Pre-clinical efficacy of FLT3/BRAF targeting with novel dual inhibitor PHI3 in FLT3/RAS co-mutated AML Free

Introduction: In AML with FLT3 mutations, FLT3 inhibitors are routinely used in combination with induction chemotherapy in fit patients, and with a low-intensity HMA/Venetoclax backbone in older patients. It was reported that relapses are commonly associated with activation of bypass signaling, most frequently through the RAS/MAPK pathway. RAS mutations occur in approximately 20% of patients progressing on gilteritinib or other FLT3 inhibitors and lead to constitutive activation of downstream pro-survival and anti-apoptotic pathways. In this study, we tested the hypothesis that dual inhibition of the downstream RAS effector protein RAF and FLT3 could be an efficient strategy to overcome RAS-mediated resistance. We have investigated the pre-clinical efficacy of a novel dual BRAF/FLT3 kinase inhibitor, PHI3, in inhibiting MAPK and FLT3 signaling in FLT3-mutant or RAS-mutant AML cells, in the in vitro and in vivo models.

Results: In vitro kinase activity assays and nanoBRET® cellular target engagement assays revealed that PHI3 potently inhibits wild-type BRAF and mutant BRAF^V600E as well as FLT3 at nanomolar concentrations. Analysis of MAPK signaling by immunoblotting demonstrated that both gilteritinib and PHI3 exposure for 1 hour blocked the phosphorylation of FLT3, MEK, and ERK at IC₅₀ of 35nM in FLT3-ITD⁺ MOLM13 and MOLM14 cells, 240nM for OCI-AML2, and 270nM for OCI-AML3 cells. In MOLM14 cells harboring NRAS^G12C mutation, the FLT3 inhibitor gilteritinib failed to inhibit MAPK signaling at 1µM, while PHI3 inhibited MEK and ERK phosphorylation at 125nM. PHI3 was unable to block MAPK signaling in cells with FLT3^D835Y mutation.

Among all cell lines tested, AML cells with FLT3-ITD mutations MOLM13, MOLM14, and MV4-11 were most sensitive to PHI3, with mean IC₅₀ for growth inhibition at 24 hr of 31nM, 34nM, and 90nM, respectively. PHI3 treatment for 24hrs induced apoptosis in MOLM14 cells as demonstrated by elevated levels of cleaved PARP and caspase-3. OCI-AML2 and OCI-AML3 (FLT3^wt and NRAS^Q61k) cells responded to PHI3 at IC₅₀ of 334nM and 340nM, respectively. SKM1 cells with KRAS^K117N mutation were highly sensitive at an IC50 of 61nM, while THP-1 cells with mutated NRAS^G12D have an IC₅₀ of 2.6µM. MOLM14 isogenic cell lines harboring NRAS mutations were resistant to the type I FLT3 inhibitor gilteritinib with IC_50s of14nM in parental vs 1µM in MOLM14 NRAS^G12C and MOLM14 NRAS^Q61K cells; however, these cells retained sensitivity to PHI3, with IC_50s of 690nM and 276nM, respectively. MOLM14 isogenic cells expressing the FLT3FLT3^F691L and FLT3^D835Y mutations showed an IC50 of 676nM and 2µM, respectively, suggesting that PHI3 functions as a type II FLT3 inhibitor. PHI3-treatment reduced the clonogenic potential, viability (IC50=190nM), and MAPK signaling of NRAS/FLT3 mutant AML primary samples (n=9).

Given the established synergy of FLT3 and MEK inhibitors with venetoclax and ongoing clinical trials of venetoclax with FLT3 inhibitors, we analyzed the combinatorial efficacy of PHI3 with venetoclax. In FLT3-ITD cells, the combination induced additive growth inhibition for MOLM13, MOLM14, and MV4-11 cells.

We next investigated the efficacy of the single agent PHI3 (60mg/kg) and gilteritinib (10mg/kg) administered daily in the in vivo MOLM14-luc and MOLM14 NRAS^G12C-luc models. In MOLM13, both treatments reduced tumor burden and extended the survival of mice compared with vehicle control (median of 29 vs 20 days, p<0.05). Gilteritinib had inferior efficacy compared with PHI3 in mice engrafted with MOLM14 NRAS^G12C, with a median survival of 20 days in vehicle-treated mice, 24 days in gilteritinib and 38 days in PHI3-treated mice. Leukemic cells isolated from the bone marrow of engrafted mice after 5 days of treatment showed a reduction in MAPK signaling for MOLM14 engrafted mice with both PHI3 and gilteritinib; however, MOLM14 NRAS^G12C engrafted mice showed MAPK signaling reduction with PHI3 but not with gilteritinib. No weight loss or other side effects were noted in mice that received PHI3 therapy.

Conclusions: Our study demonstrates the efficacy of the novel dual BRAF/FLT3 inhibitor PHI3 in FLT3-mutated AML, including those resistant to gilteritinib due to RAS mutations. These findings suggest the potential therapeutic value of dual targeting of FLT3 and RAF/MAPK signaling in FLT3-mutated AML, achieved by blocking the major resistance bypass signaling pathway.