Identifying/Targeting Mechanisms of Lymphomagenesis Driven by CREBBP Inactivation

项目总结/摘要 滤泡性淋巴瘤（FL）是发生于生殖中心B细胞的一种不可治愈的恶性肿瘤， 起源于具有少量遗传改变的肿瘤细胞祖细胞。我们最近定义了灭活 组蛋白乙酰转移酶基因CREBBP的突变是疾病发生的早期事件， BCL 2癌基因易位。原发性FL肿瘤中CREBBP的失活沉默了一个生发中心B- 细胞转录程序，该程序显著富集了参与MHC上抗原呈递的基因 II类和YY 1转录因子的靶点。抗原呈递减少与 肿瘤细胞激活CD 4 T细胞增殖的能力降低， 肿瘤微环境CREBBP通过组蛋白乙酰化在表观遗传学上调节基因表达， 通过与转录因子如CIITA的相互作用来指导，CIITA是MHC II类基因的中心调节因子 表达，和YY 1，一个关键的调节器的生发中心B细胞的发展。因此，我们假设， CREBBP失活突变通过驱动免疫逃避在促进淋巴瘤发生中起双重作用 通过减少抗原呈递，以及通过解除B细胞发育的调节， 中央B细胞期。为了研究这一点，我们开发了CREBBP失活的体外和体内模型 分别使用淋巴瘤细胞系和转基因小鼠模型的CRISPR修饰。重要的是， CRISPR修饰的细胞系重现了在原发性肝癌中观察到的MHC II类表达减少的表型。 FL肿瘤，当Crebbp缺失和Bcl 2过度表达时，转基因小鼠模型产生FL样肿瘤。 在B细胞中特异性表达。我们将使用这些模型，与原发性人类肿瘤平行， CREBBP失活在淋巴瘤发生中的假设机制作用。具体来说，我们将评估 CREBBP失活以减少MHC II类上的抗原呈递的能力，并测量其作用 这对体内和体外T细胞反应的影响。此外，我们将研究CREBBP的作用， 在降低由YY 1转录调节的生发中心B细胞基因表达中的失活 因子，并确定这是否会导致体内生发中心B细胞阶段的分化停滞， 转基因小鼠模型。我们进一步假设这些表型是由表观遗传改变驱动的， 并因此可能使用表观遗传修饰药物来纠正。因此，我们将调查 HDAC抑制剂和EZH 2抑制剂恢复抗原呈递和相关T细胞免疫应答的潜力 反应，并重建B细胞发育的正常表观遗传和转录程序。一起 这项工作将为CREBBP失活作为FL发展中的关键事件提供验证， 这些遗传事件导致免疫逃避和B细胞发育失调的机制， 并评估使用表观遗传修饰化合物来抵消CREBBP的这些特征， 失活这将有助于对FL的理解和治疗的重要进展。

Project Summary / Abstract Follicular lymphoma (FL) is an incurable malignancy of germinal center B-cells, in which sequential relapses originate from a tumor cell progenitor with a small number of genetic alterations. We recently defined inactivating mutations of a histone acetyltransferase gene, CREBBP, as an early event in disease genesis following translocation of the BCL2 oncogene. CREBBP inactivation in primary FL tumors silenced a germinal center B- cell transcriptional program that was significantly enriched for genes involved in antigen presentation on MHC class II and targets of the YY1 transcription factor. Reduced antigen presentation was associated with a decreased ability of tumor cells to activate CD4 T-cell proliferation and a reduced number of T-cells within the tumor microenvironment. CREBBP epigenetically regulates gene expression via histone acetylation that is directed via interactions with transcription factors such as CIITA, a central regulator of MHC class II gene expression, and YY1, a key regulator of germinal center B-cell development. We therefore hypothesize that inactivating mutations of CREBBP plays dual roles in promoting lymphomagenesis by driving immune evasion via decreased antigen presentation, and by deregulating B-cell development to allow stalling at the germinal center B-cell stage. To investigate this, we have developed in vitro and in vivo models of CREBBP inactivation using CRISPR-modification of lymphoma cell lines and transgenic mouse models, respectively. Importantly, CRISPR-modified cell lines recapitulate the phenotype of reduced MHC class II expression observed in primary FL tumors, and transgenic mouse models develop FL-like tumors when Crebbp is deleted and Bcl2 is over- expressed specifically within B-cells. We will use these models, in parallel with primary human tumors, to explore the hypothesized mechanistic roles of CREBBP inactivation in lymphomagenesis. Specifically, we will evaluate the capacity for CREBBP inactivation to reduce antigen presentation on MHC class II, and measure the effect that this has on T-cell responses in vitro and in vivo. In addition, we will investigate the role of CREBBP inactivation in reducing the expression of germinal center B-cell genes that are regulated by the YY1 transcription factor, and determine whether this leads to stalled differentiation at the germinal center B-cell stage in vivo using transgenic mouse models. We further hypothesize that these phenotypes are driven by epigenetic alterations, and may therefore potentially be corrected using epigenetic modifying drugs. We will therefore investigate the potential for HDAC inhibitors and EZH2 inhibitors to restore antigen presentation and associated T-cell responses, and to reestablish normal epigenetic and transcriptional programs of B-cell development. Together this work will provide validation for CREBBP inactivation as a key event in the development of FL, define the mechanism by which these genetic events contribute to immune evasion and deregulated B-cell development, and evaluate the use of epigenetic modifying compounds for counteracting these features of CREBBP inactivation. This will contribute important advances in the understanding and treatment of FL.