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Background: FGFR4 is a receptor tyrosine kinase shown to be commonly overexpressed in rhabdomyosarcoma (RMS). In fusion positive (FP) RMS, FGFR4 overexpression is driven by the PAX3-FOXO1 fusion oncogene. In fusion negative (FN) RMS, FGFR4 is often overexpressed and mutationally activated in 7.5% of patients. CRISPR knockout experiments have shown that many RMS cell lines are dependent on FGFR4 for survival. Thus, targeting FGFR4 with a small molecule inhibitor is a promising approach to treat RMS. Futibatinib, a highly selective, irreversible FGFR1-4 inhibitor that was recently granted FDA approval for cholangiocarcinoma, may be effective in treating RMS. Single agent small molecule therapy with kinase inhibitors commonly leads to acquired resistance through compensatory signaling pathways. We hypothesized that inhibition of FGFR4 would lead to suppression of negative feedback loops, resulting in activation of oncogenic kinase pathways. Furthermore, we hypothesized that dual inhibition of FGFR4 and one of these upregulated kinases would be synergistic in delaying or reversing RMS progression. Methods: Cell viability assay was used to determine the IC50 of futibatinib in RMS cell lines in which FGFR4 is mutationally activated (RMS559, FN RMS) or overexpressed (RH4, FP RMS). A high throughput kinome activity assay was used to discover kinases that were activated in RMS cells treated with futibatinib. A drug matrix screen combining futibatinib with 144 drugs was used to discover synergistic combinations. Incucyte live cell imaging was used to validate the synergy of the inhibitor combinations. Western blot was used to confirm the activity of the kinase inhibitors. Caspase activity assays were used to monitor apoptosis. Results: The IC50 of futibatinib was ~500 nM for RMS559 and ~10 μM for RH4. Western blot showed that futibatinib inhibited FGFR4 phosphorylation in a dose dependent manner. The kinome activity assay found that futibatinib treatment resulted in SFK, AKT, and IGF1R activation. Drug matrix screening in RMS559 found that futibatinib was synergistic with SFK, IGF1R, and AKT inhibitors at cell killing. Incucyte confluence measurements confirmed the synergy of futibatinib with SFK, IGF1R, or AKT inhibitors in both RMS559 and RH4. Apoptosis was significantly higher in RMS cells treated with these combinations compared to individual inhibitors. Conclusions and future directions: These results suggest that treating RMS with an FGFR4 inhibitor combined with an SFK, IGF1R, or AKT inhibitor is synergistic in both FGFR4 mutated FN RMS and FGFR4 overexpressed FP RMS. These dual inhibitor therapies may be an effective approach in overcoming resistance to monotherapy and lead to better outcomes in FGFR4-dependent RMS patients. Additionally, our approach may be beneficial for other FGFR-dependent cancers which may develop resistance to monotherapy. Animal studies are underway to determine the in vivo efficacy of these combinations, and top hits will be considered for translation to the clinic. Citation Format: Jerry Wu, Adam Cheuk, Jun S. Wei, Yong Kim, David Milewski, Xiaohu Zhang, Michele Ceribelli, Erin Beck, Paul Shinn, Carleen Klump-Thomas, Kelli M. Wilson, Crystal McKnight, Zina Itkin, Elizabeth Calleja, Volker Wacheck, Hiroshi Sotome, Hiroshi Hirai, Craig Thomas, Javed Khan. Inhibition of FGFR4 with futibatinib combined with inhibition of IGF1R, Src family kinases, or AKT is synergistic against rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB315.

Drug repurposing is a rapid approach to identifying therapeutics for the treatment of emerging infectious diseases such as COVID-19. To address the urgent need for treatment options, we carried out a quantitative high-throughput screen using a SARS-CoV-2 cytopathic assay with a compound collection of 8,810 approved and investigational drugs, mechanism-based bioactive compounds, and natural products. Three hundred and nineteen compounds with anti-SARS-CoV-2 activities were identified and confirmed, including 91 approved drug and 49 investigational drugs. Among these confirmed compounds, the anti-SARS-CoV-2 activities of 230 compounds, including 38 approved drugs, have not been previously reported. Chlorprothixene, methotrimeprazine, and piperacetazine were the three most potent FDA approved drugs with anti-SARS-CoV-2 activities. These three compounds have not been previously reported to have anti-SARS-CoV-2 activities, although their antiviral activities against SARS-CoV and Ebola virus have been reported. These results demonstrate that this comprehensive data set of drug repurposing screen for SARS-CoV-2 is useful for drug repurposing efforts including design of new drug combinations for clinical trials.