Identification of Novel Gene Regulatory Interactions Driving the Immune Evasion Program Through Transcriptional Activation of CD47 in Cancer

摘要：CD47是一种细胞表面蛋白，几乎存在于每个细胞中，它与Sirpα相互作用。 巨噬细胞抑制吞噬作用。在癌细胞中，CD47转录和蛋白表达异常 上调，保护癌细胞不被巨噬细胞识别和清除。封锁 CD47-Sirpα信号转导抗CD47或抗Sirpα单抗增强巨噬细胞 吞噬多种癌症类型。由于这一信息具有临床意义，它是至关重要的。 了解导致CD47上升的确切机制和上游监管机构 在癌细胞中表达，以开发针对转录上游组的新疗法 在白血病和实体瘤中错误调节CD47的基因或表观遗传修饰因素，以及在 通过这种方式，可以防止恶性细胞逃避免疫监视。通过执行基因组顺时态- 调节性分析，我发现在某些癌症中，两个不同的超级增强子(SE)与CD47相关 而不是在健康的对应物中。我还发现了一组活性成分增强剂，位于 在两个CD47序列中，调节不同类型癌细胞中CD47的表达：1)和 上游CD47成分增强子在T淋巴细胞白血病(T-ALL)细胞中活性，2)下游 CD47成分增强子在乳腺癌(激素阳性亚型)中是活性的，它对诱导有反应 肿瘤坏死因子-NFKB1信号通路。相比之下，我的初步数据表明，CD47在 白血病细胞系，不依赖于这种炎症途径，并依赖于所涉及的途径 在造血发育过程中。基于这些发现，我假设：在不同的癌症中 类型，独特形成的远端增强子或超级增强子(SE)，从 炎症和/或发育程序，以失调免疫逃避的靶基因(例如，CD47)。这 该提案旨在研究其他机制(包括表观遗传变化和基因组修改)， 单独或与肿瘤坏死因子炎症途径联合使用，尤其是由乳腺癌细胞和 白血病亚型通过错误调节CD47来启动免疫逃避反应。为了了解这些 癌症细胞与健康细胞的调节机制发生了变化，拟议中的实验将得到实施 主要在患者来源的实体肿瘤(特别是乳腺癌)和白血病样本中检测；结果 将与健康捐赠者样本的结果进行比较。对于这项建议，具体目标如下 执行：1)确定是否形成唯一的CD47超级增强子(SE)以响应 在激素阳性的乳腺癌中，炎症信号使免疫逃避程序失活。 2)调查CD47增强子或SE是否错误地招募输入(转录因子) 这通常会推动造血发展，引发白血病细胞中CD47的失活。

ABSTRACT: CD47 is a cell surface protein, found virtually in every cell, which interacts with SIRPα on macrophages to inhibit phagocytosis. In cancer cells, CD47 transcript and protein expression is aberrantly upregulated, protecting the cancer cells from being recognized and cleared by macrophages. Blockade of CD47-SIRPα signaling by anti-CD47 or anti-SIRPα monoclonal antibodies (mAbs) enhances macrophage phagocytosis of a variety of cancer types. Due to the clinical implications this information has, it is critical to understand the precise mechanisms and upstream regulators responsible for increasing CD47 expression in cancer cells, to develop novel therapies to target the upstream group of transcription factors, genetic or epigenetic modifications that misregulate CD47 in leukemia and solid tumors, and in this manner, prevent malignant cells from evading immunosurveillance. By performing a genomic cis- regulatory analysis, I found that two distinct super-enhancers (SEs) are associated with CD47 in certain cancer cell types and not in their healthy counterparts. I also found that a set of active constituent enhancers, located within each of the two CD47 SEs, regulate CD47 expression in different cancer cell types: while 1) an upstream CD47 constituent enhancer is active in T-cell lymphoblastic leukemia (T-ALL) cells, 2) a downstream CD47 constituent enhancer is active in breast cancer (hormone-positive subtype), and it responds to induction of the TNF–NFKB1 signaling pathway. In contrast, my preliminary data suggests that CD47 upregulation in leukemia cell lines, is independent of this inflammatory pathway, and is dependent on pathways that are involved in hematopoietic developmental processes. Based on these findings, I hypothesize that: in different cancer types, uniquely formed distal enhancers or super-enhancers (SEs), recruit inputs (transcription factors) from the inflammatory and/or developmental program to dysregulate target genes of immune evasion (e.g, CD47). This proposal aims to investigate other mechanisms (including epigenetic changes and genomic modifications) that alone or in combination with the TNF inflammatory pathway are used particularly by breast cancer cells and leukemia subtypes to initiate an immune evasion response by misregulating CD47. To understand how these mechanisms of regulation are altered in cancer versus healthy cells, the proposed experiments will be carried out mainly in patient-derived solid tumors (specifically breast cancer) and leukemia samples; and the results will be compared with results from healthy donor samples. For this proposal, the following specific aims will be performed: 1) Determine whether unique CD47 super-enhancers (SEs) are formed to respond to inflammatory signaling to misactivate the immune evasion program in hormone-positive breast cancers. 2) Investigate whether CD47 enhancers or SEs are erroneously recruiting inputs (transcription factors) that typically drive hematopoiesis development, to trigger misactivation of CD47 in leukemia cells.