Using genome wide CRISPR screening to identify synthetic lethal targets for overcoming chemoresistance in rhabdomyosarcoma

Chemotherapy resistance in Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, remains a major obstacle to improve survival outcomes, particularly in high-risk alveolar RMS (ARMS), which accounts for approximately one-third of all RMS cases and is clinically more aggressive due to its strong propensity for metastasis, with survival rates fall below 24% after relapse. Drug resistance is a primary driver of treatment failure, contributing to nearly 80-90% of cancer-related mortality. Although irinotecan remains one of the most effective chemotherapeutic agents in RMS, the emergence of irinotecan-related chemoresistance highlights the urgent need to develop rational combination therapies to target the genetic robustness and functional redundancy of RMS cells. To overcome the stagnation in survival outcomes and intrinsic drug resistance, a genome wide Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Knockout screening was performed to develop and identify novel therapeutic vulnerabilities and effective combination strategies specifically tailored to overcome irinotecan resistance in ARMS cells. For the first time, our study identified 47 candidate prognostic genes whose deletion potentially sensitizes RMS cells to irinotecan treatment. Along with several druggable targets, pathway enrichment analysis revealed that the DNA damage repair pathway is the most significantly downregulated in irinotecan-resistance in RMS cells, suggesting that targeting this pathway could induce a synthetic lethal interaction with irinotecan. Among the top hits, we validated FGFR4 and PARP1 as key modulators of resistance, and siRNA-mediated silencing of these genes sensitizes RMS cells to irinotecan treatment. Most importantly, FGFR4 was significantly enriched as a gene dependency in ARMS. In addition, pharmacological inhibition of these genes using available small-molecule inhibitors markedly enhanced irinotecan sensitivity and further reduced cell viability, both as single agents and in combination, with a favorable therapeutic window, supporting their potential for clinical translation. We are currently investigating the mechanistic basis and in vivo efficacy of these drug combinations in RMS models. This study provides a promising framework for developing personalized and targeted therapeutic strategies to improve outcomes for RMS patients.