Abstract Background: FLT3 mutations define a high-risk subset of AML, and FLT3 inhibitors are routinely used in combination with induction chemotherapy in fit patients, and with a low-intensity HMA/Venetoclax backbone in older patients. However, the majority of relapses arise from activation of bypass signaling, most commonly through the RAS/MAPK pathway. RAS mutations emerge in ∼20% of patients who progress on gilteritinib or other FLT3 inhibitors, driving sustained MAPK activation and therapy resistance. We hypothesized that simultaneous inhibition of FLT3 and the downstream RAS effector RAF would suppress MAPK reactivation and overcome this dominant resistance mechanism. Here, we characterize PHI3, a novel dual BRAF/FLT3 kinase inhibitor, and evaluate its preclinical activity in FLT3-mutant and RAS-mutant AML models. Results: Biochemical assays and nanoBRET target-engagement studies showed that PHI3 potently inhibits wild-type BRAF, BRAFV600E, and FLT3 at nanomolar concentrations. Immunoblot analysis confirmed robust suppression of FLT3, MEK, and ERK phosphorylation by PHI3 in FLT3-ITD+ MOLM13 and MOLM14 cells (IC50 ∼35 nM), with activity also observed in OCI-AML2 and OCI-AML3. In contrast to gilteritinib, PHI3 effectively inhibited MAPK signaling in MOLM14 NRASG12C cells (IC50 ∼125 nM). FLT3-ITD AML lines were most sensitive to PHI3 (IC50 31-90 nM) and underwent apoptosis within 24 h. RAS-mutant cell lines showed variable but meaningful sensitivity: OCI-AML2/3 (IC50 ∼330-340 nM), SKM1 KRASK117N (61 nM), and THP-1 NRASG12D (2.6 µM). MOLM14 isogenic NRAS mutants, resistant to gilteritinib (IC50 1 µM), retained marked sensitivity to PHI3 (IC50 276-690 nM). PHI3 also reduced clonogenic growth, viability (IC50 ∼190 nM), and MAPK signaling in primary FLT3/RAS-mutant AML specimens (n=9). PHI3 combined with venetoclax produced additive activity in FLT3-ITD models. In vivo, PHI3 and gilteritinib both reduced tumor burden and prolonged survival in MOLM13 xenografts compared with vehicle control (median of 29 vs 20 days). PHI3 had superior efficacy compared with Gilteritinib in mice engrafted with MOLM14 NRASG12C, with a median survival of 20 days in vehicle-treated mice, 24 days in gilteritinib and 38 days in PHI3-treated mice. Leukemic cells from MOLM14-engrafted mice showed MAPK suppression with both PHI3 and gilteritinib, whereas in MOLM14 NRASG12C mice, MAPK signaling was reduced only with PHI3. No weight loss or other side effects were noted in mice that received PHI3 therapy. Conclusions: PHI3 is a potent dual BRAF/FLT3 inhibitor that effectively suppresses MAPK pathway reactivation and overcomes RAS-driven resistance to FLT3 inhibitors in AML. Its strong in vivo efficacy and tolerability positions PHI3 as a promising therapeutic candidate. These findings provide a clear mechanistic and translational rationale for advancing dual FLT3/BRAF targeting in patients with FLT3-mutant and FLT3/RAS co-mutant AML. Citation Format: Shikhar Sharma, Anna Skwarska, Evangelia Matenoglou, Sovira Chaudhry, Selvam Murugan, Catherine Smith, Pamela Sung, Eunice Wang, Donald Small, Evripidis Gavathiotis, Marina Konopleva. Dual FLT3/BRAF inhibitor PHI3 overcomes RAS-driven resistance in FLT3-mutant AML abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7107.
Sharma et al. (Fri,) studied this question.