Identifying/Targeting Mechanisms of Lymphomagenesis Driven by CREBBP Inactivation

项目摘要/摘要 滤泡性淋巴瘤(FL)是一种生发中心B细胞的不可治愈的恶性肿瘤，在这种情况下会连续复发 起源于具有少量基因改变的肿瘤细胞前体。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 Bcl2癌基因易位。原发FL肿瘤中CREBBP失活使生发中心B- 细胞转录程序，显著丰富了参与MHC抗原提呈的基因 YY1转录因子的II类和靶标。抗原提呈减少与 肿瘤细胞激活CD4T细胞增殖的能力降低，体内T细胞数量减少 肿瘤微环境。CREBBP通过组蛋白乙酰化调控基因表达，即 通过与转录因子如CIITA的相互作用而定向，CIITA是MHC II类基因的中央调节因子 表达，以及YY1，生发中心B细胞发育的关键调节因子。因此，我们假设 CREBBP基因失活突变通过驱动免疫逃避促进淋巴瘤发生的双重作用 通过减少抗原提呈，以及放松对B细胞发展的管制，允许在生发期停滞 中央B细胞期。为了研究这一点，我们建立了CREBBP的体外和体内失活模型 分别采用CRISPR修饰淋巴瘤细胞系和转基因小鼠模型。重要的是 CRISPR修饰的细胞系概括了观察到的原代MHC-II表达降低的表型 FL肿瘤，当CREBBP缺失和Bcl2过度缺失时，转基因小鼠模型发生FL样肿瘤。 在B细胞中特异表达。我们将使用这些模型，与人类原发肿瘤并行，以探索 CREBBP失活在淋巴瘤发生中的假想机制作用。具体地说，我们将评估 CREBBP灭活减少MHC-II类抗原提呈的能力及其影响 这对T细胞在体外和体内的反应都有影响。此外，我们将调查CREBBP的作用 YY1转录调控生发中心B细胞基因表达的失活 因子，并确定这是否导致体内生发中心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变化驱动的， 因此，可能会使用表观遗传修饰药物进行纠正。因此，我们将调查 HDAC抑制剂和EZH2抑制剂恢复抗原提呈和相关T细胞的可能性 反应，并重建正常的表观遗传和转录程序的B细胞发展。同舟共济 这项工作将为CREBBP失活提供验证，作为FL发展中的关键事件，定义 这些遗传事件导致免疫逃避和B细胞发育失调的机制， 并评价使用表观遗传修饰化合物来对抗CREBBP的这些特征 失活。这将有助于对FL的理解和治疗取得重要进展。

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.