Chromatin regulators of stemness and therapy resistance in rhabdomyosarcoma

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PROJECT SUMMARY / ABSTRACT Rhabdomyosarcomas (RMS) are the most common childhood soft-tissue sarcomas affecting hundreds of patients in the United States annually. Current standard treatments for rhabdomyosarcoma (RMS) patients include chemotherapy, surgery, and /or radiation. However, even with these combinations of therapeis, significant subsets of patients suffer tumor recurrence, relapse, and metastasis, associated with extremely worse prognosis and dismal 5-year survival rate. This remains the major hurdle to improve the patient outcomes with rhabdomyosarcomas. To better understand the mechanisms such as tumor-propagating cells, critical molecular regulators that drives therapy resistance and tumor-relapse in RMS, researchers has employed RMS cell lines, transgenic animal models, and xenograft studies to study the potential tumor-propagating cells (TPCs) for RMS. Yet, little is known about the tumor heterogeneity and cancer cell evolution dynamics in RMS. To dissect the inter-tumoral and intra-tumoral heterogeneity, I have used the single-cell transcriptomics to profile patient-derived samples of RMS. I uncovered distinct cell states in RMS tumors, including proliferation and a mesenchymal-like subpopulations that have higher TPC potential, whereas the differentiated muscle subpopulation that barely transits towards other cell states. With this knowledge, and the innovation of barcode tracing techniques, I propose to dissect molecular mechanisms that contribute to cell state transitions, and concurrently assess the cell phenotypes changes along with its transcripts, proteins, and epigenetics alterations. One class of important and challenging molecules in regulating cancer stemness, evolution post therapies is chromatin regulators, which requires deep sequencing in limited cell line models. The technical innovation of single-cell multiomics, including single-cell RNA, single-cell ATAC, single-cell CUT&Tag, and cell lineage barcode tracing largely decrease the cost and time needed to profile cancer cell evolution along with epigenetic modifications at single-cell levels. With effective collaboration with computational biologists, I hypothesize that EZH2 and its catalytic product H3K27me3 lock RMS cells in the proliferative cell state and inhibit their transition into other differentiated states. To test this hypothesis, I will first assess the role of EZH2 in regulating cell state transitions with barcode tracing and functional stem assays in the context of EZH2 knockdown (Aim 1). Independently, I will also profile the direct targets of EZH2 and histone H3 lysine 27 trimethylation by performing single-cell CUT&Tag, and interrogate mechanism that controls cell state transition (Aim 2). In addition, I will also assess the EZH2 inhibitors in collaboration with chemotherapy and radiation utilizing the unique immune-compromised zebrafish models along with cell line and mouse xenograft studies (Aim 3). The goals of the proposed research are to investigate chromatin regulators in rhabdomyosarcoma samples while also acknowledging the tumor-heterogeneity and cell plasticity in cell state transitions. By achieving these aims, I will illustrate a comprehensive mechanism as to how RMS tumors evolve and how chromatin regulators play critical roles in controlling this process.