(P 254) THE IMPACT OF MST (HIPPO KINASE) INHIBITION ON CHROMATIN ACCESSIBILITY AND ONCOGENIC PHENOTYPES IN FUSION-POSITIVE RHABDOMYOSARCOMA

Objective: Rhabdomyosarcoma (RMS) is a cancer of skeletal muscle histogenesis and the most common soft tissue sarcoma of childhood. Five-year survival for fusion-positive (FP) RMS is < 50% and when metastatic < 8%. FP refers to the presence of the signature fusion oncogene PAX3-FOXO1 (P3F). The encoded oncoprotein P3F is a super-transcription factor and the main FP-RMS cancer driver but is refractory to pharmacologic inhibition because it is non-catalytic and highly disordered. Using human FP-RMS patient-derived cell lines, we identified a P3F-->RASSF4--|MST1 signaling axis; P3F transcriptionally upregulates the expression of the scaffold protein RASSF4 to inhibit the Hippo pathway MST tumor suppressor kinase. In an isogenic model of FP-RMS, in which normal primary human skeletal muscle myoblasts (HSMMs) are converted to tumorigenic cells by the serial expression of P3F, hTERT, and MYCN, additional expression of a dominant-negative kinase-dead MST1 (MST1DN) increased in vivo tumorigenesis of murine xenografts. Light microscopy of the xenograft sections from the MST1DN cells demonstrated nuclear dimpling and open chromatin. Since MST1 exerts its tumor suppressor function in part by promoting chromatin condensation via phosphorylation of histone H2B, and MST1 induces apoptosis by phosphorylating the forkhead domain of FOXO1, we hypothesize that MST1/2 loss of function (LOF) supports a more primitive cellular state by increasing chromatin accessibility and altering expression of key cell identity and stemness genes.

Methods: We used both unbiased and candidate approaches (ATAC-seq/RNA-seq and phosphoproteomics) to interrogate chromatin changes and signaling events underlying the P3F-->RASSF4--|MST1 axis. Promising candidates were validated with qRT-PCR and immunoblot. Complementary genetic/genomic LOF approaches (shRNA, CRISPR, and CRISPRi) are ongoing to investigate the roles of MST1 and MST2 individually in in vitro and in vivo in FP-RMS oncogenic phenotypes.

Results: FP-RMS HSMM cells harboring an MST1DN construct have increased expression of stem cell genes RUNX1/2 and CTCF, but decreased expression of myogenic transcription factors MYOD1 and MYF5, suggesting a shift to a more primitive cellular state. The chromatin remodelers HMGA1 (Serine 102 and 103) and Nucleolin (Serine 184) have increased phosphorylation at the specified serine residues in our MST1DN cells, but the significance of these phosphorylation changes has not yet been investigated.

Conclusion: The P3F-->RASSF4--|MST1 signaling axis appears to restrain MST activity and thus increase chromatin accessibility and alter cellular identity genes in FP-RMS. Future efforts will focus on the validation of top candidate genes and proteins and their potential roles in FP-RMS oncogenic phenotypes.