Overcoming AC220 resistance of FLT3-ITD by SAR302503

Activating mutations in FLT3 (Fms-like tyrosine kinase 3) by internal tandem duplication (ITD) mutations are found in approximately 30% of patients with acute myeloid leukemia (AML) and are associated with poor outcome in this patient population. Numerous FLT3 inhibitors have been tested for the treatment of AML, but these inhibitors have shown variable responses that were attributed to heterogeneity in AML and mechanistic differences associated with inhibitory mechanism employed by these inhibitors. Nevertheless, many patients showed a reduction in blast counts and hematological improvements, but these remissions were not durable that questioned the validity of FLT3 as a therapeutic target in AML. To address this, an elegant translational study designed by Neil Shah’s group has identified the AC220 resistant mutations in all FLT3-ITD relapsed patients that definitively demonstrated the validity of FLT3-ITD as a therapeutic target in human AML. Furthermore, the emergence of resistant mutations has been reported from the relapsed AML patients with FLT3-ITD treated with PKC412 and sorafenib. In addition, an in vitro resistant screening identified mutation at gatekeeper residue conferred cross-resistance to all known FLT3 inhibitors. These observations suggest that secondary mutations conferring resistance in the kinase domain will pose a significant clinical challenge, which prompted us to identify new inhibitors against the FLT3 resistant variants. SAR302503 is a rationally designed small-molecule ATP-competitive inhibitor of JAK2, and it had been shown to have a high degree of kinase selectivity for JAK2 and FLT3 in in vitro kinase assays. Given its equal potency toward FLT3 prompted us to test its efficacy against FLT3-ITD variants resistant to AC220. SAR302503 inhibited proliferation of BAF3 cells expressing FLT3 wild type and FLT3-ITD, with IC50 values of 119 and 330 nM, respectively (Figure 1a), whereas parental BAF3 cells and BAF3JAK2-V617F cells were inhibited at IC50 values of B1100 and 600 nM, respectively (Supplementary Figure 1a). In accordance to this, western blotting of phospho-STAT5 and phospho-FLT3 showed reduced phosphorylation at concentrations that parallel the concentrations required to inhibit cell proliferation (Figure 1c). These observations suggest that SAR302503 is more selective to FLT3-WT4FLT3-ITD4JAK2-V617F, and it can be exploited for therapeutic targeting of FLT3-ITD in AML (Supplementary Figure 1a). Next, we analyzed the activity of SAR302503 against five different kinase domain variants of FLT3-ITD that had been shown to confer resistance against AC220. The growth of BAF3 cells expressing resistant variants of FLT3-ITD was inhibited completely within the range of 800 nM (Figure 1a). Interestingly, two FLT3-ITD variants (D839G and Y842H) were found to be hypersensitive to the drug (Figures 1a and b) while mutations at gatekeeper residue F691L and D835F/Y showed only twofold resistance to the drug (Figure 1b). Next, we performed in vitro resistant screening as described previously, to identify patterns of drug resistance using cells expressing FLT3-ITD, FLT3-ITD-F691L and FLT3-ITD-D835Y at 3 mM (Cmax value in human ) of SAR302503. Surprisingly, we could not see the emergence of resistant clones against SAR302503 at this concentration (Supplementary Figure 1b). To rule out any off-target effect, we performed screening of BAF3 cells expressing Tel-JAK3 (having a cellular IC50 value of 2300 nm) at 2000 and 3000 nm of SAR302503, which showed growth of innumerable colonies