Serine-threonine Kinase Pim-1 Research Articles

Pim kinase inhibition sensitizes FLT3-ITD acute myeloid leukemia cells to topoisomerase 2 inhibitors through increased DNA damage and oxidative stress.

Internal tandem duplication of fms-like tyrosine kinase-3 (FLT3-ITD) is frequent (30 percent) in acute myeloid leukemia (AML), and is associated with short disease-free survival following chemotherapy. The serine threonine kinase Pim-1 is a pro-survival oncogene transcriptionally upregulated by FLT3-ITD that also promotes its signaling in a positive feedback loop. Thus inhibiting Pim-1 represents an attractive approach in targeting FLT3-ITD cells. Indeed, co-treatment with the pan-Pim kinase inhibitor AZD1208 or expression of a kinase-dead Pim-1 mutant sensitized FLT3-ITD cell lines to apoptosis triggered by chemotherapy drugs including the topoisomerase 2 inhibitors daunorubicin, etoposide and mitoxantrone, but not the nucleoside analog cytarabine. AZD1208 sensitized primary AML cells with FLT3-ITD to topoisomerase 2 inhibitors, but did not sensitize AML cells with wild-type FLT3 or remission bone marrow cells, supporting a favorable therapeutic index. Mechanistically, the enhanced apoptosis observed with AZD1208 and topoisomerase 2 inhibitor combination treatment was associated with increased DNA double-strand breaks and increased levels of reactive oxygen species (ROS), and co-treatment with the ROS scavenger N-acetyl cysteine rescued FLT3-ITD cells from AZD1208 sensitization to topoisomerase 2 inhibitors. Our data support testing of Pim kinase inhibitors with topoisomerase 2 inhibitors, but not with cytarabine, to improve treatment outcomes in AML with FLT3-ITD.

Developmental stage-specific effects of Pim-1 dysregulation on murine bone marrow B cell development.

BackgroundThe serine threonine kinase Pim-1 has documented roles in hematopoietic progenitor and B cell precursor proliferation and survival. Pim-1 is a molecular target of the transcription factor Hoxa9. Previous studies showed that Pim-1 deficiency phenocopied the hematopoietic progenitor defect in hoxa9-/- mice and forced expression of Pim-1 normalized the in vitro proliferation defect inherent to hoxa9-/- hematopoietic progenitors. Pim-1 is induced by cytokine signaling, including the early lymphoid/B lineage regulators Flt3 and IL-7, and expression levels were shown to influence the size of the B cell compartment in bone marrow (BM).ResultsIn this study, we sought to determine if transgenic expression of Pim-1, driven by the immunoglobulin enhancer, Eμ, was sufficient to rescue the lymphoid/B cell precursor defect in hoxa9 or flt3-ligand (flt3l) deficient mice. Unexpectedly, expression of Eμ − Pim1 exacerbated lymphoid progenitor deficiencies in flt3l-/-, and to a lesser extent, hoxa9-/- mice. Furthermore, Eμ − Pim1 expression alone reduced early myeloid and lymphoid, but not erythroid, progenitors. In contrast, Pim-1 deficiency had no significant effect on early lymphoid/B cell development through the Pre-Pro-B cell stage, but caused a significant reduction in IgM− B cell precursors. Importantly, loss of Pim-1 did not phenocopy hoxa9- or flt3l-deficiency on the lymphoid/early B cell progenitor pools.ConclusionsThese experimental findings demonstrate that Pim-1 overexpression has developmental-stage-specific effects on B lymphopoiesis and myelopoiesis. Importantly, these suggest that Pim-1 deficiency does not contribute significantly to the early lymphoid/B cell developmental deficiency in hoxa9-/- or flt3l-/- mice.

The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through posttranscriptional regulation of the proto-oncogene PIM1 in pancreatic cancer cells.

Previously, it has been shown that pancreatic ductal adenocarcinoma (PDA) tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO2) and decreased O2 intracellular perfusion. Chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in an understudied phenomenon known as hypoxia-induced chemoresistance. The hypoxia-inducible, pro-oncogenic, serine-threonine kinase PIM1 (Proviral Integration site for Moloney murine leukemia virus 1) has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. Although its role in therapeutic resistance has been described previously, the molecular mechanism behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present within a 38-base pair region of the PIM1 mRNA 3'-untranslated region mediate a regulatory interaction with the mRNA stability factor HuR (Hu antigen R) in the context of tumor hypoxia. Predominantly expressed in the nucleus in PDA cells, HuR translocates to the cytoplasm in response to hypoxic stress and stabilizes the PIM1 mRNA transcript, resulting in PIM1 protein overexpression. A reverse-phase protein array revealed that HuR-mediated regulation of PIM1 protects cells from hypoxic stress through phosphorylation and inactivation of the apoptotic effector BAD and activation of MEK1/2. Importantly, pharmacological inhibition of HuR by MS-444 inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and markedly enhances PDA cell sensitivity to oxaliplatin and 5-fluorouracil under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has prosurvival properties in PDA cells by enabling them with growth advantages in stressful tumor microenvironment niches. Accordingly, these studies provide evidence that therapeutic disruption of HuR's regulation of PIM1 may be a key strategy in breaking an elusive chemotherapeutic resistance mechanism acquired by PDA cells that reside in hypoxic PDA microenvironments.

Abstract A71: Post-transcriptional regulation of the proto-oncogene PIM1 by the mRNA stability factor HuR: implications for pancreatic cancer therapeutic response and cell survival

Abstract Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy characterized by its insidious development and resistance to conventional and targeted therapies. As a result of selective pressures imposed by cytotoxic agents and the tumor microenvironment, PDA cells orchestrate a potent and elusive chemoresistant mechanism. Recently, the serine-threonine kinase PIM1 has emerged as a key regulator of PDA cell survival under cancer-associated stress (e.g: cytotoxic DNA-damaging agents, hypoxia). However, the molecular mechanism behind PIM1 overexpression in PDAs is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present in the PIM1 mRNA 3’UTR mediate a regulatory interaction with the mRNA-stability factor HuR under clinically relevant hypoxic and chemotherapeutic stress. Given the previously described role of HuR in PDA cell survival and therapeutic response, we sought to determine if HuR-mediated regulation of PIM1 is essential to promote cell survival and therapeutic resistance under tumor-associated stress (e.g.: hypoxia, chemotherapy). Accordingly, PDA cells subjected to hypoxia exhibit dramatic elevated expression of PIM1 mRNA and protein. Moreover, siRNA-mediated knockdown of HuR abrogates hypoxia-mediated induction of PIM1. HuR-mediated stabilization of PIM1 is associated with high HuR cytoplasmic expression. As revealed by immunofluorescence and western blot analyses of fractionated lysates, we demonstrate that HuR translocates to the cytoplasm when PDA cells are exposed to a hypoxic microenvironment. To further ascertain HuR’s regulation of PIM1, PDA cells treated with MS-444 (Novartis), a small molecule known to disrupt the HuR mRNA-stabilizing activity by inhibiting its nuclear export, results in attenuated PIM1 expression. From a translational standpoint, we demonstrate that disruption of the HuR:PIM1 axis by HuR knockdown results in PDA cell growth inhibition and restores sensitivity to cytotoxic chemotherapeutic agents (e.g.: oxaliplatin). Similarly, pharmacological inhibition of HuR by MS-444 enhances PDA cell sensitivity to oxaliplatin and 5-Fluorouracil, key components of the promising combinatory therapy FOLFIRINOX. Taken together, these results provide continuing evidence that HuR has pro-oncogenic properties in the context of pancreatic cancer by enabling cells with selective growth advantages in stressful tumor microenvironment niches. Lastly, our studies implicate HuR’s regulation of PIM1 as a novel and targetable hallmark of PDA progression. Citation Format: Fernando F. Blanco, Nicole Meisner-Kober, Eric Londin, Isidore Rigoutsos, Jordan Winter, Charles Yeo, Jonathan Brody. Post-transcriptional regulation of the proto-oncogene PIM1 by the mRNA stability factor HuR: implications for pancreatic cancer therapeutic response and cell survival. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A71.

Abstract A05: The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through post-transcriptional regulation of the serine-threonine kinase PIM1

Abstract Pancreatic ductal adenocarcinoma (PDA) is expected to become the 2nd leading cause of cancer-related mortality in the United States by 2020. One of the reasons PDA remains one of the most lethal malignancies is due to drug resistance mechanisms to both conventional and targeted chemotherapeutic regimens. Previously, it has been shown that PDA tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO2) and decreased O2 intracellular perfusion. Importantly, chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in a widely understudied phenomenon known as hypoxia-induced chemoresistance. Recently, the hypoxia-inducible, serine-threonine, pro-oncogenic kinase PIM1 has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. While its role in therapeutic resistance has been previously described, the molecular mechanism(s) behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present in the PIM1 mRNA 3′UTR mediate a regulatory interaction with the mRNA-stability factor HuR in the context of tumor hypoxia. We have previously demonstrated that HuR, a predominantly nuclear protein, translocates to the cytoplasm upon acute cancer-associated stress and selectively stabilizes key survival-associated transcripts. Here, we demonstrate by immunofluorescence and western blot analyses of fractionated lysates that PDA cells in hypoxic conditions mobilize HuR to the cytoplasm, where it binds and stabilizes the PIM1 transcript. The functional consequence of this interaction is observed with a 5-fold increase in PIM1 protein expression (p≤0.001). PIM1 imparts its oncogenic role by phosphorylating and inactivating key apoptotic proteins (e.g., BAD) and proteins involved in the DNA repair pathway. In this regard, siRNA-mediated knockdown of HuR abrogates hypoxia-induced PIM1 expression and its downstream phosphorylation of the pro-apoptotic protein BAD. As a result, cells undergo growth arrest and succumb to apoptosis. Most importantly, we demonstrate that HuR's regulation of PIM1 modulates a potent chemoresistance phenotype. HuR-mediated stabilization of PIM1 promoted dramatic resistance to 5-Fluorouracil (5-FU) and Oxaliplatin, two critical components of the promising combination therapy FOLFIRINOX. We employed confocal microscopy to analyze DNA damage foci formation by tracking and quantifying γH2AX foci in response to 5-FU and oxaliplatin. As a result of PIM1 up-regulation in hypoxia, or forced PIM1 overexpression in normoxia, PDA cells exhibit fewer DNA damage foci (p≤0.01) due to PIM1's ability to regulate DNA repair. From a translational standpoint, we demonstrate that disruption of the HuR:PIM1 axis by HuR knockdown results in PDA cell growth inhibition and enhances sensitivity to cytotoxic chemotherapeutic agents (5-FU and oxaliplatin). Similarly, pharmacological inhibition of HuR by MS-444 (Novartis), a small molecule that inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and dramatically enhances PDA cell sensitivity to oxaliplatin and 5-FU under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has pro-oncogenic properties in PDA cells by enabling them with selective growth advantages in stressful tumor microenvironment niches. Accordingly, our studies provide evidence that therapeutic disruption of HuR's regulation of PIM1 may be a key strategy in breaking an elusive chemotherapeutic resistance mechanism acquired by PDA cells that reside in a hypoxic tumor microenvironment. Citation Format: Fernando F. Blanco, Nicole C. Meisner-Kober, Eric Londin, Isidore Rigoutsos, Makarand V. Risbud, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through post-transcriptional regulation of the serine-threonine kinase PIM1. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A05.

A Role for Polyploidy in the Tumorigenicity of Pim-1-Expressing Human Prostate and Mammary Epithelial Cells

BackgroundPolyploidy is a prominent feature of many human cancers, and it has long been hypothesized that polyploidy may contribute to tumorigenesis by promoting genomic instability. In this study, we investigated whether polyploidy per se induced by a relevant oncogene can promote genomic instability and tumorigenicity in human epithelial cells.Principal FindingsWhen the oncogenic serine-threonine kinase Pim-1 is overexpressed in immortalized, non-tumorigenic human prostate and mammary epithelial cells, these cells gradually converted to polyploidy and became tumorigenic. To assess the contribution of polyploidy to tumorigenicity, we obtained sorted, matched populations of diploid and polyploid cells expressing equivalent levels of the Pim-1 protein. Spectral karyotyping revealed evidence of emerging numerical and structural chromosomal abnormalities in polyploid cells, supporting the proposition that polyploidy promotes chromosomal instability. Polyploid cells displayed an intact p53/p21 pathway, indicating that the viability of polyploid cells in this system is not dependent on the inactivation of the p53 signaling pathway. Remarkably, only the sorted polyploid cells were tumorigenic in vitro and in vivo.ConclusionsOur results support the notion that polyploidy can promote chromosomal instability and the initiation of tumorigenesis in human epithelial cells.

Analysis of the Ribosomal Protein S19 Interactome

Ribosomal protein S19 (RPS19) is a 16-kDa protein found mainly as a component of the ribosomal 40 S subunit. Its mutations are responsible for Diamond Blackfan anemia, a congenital disease characterized by defective erythroid progenitor maturation. Dysregulation of RPS19 has therefore been implicated in this defective erythropoiesis, although the link between them is still unclear. Two not mutually exclusive hypotheses have been proposed: altered protein synthesis and loss of unknown functions not directly connected with the structural role of RPS19 in the ribosome. A role in rRNA processing has been surmised for the yeast ortholog, whereas the extracellular RPS19 dimer has a monocyte chemotactic activity. Three proteins are known to interact with RPS19: FGF2, complement component 5 receptor 1, and a nucleolar protein called RPS19-binding protein. We have used a yeast two-hybrid approach to identify a fourth protein: the serine-threonine kinase PIM1. The present study describes our use of proteomics strategies to look for proteins interacting with RPS19 to determine its functions. Proteins were isolated by affinity purification with a GST-RPS19 recombinant protein and identified using LCMS/MS analysis coupled to bioinformatics tools. We identified 159 proteins from the following Gene Ontology categories: NTPases (ATPases and GTPases; five proteins), hydrolases/helicases (19 proteins), isomerases (two proteins), kinases (three proteins), splicing factors (five proteins), structural constituents of ribosome (29 proteins), transcription factors (11 proteins), transferases (five proteins), transporters (nine proteins), DNA/RNA-binding protein species (53 proteins), other (one dehydrogenase protein, one ligase protein, one peptidase protein, one receptor protein, and one translation elongation factor), and 13 proteins of still unknown function. Proteomics results were validated by affinity purification and Western blotting. These interactions were further confirmed by co-immunoprecipitation using a monoclonal RPS19 antibody. Many interactors are nucleolar proteins and thus are expected to take part in the RPS19 interactome; however, some proteins suggest additional functional roles for RPS19.

Transgenic mice for Cre‐inducible overexpression of the oncogenes c‐MYC and Pim‐1 in multiple tissues

The transcription factor c-MYC and the serine-threonine kinase Pim-1 have multiple roles in development and cancer, including in lymphomagenesis and prostate tumorigenesis. In some cancers, MYC and Pim-1 oncogenes are co-expressed and show marked cooperativity. To facilitate the analysis of the pathological roles of MYC and Pim-1 in specific cell types and developmental stages, we generated mice carrying Cre-inducible MYC/Pim-1 transgenes. The mice carry a constitutively expressed lacZ marker and silent MYC/Pim-1 genes. Cre-mediated recombination results in deletion of the lacZ marker and concurrent activation of the MYC/Pim-1 transgene. In addition, the Pim-1 mice harbor an alkaline phosphatase gene as a positive marker for recombination. Mouse lines for each gene were established, which show distinct patterns of expression in multiple tissues. In vivo recombination was confirmed for all lines by breeding to Cre transgenic mice. These mice provide a valuable resource for investigating the significance of MYC and Pim-1 overexpression in various tissues.

The effects of benzene and the metabolites phenol and catechol on c-Myb and Pim-1 signaling in HD3 cells

Exposure to the environmental toxicant benzene has been proposed to lead to leukemogenesis. The transcription factor c-Myb plays a role in blood cell differentiation and can be regulated by the serine–threonine kinase Pim-1. Overexpressed versions of c-Myb and Pim-1 are believed to play a key role in the development of a wide variety of leukemias and tumors. In our study, we evaluated the effects of benzene and the metabolites catechol and phenol on c-Myb signaling to investigate our hypothesis that benzene exerts its toxicity by interfering with this pathway. To evaluate this hypothesis, HD3 chicken erythroblast cells were transiently transfected with a c-Myb responsive luciferase reporter plasmid and then exposed to benzene, catechol, or phenol (0–300 μM) for 1–24 h before nonproprietary dual luciferase activities were measured. Our results demonstrated that catechol exposure caused a time- and concentration-dependent increase in c-Myb activity with significance occurring at 100 and 300 μM after 24 h of exposure, which was independent of increased Pim-1 protein, but dependent on increased c-Myb phosphorylation. Benzene and phenol exposure resulted in small but significant decreases in c-Myb activity that were not dose- and time-dependent, nor was increased Pim-1 protein involved. These results are consistent with other studies, which suggest metabolite differences in benzene-mediated toxicity. More importantly, this study supports the hypothesis that benzene may mediate its toxicity through metabolite-mediated alterations in the c-Myb signaling pathway.

Reassessing the role of C-MYB in tumorigenesis.

Hematopoietic tumors in both humans and mice frequently up-regulate expression of the c-myb gene, but it is unclear whether this is a cause or a consequence of the leukemic state. Recent results placing super-activation of the c-Myb protein at the bottom of a kinase-activated signal transduction pathway indicate that it may be a downstream effector of transformation induced by other oncogenes. The relationship between c-Myb and the serine-threonine kinase pim-1, its immediate activator, is discussed, together with the possibility that c-Myb, like pim-1, may be able to synergize with c-Myc to induce tumors.