Pim-1 Kinase Activity Research Articles

Synthesis and Anticancer Activity of Novel Dual Inhibitors of Human Protein Kinases CK2 and PIM-1.

CK2 and PIM-1 are serine/threonine kinases involved in the regulation of many essential processes, such as proliferation, differentiation, and apoptosis. Inhibition of CK2 and PIM-1 kinase activity has been shown to significantly reduce the viability of cancer cells by inducing apoptosis. A series of novel amino alcohol derivatives of parental DMAT were designed and synthesized as potent dual CK2/PIM-1 inhibitors. Concomitantly with the inhibition studies toward recombinant CK2 and PIM-1, the influence of the obtained compounds on the viability of three human carcinoma cell lines, i.e., acute lymphoblastic leukemia (CCRF-CEM), human chronic myelogenous leukemia (K-562), and breast cancer (MCF-7), as well as non-cancerous cells (Vero), was evaluated using an MTT assay. Induction of apoptosis and cell cycle progression after treatment with the most active compound and a lead compound were studied by flow-cytometry-based assay. Additionally, autophagy induction in K-562 cells and intracellular inhibition of CK2 and PIM-1 in all the tested cell lines were evaluated by qualitative/quantitative fluorescence-based assay and Western blot method, respectively. Among the newly developed inhibitors, 1,1,1-trifluoro-3-[(4,5,6,7-tetrabromo-1H-benzimidazol-2-yl)amino]propan-2-ol demonstrates the highest selectivity and the most prominent proapoptotic properties towards the studied cancer cells, especially towards acute lymphoblastic leukemia, in addition to inducing autophagy in K-562 cells.

Pim1 promotes IFN-β production by interacting with IRF3

The Pim (proviral integration site for Moloney murine leukemia virus) proteins compose a serine threonine kinase family whose members regulate cell proliferation, migration and cell survival. However, whether Pim kinases participate in innate immune responses is unclear. Here, we show for the first time that Pim1 plays an essential role in the production of interferon (IFN)-β by macrophages after their Toll-like receptor (TLR) pathway is activated by pathogen-associated molecular patterns (PAMPs). Specifically, Pim1 was quickly upregulated in an NF-κB-dependent manner after TLR stimulation with PAMPs. Pim1 deficiency reduced TLR3- or TLR4-stimulated IFN-β and IFN-stimulated gene (ISG) expression but not proinflammatory cytokine expression in macrophages. Mechanistically, Pim1 specifically upregulates IRF3 phosphorylation and nuclear translocation. However, this role is not dependent on Pim1 kinase activity. Rather, Pim1 appears to promote IRF3 phosphorylation by enhancing the formation of IFN-β signaling complexes composed of TRIF, TRAF3, TBK1, and IRF3. Poly (I:C)-treated Pim1−/− mice produced less serum IFN-β and were less likely to survive than wild-type mice. These findings show for the first time that Pim1 participates in TLR-mediated IFN-β production, thus revealing a novel target for controlling antiviral innate immune responses.

Abstract 1345: Pharmacodynamic biomarkers for Pim inhibition with TP-3654 in patients with solid tumors

Abstract Proviral integration site for Moloney murine leukemia virus-1 (Pim-1) is a serine/threonine kinase downstream of Jak/Stat signaling which promotes cell growth, survival, and drug resistance. Pim-1 kinase is an important driver of tumorigenesis and tumor survival through its role in a number of downstream pathways, including inhibition of apoptosis through phosphorylation of the BH3-only protein BAD. Pim-1 is expressed at very low levels in most normal tissues, but is overexpressed in many cancers, such as prostate, colorectal, and many hematologic malignancies. Pim-1 kinase activity is constitutive and therefore directly proportional to protein expression. As such, Pim-1 is an attractive therapeutic target. TP-3654 is a second-generation, oral Pim inhibitor currently in Phase I clinical trials in solid tumors and myelofibrosis (NCT03715504 and NCT04176198). TP-3654 inhibits all three Pim kinases, with Ki values for Pim-1 (5nM), and Pim-2 and Pim-3 &lt250 nM in a biochemical assay. Treatment with TP-3654 in Jurkat and HEL cell lines showed dose-dependent modulation of the downstream targets of PIM, such as pS6K and pBad. A dose-dependent increase of TP-3654 Cmax was observed in plasma and subcutaneous PC-3 xenograft tumors in animals dosed orally at 50, 100 and 200 mg/kg with Tmax at 1 hour. Animals bearing HEL tumors were dosed similarly and Cmax values increased in a dose-dependent manner with Tmax at 2 hours. Plasma and tumor TP-3654 concentrations were above the measurement threshold out to 24 hours post dose. We further explored pharmacodynamic biomarkers of Pim inhibition (pBad, pS6K, and pS6RP) in flash frozen (FF) tumor tissue at various time points in both models by western blot and saw a maximum of 40% decrease in phosphorylated S6K at 2 hours, and an 80% decrease in pBad at 8 hrs in PC3 xenograft tumors in mice treated with 100 mg/kg of TP-3654. We hypothesized that treatment with TP-3654 would modulate Pim signaling in both cancer cells and peripheral blood mononuclear cells (PBMCs). This allows for detection of pharmacodynamic markers using repeated peripheral blood draws instead of invasive biopsies. We treated PBMCs from multiple healthy human donors with TP-3654 ex vivo at 0.3 and 3 µM to assess the movement of Pim biomarkers. Two phosphorylation markers, pS6K and pS6RP consistently exhibited dose-dependent decreases (30-70%) as measured by western blot. Inhibition of pBad was dependent on pretreatment phosphorylation state. These results were confirmed in an automated western blot system. Citation Format: Curtis A. Allred, Yuta Matsumura, Ethika Tyagi, Mark Wade, Clifford Whatcott, Jason Foulks, Adam Siddiqui, David J. Bearss, Steven L. Warner. Pharmacodynamic biomarkers for Pim inhibition with TP-3654 in patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1345.

Resveratrol suppresses gastric cancer cell proliferation and survival through inhibition of PIM-1 kinase activity

The proviral integration site for Moloney murine leukemia virus (PIM) family of serine/threonine-specific kinases consist of three isoforms, that regulate proliferation, apoptosis, metabolism, invasion, and metastasis of cancer cells. Among these, abnormally elevated kinase activity of PIM-1 contributes to the progression of gastric cancer and predicts poor prognosis and a low survival rate in gastric cancer patients. In the present study, we found that resveratrol, one of the representative chemopreventive and anticarcinogenic phytochemicals, directly binds to PIM-1 and thereby inhibits its catalytic activity in human gastric cancer SNU-601 cells. This resulted in suppression of phosphorylation of the proapoptotic Bad, a known substrate of PIM-1. Resveratrol, by inactivating PIM-1, also inhibited anchorage-independent growth and proliferation of SNU-601 cells. To understand the molecular interaction between resveratrol and PIM-1, we conducted docking simulation and found that resveratrol directly binds to the PIM-1 at the ATP-binding pocket. In conclusion, the proapototic and anti-proliferative effects of resveratrol in gastric cancer cells are likely to be mediated through suppression of PIM-1 kinase activity, which may represent a novel mechanism underlying its chemopreventive and anticarcinogenic actions.

Pim1 kinase positively regulates myoblast behaviors and skeletal muscle regeneration

Adult skeletal muscle regeneration after injury depends on normal myoblast function. However, the intrinsic mechanisms for the control of myoblast behaviors are not well defined. Herein, we identified Pim1 kinase as a novel positive regulator of myoblast behaviors in vitro and muscle regeneration in vivo. Specifically, knockdown of Pim1 significantly restrains the proliferation and accelerates the apoptosis of myoblasts in vitro, indicating that Pim1 is critical for myoblast survival and amplification. Meanwhile, we found that Pim1 kinase is increased and translocated from cytoplasm into nucleus during myogenic differentiation. By using Pim1 kinase inhibitor, we proved that inhibition of Pim1 activity prevents myoblast differentiation and fusion, suggesting the necessity of Pim1 kinase activity for proper myogenesis. Mechanistic studies demonstrated that Pim1 kinase interacts with myogenic regulator MyoD and controls its transcriptional activity, inducing the expression of muscle-specific genes, which consequently promotes myogenic differentiation. Additionally, in skeletal muscle injury mouse model, deletion of Pim1 hinders the regeneration of muscle fibers and the recovery of muscle strength. Taken together, our study provides a potential target for the manipulation of myoblast behaviors in vitro and the myoblast-based therapeutics of skeletal muscle injury.

Safety profiling of genetically engineered Pim-1 kinase overexpression for oncogenicity risk in human c-kit+ cardiac interstitial cells.

Advancement of stem cell-based treatment will involve next-generation approaches to enhance therapeutic efficacy which is often modest, particularly in the context of myocardial regenerative therapy. Our group has previously demonstrated the beneficial effect of genetic modification of cardiac stem cells with Pim-1 kinase overexpression to rejuvenate aged cells as well as potentiate myocardial repair. Despite these encouraging findings, concerns were raised regarding potential for oncogenic risk associated with Pim-1 kinase overexpression. Testing of Pim-1 engineered c-kit+ cardiac interstitial cells (cCIC) derived from heart failure patient samples for indices of oncogenic risk was undertaken using multiple assessments including soft agar colony formation, micronucleation, gamma-Histone 2AX foci, and transcriptome profiling. Collectively, findings demonstrate comparable phenotypic and biological properties of cCIC following Pim-1 overexpression compared with using baseline control cells with no evidence for oncogenic phenotype. Using a highly selective and continuous sensor for quantitative assessment of PIM1 kinase activity revealed a sevenfold increase in Pim-1 engineered vs. control cells. Kinase activity profiling using a panel of sensors for other kinases demonstrates elevation of IKKs), AKT/SGK, CDK1-3, p38, and ERK1/2 in addition to Pim-1 consistent with heightened kinase activity correlating with Pim-1 overexpression that may contribute to Pim-1-mediated effects. Enhancement of cellular survival, proliferation, and other beneficial properties to augment stem cell-mediated repair without oncogenic risk is a feasible, logical, and safe approach to improve efficacy and overcome current limitations inherent to cellular adoptive transfer therapeutic interventions.

Rational Design of a Potent Pan-Pim Kinases Inhibitor with a Rhodanine-Benzoimidazole Structure.

The serine/threonine Pim kinases are overexpressed in various types of solid carcinomas and hematological malignancies, and contribute to regulating cell-cycle progression and cell survival. The aim of this study was to discover a novel pan-Pim kinases inhibitor with potent anti-proliferative activities against cancer cell lines. We screened a panel of small molecule compounds for their ability to inhibit Pim-1 kinase activity, and the hit compound was optimized using the docking analysis to Pim-1. We evaluated kinase-inhibition activities of the rationally-designed compound against Pim-1, 2, 3 and another five kinases. Furthermore, in order to characterize the cellular activities, both solid and hematological cancer cell lines treated with the compound were subjected to anti-proliferative assay, western blotting, FACS and apoptosis assays. We discovered a pan-Pim kinases inhibitor, compound 1, with a rhodanine-benzylidene structure via Pim-1 inhibitor screening. Using docking analysis of compound 1 and Pim-1, we optimized it and found a potent- and selective-Pim kinases inhibitor, compound 2, with a rhodanine-benzoimidazole structure. Compound 2 inhibited Pim-1, 2, 3 with IC50 values of 16, 13, and 6.4 nM, respectively, and suppressed proliferation of solid and hematological cancer cell lines at submicromolar concentrations. In both types of cell lines, compound 2 inhibited phosphorylation of Pim signaling substrates and cell-cycle progression and induced apoptosis. We identified a pan-Pim kinases inhibitor, compound 2, with a rhodanine-benzoimidazole structure. Our data suggest that compound 2 can serve as a lead to novel anticancer agents, effective in the treatment of both solid carcinomas and hematological malignancies.

Biochemical changes of salivary gland adenoid cystic carcinoma cells induced by SGI-1776

Provirus integration site for Moloney murine leukemia virus 1 (Pim-1) has proved to be an oncogene and it is known that to depress Pim-1 activity may be a novel oncological treatment strategy. SGI-1776, a small molecule, is the first clinically tested inhibitor of the Pim kinase family. Here, we aimed to explore the effect of SGI-1776 on salivary adenoid cystic carcinoma (SACC). Expression of Pim-1 was confirmed in SACC and control tissues by qRT-PCR. After SGI-1776 treatment, the Pim-1 expressions and Pim-1 kinase activity in both SACC-83 and SACC-LM cell lines were measured. Cell proliferation, cell invasion, cell cycle, apoptosis and mitochondrial membrane potential were analyzed. Also, the expression of FOXO3a, p-FOXO3a, RUNX3, Bcl-2, BAD, p-BAD, Bim and p-Bim were detected by Western blot. The results showed that Pim-1 was significantly overexpressed in SACC tissues. SGI-1776 down-regulated the Pim-1 expression, inhibited Pim-1 kinase activity, reduced cell proliferation, decreased invasive ability, increased caspase-3 activity and induced apoptosis, cell cycle arrest and mitochondrial depolarization. Reduced expression was also seen in p-FOXO3a, RUNX3, Bcl-2, p-BAD and p-Bim, whereas no significant changes were observed from FOXO3a, BAD and Bim. These results confirm the pivotal role of Pim-1 in SACC and suggest that targeting Pim-1 kinase signal pathway by SGI-1776 might be a promising therapeutic modality for SACC.

QSAR STUDIES ON PYRAZOLO [1, 5-A] PYRIMIDINE-BASED PIM-1 KINASE INHIBITORS

Quantitative structure-activity relationships (QSAR) have been applied in order to explain the structural requirements of Pim-1 kinase activity of pyrazolo [1, 5-a] pyrimidine derivatives. The QSAR model was an internal predictive power (q2 = 0.7866) of 78% and a predictivity for the external test set (pred_r2 = 0.7742) of about 77%. The developed significant QSAR model indicates that HUMO energy, SsOHcount, SsNH2Count and T_O_O_4 potential of whole molecule play an important role in Pim-1 kinase inhibitor of pyrimidine. Consequently, these results can be useful in identifying the structural requirements of pyrazolo [1, 5-a] pyrimidine derivatives and helpful for better understanding the Pim-1 kinase. Eventually, they provide a beneficial basis to design new and more potent inhibitors of Pim-1 kinase.

Pim-1L Protects Cell Surface-Resident ABCA1 From Lysosomal Degradation in Hepatocytes and Thereby Regulates Plasma High-Density Lipoprotein Level.

ATP-binding cassette transporter A1 (ABCA1) exerts an atheroprotective action through the biogenesis of high-density lipoprotein in hepatocytes and prevents the formation of foam cells from macrophages. Controlling ABCA1 is a rational approach to improving atherosclerotic cardiovascular disease. Although much is known about the regulatory mechanism of ABCA1 synthesis, the molecular mechanism underpinning its degradation remains to be clearly described. ABCA1 possesses potential sites of phosphorylation by serine/threonine-protein kinase Pim-1 (Pim-1). Pim-1 depletion decreased the expression of cell surface-resident ABCA1 (csABCA1) and apolipoprotein A-I-mediated [3H]cholesterol efflux in the human hepatoma cell line HepG2, but not in peritoneal macrophages from mice. In vitro kinase assay, immunoprecipitation, and immunocytochemistry suggested phosphorylation of csABCA1 by the long form of Pim-1 (Pim-1L). Cell surface biotinylation indicated that Pim-1L inhibited lysosomal degradation of csABCA1 involving the liver X receptor β, which interacts with csABCA1 and thereby protects it from ubiquitination and subsequent lysosomal degradation. Cell surface coimmunoprecipitation with COS-1 cells expressing extracellularly hemagglutinin-tagged ABCA1 showed that Pim-1L-mediated phosphorylation of csABCA1 facilitated the interaction between csABCA1 and liver X receptor β and thereby stabilized the csABCA1-Pim-1L complex. Mice deficient in Pim-1 kinase activity showed lower expression of ABCA1 in liver plasma membranes and lower plasma high-density lipoprotein levels than control mice. Pim-1L protects hepatic csABCA1 from lysosomal degradation by facilitating the physical interaction between csABCA1 and liver X receptor β and subsequent stabilization of the csABCA1-Pim-1L complex and thereby regulates the circulating level of high-density lipoprotein. Our findings may aid the development of high-density lipoprotein-targeted therapy.

Inhibition of Pim1 kinase, new therapeutic approach in virus-induced asthma exacerbations.

Therapeutic options to treat virus-induced asthma exacerbations are limited and urgently needed. Therefore, we tested Pim1 kinase as potential therapeutic target in human rhinovirus (HRV) infections. We hypothesised that inhibition of Pim1 kinase reduces HRV replication by augmenting the interferon-induced anti-viral response due to increased activity of the janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway.Air-liquid interface (ALI) cultures of primary bronchial epithelial cells (PBECs) from healthy individuals and moderate-to-severe asthmatic volunteers were infected with HRV-16 with or without a specific Pim1 inhibitor; viral replication and induction of anti-viral responses were measured using RT-qPCR. Viral titres were measured by 50% tissue culture infective dose and release of interferon-γ-induced protein 10 (IP-10) and RANTES protein assessed by ELISA. Phosphorylation of STAT-1 was determined using western blotting.Viral replication was reduced in ALI cultures of healthy and asthmatic PBECs treated with the Pim1 inhibitor. Using cultures from healthy donors, enhanced STAT-1 phosphorylation upon inhibition of Pim1 kinase activity resulted in increased mRNA expression of interferon-β, interleukin-29, IP-10 and RANTES 12 h after infection and increased protein levels of IP-10 and RANTES 24 h after infection.We have identified Pim1 kinase as novel target to reduce viral replication in ALI cultures of PBECs. This may open new avenues for therapeutic interventions in virus-induced asthma exacerbations.

Pim1 kinase activity preserves airway epithelial integrity upon house dust mite exposure.

Most patients with allergic asthma are sensitized to house dust mite (HDM). The allergenicity of HDM largely depends on disruption of the integrity and proinflammatory activation of the airway epithelium. In this study, we hypothesized that Pim1 kinase activity attenuates HDM-induced asthma by preserving airway epithelial integrity. The effects of Pim1 kinase activity on barrier function and release of the proinflammatory mediators IL-1α and CCL20 were studied in vitro in 16HBE and primary bronchial epithelial cells (PBECs). Pim1-proficient and -deficient mice were exposed to a HDM-driven model of allergic asthma, and airway hyperresponsiveness (AHR) was measured upon methacholine challenge. Airway inflammation and proinflammatory mediators in lung tissue and BAL fluid were determined. We observed that inhibition of Pim1 kinase prolongs the HDM-induced loss of barrier function in 16HBE cells and sensitizes PBECs to HDM-induced barrier dysfunction. Additionally, inhibition of Pim1 kinase increased the HDM-induced proinflammatory activity of 16HBE cells as measured by IL-1α secretion. In line herewith, HDM exposure induced an enhanced production of the proinflammatory chemokines CCL17 and CCL20 in Pim1-deficient mice compared with wild-type controls. While we observed a marked increase in eosinophilic and neutrophilic granulocytes as well as mucus cell metaplasia and AHR to methacholine in mice exposed to HDM, these parameters were independent of Pim1 kinase activity. In contrast, levels of the Th2-cytokines IL-5 and IL-10 were significantly augmented in HDM-treated Pim1-deficient mice. Taken together, our study shows that Pim1 kinase activity maintains airway epithelial integrity and protects against HDM-induced proinflammatory activation of the airway epithelium.

Abstract 5056: PIM-1 kinase regulates vitamin D receptor signaling in human renal carcinoma cells

Abstract Dysregulation of vitamin D signaling and metabolism enzymes is a frequent change in many cancers and may confound approaches to vitamin -D based therapies. The mechanisms by which aberrant vitamin D signaling and metabolism lead to resistance to vitamin D action are poorly understood. The kidney plays a critical role in vitamin D synthesis and metabolism; in renal cell carcinoma (RCC) cells vitamin D metabolism is dysregulated. Recently, we discovered that an oncogenic protein kinase PIM-1 is involved in vitamin D-induced expression of 24-hydroxylase, a key catalytic enzyme of vitamin D encoded by CYP24A1 gene, in bladder and prostate cancer. To study the impact of PIM1 on vitamin D signaling and vitamin D-mediated anti-tumor activity in RCC, we analyzed 4 human renal cell carcinoma cell lines (Caki-1, ACHN, A498 and 786-O) and 25 samples of human RCC tumor tissue and 13 benign kidney tissues (including 10 matched tumor and benign tissue samples) by qRT-PCR, immunohistochemistry (IHC), Western blot analysis, MTT and clonogenic assays. VDR mRNA and protein were detected in all RCC cell lines. IHC showed that VDR was strongly expressed in the normal kidney tissues, but decreased in RCC tissues. As expected, VDR expression was significantly increased by treatment with 1,25-dihydroxyvitamin D3 (1,25D3) in RCC cell lines. MTT and clonogenic assays showed that 1,25D3 inhibited RCC cell growth (p<0.05). We also noted a significant increase of CYP24A1 expression induced by vitamin D3 (p<0.0001); and effect which would be expected to promote the catabolism of 1,25D3. qRT-PCR and Western blot analysis demonstrated that PIM-1 is expressed in RCC cells. PIM-1 inhibition by siRNA or PIM-1 inhibitor JP_11646 significantly reduced 1,25D3-induced CYP24A1 expression (p<0.05). Therefore, we hypothesized that targeting PIM-1 kinase activity with a PIM-1 inhibitor may be an effective strategy to reduce 1,25D3 metabolism, thereby enhancing vitamin D-mediated anti-tumor activity. Inhibition of PIM1 kinase activity by JP_11646 reduced CYP24A1 expression at the transcriptional level in RCC cells. We further evaluated the efficacy of the PIM1 inhibitor alone or in combination with 1,25D3. Inhibition of PIM1 kinase activity by JP_11646 reduced cell growth in RCC cell lines in a dose-dependent manner as measured by MTT and clonogenic assays. Furthermore, inhibition of PIM1 kinase activity enhances 1,25D3-mediated inhibitory effect on cell growth in RCC cells (p<0.05). This study indicates that PIM-1 is involved in the regulation of vitamin D signaling in RCC. Inhibition of PIM kinase activity combined with vitamin D may be an attractive new therapeutic strategy for RCC. Citation Format: Wei Luo, Yingyu Ma, Carmen Baldino, Justin Caserta, Swathi Ramakrishnan, Pili Roberto, Candace Johnson, Donald Trump. PIM-1 kinase regulates vitamin D receptor signaling in human renal carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5056. doi:10.1158/1538-7445.AM2015-5056

365 Pim-1 kinase: Validated as a therapeutic cancer target for MYC-driven tumours

Provirus integration site for Moloney murine leukemia virus 1 (Pim-1) kinase belongs to a family of constitutively active serine/threonine kinases. The overexpression of Pim-1 can contribute to tumorigenesis, thereby establishing Pim-1 as a proto-oncogene. In B-cell lymphoma, colorectal cancer, pancreatic cancer and other tumours, the over expression of Pim-1 is linked to poor prognosis. Proviral integration experiments suggested the cooperation between Pim-1 and the protooncogene c-myc that was subsequently confirmed by double transgenic mice. Pim-1 acts synergistically with the transcription factor c-myc by phosphorylation and by interaction with c-myc on the chromatin level, leading to elevated trancription of c-myc target genes and cell proliferation. Pim-1 also promotes cell cycle progression. Pim-1-mediated phosphorylation of p27 leads to its binding to 14−3−3 protein, its nuclear export and proteasome-dependent degradation. The aim of our study was to analyze the effect of kinase-inhibition of Pim-1 on tumour growth in a mouse model of haematological malignancy. To this end, we constructed a conditional knock-in mouse model in which a single amino acid exchange can be induced by expression of the Cre-recombinase, leading to expression of an inactive form of Pim-1 kinase. These Pim-1 kinase-dead (KD) mice do not display phenotypic abnormalities or reduced live span even after homozygous exchange by CMV-Cre expression of the wild type form of Pim-1 against the mutated form. As expected, elevated levels of the cell cycle inhibitor p27 can be observed in the organs of Pim-1 kinase-dead mice by immunohistochemistry (IHC). In order to study the effect of the Pim-1 kinase inhibition on tumour development driven by c-myc, we crossed the Pim-1 KD mice with the Em-cmyc transgenic mouse line, which expresses high levels of the oncoprotein c-myc leading to the development of a rapidly growing B-cell lymphoma that resembles the human Burkitt-lymphoma. Even the heterozygous switch to the kinase-dead form of Pim-1 doubles the survival of the Em-c-myc mice; the homozygous switch brings survival back close to normal. On the molecular level, we observed elevated p27-levels in tumours of heteroand homozygous Pim-1 kinase-dead mice carrying the Em-c-myc transgene. The tumours of these mice also displayed areas with reduced levels of c-myc (Western blot and IHC). Based upon these results, we set-up an allogenic Em-c-myc mouse model that allowed us to study the effect of kinase inhibitors in an efficient and standardized way. We can show that novel, selective and potent Pim-1 kinase inhibitors that have been developed in our department are able to efficiently inhibit c-myc-driven tumour growth, as proposed by the genetic ablation of Pim-1 kinase activity. In summary, the presented study proofs inhibition of Pim-1 kinase activity to be a promising strategy to inhibit cancer growth driven by c-myc, one of the most frequently deregulated oncogenes in human cancer.

Abstract 607: Targeting PIM1 kinase enhances 1,25-dihydroxyvitamin D3-mediated anti-tumor activity in bladder cancer cells

Abstract Bladder cancer is the 5th most common in the United States. 30% of cases are advanced stage requiring multimodality therapy at diagnosis and many which are first diagnosed when superficial will progress to more advanced disease. High recurrence and progression rate and drug resistance highlight the need for new therapeutic strategies to improve clinical outcomes. 1,25-dihydroxyvitamin D3 (1,25D3), the most active vitamin D metabolite, has demonstrated broad spectrum antitumor activities in vitro and in vivo. In addition, 1,25D3 potentiates the antitumor effects of common chemotherapeutic agents in several cancer cell types including bladder cancer. 24-hydroxylase, encoded by the CYP24A1 gene, plays a key role in degrading 1,25D3. Increased expression of CYP24A1 has been found in several human tumors. Several studies reveal that the protein kinase signaling pathways including PIM1 are involved in the regulation of CYP24A1 expression. The PIM kinases are a family of serine/threonine kinases (PIM-1, PIM-2, PIM-3) that have been associated with tumorigenesis and drug resistance. PIM1 has been extensively studied in lymphoma and upregulation of PIM kinase expression has been reported in many malignancies. We analyzed mRNA expression of CYP24A1 and PIM1 in 17 bladder cancer cell lines by qRT-PCR. We found that CYP24A1 expression is increased in 12 out of 17 bladder cancer cell lines and PIM1 expression is increased in 8 out of 17 bladder cancer cell lines compared to expression in a benign bladder cancer cell line. Therefore, we hypothesized that inhibition of PIM kinase activity would suppress CYP24A1 expression and thus enhancing 1,25D3-mediated anti-tumor activity. Using the PIM1 inhibitor, JP11646, we found that inhibition of PIM1 kinase activity reduces CYP24A1 expression at transcriptional level in bladder cancer cells RT112 and RT112D21 which express high level of PIM1 and CYP24A1. We further evaluated the efficacy of the PIM1 inhibitor alone and in combination with 1,25D3. Inhibition of PIM1 kinase activity by JP11646 reduces cell growth in a panel of bladder cancer cell lines in a dose-dependent manner as measured by MTT assays. MTT assays detected a significant inhibition of cell proliferation by 50% compared to the control when cells were treated with 128 nM of JP11646 for 72 h in 6 of bladder cancer cell lines tested. Furthermore, inhibition of PIM1 kinase activity enhances 1,25D3-mediated inhibitory effect on cell growth in bladder cancer cells RT112 and RT112D21. This study provides the first evidence that inhibition of PIM kinase activity may be an attractive therapeutic strategy to facilitate the optimization of antitumor therapy of vitamin D in bladder cancer. This study was supported by NIH/NCI grants 5R01CA067267 and 5R01CA095045 Citation Format: Wei Luo, Carmen M. Baldino, Justin Caserta, Yingyu Ma, Candace Johnson, Donald L. Trump. Targeting PIM1 kinase enhances 1,25-dihydroxyvitamin D3-mediated anti-tumor activity in bladder cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 607. doi:10.1158/1538-7445.AM2014-607

SGI-1776, an imidazo pyridazine compound, inhibits the proliferation of ovarian cancer cells by inactivating Pim-1.

To investigate the antitumor effect of SGI-1776 on human ovarian cancer HO-8910 cells and its molecular mechanism. HO-8910 cells were cultured in vitro, and the proliferation inhibitory effects of SGI- 1776 were determined by MTT assay and colony formation assay. The effect of SGI-1776 on the distribution of cell cycle phase was observed by flow cytometry with propidium iodide (PI) staining. The inhibition rate of migration and invasion were valued by transwell cell assay. Multiple molecular techniques, such as ELISA, Western blot, siRNA and cDNA transfection were used to explore the molecular mechanism. SGI-1776 presented dramatic anti-tumor activity against HO-8910 cells in vitro, inhibited the cells proliferation and colony formation, and attenuated the migration and invasion in a dosedependent manner, accompanied by cell cycle arrest in G1 phase. SGI-1776 caused the proliferation inhibition with concomitant decrease in Pim-1 kinase activity, down-regulated the expression of Pim-1 protein and and its downstream genes, such as CDK6, pCDK6, CDK4, pCDK4, CDK2 and pCDK2, and increased the expression of P21 and P27. Down-regulation expression of Pim-1 by siRNA followed SGI-1776 treatment resulted in enhanced cell proliferation inhibition rate and attenuated migration/invasion. Up-regulation of Pim-1 by cDNA transfection attenuated SGI- 1776-induced cell proliferation inhibition and its migration/invasion. Pim-1 mediates the biological effect of SGI-1776 in human ovarian cancer HO-8910 cells, suggesting Pim-1 might be a novel target for human ovarian cancer.

The Pim-1 protein kinase is an important regulator of MET receptor tyrosine kinase levels and signaling.

MET, the receptor for hepatocyte growth factor (HGF), plays an important role in signaling normal and tumor cell migration and invasion. Here, we describe a previously unrecognized mechanism that promotes MET expression in multiple tumor cell types. The levels of the Pim-1 protein kinase show a positive correlation with the levels of MET protein in human tumor cell lines and patient-derived tumor materials. Using small interfering RNA (siRNA), Pim knockout mice, small-molecule inhibitors, and overexpression of Pim-1, we confirmed this correlation and found that Pim-1 kinase activity regulates HGF-induced tumor cell migration, invasion, and cell scattering. The novel biochemical mechanism for these effects involves the ability of Pim-1 to control the translation of MET by regulating the phosphorylation of eukaryotic initiation factor 4B (eIF4B) on S406. This targeted phosphorylation is required for the binding of eIF4B to the eIF3 translation initiation complex. Importantly, Pim-1 action was validated by the evaluation of patient blood and bone marrow from a phase I clinical trial of a Pim kinase inhibitor, AZD1208. These results suggest that Pim inhibitors may have an important role in the treatment of patients where MET is driving tumor biology.

PIM1 kinase inhibitors induce radiosensitization in non-small cell lung cancer cells

Radiotherapy plays a critical role in the treatment of non-small cell lung cancer (NSCLC). However, radioresistance is a major barrier against increasing the efficiency of radiotherapy for NSCLC. To understand the mechanisms underlying NSCLC radioresistance, we previously focused on the potential involvement of PIM1, PRAS40, FOXO3a, 14-3-3, and protein phosphatases. Among these proteins, PIM1 functioned as an oncogene and was found to act as a crucial mediator in radioresistant NSCLC cells. Therefore, we investigated the use of PIM1-specific inhibitors as novel therapeutic drugs to regulate radiosensitivity in NSCLC. After structure-based drug selection, SGI-1776, ETP-45299, and tryptanthrin were selected as candidates of PIM1 inhibitors that act as radiosensitizers. With irradiation, these drugs inhibited only PIM1 kinase activity without affecting PIM1 mRNA/protein levels or cellular localization. When PIM1 kinase activity was suppressed by these inhibitors, PRAS40 was not phosphorylated. Consequently, unphosphorylated PRAS40 did not form trimeric complexes with 14-3-3 and FOXO3a, leading to increased nuclear localization of FOXO3a. Nuclear FOXO3a promoted the expression of pro-apoptotic proteins such as Bim and FasL, resulting in a radiosensitizing effect on radioresistant NSCLC cells. Moreover, an in vivo xenograft mouse model confirmed this radiosensitizing effect induced by PIM1 inhibitors. In these model systems, tumor volume was significantly reduced by a combinational treatment with irradiation and PIM1 inhibitors compared to irradiation alone. Taken together, our findings provided evidence that PIM1-specific inhibitors, SGI-1776, ETP-45299, and tryptanthrin, can act as novel radiosensitizers to enhance the efficacy of radiotherapy by inhibiting irradiation-induced signaling pathway associated with radioresistance.

The role of PIM kinases in human and mouse CD4+ T cell activation and inflammatory bowel disease

PIM kinases are a family of three serine/threonine kinases expressed following T cell activation. Using potent selective small molecule antagonists of PIM-1/3 kinases, we demonstrate a potential role for these enzymes in naïve and effector CD4+ T cell activation. PIM-1/3 inhibition prevented CD4+ T cell proliferation by inducing a G0/G1 cell cycle arrest without affecting cellular survival. In the absence of PIM-1/3 kinase activity, naïve CD4+ T cells failed to fully differentiate into effector cells both in vitro and in vivo. Therapeutic dosing of a PIM-1/3 inhibitor was efficacious in a CD4+ T cell-mediated model of inflammatory bowel disease suggesting that PIM-1 and PIM-3 kinase activity contributes to sustained disease severity. These results demonstrate that PIM-1/3 kinases have an important role in CD4+ T cell responses and inhibition of this activity may provide a therapeutic benefit in T cell-mediated diseases.

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

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