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

恶性淋巴瘤是世界上第五大最常见的癌症，约占所有肿瘤的5%。它们可能来自b细胞或t细胞谱系，本质上通常是成熟的。淋巴瘤的预后变化很大，取决于疾病的分期和组织学。尽管高达60%的侵袭性弥漫性大b细胞淋巴瘤（DLBCL）的年轻患者（小于60岁）通过联合免疫化疗治愈，但老年患者的前景相当黯淡。此外，虽然单克隆抗体和小分子抑制剂可以提高低级别惰性疾病（如滤泡性淋巴瘤）患者的生存率，但这些疾病通常是无法治愈的。此外，上述所有疗法都是有毒的，可能导致淋巴瘤患者显著的发病率和偶尔的死亡率。令人担忧的是，恶性淋巴瘤的发病率继续上升，每年3-4%的发病率似乎远远超过了单纯因人口老龄化而预期的发病率。因此，确定淋巴瘤发生的关键介质和针对它们的新疗法是这一日益常见的恶性肿瘤和未满足的医疗需求的优先事项。尽管淋巴瘤具有重要的临床意义，但实际上我们对淋巴瘤的分子和细胞生物学知之甚少。下一代测序（NGS）的努力开始揭示发生在淋巴瘤中的突变，然而这些突变的顺序以及它们如何协同产生淋巴瘤仍然是一个谜。同样，基因表达研究已经确定了驱动淋巴瘤的关键基因，但对于其他途径，我们不知道这些是恶性转化的原因还是结果。此外，我们知道正常的b细胞存活依赖于细胞表面的单化事件。此外，改变细胞单链的突变经常发生在淋巴瘤中，这表明淋巴瘤也依赖于异常单链事件。然而，在淋巴瘤的发展过程中，这种情况是如何发生的，在哪里发生的，以及它在这种演变中所起的作用是未知的。此外，淋巴瘤的细胞生物学特性还很差。尽管在类似的b细胞恶性肿瘤慢性淋巴细胞白血病（CLL）、毛细胞白血病（HCL）和一些t细胞淋巴瘤中已经发现了造血干细胞和祖细胞的初始转化步骤，但我们对淋巴瘤最初产生的细胞以及这种细胞类型在淋巴瘤最终表型中可能发挥的作用知之甚少。我们将在这个应用程序中使用小鼠模型、人类细胞系和强大的全基因组分析的组合来解决所有这些问题。我们将使用我们开发的独特的小鼠模型，在B细胞发育的特定时间产生Crebbp的损失，并将在这些模型中通过设计一个突变诱导系统来产生和识别进一步的进展事件，该系统可以在B细胞中特异性地跳跃DNA序列。该系统将使我们能够有力地回答初始细胞类型对淋巴瘤结果和表型的贡献的问题。利用模型中存在的独特但易于识别的癌前期，我们还将通过结合最先进的基因组技术来表征淋巴瘤进化过程中发生的基因表达和信号传导的变化。最后，我们将测试这些CREBBP -/-淋巴瘤中对特定DNA修复途径和残留CREBBP/P300功能的需求的先验假设，并进行全基因组筛选以确定CREBBP突变淋巴瘤细胞的进一步脆弱性，其中一部分将在我们的体内模型中进一步测试

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