PIM: An Integral Component of FLT3 Signaling and a Potential Therapeutic Target in Acute Myeloid Leukemia.
Authors: Fathi Amir T. , Swinnen Ian , Rajkhowa Trivikram , Small Donald , Marmsater Fredrik , Robinson John E , Gross Stefan David , Martinson Matthew , Allen Shelley , Kallan Nick , Levis Mark 
Published: October 2019

Abstract 1735

Poster Board I-761

Acute myelogenous leukemia (AML) continues to be a frequently fatal diagnosis, and traditional induction regimens do not provide satisfactory outcomes in most cases. Targeted therapies that would enhance options for therapy for AML are being actively investigated. PIM is a serine/threonine kinase, widely expressed as multiple subtypes, and noted to be up-regulated in several malignancies, including AML. It appears to play an important role in FLT3 mediated leukemogenesis and inhibition of apoptosis. AR29 is a small molecule inhibitor of PIM 1 and 3 (Array Biopharma Inc.). We have used this compound to investigate the potential utility of PIM inhibition in AML, and in particular, FLT3-mutant AML. We first used the FLT3-mutant cell lines MV4-11 and Molm14, as well as a FLT3/ITD-transfected line (TF/ITD), to study the cytotoxic effects of AR29. AR29 induced cytotoxicity in all 3 cell lines with IC50 values ranging from 115 nM to 583 nM. We also examined AR29's effects in a series of 20 primary blast samples from patients with AML. AR29 induced a dose-dependent cytotoxic effect (over the same dose range as seen in the cell lines) in most FLT3-mutant AML samples, and a limited number of wild type FLT3 samples. These same samples were treated in parallel with AR46, a potent FLT3 inhibitor with an IC50 of 1-2 nM (Array Biopharma Inc). Interestingly, the FLT3/ITD samples that responded to AR46 were the exact same ones that responded to AR29, implying that both drugs target the same signaling pathway in these samples. To further investigate the interaction between PIM and FLT3 in AML, we used immunoblot analysis to demonstrate that AR29 suppresses phosphorylation of Akt and STAT5 in Molm14, MV411, and TF/ITD cells at dosages that correlate with its cytotoxicity, but has no effect on FLT3 autophosphorylation. We have found that both PIM-1 and PIM-2 proteins co-precipitate with Akt, STAT5, and FLT3 in AML cell lines, suggesting the possible existence of an important signaling complex comprised of these proteins. When AR29 was combined with the FLT3 inhibitor, AR46, the cytotoxic effect was only additive rather than synergistic in leukemia cell lines and primary patient samples, suggesting again that the PIM and FLT3 inhibitors may be targeting the same signaling complex to induce cytotoxicity. In summary, we confirm that PIM proteins have an integral role in FLT3 mutant signaling, and that the PIM inhibitor AR29 has cytotoxic activity against AML cells, particularly those with FLT3/ITD mutations. These findings provide a proof-of-concept for the potential role of PIM inhibition as a new therapy for AML, and for overcoming resistance to FLT3 inhibitors by interacting at an alternate site in the FLT3 signaling mechanism.

Disclosures

Marmsater: Array Biopharma: Employment, Equity Ownership. Robinson:Array BioPharma: Employment. Gross:Array Biopharma: Employment, Equity Ownership. Martinson:Array Biopharma: Employment, Equity Ownership. Allen:Array Biopharma: Employment, Equity Ownership. Kallan:Array BioPharma: Employment.