Elucidation of cellular and molecular mechanisms of lymphoma induction and evolution to identify therapeutic targets

Malignant lymphomas are the 5th most common cancers in the world, accounting for ~5% of all tumours. They may be derived from the B-Cell or T-cell lineage and are generally mature in nature. The prognosis of lymphomas greatly varies and is dependent on the stage and histology of the disease. Although up to 60% of young patients (<60y) with aggressive Diffuse Large B-Cell Lymphoma (DLBCL) are cured by combination immune-chemotherapy, older patients have a fairly dismal outlook. In addition, although monoclonal antibodies and small molecule inhibitors have improved survival in patients with low-grade indolent disease such as Follicular lymphoma (FL), these diseases are commonly incurable. In addition, all of the aforementioned therapies are toxic and may lead to significant morbidities and occasional mortalities in lymphoma patients. Worryingly, the incidence of malignant lymphomas continues to rise, and at 3-4% per year4 appears far in excess of what would be expected simply from the ageing of the population. The identification of critical mediators of lymphomagenesis and novel therapies that target them is therefore a priority for this increasingly common malignancy and unmet medical need.

Despite their clinical importance, we actually know very little about the molecular and cellular biology of lymphomas. Next generation sequencing (NGS) efforts are beginning to uncover mutations that occur in lymphomas, however the order of these mutations and how they collaborate to generate lymphomas remains mysterious. Likewise gene expression studies have identified critical genes that are drive lymphoma but for other pathways we do not know if these are cause or consequence of malignant transformation. In addition, we know that normal B-cell survival is dependent upon t singling events at the cell surface. Furthermore mutations that alter cell singling often occur in lymphomas suggesting that lymphomas are also dependent upon abnormal singling events. However, exactly how and where this occurs during the development of the lymphoma and the role that it plays in this evolution is unknown. Moreover, the cell biology of lymphoma is poorly characterized. Our knowledge of the cells from which lymphomas derive initially and the role that this cell typemight play in the eventual phenotype of the lymphoma are poorly understood, although initial transforming steps in haematopoietic stem and progenitor cells have been shown for the similar B-cell malignancies chronic lymphocytic leukaemia (CLL), hairy cell leukaemia (HCL) and for some T-cell lymphomas. We will address all of these questions within this application using a combination of mouse models, human cell lines and powerful genome-wide analysis.

We will use unique mouse models that we have developed that generate loss of Crebbp at defined times within B-cell development, and will engineer within these models the ability to generate and identify further progression events by engineering a mutation inducing system that jumps around DNA sequences specifically in B cells. This system will allow us to robustly answer the question of the contribution of the initial cell-type to outcome and phenotype of lymphoma. Using the presence of a unique but easily identifiable premalignant phase within the model, we will also characterize alterations gene expression and signaling that occur during lymphoma evolution, through a combination of stat-of-the-art genomic techniques. Finally we will test a priori hypotheses about the requirement for specific DNA repair pathways and residual CREBBP/P300 function in these Crebbp-/- lymphomas, as well as performing a genomewide screen to identify further vulnerabilities in CREBBP mutated lymphoma cells, a subset of which will be further tested in our in vivo model

Malignant lymphomas are the 5th most common cancers in the world, accounting for ~5% of all tumours. They may be derived from the B-Cell or T-cell lineage and are generally mature in nature. The prognosis of lymphomas greatly varies and is dependent on the stage and histology of the disease. Although up to 60% of young patients (<60y) with aggressive Diffuse Large B-Cell Lymphoma (DLBCL) are cured by combination immune-chemotherapy, older patients have a fairly dismal outlook. However, low-grade indolent disease such as Follicular lymphoma (FL are commonly incurable. Worryingly, the incidence of malignant lymphomas continues to rise, and at 3-4% increase per year appears far in excess of what would be expected simply from the ageing of the population. The identification of critical mediators of lymphomagenesis and novel therapies that target them is therefore a priority for this increasingly common malignancy and unmet medical need.

To address these questions, we will use unique mouse models that we have developed engineering in the ability to generate and record further progression events through insertional mutagenesis, specifically within B-cells. This system will allow us to robustly answer the question of the contribution of the initial cell-type to outcome and phenotype of lymphoma. Using the presence of a unique premalignant phase within the model, we will also characterize alterations of transcription, epigenetic state and signaling that occur during lymphoma evolution, through a combination of single cell and bulk RNA-Seq, ChIP-Seq and ATATC-Seq, promoter based capture HiC and intracellular flow cytometry. Finally we will test a priori hypotheses about the requirement for specific DNA repair pathways and residual CREBBP/P300 function in these Crebbp-/- lymphomas, as well as performing a genomewide CRISPR/Cas9 dropout screen to identify further genotype-specific vulnerabilities, a subset of which will be further tested in our in vivo model.

Malignant lymphomas are the 5th most common cancers and are increasing in incidence in excess of what might be expected simply from an ageing population. Around 16,000 new cases occur annually in the UK with around 40% fatal. Malignant lymphomas are therefore an unmet medical need. In addition to this social burden to the UK and beyond, it places a significant financial burden on the NHS. Multiple beneficiaries will benefit from this application as users of the research outputs. Amongst these beneficiaries are specific research charities dedicated to improving outcomes in haematological and other malignancies, examples of which are Bloodwise, the Kay Kendall Leukaemia Fund, Leukaemia Care and Cancer Research UK. In addition, there will likely be beneficiaries in the commercial sector, particularly those drug companies and biotechs with an interest in targeting lymphomas and allied lymphoid malignancies. Our research may also benefit policy makers, agencies and regulators interested in the national treatment of lymphomas. Finally the most obvious group of beneficiaries will be those researchers and physicians interested in the biology, evolution and treatment of lymphomas and similar lymphoid malignancies.

These groups will benefit in a number of different ways. The academic beneficiaries will utilise our data to inform their own studies of lymphoma biology and may make future use of our models and datasets to perform further studies or may use them in comparison to their own data. The policy makers may use our data to help improve treatments and outcomes in lymphoma. In addition, as we are studying the initiation and evolution of these disorders, our work may have implications for earlier detection and intervention that would also be predicted to improve patient outcomes. Charities would use our work to advertise the need for improved therapies and hope to generate awareness and further finance to fund research and treatment into lymphomas. Finally our work would be of particular interest to pharmaceutical companies; both the results of our directed testing of inhibitors of the DNA damage response and CREBBP HAT activity as well as in our agnostic screens of genetic vulnerabilities form the CRISPR dropout screen would have implications for the marketing of existing therapeutics as well as the direction of future drugs and lead compounds.