Acute myeloid leukemia (AML) is an important health concern for veterans, due to its high incidence and frequent poor response to treatment. Notably, internal tandem duplication (ITD) mutations of the fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase genes are present AML cells in a third of AML patients and are associated with rapid relapse following chemotherapy. FLT3 inhibitors have activity, but responses are limited and transient. Pim-1 kinase is a serine threonine kinase that is expressed in AML cells and is overexpressed downstream of FLT3-ITD in AML cells with this molecular abnormality. Pim-1 kinase regulates diverse proteins involved in proliferation, cell cycle, apoptosis and drug resistance, including, among others, c-MYC, the pro-apoptotic protein Bad and the anti-apoptotic protein Mcl-1, and is also implicated in DNA repair. We demonstrated that Pim-1 also phosphorylates FLT3 and promotes its aberrant signaling in a positive feedback loop in FLT3-ITD AML cells, and that Pym kinase inhibitors sensitize FLT3-ITD AML cells to apoptosis induction by FLT3 inhibitors. We then found that Pim kinase inhibitors also sensitize FLT3-ITD AML cells to apoptosis induction by chemotherapy drugs used to treat AML. Pim-1 substrate proteins are also expressed in key pathways in AML stem cells, and Pim-1 phosphorylation of these proteins may protect AML stem cells from the effects of chemotherapy and of FLT3 inhibitors. Pim kinase inhibitors are in current preclinical and clinical development. 1. The overall hypotheses of this grant proposal is that Pim-1 plays a key role in resistance of FLT3-ITD AML to available treatments, and that inhibition of Pim kinase will improve treatment outcomes in FLT3-ITD AML by abrogating multiple mechanisms of drug resistance and of disease progression.
The Specific Aims of the proposed work are: 1. To determine the mechanisms by which Pim kinase inhibition sensitizes FLT3-ITD AML cells to induction of apoptosis by chemotherapy drugs and by FLT3 inhibitors; 2. To optimize scheduling of administration of Pim kinase inhibitors, chemotherapy drugs and by FLT3 inhibitors in FLT3-ITD AML; and 3. To test the effects of in vivo administration of Pim kinase inhibitors with chemotherapy drugs and with FLT3 inhibitors on FLT3-ITD AML cells and AML stem cells and on normal hematopoietic cells. The effects of Pim-1 kinase inhibition will be studied in AML cell lines and patient samples using both in vitro culture systems and an in vivo model.
In Aim 1, we will test the hypotheses that Pim kinase inhibition sensitizes FLT3-ITD cells to apoptosis by inhibiting FLT3-ITD signaling, altering expression and activation of pro- and anti-apoptotic proteins, promoting generation of reactive oxygen species and/or inhibiting repair of DNA damage. Mechanistic insights achieved in Aim 1 will guide design of scheduling of Pim kinase inhibition in relation to exposure to chemotherapy drugs and to FLT3 inhibitors. Approaches to scheduling will be tested in vitro, with apoptosis as the readout. In addition, effects of Pim kinase inhibition on cell cycle could result in kinetic resistance to drugs, and notably cytarabine, and will be tested. Finally, effects on normal hematopoietic cells will be studied to establish a therapeutic index.
In Aim 3, strategies optimized in vitro in Aim 2 will be tested in vivo an immunodeficient mouse model. It is anticipated that the proposed work will lead to development and optimization of regimens incorporating Pim kinase inhibitors in the treatment of FLT3-ITD AML, with the goal of improved outcomes in this unfavorable AML subset...

Public Health Relevance

Acute myeloid leukemia (AML) is an important health concern for Veterans due to its high incidence and frequent poor response to treatment in this population. AML accounts for 80% of adult acute leukemia. The incidence increases with age, with a median age of 66 years at diagnosis, and is higher in males than females. Importantly, AML arises not only de novo, but also following chemotherapy and/or radiation therapy of other cancers, including those common in Veterans. AML also arises as a consequence of environmental exposures, including those during military service. The proposed work explores an approach to improving treatment outcomes for patients with AML. As such, it has the potential to favorably impact outcomes in Veterans who develop this disease, or are at risk for this disease, including following medical and environmental exposures.

National Institute of Health (NIH)
Veterans Affairs (VA)
Non-HHS Research Projects (I01)
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Hematology (HEMA)
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Baltimore VA Medical Center
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Larrosa-Garcia, Maria; Baer, Maria R (2017) FLT3 Inhibitors in Acute Myeloid Leukemia: Current Status and Future Directions. Mol Cancer Ther 16:991-1001
Muvarak, Nidal E; Chowdhury, Khadiza; Xia, Limin et al. (2016) Enhancing the Cytotoxic Effects of PARP Inhibitors with DNA Demethylating Agents - A Potential Therapy for Cancer. Cancer Cell 30:637-650
Doshi, Kshama A; Trotta, Rossana; Natarajan, Karthika et al. (2016) Pim kinase inhibition sensitizes FLT3-ITD acute myeloid leukemia cells to topoisomerase 2 inhibitors through increased DNA damage and oxidative stress. Oncotarget 7:48280-48295