Enhancing FLT3 inhibitor efficacy in acute myeloid leukemia with FLT3-ITD

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Acute myeloid leukemia (AML), the common type of acute leukemia in adults, is an important health concern for Veterans due to its high incidence and frequent poor response to treatment. Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase is present in AML cells of 30% of patients and is associated with poor treatment outcomes. FLT3 inhibitors have activity, but responses are limited and transient, with rapid development of resistance occurring by diverse mechanisms. FLT3-ITD causes constitutive FLT3 signaling, which is also aberrant. In addition to activating the PI3K/AKT/mTOR and MEK/ERK/RAS pathways, which are activated by signaling of wild-type FLT3, FLT3-ITD activates signal transducer and activation of transcription (STAT) 5, which transcriptionally upregulates the oncogenic serine threonine kinase Pim-1. Pim-1 contributes directly to the proliferative and anti-apoptotic effects of FLT3-ITD, and also phosphorylates and stabilizes FLT3, promoting STAT5 signaling in a positive feedback loop in cells with FLT3-ITD. Cells with FLT3-ITD also exhibit inactivation of the serine/threonine phosphatase protein phosphatase 2A (PP2A), which has tumor suppressor activity. The transcription factor c- Myc is transcriptionally upregulated downstream of FLT3-ITD, along with Pim-1, and knockdown of c-Myc or Pim-1 has anti-proliferative effects in cells with FLT3-ITD. In addition, PP2A and Pim-1 both regulate c-Myc post-translationally. We hypothesize that concurrent targeting of aberrant signaling pathways will enhance the efficacy of FLT3 inhibitors and prevent development of resistance to FLT3 inhibitors, and overcome resistance. Mechanisms of acquired resistance to FLT3 inhibitors include development of point mutations in the FLT3 kinase domain, a FLT3-dependent mechanism, and of RAS mutations - a FLT3-independent mechanism. Expression of Pim kinases is also further upregulated in cells with FLT3-ITD with FLT3 inhibitor resistance. Work in our prior VA Merit Award focused on cellular and molecular effects of pan-Pim kinase inhibition in AML cells with FLT3-ITD. Pim inhibition has little effect by itself, but potentiates the anti-proliferative and pro- apoptotic effects of FLT3 inhibitors and of chemotherapy drugs in cells with FLT3-ITD. We found that Pim inhibition enhances induction of apoptosis of AML cells with FLT3-ITD by FLT3 inhibitors in vitro and in vivo via post-translational downregulation of the anti-apoptotic protein Mcl-1 and its deubiquitinase USP9X. We subsequently found that post-translational c-Myc downregulation precedes these changes. We also found that concurrent treatment of cells with FLT3-ITD with PP2A-activating drugs and FLT3 inhibitors causes post-translational downregulation of c-Myc and Pim-1; and that these effects are mediated by AKT inactivation-dependent activation of GSK-3 which phosphorylates both c-Myc and Pim-1, enhancing their proteasomal degradation. The overall hypothesis of this Merit award proposal is that concurrent treatment with PP2A activating drugs or Pim kinase inhibitors enhances the efficacy of FLT3 inhibitors in AML with FLT3-ITD, abrogates or delays development of FLT3 inhibitor resistance, and has the potential to re-sensitize cells with FLT3 inhibitor resistance occurring by diverse mechanisms. Our Specific Aims are: 1. To test the hypothesis that PP2A-activating drugs and Pim kinase inhibitors increase FLT3 inhibitor efficacy through GSK-3-mediated post-translational c-Myc downregulation; 2. To test the efficacy of PP2A-activating drug or Pim inhibitor co-treatment in preventing FLT3 inhibitor resistance; and 3. To determine the efficacy of Pim inhibitor or PP2A-activating drug co-treatment in re-sensitizing AML cells with FLT3-ITD with FLT3 inhibitor resistance occurring by diverse mechanisms. The long-term objective is to develop clinical trials, with the ultimate goal of improving outcomes for patients with AML with FLT3-ITD, a common AML subtype with poor treatment outcome.