A Novel Inhibitor of FLT3 and Its Drug-Resistant Mutants with Superior Activity to Gilteritinib in Molm-13 Preclinical Acute Myeloid Leukemia Xenograft Model

Acute Myeloid Leukemia (AML) is a complex heterogenous disease characterized by the aberrant growth of undifferentiated myeloid progenitor cells typically driven by a combination of epigenetic and signaling mutations. In particular, fms-like tyrosine kinase 3 (Flt3) internal tandem duplication (FLT3-ITD) is associated with poor relapse-free and overall survival. Several FLT3 inhibitors have been developed such as the first generation multikinase inhibitor Midostaurin which is approved for newly diagnosed FLT3 mutant AML in combination with induction chemotherapy. Second generation inhibitors such as gilteritinib are more selective and potent and are also approved for relapsed/refractory FLT3 mutant AML. Although initial response has been positive with this class of agents, prolonged remission is rarely achieved, and diverse mechanisms of resistance often arise with FLT3 inhibitors representing an ongoing challenge. Common mechanisms of resistance to FLT3 inhibitors includes the acquisition of tyrosine kinase domain (TKD) secondary mutations such as the gatekeeper F691L TKD mutation, or the emergence of additional mutations in alternative signaling pathways such as RAS/MAPK. Thus, it is important to continue developing novel inhibitors capable of circumventing these resistance mechanisms. Herein we describe 2082-0047, a novel tyrosine kinase inhibitor with sub-nanomolar potency against FLT3 mutant AML, including TKD mutations. Compound 2082-0047 is a highly selective pan-FLT3 kinase inhibitor, as in both scanMAX and KdELECT KINOMEscan™ Profiling assays (Eurofins DiscoverX) it has shown 400-1000 fold selectivity.

In our initial screening, 2082-0047 was only effective in FLT3-ITD mutant cells, however the KINOMEscan™ Profiling data described above demonstrates that 2082-0047 is potent against FLT3, FLT3 ITD, D835H, D835V, D835Y, D835V, K663Q, N841I, and R834Q FLT3 TKD mutations. We confirmed this data in a cellular assay using BaF3 cell lines expressing the TKD mutations D835Y, D835H or F691L alone or with Flt3-ITD. Next, we confirmed using immunoblot analysis of phosphorylated Flt3 that 2082-0047 is a potent inhibitor of FLT3-ITD signaling (superior to gilteritinib) in FLT3-ITD mutant AML cell lines.

Using wildtype B6 mice, we conducted pharmacokinetic (PK) studies and found that 2082-0047 had 79 % oral bioavailability, achieved 1.2 μΜ C_{max ­}after 4 hours, and had half-life of 4.7 hours. Next, our absorption, distribution, metabolism and excretion (ADME) studies showed lack of CYP inhibition of major human isoforms.

Overall, our PK studies demonstrated overall favorable drug like properties enabling in vivo investigation of therapeutic activity. Therefore, we proceeded with using the MOLM-13 disseminated xenograft model in NCG recipient mice to determine the overall survival of 2082-0047 compared to gilteritinib. We enrolled mice 10 days post engraftment to receive daily oral vehicle, 3, 10, 20 or 30 mg/kg of 2082-0047 or 30 mg/kg of gilteritinib and found that 10 and 20 mg/kg of 2082-0047 were well tolerated and superior to gilteritinib at 30 mg/kg (Figure 1).

In summary, we describe a novel and potent inhibitor of FLT3, including both internal tandem duplication and drug-resistant tyrosine kinase domain (TKD) mutants, with promising preclinical activity which is superior to the most potent FDA approved FLT3 inhibitor gilteritinib in an aggressive AML in vivo model. Our non-GLP in vivo safety studies confirmed a broad therapeutic window of 2082-0047. Ongoing Investigational New Drug (IND) enabling studies will include comparing the effect of 2082-0047 to gilteritinib in an immunocompetent syngeneic AML murine model harboring both a Flt3-ITD and F691 TKD gatekeeper mutation.