Functional impact and targetability of , , and mutations in a spindle cell rhabdomyosarcoma with MYOD1 L122R mutation.

Spindle cell/sclerosing rhabdomyosarcoma (ssRMS) is a rare subtype of rhabdomyosarcoma, commonly harboring a gain-of-function L122R mutation in the muscle-specific master transcription factor MYOD1. -mutated ssRMS is almost invariably fatal, and development of novel therapeutic approaches based on the biology of the disease is urgently needed. MYOD1 L122R affects the DNA-binding domain and is believed to confer MYC-like properties to MYOD1, driving oncogenesis. Moreover, the majority of the -mutated ssRMS harbor additional alterations activating the PI3K/AKT pathway. It is postulated that the PI3K/AKT pathway cooperates with MYOD1 L122R. To address this biological entity, we established and characterized a new patient-derived ssRMS cell line OHSU-SARC001, harboring MYOD1 L122R as well as alterations in , , and We explored the functional impact of these aberrations on oncogenic signaling with gain-of-function experiments in C2C12 murine muscle lineage cells. These data reveal that PIK3CA, the novel PIK3CA variant discovered in this patient specimen, is a constitutively active kinase, albeit to a lesser extent than PI3KCA, a hotspot oncogenic mutation. Furthermore, we examined the effectiveness of molecularly targeted PI3K/AKT/mTOR and RAS/MAPK inhibitors to block oncogenic signaling and suppress the growth of OHSU-SARC001 cells. Dual PI3K/mTOR (LY3023414, bimiralisib) and AKT inhibitors (ipatasertib, afuresertib) induced dose-dependent reductions in cell growth. However, mTOR-selective inhibitors (everolimus, rapamycin) alone did not exert cytotoxic effects. The MEK1/2 inhibitor trametinib did not impact proliferation even at the highest doses tested. Our data suggest that molecularly targeted strategies may be effective in PI3K/AKT/mTOR-activated ssRMS. Taken together, these data highlight the importance of utilizing patient-derived models to assess molecularly targetable treatments and their potential as future treatment options.