PIM is a family of protein kinases activated by JAK/STAT through interleukins stimulus that results in proproliferative and prosurvival actions through p21/p27 deregulation and proapoptotic protein inactivation. PIM kinases represent a potential target in various neoplasm types due to its critical role in tumorigenesis. Thus, the goal of the present research was to investigate the PIMgene and protein expressions in AML patients as well as the effects of PIM protein inhibition in leukemia cells.

Analysis of PIM genes expression was performed in a cohort of 163 AML patients (median age 57.5 years [range: 18-88])(TCGA AML study). Acute leukemia cell lines, both myeloid (OCI-AML3, U937) and lymphoid (RS4;11, Jurkat) were treated with increasing concentrations of the PIM inhibitor compound PIM447 (Novartis) for 48h for analysis of the 50% inhibiting concentration (IC50). Apoptosis (annexin-V stain), reactive oxygen species (ROS) production (DFCHA dye), nitric oxide (NO) (DAF dye), and mitochondrial membrane potential (DilC1 probe) were assessed by flow cytometry, after 48h of treatment. Statistical analyzes were performed using Kruskal Wallis, ANOVA or Mann-Whitney tests. p-value <.05 was considered significant.

Increased PIM genes expression in patients with erythroid and megakaryoblastic AML (p < 0.001) was observed. Our results demonstrated a dose-dependent sensitivity after treatment with PIM447, with IC50 values of 12.2μM in OCI-AML3; 22.3μM in U937; 16.1μM in MOLM13; 20.9μM in RS4;11 and 15.1μM in Jurkat. Treatment with PIM447 (2-20μM) in OCI-AML3, U937, MOLM13, RS4;11 and Jurkat cells significantly reduced cell viability and increased apoptosis (ford-increase (FI): 1.0-5.0, 1.0-8.1, 1.7-9.7, 1.0-5.1, and 1.5-9.4, respectively) in a concentration-dependent manner. PIM447 was able to reduce ROS (fold-decrease (FD): 1.6; 1.1; 1.3; 1.8; 2.8) and NO (FD: 1.2; 0.8; 2.2; 1.1; 1.4) production and mitochondrial membrane potential (FD: 4.0; 8.0; 5.1; 2.5; 9.8), respectively, for OCI-AML3, U937, MOLM13, RS4;11 and Jurkat (p < .05).

The PIM genes expression was associated with a poor prognosis of AML patients. The TGCA data bank analysis showed higher PIM genes expression in patients with rare forms of leukemia. Our in vitro studies demonstrated that the PIM inhibitor diminished viability and increased cell death of leukemia cells, possibly by action on the intrinsic pathway of apoptosis due to deregulation of ROS and NO production and unbalanced mitochondrial membrane potential. In this scenario, our findings provide important insights for further investigations into a protein that is part of a complex network that regulates tumor survival, representing a potential therapeutic strategy.

São Paulo Research Foundation (FAPESP) grants #2017/21801-2, #2019/25247-5, #2021/05320-0.