AMG925, a Dual FLT3-CDK4/6 Inhibitor, Disrupts Survival Signaling and Triggers Apoptosis in AML Progenitor/Stem Cells

FLT3 mutations are detected in one third of patients diagnosed with acute myeloid leukemia (AML) and confer poor outcomes. Selective FLT3 inhibitors have been developed and are being tested at different stages of clinical development. Drug resistance triggered by inhibitor-induced secondary mutations is often observed, and is a major cause of treatment failure. AMG925 (FLX925) is a small molecule dual FLT3-CDK4/6 inhibitor, currently in clinical development. Because AMG925 concomitantly inhibits both FLT3 and CDK4/6, AMG925 treatment rarely causes development of secondary drug-resistant FLT3 mutations (Li C et. al Mol. Cancer Ther. 2015). In this study, we examined the anti-leukemic effect of AMG925 on AML, focusing on AML progenitor/stem cells. Utilizing the high-throughput technology of time-of-flight mass cytometry (CyTOF) and reverse phase protein array (RPPA), we investigated alterations of the FLT3-mediated signaling network trigged by AMG925 at the single cell level. While AMG925 caused cell growth inhibition and apoptosis in both, FLT3-mutated (MOLM13, MV4;11, Ba/F3-ITD, Ba/F3-D835Y and Ba/F3-ITD/D835Y) and -wild type (Ba/F3-wt, OCI-AML3 and U937) cell lines and primary AML blasts, it elicited maximal response in cells carrying ITD, D835Y and ITD/D835Y double mutations. Mechanistically, AMG925 attenuates CDK4 phosphorylation of retinoblastoma protein (Rb), inhibits FLT3 activation and abrogates FLT3-dependent and independent AKT/mTOR, MEK/ERK and STAT5 signaling. AMG925 at 10nM effectively inhibited colony formation of PDX-derived leukemic stem cells originating from 2 AML patients carrying FLT3-ITD mutation (54±8.7% inhibition, n=4). CyTOF analysis of cell surface and intracellular molecules measured by a panel of 36 antibodies revealed that the inhibitory effect of AMG925 on p-AKT, p-ERK and p-STAT5 is operational primarily in subpopulations expressing the stem cell markers CD123, CD117, CD34, CD133 and CD47. Using RPPA, we studied the expression and phosphorylation changes of 273 proteins from the same 2 AML-ITD xenograft samples in response to AMG925 ex-vivo. Heterogeneous expression signatures were observed in all samples isolated from bone marrows (n=1) and spleens (n=4). AMG925 treatment altered multiple signaling networks by inhibiting key molecules in the AKT/mTOR, cyclin D family, MEK/ERK and Src pathways, and triggered a stress-response by up-regulating STAT3 and TSC1 in all samples examined. The combinational consequences of targeting FLT3-CDK4/6 and the proteins that contribute to resistance are under investigation. Conclusions: combined FLT3-CDK4/6 inhibition by AMG925 results in growth inhibition and apoptosis of AML cells with or without FLT3 mutations and selectively inhibits signaling within PDX and phenotypically defined AML stem/progenitor cell populations. CyTOF analysis confirmed on-target effects of AMG925 on FLT3-regulated signaling in AML stem cell subsets. RPPA profiling of AML-ITD xenografts exposed ex-vivo to AMG925 revealed alterations in multiple pro-survival signaling pathways and identified the upregulation of alternative pathways potentially conferring resistance to AMG925. While our results indicate efficacy of combined FLT3-CDK4/6 inhibition it identifies unexpected resistance mechanisms that provide rationale for the additional mechanism-based combinatorial approaches to increase the therapeutic efficacy in AML