PIM Kinase Family Research Articles

Regulation ofC. eleganspresynaptic differentiation and neurite branching via a novel signaling pathway initiated by SAM-10

Little is known about transcriptional control of neurite branching or presynaptic differentiation, events that occur relatively late in neuronal development. Using the Caenorhabditis elegans mechanosensory circuit as an in vivo model, we show that SAM-10, an ortholog of mammalian single-stranded DNA-binding protein (SSDP), functions cell-autonomously in the nucleus to regulate synaptic differentiation, as well as positioning of, a single neurite branch. PLM mechanosensory neurons in sam-10 mutants exhibit abnormal placement of the neurite branch point, and defective synaptogenesis, characterized by an overextended synaptic varicosity, underdeveloped synaptic morphology and disrupted colocalization of active zone and synaptic vesicles. SAM-10 functions coordinately with Lim domain-binding protein 1 (LDB-1), demonstrated by our observations that: (1) mutations in either gene show similar defects in PLM neurons; and (2) LDB-1 is required for SAM-10 nuclear localization. SAM-10 regulates PLM synaptic differentiation by suppressing transcription of prk-2, which encodes an ortholog of the mammalian Pim kinase family. PRK-2-mediated activities of SAM-10 are specifically involved in PLM synaptic differentiation, but not other sam-10 phenotypes such as neurite branching. Thus, these data reveal a novel transcriptional signaling pathway that regulates neuronal specification of neurite branching and presynaptic differentiation.

PIM Kinase Inhibitors Downregulate STAT3Tyr705 Phosphorylation

Using a cell-based high-throughput screen designed to detect small chemical compounds that inhibit cell growth and survival, we identified three structurally related compounds, 21A8, 21H7, and 65D4, with differential activity on cancer versus normal cells. Introduction of structural modifications yielded compound M-110, which inhibits the proliferation of prostate cancer cell lines with IC(50)s of 0.6 to 0.9 μmol/L, with no activity on normal human peripheral blood mononuclear cells up to 40 μmol/L. Screening of 261 recombinant kinases and subsequent analysis revealed that M-110 is a selective inhibitor of the PIM kinase family, with preference for PIM-3. The prostate cancer cell line DU-145 and the pancreatic cancer cell line MiaPaCa2 constitutively express activated STAT3 (pSTAT3(Tyr705)). Treatment of DU-145 cells with M-110 or with a structurally unrelated PIM inhibitor, SGI-1776, significantly reduces pSTAT3(Tyr705) expression without affecting the expression of STAT3. Furthermore, treatment of DU-145 cells with M-110 attenuates the interleukin-6-induced increase in pSTAT3(Tyr705). To determine which of the three PIM kinases is most likely to inhibit expression of pSTAT3(Tyr705), we used PIM-1-, PIM-2-, or PIM-3-specific siRNA and showed that knockdown of PIM-3, but not of PIM-1 or PIM-2, in DU-145 cells results in a significant downregulation of pSTAT3(Tyr705). The phosphorylation of STAT5 on Tyr694 in 22Rv1 cells is not affected by M-110 or SGI-1776, suggesting specificity for pSTAT3(Tyr705). These results identify a novel role for PIM-3 kinase as a positive regulator of STAT3 signaling and suggest that PIM-3 inhibitors cause growth inhibition of cancer cells by downregulating the expression of pSTAT3(Tyr705).

Abstract 3104: The role of Pim-3 kinase in NF-κB signaling and transformation of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 4th most common cause of cancer deaths in the United States. Due to the aggressive nature of this cancer and the lack of biomarkers for early detection, the incidence and mortality rates for PDAC are nearly equivalent. A better understanding of the molecular mechanisms leading to the development of pancreatic cancer remains a major goal for defining appropriate treatment strategies. Past studies have shown that the proto-oncogene Pim-3 kinase is expressed in pancreatic cancer and is able to phosphorylate the pro-apoptotic protein Bad to block Bad-mediated apoptosis in human pancreatic cancer cell lines. With these and other observations implicating Pim kinases as oncogenes and inhibitors of apoptosis, the purpose of our study is to further characterize the functional significance of Pim-3 in PDAC growth as well as identify possible downstream targets. We hypothesize that inhibition of Pim-3 signaling will be an effective approach for antagonizing the aberrant growth of pancreatic cancer. Initially, we confirmed by immunohistochemistry that Pim-3 protein is aberrantly expressed in human pancreatic cancer and pancreatitis tissue and is constitutively expressed in a panel of human pancreatic cancer cell lines. Also, we found that suppression of Pim-3 by shRNAs significantly decreased cell growth and caused a reduction in invasion of pancreatic cancer cells in Matrix-gel. To our knowledge, this is the first time that any of the Pims have been associated with invasion in pancreatic cancer. Most recently, other members of the Pim kinase family have been shown to activate NF-κB in cancer. From our studies, we observed that suppression of Pim-3 upregulated IκBα and decreased phospho-NF-κB p65 (Ser536), suggesting an effect on NF-κB transcription activity. Our results indicate the importance of Pim-3 in various aspects of transformation of pancreatic cancer and how NF-κB may be a potential downstream target of Pim-3 signaling in PDAC. These findings will allow us to critically validate Pim kinases as novel biomarkers and therapeutic targets for pancreatic cancer. 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 3104.

Abstract A242: Identification of novel and specific Pim-1 kinase inhibitors with potent anticancer activity in hematological and solid tumor malignancies

Abstract The Pim serine-threonine kinase family is composed of three members which play an important role in intracellular signaling and contribute to pathways involved in cell survival, proliferation, stress response and cellular motility. Out the three family members, Pim-1 has been studied most extensively and was shown to be a crucial downstream effector of several oncogenes like Jak2 and FLT3 kinases. Pim-1 overexpression has been also reported in a variety of cancers such as diffuse B cell lymphoma, chronic lymphocytic leukemia, Flt3-mediated acute myelogenous leukemia and solid tumors such as prostate and pancreatic cancers. For this reason, Pim-1 kinase emerged as a novel and interesting target of significant potential for therapeutic intervention. In the current study we have performed lead optimization of a new small molecule Pim-1 kinase inhibitor. Initial compound exerted moderate potency in Pim-1 kinase inhibition accompanied by high selectivity for the Pim kinase family. In parallel to biochemical characterization, the original compound had been extensively tested in vitro and in vivo for anticancer activity. In order to improve potency and selectivity of the original compound a series of new derivatives was synthesized and screened for Pim-1 inhibition. Among the newly synthesized compounds several exerted increased potency in Pim-1 inhibition with IC50 values as low as 4,8 nM. Further biochemical profiling of best inhibitors on a 440 KINOMEscanTM Max kinase panel revealed superior selectivity in binding and inhibition of the Pim-1 kinase with over 20-fold off-target selectivity. Anticancer effect of new derivatives was investigated in several cancer cell lines of hematological and solid tumor origin where the compounds was shown to induce of apoptosis. As Pim-1 kinase was shown to regulate cell cycle progression by phosphorylation of several key proteins such as p27 and cdc25A in the G1/S checkpoint and cdc25C and c-TAK-1 in the G2/M checkpoint, we investigated cell cycle effects after compounds administration. We could observe two phenotypes in cell that did not undergo apoptosis, namely a time and dose dependent arrest of the cells either in the G0/G1 or G2/M phase, depending on the cell line. To confirm that the observed cytotoxic and cell cycle effects were due to inhibition of the Pim-1 kinase by the new derivatives, a biomarker analysis was performed. Pim-1 kinase was shown to phosphorylate p27KIP1 at threonine 198 both in vitro and in the cells. As soon as 1h after treatment with the compounds, a dramatic inhibition of phospho-p27KIP1 was observed on K562 cells proving the expected mechanism of action of Pim-1 kinase inhibiting compounds in the cells. Results of the Selvita's Pim-1 inhibitor lead optimization efforts are shown and discussed, supporting further development of this class of inhibitors in oncology indications. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A242.

Abstract A246: Discovery of selective small molecule Pan-Pim kinase inhibitors with potent oral efficacy in murine xenograft models

Abstract The PIM family of serine/threonine kinases are pro-proliferative kinases activated by multiple cytokines and growth factor signaling. The Pim kinases are unusual in that they are regulated primarily by transcription and not by membrane recruitment or phosphorylation like other serine/threonine kinases. Activated cytokine receptors recruit JAKs to induce STAT-dependent transcription of the Pim genes. They are proto-oncogenes and have been implicated in the process of lymphomagenesis and malignant transformation. Pim overexpression has been reported in diffuse B cell lymphoma, chronic lymphocytic leukemia, FLT3-mediated acute myelogenous leukemia and prostate cancer. Pim-2 is over expressed in leukemias and lymphomas, whereas Pim-3 overexpression has been observed in melanoma, pancreatic and gastric tumors. The recent reports of elevated levels of Pim-1 expression in human prostate tumor biopsies implicate the Pim family of protein kinases in the progression of human prostate tumors. Further, in transgenic animal models, Pim-1 expression has been shown to be elevated in prostate tumors that are caused by overexpression of the c-myc oncogene. Recent evidence reveals the overlapping and compensatory nature of Pim-1 and Pim-2 phosphorylation and highlights the importance of inhibiting all isoforms. The emerging role of the PIM kinase family in hematological malignancies and solid tumors and the druggable nature of their ATP binding pocket make them attractive targets for anticancer drug development. Utilizing a highly distinct molecular scaffold, CX-6258 was developed as a selective and potent small molecule pan-PIM kinase inhibitor. CX-6258 inhibits Pims 1, 2 and 3 with IC50 values in the low nanomolar range and high selectivity as evidenced in a screening panel of over 100 kinases. CX-6258 demonstrates potent in vitro antiproliferative activity, particularly in leukemia derived cell lines expressing the FLT3-ITD. Moreover, CX-6258 inhibits the phosphorylation of BAD and 4EBP1, known substrates for PIMs 1, 2 and 3. When delivered orally, this pan-Pim inhibitor is well tolerated and demonstrates potent antitumor activity in murine xenograft models of PIM driven cancer. Using CX-6258 as our “path finder” molecule, we have created four additional unique chemical scaffolds as pan-Pim inhibitors, and certain molecules from these scaffolds can inhibit Pims 1, 2 and 3 in the picomolar range while exhibiting no inhibitory activity of the Flt3 protein kinase. The in vivo and in vitro profiles of these chemically diverse series are indicative of an effective and potent anti-cancer mechanism mediated through the selective inhibition of PIM kinase activity. Together, these findings exemplify that we have created multiple proprietary chemical series of pan-Pim inhibitors exhibiting picomolar potency and discerning selectivity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A246.

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.

The PIM1 Oncogene Accelerates TCL1 Driven Lymphomagenesis in a Double-Transgenic Murine Model.

Abstract 2968Poster Board II-944Previously, we identified and validated PIM1 as a differentially expressed gene in mantle cell lymphoma (MCL) patient samples. Further, we have shown PIM1 to be a significant prognostic biomarker in MCL. PIM1 is a serine/threonine kinase that is transcriptionally regulated by cytokines, mitogens, and numerous growth factors. PIM1 cooperates with other oncogenes in tumorigenesis and has been implicated in the development of leukemias, lymphomas, late progression events, and most recently in prostate cancer. PIM1 is overexpressed in aggressive lymphomas, such as the blastoid variant of MCL, and the ABC-subtype of diffuse large B-cell lymphomas (DLBCL). Here, we tested the in vivo cooperation of PIM1 with TCL1 in murine lymphomagenesis by producing double transgenic murine strains. PIM1 transgenic mice overexpress murine PIM1 under the control of the immunoglobulin enhancer Eμ. TCL1 transgenic mice (pEμ-B29-TCL1) fail to down-regulate TCL1 expression in mature B and T cells and provide a unique model for mature B-cell malignancies. We hypothesized that PIM1 would either accelerate TCL1-driven lymphomagenesis, result in the development of immature lymphomas, or both. Lymphoid malignancies were examined by immunohistochemistry and classified by a hematopathologist according to “Mouse Models of Human Blood Cancers” (Li et al., 2008). A Kaplan-Meier plot demonstrated statistically significant acceleration of lymphomagenesis in the PIM1/TCL1 transgenic mice when compared with the single transgenic strains. The median lymphoma-free survival for TCL1 single transgenic mice, PIM1 single transgenic mice, or PIM1/TCL double transgenic mice were 10.0 months, 16.0 months, and 8.5 months, respectively. The results were statistically significant: TCL1 vs. PIM1/TCL1 (p=0.0008), PIM1 vs. PIM1/TCL1 (p<0.0001). PIM1 transgenic mice developed rare [1 of 31 (3.2%)] and early (< 7 months of age) T-cell lymphoblastic lymphomas and more commonly developed late (>12 months of age) B-cell lymphomas. The most common lymphomas in PIM1 single transgenic were low-grade B-cell lymphomas [12 of 31 (38.7%)], mainly follicular lymphomas. A minority of PIM1 single transgenic mice developed aggressive lymphomas [6 of 31 (19.4%)], including DLBCL and Burkitt's lymphoma. In contrast, the majority of TCL1 transgenic mice developed aggressive B-cell lymphomas [21 of 36 (58.3%)], mainly DLBCL, lymphohistiocytic subtype. The majority of PIM1/TCL1 double transgenic mice also developed aggressive B-cell lymphomas [20 of 34 (58.8%)], mainly DLBCL, lymphohistiocytic subtype. The low-grade lymphomas that developed in PIM1/TCL1 mice included 5 cases of lymphoplasmacytic lymphoma (LPL); one of these cases had transformed in addition to a DLBCL. Further, endogenous expression of PIM kinase family members was investigated in a human lymphoma cell line bank (n=40) by quantitative real-time PCR. PIM1, PIM2, and PIM3 were found to be overexpressed in cell lines derived from human lymphoid malignancies of multiple histologies. In summary, aberrant PIM1 overexpression in TCL1 transgenic mice accelerated the development of mature, aggressive B-cell lymphomas. The classification of lymphomas in PIM1/TCL1 mice revealed similar histologies as in TCL1 single transgenic mice, mainly DLBCL. Single transgenic PIM1 mice developed low-grade B-cell lymphomas after prolonged observation time. The expression of all 3 PIM kinase family members in human lymphomas implies that pan-PIM kinase inhibitors should be developed as a potential mechanism of drug resistance to more restricted PIM inhibitors could be compensatory overexpression of the non-targeted Pim family members. A clinical trial with a pan-PIM inhibitor is currently ongoing. Disclosures:No relevant conflicts of interest to declare.

PIM as a Rational Target for B-Cell Lymphomas.

Abstract 3946Poster Board III-882 INTRODUCTIONThe Pim kinases are a family of serine/threonine kinases composed by three members: Pim1, Pim2 and Pim3, involved in the phosphorylation and regulation of several proteins that are essential for cell cycle progression, metabolism or apoptosis (BAD, p21, p27KIP, AKT, Mdm2 and cMyc, among them). Overexpression, translocation or amplification of Pim family have been described in many human cancers, including B-cell Non Hodgkin's Lymphoma, Multiple Myeloma, Prostate cancer and Pancreatic cancer. In addition, 50% of patients diagnosed with diffuse large B-cell Lymphoma (DLBCL) present somatic mutations in Pim1. Despite of its important role in cancer progression, very few chemical inhibitors have been described in the literature, being effective all of them in the high micromolar range. PURPOSEValidating PIM as a rational therapeutic target in B-cell lymphoma, developing tools for patient stratification and pharmacodynamic studies on PIM inhibition. MATERIAL AND METHODSGene expression profiling and Copy Number data were obtained from a series of 94 B-cell Non-Hodgkin Lymphoma patients (DLBCL, FL, MALT, MCL and NMZL). The effect of Pim inhibition was checked on cell lines by using a novel specific inhibitor for the Pim family (ETP-39010). Newly produced antibodies and RT-PCR primers and protocols were standarized. RESULTSGene expression data revealed high Pim isoforms expression in a subset of patients with Mantle cell lymphoma (MCL), and Diffuse Large B-cell lymphoma (DLBLC)-ABC type. CGH analysis focused on chromosomal regions containing Pim family and its main regulatory upstream pathway (JAK/STAT) was performed. Heterozygous gains of Pim1 (6p21.2) and Pim3 (22q13.33) were identified in 13.6% of DLBCL patients and in 4.2% of MCL. Alterations in JAK/STAT pathway were also detected in 59.1% of DLBCL patients, and 37.5% of MCL patients presented any alteration in JAK/STAT pathway, being frequent losses of JAK2 chromosomal region. Analysis of additional pathways involved in the up-stream regulation of Pim family disclosed heterozygous gains of PIK3C3 in 40.9% of DLBCL patients, and gains of PIK3CA in 45.9% of MCL patients.Lymphoma cell lines (15) derived from both MCL (9) and ABC-DLBLC (6) subtype, have been analyzed by qRT-PCR and Western-blot, showing variable expression levels of Pim1, Pim2 and Pim3. IC50 obtained for the ETP-39010 compound is in the low micromolar range for the MCL (0.7-8.7 micromolar) and DLBCL-ABC (0.8-10.3 micromolar) cell lines. Since Pim kinase family phosphorilate multiple sites of Bad and AKT, we have checked the inhibition of its phosphorilation as molecular biomarkers for the ETP-39010 effect. Our data show an inhibition of at least 20% of pBad (S112) and almost a complete inhibition of pAKT (S473) 4h after treatment. In addition, cell cycle arrest at G1 and induction of apoptosis were observed 24h after the treatment. CONCLUSIONPim family genes are a rational therapeutic target in MCL and DLBCL-ABC lymphoma subtypes. Stratification and pharmacodynamic markers have been developed for PIM inhibition using a novel specific inhibitor compound -ETP-39010-. Disclosures:No relevant conflicts of interest to declare.

Discovery of Selective Small Molecule Pan-Pim Kinase Inhibitors with Potent Oral Efficacy in Murine Xenograft Models.

Abstract 1726Poster Board I-752The PIM family of serine/threonine kinases are pro-proliferative kinases activated by multiple cytokines and growth factor signaling. The Pim kinases are unusual in that they are regulated primarily by transcription and not by membrane recruitment or phosphorylation like other serine/threonine kinases. Activated cytokine receptors recruit JAKs to induce STAT-dependent transcription of the Pim genes. They are proto-oncogenes and have been implicated in the process of lymphomagenesis and malignant transformation. Pim overexpression has been reported in diffuse B cell lymphoma, chronic lymphocytic leukemia, FLT3-mediated acute myelogenous leukemia and prostate cancer. Pim-2 is over expressed in leukemias and lymphomas, whereas Pim-3 overexpression has been observed in melanoma, pancreatic and gastric tumors. The recent reports of elevated levels of Pim-1 expression in human prostate tumor biopsies implicate the Pim family of protein kinases in the progression of human prostate tumors. Further, in transgenic animal models, Pim-1 expression has been shown to be elevated in prostate tumors that are caused by overexpression of the c-myc oncogene. Recent evidence reveals the overlapping and compensatory nature of Pim-1 and Pim-2 phosphorylation and highlights the importance of inhibiting all isoforms. The emerging role of the PIM kinase family in hematological malignancies and solid tumors and the druggable nature of their ATP binding pocket make them attractive targets for anticancer drug developmentUtilizing a highly distinct molecular scaffold, CX-6258 was developed as a selective and potent small molecule pan-PIM kinase inhibitor. CX-6258 inhibits Pims 1, 2 and 3 with IC50 values in the low nanomolar range and high selectivity as evidenced in a screening panel of over 100 kinases. CX-6258 demonstrates potent in vitro antiproliferative activity, particularly in leukemia derived cell lines expressing the FLT3-ITD. Moreover, CX-6258 inhibits the phosphorylation of BAD and 4EBP1, known substrates for PIMs 1, 2 and 3. When delivered orally, this pan-Pim inhibitor is well tolerated and demonstrates potent antitumor activity in murine xenograft models of PIM driven cancer. Using CX-6258 as our “path finder” molecule, we have created four additional unique chemical scaffolds as pan-Pim inhibitors, and certain of these scaffolds can inhibit Pims 1, 2 and 3 in the picomolar range while exhibiting no inhibitory activity of the Flt3 protein kinase. The in vivo and in vitro profiles of these chemically diverse series are indicative of an effective and potent anti-cancer mechanism mediated through the selective inhibition of PIM kinase activity. Together, these findings exemplify that we have created multiple proprietary chemical series of pan-Pim inhibitors exhibiting picomolar potency and high selectivity. DisclosuresNo relevant conflicts of interest to declare.

Crystal Structure of the PIM2 Kinase in Complex with an Organoruthenium Inhibitor

BackgroundThe serine/threonine kinase PIM2 is highly expressed in human leukemia and lymphomas and has been shown to positively regulate survival and proliferation of tumor cells. Its diverse ATP site makes PIM2 a promising target for the development of anticancer agents. To date our knowledge of catalytic domain structures of the PIM kinase family is limited to PIM1 which has been extensively studied and which shares about 50% sequence identity with PIM2.Principal FindingsHere we determined the crystal structure of PIM2 in complex with an organoruthenium complex (inhibition in sub-nanomolar level). Due to its extraordinary shape complementarity this stable organometallic compound is a highly potent inhibitor of PIM kinases.SignificanceThe structure of PIM2 revealed several differences to PIM1 which may be explored further to generate isoform selective inhibitors. It has also demonstrated how an organometallic inhibitor can be adapted to the binding site of protein kinases to generate highly potent inhibitors.Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Synthesis, Kinase Inhibitory Potencies, and in Vitro Antiproliferative Evaluation of New Pim Kinase Inhibitors

Members of the Pim kinase family have been identified as promising targets for the development of antitumor agents. After a screening of pyrrolo[2,3-a]- and [3,2-a]carbazole derivatives toward 66 protein kinases, we identified pyrrolo[2,3-a]carbazole as a new scaffold to design potent Pim kinase inhibitors. In particular, compound 9 was identified as a low nM selective Pim inhibitor. Additionally, several pyrrolo[2,3-a]carbazole derivatives showed selectivity for Pim-1 and Pim-3 over Pim-2. In vitro antiproliferative activities of 9 and 28, the most potent Pim inhibitors identified, were evaluated toward three human solid cancer cell lines (PA1, PC3, and DU145) and one human fibroblast primary culture, revealing IC50 values in the micromolar range. Finally, the crystal structure of Pim-1 complexed with lead compound 9 was determined. The structure revealed a non-ATP mimetic binding mode with no hydrogen bonds formed with the kinase hinge region and explained the selectivity of pyrrolo[2,3-a]carbazole derivatives for Pim kinases.

The PIM1 Oncogene Accelerates TCL1 Driven Lymphomagenesis in a Double-Transgenic Murine Model.

Previously, we identified and validated PIM-1 as a differentially expressed gene in mantle cell lymphoma (MCL) patient samples. Further, we have shown PIM-1 to be a significant prognostic biomarker in MCL. PIM-1 is an oncogenic serine/threonine kinase that is transcriptionally regulated by cytokines, mitogens, and numerous growth factors. It cooperates with other oncogenes in tumorigenesis and has been implicated in the development of leukemias, lymphomas, late progression events, and most recently in prostate cancer. PIM-1 is overexpressed in aggressive lymphomas, such as the blastoid variant of MCL, and the ABC-type of DLBCL. Here we tested the in vivo cooperation of PIM-1 with TCL1 in murine lymphomagenesis by producing double transgenic murine strains. PIM-1 transgenic mice overexpress murine PIM-1 under the control of the immunoglobulin enhancer Eμ. TCL1 transgenic mice (pEμ -B29-TCL1) fail to downregulate TCL1 expression in mature B and T cells and provide a unique model for mature B-cell malignancies, including Burkitt-like lymphoma (BLL), DLBCL, marginal zone lymphoma, and B-cell chronic lymphocytic leukemia. We hypothesized that PIM-1 would either accelerate TCL1-driven lymphomagenesis, result in the development of immature lymphomas, or both. Lymphoid malignancies were examined by immunohistochemistry and flow cytometry and classified according to the mouse models of human cancer consortium (MMHCC) ‘Bethesda’ classification scheme. Forty double transgenic mice (PIM-1/TCL1) have been generated and observed for a median follow-up of 9 months. To date, 8/40 (20 %) of the PIM-1/TCL1 mice developed lymphomas, in contrast to 9/88 (10%) PIM-1 and 11/49 (22%) TCL1 transgenic mice, with a median follow-up of 7 and 15 months, respectively. A Kaplan-Meyer plot demonstrated statistically significant acceleration of lymphomagenesis in the PIM-1/TCL1 transgenic mice when compared with single TCL1 transgenic mice (p=0.037). PIM-1 transgenic mice developed early (< 7 months of age) T-cell lymphoblastic lymphomas and late (> 20 months of age) DLBCL. TCL1 transgenic mice developed DLBCL, with single occurrences of lymphoblastic, lymphocytic and Burkitt lymphomas. PIM-1/TCL1 transgenic mice developed DLBCL, frequently with extranodal involvement (spleen, liver and lung). A single case of follicular lymphoma was seen. In addition, endogenous expression of PIM kinase family members was investigated in a human lymphoma cell line bank (n=40) by quantitative real-time PCR. PIM-1, PIM-2, and also PIM-3 were found to be overexpressed in cell lines derived from human lymphoid malignancies of multiple histologies. In summary, aberrant PIM-1 overexpression in TCL1 transgenic mice accelerated the development of mature B-cell lymphomas. To date, the classification of lymphomas in PIM-1/TCL1 mice revealed similar histologies as in TCL1 single transgenic mice, mainly DLBCL. The expression of all 3 PIM kinase family members in lymphomas implies that pan-PIM kinase inhibitors should be developed as a potential mechanism of resistance to more restricted PIM inhibitors could be compensatory overexpression of the non-targeted family members.

Targeting PIM Kinases Impairs Survival of Hematopoietic Cells Transformed by Kinase Inhibitor–Sensitive and Kinase Inhibitor–Resistant Forms of Fms-Like Tyrosine Kinase 3 and BCR/ABL

Previous studies have shown that activation of the signal transducer and activator of transcription 5 (STAT5) plays an essential role in leukemogenesis mediated through constitutive activated protein tyrosine kinases (PTK). Because PIM-1 is a STAT5 target gene, we analyzed the role of the family of PIM serine/threonine kinases (PIM-1 to PIM-3) in PTK-mediated transformation of hematopoietic cells. Ba/F3 cells transformed to growth factor independence by various oncogenic PTKs (TEL/JAK2, TEL/TRKC, TEL/ABL, BCR/ABL, FLT3-ITD, and H4/PDGFbetaR) show abundant expression of PIM-1 and PIM-2. Suppression of PIM-1 activity had a negligible effect on transformation. In contrast, expression of kinase-dead PIM-2 mutant (PIM-2KD) led to a rapid decline of survival in Ba/F3 cells transformed by FLT3-ITD but not by other oncogenic PTKs tested. Coexpression of PIM-1KD and PIM-2KD abrogated growth factor-independent growth of Ba/F3 transformed by several PTKs, including BCR/ABL. Targeted down-regulation of PIM-2 by RNA interference (RNAi) selectively abrogated survival of Ba/F3 cells transformed by various Fms-like tyrosine kinase 3 (FLT3)-activating mutants [internal tandem duplication (ITD) and kinase domain] and attenuated growth of human cell lines containing FLT3 mutations. Interestingly, cells transformed by FLT3 and BCR/ABL mutations that confer resistance to small-molecule tyrosine kinase inhibitors were still sensitive to knockdown of PIM-2, or PIM-1 and PIM-2 by RNAi. Our observations indicate that combined inactivation of PIM-1 and PIM-2 interferes with oncogenic PTKs and suggest that PIMs are alternative therapeutic targets in PTK-mediated leukemia. Targeting the PIM kinase family could provide a new avenue to overcome resistance against small-molecule tyrosine kinase inhibitors.

Pim kinases phosphorylate multiple sites on Bad and promote 14-3-3 binding and dissociation from Bcl-XL

BackgroundPim-1, 2 and 3 are a group of enzymes related to the calcium calmodulin family of protein kinases. Over-expression of Pim-1 and Pim-2 in mice promotes the development of lymphomas, and up-regulation of Pim expression has been observed in several human cancers.ResultsHere we show that the pim kinases are constitutively active when expressed in HEK-293 cells and are able to phosphorylate the Bcl-2 family member Bad on three residues, Ser112, Ser136 and Ser155 in vitro and in cells. In vitro mapping showed that Pim-2 predominantly phosphorylated Ser112, while Pim-1 phosphorylated Ser112, but also Ser136 and Ser155 at a reduced rate compared to Ser112. Pim-3 was found to be the least specific for Ser112, and the most effective at phosphorylating Ser136 and Ser155. Pim-3 was also able to phosphorylate other sites in Bad in vitro, including Ser170, another potential in vivo site. Mutation of Ser136 to alanine prevented the phosphorylation of Ser112 and Ser155 by Pim kinases in HEK-293 cells, suggesting that this site must be phosphorylated first in order to make the other sites accessible. Pim phosphorylation of Bad was also found to promote the 14-3-3 binding of Bad and block its association with Bcl-XL.ConclusionAll three Pim kinase family members predominantly phosphorylate Bad on Ser112 and in addition are capable of phosphorylating Bad on multiple sites associated with the inhibition of the pro-apoptotic function of Bad in HEK-293 cells. This would be consistent with the proposed function of Pim kinases in promoting cell proliferation and preventing cell death.

Structure and Substrate Specificity of the Pim-1 Kinase

The Pim kinases are a family of three vertebrate protein serine/threonine kinases (Pim-1, -2, and -3) belonging to the CAMK (calmodulin-dependent protein kinase-related) group. Pim kinases are emerging as important mediators of cytokine signaling pathways in hematopoietic cells, and they contribute to the progression of certain leukemias and solid tumors. A number of cytoplasmic and nuclear proteins are phosphorylated by Pim kinases and may act as their effectors in normal physiology and in disease. Recent crystallographic studies of Pim-1 have identified unique structural features but have not provided insight into how the kinase recognizes its target substrates. Here, we have conducted peptide library screens to exhaustively determine the sequence specificity of active site-mediated phosphorylation by Pim-1 and Pim-3. We have identified the major site of Pim-1 autophosphorylation and find surprisingly that it maps to a novel site that diverges from its consensus phosphorylation motif. We have solved the crystal structure of Pim-1 bound to a high affinity peptide substrate in complexes with either the ATP analog AMP-PNP or the bisindolylmaleimide kinase inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide HCl. These structures reveal an unanticipated mode of recognition for basic residues upstream of the phosphorylation site, distinct from that of other kinases with similar substrate specificity. The structures provide a rationale for the unusually high affinity of Pim kinases for peptide substrates and suggest a general mode for substrate binding to members of the CAMK group.