2BP3 expression. Presumably, this leads to an increase in their numbers and differentiation into more committed downstream progenitors (CLPs and Hardy fractions A and B). Hence, the enforced expression of IGF2BP3 causes a preferential increase in numbers and proliferation of early progenitor populations, leading to the observed B cell– and myeloid biased leukocytosis seen in the periphery.

Analysis of BM progenitor populations from IGF2BP3-overexpressing mice. (A) Enumeration (left panel) and representative flow cytometry histograms to define HSCs from control vector– (second panel from left), human IGF2BP3– (second from right), and murine IGF2BP3–overexpressing mice (right panel). (B and C) Analysis for LMPPs and CLPs from mice noted as in A. Statistically significant differences were found in LMPPs and CLPs. (D) Intracellular Ki67 staining and FACS-based analyses, depicted in the same manner, with enumeration on the left hand side, within the LSK population enriched for HSCs. Significant differences in the high Ki67-expressing population were found. (E) Intracellular Ki67 staining and FACS analysis of proliferation in the LMPP population shows significant differences in the proliferative fraction. All comparisons used one-way ANOVA followed by Bonferroni’s test. *P < 0.05 **P < 0.01 ***P < 0.001 ****P < 0.0001. LSK, Lin – Sca1 hi c-Kit hi . Three separate BMT experiments were completed for validation. Data represent mean ±SD. See also Supplemental Figure 3. hI3, human IGF2BP3 mI3, murine IGF2BP3.

IGF2BP3 increases the number of B cells in the thymus and myeloid cells in the spleen. The normal mouse thymus is composed mostly of T cell progenitors, but in many murine models of leukemia and lymphoma, it becomes enlarged and overrun by malignant leukocytes ( 26 ). On microscopic examination, IGF2BP3 caused thymic medullary expansion with infiltration by large cells. One of the thymi expressing human IGF2BP3 had complete ablation of the cortico-medullary junction (Figure 5A). Hence, IGF2BP3 expression may serve as a precursor to malignant transformation. We observed a significantly higher percentage of GFP + B220 + B cells in the thymus when human or mouse IGF2BP3 was overexpressed, with the effect being more pronounced with mouse IGF2BP3 (Figure 5, B and C). Mice with enforced expression of mouse IGF2BP3 also showed a substantial decrease in the number of CD3ε + T cells. There was no significant difference in the level of GFP + cells in these thymi, indicating a lineage-specific expansion of B cells (Supplemental Figure 4, G–I). Four of 8 of the thymi overexpressing human IGF2BP3 and 1/8 of the thymi overexpressing mouse IGF2BP3 weighed over 50 mg, with no such increase in control mice (data not shown). Interestingly, the spleens were also enlarged following enforced expression of IGF2BP3. Differences in splenic weight were statistically significant for mice with overexpression of human IGF2BP3 with a trend noted for the mouse IGF2BP3 group (Supplemental Figure 4A). IGF2BP3 led to an increase in the number of myeloid cells in the spleen with a significant decrease in the number of CD3ε + T cells (Supplemental Figure 4, B–F). Overall, IGF2BP3 appears to tilt the hematopoietic developmental program toward the B cell and myeloid lineages. Hence, the changes seen in the BM — increased numbers and proliferation of B-lymphoid and myeloid progenitors — may result in alterations in hematopoietic homeostasis in the periphery.

Analysis of thymic cellular composition and competitive repopulation advantage from IGF2BP3-overexpressing mice. (A) Histologic images of thymic sections from mice with enforced expression of IGF2BP3. H&E staining. Scale bar: 40 μm. (B and C) Representative FACS plots and enumeration showing an increase in B220 + cells in the thymus of mice with enforced expression (one-way ANOVA with Bonferroni’s test **P < 0.01). See also Supplemental Figure 4. n = 8 for all 3 groups. Three separate BMT experiments were completed for validation. (DH) Competitive repopulation study. (D) Quantitation of GFP expression in the PB between 4 and 20 weeks after transplant in competitive repopulation study of IGF2BP3. (EG) FACS of PB (E), BM (F), and thymus (G) done at 20 weeks after BMT, showing CD45.2 and GFP positivity (one-way ANOVA followed by Bonferroni’s test **P < 0.01, ***P < 0.001). (H) qPCR confirmation of overexpression of IGF2BP3 in mouse BM (t test ***P = 0.0006). n = 8 (MIG), n = 8 (Hoxa9), n = 5 (hI3), and n = 4 (100% CD45.1). Competitive repopulation study was completed 3× for validation. Data represent mean ±SD. hI3, human IGF2BP3 mI3, murine IGF2BP3 PB, peripheral blood.

IGF2BP3 provides hematopoietic progenitors with a survival advantage. To confirm that IGF2BP3 overexpression equipped the BM progenitors with an advantage while repopulating the irradiated host mouse BM, we performed a formal competitive repopulation transplant assay. Fifty percent of CD45.1 BM cells were mixed with 50% of MIG or IGF2BP3- or HOXA9-overexpressing CD45.2 BM cells and injected into lethally irradiated mice. The IGF2BP3-overexpressing CD45.2 cells had a clear advantage over the MIG- or MIG-HOXA9–expressing cells in engraftment in the peripheral bleeds over time (Figure 5, D and E). Harvesting of the BM revealed that IGF2BP3 conferred a competitive advantage to cells in the BM (Figure 5F). This was also reflected in the thymus (Figure 5G). This corroborates our earlier data showing IGF2BP3 overexpression (Figure 5H) leading to an increase in BM progenitor numbers as well as proliferation rate.

iCLIP identifies the IGF2BP3-RNA interactome in B-ALL cells. The molecular basis of the action of RBPs has recently been investigated using iCLIP and high throughput sequencing. To gain insight into the role of IGF2BP3 in cell growth and MLL-driven leukemogenesis, we performed an iCLIP assay with this protein. iCLIP exploits the photoreactivity of nucleic acid and protein residues and nuclease fragmentation of protein-bound transcripts to capture protein-RNA interactions occurring in situ. Antibodies against IGF2BP3 were used to immunoprecipitate protein-RNA complexes from control or UV-irradiated RS411 and REH cells (input shown in Supplemental Figure 5, A and C). As expected, the immunoprecipitated material was antibody dependent and UV dependent, and the electrophoretic mobility of the complex was nuclease sensitive, as predicted for a protein-RNA complex (Supplemental Figure 5, B and D). Coprecipitated RNA was converted to cDNA libraries (Supplemental Figure 5E) and subject to high throughput sequencing. After accounting for PCR duplications, we obtained about 1 million reads per replicate, of which >70% mapped uniquely to the human genome (Supplemental Table 1). Replicate iCLIP sequences from both RS411 and REH cells were highly reproducible (Supplemental Figure 6, D and E). Compared with iCLIP cross link sites for heterogeneous ribonucleoprotein A1 (hnRNPA1 HEK cells) and with simulated data drawn randomly from the genome, IGF2BP3 cross-link sites, located at the 5′ end of the iCLIP sequences, were enriched in exons (Figure 6A).

iCLIP analysis of IGF2BP3 in human leukemia cell lines. (A) Proportion of IGF2BP3 (REH and RS411 cells), hnRNPA1(HEK cells), and simulated (Genome) cross-linking sites observed in exons, introns, or unannotated regions of the human genome. (B) Proportion of IGF2BP3 (REH and RS411 cells), hnRNPA1 (HEK cells), and simulated (mRNA background) binding sites in coding and noncoding exons. (C) Tetramer sequence enrichment at IGF2BP3–cross-linking sites in RS411 and REH cells (upper and lower panel, respectively). (D) IGF2BP3 (REH, RS411) and hnRNPA1 (HEK cells) cross-link site density relative to termination codons. (E) IGF2BP3 cross-linking density from REH (dark blue line) and RS411 (light blue line) cell lines mapped relative to annotated miR target sites. hnRNPA1 (black line) cross-linking sites from HEK293 cells are included as a control. (F and G) UCSC Genome Browser snapshot of the CDK6 and MYC 3′UTR loci, respectively. Each panel shows the exon-intron structure of the gene, sequence conservation across vertebrate species, and unique read coverage from 2 iCLIP replicates from each cell line. The maximum number of reads at each position is indicated to the left of each histogram. See also Supplemental Figures 5 and 6. (H) Western blot of protein samples from IGF2BP3 RIP. Input refers to RS411 cell lysate used for immunoprecipitation. FT is flowthrough of immunoprecipitation from either control (mouse IgG) or IGF2BP3 RIP. RIP is RNA immunoprecipitation from control (mouse IgG) or α-IGF2BP3 antibody (D-7). (I) Scatter bar plots comparing the fold-enrichment for MYC (n = 4, t test **P < 0.01) and CDK6 (n = 3, t test *P < 0.05) in control (mouse IgG) and α-IGF2BP3 antibody RNA immunoprecipitations. Levels of MYC and CDK6 are normalized to input levels from total RNA with 18s rRNA as reference.

We identified peaks using a negative binomial model (see Methods) in each biological replicate from RS411 and REH cells. In total, 849 peaks in 669 genes and 1,937 peaks in 1,149 genes were identified in REH and RS411 iCLIP experiments, respectively. Of the peaks called within mRNA sequences, the majority were located within the 3′UTRs (Figure 6B and Supplemental Figure 6, A–C). A search for sequence specificity in cross-linked regions revealed an 8- to 16-fold enrichment of GCAC tetramer–containing motifs over background in both the REH and RS411 datasets (Figure 6C). Given the apparent bias of IGF2BP3 binding sites in 3′UTR of target transcripts, we investigated the positional bias of cross-linking sites at a single nucleotide resolution relative to mRNA stop codons. The cross-link density of IGF2BP3 differed from hnRNPA1 in the 3′UTR, reaching its apex just downstream of the stop codon. These data suggested that IGF2BP3 binding sites within the 3′UTR specifically target sequences close to the stop codon in both B-ALL cell lines (Figure 6D).

Based on the distribution of IGF2BP3 cross-linking sites within 3′UTRs (Figure 6D), we hypothesized that IGF2BP3 binding sites may overlap with cis-regulatory features associated with 3′UTR-mediated gene regulation. To investigate this possibility, we examined the distribution of IGF2BP3 cross-linking sites relative to miR target sites in the 2 different cell lines. After correcting for the uniform background distribution of simulated cross-link sites, we found that IGF2BP3 cross-link density in both REH and RS411 cell lines is highly enriched within a 25-bp window centered on predicted miR target sequences (Figure 6E). By contrast, the density of hnRNPA1 cross-linking sites were uniformly distributed relative to miR target sites. Among the genes with a strong signal by iCLIP-sequencing (CLIP-Seq) were CDK6 and MYC. To confirm our findings demonstrating interaction of IGF2BP3 with these 2 targets by iCLIP (Figure 6, F and G), we utilized RNA immunoprecipitation (RIP Figure 6H), which showed enrichment of MYC and CDK6 (Figure 6I) in IGF2BP3 RIPs over mouse IgG control. These data demonstrate, for the first time to our knowledge, a comprehensive IGF2BP3 RNA interaction site atlas from human leukemia cells. This extensive interaction map reveals a strong preference of IGF2BP3 binding nonuniformly to 3′UTRs with a preference for a GCAC-rich consensus motif near miR target sites.

IGF2BP3 modulates the expression of its targets. The ENRICHR tool ( 27 ) was used to functionally classify IGF2BP3 mRNA targets in REH and RS411 cells. In both cell lines, we found that target transcripts were enriched for KEGG pathways related to ribosome biogenesis and translation (Supplemental Table 2). By contrast, transcripts classified by ENRICHR as involved in pathogenic E. coli infection, and perhaps most importantly, chronic myeloid leukemia (CML) were enriched in RS411 but not the REH dataset (Supplemental Table 2). Hence, we wanted to further explore the functional consequences of IGF2BP3 binding on gene expression at the global level. To determine if IGF2BP3 iCLIP targets (Supplemental Table 3) are regulated by IGF2BP3 expression levels, we performed RNA-Seq on control and IGF2BP3-depleted RS411 cells (Supplemental Table 4). Cross-validation of genes differentially expressed by at least 1.5-fold with RS411-specific IGF2BP3 iCLIP targets found 216 common genes. Of these targets, the majority showed decreased expression of IGF2BP3 iCLIP targets with IGF2BP3 depletion (157 decreased vs. 59 increased Figure 7A). Using the ENRICHR tool to classify the common genes cohort, OMIM disease gene ontology analysis (GO analysis) revealed genes associated with leukemogenesis (Figure 7A black circles), including CDK6 and MYC. GO analysis for downregulated IGF2BP3 iCLIP targets revealed genes associated with posttranscriptional control, hematopoietic cell differentiation, and chromatin modification (Figure 7C). KEGG Pathway analysis also suggested these targets are involved in cell cycle and a variety of cancer pathways (Figure 7C). By contrast, the upregulated IGF2BP3 iCLIP targets revealed genes primarily associated with translation and protein localization (Figure 7B).

Cross-validation of IGF2BP3 iCLIP targets with IGF2BP3-sensitive differentially expressed genes. (A) Volcano plot of differentially expressed genes (blue dots) determined using DESeq analysis on RNA-Seq samples from control and IGF2BP3 knockdown RS411 cells (as described in Figure 2). Differentially expressed genes identified as IGF2BP3 targets by iCLIP are highlighted (orange dots). Dots demarcated by black outlines are leukemogenic genes by GO analysis of OMIM-associated disease pathways. Dotted lines represent 1.5-fold–change in expression (vertical lines) and P < 0.05 cutoff (horizontal line). (B and C) GO analysis of gene subgroups showing increased expression (B) and decreased expression (C) with IGF2BP3 knockdown using ENRICHR gene list enrichment analysis webtool. Term lists used in this analysis were GO_Biological_Processes and KEGG to determine enriched processes and pathways from our cross-validated list of 269 IGF2BP3-targeted and -sensitive genes. Vertical dotted lines represent P value cutoff (P < 0.05). KD, knockdown.

CDK6 and MYC mRNAs are targets of IGF2BP3 in vitro and in vivo. Among the many IGF2BP3 mRNA targets, CDK6 and MYC are very important in the pathogenesis of MLL-translocated B-ALL ( 6 , 7 ). In our patient data set, MLL-translocated cases of B-ALL demonstrated high levels of these genes, in line with their proposed role as targets bound by and regulated by IGF2BP3 (Supplemental Figure 1, A and B). To test the hypothesis that IGF2BP3 posttranscriptionally regulates the expression of CDK6 and MYC via binding sites in their 3′UTR, we generated a series of vectors using a dual-luciferase reporter system containing the respective UTRs (Figure 8A). The CDK6 3′UTR (10 kb) was cloned in 5 separate pieces (CDK6-1 to CDK6-5). Cotransfection of the luciferase vectors with IGF2BP3 resulted in increased luciferase activity in CDK6-3 to CDK6-5, as well as the MYC 3′UTR reporters (Figure 8B). This, along with the iCLIP and RNA-Seq data, confirms IGF2BP3 binding to the 3′UTRs of these genes may stabilize the mRNA and/or enhance translation. Mutation of one of the binding sites within the MYC 3′UTR, designated MYC Δ3, resulted in a small but reproducible attenuation of IGF2BP3-dependent enhancement of the MYC 3′UTR reporter (Figure 8C). However, mutation of individual IGF2BP3 binding sites in the CDK6 3′UTRs, followed by IGF2BP3 overexpression and a luciferase assay, did not show any significant difference (data not shown). These findings may result from one of the following possibilities: (i) binding may not be sufficient for alterations of gene expression, or (ii) cooperative binding at multiple sites may be required for an effect on gene expression. Indeed, some parallels have been seen in the case of miRs, where increased repression is seen in targets with multiple binding sites ( 28 ). Nonetheless, IGF2BP3 binding would be predicted to lead to increased levels of CDK6 and MYC in cells overexpressing IGF2BP3 and to decreased levels in knockdown cells.

CDK6 and MYC are targeted by IGF2BP3. (A) Schematic of the luciferase assay used. (B) Luciferase assay showing targeting of the CDK6 and MYC 3′UTRs by IGF2BP3 (t test *P = 0.05 P = 0.0250 CDK6-4, P = 0.0475 CDK6-5, P = 0.0165 MYC). (C) Deletion of the MYC 3′UTR binding sites of IGF2BP3 led to modest but significantly decreased luciferase activity (t test *P = 0.05 P = 0.0120 MYC, P = 0.0294 MYC Δ3). (D and E) CDK6 analysis of BM progenitors shows a significantly increased amount of CDK6 protein in the GFP + BM cells (t test *P = 0.0213) (D) but not in the GFP – (E) cells. (F and G) Intracellular staining for MYC reveals significantly increased levels in the GFP + BM cells after IGF2BP3-enforced expression (t test ****P < 0.0001) (F) but not in the GFP – (G) cells. n = 8 for all 3 groups. (H and I) After Igf2bp3 knockdown in 7OZ/3 cells (t test IGF2BP3 si1 and si2, respectively ***P = 0.0007, ***P = 0.0005), there is reduced expression of CDK6 and MYC protein (H). In the RS411 cell line, Western blot confirmed knockdown of IGF2BP3 protein and reduced expression of CDK6 protein (I). Experiments were conducted 3× for validation. Data represent mean ±SD. See also Supplemental Figure 7. hI3, human IGF2BP3 PE, phycoerythrin.

To elucidate whether IGF2BP3 targets CDK6 and MYC in vivo, we performed intracellular staining in BM cells from mice with enforced IGF2BP3 expression and measured mean fluorescence intensity (MFI) by flow cytometry. BM GFP + cells derived from human IGF2BP3–expressing mice had increased MFI for CDK6 and MYC (Figure 8, D and F). As a control, GFP – cells of both groups did not show any difference (Figure 8, E and G). To complement these in vivo data, we analyzed CDK6 and MYC protein levels in cell lines where IGF2BP3 was knocked down using siRNAs previously. There was a substantial decrease in CDK6 protein levels in RS411 cells after IGF2BP3 knockdown (Figure 8I). Similarly, Igf2bp3 knockdown in the murine pre–B 7OZ/3 cell line led to a reduction in CDK6 and MYC protein (Figure 8, H and I). These findings are in agreement with the RNA-Seq data and demonstrate a conserved function for IGF2BP3 (Figure 7A).

At the mRNA level, IGF2BP3 overexpression led to a slight but significant increase in Myc mRNA levels, but not in Cdk6, in bulk BM (Supplemental Figure 7, A and B). It is possible that an increase in Cdk6 mRNA may not be detected due to the heterogeneity of the BM cells or because effects on mRNA stability by IGF2BP3 may be mRNA specific. Similarly, murine 7OZ/3 cells showed a modest increase in the Cdk6 mRNA levels when murine IGF2BP3 was overexpressed (Supplemental Figure 7, C–E). These findings corroborate IGF2BP3 binding and subsequent translational augmentation of these target genes. Overall, these experiments demonstrate targeting of CDK6 and MYC by IGF2BP3 in a variety of systems, and that may underlie the observed phenotypic effects of enforced expression.

Mutated IGF2BP3 does not increase hematopoietic progenitor numbers in vivo. IGF2BP3 has 6 RNA-binding domains: 2 RNA recognition motif domain (RRM) and 4 K homology domain (KH) domains that are predicted to mediate the RNA-binding function of IGF2BP3. We created 2 deletion mutants: the KH mutant, containing only the 4 KH domains and devoid of both the RRM domains, and the RRM mutant, containing the 2 RRM domains and lacking the 4 KH domains (Figure 9, A and F). A murine BM transplant was performed with MIG, WT IGF2BP3, KH, and RRM mutants into lethally irradiated recipients. Unlike the WT protein, enforced expression of these mutant proteins failed to cause enhanced hematopoiesis or the skewing toward the B cell and myeloid lineages that we observed previously. The mutant recipient mice showed reduced engraftment (Figure 9, B and C). B cell and myeloid cell counts in the periphery showed a trend toward normalization in IGF2BP3 mutant mice (Figure 9, D and E). A luciferase assay using the MYC 3′UTR and comparing WT and mutant IGF2BP3 demonstrated decreased luciferase activity, confirming the idea that these mutants no longer bind to and/or stabilize target mRNAs (Figure 9G). Interestingly, one study reported that the role of KH domains is not well understood in IGF2BP3, among the various IGF2BP family members ( 29 ). Although the precise determinants of RNA binding are not known, our findings provide an impetus to perform detailed structure-function analyses to elucidate domains and residues important for IGF2BP3 function. Together with our high throughput data, these experiments provide some of the first comprehensive studies that link the RNA binding function of this protein to organism-level phenotypes.

Expression of IGF2BP3 RNA-binding domain mutants in vivo and in vitro. (A) Schematic of IGF2BP3 with its binding domains and the respective mutants (KH and RRM). (B) Time course of normalized engraftment to MIG in PB between 4 and 20 weeks after transplant. (C) FACS of PB done at 4 weeks after BMT, showing CD45.2 and GFP positivity (one-way ANOVA followed by Bonferroni’s test ***P < 0.001). (D) B cells in PB 16 weeks after transplant. (E) Myeloid cells in PB 12 weeks after transplant. n = 8 for all groups. Mutant BMT experiment was completed twice for validation. (F) Western blot confirmed expression of IGF2BP3 (64 kDa), KH (47 kDa), and RRM (22 kDa) proteins in 7OZ/3 using anti-T7 (top panel) and anti-IGF2BP3 (bottom) antibodies. Actin used as a loading control. (G) Luciferase assay shows increased luciferase activity for the MYC 3′UTR when cotransfected with hI3 and decreased luciferase activity for the MYC 3′UTR when cotransfected with KH and RRM mutants (t test hI3, K,H and RRM ***P < 0.001 ****P < 0.0001). Experiment was completed 3×. Data represent mean ±SD. hI3, human IGF2BP3 PB, peripheral blood.

The molecular mechanism of leukemogenesis mediated by MLL-fusion proteins is not completely understood, despite knowledge of the translocation and the resulting fusion proteins for 2 decades. Increasingly, it is recognized that secondary, nongenetic changes are necessary for elaboration of leukemia. For example, deregulation of epigenetic marks by DOT1L, which is recruited by the MLL-AF4 fusion protein, is a key oncogenic mechanism ( 30 ). In this study, we found that an RBP, IGF2BP3, is overexpressed in cases of B-ALL that carry a translocation of the MLL gene. We propose that posttranscriptional gene expression dysregulation may also play an important role in leukemogenesis. This is borne out by prior studies of another RBP, musashi-2, which demonstrated that it was highly expressed in acute myeloid leukemia and that its overexpression could collaborate with BCR-ABL to promote myeloid leukemogenesis ( 31 ). Another recent example is the HuR protein, which is upregulated in multiple epithelial malignancies and is known to stabilize its targets by binding to AU-rich elements within the 3′UTR of target mRNAs ( 32 ). Our findings here extend the repertoire of dysregulated RBP expression to B-ALL, providing new insights into this disease.

The mechanism of IGF2BP3 upregulation in MLL-AF4–expressing leukemia is an important question. Our studies showed that a BET domain inhibitor could downregulate IGF2BP3 in RS411, and this is thought to specifically target MLL-mediated transcription at low doses ( 23 ). Previous reports have also shown IGF2BP3 to be upregulated after MLL-AF4 overexpression in murine BM cells ( 4 ). Interestingly, an indirect ChIP-Seq assay done in SEM cells (which carry the MLL-AF4 translocation) showed binding of the fusion protein to the genomic locus containing IGF2BP3 ( 33 ). Therefore, it is tempting to speculate that IGF2BP3 is a direct transcriptional target of MLL fusion proteins. However, IGF2BP3 is also overexpressed in most of the B-ALL cell lines tested, various mature B cell neoplasms ( 34 – 37 ), and a number of epithelial malignancies. Furthermore, differential regulation of this protein has been observed in B-ALL ( 38 ). Hence, the mechanism of its upregulation may include other oncogenic pathways.

Previously, knockdown of IGF2BP3 in epithelial cell lines has been shown to reduce cell proliferation and cause apoptosis ( 18 , 37 , 39 ). We found that knockdown or deletion of IGF2BP3 by siRNA or by the CRISPR-Cas9 system led to reduced cell proliferation and increased apoptosis in RS411, an MLL-AF4–expressing cell line, and NALM6, another B-ALL cell line that shows high levels of IGF2BP3. These findings highlight the important role that posttranscriptional gene regulation can play in maintaining the malignant behavior of B-ALL cells. It will be of great interest to study whether B-ALL with low expression levels of IGF2BP3 demonstrate an altered RNA-binding repertoire, with an aim toward illuminating key mRNAs that mediate downstream effects of IGF2BP3.

To study the pathogenetic function of this protein, we also created the first in vivo model of IGF2BP3-enforced expression in the murine hematopoietic system. We found that IGF2BP3 increases the number of HSCs, LMPPs, and CLPs in the BM with a concomitant increase in the proliferation rate of HSCs and LMPPs. IGF2BP3 conferred a competitive reconstitution advantage and skewed mouse hematopoiesis toward the B cell and myeloid lineage in the periphery, with leukocytosis in the peripheral blood, atypical B cell infiltration into the thymic medulla, and increased myeloid cells in the spleen. Although the mice did not develop overt leukemia by 6 months after transplantation, these abnormal developmental features are similar to those seen early in MLL-driven leukemogenesis, which include the expansion of the B cell and myeloid lineage. Such IGF2BP3-driven effects on stem and progenitor cells and differentiation fates may extend beyond the hematopoietic system. IGF2BP3 can cause remodeling of the exocrine pancreas when specifically overexpressed in that tissue and is highly expressed in cancer stem cells/tumor-initiating cells in hepatocellular carcinoma ( 40 , 41 ).

It has been known that IGF2BP3 targets mRNAs for IGF2, CD44, and the transcription factor HMGA2 ( 16 , 42 ). Further reports on HMGA2 and IGF2BPs have suggested that they may play a role in the self-renewal potential of fetal HSCs ( 43 , 44 ). Let-7 miR has been known to inhibit tumor cell migration and invasion by targeting IGF2BP3 ( 45 ). Thus, IGF2BP3 may function in regulating both developmental and oncogenic processes. To examine the molecular mechanism behind the observed disruptions in cellular and hematopoietic homeostasis, we performed iCLIP-Seq analyses. Remarkably, we identified numerous RNA targets of IGF2BP3 in B-ALL cell lines that were known targets of MLL, including CDK6 and MYC. CDK6 has recently been implicated as a highly important target in B-ALL, and inhibition of CDK6 may form the basis of a new therapeutic intervention in B-ALL ( 7 ). MYC is a quintessential oncogene, and its overexpression plays a direct, causative role in many B cell leukemias and in lymphoma. Reporter assays confirmed that the interaction of IGF2BP3 with the 3′UTRs of MYC and CDK6 are functionally important. IGF2BP3 with deletions of RNA-binding domains failed to bind and stabilize MYC mRNA in the luciferase reporter assay. However, when single IGF2BP3 binding sites were deleted in the MYC and CDK6 3′UTR, the majority of these deletions failed to reverse the phenotype in the reporter assay. The complexity of 3′UTR targeting is illustrated by the variety of RBPs and noncoding RNAs that have been reported to bind to the MYC and CDK6 3′UTRs ( 46 – 49 ). Hence, RBP action on its cognate targets is likely dose dependent and cooperative multiple RBPs have to bind to the same 3′UTR at different locations to exert an effect. In vivo and in vitro targeting of CDK6 and MYC protein was confirmed in the mouse BM and leukemia cell lines, in both the loss- and gain-of-function settings. Hence, we have validated iCLIP as a powerful technique for uncovering disease-relevant targets. It is important to point out that there are likely mRNAs other than MYC and CDK6 that interact with IGF2BP3, including those we identify here, that play important roles in cellular proliferation and/or differentiation. This is illustrated by our attempt to rescue IGF2BP3 knockdown–mediated cell death by cotransducing RS411 cells with MYC constructs containing or lacking the 3′UTR. MYC alone was not sufficient to rescue changes in the cell cycle engendered by the loss-of-function of IGF2BP3 (data not shown).

The iCLIP-Seq analyses provide a global view of the transcriptome regulated by IGF2BP3. Prior studies have demonstrated single or a few targets for this protein, but our work here shows several hundred mRNAs bound by this protein. To refine our list of possible transcripts, we combined this biochemical target identification with gene expression studies in cells with IGF2BP3 loss of function. Distinct sets of genes that were bound by IGF2BP3 showed upregulation or downregulation, with downregulated genes demonstrating enrichment for pathways related to cell cycle and leukemia. These findings suggest that IGF2BP3 upregulation in B-ALL serves to stabilize leukemogenic gene expression. Like other modes of posttranscriptional gene expression regulation, the actions of IGF2BP3 are dependent on the cellular transcription program. IGF2BP3 is itself induced by MLL-AF4, and it binds to and upregulates several mRNAs (e.g., CDK6 and MYC) that are also induced by MLL-AF4. In this way, IGF2BP3 may reinforce certain aspects of the gene expression program, thereby sustaining oncogenesis. Interestingly, however, enforced expression of this protein alone also led to proliferation of hematopoietic progenitor cells, suggesting that specificity of RNA stabilization and/or translational enhancement can direct development and influence lineage choice and proliferation.

As oncogenic mutations and translocations are being catalogued via various high throughput sequencing approaches, it is also becoming apparent that single genetic abnormalities are insufficient to cause oncogenesis. Prior work has implicated nongenetic mechanisms, including epigenetic regulation, as key factors in the pathway to full-blown oncogenesis. Here, our studies have uncovered a posttranscriptional mechanism of stabilizing oncogenic gene products such as CDK6 and MYC. This mechanism requires further study and clarification and is likely to yield important insights into the nature of gene expression regulation in leukemogenesis. With targeted therapies emerging against CDK6 and MYC, it will become critical to consider the role of posttranscriptional mechanisms in regulating oncogene-mediated gene expression programs. Moreover, therapeutic avenues are also suggested by the current study, including the generation of sink RNAs to block the binding of IGF2BP3 to its targets or small molecule inhibitors of this protein designed to block its RNA-binding function. Given the expression of IGF2BP3 in many different types of cancer, it will be of great interest to define whether the repertoire of bound mRNAs is similar in tumors of distinct histogenesis and whether conserved oncogenic pathways can be targeted in hematologic and nonhematologic malignancies.

Patient samples, CD19 + cell isolation, and microarray data analysis. All procedures and protocols related to these have been previously described, and the microarray data set has been made publically available (NCBI’s Gene Expression Omnibus [GEO GSE65647]) ( 21 ).

Apoptosis, proliferation, and cell cycle analysis. To measure cell proliferation, 2,000–4,000 cells per well were cultured in 96-well plates. MTS reagents were added according to the manufacturer’s instructions (Promega CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit) and cells were incubated at 37°C, 5% CO2 for 4 hours before absorbance was measured at 490 nm. To measure apoptosis, cells were stained with APC-tagged annexin V and analyzed by flow cytometry. For cell cycle analysis, cells were fixed with 70% ethanol and stained with 1× propidium iodide solution in PBS and analyzed using flow cytometry.

qPCR. RNA collected from human samples was reverse transcribed using iScript reagent (Quanta BioSciences). RNA from cell lines was reverse transcribed using qScript (Quanta BioSciences). qPCR was performed with the StepOne Plus Real-Time PCR System (Applied Biosystems) using PerfeCTa SYBR Green FastMix reagent (Quanta BioSciences). The qPCR primer sequences used are listed in Supplemental Table 5.

Western blot. Cells were lysed in RIPA buffer (Boston BioProducts) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Equal amounts of protein lysate (as quantified by using bicinchoninic acid protein assay, BCA [Thermo Scientific]) were electrophoresed on a 5%–12% SDS-PAGE and electroblotted onto a nitrocellulose membrane. Antibodies used were c-MYC rabbit polyclonal (catalog 9402, Cell Signaling Technology), CDK6 rabbit monoclonal (catalog DCS83, Cell Signaling Technology), IGF2BP3 goat polyclonal (catalog sc-47893, Santa Cruz Biotechnology Inc.), T7 rabbit polyclonal (catalog AB3790, Millipore), and β-actin (catalog AC15, Sigma-Aldrich) mouse monoclonal. Secondary HRP-conjugated antibodies (Santa Cruz Biotechnology Inc.) and SuperSignal West Pico kit (Pierce Biotechnology) were used for enhanced chemiluminescence-based detection.

Cell culture, plasmids, and spin infection. mmu-miR-155 or hsa-miR-21 formatted siRNAs were cloned between NotI and BamHI sites of a pHAGE6 lentiviral vector (pHAGE6-CMV-siRNA-UBC-ZsGreen) or between NotI and BamHI sites of a modified pHAGE6 vector downstream of GFP (CMV-GFP-siRNA-UBC-Puromycin). In mouse cell lines, siRNAs and protein-coding inserts were cloned into MGP and MIG vectors ( 50 ). The WT IGF2BP3 and deletion mutants (KH and RRM) with a T7 epitope tag were cloned between BglII and XhoI sites in a MIG-based vector (MSCV-T7 tag-Mutant CDS-IRES-GFP See Supplemental Table 5). For CRISPR-Cas9–mediated targeting, guide RNAs were designed using the Zhang lab website (http://crispr.mit.edu/) and cloned into the LentiCRISPR vector ( 24 ). RS411 cells were spin-infected at 30°C for 90 minutes in the presence of polybrene. Cells were selected with 5 μg/ml of puromycin for 7 days and used for cell proliferation and apoptosis assays. The human B-ALL cell lines RS411 (MLL-AF4 translocated ATCC CRL-1873), NALM6 (gift from K. Sakamoto, Stanford University, Stanford, California, USA), 697 (E2A-PBX1-translocated gift from K. Sakamoto), Reh (TEL-AML1-translocated ATCC CRL-8286), murine pre-B leukemic cell line 7OZ/3 (ATCC TIB-158), and HEK 293T cell line (ATCC CRL-11268) were grown in their corresponding media at 37°C in a 5% CO2 incubator. Lentiviruses and retroviruses were generated as previously described ( 50 , 51 ).

BM transplant and competitive repopulation assay. BM was harvested and spin-infected from 8-week-old CD45.2 + donor C57BL/6J female mice as previously described ( 50 ). We also transplanted donor mice with T7 epitope–tagged IGF2BP3 and mutant constructs, including deletion mutants lacking RNA-bining domains. Eight-week-old CD45.1 + recipient B6.SJL-Ptprc-Pep3/BoyJ female mice were lethally irradiated and injected with donor BM 6 hours after irradiation. Eight mice were used per group. In some experiments, the normalized engraftment was calculated as the percent engraftment/transduction efficiency. For competitive repopulation experiments, 8-week-old CD45.1 + donor B6.SJL-Ptprc-Pep3/BoyJ female mice and 8-week-old CD45.2 + donor C57BL/6J female mice were harvested for BM. CD45.2 + BM was infected with viruses overexpressing MIG, HOXA9, or IGF2BP3. CD45.1 + and CD45.2 + BM cells were mixed in a ratio of 1:1 and injected into lethally irradiated 8-week-old CD45.2 + recipient C57BL/6J female mice. A negative control group had 100% CD45.1 + BM cells injected into lethally irradiated 8-week-old CD45.2 + recipient C57BL/6J female mice. Mice were bled at 4, 8, 12, 16, and 20 weeks after BM injection for analysis of the peripheral blood. All mice were purchased from the Jackson Laboratory and housed under pathogen-free conditions at UCLA.

Flow cytometry. Blood, BM, thymus, and spleen were collected from the mice under sterile conditions at 27 weeks after transplant. Single cell suspensions were lysed in red blood cell lysis buffer. Fluorochrome-conjugated antibodies were used for staining. The list of antibodies used is provided in Supplemental Table 6. For intracellular staining, after initial staining with surface marker antibodies and fixation with 1% paraformaldehyde (PFA), cells were incubated with antibodies against intracellular antigens (Ki67, Cdk6, and Myc) in 1% Triton containing MACS buffer. After 30 minutes of staining at 4°C, cells were washed twice with PBS and fixed with 1% PFA. Flow cytometry was performed at the UCLA JCCC and at the Broad Stem Cell Research Flow Core. Analysis was performed using FlowJo software.

Histopathology. Fixation and sectioning has been described previously ( 51 ). Analysis was performed by a board certified hematopathologist (D.S. Rao).

iCLIP. iCLIP was performed as previously described ( 52 ). Briefly, RS411 or REH cells were irradiated with UV-C light to form irreversible covalent cross-link proteins to nucleic acids in vivo. After cell lysis, RNA was partially fragmented using micrococcal nuclease, and IGF2BP3-RNA complexes were immunopurified with anti-IGF2BP3 antibody (MBL International Corporation) immobilized on protein A–coated magnetic beads (Invitrogen). After stringent washing and dephosphorylation (FastAP, Fermentas), RNAs were ligated at their 3′ ends to a 3′ preadenylated RNA adaptor, radioactively labeled, run using MOPS-based protein gel electrophoresis, and transferred to a nitrocellulose membrane. Protein-RNA complexes 15–80 kDa above free protein were cut from the membrane, and RNA was recovered by proteinase K digestion under denaturing (3.5 M urea) conditions. The oligonucleotides for reverse transcription contained 2 inversely oriented adaptor regions adapted from the Bioo NEXTflex small RNA library preparation kit (Bioo Scientific), separated by a BamHI restriction site and a barcode region at their 5′ end containing a 4-nt experiment-specific barcode within a 5-nt random barcode to mark individual cDNA molecules. cDNA molecules were size purified using denaturing PAGE, circularized by CircLigase II (Epicenter, Illumina), annealed to an oligonucleotide complementary to the restriction site and cut using BamHI (New England Biolabs Inc.). Linearized cDNAs were then PCR-amplified using Immomix PCR Master Mix (Bioline) with primers (Bioo Scientific) complementary to the adaptor regions and subjected to high throughput sequencing using Illumina HiSeq. A more detailed description of the iCLIP protocol has been published ( 53 ). The data discussed in this publication have been deposited in NCBI’s GEO ( 54 ) (GSE76931).

iCLIP data analysis. Following transcriptomic and genomic alignment with “TopHat2” ( 55 ), individual reads were truncated to their 5′ ends to represent the site of cross-linking. To denote specific sites of protein-RNA interaction, 30-bp regions of enrichment over background were determined using “Piranha” ( 56 ) in zero-truncated negative binomial mode with a custom local covariate. The covariate was calculated by uniformly distributing the cross-link number of each 30-bp bin across the neighboring 6 bins (3 on each side) to control for regions of overall higher depth as an indication of protein-RNA interaction. Adjacent bins with significant P values from Piranha (α < 0.05) were combined into single regions. Only overlapping peak regions found to be statistically significant in all 3 replicates were considered biologically reproducible candidates for further analysis. To derive the intragenic distributions of iCLIP-Seq sites, we queried the UCSC Genome Browser MySQL database for hg19 ( 57 ) and determined the nearest overlapping gene based first on CCDS gene annotations ( 58 ) to determine the canonical ORF and second on Gencode V19 comprehensive for other features ( 59 ). Following this, the nearest intragenic anchor (transcription start site, start codon, 5′ splice site, 3′ splice site, stop codon, and polyadenylation site) was recorded, and the spliced distance to the nearest ORF boundary was calculated. As a background control, uniformly distributed cross-link sites were simulated by pseudo-random intervals from hg19 using “bedtools” ( 60 ). The intragenic distribution of these sites was determined following the same methodology as the iCLIP cross-link sites.

To determine the binding specificity of IGF2BP3, a 10-nt window surrounding each cross-link site occurring within a biologically reproducible and statistically significant peak from a last exon was extracted, and the counts of each n-mer (4 through 6) were calculated. Random 20-bp intervals from a window of 100–300 nucleotides adjacent to each cross-link site were included as a normalized frequency of n-mers to represent the background probability (p) of observing each n-mer. For each n-mer size, the probability of observing k occurrences of some n-mer out of N total observations is binomially distributed (k

Bin[p, N]) and Poisson approximated, given sufficiently large values for N (>1,000) and small values of P (<0.01). Individual n-mers with Poisson-approximated P values significant at a 5% false discovery rate ( 61 ) were then aligned with one another and grouped into motifs using k-medoid clustering for optimal values of k (using average silhouette width). The frequency of occurrence of each nucleotide was then plotted in a position-specific manner for each motif cluster.

RNA immunoprecipitation. Protein A Dynabeads (Invitrogen) in 100 mM sodium phosphate buffer (pH 8.1) were treated with either IgG mouse antibody (Jackson ImmunoResearch Laboratories Inc.) or α-IGF2BP3 (Santa Cruz Biotechnology Inc.) for 1 hour at 4°C. Beads were then washed 3× with RSB-100 buffer (10 mM Tris-Cl [pH 7.4], 100 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40). Cytoplasmic lysates were prepared from suspension RS411 cells in RSB-100 containing RNase inhibitors. Lysate supernatants were combined with the bead/antibody, rotated at 4°C overnight, and washed, and a portion was removed for Western blot analysis. Pelleted beads were resuspended in Proteinase K buffer (100 mM Tris-HCl [pH 7.4], 50 mM NaCl, 10 mM EDTA [pH 8.0]). Samples were treated with RQ DNase I (Promega) and then treated with Proteinase K (Ambion) both were incubated at 37°C. The RNA was extracted with acid phenol–chloroform and precipitated with sodium acetate, absolute ethanol, and coprecipitate GlycoBlue (Ambion). RNA was recovered by centrifugation, washed, and resuspended in RNase-free water. Quantity and quality were checked with Nanodrop and Total RNA NanoBioanalyzer kit (Agilent Technologies). RNA samples (200 μg of each sample triplicates of both IgG controls and α-IGF2BP3 immunoprecipitations) were subjected to reverse transcription using the High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific) and subsequent qPCR using the Lightcycler 480 (Roche Diagnostics).

RNA sequencing experiments. RNA was purified from both cytosolic fractions of IGF2BP3-depleted or control RS411 cells using TRI-Reagent LS (Sigma-Aldrich), converted to double-stranded libraries using the NEXTflex Rapid Directional qRNA-Seq Library Prep Kit (Bioo Scientific), and sequenced using Illumina HiSeq 2500 platform. High throughput RNA sequencing data generated by Illumina HighSeq 2500 and corresponded to approximately 30–115 million reads per sample (Supplemental Table 4) was mapped to hg19 build of the human genome (Feb. 2009 GRCh37, NCBI Build 37.1) using Bowtie and TopHat software ( 62 , 63 ). All data collection and parsing was performed with Perl, and statistical analyses with R, version 2.14.1. All external library packages used are available on CPAN or CRAN. Differentially expressed genes were identified using DESeq. The data are accessible through NCBI’s GEO (GSE76931).

Luciferase assays. The CDK6 3′UTR is approximately 10-kb long while the MYC 3′UTR is approximatley 300 bp. MYC Δ3 and MYC Δ4 are mutant MYC 3′UTRs lacking IGF2BP3 binding sites determined from iCLIP data. Primers were designed to exclude these binding sites, fusion PCR was completed, and MYC and MYC Δ 3′UTRs were cloned downstream of firefly luciferase in the pmirGlo vector between the SacI and XhoI sites ( 64 ). The CDK6 3′UTR was divided into 5 pieces (CDK6 1–5

2 kb each) and cloned individually downstream of the firefly luciferase. 293T cells were transfected with the pmirGlo, 3′UTR, or Δ 3′UTR containing reporter vectors along with the MIG empty vector, MIG-hIGF2BP3 overexpression vector, MIG-T7-hIGF2BP3 overexpression vector, or MIG-T7-KH/RRM mutant vectors at a 1:10 ratio (50:500 ng). Cotransfections were performed with Lipofectamine 2000 (Invitrogen) as per the manufacturer’s instructions. Cells were lysed after 24 hours, substrate was added, and luminescence was measured on a GloMax-Multi Jr (Promega). The ratio of firefly to Renilla luciferase activity was calculated for all samples. The hIGF2BP3/MIG or mutant/MIG luminescence for the pmirGlo empty vector was used as a normalization control.

Statistics. Data represent mean ±SD for continuous numerical data. One-way ANOVA followed by Bonferroni’s multiple comparisons test or 2-tailed Student’s t tests were performed using GraphPad Prism software and applied to each experiment as described in the figure legends. A P value less than 0.05 was considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Study approval. Written informed consent was obtained from all of the parents of the patients by the Italian Association of Pediatric Hematology and Oncology (AIEOP) and the Berlin-Frankfurt-Muenster (BFM) ALL-2000 trial. The University of Padova IRB approved all procedures, and the study was considered exempt from review at UCLA. Peripheral blood mononuclear cells derived from anonymized donors were obtained from the Center for AIDS Research Virology Core Lab at UCLA or following diagnostic work from the UCLA Department of Pathology and Laboratory Medicine with written consent and IRB approval. All mouse experimental procedures were conducted with the approval of the UCLA Chancellor’s Animal Research Committee (ARC).

JKP designed the study, performed the experiments, analyzed the data, and wrote the paper. JRS and DSR designed the study, analyzed the data, and wrote the paper. TMT and JMH performed the experiments, analyzed the data, and wrote the paper. JRC and TRF performed the experiments. TSW, SK, MT, WY, MP, and GB analyzed the data.

We thank members of the Rao lab for their helpful discussions. We thank Parth Patel, May Paing, Norma Iris Rodriguez-Malave, Jennifer King, Jaspal Bassi, Kim Pioli, Nolan Ung, and Jaime Anguiano for their technical support. This work was supported by NIH grant R01 CA166450, a seed grant from the UCLA JCCC, and a Career Development Award K08CA133521 (to D.S. Rao), as well as by NIH grants R01GM109146 and R21AG042003 (to J.R. Sanford). J.R. Contreras was supported by the Tumor Immunology Training Grant, NIH T32CA009120. T.R. Fernando was supported by Tumor Biology Training Grant NIH T32CA009056. J.M. Howard was supported by R41HG007336. Flow cytometry was performed in the UCLA JCCC/CFAR Flow Cytometry Core Facility that is supported by NIH AI-28697, P30CA016042, the JCCC, the UCLA AIDS Institute, and the David Geffen School of Medicine at UCLA.

Jayanth Kumar Palanichamy’s present address is: Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2016126(4):1495–1511. doi:10.1172/JCI80046.


9.12C: RNA Oncogenic Viruses - Biology

Perhaps no disease is more strongly identified with the late twentieth century than acquired immunodeficiency syndrome, commonly known as AIDS . Yet, according to a 2004 United Nations report on the state of the global AIDS epidemic, the disease has not yet begun to reduce its grip on the world population despite the fact that AIDS does not generally receive the same amount of public attention as it once did. On the contrary, infections are on the rise in many countries, including high income nations such as the United States. In 2003, nearly five million people contracted the human immunodeficiency virus (HIV) that causes AIDS, the greatest number of new infections in a single year since AIDS was first officially recognized as a disease in 1981.

The virus responsible for HIV was first isolated in 1983 by Robert Gallo of the United States and French scientist Luc Montagnier. Since that time, a tremendous amount of research focusing upon the causative agent of AIDS has been carried out and much has been learned about the structure of the virus and its typical course of action. HIV is one of a group of atypical viruses called retroviruses that maintain their genetic information in the form of ribonucleic acid ( RNA ). Through the use of an enzyme known as reverse transcriptase, HIV and other retroviruses are capable of producing deoxyribonucleic acid (DNA) from RNA, whereas most cells carry out the opposite process, transcribing the genetic material of DNA into RNA. The activity of the enzyme enables the genetic information of HIV to become integrated permanently into the genome (chromosomes) of a host cell.

The primary hosts for HIV are the white blood cells variously called helper T lymphocytes, helper T cells, or CD4+ T cells. These cells are important components of the immune system that are normally responsible for activating the responses of many other immune cells. Helper T cells that become infected with HIV rapidly die. Soon after primary infection, the body is usually able to compensate for the loss of infected T cells by producing them in greater quantities, at which time individuals infected with HIV are asymptomatic. Over time, however, the virus becomes increasingly acute and there is a slow decline of helper T cells. Consequently, the number of helper T cells in the body (termed the CD4 count ) is generally utilized to gauge the advance of the virus. A CD4 count of less than approximately 200 cells per microliter of blood may be accompanied by a variety of opportunistic infections and is considered the final stage of infection. The persistent barrage of such infections is what typically leads to the death of AIDS patients.

HIV is a relatively complex virus that is able to infect helper T cells chiefly due to a glycoprotein embedded in its envelope called gp120 (see Figure 1) that attaches to CD4 , a protein found on the surfaces of the T cells. Entry of HIV into a host cell is also thought to involve a co-receptor on the cell surface, either CCR5 or CXCR4 , which typically function as receptors of chemokines. The HIV virus envelope is a derivative of the plasma membrane of a host cell, obtained via budding. When HIV attempts to enter a cell, interactions between cell surface molecules and viral envelope proteins allow the envelope to fuse with the cell membrane. The envelope protein called gp41 is known to play an important role in this process.

In an infected cell, HIV undergoes reverse transcription, producing double-stranded DNA from its RNA with the help of reverse transcriptase, a specialized virus-specific enzyme that transcribes DNA from an RNA template. Once in DNA form, the genetic information of HIV is incorporated as a provirus with the host cell DNA. The proviral genome can subsequently be transcribed into viral RNA that functions as mRNA for translation into HIV proteins and as genomes for the subsequent generation of the virus. Capsids form around the viral genomes and proteins before they are budded from the cell membrane, further encapsulating the material into envelopes.

The replication process of HIV is associated with a very high mutation rate because reverse transcription does not allow for correction of errors in nucleotide incorporation. HIV evolves at rate a million times more rapid than the evolution of the human genome, making it extremely difficult for the immune system to recognize and effectively combat. For similar reasons, the development of drugs or vaccines for HIV is a complicated endeavor, the virus not only varying greatly from patient to patient, but even exhibiting significant genomic discrepancies in individuals. A cure for HIV continues to remain elusive, but significant advances have greatly improved the quality of care that patients can receive. Highly active antiretroviral therapy ( HAART ), which involves the use of multiple reverse transcriptase and protease inhibitors, has proved a highly beneficial form of treatment for many patients, but the cost of the therapy has prevented its widespread use in many parts of the world hardest hit by HIV and AIDS.


9.12C: RNA Oncogenic Viruses - Biology

Retroviruses comprise a diverse family of enveloped RNA viruses, remarkable for their use of reverse transcription of viral RNA into linear double stranded DNA during replication and the subsequent integration of this DNA into the genome of the host cell. Members of this family include important pathogens such as HIV-1, feline leukemia, and several cancer-causing viruses. However interest in these viruses extends beyond their disease causing capabilities. For example, research in this area led to the discovery of oncogenes, a major advance in the field of cancer genetics. Studies of retroviruses have contributed greatly to our understanding of mechanisms that regulate eukaryotic gene expression. In addition retroviruses are proving to be valuable research tools in molecular biology and have been used successfully in gene therapy (e.g. to treat X-linked severe combined immunodeficiency).

Written by the top retroviral specialists, this book reviews the genomics, molecular biology, and pathogenesis of these important viruses, comprehensively covering all the recent advances. Topics include: host and retroelement interactions, endogenous retroviruses, retroviral proteins and genomes, viral entry and uncoating, reverse transcription and integration, transcription, splicing and RNA transport, pathogenesis of oncoviral infections, pathogenesis of immunodeficiency virus infections, retroviral restriction factors molecular vaccines and correlates of protection, gammaretroviral and lentiviral vectors, non-primate mammalian and fish retroviruses, simian exogenous retroviruses, and HTLV and HIV. Essential reading for every retrovirologist and a recommended text for all virology and molecular biology laboratories.

"excellent chapters on non-primate mammalian retroviruses, simian retroviruses, fish retroviruses, use of retoviral vectors, and cellular factors that restrict retroviral infection. All the chapters are beautifully illustrated and written by some of the most respected authorities in the field. I highly recommend K and B's "Retroviruses" book to both students and expert colleagues." from Retrovirology blog

"impressive work . a substantial resource to the field . thorough state of research coverage by leading specialists . essential reading for veterinary scientists, clinicians, virologists, and graduate students in the field." from SciTech Book News (March 2010)

"a succinct, state-of-the-art summary of the biology not only of retroviruses but also other retroelements . comprehensive, convenient and satisfying reference work" from Microbiology Today (2010)

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(EAN: 9781904455554 9781912530977 Subjects: [virology] [microbiology] [medical microbiology] [molecular microbiology] [genomics] )


To Be or Not to Be Infected: EVs in Pro- and Antiviral Strategies

In vivo, EVs can interact with viruses and with each other either directly or via modulation of host responses, thus participating in a “War and Peace” between viruses and host (49, 50). Some viruses induce the infected cells to release modified EVs that facilitate infection by increasing the pool of susceptible target cells (e.g., by increasing the number of activated cells) or their susceptibility to viral infection or by serving as decoys that absorb antiviral antibodies, thereby compromising antiviral immunity. In contrast, EVs carrying viral proteins can also be beneficial to the host, for example, by providing dendritic cells with viral antigens to facilitate the initiation of adaptive immune responses. Hypothetically, the capacity of EVs to regulate the lifespan of permissive cells and to modify antiviral immune responses may give additional flexibility to the host in responding to viral infection. Thus, EVs formed during viral infection may play either pro- or counter-viral roles (Fig. 3). It is currently unknown whether the diverse functions ascribed to virus-induced EV may in part be explained by differences in the purity of EV populations used in various studies. A general understanding of parameters that determine the net effect of EVs on viral infections is therefore still lacking.

Proviral and antiviral effects of EVs released by retrovirus-infected cells. Retrovirus infection can lead to the release of modified EVs that either facilitate or suppress infection. Potential antiviral effects include (A) EV-mediated delivery of antiviral components, such as APOBEC3G, to increase resistance to infection (B) spread of TLR ligands, such as viral RNA, via EVs to warn nonsusceptible neighboring cells of the presence of viral infection and (C) provision of antigen presenting cells with viral antigen to facilitate the initiation of adaptive immune responses. Potential proviral effects include (D) inhibition of the neutralizing effect of EV, leading to decreased binding of EV to virions and an increase in the number of virions that may infect other cells (E) EV-mediated delivery of viral components (e.g., Nef) that induce induce cell senescence or death of antiviral immune cells (F) EV-mediated delivery of viral components that suppress the function of immune cells (e.g., Nef-induced down-regulation of antibody production by B cells) and (G) increase of the pool of virus-susceptible cells, e.g., by transference of coreceptors for virus binding to other cells.

EVs Facilitate Viral Infection.

Several HIV proteins and RNAs have been detected in EVs released from HIV-infected cells. One of the viral components released via EVs is the HIV transactivation response element (TAR) RNA (51). TAR is an RNA stem-loop structure located at the 5′ ends of HIV-1 transcripts, which in infected cells can be bound by Tat, thereby facilitating recruitment of elongation factors and increased production of viral RNA (52). When transferred via EVs, TAR RNA can increase the population of susceptible target cells. Inside EV-targeted cells, the full-length TAR RNA is processed into miRNAs, which silence mRNA coding for Bcl-2 Interacting Protein. The consequent increase in resistance to apoptosis allows the cell to produce virus for a longer period, thereby facilitating HIV infection (51).

In addition, EVs released by HIV-infected cells selectively incorporate the HIV virulence factor Nef via interaction of the Nef secretion modification region with mortalin, a member of the Hsp70 family of chaperones involved in cellular protein trafficking (53). Delivery of the EV-associated Nef to T cells affects these cells in several ways. First, the transferred Nef may activate T cells, rendering them more susceptible to HIV infection (54). Second, EVs can deliver Nef to some of the bystander CD4 + T cells and induce cell senescence or death (55). This mechanism can contribute to the high level of T-cell deaths during the early stages of HIV infection, when viral load is still low (55). Finally, intercellular transfer of Nef by EVs may facilitate evasion of the humoral immune response by suppressing IgG2 and IgA production in B cells, as has also been shown for Nef transfer by HIV-infected macrophages to B cells via intercellular conduits (56). In in vitro systems, it has been shown that EVs can transfer the HIV coreceptors CCR5 and CXCR4 to other cells, thus making them prone to HIV infection (57, 58). This EV-mediated process may expand the spectra of HIV-infected cells, but it is yet unknown whether such a phenomenon plays an important role in vivo.

EVs Suppress Viral Infection.

In in vitro experiments, it has been shown that T cells can produce EVs containing the HIV receptor CD4. These EVs can attach to viral particles, thereby decreasing the numbers of virions that would otherwise infect CD4 + T cells (59). However, HIV can counteract this by stimulating the incorporation of HIV-Nef into these EVs, leading to the inhibition of CD4 incorporation in EVs and a decreased effectiveness of the above-described host antiviral response (59).

Another EV-mediated host cell protection mechanism against HIV involves the EV-mediated transport of the host antiviral protein APOBEC3G. This cytidine deaminase is usually incorporated into virions together with retroviral RNA and inhibits viral replication by creating G-to-A mutations in the transcribed viral DNA (60). The antiviral action of APOBEC3G is counteracted by the HIV-encoded protein Vif, which interferes with APOBEG3G incorporation into virions. Delivery of APOBEC3G without Vif via EVs can counteract the effect of Vif and thus increase resistance of EV-targeted cells to HIV infection (34). Similarly, recent data indicate that the second messenger cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) (induced by cGAMP synthase) is enclosed both in HIV particles and in EVs that are released from infected cells. Intercellular transfer of cGAMP, although accomplished more efficiently by viruses than by EV, triggers antiviral IFN responses in newly infected cells in a stimulator of interferon genes (STING)-dependent manner (35, 36).

EVs from virus-infected cells not only contain endogenous (mi)RNAs but have also been shown to be selectively enriched in viral RNAs (e.g., in the case of HCV-induced EVs) (38). The PRRs in EV-targeted cells may recognize such RNAs as pathogen-associated molecular patterns (PAMPs) and respond by triggering the innate antiviral response (38, 61). HIV-infected macrophages also release EV containing viral RNAs (viral miRNAs vmiR88 and -99) that trigger endosomal TLR8 and NF-κB signaling in EV-targeted bystander macrophages (61). The subsequent production of proinflammatory cytokines (e.g., TNFα) contributes to the initiation of the immune response against HIV. Dissemination of viral RNA via EVs provides a strategy to warn nonsusceptible neighboring cells of the presence of viral infection. During HCV infection, for example, plasmacytoid DC are targeted by viral RNA containing EVs and, as a result, initiate an inflammatory response (38). In addition, EVs containing host miRNA produced by virus-resistant cells can confer resistance to other cells. This has been demonstrated for trophoblasts, which are largely resistant to infection by various viruses, including HIV, probably contributing to in vivo fetus protection. EVs produced by these cells in vitro carry host miRNAs and deliver them to virus-susceptible cells, making them resistant to virus infection (62).


John Joseph Karijolich, Ph.D.

The association between infection with viruses and neoplasia is well established for a variety of cancers. In fact, approximately 12% of human cancers worldwide are caused by oncogenic viral infections, with more than 80% of cases occurring in the developing world. Despite their prevalence and public health importance, our understanding and ability to manage viral-induced cancers is still limited. This is in part due to the complexity of host-virus interactions leading to cellular transformation.

Research in our laboratory is focused on defining the host-virus interaction in the context of gammaherpesviral infection. ¿-herpesviruses, which include the human oncogenic viruses Kaposi Sarcoma-associated herpesvirus (KSHV) and Epstein Barr virus (EBV), are a family of large double-stranded DNA lymphotrophic viruses that are the causative agents of a variety of disorders, including lymphoproliferative diseases, lymphomas, as well as other nonlymphoid cancers in mammals. Studies of the host response to viral infection have historically focused on protein-coding genes, thus our understanding of how the non-protein-coding transcriptome, including both viral- and host-derived noncoding RNAs, impacts host-virus interactions is limited. Along this line, our primary research goals are directed towards understanding how noncoding RNAs and their RNA-binding proteins are integrated in to the regulation of gene expression and modulation of the host immune response during ¿-herpesviral infection. To accomplish this we undertake a multidisciplinary approach combining virology, immunology, RNA biochemistry, proteomics, and genomics/transcriptomics. Major thematic questions of the lab include:

1) What are the endogenous and exogenous noncoding RNA species that contribute to innate immune modulation and how are these functions mediated?

2) How does the cell intrinsic innate immune system shape the ¿-herpesviral lifecycle and what are the host and viral entities at play?

3) What is the contribution of endogenous retroviruses and transposons to the host-virus interaction?

Ye X, Zhao Y, Karijolich J. The landscape of transcription initiation across latent and lytic KSHV genomes. PLoS Pathog. 2019 Jun 15(6): e1007852. PMID: 31188901, PMCID: PMC6590836, PII: PPATHOGENS-D-19-00162, DOI: 10.1371/journal.ppat.1007852, ISSN: 1553-7374.

Heinrich MJ, Purcell CA, Pruijssers AJ, Zhao Y, Spurlock CF, Sriram S, Ogden KM, Dermody TS, Scholz MB, Crooke PS, Karijolich J, Aune TM. Endogenous double-stranded Alu RNA elements stimulate IFN-responses in relapsing remitting multiple sclerosis. J. Autoimmun [print-electronic]. 2019 Jun 100: 40-51. PMID: 30826177, PMCID: PMC6513682, PII: S0896-8411(18)30699-1, DOI: 10.1016/j.jaut.2019.02.003, ISSN: 1095-9157.

Zhao Y, Ye X, Dunker W, Song Y, Karijolich J. RIG-I like receptor sensing of host RNAs facilitates the cell-intrinsic immune response to KSHV infection. Nat Commun. 2018 Nov 11/19/2018 9(1): 4841. PMID: 30451863, PMCID: PMC6242832, PII: 10.1038/s41467-018-07314-7, DOI: 10.1038/s41467-018-07314-7, ISSN: 2041-1723.

Dunker W, Song Y, Zhao Y, Karijolich J. FUS Negatively Regulates Kaposi's Sarcoma-Associated Herpesvirus Gene Expression. Viruses. 2018 Jul 7/6/2018 10(7): PMID: 29986386, PMCID: PMC6070805, PII: v10070359, DOI: 10.3390/v10070359, ISSN: 1999-4915.

Hesser CR, Karijolich J, Dominissini D, He C, Glaunsinger BA. N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection. PLoS Pathog. 2018 Apr 14(4): e1006995. PMID: 29659627, PMCID: PMC5919695, PII: PPATHOGENS-D-17-02239, DOI: 10.1371/journal.ppat.1006995, ISSN: 1553-7374.

Zhao Y, Dunker W, Yu YT, Karijolich J. The Role of Noncoding RNA Pseudouridylation in Nuclear Gene Expression Events. Front Bioeng Biotechnol. 2018 6: 8. PMID: 29473035, PMCID: PMC5809436, DOI: 10.3389/fbioe.2018.00008, ISSN: 2296-4185.

Dunker W, Zhao Y, Song Y, Karijolich J. Recognizing the SINEs of Infection: Regulation of Retrotransposon Expression and Modulation of Host Cell Processes. Viruses. 2017 Dec 12/18/2017 9(12): PMID: 29258254, PMCID: PMC5744160, PII: v9120386, DOI: 10.3390/v9120386, ISSN: 1999-4915.

Ge J, Karijolich J, Zhai Y, Zheng J, Yu YT. 5-Fluorouracil Treatment Alters the Efficiency of Translational Recoding. Genes (Basel). 2017 Oct 10/31/2017 8(11): PMID: 29088058, PMCID: PMC5704208, PII: genes8110295, DOI: 10.3390/genes8110295, ISSN: 2073-4425.

Karijolich J, Zhao Y, Alla R, Glaunsinger B. Genome-wide mapping of infection-induced SINE RNAs reveals a role in selective mRNA export. Nucleic Acids Res [print-electronic]. 2017 Mar 3/15/2017 PMID: 28334904, PII: 3071714, DOI: 10.1093/nar/gkx180, ISSN: 1362-4962.

Zhao Y, Karijolich J, Glaunsinger B, Zhou Q. Pseudouridylation of 7SK snRNA promotes 7SK snRNP formation to suppress HIV-1 transcription and escape from latency. EMBO Rep [print-electronic]. 2016 Aug 8/24/2016 PMID: 27558685, PII: embr.201642682, DOI: 10.15252/embr.201642682, ISSN: 1469-3178.

Huang C, Karijolich J, Yu YT. Detection and quantification of RNA 2'-O-methylation and pseudouridylation. Methods [print-electronic]. 2016 Jul 7/1/2016 103: 68-76. PMID: 26853326, PMCID: PMC4921259, PII: S1046-2023(16)30021-4, DOI: 10.1016/j.ymeth.2016.02.003, ISSN: 1095-9130.

Karijolich J, Yi C, Yu YT. Transcriptome-wide dynamics of RNA pseudouridylation. Nat. Rev. Mol. Cell Biol [print-electronic]. 2015 Oct 16(10): 581-5. PMID: 26285676, PII: nrm4040, DOI: 10.1038/nrm4040, ISSN: 1471-0080.

Karijolich J, Yu YT. The new era of RNA modification. RNA. 2015 Apr 21(4): 659-60. PMID: 25780180, PMCID: PMC4371322, PII: 21/4/659, DOI: 10.1261/rna.049650.115, ISSN: 1469-9001.

Karijolich J, Abernathy E, Glaunsinger BA. Infection-Induced Retrotransposon-Derived Noncoding RNAs Enhance Herpesviral Gene Expression via the NF-¿B Pathway. PLoS Pathog. 2015 11(11): e1005260. PMID: 26584434, PMCID: PMC4652899, PII: PPATHOGENS-D-15-02010, DOI: 10.1371/journal.ppat.1005260, ISSN: 1553-7374.

Karijolich J, Yu YT. Therapeutic suppression of premature termination codons: mechanisms and clinical considerations (review). Int. J. Mol. Med [print-electronic]. 2014 Aug 34(2): 355-62. PMID: 24939317, PMCID: PMC4094583, DOI: 10.3892/ijmm.2014.1809, ISSN: 1791-244X.

Karijolich J, Zhao Y, Peterson B, Zhou Q, Glaunsinger B. Kaposi's sarcoma-associated herpesvirus ORF45 mediates transcriptional activation of the HIV-1 long terminal repeat via RSK2. J. Virol [print-electronic]. 2014 Jun 88(12): 7024-35. PMID: 24719417, PMCID: PMC4054375, PII: JVI.00931-14, DOI: 10.1128/JVI.00931-14, ISSN: 1098-5514.

Lim SJ, Scott A, Xiong XP, Vahidpour S, Karijolich J, Guo D, Pei S, Yu YT, Zhou R, Li WX. Requirement for CRIF1 in RNA interference and Dicer-2 stability. RNA Biol. 2014 11(9): 1171-9. PMID: 25483042, PMCID: PMC4615304, DOI: 10.4161/rna.34381, ISSN: 1555-8584.

Karijolich JJ, Hampsey M. The Mediator complex. Curr. Biol. 2012 Dec 12/18/2012 22(24): R1030-1. PMID: 23257183, PII: S0960-9822(12)01320-6, DOI: 10.1016/j.cub.2012.11.011, ISSN: 1879-0445.

Karijolich J, Yu YT. Converting nonsense codons into sense codons by targeted pseudouridylation. Nature. 2011 Jun 6/16/2011 474(7351): 395-8. PMID: 21677757, PMCID: PMC3381908, PII: nature10165, DOI: 10.1038/nature10165, ISSN: 1476-4687.

Huang C, Karijolich J, Yu YT. Post-transcriptional modification of RNAs by artificial Box H/ACA and Box C/D RNPs. Methods Mol. Biol. 2011 718: 227-44. PMID: 21370052, PMCID: PMC4161979, DOI: 10.1007/978-1-61779-018-8_14, ISSN: 1940-6029.

Karijolich J, Yu YT. Spliceosomal snRNA modifications and their function. RNA Biol [print-electronic]. 2010 Mar 7(2): 192-204. PMID: 20215871, PMCID: PMC4154345, PII: 11207, ISSN: 1555-8584.

Karijolich J, Kantartzis A, Yu YT. Quantitative analysis of RNA modifications. Methods Mol. Biol. 2010 629: 21-32. PMID: 20387140, PMCID: PMC4154561, DOI: 10.1007/978-1-60761-657-3_2, ISSN: 1940-6029.

Karijolich J, Kantartzis A, Yu YT. RNA modifications: a mechanism that modulates gene expression. Methods Mol. Biol. 2010 629: 1-19. PMID: 20387139, PMCID: PMC4154353, DOI: 10.1007/978-1-60761-657-3_1, ISSN: 1940-6029.

Karijolich J, Yu YT. Insight into the Protein Components of the Box H/ACA RNP. Curr Proteomics. 2008 Jul 7/1/2008 5(2): 129-37. PMID: 19829749, PMCID: PMC2760984, DOI: 10.2174/157016408784911936, ISSN: 1570-1646.

Moelling C, Oberschlacke R, Ward P, Karijolich J, Borisova K, Bjelos N, Bergeron L. Metal-dependent repression of siderophore and biofilm formation in Actinomyces naeslundii. FEMS Microbiol. Lett [print-electronic]. 2007 Oct 275(2): 214-20. PMID: 17825071, PII: FML888, DOI: 10.1111/j.1574-6968.2007.00888.x, ISSN: 0378-1097.

Karijolich J, Stephenson D, Yu YT. Biochemical purification of box H/ACA RNPs involved in pseudouridylation. Meth. Enzymol. 2007 425: 241-62. PMID: 17673087, PII: S0076-6879(07)25011-6, DOI: 10.1016/S0076-6879(07)25011-6, ISSN: 0076-6879.

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Shou-Jiang (SJ) Gao, Ph.D.

The main research area in Dr. Gao’s laboratory has been on viral oncogenesis with current focus on Kaposi’s sarcoma-associate herpesvirus (KSHV) and AIDS-related malignancies. The lab has been engaging in a multidisciplinary effort and expanding the research program into translational and cancer therapeutics, cancer metabolism, microbiota, inflammation, angiogenesis, innate immunity, and microRNAs. The lab has applied state-of-art approaches and technologies in genomics, epigenetics, RNA epigenetics, metabolomics, high-throughput genomic screening, high-throughput drug screening, and systems biology to tackle these forefront biomedical issues. In particular, the lab has recently invested in drug screening and discovery as a result of the identification of new therapeutic targets and development of new model systems for infections and cancers. Dr. Gao’s laboratory is well supported by extramural funding. There are currently 6 active R01s and 1 Sub-project of an active PPG. Here are some of the ongoing research directions and projects:

1. Drug screening and discovery: The lab has developed several cancer and infection models, and used them in Crispr-Cas9 mediated genome-wide screening and drug screening. The lab has already identified numerous new targets and agents, which have been shown to be effective in both in vitro and in vivo models.

2. Systems biology: The lab has been engaging in works in epigenetics, RNA epigenetics and metabolomics in cancer and infections.

3. Infections, inflammation and innate immunity: The lab continues to study microRNAs, inflammation, angiogenesis, innate immunity and infections.

Representative Publications

Graffaz M, Zhou SH, Vasan K, Rushing T, Michael QL, Lu C, Jung JU, Gao S-J. Repurposing cytarabine for treating primary effusion lymphoma by targeting KSHV latent and lytic replications. mBio, 2018, 9: e00756-18.

Tan B, Liu H, da Silva SR, Yuan HF, Sorel O, Zhang L, Meng J, Huang YF, Gao SJ. Viral and cellular N6-methyladenosine and N6,2'-O-dimethyladenosine epitranscriptomes in the KSHV life cycle. Nature Microbiology, 2018, 3:108-120

Graffaz M, Vasan K, Tan B, da Silva SR, Gao SJ. TLR4-mediated inflammation promotes KSHV-induced cellular transformation and tumorigenesis by activating the STAT3 pathway. Cancer Research, 2017, 77:7094-7108.

Zhu Y, Li TT, da Silva SR, Jae-Jin Lee, Lu C, Hyungjin Eoh, Jung JU, Gao SJ. A critical role of glutamine and asparagine γ-nitrogen for nucleotide biosynthesis in cancer cells hijacked by an oncogenic virus. mBio, 2017, 8. pii: e01179-17. doi: 10.1128/mBio.01179-17.

Zhu Y, da Silva SR, Liang QM, Lu C, Feng PH, Jung JU, Gao SJ. Suppression of glycolysis by KSHV promotes cell survival and oncogenic transformation. PLoS Pathogens, 2016, 12: e1005648.

Lee MS, Jones T, Song DY, Jang JH, Jung JU, Gao S-J. Exploitation of complement system by oncogenic Kaposi’s sarcoma-associated herpesvirus for cell survival and persistent infection. PLoS Pathogens, 2014, 10: e1004412.

Moody R, Zhu Y, Huang YF, Cui XD, Jones T, Bedolla R, Lei XF, Bai ZQ, Gao S-J. KSHV microRNAs mediate cellular transformation and tumorigenesis by redundantly targeting cell growth and survival pathways. PLoS Pathogens, 2013, 9: e1003857.

Greene W, Zhang W, He ML, Witt C, Ye FC and Gao S-J. The ubiquitin/proteasome system regulates Kaposi's sarcoma-associated herpesvirus entry into endothelial cells. PLoS Pathogens, 2012, 8: e1002703.

Jones T, Ye FC, Bedolla RG, Huang YF, Meng J, Qian LW, Pan HY, Zhou FC, Moody R, Wagner, B, Arar M and Gao S-J. Direct efficient cellular transformation of primary rat metanephric mesenchymal cells by KSHV. Journal of Clinical Investigation, 2012, 122, 1076-1081.

Lei XF, Bai ZQ, Ye FC, Xie JP, Kim CG, Huang YF and Gao S-J. Regulation of NF-kB inhibitor IkBa and viral replication by a KSHV microRNA. Nature Cell Biology, 2010, 12, 193-199.



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