Identification of oncogenic drivers in aggressive B cell Lymphoma by ribosome profiling and a novel primary human lymphocyte transformation assay

Diffuse Large B Cell Lymphoma (DLBCL) is the most common form of non-Hodgkin Lymphoma. It is an aggressive and devastating form of cancer. Although potentially curable with combination chemotherapy, more than a third of patients will succumb to their disease. Importantly, the incidence of DLBCL increases with age and many older patients are simply unable to tolerate the required chemotherapy. Certain subtypes of DLBCL have been identified that respond particularly poorly to all existing therapies. Thus there is a pressing need for the development of more effective and better tolerated, "targeted" treatments.

Over the last decade there have been considerable advances in our understanding of underlying biology of this disease. Much of this has arisen from studies that examine how the activity of cohorts of genes differs between different subtypes of lymphoma. These "gene expression " studies have predominantly examined the total amount mRNA in each cell. mRNA is a messenger molecule that carries instructions from the gene's DNA before being translated in to the final active product, termed the protein. In general more active genes make more mRNA, which is why mRNA has generally been used as a proxy for gene activity. However, recently it has become clear that not all mRNA molecules are "translated" into protein with equal efficiency. Furthermore, a frequent finding in cancer is that certain mRNA molecules that carry instructions advantageous to the cancer may be preferentially translated by the tumor. Recent technology now allows the opportunity to measure the translational rate for each individual mRNA. I will use this technique to identify those mRNAs that are preferentially translated by the different subtypes of DLBCL. Those genes / mRNAs with the greatest changes in translational rate will be tested in cell culture systems to identify those that contribute most to the development and growth of the lymphoma. Ultimately drugs designed to inhibit these genes may prove useful in the treatment of lymphoma. I anticipate that this approach will identify new targets for the development of anti-lymphoma drug treatments.

In addition I will investigate the mechanism by which some mRNA is preferentially translated by lymphoma cells. I will use a computational approach to screen preferentially translated mRNA to identify common sequences that the lymphoma cells may use to control the translation. I will then use these sequences as bait to identify the "translation factors' responsible for the altered translation in lymphoma. I will investigate how the activity of these translation factors is controlled, either by lymphoma specific signaling pathways or by mutation. Indeed, mutations have already been identified in factors known to regulate translation in lymphoma cells but the functional significance of these mutations is yet to be investigated.

Overall this project will provide a detailed understanding of how lymphoma cells corrupt the normally tightly regulated activity of their genes. In doing so this project may reveal opportunities for new forms of targeted treatment in lymphoma. In addition much of what we learn about the mechanisms of corrupted translational regulation in lymphoma may prove to be of broader relevance to other diseases, in particular other types of cancer.

Deregulated gene expression is fundamental to the development of malignant disease. Much of our current understanding of lymphoma biology comes from transcriptional profiling and the study of mutations in transcription factors and components of the signalling pathways that regulate transcription. Recent work suggests that translational regulation may be of equivalent magnitude to transcriptional regulation and that translational deregulation is a prominent feature of malignant disease including lymphoma. This study proposes to use the ribosome footprinting technique to profile translation across three major classes of aggressive B cell lymphoma; ABC DLBCL, GCB DLBCL and Burkitt lymphoma using both cell lines, primary tumour biopsy tissue and non-malignant, "normal" human germinal centre B cells. Modifications of this assay will also identify non-canonical translational start sites that either inhibit translation of the true ORF or generate altered protein isoforms or translated "micropeptides". Mechanisms of translational deregulation will be investigated by bioinformatics screening of deregulated transcripts for enriched motifs. Biotinylated bait RNA pull-down combined with mass spectrometry will identify potential RNA binding proteins that acts as "translation factors". The functional significance of transcripts deregulated at the level of translation, non-canonical translation initiation sites, putative translation factors and micropeptides will be tested using a transformation assay that uses semi-immortalized human germinal centre B cells cultured in vitro. I will also use this system to investigate the significance of potential translation factors (such as ZFP36L1) known to be mutated in DLBCL from existing RNA-seq data. Finally, I will use a combination of shRNA knockdown, over-expression and pharmacological agents to test the interplay between known lymphoma signalling pathways (eg BCR and TLR signalling) and translational regulation.

It is clear that existing therapeutic options for aggressive lymphoma are inadequate. More than 30% of treated patients die from their lymphoma (most within the first 18 months of diagnosis) and many elderly patients are unable to tolerate current immunochemotherapy and are treated palliatively from the outset. Furthermore Diffuse Large B Cell Lymphoma is the most common of all haematological malignancies with more than 3000 new cases per year in England and Wales. It is also clear that treatment breakthroughs are most likely to arise from a detailed understanding of the underlying biology. This project will provide a detailed and novel study of the regulation translation in this disease. It will identify genes and pathways aberrantly activated or inactivated in lymphoma, and the mechanism and functional significance thereof. As such it will identify potential targets for new drug development. It may also identify genes and pathways already targetable by drugs that have been developed for other indications. By investigating how gene expression becomes corrupted at the translational level as a B lymphocyte transforms to a malignant lymphoma cell the results may be highly informative to those studying other forms of cancer, B cell biology and autoimmune disease.

Ultimately the expected beneficiary is patients suffering from lymphoma for whom a more effective and less toxic, targeted therapy will be of clear benefit in terms of health and quality of life. In particular, elderly patients, who tolerate existing therapy poorly, stand to benefit the most from this form of research. As the UK population steadily ages the health of this population of patients is becoming increasingly important.

In particular I anticipate that pharmaceutical companies may take forward the results of this project in terms of the design and testing of small molecules designed to target pathways identified from this research. The Addenbrooke's site in Cambridge is rapidly becoming a major bio-tech hub and this will provide genuine opportunity for this type of interaction with biotech companies in the latter stages of this project.

In addition I and the research associate working on this project will benefit; the project will employ cutting edge technology and will be based at the University of Cambridge, one of the best academic institutions in the world. The training, experience and opportunity for both formal and informal collaboration will leave me well placed to move forward to the next stage of my career as a clinical academic and independent research group leader.