PIM Kinase Expression Research Articles

PI3K-like kinases restrain Pim gene expression in endothelial cells

Pim kinases contribute to tumor formation and development of lymphoma, which shows enhanced DNA replication, DNA recombination and repair. Endothelial cells^(ECs) express all the three members of Pim kinase gene family. We hypothesized that DNA repair gene would regulate Pim expression in ECs. Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium. The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining. The siRNA fragments were synthesized and transfected by using Lipofectamine LTX. The total cellular RNA was extracted from the cells by using Trizol reagent. cDNAs were quantified by semi-quantity PCR. The effects of LY294002 and wortmannin on RNA stability in ECs were also examined. Our data showed that LY294002 and wortmannin, phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors, increased Pim mRNA expression in ECs without altering the mRNA stability. RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1, respectively. Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs. But etoposide, a nucleoside analogue, which could activate DNA-PKcs and ATM, increased Pim expression in ECs. Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.

Regulation and Functional Role of Beta2-Adrenergic Receptor in Acute Myelogenous Leukemia

Abstract 2563Acute myelogenous leukemia (AML) occurs with an incidence of 2.7 per 100,000 population in the year of 2009, and is associated with significant mortality and morbidity. Despite recent advances in molecular and cytogenetic analytic technologies, initial treatment for AML patients has remained essentially the same over the last 30 years; and the treatment outcome is dreadful with a 5-year survival rate of ∼25%. In an effort to gain a better understanding of AML cell biology and to develop more effective treatments for AML, we have been exploring the roles of the b2-adrenergic receptor (B2-AR) in AML. B2-AR is a G-protein- coupled catecholamine receptor and was recently found to play a direct role in the engraftment, proliferation and migration of hematopoietic stem cells [Spiegel, A., et al. Nat Immunol8, 1123–1131 (2007)]. However, very little is known about the roles of B2-AR in AML. We first screened a number of leukemia/hematopoietic cell lines, including K562, NBAL3, Jurkat, RPMI8226, U937, HEL, HL60, NB4, THP-1, and MV4;11, for B2-AR expression using flow cytometry. We found that B2-AR expression was not uniform in the leukemia/hematopoietic cell lines we tested. B2-AR was absent in acute non-myelogenous leukemia/hematopoietic cell lines such as K562, NBAL3, Jurkat or RPMI8226. In contrast, B2-AR was expressed on all acute myelogenous leukemia cell lines tested, with the highest expression on 2 myelomonocytic leukemia cell lines (THP-1 and MV4;11). We next examined the roles of B2-AR in leukemia cell survival, in leukemia cell responses to chemotherapy, and in leukemia cell migration in response to stromal derived factor-1 (SDF-1). We found that treatment with a B2-AR antagonist (ICI115881) modestly inhibited leukemia cell growth. Interestingly, treatment with a B2-AR agonist (i.e., isoproteronol or clenbuterol) inhibited leukemia cell migration to SDF-1. Additionally, combined treatment of MV4-11 cells with Isoproterenol and SDF-1 increased downstream ERK phosphorylation synergistically, suggesting a potential interaction or reciprocal regulation between B2-AR and CXCR4 receptor. To further understand the regulation and functional role of B2-AR in AML cells, we have been focusing on two biphenotypic leukemia cell lines, that is, MV4;11 and RS4;11 cells. Both MV4;11 and RS4;11 cells carry the t(4;11)(q21;q23) chromosomal translocation. While RS4;11 cells bear only the wild-type version of the Flt3 gene, MV4;11 cells express exclusively the mutated Flt3-ITD gene. Interestingly, we found that in contrast to MV4;11 leukemia cells, RS4;11 cells did not express surface B2-AR (panel A), although the total amount of B2-AR in the whole cell lysate was comparable between these two cell lines (panel B). This significantly different B2-AR expression pattern between these 2 cell lines may be related to the difference in the expression level of proviral insertion in murine lymphoma (Pim) kinases (panel B); RS4;11 has reduced/absent expression of Pim kinases compared to MV4;11 cells. Consistent with the potential regulation of B2-AR expression by Pim kinases, we found that Pim 2 and 3 double knockout mice had reduced B2-AR surface expression in peripheral blood mononucleated cells. Additional experiments are currently ongoing to further dissect the interaction between B2-AR and Pim kinases. Taken together, our current studies demonstrated a novel role of B2-AR in AML and a potential functional interaction between B2-AR, CXCR4, Pim kinase, and Flt3 gene.This work is supported by MUSC Hollings Cancer Center Startup Fund, Hollings Cancer Center ACS IRG, and ASCO Conquer Cancer Foundation Career Development Award [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Targeting PIM kinase enhances the activity of sunitinib in renal cell carcinoma

Background:Upregulation of PIM kinase expression has been reported in many malignancies, suggesting that inhibition of PIM kinase activity may be an attractive therapeutic strategy. We hypothesised that inhibition of PIM kinase activity with SGI-1776, a novel small molecule inhibitor of PIM kinase activity, would reduce the viability of renal cell carcinoma (RCC) cells and enhance the activity of sunitinib.Methods:Immunoblotting, qRT–PCR, and gene expression arrays were carried out to identify genes modulated by SGI-1776 treatment. The anticancer activity of SGI-1776 and sunitinib was determined by viability and apoptosis assays and in tumour xenografts in vivo.Results:Treatment with SGI-1776 led to a decrease in phosphorylated and total c-Myc levels, which resulted in the modulation of c-Myc target genes. SGI-1776 in combination with sunitinib induced a further reduction in c-Myc levels, which was associated with enhanced anticancer activity. siRNA-mediated knockdown of c-Myc demonstrated that its expression has a key role in regulating the sensitivity to the combination of SGI-1776 and sunitinib. Importantly, the combination significantly reduced tumour burden in two RCC xenograft models compared with single-agent therapy and was very well tolerated.Conclusion:These data indicate that targeting PIM kinase signalling is a promising treatment strategy for RCC.

Targeting cap-dependent translation blocks converging survival signals by AKT and PIM kinases in lymphoma.

New anticancer drugs that target oncogenic signaling molecules have greatly improved the treatment of certain cancers. However, resistance to targeted therapeutics is a major clinical problem and the redundancy of oncogenic signaling pathways provides back-up mechanisms that allow cancer cells to escape. For example, the AKT and PIM kinases produce parallel oncogenic signals and share many molecular targets, including activators of cap-dependent translation. Here, we show that PIM kinase expression can affect the clinical outcome of lymphoma chemotherapy. We observe the same in animal lymphoma models. Whereas chemoresistance caused by AKT is readily reversed with rapamycin, PIM-mediated resistance is refractory to mTORC1 inhibition. However, both PIM- and AKT-expressing lymphomas depend on cap-dependent translation, and genetic or pharmacological blockade of the translation initiation complex is highly effective against these tumors. The therapeutic effect of blocking cap-dependent translation is mediated, at least in part, by decreased production of short-lived oncoproteins including c-MYC, Cyclin D1, MCL1, and the PIM1/2 kinases themselves. Hence, targeting the convergence of oncogenic survival signals on translation initiation is an effective alternative to combinations of kinase inhibitors.

Targeting PIM kinase activity significantly augments the efficacy of cytarabine

Targeting PIM kinase activity significantly augments the efficacy of cytarabine

Abstract 586: Targeting Pim kinases by SGI-1776 in mantle cell lymphoma

Abstract Proviral integration site for Moloney murine leukemia virus (PIM) kinases are oncoproteins that promote tumor progression. Pim-1, -2 and -3 are highly conserved Ser/Thr kinases that are important for normal B-cell development and are over-expressed in B-cell malignancies such as mantle cell lymphoma (MCL). Pim kinases phosphorylate a number of substrates; including: cMyc and Histone H3, which drive transcription, eukaryotic elongation factor 4E-BP-1 that regulates translation and Bad that activates cell survival. Furthermore, cell cycle protein activation by Pim kinase is involved with proliferation. An imidazo-[1, 2-b]-pyridazine compound, SGI-1776 (SuperGen) has high inhibitory specificity to Pim kinases at nM levels. Based on this knowledge we hypothesized that Pim kinase are druggable targets in MCL and inhibition of Pim kinase-mediated phosphorylation of downstream substrates will disrupt transcriptional and translational processes and trigger apoptosis. To test this hypothesis, Pim kinase expression profile and actions of SGI were studied in MCL cell lines, Jeko and Mino. All three Pim kinase were expressed in both cell lines. SGI was used in concentration (0.1 to 10 µM) and time dependent (up to 24 hrs) fashion. Treatment with SGI resulted in decreased Pim-1 and -3 protein levels in both cell lines; however Pim-2 protein was declined only in Jeko. Pim kinase pathways were investigated to identify mechanism of action of SGI. In both Jeko and Mino, total and phospho cMyc (Ser62) protein expression decreased at 3 and 10 µM, respectively. Interestingly, Histone H3 (Ser10) phosphorylation decreased only in Jeko. Consistent with cMyc data, there was 50% decrease in RNA synthesis (uridine incorporation) at 5 µM SGI in Jeko and 10 µM in Mino. When evaluated in Mino cells, 4E-BP-1 was not affected up to 3 µM of SGI. SGI inhibition on transcription/translation in principle would affect short-lived proteins such as Mcl-1 that are survival factors. Significant decreases of Mcl-1 mRNA (80% and 50%) and protein (50% and 40%) levels were observed in Jeko and Mino at 3 and 10 µM, respectively. In contrast, other anti-apoptotic proteins Bcl-2, Bcl-XL and XIAP were not affected by SGI treatment. In concordance with the survival role of Mcl-1, apoptosis (Annexin V+) was induced at 3, 5 and 10 µM SGI (15%, 30% and 80%) in both cell lines. Similarly, PARP cleavage was detected. Total and phospho Bad (Ser112) protein was decreased at 3 µM in Jeko, suggesting a role of this Pim substrate in apoptosis. Consistent with the observations in cell lines, there was induction of cell death in primary MCL cells from patients (n = 5). Preliminary data suggest inhibition of RNA synthesis in these malignant lymphocytes. Taken together, Pim kinases are potential therapeutic targets in MCL and SGI-1776 is effective to inhibit global RNA synthesis, as well as inducing apoptosis. MCl-1 appears important factor in Pim kinase pathway and warrant more detailed investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 586. doi:10.1158/1538-7445.AM2011-586

Abstract B272: The novel PIM kinase inhibitor SGI-1776 significantly enhances the preclinical activity of sunitinib in renal cell carcinoma

Abstract The PIM kinases (PIM1, PIM2, and PIM3) are constitutively active serine/threonine kinases that promote tumorigenesis in mouse models and are associated with drug resistance. Upregulation of PIM kinase expression has been reported in many malignancies including renal cell carcinoma (RCC), suggesting that inhibition of PIM kinase activity may be an attractive therapeutic strategy. SGI-1776 is a novel orally available small molecule inhibitor of PIM kinase activity that is currently in Phase I clinical trials. We hypothesized that inhibition of PIM kinase activity would reduce the viability of RCC cells and enhance the activity of the multi-targeted receptor tyrosine kinase (RTK) inhibitor sunitinib. Cell viability and apoptosis assays demonstrated that SGI-1776 possessed significant in vitro activity in a panel of RCC cell lines. One of the mechanisms by which the PIM kinases have been reported to inhibit apoptosis is by phosphorylating the BH3-only protein Bad to abrogate its pro-apoptotic function. Accordingly, SGI-1776-mediated apoptosis was associated with decreased levels of the phosphorylated form of Bad. Moreover, targeted knockdown of Bad expression significantly decreased sensitivity to SGI-1776 indicating that Bad is an important mediator of the anticancer activity of this agent. Treatment with SGI-1776 also led to a markedly decrease in phosphorylated and total c-Myc levels and enhanced the activity of sunitinib in RCC cells. The addition of sunitinib to SGI-1776 further reduced c-Myc expression. Silencing of the gene suggested that c-Myc plays a significant role in sensitivity to the SGI-1776/sunitinib combination. To further evaluate this drug combination, Caki-1 and 786-O RCC xenografts were established in nude mice and they were treated orally with SGI-1776, sunitinib, or the combination. The SGI-1776/sunitinib combination significantly reduced tumor burden in both xenograft models compared to single agent therapy and was very well tolerated. Analysis of the tumors revealed that the drug combination strongly decreased tumor proliferation and induced apoptosis as measured by PCNA and TUNEL immunohistochemistry, respectively. These data indicate that PIM kinase signaling has a significant role in the biology of RCC and warrants further investigation. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B272.

Inhibition of T cell function in vitro and in vivo using small molecule antagonists of PIM kinases (35.33)

Abstract The PIM family consists of three highly conserved serine/threonine kinases whose activity is regulated at the level of expression. PIM kinase expression has been shown to be stimulated by a variety of cytokines, hormones, and mitogens and it functions in the regulation of cellular proliferation, differentiation, apoptosis and tumorigenesis. The role of PIM-induced oncogenic transformation has been extensively studied in hematopoietically derived tumors; however the function of PIM in normal immune cells has yet to be fully defined. We sought to elucidate the role of PIM in activation of primary murine T cells by utilizing a highly selective, small molecule PIM inhibitor. We found that inhibition of PIM activity resulted in decreased anti-CD3 and antigen-induced CD4+ T cell proliferation. These effects were observed under Th0, Th1, Th2 and Th17 polarizing conditions, and were not due to increased cell death, but appear to be mediated by a G0/G1 cell cycle arrest. Inhibition of PIM during polarization prevented the production of Th1, Th2, and Th17 effector cytokines suggesting that PIM expression is required for naïve T cell differentiation. Consistent with the in vitro data, mice treated with the PIM inhibitor prior to immunization had a significant reduction in ex vivo recall responses. These results support a key role for PIM in the activation and differentiation of murine CD4+ T cells.

14-3-3 Binding to Pim-phosphorylated Ser166 and Ser186 of human Mdm2 – Potential interplay with the PKB/Akt pathway and p14 ARF

Here we show that 14-3-3 proteins bind to Pim kinase-phosphorylated Ser166 and Ser186 on the human E3 ubiquitin ligase mouse double minute 2 (Mdm2), but not protein kinase B (PKB)/Akt-phosphorylated Ser166 and Ser188. Pim-mediated phosphorylation of Ser186 blocks phosphorylation of Ser188 by PKB, indicating potential interplay between the Pim and PKB signaling pathways in regulating Mdm2. In cells, expression of Pim kinases promoted phosphorylation of Ser166 and Ser186, interaction of Mdm2 with endogenous 14-3-3s and p14 ARF, and also increased the amount of Mdm2 protein by a mechanism that does not require Pim kinase activities. The implications of these findings for regulation of the p53 pathway, oncogenesis and drug discovery are discussed. Structured summary MINT- 6823587: PIM3 (uniprotkb: Q86V86) phosphorylates (MI: 0217) MDM2 (uniprotkb: Q00987) by protein kinase assay (MI: 0424) MINT- 6823623: MDM2 (uniprotkb: Q00987) physically interacts (MI: 0218) with p14ARF (uniprotkb: Q8N7268N726) by coimmunoprecipitation (MI: 0019) MINT- 6823537: PKB (uniprotkb: P31749) phosphorylates (MI: 0217) MDM2 (uniprotkb: Q00987) by protein kinase assay (MI: 0424) MINT- 6823574: PIM2 (uniprotkb: QP1W9) phosphorylates (MI: 0217) MDM2 (uniprotkb: Q00987) by protein kinase assay (MI: 0424) MINT- 6823555: PIM1 (uniprotkb: P11309)P phosphorylates (MI: 0217) MDM2 (uniprotkb: Q00987) by protein kinase assay (MI: 0424)

Pim Kinase Inhibitor, SGI-1776, Induces Apoptosis in CLL Lymphocytes

We report investigational studies of a Pim-1 kinase inhibitor, SGI-1776, as a therapeutic agent for the treatment of chronic lymphocytic leukemia (CLL). Pim family proteins are serine/threonine kinase inhibitors of apoptosis, and elevated expression of Pim kinases have been detected in hematological malignancies and in certain solid tumors. Three Pim kinases have been identified to date, Pim-1, -2 and -3. Pim-1 has been shown to synergize with c-Myc in tumorigenesis, and increased expression of the human Pim-2 gene is observed in CLL and non-Hodgkin lymphomas. Specifically, Pim-1 and Pim-2 have been shown to be required for efficient pre–B-cell transformation by v-Abl oncogene. Small molecule SGI-1776 was evaluated using Ambit Biosciences' KINOMEscan and was found to have IC50s in the nanomolar range for Pim-1, Pim-2, and Pim-3 using the Millipore IC50 Profiler. SGI-1776 was screened against a panel of kinases utilizing radiolabeled biochemical assays, and was found to be highly selective for Pim kinases without any effects on CDKs, Chk1, IKK, JNK, Abl, Raf, MAP kinases and protein kinase A and B. Specifically, the IC50 were measured as follows: Pim-1 (7 nM), Pim-2 (363 nM) and Pim-3 (69 nM.) The other two enzymes affected at nM concentration of SGI-1776 were Flt-2 and haspin. Using primary samples obtained from patients with CLL, we evaluated the potential for SGI-1776 to induce cell death. In vitro incubation of primary CLL cells (n=7), with 1, 3, and 10 μM SGI-1776 for 24 h resulted in an average increase in apoptosis of 10%, 22% and 38% respectively, compared with untreated cells. SGI-1776-induced apoptosis was observed in heterogeneous patient populations, and there was disparity in the expression levels of traditional CLL prognostic markers including ZAP-70, b2-microglobulin, IgVH mutation status, Rai stage and number of prior treatments. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. Phosphorylation of traditional Pim-1 kinase targets, phospho-Bad (Ser112) and histone H3 (Ser10) were not affected by SGI-1776 treatment in CLL, unlike in replicating cell types, suggesting an alternative mechanism in CLL. We then evaluated the potential inhibition of c-Myc driven transcription by measuring total RNA synthesis. Following treatment with 3 or 10 μM SGI-1776, there was a decrease in total RNA synthesis to approximately 50% of control, measured using a radioactive uridine incorporation assay (n = 3). There was also a reduction in Mcl-1 and c-Myc protein level, both of which have short transcript half-lives. The reduction in Mcl-1 protein was not due to apoptosis or cleavage by caspases, since Mcl-1 reduction occurred in the presence of caspase inhibitor ZVAD, nor was there an increase in cleaved Mcl-1. In contrast, there was no change in anti-apoptotic proteins Bcl-2, XIAP, survivin, stabilization of p53 or p21. Taken together, SGI-1776 consistently induced apoptosis in CLL cells. Although its mechanism of action is not fully elucidated, inhibition of RNA synthesis and reduction of Mcl-1 and c-Myc protein levels are associated with SGI-1776-induced apoptosis.

Pim Kinases Promote Cell Cycle Progression by Phosphorylating and Down-regulating p27Kip1 at the Transcriptional and Posttranscriptional Levels

The serine/threonine kinase Pim is known to promote cell cycle progression and to inhibit apoptosis leading to tumorigenesis. However, the precise mechanisms remain unclear. We show, herein, that all the Pim family members (Pim1, Pim2, and Pim3) bind to and directly phosphorylate the cyclin-dependent kinase inhibitor p27(Kip1) at threonine-157 and threonine-198 residues in cells and in vitro. The Pim-mediated phosphorylation induced p27(Kip1) binding to 14-3-3 protein, resulting in its nuclear export and proteasome-dependent degradation. Ectopic expression of Pim kinases overcome the G(1) arrest mediated by wild-type p27(Kip1) but not by phosphorylation-resistant T157A-p27(Kip1) or T198A-p27(Kip1). In addition to the posttranslational regulations, p27(Kip1) promoter assay revealed that Pim kinases also had the ability to suppress p27(Kip1) transcription. Pim-mediated phosphorylation and inactivation of forkhead transcription factors, FoxO1a and FoxO3a, was involved in the transcriptional repression of the p27(Kip1) gene. In contrast, inhibition of Pim signaling by expressing the dominant-negative form of Pim1 increased nuclear p27(Kip1) level and attenuated cell proliferation. Because the CDK inhibitor p27(Kip1) plays a crucial role in tumor suppression by inhibiting abnormal cell cycle progression, Pim kinases promote cell cycle progression and tumorigenesis by down-regulating p27(Kip1) expression at both transcriptional and posttranslational levels.

Developmental expression of pim kinases suggests functions also outside of the hematopoietic system.

We have cloned a novel quail cDNA with strong homology to the pim family of proto-oncogenes. The deduced amino acid (aa) sequence of the cDNA, named qpim, is more closely related to Xenopus Pim and to the recently identified rat Pim-3 than to human or rodent Pim-1 or Pim-2. The protein encoded by the qpim cDNA can autophosphorylate itself and share substrates with murine Pim-1, suggesting functional redundancy to other Pim family serine/threonine kinases. We have compared the expression of qpim in avian embryos to mouse pim-1, -2 and -3 by in situ hybridization. qpim shows a highly dynamic expression pattern, particularly at early developmental stages. Surprisingly, its expression pattern is not identical to any of the murine pim genes, which show complementary and/or partially overlapping expression sites both in- and outside of the hematopoietic system. Altogether, our results suggest novel functions for Pim family kinases during embryonic development, in particular in epithelia and in the central nervous system.

Pim kinase expression is induced by LTP stimulation and required for the consolidation of enduring LTP.

In animals and several cellular models of synaptic plasticity, long-lasting changes in synaptic strength are dependent on gene transcription and translation. Here we demonstrate that Pim-1, a serine/threonine kinase closely related to Pim-2 and Pim-3, is induced in hippocampus in response to stimuli that evoke long-term potentiation (LTP). Mice deficient for Pim-1 show normal synaptic transmission and short-term plasticity. However, they fail to consolidate enduring LTP even though Pim-2 and Pim-3 are constitutively expressed in the hippocampus and Pim-3 expression is similarly induced by synaptic activity. Thus, expression of Pim-1 is required for LTP. Its level of expression and, consequently, its capacity to phosphorylate target proteins in dendritic and nuclear compartments of stimulated neurons might be a determining factor for the establishment of long-lasting changes in synaptic strength.