The MEK/MYC/p21 Axis Drives Senescence Bypass and BCL-xL Dependence in Vincristine-Resistant Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma, with outcomes for relapsed or metastatic disease remaining poor, with 3-year survival rates below 30%. Despite aggressive multimodal therapy, no new effective treatments have been introduced for relapsed RMS in more than three decades, underscoring the urgent need to elucidate mechanisms of therapy resistance. We generated vincristine-resistant fusion-negative (FN) RMS cell lines through long-term dose escalation and identified enhanced MAPK pathway activation, evidenced by increased ERK phosphorylation in vitro and a major subset of post-chemotherapy patient tumor specimens. Pharmacologic MEK inhibition with trametinib or genetic suppression of MEK1 significantly impaired the growth of resistant cells and enhanced vincristine efficacy in a KRAS(G12D)-induced zebrafish FN RMS model, leading to reduced tumor burden and delayed relapse. Mechanistically, trametinib induced a senescence-associated cell-cycle arrest in resistant cells through a MYC–p21 regulatory axis, wherein MYC directly represses CDKN1A (p21) expression by binding to its promoter, and MYC depletion or trametinib-mediated MYC suppression result in p21 upregulation and senescence induction. Importantly, MYC overexpression rescued cells from trametinib-induced senescence, confirming its role in senescence bypass. Vincristine-resistant cells also exhibited increased dependence on the anti-apoptotic protein BCL-xL for survival, demonstrating heightened sensitivity to the selective BCL-xL inhibitor A-1155463. Accordingly, combined inhibition of MEK and BCL-xL using trametinib and A-1155463 significantly suppressed tumor growth and induced apoptosis in vincristine-resistant cells, producing significant antitumor activity in both cell line and zebrafish xenograft models of vincristine-resistant tumors. Collectively, these findings identify enhanced MAPK signaling and MEK/MYC/p21-mediated senescence bypass as key drivers of vincristine-resistant FN RMS and reveal a therapeutic vulnerability to combined MAPK and BCL-XL targeting, providing a rational therapeutic strategy and preclinical rationale for combination therapy in treatment-refractory RMS.