Targeting AML Resistance with LY3009120-Sapanisertib and Ruxolitinib-Ulixertinib Combinations Demonstrate Superior Efficacy in <i>FLT3</i> , <i>TP53,</i> and <i>MUC4</i> mutations

Abstract Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy characterized by the clonal expansion of myeloid precursor cells. Advances in genomic profiling have enhanced our understanding of AML pathogenesis, leading to the identification of recurrent mutations, including TP53, FLT3, MUC4, RAS, and IDH1/2 . These mutations significantly influence treatment response and prognosis, with TP53 mutations conferring poor outcomes and resistance to conventional therapies. Despite the advent of venetoclax-based regimens, resistance mechanisms persist, necessitating the development of novel therapeutic strategies. This study aims to investigate the efficacy of drug combinations for the treatment of AML using both in vitro AML cell lines and an in vivo zebrafish embryo xenograft model. Specifically, we focus on two drug combinations; the pan-RAF inhibitor LY3009120 combined with the mTOR inhibitor sapanisertib (designated as LS), and the JAK1/2 inhibitor ruxolitinib combined with the ERK inhibitor ulixertinib (designated as RU). The study integrates real-time cell viability assays, xenograft imaging, and genomic analyses to assess drug efficacy and explore correlations between treatment responses and mutational profiles, particularly TP53, FLT3, and MUC4 mutations. Both combinations, LS and RU, demonstrated superior efficacy compared to venetoclax-based treatments in reducing cell viability across AML cell lines. The LS combination showed significant reductions in cell viability in MOLM16 and SKM cells, while RU exhibited comparable efficacy with lower toxicity profiles. In zebrafish embryos, the LS combination effectively inhibited the proliferation of xenografted human AML cells, as evidenced by decreased fluorescence signals, indicating cell death. The RU combination also disrupted survival signaling pathways, showing promise as a therapeutic strategy. Furthermore, a correlation was identified between drug response and mutational profiles, with TP53, FLT3 and MUC4 mutations significantly influencing sensitivity to the LS and RU combinations. These findings support the further development of LS and RU as effective alternatives to current clinical regimens, with implications for personalized AML treatment.