Activity of Allosteric, Switch-Pocket, ABL/FLT3 Kinase Inhibitor DCC2036 Against Cultured and Primary AML Progenitors with FLT-ITD or FLT3 Kinase Domain Mutations

Abstract 2611

Approximately 30% of acute myeloid leukemia (AML) patients have activating mutations in FLT3, commonly internal tandem duplication (ITD) mutations, which are associated with poor survival. Although FLT3 tyrosine kinase inhibitors (TKI) such as AC220 can induce remissions, resistance-causing mutations in FLT3-ITD are known to impair the in vitro activity of first and second generation FLT3 TKIs. DCC2036 is a unique switch-pocket, non-ATP competitive (allosteric) inhibitor with low nanomolar inhibitory concentration 50 (IC50) activity against a number of tyrosine kinases including FLT3 (1.7 nM), TRKA (7.0 nM), TIE-2 (2.7 nM) and BCR-ABL (2.0 nM). DCC2036 has shown promising activity in a phase I/II clinical trial in chronic myeloid leukemia (CML), where plasma concentrations of 350 nM of DCC2036 have been safely achieved. DCC-2036 has induced clinical and molecular remissions in patients with TKI-resistant CML expressing the ‘gate-keeper’ T315I BCR-ABL mutation, as well as demonstrated activity against mutations that cause BCR-ABL conformational escape resistant (Cancer Cell. 2011;19:556). Here, we evaluated the in vitro activity of DCC2036 against FLT3-ITD in cell line model systems. In the FLT3-ITD expressing human leukemia MV4-11 and MOLM-13 cells, treatment with DCC2036 (20 to 500 nM for 24 hours) dose-dependently induced cell cycle G1-phase accumulation with decline in the S and G2/M phases. Exposure to 50 to 500 nM DCC2036 for 48 to 72 hours also dose-dependently induced apoptosis of 30 to 80 % of MV4-11 and MOLM-13, as well as induced 30 to 50% apoptosis of patient-derived primary AML cells with FLT3-ITD (n =4). This was associated with dose-dependent decline in the levels of p-FLT3, p-STAT5, p-AKT, p-ERK1/2 and Bcl-xL levels but increase in the levels of BIM and p27. In contrast, treatment with DCC2036 induced significantly lower level of apoptosis (<15%) in either normal CD34+ progenitor cells or AML cells without FLT3-ITD. We next evaluated the in vitro activity of DCC2036 against AC220-resistant mutations in FLT3-ITD (F691L, D835V/Y and Y842C/H) that we identified in a pre-clinical screen and in patients with loss of response to AC220 (Smith et al, ASH 2011, submitted), as well as against FLT3-ITD/F691I, analogous to the BCR-ABL/T315I mutation. DCC2036 potently inhibited proliferation of FLT3-ITD transduced Ba/F3 cells with an IC50 of 14.5 nM (IC50 of parental Ba/F3 cells in the presence of IL-3 >1000nM). DCC2036 retained some activity against the clinically relevant FLT3-ITD gatekeeper mutation F691L and F691I (IC50 49 nM and 34 nM), and was similarly active against the activation loop mutations Y842C and Y842H (IC50 26–28 nM). The activation loop mutations D835V and D835Y, which are commonly detected in patients with loss of response to AC220 and are hypothesized to destabilize the kinase inactive “DFG-out” conformation, were substantially less sensitive to DCC2036 (IC50 233 nM and 196 nM, respectively). Based on our previous findings (Blood. 2005;105:1768) that FLT3-ITD is a heat shock protein (hsp) 90 client oncoprotein, we also determined the effect of co-treatment with the non-geldanamycin hsp90 inhibitor AUY922 (5 to 10 nM) (Novartis Pharmaceuticals) against the cultured and primary FLT3-ITD expressing AML cells. Co-treatment with AUY922 significantly improved the activity of DCC2036 against primary AML cells (p < .05). These findings demonstrate that DCC2036 exhibits potent activity against cultured and primary AML cells with FLT-3-ITD, as well as against cellular models of FLT3-ITD with AC220-resistant gatekeeper and select activation loop mutations. The molecular basis of resistance to DCC2036 conferred by activation loop mutations at D835 is under investigation. Co-treatment with DCC-2036 and the hsp90 inhibitor AUY922 exerted higher lethal activity against cultured and primary AML cells with FLT3-ITD.