UM171 cooperates with PIM1 inhibitors to restrict HSC expansion markers and suppress leukemia progression

Abstract

The pyrimido-indole derivative UM171 promotes human Hematopoietic Stem Cells Expansion (HSCE), but its impact on leukemia is not known. Herein, we show in a mouse model of erythroleukemia that UM171 strongly suppresses leukemia progression. UM171 inhibits cell cycle progression and apoptosis of leukemic cells in culture. The effect of UM171 on leukemia differentiation was accompanied by increased expression of HSCE markers. RNAseq analysis combined with Q-RT-PCR and western blotting revealed that the PIM1 protein kinase is highly elevated in response to UM171 treatment. Moreover, docking analysis combined with immunoprecipitation assays revealed high binding affinity of UM171 to PIM1. Interestingly, pan-PIM kinase inhibitors counteracted the effect of UM171 on HSCE marker expression and PIM1 transcription, but not its suppression of leukemic cell growth. Moreover, combination treatment with UM171 and a pan-PIM inhibitor further suppressed leukemic cell proliferation compared to each drug alone. To uncover the mechanism of growth inhibition, we showed strong upregulation of the cyclin-dependent kinase inhibitor P21^CIP1 and the transcription factor KLF2 by UM171. In accordance, KLF2 knockdown attenuated growth inhibition by UM171. KLF2 upregulation by UM171 is also responsible for the activation of P21^CIP1 in leukemic cells leading to a G1/S arrest and suppression of leukemogenesis. Thus, suppression of leukemic growth by UM171 through KLF2 and P21^CIP1 is thwarted by PIM-mediated expansion of leukemic stemness, uncovering a novel therapeutic modality involving combined UM171 plus PIM inhibitors.

Fig. 1. Inhibition of leukemogenesis by UM171 in culture and in vivo.

A Structure of UM171. B UM171 at indicated concentration inhibits proliferation of HEL cells in culture. C IC₅₀ analysis of UM171 for the indicated cell lines. D Macroscopic image of apoptotic cells after treatment of HEL cells with 1.6 μM of UM171 (original magnification X20, scale bars, 100 μm). E UM171 induces apoptosis of HEL cells at the indicated concentrations. F UM171 induces G₁/S cell cycle arrest of HEL cell at indicated concentrations. G Newborn BALB/c mice infected with F-MuLV were treated for two weeks with UM171 (3 mg/ Kg), 5 weeks postviral infection, and plotted after all mice succumbed to leukemia. H, I UM171 treated mice with their hematocrit values (H) and tumor volume (I) at the time of death.

Fig. 2. Induction of stem cell markers by UM171 in HEL cells.

A–D The Bi-potential Megakaryocytic Erythroid Progenitor (MEP) derived HEL cell line was treated with UM171 (6 μM), DMSO for 24 hours and subjected to flow cytometry analysis for the stem cell markers CD34 (A), c-KIT (B), CD69 (C) and CD44 (D). E, F Q-RT-PCR analysis of HEL cell treated with UM171 (3 μM) and DMSO for expression of the stem cell markers CD34, c-KIT, CD69, CD44, ERG (E) and CD40, CD41, CXCL8 and GFI1 (F).

Fig. 3. UM171 binds to PIM1 and activates its phosphorylation.

A Q-RT-PCR analysis of HEL cells treated with UM171 (6 μM) for expression of the PIM1. B Q-RT-PCR analysis of HEL cells treated with UM171 (6 μM) for expression of the PIM2, and PIM3 genes. C Western blot of HEL cells treated with the indicated concentration of UM171 compound. GAPDH was used as a loading control. D Molecular docking of the compound UM171 and PIM1 (PDB:5O12). The phosphorylation site of PIM1 kinase was shown as insert. E The molecular binding energy between PIM1 and UM171 as well as pan-PIM inhibitors LGH447, TP3654, SGI1776 ad AZ1208. F Binding of PIM1 to UM171 in pull-down experiment using affinity ES6B beads. G Q-RT-PCR analysis of HEL cells treated with UM171 (6 uM), LGH447(5 μM), and UM171 + LGH447 for 24 hours for expression of PIM1. H Western blot of HEL cells treated with UM171 and DMSO for expression of LSD1.

Fig. 4. UM171 induces markers of HSCs in HEL cells.

A–E HEL cells were treated with UM171 (6 μM), LGH447(5 μM), and UM171 + LGH447 for 24 hours and subjected to Q-RT-PCR analysis for the indicated genes. F HEL cells were treated with UM171 (6 μM), LGH447(5 μM), and UM171 + LGH447 for 24 hours and subjected to western blot analysis for detection of ERK and Phospho-ERK. GAPDH was used as a loading control. G–I Q-RT-PCR analysis of HEL cells treated with the indicated compounds for expression of HBA1 (G), HBA2 (H), ZFP36L1 (I).

Fig. 5. UM171 upregulates expression of the cell cycle inhibitor p21^CIP1 independent of PIM1 activation.

A HEL cells were treated with indicated doses of UM171 and subjected to western blot for P21^CIP1 and PIM1 expression. B, C HEL cells were treated with UM171, LGH447, and UM171 + LGH447 for 24 hours and subjected to Q-RT-PCR (B) or western blot (C) analysis for the indicated genes. D, E HEL cells were treated with UM171, GSK-LDS1 (10 μM), and P21^CIP1 levels were determined by Q-RT-PCR (D) and western blotting (E). F Cell cycle arrest of HEL cells treated with UM171, LGH447 and UM171 + LGH447. Right panel shows the percentage of cells in different phases of the cell cycle.

Fig. 6. Induction of KLF2 by UM171 inhibit cell proliferation in culture.

A Treatment of HEL cells with UM171 (6 μM) induces KLF2 expression, as determined by Q-RT-PCR. B KLF2 induction was partially blocked by treatment with LGH447 (5 μM), as determined by western blot. C Inhibition of KLF2 expression in HEL cells using shRNA constructs (KLF2-sh1, KLF2-sh2, KLF2-sh3), as determined by Q-RT-PCR. D Induction of KLF2 by UM171 was moderated in KLF2-sh2 cells, determined by Q-RT-PCR. E KLF2 knockdown moderated suppression of KLF2-sh2 cell proliferation by UM171 when compared to the control scrambled cells. F Growth suppression by UM171 was further accelerated when co-treated with LGH447(5 μM) in the culture of HEL cells. G Depicted diagram of growth suppression by UM171. UM171 through binding to PIM1 activates HSCE markers and cancer stemness by increasing cancer stem cells (CSCs) and via unknown target increases the expression of P21^cip1 and KLF2, leading to growth suppression. Cancer stemness induction by UM171 may partially interfere with growth suppression by UM171 and blocking CSC by a pan-PIM inhibitor should further accelerate leukemia inhibition by the compound.