Pim-1 Activity Research Articles

Elevation of Receptor Tyrosine Kinases by Small Molecule AKT Inhibitors in Prostate Cancer Is Mediated by Pim-1

The PI3K/AKT pathway is hyperactivated in prostate cancer but its effective therapeutic targeting has proven difficult. In particular, the antitumor activity of AKT inhibitors is attenuated by upregulation of receptor tyrosine kinases (RTK) through an uncharacterized feedback mechanism. In this report, we show that RNA interference-mediated silencing or pharmacologic inhibition of Pim-1 activity curtails AKT inhibitor-induced upregulation of RTKs in prostate cancer cells. Although Pim kinases have been implicated in cap-dependent translational control, we find that in the context of AKT inhibition, the expression of RTKs is controlled by Pim-1 in a cap-independent manner by controlling internal ribosome entry. Combination of Pim and AKT inhibitors resulted in synergistic inhibition of prostate tumor growth in vitro and in vivo. Together, our results show that Pim-1 mediates resistance to AKT inhibition and suggest its targeting to improve the efficacy of AKT inhibitors in anticancer therapy.

Inhibition of Pim1 expression and activity impairs the proliferation of rat bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cell (BMMSC) is one of the ideal seed cells in stem cell‐based regenerative medicine such as the myocardial infarction repairing; however, most of the infused BMMSCs would expire within 48h post cell transplantation. To further explore the mechanism involved in regulating the life span of BMMSC itself would be beneficial to understand the stem cell‐based regenerative medicine. Pim1 is one of the serine/threonine kinases involved in cell growth, however, little is known about the role of Pim1 in the proliferation of BMMSC. Here, Immunoblot and quantitative RT‐PCR revealed that Pim1 was decreased in SD rat BMMSCs induced by TGF¦Â1 (5 ng/ml) for 7 days while compared with the cells in the undifferentiated status. Treatment the cells with Pim1 inhibitor, quercetagetin (5.5 ¦ÌM), led to Pim1 changes in its activity but not expression; Transduction the cells with Pim1 shRNA resulted in obvious decrease of Pim1 expression and activity. Treatment with both the quercetagetin and Pim1 shRNA led to significant decrease in cell viability and cell numbers in day 7, and the proportion of G0/G1 in cell cycles also increased after such treatment for 7 days. Thus, our results indicate that the maintenance of Pim1 expression in rat BMMSC, though in a low level, is crucial for cell proliferation. Our results support the rational that Pim1 may be as a potential molecular target in stem cell‐based regenerative medicine.

Pim-1 preserves mitochondrial morphology by inhibiting dynamin-related protein 1 translocation

Mitochondrial morphological dynamics affect the outcome of ischemic heart damage and pathogenesis. Recently, mitochondrial fission protein dynamin-related protein 1 (Drp1) has been identified as a mediator of mitochondrial morphological changes and cell death during cardiac ischemic injury. In this study, we report a unique relationship between Pim-1 activity and Drp1 regulation of mitochondrial morphology in cardiomyocytes challenged by ischemic stress. Transgenic hearts overexpressing cardiac Pim-1 display reduction of total Drp1 protein levels, increased phosphorylation of Drp1-(S637), and inhibition of Drp1 localization to the mitochondria. Consistent with these findings, adenoviral-induced Pim-1 neonatal rat cardiomyocytes (NRCMs) retain a reticular mitochondrial phenotype after simulated ischemia (sI) and decreased Drp1 mitochondrial sequestration. Interestingly, adenovirus Pim-dominant negative NRCMs show increased expression of Bcl-2 homology 3 (BH3)-only protein p53 up-regulated modulator of apoptosis (PUMA), which has been previously shown to induce Drp1 accumulation at mitochondria and increase sensitivity to apoptotic stimuli. Overexpression of the p53 up-regulated modulator of apoptosis-dominant negative adenovirus attenuates localization of Drp1 to mitochondria in adenovirus Pim-dominant negative NRCMs promotes reticular mitochondrial morphology and inhibits cell death during sI. Therefore, Pim-1 activity prevents Drp1 compartmentalization to the mitochondria and preserves reticular mitochondrial morphology in response to sI.

Abstract 14: Pim-1 Preserves Mitochondrial Morphology by Inhibiting Drp1 Translocation

Rationale: Mitochondrial morphological dynamics affect the outcome of ischemic heart damage. Mitochondrial fission protein Dynamin Related Protein 1 (Drp1) is a mediator of mitochondrial morphological changes and cell death during ischemic injury. Mitochondrial integrity is maintained by cardioprotective kinase Pim1, which enhances resistance to apoptotic challenge and ischemia reperfusion injury. In this study we examine the relationship between Pim1 activity and Drp1 regulation of mitochondrial morphology in cardiomyocytes challenged by ischemia. Objective: To demonstrate that Pim1 inhibits Drp1 translocation to the mitochondria in response to ischemic injury. Methods and Results: Simulated ischemia and simulated ischemia reperfusion (sI & sI/R) induced mitochondrial fragmentation and cell death in neonatal rat cardiac myocytes (NRCM), respectively. Mitochondrial fragmentation accompanied Drp1 translocation to the mitochondria in NRCM. Inhibition of Drp1 by mdivi1 preserved mitochondrial reticular morphology and inhibited apoptotic cell death. Mice subjected to sI/R injury displayed Drp1 mitochondrial localization, while exposure to mdivi1 led to reduced infarct size. Interestingly, transgenic hearts overexpressing Pim1 decreased total Drp1 levels, increased phosphorylation of Drp1 at serine 637, and inhibited Drp1 localization to mitochondria while preserving reticular morphology after sI. In contrast, Pim1 dominant negative (PDN) transgenic hearts and NRCM exhibit increased Drp1 translocation to mitochondria and fragmented mitochondria. PDN hearts exhibit decreased phosphorylation of serine 637 and upregulation of BH3 protein PUMA, inducing Drp1 accumulation at mitochondria and increased sensitivity to apoptotic stimuli. In PDN NRCMs, overexpression of Puma dominant negative (PumaDN) attenuated localization of Drp1 to mitochondria and inhibited cell death during sI. Conclusion: Pim1 activity prevents Drp1 compartmentalization to the mitochondria and preserves reticular mitochondrial morphology in response to simulated ischemia. Therefore, selective manipulation of Pim1 should be pursued as a therapeutic target to maintain mitochondrial morphology for cardioprotection.

IL-6 Stimulates STAT3 and Pim-1 Kinase in Pancreatic Cancer Cell Lines

We investigated the signaling pathways activated in response to interleukin 6 (IL-6) in pancreatic cell lines, with a focus on signal transducer and activator of transcription 3 (STAT3) and proto-oncogene serine/threonine-protein (Pim-1) kinase. Interleukin 6 receptor (IL-6R) expression and IL-6-induced cell signaling was measured by Western blotting in human pancreatic cell lines. Cucurbitacin I was used as a pharmacological tool to investigate the role of STAT3 in Pim-1 activation. Stably overexpressing Pim-1 kinase cell lines were characterized for their response to IL-6 in vitro and for their growth rate as flank tumors in scid mice. Interleukin 6 receptor was expressed across multiple cancer cell lines. In Panc-1 cells, IL-6 treatment increased expression of phosphorylation of signal transducer and activator of transcription 3 and Pim-1 kinase. Cucurbitacin I treatment alone increased pErk1/2 expression in wild-type and Pim-1-overexpressing cell lines and resulted in exaggerated Pim-1 kinase protein levels in control and IL-6-stimulated cells, suggesting that up-regulation of Pim-1 may be partially STAT3 independent. Pim-1 overexpression did not significantly affect growth rate in vitro or in vivo in Panc-1 or MiaPaCa2 cell lines. Interleukin 6 activates STAT3 and stimulates Pim-1 kinase in pancreatic cell line models. The regulation and consequence of Pim-1 expression seems to be highly context dependent.

Abstract 1270: Identification and functional analysis of Pim-1 kinase substrates in prostate cancer

Abstract Protein kinases constitute one of the largest and most pleiotropic families of proteins. Mutations leading to activation of protein kinases and loss of function of phosphatases can have deleterious consequences that lead to disease. Pim-1, a serine/threonine kinase, is one of many kinases that may play a role in the etiology of malignant diseases including lymphoma and prostate cancer. Recent studies indicate Pim-1 is overexpressed in prostate cancer and is a highly plausible therapeutic target. Paradoxically, a decrease in Pim-1 expression in later stages of prostate cancer correlates with poor prognosis. Pim-1 has been known to be an oncogene for more than two decades, however, our understanding of how it functions is incomplete. A key underlying factor in this lack of knowledge is that, to date, fewer than fifteen Pim-1 substrates have been identified. The identification and validation of novel Pim-1 substrates could elucidate biomarkers of Pim-1 activity and provide insight into the molecular basis of Pim-1 function. A recent, innovative kinase-substrate profiling method termed the Reverse In-gel Kinase Assay (RIKA) reverses the role of kinase and substrate in a traditional in-gel kinase assay. The assay involves the partial immobilization of an active kinase in a denaturing polyacrylamide gel to determine which substrates from cell lysates can be phosphorylated by the kinase within the gel. The potential substrates are excised from the gel and identified using mass spectrometry. Using a Pim-1 RIKA approach, 40S Ribosomal Protein 7 (RPS7) was identified as a potential novel Pim-1 substrate. RPS7 was expressed in E.coli to conduct preliminary in vitro studies. These in vitro studies demonstrated that Pim-1 robustly phosphorylates RPS7. Further studies were performed to identify sites of phosphorylation on RPS7, and alanine-substitution mutants were generated that completely abolished phosphorylation of RPS7 by Pim-1. These data indicated that RPS7 may be a novel Pim-1 substrate. Studies to validate RPS7 as a direct target of Pim-1 in vivo, and to determine the functional consequences of Pim-1 phosphorylation of RPS7 are in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1270. doi:1538-7445.AM2012-1270

Pim-1 knockdown potentiates paclitaxel-induced apoptosis in human hormone-refractory prostate cancers through inhibition of NHEJ DNA repair

The knockdown of Pim-1 or inhibition of Pim-1 activity significantly increased γ-H2A.X expression. The effect was correlated to apoptosis and was attributed to the inhibition of nonhomologous DNA-end-joining (NHEJ) repair activity supported by the following observations: (1) inhibition of ATM and DNA-PKcs activities, (2) down-regulation of Ku expression and nuclear localization and (3) decrease of DNA end-binding of both Ku70 and Ku80. The data suggest that Pim-1 plays a crucial role in the regulation of NHEJ repair. In the absence of Pim-1, the ability of DNA repair significantly decreases when exposed to paclitaxel, leading to severe DNA damage and apoptosis.

Abstract P313: Genetic Deletion of Pim Causes Premature Cardiac Senescence

Rationale: Cardiac aging is defined as increased cellular senescence and loss of survival signaling within cardiomyocytes and the cardiac stem cell population. To prevent myocardial, aging it is important to reverse the characteristic aging phenotype. Pim-1 is a cardioprotective kinase downstream of AKT, and has been shown to be pro-proliferative and anti-apoptotic. Pim-1 mediated antagonism of myocardial aging will lead to a decrease in cardiac senescence, maintain contractile performance, and prolong the lifespan of an organism. Objective: Demonstrate that myocardial senescence is antagonized by Pim-1 mediated signaling and loss of Pim-1 activity leads to premature aging of the myocardium and associated decline in cardiac performance. Methods and Results: Pim Triple KnockOut (PTKO) mice, in which all three isoforms of Pim are genetically deleted, develop premature phenotypic hallmarks of myocardial senescence. Elevated levels of the tumor suppressor p53 and senescence marker p16 are present in hearts of PTKO mice at two months of age. Increased p16 expression is consistent with observed induction of Ets-1, a transcription factor that activates p16 transcription in PTKO mice. Levels of Id1 and Id2, positive transcriptional regulators of the cell cycle, are decreased in hearts of PTKO mice. Hemodynamic analyses reveal impaired cardiac function in PTKO mice at two months of age. Conversely, cardiac specific overexpression of Pim1-kinase attenuates the senescent phenotype. Hypoxia treatment of neonatal rat cardiomyocytes infected with Pim-1 encoding adenovirus results in reduction of p16 accumulation as seen by qRT-PCR. Pim-1 overexpression increases expression of c-myc, thereby upregulating Id2 and inhibits p16 induced senescence. Cardiac Pim-1 transgenic mice show a delay in the onset of senescence as evidenced by decreased frequency of p16 positive cells in the myocardium. Conclusion: Pim-1 inhibits cardiac senescence and is important for maintaining cardiac function. Therefore, selective manipulation of Pim-1 should be pursued as a therapeutic target to promote and extend myocardial cellular lifespan and function.

RAGE-Dependent Activation of the Oncoprotein Pim1 Plays a Critical Role in Systemic Vascular Remodeling Processes

Vascular remodeling diseases (VRD) are mainly characterized by inflammation and a vascular smooth muscle cells (VSMCs) proproliferative and anti-apoptotic phenotype. Recently, the activation of the advanced glycation endproducts receptor (RAGE) has been shown to promote VSMC proliferation and resistance to apoptosis in VRD in a signal transducer and activator of transcription (STAT)3-dependant manner. Interestingly, we previously described in both cancer and VRD that the sustainability of this proproliferative and antiapoptotic phenotype requires activation of the transcription factor NFAT (nuclear factor of activated T-cells). In cancer, NFAT activation is dependent of the oncoprotein provirus integration site for Moloney murine leukemia virus (Pim1), which is regulated by STAT3 and activated in VRD. Therefore, we hypothesized that RAGE/STAT3 activation in VSMC activates Pim1, promoting NFAT and thus VSMC proliferation and resistance to apoptosis. Methods/Results- In vitro, freshly isolated human carotid VSMCs exposed to RAGE activator Nε-(carboxymethyl)lysine (CML) for 48 hours had (1) activated STAT3 (increased P-STAT3/STAT3 ratio and P-STAT3 nuclear translocation); (2) increased STAT3-dependent Pim1 expression resulting in NFATc1 activation; and (3) increased Pim1/NFAT-dependent VSMC proliferation (PCNA, Ki67) and resistance to mitochondrial-dependent apoptosis (TMRM, Annexin V, TUNEL). Similarly to RAGE inhibition (small interfering RNA [siRNA]), Pim1, STAT3 and NFATc1 inhibition (siRNA) reversed these abnormalities in human carotid VSMC. Moreover, carotid artery VSMCs isolated from Pim1 knockout mice were resistant to CML-induced VSMC proliferation and resistance to apoptosis. In vivo, RAGE inhibition decreases STAT3/Pim1/NFAT activation, reversing vascular remodeling in the rat carotid artery-injured model. RAGE activation accounts for many features of VRD including VSMC proliferation and resistance to apoptosis by the activation of STAT3/Pim1/NFAT axis. Molecules aimed to inhibit RAGE could be of a great therapeutic interest for the treatment of VRD.

Abstract 2930: IL-6 stimulates STAT-3 and Pim-1 kinase in pancreatic cancer cell lines

Abstract Introduction: IL-6 has both diagnostic and prognostic significance in pancreatic cancer. We investigated the signaling pathways activated in response to IL-6 in pancreatic cell lines, with a focus on STAT-3 and Pim-1 kinase. Methods: IL-6 receptor (IL-6R) expression was evaluated by Western blot analysis across a panel of human pancreatic cell lines. Panc-1 and MiaPaCa2 cell lines were serum starved for 24 hrs and then treated with 100 ng/mL of IL-6 for various time courses from 0 to 6 hr. STAT-3 activation and Pim-1 kinase protein levels were assessed in the presence and absence of IL-6 by Western blot analysis. The ability of cucurbitacin B to block both STAT-3 and Pim-1 activation, and inhibit cell proliferation was investigated. Stably transfected Pim-1 kinase over-expressing cell lines were generated and characterized for their response to IL-6 in vitro, and their growth rate in vivo as flank tumors in scid mice. Results: IL-6R is broadly expressed across multiple cancer cell lines, with the highest expression observed in the Panc-1 cell line. IL-6 stimulation resulted in increased STAT-3 phosphorylation in Panc-1, and MiaPaCa2 tumor cell lines as well as the HP-DEV transformed pancreatic ductal cell line. Treatment with IL-6 resulted in increased Pim-1 protein expression in the Panc-1 but not the MiaPaCa2 parent cell lines. Induction of Pim-1 protein was also observed in both Panc-1 and MiaPaCa2 stably transfected over-expressing cell lines. Pre-treatment with the STAT-3 inhibitor cucurbitacin B resulted in inhibition of STAT-3 phosphorylation and decreased cell proliferation, but was not associated with suppression of Pim-1 protein expression in the Panc-1 cell lines. Over-expression of Pim-1 in the Panc-1 cell line was associated with no significant changes in basal apoptosis rate, cell growth rate in vitro or in flank xenografts, or IL-6 induced expression of VEGF. Conclusions: IL-6 stimulation activates both STAT-3 and Pim-1 kinase expression in pancreatic cell line models. Induction of Pim-1 kinase was cell line specific, suggesting that the molecular context is essential for this pathway to be activated. Suppression of STAT-3 with a pharmacological inhibitor resulted in exaggerated Pim-1 expression in over-expressing cell lines implying Pim-1 up-regulation may be one of the compensatory and/or stress pathways activated when STAT-3 is inhibited. Stable over-expressing Pim-1 kinase Panc-1 and MiaPaCa2 cells did not exhibit significant changes in growth or apoptosis, unlike previously reported studies in the literature with other pancreatic cancer cell line models. This may be due to the multiple pathways that are dysregulated in these cell lines. Both STAT-3 and Pim-1 kinase have previously been identified as drug targets in multiple malignancies, including leukemia, lymphoma, prostate, and pancreatic cancer. Our study suggests that both pathways may be activated in patients with elevated IL-6 levels. 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 2930. doi:10.1158/1538-7445.AM2011-2930

The oncogenic kinase Pim-1 is modulated by K-Ras signaling and mediates transformed growth and radioresistance in human pancreatic ductal adenocarcinoma cells

Oncogenic Pim-1 kinase is upregulated in multiple solid cancers, including human pancreatic ductal adenocarcinoma (PDAC), a highly lethal disease with few useful treatment options. Pim-1 is also transcriptionally induced upon oncogenic K-Ras-mediated transformation of the human pancreatic ductal epithelial (HPDE) cell model of PDAC. Given the near ubiquitous presence of mutant K-Ras in PDAC and its critical role in this disease, we wished to study the effects of oncogenic K-Ras signaling on Pim-1 expression, as well as the role of Pim-1 in growth transformation of PDAC cells. Pim-1 protein levels were upregulated in both PDAC cell lines and patient tumor tissues. Furthermore, ectopic oncogenic K-Ras increased Pim-1 expression in human pancreatic nestin-expressing (HPNE) cells, a distinct immortalized cell model of PDAC. Conversely, shRNA-mediated suppression of oncogenic K-Ras decreased Pim-1 protein in PDAC cell lines. These results indicate that oncogenic K-Ras regulates Pim-1 expression. The kinase activity of Pim-1 is constitutively active. Accordingly, shRNA-mediated suppression of Pim-1 in K-Ras-dependent PDAC cell lines decreased Pim-1 activity, as measured by decreased phosphorylation of the pro-apoptotic protein Bad and increased expression of the cyclin-dependent kinase inhibitor p27Kip1. Biological consequences of inhibiting Pim-1 expression included decreases in both anchorage-dependent and -independent cell growth, invasion through Matrigel and radioresistance as measured by standard clonogenic assays. These results indicate that Pim-1 is required for PDAC cell growth, invasion and radioresistance downstream of oncogenic K-Ras. Overall, our studies help to elucidate the role of Pim-1 in PDAC growth transformation and validate Pim-1 kinase as a potential molecular marker for mutated K-Ras activity.

Activation of a non-genomic Pim-1/Bad-P ser75 module is required for an efficient pro-survival effect of Bcl-xL induced by androgen in LNCaP cells

The present report investigated the pathway(s) involved in the inhibition of apoptosis by the synthetic androgen, R1881 in serum-starved LNCaP cells exposed to the pi3K inhibitor, LY294002. R1881 blocked LY294002-induced apoptosis through the inhibition of Bak activation via an increase in Bcl-xL transcription and protein expression. In addition, R1881 treatment enhanced the stability of the Pim-1 kinase, resulting in the inhibition of the activation of the BH3-only protein Bad through its phosphorylation at ser75. Pharmacological inhibition of the Pim-1 kinase activity with quercetagetin, a highly selective Pim-1 inhibitor, prevented R1881-mediated increase in Bad phosphorylation and restored cell sensitivity to LY294002-induced apoptosis despite the increase in Bcl-xL expression. These results demonstrate for the first time that the inhibition of LY294002-induced apoptosis by androgen is a function of an androgen receptor-dependent genomic signaling pathway leading to an increase in Bcl-xL expression as well as a non-genomic, Pim-1-dependent, signaling pathway mediated via phosphorylation of Bad at ser75.

Inhibition of the Serine/Threonine Kinase Pim-1 Has Anti-Proliferative Effects In Acute Myeloid Leukemia (AML) and Sensitizes Multidrug Resistant Cells to Chemotherapy

AbstractAbstract 1832Poster Board I-812The serine/threonine kinase Pim-1, encoded by a proto-oncogene originally identified as the proviral integration site in Moloney murine leukemia virus lymphomagenesis, phosphorylates and thereby increases expression of multiple cellular proteins, including the pro-apoptotic protein BAD, the cell cycle regulatory proteins p21, p27, Cdc25A and Cdc25C, the transcription factors SOCS-1, RUNX3 and c-myc and, as we recently demonstrated, the drug resistance-associated ATP-binding cassette (ABC) proteins P-glycoprotein (Pgp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2). Pim-1 is synthesized in an active form by virtue of its hinge structure, and its activity is therefore regulated solely by its level of expression. Pim-1 is overexpressed downstream of FLT3 in AML cells with FLT3-ITD, but less is known about its expression and role in AML with wild-type (wt) FLT3. We studied Pim-1 expression and the effects of Pim-1 inhibition on AML cell survival, proliferation, apoptosis and chemosensitivity. Cell lines studied included HL60, K562, U937, Kasumi-1 and EOL-1 FLT3-wt cells and MV4-11 and MOLM-14 FLT3-ITD cells, as well as Pgp+ HL60/VCR and BCRP+ 8226/MR20 and parental 8226 myeloma cells as a model for BCRP-mediated drug resistance. Expression of Pim-1 and of phospho-BAD at S112, a measure of Pim-1 activity, was studied by Western blot analysis, normalized to GAPDH expression. Effects of the Pim-1 inhibitor SGI-1776 (SuperGen, Inc., Dublin, CA) on survival, cell cycle, apoptosis and colony growth were measured in WST-1 cell survival, flow cytometric cell cycle and apoptosis, and methylcellulose colony formation assays, respectively. SGI-1776 inhibits Pim-1 at a concentration of 7 +/− 1.8 nM, but is more than 90% bound to human plasma protein, so that its Pim-1 inhibitory concentration in cell culture-based assays is in μM range. Of note, SGI-1776 also inhibits FLT3 in this concentration range. Pim-1 was expressed in all cell lines studied, and expression of Pim-1 and of phopho-BAD did not differ between FLT3-ITD and FLT3-wt cells, nor between drug-resistant and parental cells. SGI-1776 decreased viable cell numbers in 96-hour WST-1 cell viability assays, with IC50's of 5 to 7 μM in FLT-wt cells, while IC50's were 20 and 65 nM, respectively, in MV4-11 and MOLM-14 FLT3-ITD cells. SGI-1776 IC50's did not differ between Pgp+ or BCRP+ cells and parental cells. In FLT3-wt cells, SGI-1776 had no effect on cell cycle at concentrations up to 5 μM, and caused apoptosis at 10 μM, while in FLT3-ITD cells, G1 arrest and apoptosis occurred at 100 nM. HL60 colony formation was completely inhibited by 5 μM SGI-1776, while MOLM-14 colony formation inhibition occurred at 500 nM. Finally, SGI-1776 sensitized multidrug resistant, but not parental, cells to multidrug resistance protein substrate, but not non-substrate, drugs. SGI-1776 at 1 μM decreased the IC50 of the Pgp substrate chemotherapy drug daunorubicin in Pgp+ HL60/VCR cells 7-fold, but had no effect on daunorubicin IC50 in HL60 cells, nor on IC50 of the non-Pgp substrate cytarabine in either cell line. SGI-1776 at 1 μM also decreased the IC50 of the BCRP substrate chemotherapy drug mitoxantrone in BCRP+ 8226/MR20 cells 7-fold. SGI-1776 at 1 μM doubled the percentage of apoptotic cells among HL60/VCR, but not HL60, cells exposed to daunorubicin and 8226/MR20 cells exposed to mitoxantrone. Finally, SGI-1776 at 1 μM decreased HL60/VCR colony formation in the presence of daunorubicin, but not cytarabine, but had no effect in HL60 cells, and also decreased 8226/MR20 colony formation in the presence of mitoxantrone, but not cytarabine. Thus the Pim-1 inhibitor SGI-1776 has anti-proliferative effects in AML cells with wt FLT3 as well FLT3-ITD, and sensitizes Pgp+ and BCRP+ multidrug resistant cells to chemotherapy. These data support clinical trials of SGI-1776 in AML with wt FLT3 as well FLT3-ITD, as a single agent and in combination with chemotherapy in multidrug resistant AML. Disclosures:No relevant conflicts of interest to declare.

PIM1 kinase is destabilized by ribosomal stress causing inhibition of cell cycle progression

PIM1 is a constitutively active serine/threonine kinase regulated by cytokines, growth factors and hormones. It has been implicated in the control of cell cycle progression and apoptosis and its overexpression has been associated with various kinds of lymphoid and hematopoietic malignancies. The activity of PIM1 is dependent on the phosphorylation of several targets involved in transcription, cell cycle and apoptosis. We have recently observed that PIM1 interacts with ribosomal protein (RP)S19 and cosediments with ribosomes. Defects in ribosome synthesis (ribosomal stress) have been shown to activate a p53-dependent growth arrest response. To investigate if PIM1 could have a role in the response to ribosomal stress, we induced ribosome synthesis alterations in TF-1 and K562 erythroid cell lines. We found that RP deficiency, induced by RNA interference or treatment with inhibitor of nucleolar functions, causes a drastic destabilization of PIM1. The lower level of PIM1 induces an increase in the cell cycle inhibitor p27(Kip1) and blocks cell proliferation even in the absence of p53. Notably, restoring PIM1 level by transfection causes a recovery of cell growth. Our data indicate that PIM1 may act as a sensor for ribosomal stress independently of or in concert with the known p53-dependent mechanisms.

Abstract 5659: Down-regulation of hTERT sensitizes chemotherapeutic effects of docetaxel in human prostate cancer cells via suppression of PIM-1 oncogene

Abstract Taxanes comprise some of the most widely used cancer chemotherapeutic agents for various cancers. Docetaxel is an effective first-line treatment for hormone-refractory prostate cancer. However, despite the recently demonstrated survival benefits of docetaxel-based chemotherapy, most patients will move from response to progression within 6 months. Moreover, effective concentrations of docetaxel have side effects including localized erythema of the extremities with edema followed by desquamation, severe neuropathy, fatigue, and weakness which can have debilitating effects in patients with prostate cancer. One reason for disease progression is the up-regulation of PIM-1 expression following docetaxel treatment of prostate cancer cells. PIM-1 is a serine-threonine kinase that promotes survival by inhibiting the activity of apoptotic proteins. In a recent study, we observed that a telomerase suppressing small molecule MST-312, and hTERT siRNA down-regulated PIM-1 protein level in prostate cancer cells. These results prompted us to investigate whether combination of docetaxel with inhibition of telomerase by MST-312 or TERT siRNA would have synergistic effects on prostate cancer cell growth and induce death. Treatment of PC3 cells with 1 nM of docetaxel for 3 days substantially promoted the viability of cells compared to the control, with a 27% increase in cell number. This increase was associated with the up-regulation of PIM-1. Treatment of telomerase inhibitory concentration (2 μM) of MST-312 over 3 days resulted in a 51% decrease in cell viability relative to the 1 nM docetaxel treated groups, associated with a decrease in PIM-1 protein levels. More interestingly, treatment of 2 μM MST-312 with 1 nM docetaxel for 3 days resulted in 72% reduction in cell viability compared to docetaxel alone and 43% reduction in cell viability compared to MST-312 alone. This combined treatment also resulted in lower levels of PIM-1 activity than MST-312 or docetaxel treatment alone. These results suggest that there is synergy between the combination of telomerase inhibitor MST-312 with docetaxel in the treatment of metastatic prostate cancer. This can be explained by the down-regulation of PIM-1 kinase that promotes resistance to docetaxel treatment. These findings suggest potential clinical benefit by the combined use of docetaxel and telomerase inhibitors for prostate cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5659.

Pim-1 kinase protects mitochondrial integrity in cardiomyocytes.

Cardioprotective signaling mediates antiapoptotic actions through multiple mechanisms including maintenance of mitochondrial integrity. Pim-1 kinase is an essential downstream effector of AKT-mediated cardioprotection but the mechanistic basis for maintenance of mitochondrial integrity by Pim-1 remains unexplored. This study details antiapoptotic actions responsible for enhanced cell survival in cardiomyocytes with elevated Pim-1 activity. The purpose of this study is to demonstrate that the cardioprotective kinase Pim-1 acts to inhibit cell death by preserving mitochondrial integrity in cardiomyocytes. A combination of biochemical, molecular, and microscopic analyses demonstrate beneficial effects of Pim-1 on mitochondrial integrity. Pim-1 protein level increases in the mitochondrial fraction with a corresponding decrease in the cytosolic fraction of myocardial lysates from hearts subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion. Cardiac-specific overexpression of Pim-1 results in higher levels of antiapoptotic Bcl-X(L) and Bcl-2 compared to samples from normal hearts. In response to oxidative stress challenge, Pim-1 preserves the inner mitochondrial membrane potential. Ultrastructure of the mitochondria is maintained by Pim-1 activity, which prevents swelling induced by calcium overload. Finally, mitochondria isolated from hearts created with cardiac-specific overexpression of Pim-1 show inhibition of cytochrome c release triggered by a truncated form of proapoptotic Bid. Cardioprotective action of Pim-1 kinase includes preservation of mitochondrial integrity during cardiomyopathic challenge conditions, thereby raising the potential for Pim-1 kinase activation as a therapeutic interventional approach to inhibit cell death by antagonizing proapoptotic Bcl-2 family members that regulate the intrinsic apoptotic pathway.

Pim‐1 kinase as activator of the cell cycle pathway in neuronal death induced by DNA damage

DNA damage is a critical component of neuronal death underlying neurodegenerative diseases and injury. Neuronal death evoked by DNA damage is characterized by inappropriate activation of multiple cell cycle components. However, the mechanism regulating this activation is not fully understood. We demonstrated previously that the cell division cycle (Cdc) 25A phosphatase mediates the activation of cyclin-dependent kinases and neuronal death evoked by the DNA damaging agent camptothecin. We also showed that Cdc25A activation is blocked by constitutive checkpoint kinase 1 activity under basal conditions in neurons. Presently, we report that an additional factor is central to regulation of Cdc25A phosphatase in neuronal death. In a gene array screen, we first identified Pim-1 as a potential factor up-regulated following DNA damage. We confirmed the up-regulation of Pim-1 transcript, protein and kinase activity following DNA damage. This induction of Pim-1 is regulated by the nuclear factor kappa beta (NF-kappaB) pathway as Pim-1 expression and activity are significantly blocked by siRNA-mediated knockdown of NF-kappaB or NF-kappaB pharmacological inhibitors. Importantly, Pim-1 activity is critical for neuronal death in this paradigm and its deficiency blocks camptothecin-mediated neuronal death. It does so by activating Cdc25A with consequent activation of cyclin D1-associated kinases. Taken together, our results demonstrate that Pim-1 kinase plays a central role in DNA damage-evoked neuronal death by regulating aberrant neuronal cell cycle activation.

Abstract A249: Potent protein kinase inhibitors block Pim kinase-mediated increase in prostate epithelial cell migration, regulation of p27 protein half-life, and secretion of hepatocyte growth factor

Abstract Pim-1 proto-oncogene encodes a serine/threonine protein kinase that regulates apoptosis, cell cycle progression, and transcription. The expression of this protein kinase is elevated in both prostate intraepithelial neoplasia (PIN) and prostatic adenocarcinoma, suggesting an important role for Pim-1 kinase in prostate cancer development and growth. To investigate the role of Pim-1 in controlling tumor growth we have synthesized novel benzylidene-thiazolidine2, 4-dione (J. Med. Chem. (2009) 52:74) inhibitors of this kinase. The most potent members of this chemotype have IC50s of 13 nM for Pim-1. To examine the activity of these agents in prostate cancer we have first set out to define the biochemical activity of Pim-1 in epithelial cells. We have expressed Pim-1 in a mouse prostate epithelial cell (MPECs) line that demonstrates stem cell characteristics. We find that Pim-1 expressing cells produce and secrete markedly increased levels of hepatocyte growth factor/scatter factor (HGF/SF), and this protein kinase stimulates increases in HGF/SF mRNA. Additionally, expression of Pim-1 markedly increases HGF/SF induced migration of MPECs. The contribution of Pim-1 kinase to this biochemical pathway is confirmed in murine embryonic fibroblasts (MEFs) that are deficient for all three Pim protein kinases (TKO) and evidence reduced level of HGF mRNA in TKO versus wild type MEFs. The Pim-1-stimulated migration of MPECs is inhibited by two benzylidene-thiazolidine-2, 4-diones, SMI-4a and 16a, as well as known inhibitors of the HGF receptor, c-Met. HGF treatment of MPECs induced p27 upregulation that could be inhibited by the expression of Pim-1, thus allowing cell cycle progression. Further studies showed that expression of Pim-1 did not alter the mRNA level of p27, but enhanced the cell cycle-dependent degradation and thus decreased the half-life of the p27 protein. Consistent with this finding, p27 ubiquitination assays showed that Pim-1 increases this modification in vivo. We found that the Pim-1-mediated ubiquitination is regulated by complex formation between Pim-1 and Skp2, a protein component of the SCF complex, which is known to regulate the ubiquitination and degradation of p27. Pim-1 does not affect Skp2's E3 ligase activity, but appears to inhibit the degradation of Skp2 through phosphorylation. Incubation of these cells with the Pim protein kinase inhibitor, SMI-4a, decreased Skp2 expression and increased p27 and cyclin E expression. Together our data demonstrates the complex pathway by which Pim-1 protein kinase regulates HGF/SF and p27 levels, thus controlling cell migration, proliferation, and potentially transformation. Potent Pim kinase inhibitors block these two signaling pathways thus inhibiting prostate epithelial cell migration and growth. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A249.

The Serine/Threonine Kinase Pim-1 Stabilizes 130 Kilodalton FLT3 and Promotes Aberrant Signaling through STAT5 in Acute Myeloid Leukemia Cells with FLT3 Internal Tandem Duplication: Synergistic Apoptosis Induction by Pim-1 and FLT3 Inhibitors.

Abstract 942▪▪This icon denotes an abstract that is clinically relevant.Fms-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) results in FLT3 constitutive activation and aberrant signaling in acute myeloid leukemia (AML) cells. FLT3-ITD is associated with adverse treatment outcome in AML, but FLT3 inhibitors have had limited therapeutic efficacy. The oncogenic serine/threonine kinase Pim-1 is upregulated in AML cells with FLT3-ITD. Pim-1 inhibitors are entering clinical trials, and we sought to characterize the role of Pim-1 and the effects of Pim-1 inhibition in FLT3-ITD cells. Wild-type (WT) FLT3 exists predominantly in a 150 kDa complex glycosylated form. In contrast, FLT3-ITD is partially retained in the endoplasmic reticulum (ER) as a misfolded 130 kDa underglycosylated, or high-mannose, species in association with the ER transmembrane chaperone calnexin. In addition, FLT3-ITD also associates with and is stabilized by the cytosolic chaperone heat shock protein (HSP) 90. FLT3-ITD activates signal transducer and activation of transcription (STAT) 5 and upregulates the STAT5 downstream target Pim-1. FLT3 contains a putative Pim-1 substrate consensus serine phosphorylation site, and we hypothesized that FLT3 might be a Pim-1 substrate. FLT3-ITD cell lines studied included MV4-11, MOLM-14 and transfected Ba/F3-ITD, and FLT3 WT cells included BV173, EOL-1 and transfected Ba/F3-WT. Pim-1 activity was measured by an in vitro kinase assay of BAD phosphorylation at serine 112, and Pim-1 expression, FLT3 expression, phosphorylation and co-immunoprecipitation, and STAT5 phosphorylation and expression by Western blot analysis. Pim-1 knockdown was accomplished by infection with lentivirus containing Pim-1 small hairpin RNA (shRNA) or non-target control, and Pim-1 kinase inhibition by incubation with the Pim-1-selective inhibitor quercetagetin. Pim-1 was found to directly interact with and serine-phosphorylate FLT3 from FLT3-ITD, but not FLT3-WT, cells in vitro. Inhibition of Pim-1 kinase disrupted binding of FLT3 to its chaperones calnexin and HSP90, and resulted in decreased expression and half-life of 130 kDa FLT3 and increased expression and half-life of 150 kDa FLT3. The decrease in expression and half-life of 130 kDa FLT3 was partially abrogated by co-incubation with the proteasome inhibitor MG132. Moreover, the increase in 150 Kda FLT3 was abrogated by co-incubation with the glycosylation inhibitor 2-deoxy-D-glucose. Thus Pim-1 maintains FLT3 as a 130 kDa species by enhancing its binding to its chaperones calnexin and HSP90, protecting it from proteasomal degradation and inhibiting its glycosylation to form 150 kDa FLT3. Inhibition of Pim-1 kinase activity also decreased phosphorylation of FLT3 at tyrosine 591, a docking site for binding of FLT3-ITD, but not FLT3-WT, to STAT5, and decreased both STAT5 phosphorylation and expression of Pim-1 itself. In contrast, Pim-1 inhibition had no effect on FLT3 tyrosine kinase activity nor on expression of Pim-2, another Pim kinase family member implicated in promoting survival of FLT3-ITD cells. Finally, the Pim-1 kinase inhibitor quercetagetin and the FLT3 inhibitor PKC412 had a synergistic effect in inducing apoptosis of Ba/F3-ITD cells: [Display omitted] We conclude that Pim-1, which is transcriptionally upregulated through STAT5 in FLT3-ITD cells, serine-phosphorylates FLT3-ITD, thereby maintaining it in an underglycosylated form, and promotes STAT5 signaling, and that inhibition of Pim-1 and of FLT3 is synergistic in inducing apoptosis of FLT3-ITD cells. Thus Pim-1 inhibitors should inhibit aberrant signaling upstream as well as downstream of Pim-1 in FLT3-ITD cells, and have the potential to enhance the therapeutic efficacy of FLT3 inhibitors in patients with AML with FLT3-ITD Disclosures:No relevant conflicts of interest to declare.

Oncogenic Pim Kinase Activity Provides Resistance to Mtor Inhibition in Vitro and In Vivo.

Abstract 3974Poster Board III-910The PI3K/Akt/mTor pathway is among the most frequently deregulated in human cancers, including many leukemias and lymphomas, and inhibitors targeting it at multiple levels are either clinically available or under development. The Pim family proteins are oncogenic serine/threonine kinases expressed in many malignancies and that have numerous overlapping downstream targets and functional consequences with PI3K/Akt/mTor. We here investigate the role of Pim proteins in oncogenesis and their ability in particular to mediate resistance to mTor inhibition. We find over-expression and genomic amplification of Pim1 and Pim2 in tumor samples and cell lines derived from patients with multiple B lymphomas. We show that both Pim1 and Pim2 powerfully mediate resistance to apoptosis in the murine pro-B cell line FL5-12 when withdrawn from IL-3. In murine tumor cells with constitutive activation of mTor, Pim1 and Pim2 provide resistance to treatment with the mTor inhibitor rapamycin. In vivo, we find that murine Pim2 (mPim2) accelerates tumorigenesis in the Eu-Myc transgenic model of Burkitt's lymphoma in a manner highly similar to Akt. However, unlike Akt, mPim2 leads to tumors that resist sensitization to chemotherapy by co-administration of rapamycin. We also investigate Pim1's role as a target of aberrant somatic hypermutation (ASHM), the process by which the enzyme activation induced deamidase (AID) introduces mutations into the coding and non-coding regions of the Pim1 locus and other proto-oncogenes in many B-cell malignancies. We find that structural Pim1 mutants found in patient tumors samples retain their ability to accelerate tumorigenesis in the Eu-Myc model in vivo and to mediate resistance to apoptosis and rapamycin in vitro. In conclusion, Pim activity is clinically significant, highly oncogenic, and provides resistance to mTor inhibition. Pim inhibition is therefore an attractive therapeutic approach, especially in combination with PI3K/Akt/mTor inhibition, even as ASHM provides a potential mechanism for B-cell tumors to escape it. Disclosures:No relevant conflicts of interest to declare.