Abstract 681: Translational pharmacology approaches to explore the novel mechanism of a pan-PIM kinase inhibitor, JP-11646, in acute myeloid leukemia
Authors: Pundt Krista E. , Baldino Carmen , Caserta Justin , Pitzonka Laura B. , Lee Kelvin , Adjei Alex , Fetterly Gerald J. 
Published: August 2015

Abstract
Leukemia, an aggressive immature hematological malignancy, is the sixth leading cause of cancer death in the U.S., with an overall cure rate of only 25%. PIM kinases are serine/threonine kinases that regulate cell cycle and survival. In hematological malignancies, PIM kinases are overexpressed, constitutively active, and associated with drug resistance. Thus, PIM kinases are novel therapeutic targets. JP-11646 is a pan-PIM inhibitor that has shown antitumor activity in various tumor types, with an increased potency against PIM2. In these studies, a comparison of three pan-PIM inhibitors on cell proliferation and PIM expression, along with the effect of JP-11646 on cellular signaling were investigated in human acute leukemia (AML).
An in vitro MTT assay was used to determine the overall anti-proliferative effects of various pan-PIM inhibitors in human AML (HEL, and MV(4;11)) cell lines. Cells were exposed to various concentrations (0.001 to 100 μM) of JP-11646, AZD1208 and LGB321. An Emax model was used to estimate the overall potency (IC50) and maximal inhibition (Imax) of the three inhibitors (0.06 μM, 0.20 μM and 0.43 μM and 89, 35 and 46 percent, respectively). It was determined that JP_11646 is 3-100 times more potent than AZD1208 and LGB321. Following inhibitor treatment (1, 2, 4, 6 and 24 hrs), PIM2 protein expression decreases by 50% at 6 hrs following 0.1 μM JP-11646; while both AZD1208 (0.45 μM) and LGB321 (0.20 μM) increase PIM2 protein expression by 1.5-fold following 2-6 hours of treatment. In conjunction with PIM 2 protein inhibition, real-time PCR results show that JP_11646 treatment results in a time- and concentration-dependent decrease of PIM1, 2 and 3 mRNA. In HEL cells, JP_11646 is more potent against PIM2, with PIM2 mRNA loss occurring as early as 1-2 hours post treatment. To determine the consequence of PIM inhibition via JP-11646, p-4EBP-1 and cleaved caspase protein expression were examined. Following 24 hours of JP-11646 treatment, p-4EBP-1 protein decreases in a concentration-dependent manner, suggesting the effect of JP-11646 on AML cell proliferation may be due to a disruption in p-4EBP-1 function in translation. Similarly, cleaved caspase increases and caspase 3 decreases following JP-11646 treatment, suggesting JP-11646 induces apoptosis. PE annexin V staining with FACS analysis confirmed that cells treated with JP-11646 undergo apoptotic or necrotic cell death in a time and concentration dependent manner, which is a unique characteristic of JP-11646 versus other known pan-PIM inhibitors.
In summary, the novel PIM kinase inhibitor, JP-11646, demonstrated more potent anti-leukemic activity than other pan-PIM kinase inhibitors in human AML cell lines. The modulation of PIM2 kinase, additional downstream targets, and the induction of apoptosis at nM concentrations by JP-11646 suggests JP-11646 is a novel and unique pan-PIM inhibitor.
Citation Format: Krista E. Pundt, Carmen Baldino, Justin Caserta, Laura B. Pitzonka, Kelvin Lee, Alex Adjei, Gerald J. Fetterly. Translational pharmacology approaches to explore the novel mechanism of a pan-PIM kinase inhibitor, JP-11646, in acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 681. doi:10.1158/1538-7445.AM2015-681