Abstract LB251: Vertical inhibition of the RAF MEK ERK cascade induces myogenic differentiation, apoptosis and tumor regression in H/NRAS Q61X mutant rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most commonly diagnosed soft tissue sarcoma in children. While the 5-year event-free survival for localized rhabdomyosarcoma is over 70%, it is less than 30% for patients presenting with metastatic disease. Given that the current treatment modalities for RMS patients remain cytotoxic therapies with profound life-long lasting complications, identification of less toxic and more effective therapies is highly desired. Whole-genome and RNA sequencing of human rhabdomyosarcoma have identified frequent RAS genes oncogenic mutations in fusion-negative rhabdomyosarcoma (FN-RMS), which comprises 65% of RMS. This high incidence of RAS mutations present opportunity for design of targeted therapeutic strategies for FN-RMS. By siRNA-mediated knock down of individual RAS isoforms we showed that H/NRAS Q61X-mutant FN-RMS cell lines exhibited oncogenic RAS dependency and that knockdown of the Q61X-mutant H/NRAS inhibited ERK MAPK, but not PI3K-AKT pathway activity. Knockdown of the Q61X-mutant H/NRAS phenocopied ERK inhibitor treatment by inducing myogenic differentiation. Further, as compared to RAS-wild type RMS cells, H/NRAS-Q61X FN-RMS cell lines were preferentially more sensitive to MEK or ERK, but not PI3K or AKT inhibitors. RNA sequencing analysis revealed significant overlap of genes signatures associated with NRAS-Q61H knockdown and ERK inhibitor treatment in the FN-RMS cell line RD. These results indicated a central role of the ERK MAPK pathway in mediating H/NRAS Q61X-dependency in FN-RMS. However, in vivo evaluation of clinically relevant exposers with the MEK inhibitor trametinib demonstrated poor response, which correlated with inefficient ERK inhibition in pharmacodynamic assays. A trametinib-sensitizing CRISPR screen pointed to vertical inhibition of the RAF-MEK-ERK cascade by co-suppression of MEK and either CRAF or ERK. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically blocked ERK activity and induced cell cycle arrest, myogenic differentiation and cell death. In vivo evaluation of low dose pan-RAFi + ERKi or MEKi + ERKi potently suppressed H/NRAS Q61X RMS tumor growth, with pan-RAFi + ERKi being more effective and better tolerated, suggesting wider therapeutic window. These results support the clinical translation of pan-RAFi + MEKi/ERKi for H/NRAS Q61X-mutant FN-RMS. Citation Format: Natalia Garcia, Vanessa Del-Pozo, Marielle Yohe, Craig Goodwin, Terry Shackleford, Long Wang, Kunal Baxi, Yidong Chen, Myron Ignatius, Kris Wood, Peter Houghton, Angelina Vaseva. Vertical inhibition of the RAF MEK ERK cascade induces myogenic differentiation, apoptosis and tumor regression in H/NRAS Q61X mutant rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB251.