Transcription and translation are primary targets of Pim kinase inhibitor SGI-1776 in mantle cell lymphoma

Abstract

Proviral integration site for Moloney murine leukemia virus (Pim) kinases are serine/threonine/tyrosine kinases and oncoproteins that promote tumor progression. Three isoforms of Pim kinases have been identified and are known to phosphorylate numerous substrates, with regulatory functions in transcription, translation, cell cycle, and survival pathways. These kinases are involved in production, proliferation, and survival of normal B cells and are overexpressed in B-cell malignancies such as mantle cell lymphoma (MCL). SGI-1776 is a small molecule and Pim kinase inhibitor with selectivity for Pim-1. We hypothesize that Pim kinase function can be inhibited by SGI-1776 in MCL and that inhibition of phosphorylation of downstream substrates will disrupt transcriptional, translational, and cell cycle processes and promote cell death. SGI-1776 treatment in 4 MCL cell lines resulted in apoptosis induction. Phosphorylation of transcription (c-Myc) and translation targets (4E-BP1), tested in Jeko-1 and Mino, was declined. Consistent with these data, Mcl-1 and cyclin D1 protein levels were decreased. Importantly, similar to cell line data, MCL primary cells but not normal cells showed similar inhibition of substrate phosphorylation and cytotoxicity from SGI-1776 treatment. Genetic knockdown of Pim-1/Pim-2 affected similar proteins in MCL cell lines. Collectively these data demonstrate Pim kinases as therapeutic targets in MCL.

Figure 1

Induction of apoptosis by SGI-1776 treatment in MCL cell lines. (A) Dose-dependent induction of SGI-1776–mediated annexin V and PI positivity in MCL cells. MCL cell lines (JeKo-1, Mino, Granta 519, and SP-53) were treated with DMSO alone or with 0.1, 1, 3, 5, or 10μM SGI-1776 for 24 hours, stained with annexin V and PI, and analyzed by flow cytometry. (B) Apoptosis in JeKo-1 and Mino was confirmed by PARP cleavage assay. MCL cells were treated with SGI-1776 for 24 hours in the concentrations mentioned in panel A for immunoblots. (C) Time-dependent induction of SGI-1776–mediated annexin V/PI–positive MCL cells. JeKo-1 and Mino cells were treated with DMSO alone or with 10μM SGI-1776 for 0.5, 1, 2, 4, 8, 16, or 24 hours, stained with annexin V and PI, and analyzed by flow cytometry. (D) Apoptosis in JeKo-1 and Mino was confirmed by PARP cleavage assay. MCL cells were treated with 10μM SGI-1776 for time points mentioned above for immunoblots. All experiments were performed in triplicate.

Figure 2

Expression Pim kinase protein in cell lines and lymphoma patient tissue samples. (A) Pim-1, -2, and -3 expressions in JeKo-1 and Mino cells. Cells were either untreated or treated with DMSO, and immunoblots were prepared for Pim protein expressions. Note: multiple bands in Pim-1 and Pim-2 are expected because of multiple translation initiation sites. (B) Histologic section of case of MCL stained with hematoxylin and eosin, and immunohistochemical staining of MCL patient tissue samples, stained with rabbit anti–Pim-1, -2, and -3 antibodies and counterstained with hematoxylin.

Figure 3

Effects of SGI-1776 treatment on Pim kinases targets. Effect of SGI-1776 on phospho–c-Myc (Ser62) and total c-Myc (A) phospho-Histone H3 (Ser10) and total Histone H3 (B), phospho-Bad (Ser112) and total Bad (C), and phospho–4E-BP1(Thr37/46) and total 4E-BP1 (D) protein expressions. JeKo-1 and Mino cell were treated with 0.1, 1, 3, 5, or 10μM SGI-1776 for 24 hours, and then cell lysates were analyzed via immunoblot. Quantitation of immunoblots with phosphorylation targets (c-Myc, Histone H3, Bad, and 4E-BP1) is shown in panel E for JeKo-1 cells and in panel F for Mino cells. Phospho-to-total protein ratios were calculated, and data are presented as means of 3 independent experiments ± SEM.

Figure 4

Reduction of global RNA synthesis and Mcl-1 mRNA and protein expression in MCL cell lines by SGI-1776. (A) Dose-dependent effects of SGI-1776 treatment on global transcription level in JeKo-1, Mino, Granta 519, and SP-53 cells. Cells were treated with DMSO alone or with 0.1, 1, 3, 5, or 10μM SGI-1776 for 24 hours and then incubated with uridine for 30 minutes, and radioactive incorporation was measured via scintillation counter. (B) Time-dependent effects of SGI-1776 treatment on global transcription level in JeKo-1 and Mino cells. Cells were treated with DMSO alone or with 10μM SGI-1776 for 0.5, 1, 2, 4, 8, 16, or 24 hours and then prepared for uridine incorporation as described in panel A. Dose-dependent (C) and time-dependent (D) effects of SGI-1776 on MCL1 mRNA expressions in JeKo-1 and Mino cells. Cells were treated with SGI-1776 in dose-dependent or time-dependent manner as mentioned in Panels A and B, and RNA was isolated and analyzed using real-time RT-PCR. Impact of SGI-1776 in dose-dependent (E) and time-dependent (F) treatments on Mcl-1 protein expression in JeKo-1 and Mino cells. Mcl-1 protein expression was measured by immunoblots in both dose- and time-dependent SGI-1776 treatments (see panels A and B) of JeKo-1 and Mino cells. Quantitation of immunoblot with dose-dependent SGI-1776 treatment in JeKo-1 (G) and Mino (H) cells. Mcl-1-to-GAPDH ratio was calculated, and data are presented as means of 3 independent experiments ± SEM.

Figure 5

Effect of PIM1, PIM2 siRNAs on PIM1, PIM2 and MCL1 mRNA and Pim kinase targets in MCL cell lines. Effects of PIM1, PIM2 siRNAs and their combination on PIM1, PIM2 and MCL1 mRNA expressions in JeKo-1 (A) and Mino (B) cells. Cells were treated with 150nM scramble, siGLO, PIM1, PIM2 siRNA as well as PIM1 and PIM2 siRNA combination (150nM for each) for 24 hours. Then, RNA was extracted and analyzed using RT-PCR. (C) Effects of PIM1, PIM2 siRNA and their combination on total and phospho–c-Myc (Ser62), total and phospho–4E-BP1 (Thr37/46), as well as Mcl-1 protein expression were measured by immunoblots. (D) Quantitation of immunoblots in cells with siRNA treatments. Phospho- and total protein ratios or Mcl-1-to-GAPDH ratios were calculated, and data are presented as means of 3 independent experiments ± SEM. Percentage of cell death is marked under each lane of immunoblot measured by annexin V and PI positivity.

Figure 6

Effects of SGI-1776 on MCL malignant lymphocytes and normal PBMCs. Effects of SGI-1776 on phospho–c-Myc (Ser62) and total c-Myc, Mcl-1, cyclin D1 and phospho–4E-BP1 (Thr37/46), and total 4E-BP1 protein expressions in MCL malignant lymphocytes (A) and normal PBMCs from MCL patients and apparently healthy donors (C). Primary patient cells were treated with 0.1, 1, 3, 5, or 10μM, and normal PBMCs were treated with 1, 3, or 10μM SGI-1776 for 24 hours, and then cell lysates were analyzed via immunoblot. Untreated Mino cells were positive control. Apoptosis levels measured by annexin V and PI positivity is marked under each lane of the immunoblot in each primary sample. Effects of SGI-1776 on MCL1 mRNA expression levels in primary MCL malignant lymphocytes (B) and normal PBMCs from MCL patients and healthy donors (D). Primary PBMCs were treated with SGI-1776 using concentrations mentioned in this legend; RNA was isolated and analyzed using real-time RT-PCR.

Figure 7

Association between transcription and translation inhibition and cell death. (A) Global RNA synthesis inhibition versus apoptosis levels and its linear regression. RNA synthesis was measured in percentages and apoptosis was defined by annexin V positivity. Values were taken from dose-dependent SGI-1776 treatments in JeKo-1, Mino, SP-53, and Granta-519 cell lines. In experiments performed in JeKo-1 and Mino after dose-dependent SGI-1776 treatments for 24 hours, the following plots were generated: c-Myc (Ser62) phosphorylation versus global RNA synthesis, where c-Myc (Ser62) levels were normalized to total c-Myc to obtain the ratios (B); c-Myc (Ser62) phosphorylation versus MCL1 mRNA expressions, where c-Myc (Ser62) levels were normalized to total c-Myc and MCL1 mRNA was normalized to 18S rRNA controls to obtain the ratios (C); inhibition of 4E-BP1 (Thr37/46) phosphorylation versus apoptosis, where 4E-BP1 (Thr37/46) phosphorylation levels were normalized to total 4E-BP1 to obtain the ratios (D); and 4E-BP1 (Thr37/46) phosphorylation versus Mcl-1 protein expression, where phospho–4E-BP1 (Thr37/46) was normalized to total 4E-BP1 protein and Mcl-1 protein was normalized to GAPDH levels (E). Linear regression was performed, and r² values and P values were calculated using the GraphPad Prism Version 5 software.