Activating RAS mutations are found in a subset of fusion-negative rhabdomyosarcoma (RMS), and therapeutic strategies to directly target RAS in these tumors have been investigated, without clinical success to date. A potential strategy to inhibit oncogenic RAS activity is the disruption of RAS prenylation, an obligate step for RAS membrane localization and effector pathway signaling, through inhibition of farnesyltransferase (FTase). Of the major RAS family members, HRAS is uniquely dependent on FTase for prenylation, whereas NRAS and KRAS can utilize geranylgeranyl transferase as a bypass prenylation mechanism. Tumors driven by oncogenic HRAS may therefore be uniquely sensitive to FTase inhibition. To investigate the mutation-specific effects of FTase inhibition in RMS we utilized tipifarnib, a potent and selective FTase inhibitor, in in vitro and in vivo models of RMS genomically characterized for RAS mutation status. Tipifarnib reduced HRAS processing, and plasma membrane localization leading to decreased GTP-bound HRAS and decreased signaling through RAS effector pathways. In HRAS-mutant cell lines, tipifarnib reduced two-dimensional and three-dimensional cell growth, and in vivo treatment with tipifarnib resulted in tumor growth inhibition exclusively in HRAS-mutant RMS xenografts. Our data suggest that small molecule inhibition of FTase is active in HRAS-driven RMS and may represent an effective therapeutic strategy for a genomically-defined subset of patients with RMS.

Directly targeting RAS is a promising approach for the treatment of RAS-altered malignancies. Recently, several groups have developed mutation-specific agents such as sotorasib and adagrasib, which directly target KRAS by covalently modifying KRAS(G12C). KRAS is commonly altered in adult malignancies, such as lung, pancreatic, and colorectal adenocarcinomas, but is less commonly altered in pediatric cancers. However, rare pediatric solid tumors harboring KRAS(G12C), HRAS(G12C), or NRAS(G12C) have been observed, including rhabdomyosarcoma and neuroblastoma tumors, as well as leukemias. The efficacy of KRAS(G12C) inhibitors in pediatric malignancies is currently unknown, and the ability of these drugs to modify HRAS(G12C) and NRAS(G12C) has not been completely characterized. In this study, we show that sotorasib, adagrasib, and the RAS-ON inhibitor RMC-6291 are effective in a neuroblastoma cell line altered by KRAS(G12C). We further demonstrated that sotorasib and adagrasib inhibited SOS-mediated guanine nucleotide exchange on HRAS(G12C) and NRAS(G12C). Sotorasib and RMC-6291 decreased ERK phosphorylation in cells expressing HRAS(G12C), KRAS(G12C), or NRAS(G12C). Importantly, sotorasib also decreased ERK phosphorylation in a NRAS(G12C)-altered cell line xenograft model; however, this treatment did not prolong survival as a single agent. These results suggest that combinations of targeted agents that include sotorasib may be required for clinical benefit in pediatric patients with HRAS(G12C)- or NRAS(G12C)-altered malignancies, in addition to those with KRAS(G12C)-altered malignancies.

Despite comprehensive and multi-modal therapy, outcomes for children and adolescents with rhabdomyosarcoma (RMS) have plateaued over the past four decades. This is not for a lack of progress in the basic and translational studies of RMS. Indeed, advances in animal models and/or patient tissue sample acquisition and analysis have improved our understanding of RMS biology. Large-scale sequencing efforts have generated transcriptomic, genomic, and epigenomic datasets that highlight the heterogeneity of RMS and have the potential to improve prognostication and the application of precision medicine in patients with RMS. However, few of these discoveries have been clinically translated, and limitations to the accessibility, uniformity, and application of these new models and datasets hinder their utility. Here, we discuss how advances in understanding RMS biology, optimization of preclinical models, and strategies for translating basic science discoveries to the clinic can potentially improve outcomes for patients with RMS.