Overexpression Of Pim-1 Research Articles

Abstract B232: PIM1 kinase is essential for the growth of MYC-overexpressing triple-negative breast tumors and is an efficacious therapeutic target.

Abstract While de-regulated MYC signaling has been identified in a wide variety of human malignancies, no targeted therapeutic strategies have been established to target tumors with elevated MYC expression. We have previously reported that receptor “triple negative” (TN) breast cancers, which lack predictive biomarkers of response (i.e., estrogen/progesterone receptors, human epidermal growth factor receptor 2), exhibit significantly elevated MYC expression as well as MYC pathway activation (Horiuchi, D., et al. 2012 JEM). In an effort to uncover the potential Achilles’ heels of TN tumors, we performed a kinome, synthetic-lethal shRNA screen in human mammary epithelial cells in which MYC activity can be controlled (i.e., HMEC-MycER cells). The screen yielded 13 potential synthetic lethal partners of MYC, including recently described AMPK-related kinase 5 (Liu, L., et al. 2012. Nature). One of our top hits is the proto-oncogene PIM1, previously shown by others to be a genetic enhancer of MYC in transgenic mouse models of lymphomas and prostate cancer. Our bioinformatics studies performed on four independent clinical cohorts (n: 146 ∼ 683 patients each) show that in all the cohorts both MYC and PIM1 are significantly enriched in the TN populations. MYC and PIM1 expression are correlated in three out of the four cohorts. Furthermore, we find that PIM1 overexpression is a poor prognostic factor associated with diminished recurrence-free survival. Our in vivo efficacy studies using conventional as well as novel “human-in-mouse” orthotopic xenograft models demonstrate that PIM1 is required for the growth of MYC-overexpressing human TN tumors. We are currently investigating the cellular mechanisms by which PIM kinase inhibition causes such growth defects. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B232. Citation Format: Dai Horiuchi, Alicia Y. Zhou, Alexandra N. Corella, Christina Yau, Devon A. Lawson, Alexey V. Bazarov, Paul Yaswen, Michael T. McManus, Alana L. Welm, Zena Werb, Andrei Goga. PIM1 kinase is essential for the growth of MYC-overexpressing triple-negative breast tumors and is an efficacious therapeutic target. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B232.

Pim1 permits generation and survival of CD4+T cells in the absence of γc cytokine receptor signaling

γ-Chain (γc) cytokine receptor signaling is required for the development of all lymphocytes. Why γc signaling plays such an essential role is not fully understood, but induction of the serine/threonine kinase Pim1 is considered a major downstream event of γc as Pim1 prevents apoptosis and increases metabolic activity. Consequently, we asked whether Pim1 overexpression would suffice to restore lymphocyte development in γc-deficient mice. By analyzing Pim1-transgenic γc-deficient mice (Pim1(Tg) γc(KO) ), we show that Pim1 promoted T-cell development and survival in the absence of γc. Interestingly, such effects were largely limited to CD4(+) lineage αβ T cells as CD4(+) T-cell numbers improved to near normal levels but CD8(+) T cells remained severely lymphopenic. Notably, Pim1 over-expression failed to promote development and survival of any T-lineage cells other than αβ T cells, as we observed complete lack of γδ, NKT, FoxP3(+) T regulatory cells and TCR-β(+) CD8αα IELs in Pim1(Tg) γc(KO) mice. Collectively, these results uncover distinct requirements for γc signaling between CD4(+) αβ T cells and all other T-lineage cells, and they identify Pim1 as a novel effector molecule sufficient to drive CD4(+) αβ T-cell development and survival in the absence of γc cytokine receptor signaling.

Abstract 5185: RNAi screen identifies a synthetic lethal interaction between Pim1 overexpression and Plk1 inhibition.

Abstract PIM1 kinase is overexpressed in many tumor types including lymphomas and prostate cancer, where it is known to cooperate with the MYC oncogene in promoting tumorigenicity. PIM1 enhances MYC stability and transcriptional activity. Knowledge of the vulnerabilities of Pim1-expressing tumor cells will be of great value in efforts to develop novel anti-cancer therapeutics. Here we have used RNAi screening to identify genes whose depletion is detrimental to Pim1-overexpressing cells. We screened a collection of 570 siRNAs targeting cell cycle, apoptosis, serine-threonine kinase and tyrosine kinase genes for their effects on the viability of Pim1-expressing RWPE1 prostate epithelial cells. We identified the mitotic regulator polo-like kinase (PLK1) as a gene whose depletion is particularly detrimental to the viability of Pim1-expressing prostate cancer cells in vitro and in vivo. Inhibition of PLK1 by shRNA or by the chemical inhibitor BI2536 in prostate cancer xenograft model resulted in a dramatic inhibition of tumor progression in Pim1-overexpressing cells. Interestingly Pim1-overexpressing cells are more prone to mitotic arrest followed by apoptosis. Furthermore, inhibition of Plk1 activity led to the reduction of MYC protein levels both in vitro and in vivo. These results suggest that PLK1 inhibition may be particularly effective against PIM1-expressing tumors due, at least in part, to the fact that PLK1 inhibition reduces MYC protein levels. Citation Format: Sarki Abdulkadir, Riet Vander Meer, Meejeon Roh. RNAi screen identifies a synthetic lethal interaction between Pim1 overexpression and Plk1 inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5185. doi:10.1158/1538-7445.AM2013-5185 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2353: Novel inhibitors of PIM-1, PIM-2, and PIM-3 protein kinases: medicinal chemistry leading to AZD1208.

Abstract The PIM serine/threonine kinase family, composed of three highly homologous members, PIM-1, PIM-2 and PIM-3, are upregulated in leukemias and lymphomas, including AML, NHL and CLL, highlighting the potential of these kinases as therapeutic targets in these indications. Over-expression of PIM-1 or PIM-3 has also been observed several solid tumors, in particular prostate, pancreatic, gastric, bladder and hepatocellular cancers. PIM kinases are downstream effectors of many cytokine and growth factor signaling pathways and are direct transcriptional targets of STAT transcription factors activated by these pathways, thereby mediating cell proliferation and survival. We have identified novel, potent and highly selective inhibitors of the PIM family kinases. The synthesis, X-ray crystallographic binding mode, and SAR of this benzylidene-1,3-thiazolidine-2,4-dione series are described. Examples from this series exhibit single digit nanomolar potency against all three PIMs, and have been shown to be selective across a panel of more than 440 kinases, with inhibition found for only approximately 3% of the panel, and at least 10-fold selectivity over kinases outside the PIM family. The compounds additionally are stable in rat microsomes, have high aqueous solubilities, and are not potent against the hERG ion channel. The series has antiproliferative activity in a panel of AML cells, has excellent pre-clinical pharmacokinetic properties, and lead compounds have shown strong tumor growth inhibition in vivo in mouse AML xenograft models. From this series, AZD1208 has recently entered Phase I clinical trials for AML and solid tumors. Citation Format: Michelle L. Lamb, Les A. Dakin, Michael H. Block, Huawei Chen, Erin Code, James E. Dowling, Xiaomei Feng, Andrew D. Ferguson, Isabelle Green, Alexander W. Hird, Tina Howard, Dennis Huszar, Erika K. Keeton, Paul D. Lyne, Hannah Pollard, Michael Rooney, Jon Read, Allan J. Wu, Tao Zhang, Xiaolan Zheng. Novel inhibitors of PIM-1, PIM-2, and PIM-3 protein kinases: medicinal chemistry leading to AZD1208. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2353. doi:10.1158/1538-7445.AM2013-2353

Abstract 2187: RNAi- and U1 small nuclear interference (U1i)-mediated gene knockdown reveals the functional relevance of Pim-1 kinase in colon carcinoma and glioblastoma.

Abstract Pim-1 is a constitutively active serine/threonine kinase overexpressed in various tumors. Its role as proto-oncogene is based on several Pim-1 target proteins involved in pivotal cellular processes, and Pim-1 overexpression has been linked to poor prognosis. RNAi-based knockdown approaches were used to inhibit Pim-1 in colon carcinoma cells. We demonstrate anti-proliferative, pro-apoptotic and overall antitumor effects of Pim-1 inhibition. The analysis of the molecular effects of Pim-1 inhibition reveals a complex regulatory network, with therapeutic Pim-1 repression leading to major changes in oncogenic signal transduction with regard to p21Cip1/WAF1, STAT3, JNK, c-Myc and survivin, and in the levels of apoptosis-related proteins Puma, Bax and Bcl-xL. Furthermore, Pim-1 knockdown sensitizes tumor cells towards 5-FU treatment, thereby antagonizing a 5-FU-triggered Pim-1 upregulation. This effect is mediated through decreased miR-15b levels, and our studies identify miR-15b and miR-33a to regulate Pim-1. There is first evidence of Pim-1 overexpression also in glioblastoma, but the functional relevance is so far unknown. U1 small nuclear interference (U1i) has recently been described as novel gene silencing mechanism. It employs short oligonucleotides, so-called U1 adaptors, for specific gene knockdown. We generated Pim-1 specific U1 adaptors and demonstrate their ability to induce Pim-1 knockdown in GBM cells. U1 adaptor transfection leads to anti-proliferative and pro-apoptotic effects. To explore therapeutic in vivo applications, we complexed siRNAs or U1 adaptors with a low molecular weight polyethylenimine (PEI) that mediates protection and cellular internalization. Treatment of tumor xenograft-bearing mice with PEI/siRNA or PEI/adaptor nanoparticles exhibits antitumor effects based on the knockdown of Pim-1, thus offering novel therapeutic strategies. Conclusions: We demonstrate that Pim-1 plays a pivotal role in several tumor-relevant signalling pathways, and establish the functional relevance of Pim-1 in colon carcinoma and in glioblastoma. Our results also substantiate the RNAi-mediated Pim-1 knockdown based on polymeric PEI/siRNA nanoparticles as a promising therapeutic approach, and we show that U1i represents an alternative to RNAi for the therapeutic knockdown of pathologically upregulated genes. Citation Format: Ulrike Weirauch, Maren Thomas, Arnold Grünweller, Roland K. Hartmann, Achim M. Aigner. RNAi- and U1 small nuclear interference (U1i)-mediated gene knockdown reveals the functional relevance of Pim-1 kinase in colon carcinoma and glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2187. doi:10.1158/1538-7445.AM2013-2187

Abstract 3245: Identification of potent, dual PIM/FLT3 kinase inhibitors for AML treatment.

Abstract Acute myeloid leukemia (AML) is a highly heterogeneous disease with numerous signaling pathways that contribute to its pathogenesis. Advances in our understanding of molecular mechanisms of AML pathogenesis and prognosis so far did not translate into significant clinical improvements. AML is still the highest unmet medical need within hematological malignancies area, particularly in case of the elderly patients. FLT3 inhibitors were investigated in the recent years as possible therapeutic agents, however to date the clinical trials of FLT3 inhibitors have yielded disappointing results. On the other hand, PIM kinases have been identified in the last years as critical downstream components of FLT3 signaling, especially in the case of oncogenic FLT3 mutants. PIM kinases are being overexpressed in a range of hematopoietic malignancies and solid cancers and the overexpression of PIMs is associated with a poor prognosis and decreased survival of patients suffering from cancer. In many cases also, PIM overexpression was associated with development of drug resistance. As dual and simultaneous inhibition of various pathway components is an emerging therapeutic idea, exemplified by several compounds in development for cancer treatment, we have developed a series of dual PIM/FLT3 small molecule inhibitors to investigate this concept. Similarly to examples of small molecule inhibitors from the JAK/STAT or PI3K/AKT pathways, where compounds are being developed as a way to improve efficacy, resistance development and overcoming the negative feedback loops, often seen after single target inhibition, we have observed increased potency of compounds developed in this series of dual PIM/FLT3 inhibitors. Synthesized inhibitors showed higher activity towards mutated FLT3 (FLT3-ITD and other FLT3 mutants) than wild type kinase and the selectivity profile on a panel of 450 kinases was comparable to best clinical examples of kinase inhibitors. In contrast to selective PIM inhibitors, the treatment with dual PIM/FLT3 inhibitors showed potent apoptosis induction as a results of Erk and S6 phosphorylation inhibition. The in vitro activity in FTL3-ITD positive cells was also confirmed in vivo in a PK/PD xenograft experiment, where sustained biomarker inhibition was observed already after single compounds administration. Oral administration of dual PIM/FLT3 inhibitors led to potent effect in vivo and in certain cases also to remissions in subcutaneous xenograft models. Observed activity profile and synergistic effects observed with other targeted therapies and standard of care compounds, makes dual PIM/FLT3 inhibitors an exciting approach for treatment of FLT3-mutant positive AML patients with high chances of clinical success. Citation Format: Wojciech Czardybon, Michal Galezowski, Renata Windak, Magdalena Salwińska, Izabela Dolata, Ewa Trebacz, Radosław Obuchowicz, Pawel Guzik, Magdalena Zawadzka, Ewelina Wincza, Katarzyna Wiklik, Mariusz Milik, Malgorzata Zurawska, Karolina Krawczynska, Krzysztof Brzózka. Identification of potent, dual PIM/FLT3 kinase inhibitors for AML treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3245. doi:10.1158/1538-7445.AM2013-3245

Conditional transgenic expression of PIM1 kinase in prostate induces inflammation-dependent neoplasia.

The Pim proteins are a family of highly homologous protein serine/threonine kinases that have been found to be overexpressed in cancer. Elevated levels of Pim1 kinase were first discovered in human leukemia and lymphomas. However, more recently Pim1 was found to be increased in solid tumors, including pancreatic and prostate cancers, and has been proposed as a prognostic marker. Although the Pim kinases have been identified as oncogenes in transgenic models, they have weak transforming abilities on their own. However, they have been shown to greatly enhance the ability of other genes or chemical carcinogens to induce tumors. To explore the role of Pim1 in prostate cancer, we generated conditional Pim1 transgenic mice, expressed Pim1 in prostate epithelium, and analyzed the contribution of PIM1 to neoplastic initiation and progression. Accordingly, we explored the effect of PIM1 overexpression in 3 different settings: upon hormone treatment, during aging, and in combination with the absence of one Pten allele. We have found that Pim1 overexpression increased the severity of mouse prostate intraepithelial neoplasias (mPIN) moderately in all three settings. Furthermore, Pim1 overexpression, in combination with the hormone treatment, increased inflammation surrounding target tissues leading to pyelonephritis in transgenic animals. Analysis of senescence induced in these prostatic lesions showed that the lesions induced in the presence of inflammation exhibited different behavior than those induced in the absence of inflammation. While high grade prostate preneoplastic lesions, mPIN grades III and IV, in the presence of inflammation did not show any senescence markers and demonstrated high levels of Ki67 staining, untreated animals without inflammation showed senescence markers and had low levels of Ki67 staining in similar high grade lesions. Our data suggest that Pim1 might contribute to progression rather than initiation in prostate neoplasia.

The Pim kinase inhibitor SGI-1776 decreases cell surface expression of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and drug transport by Pim-1-dependent and -independent mechanisms

Overexpression of the ATP-binding cassette (ABC) drug efflux proteins P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) on malignant cells is associated with inferior chemotherapy outcomes. Both, ABCB1 and ABCG2, are substrates of the serine/threonine kinase Pim-1; Pim-1 knockdown decreases their cell surface expression, but SGI-1776, the first clinically tested Pim inhibitor, was shown to reverse drug resistance by directly inhibiting ABCB1-mediated transport. We sought to characterize Pim-1-dependent and -independent effects of SGI-1776 on drug resistance. SGI-1776 at the Pim-1-inhibitory and non-cytotoxic concentration of 1μM decreased the IC50s of the ABCG2 and ABCB1 substrate drugs in cytotoxicity assays in resistant cells, with no effect on the IC50 of non-substrate drug, nor in parental cells. SGI-1776 also increased apoptosis of cells overexpressing ABCG2 or ABCB1 exposed to substrate chemotherapy drugs and decreased their colony formation in the presence of substrate, but not non-substrate, drugs, with no effect on parental cells. SGI-1776 decreased ABCB1 and ABCG2 surface expression on K562/ABCB1 and K562/ABCG2 cells, respectively, with Pim-1 overexpression, but not HL60/VCR and 8226/MR20 cells, with lower-level Pim-1 expression. Finally, SGI-1776 inhibited uptake of ABCG2 and ABCB1 substrates in a concentration-dependent manner irrespective of Pim-1 expression, inhibited ABCB1 and ABCG2 photoaffinity labeling with the transport substrate [125I]iodoarylazidoprazosin ([125I]IAAP) and stimulated ABCB1 and ABCG2 ATPase activity. Thus SGI-1776 decreases cell surface expression of ABCB1 and ABCG2 and inhibits drug transport by Pim-1-dependent and -independent mechanisms, respectively. Decrease in ABCB1 and ABCG2 cell surface expression mediated by Pim-1 inhibition represents a novel mechanism of chemosensitization.

Investigational agent MLN9708/2238 targets tumor-suppressor miR33b in MM cells

AbstractmiRs play a critical role in tumor pathogenesis as either oncogenes or tumor-suppressor genes. However, the role of miRs and their regulation in response to proteasome inhibitors in multiple myeloma (MM) is unclear. In the current study, miR profiling in proteasome inhibitor MLN2238-treated MM.1S MM cells shows up-regulation of miR33b. Mechanistic studies indicate that the induction of miR33b is predominantly via transcriptional regulation. Examination of miR33b in patient MM cells showed a constitutively low expression. Overexpression of miR33b decreased MM cell viability, migration, colony formation, and increased apoptosis and sensitivity of MM cells to MLN2238 treatment. In addition, overexpression of miR33b or MLN2238 exposure negatively regulated oncogene PIM-1 and blocked PIM-1 wild-type, but not PIM-1 mutant, luciferase activity. Moreover, PIM-1 overexpression led to significant abrogation of miR33b- or MLN2238-induced cell death. SGI-1776, a biochemical inhibitor of PIM-1, triggered apoptosis in MM. Finally, overexpression of miR33b inhibited tumor growth and prolonged survival in both subcutaneous and disseminated human MM xenograft models. Our results show that miR33b is a tumor suppressor that plays a role during MLN2238-induced apoptotic signaling in MM cells, and these data provide the basis for novel therapeutic strategies targeting miR33b in MM.

Overexpression of Osteopontin, αvβ3 and Pim-1 Associated with Prognostically Important Clinicopathologic Variables in Non-Small Cell Lung Cancer

In this study, we examined the expression of osteopontin (OPN), αvβ3 and Pim-1 in non-small cell lung cancer (NSCLC) and investigated the potential clinical implications of their expression patterns in NSCLC. Immunohistochemical assays were used to examine the protein expression of OPN, αvβ3 and Pim-1 in 208 NSCLC samples and their adjacent normal lung tissue specimens. Statistical analyses were performed to evaluate the relationships between OPN, αvβ3 and Pim-1 expression patterns, and their association with the clinical-pathological parameters of NSCLC patients. In NSCLC tissues, the positive rates of OPN, αvβ3 and Pim-1 expression were 67.8% (141/208), 76.0% (158/208) and 58.7% (122/208), respectively. However, in the adjacent normal lung tissues, the positive rates of OPN, αvβ3 and Pim-1 were 20.2% (42/208), 24.0% (50/208) and 14.9% (31/208), respectively. The differences in the positive expression rates of OPN, αvβ3 and Pim-1 between NSCLCs and the adjacent normal lung tissues were all significant (P<0.01). Additionally, the positive expression of OPN, αvβ3 and Pim-1 in NSCLCs was associated with an increase in pathological grade, lymph node metastasis and advanced clinical stage (all P<0.01). Furthermore, associations between the expression of OPN and αvβ3, OPN and Pim-1, and αvβ3 and Pim-1 were also observed in our NSCLC cohort (all P<0.01). The OPN, αvβ3 and Pim-1 proteins are frequently overexpressed in NSCLC and are associated with some clinicopathologic variables that are of known prognostic importance in NSCLC, suggesting that they may play an important role in the development and/or progression of NSCLC.

Increased Mitotic Rate Coincident with Transient Telomere Lengthening Resulting from Pim‐1 Overexpression in Cardiac Progenitor Cells

Cardiac regeneration following myocardial infarction rests with the potential of c-kit+ cardiac progenitor cells (CPCs) to repopulate damaged myocardium. The ability of CPCs to reconstitute the heart is restricted by patient age and disease progression. Increasing CPC proliferation, telomere length, and survival will improve the ability of autologous CPCs to be successful in myocardial regeneration. Prior studies have demonstrated enhancement of myocardial regeneration by engineering CPCs to express Pim-1 kinase, but cellular and molecular mechanisms for Pim-1-mediated effects on CPCs remain obscure. We find CPCs rapidly expand following overexpression of cardioprotective kinase Pim-1 (CPCeP), however, increases in mitotic rate are short-lived as late passage CPCePs proliferate similar to control CPCs. Telomere elongation consistent with a young phenotype is observed following Pim-1 modification of CPCeP; in addition, telomere elongation coincides with increased telomerase expression and activity. Interestingly, telomere length and telomerase activity normalize after several rounds of passaging, consistent with the ability of Pim-1 to transiently increase mitosis without resultant oncogenic transformation. Accelerating mitosis in CPCeP without immortalization represents a novel strategy to expand the CPC population in order to improve their therapeutic efficacy. Stem Cells2012;30:2512–2522

Phosphorylation of the androgen receptor by PIM1 in hormone refractory prostate cancer

Integration of cellular signaling pathways with androgen receptor (AR) signaling can be achieved through phosphorylation of AR by cellular kinases. However, the kinases responsible for phosphorylating the androgen receptor at numerous sites and the functional consequences of AR phosphorylation are only partially understood. Bioinformatic analysis revealed AR serine 213 (S213) as a putative substrate for PIM1, a kinase overexpressed in prostate cancer. Therefore, phosphorylation of AR serine 213 by PIM1 was examined using a phosphorylation site-specific antibody. Wild type PIM1, but not catalytically inactive PIM1, specifically phosphorylated AR but not an AR serine to alanine mutant (S213A). In vitro kinase assays confirmed that PIM1 can phosphorylate AR S213 in a ligand independent manner and cell type specific phosphorylation was observed in prostate cancer cell lines. Upon PIM1 overexpression AR phosphorylation was observed in the absence of hormone and was further increased in the presence of hormone in LNCaP, LNCaP-abl, and VCaP cells. Moreover, phosphorylation of AR was reduced in the presence of PIM kinase inhibitors. An examination of AR mediated transcription showed that reporter gene activity was reduced in the presence of PIM1 and wild type AR, but not S213A mutant AR. Androgen mediated transcription of endogenous PSA, Nkx3.1, and IGFBP5 was also decreased in the presence of PIM1 whereas IL6, cyclin A1, and caveolin 2 were increased. Immunohistochemical analysis of prostate cancer tissue microarrays showed significant P-AR S213 expression that was associated with hormone refractory prostate cancers, likely identifying cells with catalytically active PIM1. In addition, prostate cancers expressing a high level of P-AR S213 were twice as likely to be from biochemically recurrent cancers. Thus, AR phosphorylation by PIM1 at S213 impacts gene transcription and is highly prevalent in aggressive prostate cancer.

Abstract 160: Pim-1 Preserves Cardiac Stem Cell Proliferation with Age

Rationale Cardiac stem cells (CSC) regulate cardiomyogenesis and support regenerative processes in the heart, but aging adversely affects stem cell repair capacity. Aging is a primary cause of impaired cardiac function characterized by accumulation of senescent cells. CSC senescence is associated with permanent growth arrest that decreases survival signaling and cellular replacement, inevitably diminishing the capacity of the heart to maintain tissue homeostasis. Therefore, promoting CSC growth may improve cardiac performance with age. Pim-1 kinase exhibits protective and proliferative effects in the myocardium but the role of Pim-1 in cardiac aging has not been thoroughly studied. Objective Demonstrate that Pim-1 promotes stem cell growth in the aged myocardium correlating with increased expression of centromere protein A (CENP-A), a kinetochore-associated protein known to support cell proliferation in numerous species and cell types. Methods &amp; Results CENP-A expression levels were evaluated from murine myocardial tissue samples ranging in age from 11 days post coitum to 4 months of age with analysis by immunoblot as well as quantitative PCR. CENP-A expression was colocalized with c-kit as a marker of CSC by immunohistochemical labeling, revealing a decline in CENP-A expression over the time course of postnatal myocardial maturation. The impact of Pim-1 upon CENP-A level was assessed by comparative analysis of non-transgenic mice versus genetically modified transgenic mouse lines expressing either Pim-1 (wild type) or a dominant negative functionally dead Pim-1 mutant. Pim-1 overexpression increases persistence of CENP-A in CSCs with age, as well as the prevalence of cycling CSCs as marked by phosph-H3 expression, while the functionally dead mutant accelerates CENP-A diminution and decreases CSC proliferation. Conclusion CENP-A decline in c-kit positive cells with age provides intriguing evidence of a potential mechanism for the diminished capacity of CSCs to maintain tissue homeostasis. Pim-1 mitigates CENP-A diminution, demonstrating the promising potential of Pim-1 to promote cardiac growth and repair with age.

Inhibition of Pim–1 attenuates the proliferation and migration in nasopharyngeal carcinoma cells

ObjectiveTo explore the role of proto-oncogene Pim-1 in the proliferation and migration of nasopharyngeal carcinoma (NPC) cells. MethodsPim-1 expressions in NPC cell lines CNE1, CNE1-GL, CNE-2Z and C666-1 were examined by RT-PCR, western blotting and immunoflucesence, respectively. After CNE1, CNE1-GL and C666-1 cells were treated with different concentrations of Pim-1 special inhibitor, quercetagetin, the cell viability, colony formation rate and migration ability were analyzed. ResultsPim-1 expression was negative in well-differentiated CNE1 cells, whereas expressed weakly positive in poor-differentiated CNE-2Z cells and strongly positive in undifferentiated C666-1 cells. Interestingly, CNE1-GL cells that derived from CNE1 transfected with an Epstein Barr virus latent membrane protein-1 over-expression plasmid displayed stronger expression of Pim-1. Treatment of CNE1-GL and C666-1 cells with quercetagetin significantly decreased the cell viability, colony formation rate and migration ability but not the CNE1 cells. ConclusionsThese findings suggest that Pim-1 overexpression contributes to NPC proliferation and migration, and targeting Pim-1 may be a potential treatment for anti-Pim-1-expressed NPCs.

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 2796: Efficacy and biomarker modulation by AZD1208, a novel, potent and selective pan-Pim kinase inhibitor, in models of acute myeloid leukemia

Abstract The Pim serine/threonine kinase family is composed of three highly homologous members; Pim-1, Pim-2 and Pim-3, identified by the ability of the prototype member Pim-1 to drive lymphomagenesis in mice. Upregulation of Pim-1 and Pim-2 is observed in leukemias and lymphomas, including AML, NHL and CLL, highlighting the potential of these kinases as therapeutic targets in these indications. Overexpression of Pim-1 or Pim-3 has also been observed in prostate, pancreatic, gastric, bladder and hepatocellular cancers. Pim kinases are downstream effectors of many cytokine and growth factor signaling pathways and are direct transcriptional targets of STAT transcription factors activated by these pathways. Pims can phosphorylate multiple substrates to mediate cell proliferation and survival. Here we describe the activity of AZD1208, an orally available, potent and highly selective Pim inhibitor that effectively inhibits all three isoforms. AZD1208 inhibits the growth of several AML cell lines and sensitivity correlates with the level of Pim-1 expression, STAT5 activation and presence of protein tyrosine kinase mutation. AZD1208 causes cell cycle arrest and apoptosis in MOLM-16 cells in culture. This is accompanied by a dose-dependent reduction in phosphorylation of BAD, 4EBP1 and p70S6K. AZD1208 suppresses the growth of MOLM-16 and KG-1a xenograft tumors in vivo in a dose proportional manner. In addition, AZD1208 leads to potent inhibition of colony growth of primary AML cells from bone marrow aspirates and downregulates phosphorylation of Pim targets. These results underscore the therapeutic potential of Pim kinase inhibition by AZD1208 for the treatment of AML. They also further support investigation of this inhibitor in other hematological and solid tumor malignancies where PIM signaling may play a role in tumorigenesis and survival. 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 2796. doi:1538-7445.AM2012-2796

Abstract 3762: Second generation PIM inhibitors exhibit improved activity in solid tumor models

Abstract The proto-oncogene PIM kinase family (PIM-1, -2 and -3) includes constitutively active serine/threonine kinases upregulated in multiple cancer indications, including lymphomas, leukemias, multiple myeloma, prostate and bladder cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The PIM kinases function by inhibiting apoptosis in MYC-driven tumors, and promoting tumor cell survival and proliferation. In the HEK-293T cell line, enhanced PIM kinase substrate BAD phosphorylation (pBAD) was observed following PIM and BAD overexpression. Enhancement of pBAD was inhibited by SGI-1776, a well-described PIM inhibitor, and more effectively by second generation PIM inhibitors exhibiting 4-10 fold improved potency against the PIM kinase family. The current PIM inhibitors display sub-µM activity in pharmacodynamic marker, proliferation and 2D colony formation assays using the UM-UC-3 human bladder cancer cell line. PIM1 and PIM2 overexpression models were established in the human prostate cancer cell line 22RV-1 and the non-tumorigenic mouse NIH-3T3 cell background. Overexpression of PIM kinases led to enhanced cell growth and tumorigenicity in both NIH-3T3 and 22RV-1 cell lines. In vivo xenograft studies using both PIM overexpression models and a clinically relevant solid tumor model facilitated identification of a lead candidate with demonstrated efficacy and favorable toxicity. IND-enabling studies with a lead candidate are underway. 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 3762. doi:1538-7445.AM2012-3762

PIM1‐activated PRAS40 Regulates Radioresistance in Non‐small Cell Lung Cancer Cells through Interplay with FOXO3a, 14‐3‐3, and Protein Phosphatases

Resistance of cancer cells to ionizing radiation (IR) plays an important role in the clinical setting of lung cancer treatment. To date, however, the exact molecular mechanism of radiosensitivity has not been well explained. In this study, we compared radioresistance in two types of non‐small cell lung cancer (NSCLC) cells, NCI‐H460 and A549, and investigated the signaling pathways that confer radioresistance. In radioresistant cells, IR led to overexpression of PIM1 and reduction of protein phosphatases (PP2A and PP5), which induced translocation of PIM1 into nucleus. Increased nuclear PIM1 phosphorylated PRAS40. Consequently, pPRAS40 made a trimeric complex with 14‐3‐3 and AKT‐activated pFOXO3a, which then moved rapidly to cytoplasm. Cytoplasmic retention of FOXO3a was associated with down‐regulation of pro‐apoptotic genes and possibly radioresistance. On the other hand, a suppressive effect of IR on protein phosphatases was not detected and, concomitantly, protein phosphatases down‐regulated PIM1 in radiosensitive cells. In this setting, PIM1‐activated pPRAS40, AKT‐activated pFOXO3a, and their complex formation with 14‐3‐3 could be key regulators of the IR‐induced radioresistance in NSCLC cells.

Asymmetric Chromatid Segregation in Cardiac Progenitor Cells Is Enhanced by Pim-1 Kinase

Cardiac progenitor cells (CPCs) in the adult heart are used for cell-based treatment of myocardial damage, but factors determining stemness, self-renewal, and lineage commitment are poorly understood. Immortal DNA strands inherited through asymmetric chromatid segregation correlate with self-renewal of adult stem cells, but the capacity of CPCs for asymmetric segregation to retain immortal strands is unknown. Cardioprotective kinase Pim-1 increases asymmetric cell division in vivo, but the ability of Pim-1 to enhance asymmetric chromatid segregation is unknown. We aimed to demonstrate immortal strand segregation in CPCs and the enhancement of asymmetric chromatid distribution by Pim-1 kinase. Asymmetric segregation is tracked by incorporation of bromodeoxyuridine. The CPC DNA was labeled for several generations and then blocked in second cytokinesis during chase to determine distribution of immortal versus newly synthesized strands. Intensity ratios of binucleated CPCs with bromodeoxyuridine of ≥70:30 between daughter nuclei indicative of asymmetric chromatid segregation occur with a frequency of 4.57, and asymmetric chromatid segregation is demonstrated at late mitotic phases. Asymmetric chromatid segregation is significantly enhanced by Pim-1 overexpression in CPCs (9.19 versus 4.79 in eGFP-expressing cells; P=0.006). Asymmetric segregation of chromatids in CPCs is increased nearly two-fold with Pim-1 kinase overexpression, indicating that Pim-1 promotes self-renewal of stem cells.

Pim1 and Myc reversibly transform murine precursor B lymphocytes but not mature B lymphocytes

The proto-oncogenes Myc and Pim1, which are deregulated in many types of cancers, are known to cooperate in B lymphoma development. Here we show that overexpression of retrovirally transduced, doxycycline-inducible Myc alone in IL-7-deprived, growth-arrested pre-B cells enhanced cell cycle entry without impairing apoptosis. Overexpression of Pim1 decreased apoptosis, but had no effect on cell cycle entry. Co-expression of Pim1 and Myc inhibited apoptosis and led to IL-7-independent proliferation of the transduced pre-B cells in vitro, while blocking their differentiation to IgM(+) immature cells. Transplantation of Pim1/Myc overexpressing pre-BI cells into B-cell-deficient mice expanded the pre-B-cell compartments up to 100-fold within 4-8 weeks. Transformation remained dependent on the expression of both oncogenes, as removal of doxycycline in vitro and in vivo terminated proliferation and induced differentiation to IgM(+) B cells. In contrast, Pim1/Myc-transduced mature B cells that developed from the oncogene-transduced pre-BI cells in the absence of oncogene overexpression in vivo were not capable of long-term proliferation after induction of Pim and Myc overexpression, neither in vivo nor in vitro, neither with nor without stimulation by polyclonal activators.