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

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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.