Dual Targeting of MAPK Signaling and Senescence-Associated Survival Pathways Overcomes Vincristine Resistance in Fusion-Negative Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma, with limited therapeutic options for relapsed or metastatic disease. Fusion-negative (FN) RMS, the predominant subtype, frequently harbors mutations activating the RAS/MAPK pathway, yet the mechanisms underlying chemoresistance remain poorly defined. Here, we generated vincristine-resistant FN RMS cell lines through long-term dose escalation and identified increased MAPK signaling activity, evidenced by elevated phosphorylated ERK levels in vitro and in patient tumors post-chemotherapy. MEK inhibition via trametinib or MEK1 knockdown significantly impaired growth of resistant cells and enhanced vincristine efficacy in zebrafish tumor models, reducing tumor burden and relapse. Mechanistically, trametinib induced cell cycle arrest and senescence--marked by p21 upregulation--in resistant cells. This effect was dependent on suppression of MYC, as MYC disruption promoted senescence while MYC overexpression rescued the phenotype. Senescent cells remained partially viable via BCL-XL-mediated survival, rendering them sensitive to the BCL-XL inhibitor, A-1155463. Combined trametinib and A-1155463 significantly reduced growth of resistant RMS cells. Our findings highlight the MEK/MYC/p21 axis and BCL-XL dependency as key vulnerabilities in therapy-resistant FN RMS and support dual targeting of MAPK and senescence-survival pathways as a rational therapeutic strategy. STATEMENT OF SIGNIFICANCETherapeutic resistance remains a major barrier to improving survival outcomes in fusion-negative rhabdomyosarcoma (FN RMS), the most common pediatric soft tissue sarcoma. This study identifies enhanced MAPK signaling and MYC-driven suppression of p21 as key mechanisms by which FN RMS cells evade vincristine-induced senescence. Moreover, we reveal that senescent-resistant cells rely on BCL-XL for survival, uncovering a new vulnerability that can be exploited therapeutically. Dual targeting of the MEK/MYC/p21 axis and BCL-XL- mediated survival pathways significantly suppresses growth of vincristine-resistant FN RMS cells. These findings provide a compelling rationale for combination therapies that simultaneously disrupt oncogenic signaling and senescence escape mechanisms in treatment-refractory FN RMS.