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.