Overexpression Of Pim-1 Research Articles

Kinase PIM1 governs ferroptosis to reduce retinal microvascular endothelial cell dysfunction triggered by high glucose.

Previous studies have implicated targeting Pim-1 proto-oncogene, serine/threonine kinase (PIM1) as a preventive measure against high glucose-induced cellular stress and apoptosis. This study aimed to reveal the potential role and regulatory mechanism of PIM1 in diabetic retinopathy. Human retinal microvascular endothelial cells (hRMECs) underwent high glucose induction, and fluctuations in PIM1 levels were assessed. By overexpressing PIM1, its effects on the levels of inflammatory factors, oxidative stress indicators, migration and tube formation abilities, tight junction protein expression levels, and ferroptosis in hRMECs were identified. Afterwards, hRMECs were treated with the ferroptosis-inducing agent erastin, and the effect of erastin on the above PIM1 regulatory functions was focused on. PIM1 was downregulated upon high glucose, and its overexpression inhibited the inflammatory response, oxidative stress, cell migration, and tube formation potential in hRMECs, whereas elevated tight junction protein levels. Furthermore, PIM1 overexpression reduced intracellular iron ion levels, lipid peroxidation, and levels of proteins actively involved in ferroptosis. Erastin treatment reversed the impacts of PIM1 on hRMECs, suggesting the mediation of ferroptosis in PIM1 regulation. The current study has yielded critical insights into the role of PIM1 in ameliorating high glucose-induced hRMEC dysfunction through the inhibition of ferroptosis.

Glycyrrhetinic acid alleviates atrial fibrillation by inhibiting the expression of PIM1

AbstractGlycyrrhetinic acid (GA) is known to exert a cardioprotective effect, while its mechanism in atrial fibrillation (AF) remains poorly defined. Accordingly, isoprenaline (ISO) was applied to establish the rat AF model, and then GA (5, 10, and 20 mg/kg/day) was administrated to these rats for a total of 21 days. The fibrosis and pathological conditions of rat atrial tissues were observed. Quantitative real‐time polymerase chain reaction (qRT‐PCR) was adopted to calculate the levels of proto‐oncogene serine/threonine‐protein kinase Pim1 (PIM1) and Interleukin (IL)‐4/6/13/17. For in‐vitro experiments, isolated cardiac fibroblasts received the transfection with PIM1‐specific overexpression plasmid or siPIM1 and the treatment with ISO. Subsequently, cell viability was gauged by cell counting kit‐8 assay. The protein levels of alpha‐smooth muscle actin (α‐SMA), collagen I and III, transforming growth factor‐beta1 (TGF‐β1) and mothers against decapentaplegic homolog 7 (Smad7) were measured by western blot. GA led to the shortened duration of AF and alleviated myocardial fibrosis and atrial tissue lesions in ISO‐treated AF rats in vivo. Meanwhile, GA inhibited the expressions of PIM1 and IL‐4/6/13/17 in the atrial tissue of AF model rats. In the meantime, enhanced cell viability, inhibited expressions of PIM1, IL‐4/6/13/17, α‐SMA, collagen‐I, collagen‐III and TGF‐β1, and promoted Smad7 expression were observable in ISO‐treated cardiac fibroblasts in vitro. Oppositely, the effects of GA were reversed following PIM1 overexpression, while knockdown of PIM1 enhanced the effects of GA on cardiac fibroblasts. Collectively speaking, GA alleviates AF through suppressing the expression of PIM1.

Exploring the potential of baicalin and resveratrol as PIM-1 kinase inhibitors: Therapeutic targeting of prostate and breast cancers

PIM-1 kinase is a member of the Ser/Thr kinases family, a well-studied therapeutic target for cancer therapy. The overexpression of PIM-1 is associated with prostate and breast cancer progression. Therapeutic targeting of PIM-1 disrupts crucial signaling pathways which contributes to cancer growth and resistance to conventional treatments. Here, we screened 100 natural compounds with known antioxidant properties to discover potential inhibitors of PIM-1. Two natural compounds, baicalin, and resveratrol, were investigated for their binding affinity and PIM-1 inhibitory potential using integrated computational and experimental approaches. Baicalin and resveratrol bind to PIM-1 with a docking score of −10.2 and −7.5 kcal/mol, respectively. Subsequently, fluorescence binding studies showed an excellent affinity with Ka values, 5.9 × 105 M−1 and 1.7 × 107 M−1 for baicalin and resveratrol, respectively. In addition, baicalin and resveratrol strongly inhibited the kinase activity of PIM-1 with IC50 values of 36.8 μM and 75 μM, respectively. Furthermore, both baicalin and resveratrol suppress the proliferation of cancer cells in a dose-dependent manner, with baicalin’s IC50 value for LNCaP and MDA-MB-231 cells being 34.8 µM and 35.6 µM, respectively, and resveratrol’s being 15.2 µM and 41.6 µM, respectively. We conclude that baicalin and resveratrol may be considered potent PIM-1 inhibitors, opening promising possibilities for the development of anti-cancer therapy via targeting PIM-1 for prostate and breast cancer. Finally, utilizing natural compounds that offer a safer alternative to drive the development of more potent, specific, and pharmacokinetically optimized derivatives for therapeutic targeting of emerging diseases.

Abstract C144: MEN1703/SEL24, a potent PIM inhibitor, demonstrates promising anti-tumour activity in activated B cell like diffuse large B cell lymphoma, mantle cell lymphoma and marginal zone lymphoma cells

Abstract Background. PIM kinases (PIM1, PIM2, PIM3) are serine threonine kinases that function in multiple biological processes including cell cycle regulation, cell survival and proliferation. They also mediate resistance to anti-tumour agents comprising PI3K inhibitors. PIM1 overexpression and mutations are common in diffuse large B cell lymphoma (DLBCL) and PIM1 overexpression has been validated as a rational therapeutic target particularly in ABC and MCD subtypes. PIM1 overexpression is also found in mantle cell lymphoma (MCL). We investigated the pharmacological inhibition of PIM using the small molecule MEN1703/SEL24 in a large panel of lymphoma cell lines. Methods. Anti-proliferative activity was assessed by MTT assay at 72h: MEN1703/SEL24 was tested in 7 ABC-DLBCL, 19 germinal centre-B cell-like (GCB)-DLBCL, nine MCL, six marginal zone lymphoma (MZL), one primary mediastinal, four MZL ibrutinib-and PI3K-resistant models and one canine lymphoma. Cell cycle and apoptosis assays were done by flow cytometry. RNA-Seq was performed on two ABC- and two GCB-DLBCL cell lines treated with MEN1703/SEL24 or DMSO for 4, 8, 12h, followed by limma and functional annotation analysis with gene set enrichment analysis (GSEA). Results. MEN1703/SEL24 showed anti-proliferative activity in all lymphoma histotypes tested (IC50 median = 593 nM; range = 30 - 10,193 nM). MCL (IC50 median = 108 nM; range = 53 - 412 nM) and ABC-DLBCL (IC50median = 994 nM; range = 30 - 1756 nM) were the most sensitive histotypes; among them OCI-Ly-10 (30 nM) and OCI-Ly-3 (142 nM) had mutated PIM1. GCB-DLBCLs comprised the least sensitive cell lines (IC50 median = 961 nM; range = 252 - 10,193 nM) accounting for 8/11 cell lines with IC50 in the upper percentile. More importantly, MEN1703/SEL24 was active in MZL cell lines with acquired resistance to idelalisib, copanlisib and ibrutinib (IC50 median = 367 nM; range = 273 - 410 nM). MEN1703/SEL24 induced apoptosis in 6/6 cell lines and minor G1 arrest in 1/6 cell lines. In DLBCL cell lines more sensitive to MEN1703/SEL24 treatment, RNA-Seq and GSEA revealed differential modulation of immune, MYC and E2F targets. Conclusions. MEN1703/SEL24 shows robust preclinical activity in B cell lymphomas of different histotypes. RNA-Seq indicates that MEN1703/SEL24 modulates the transcriptome of highly responsive DLBCL cell lines differently from poorly responsive cells, providing novel clues to mechanisms involved in sensitivity to PIM inhibitors and supporting a potential for clinical development in this indication. Citation Format: Afua A. Mensah, Alessandro Ghiringhelli, Giulio Sartori, Filippo Spriano, Chiara Tarantelli, Luciano Cascione, Andrea Rinaldi, Alberto A. Arribas, Daniela Bellarosa, Monica Binaschi, Francesco Bertoni. MEN1703/SEL24, a potent PIM inhibitor, demonstrates promising anti-tumour activity in activated B cell like diffuse large B cell lymphoma, mantle cell lymphoma and marginal zone lymphoma cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C144.

PIM1 attenuates cisplatin-induced AKI by inhibiting Drp1 activation

Cisplatin, an effective anti-cancer drug, always causes acute kidney injury (AKI) by inducing mitochondrial damage. PIM1 is a serine/threonine kinase, which has been shown to regulate mitochondrial function. However, the role and mechanisms of PIM1 in cisplatin-induced AKI remain unexplored. This study aimed to investigate the effects of PIM1 in cisplatin-induced AKI and its underlying mechanisms. To established Cisplatin-induced AKI model, mice were given a single intraperitoneal injection(20 mg/kg) and BUMPT cells were treated with cisplatin(20 μM). PIM1 inhibitor AZD1208 was used to inhibit PIM1 and PIM1-experssing adenovirus was used to overexpress PIM1. Drp1 inhibitor P110 and pcDNA3-Drp1K38A were used to inhibit the activation of Drp1 and mitochondrial fission. The indicators of renal function, renal morphology, apoptosis and mitochondrial dysfunction were assessed to evaluate cisplatin-induced nephrotoxicity. We observed that PIM1 was activated in cisplatin-induced AKI in vivo and cisplatin-induced tubular cells injury in vitro. PIM1 inhibition aggravated cisplatin-induced AKI in vivo, while PIM1 overexpression attenuated cisplatin-induced kidney injury in vivo and in vitro. Moreover, inhibiting PIM1 exacerbated mitochondrial damage in mice, but overexpressing PIM1 relieved mitochondrial damage in mice and BUMPT cells. In mice and BUMPT cells, inhibiting PIM1 deregulated the expression of p-Drp1S637, overexpressing PIM1 upregulated the ex-pression of p-Drp1S637. And inhibiting Drp1 activity alleviated cell damage in BUMPT cells with PIM1 knockdown or inhibition. This study demonstrated the protective effect of PIM1 in cisplatin-induced AKI, and regulation of Drp1 activation might be the underlying mechanism. Altogether, PIM1 may be a potential therapeutic target for cisplatin-induced AKI.

Characterizing the role of PIM kinases in the prostate tumor immune microenvironment.

e17080 Background: The Proviral Integration site for Moloney murine leukemia virus (PIM) kinases, a family of pro-survival kinases ( PIM1, PIM2, and PIM3), are frequently overexpressed in prostate cancer (PC) and associated with PC aggressiveness and immune evasion. Preclinical data indicate that PIM1 overexpression increases macrophage infiltration and survival using in vivo models of PC. Here, we characterized the association of PIM1, PIM2, and PIM3 with PC immune signatures. Methods: 77 primary prostate and 11 metastatic lymph node samples from treatment-naive metastatic hormone-sensitive PC pts were analyzed by DNA next-generation sequencing (592-gene, NextSeq; WES, NovaSeq) and Whole Transcriptome Sequencing (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ). PC with PIM1/PIM2/PIM3-high(H) and -low(L) expression were classified by top and bottom quartile, respectively. Androgen receptor (AR) signature and Neuroendocrine Prostate Cancer (NEPC) score were calculated based on the expression level of previously defined gene signatures (Hieronymus et al. 2006, Beltran et al. 2016). Pathway enrichment was determined by GSEA (Broad Inst). Immune cell fractions were calculated by deconvolution of WTS using Quantiseq. Statistical significance was determined by chi-square and Mann-Whitney U (p < 0.05) and adjusted for multiple comparisons (q < 0.05). Results: PIM1/PIM2/PIM3-H PC had a higher median MAPK activation score (MPAS) compared to PIM1/PIM2/PIM3-L PC (0.9, 1.3, 0 vs -1.6, -1.4, -1.1 respectively, all p < 0.05). There was no difference in AR signature or NEPC score between PIM-H and PIM-L PC. PIM3-H PC exhibited higher PSA expression (3.15-fold, p < 0.05). PIM1/PIM2-H PC had higher AR expression (1.9 and 3.5-fold, respectively, all p < 0.05). PIM2/PIM3-H PC had enrichment of PI3K/AKT/mTOR, IL2/STAT5, IL6/JAK/STAT3, KRAS, TGFβ, NOTCH, hypoxia signaling, and ROS, P53, OXPHOS pathway and EMT (NES: 1.3-1.5, all FDR < 0.25). PIM1/PIM2-H PC had higher expression of immunostimulatory genes ( IL1β, TNF, and TNFSF13, FC: 1.1-3.1, p < 0.05). PIM1/PIM2/PIM3-H PC had higher expression of MHC class I ( HLA-A, HLA-B, HLA-C, B2M, FC: 1.3-2.5, all p < 0.05) and MHC class II ( HLA-DPA1, HLA-DRB1, HLA-DPB2, TAP1, TAP2, FC: 1.1-3.2, all p < 0.05) genes. PIM3-H PC had increased infiltration of M2 macrophages (8% vs 5.2%) and NK cells (5.2% vs 3.3%), while PIM2-H PC had increased Tregs (1.7% vs 0%) (all p < 0.05). Moreover , PIM1/PIM2/PIM3-H PC had a high T cell inflamed score compared to PIM1/PIM2/PIM3-L PC (66, 80, 56 vs -143, -157, -136, respectively, all p < 0.05). Conclusions: Our data suggest a strong association between PIM expression and increased MAPK activation score, T cell inflamed score, inflammatory, MHC class I and MHC class II gene expression, and differential immune cell infiltration. A better understanding of these differences will provide a rationale for tailored therapeutic approaches for PIM-expressing PC.

PIM1 phosphorylates ABI2 to enhance actin dynamics and promote tumor invasion.

Distinguishing key factors that drive the switch from indolent to invasive disease will make a significant impact on guiding the treatment of prostate cancer (PCa) patients. Here, we identify a novel signaling pathway linking hypoxia and PIM1 kinase to the actin cytoskeleton and cell motility. An unbiased proteomic screen identified Abl-interactor 2 (ABI2), an integral member of the wave regulatory complex (WRC), as a PIM1 substrate. Phosphorylation of ABI2 at Ser183 by PIM1 increased ABI2 protein levels and enhanced WRC formation, resulting in increased protrusive activity and cell motility. Cell protrusion induced by hypoxia and/or PIM1 was dependent on ABI2. In vivo smooth muscle invasion assays showed that overexpression of PIM1 significantly increased the depth of tumor cell invasion, and treatment with PIM inhibitors significantly reduced intramuscular PCa invasion. This research uncovers a HIF-1-independent signaling axis that is critical for hypoxia-induced invasion and establishes a novel role for PIM1 as a key regulator of the actin cytoskeleton.

PIM1 attenuates renal ischemia–reperfusion injury by inhibiting ASK1-JNK/P38

Renal ischemia-reperfusion injury (IRI) is the main cause of acute kidney injury (AKI), yet therapeutic approaches to alleviate IRI remain limited. PIM1 (provirus integration site for Moloney murine leukemia virus 1) is a constitutive serine threonine kinase that phosphorylates various substrates to regulate cell death and survival. However, the role of PIM1 in renal IRI remains unclear. This study aims to investigate the effect of PIM1 on renal IRI and explore its downstream regulatory mechanism. In this study, we inhibited or overexpressed PIM1 in mice and cultured proximal tubular cells, and then induced renal IRI model in vivo and hypoxia reoxygenation (HR) model in vitro. Renal function, renal structure injuries and cellular death were assessed to reflect the extent of IRI. The expression of PIM1 and the levels of ASK1, MAPK and their phosphorylated forms were detected by immunoblot. RNA sequencing of kidney cortex was performed to analyze downstream pathway of PIM1 in renal IRI. The results showed that PIM1 expression was significantly upregulated in renal IRI mouse model and in renal tubular cell HR model. AZD1208 (a PIM1 inhibitor) aggravated renal IRI, while PIM1 overexpression ameliorated renal IRI. This was involved in the regulation of the ASK1-MAPK pathway. Moreover, results demonstrated that ASK1 was a downstream target of PIM1 by administering Selonsertib (an inhibitor of ASK1 activity), and inhibiting ASK1 alleviated cell death after HR in PIM1 knockdown cells by reducing JNK/P38 activation. In conclusion, this study elucidated the protective effect of PIM1 on renal IRI, and the underlying mechanism may be related to ASK1-JNK/P38 signaling pathway. Taken together, PIM1 may be a potential therapeutic target for renal IRI.

Pim-1 kinase is a positive feedback regulator of the senescent lung fibroblast inflammatory secretome.

Cellular senescence is emerging as a driver of idiopathic pulmonary fibrosis (IPF), a progressive and fatal disease with limited effective therapies. The senescence-associated secretory phenotype (SASP), involving the release of inflammatory cytokines and profibrotic growth factors by senescent cells, is thought to be a product of multiple cell types in IPF, including lung fibroblasts. NF-κB is a master regulator of the SASP, and its activity depends on the phosphorylation of p65/RelA. The purpose of this study was to assess the role of Pim-1 kinase as a driver of NF-κB-induced production of inflammatory cytokines from low-passage IPF fibroblast cultures displaying markers of senescence. Our results demonstrate that Pim-1 kinase phosphorylates p65/RelA, activating NF-κB activity and enhancing IL-6 production, which in turn amplifies the expression of PIM1, generating a positive feedback loop. In addition, targeting Pim-1 kinase with a small molecule inhibitor dramatically inhibited the expression of a broad array of cytokines and chemokines in IPF-derived fibroblasts. Furthermore, we provide evidence that Pim-1 overexpression in low-passage human lung fibroblasts is sufficient to drive premature senescence, in vitro. These findings highlight the therapeutic potential of targeting Pim-1 kinase to reprogram the secretome of senescent fibroblasts and halt IPF progression.

Inhibition of CEBPB Attenuates Lupus Nephritis via Regulating Pim-1 Signaling.

Systemic lupus erythematosus (SLE) is an autoimmune disease leading to inflammatory damage in multiple target organs, and lupus nephritis (LN) is one of the most life-threatening organ manifestations. CCAAT/enhancer-binding protein β (CEBPB) regulates the NLRP3 inflammasome and is involved in the pathogenesis of SLE. However, the role and mechanism of CEBPB in LN remains unclear. MRL/lpr mice and lipopolysaccharides (LPS) combined with adenosine triphosphate- (ATP-) treated glomerular podocytes were used as models of LN in vivo and in vitro, respectively. In vivo, we investigated the expressions of CEBPB during the development of MRL/lpr mice. Then we assessed the effect of CEBPB inhibition on renal structure and function through injecting shCEBPB lentivirus into MRL/lpr mice. In vitro, glomerular podocytes were treated with Pim-1-OE and siCEBPB to explore the relation between CEBPB and Pim-1. The progression of LN in mice was associated with the increased level of CEBPB, and the inhibition of CEBPB ameliorated renal structure impairments and improved renal function damage associated with LN. Knockdown of CEBPB could suppress the activation of NLRP3 inflammasome and the secretion of IL-1β and IL-6. Furthermore, the knockdown of CEBPB could inhibit NLRP3 inflammasome activation and pyroptosis via binding to Pim-1 promoter to downregulate its expression, and the overexpression of Pim-1 reversed the effects of CEBPB deficiency. The regulation of CEBPB on Pim-1 facilitated pyroptosis by activating NLRP3 inflammasome, thereby promoting the development of LN.

Abstract 263: PIM kinase regulates actin dynamics in hypoxia

Abstract One of the leading factors in prostate cancer mortality is the onset of metastatic events. This is seen by the drastic decline in survival rates following tumor escape from the primary site. A key promoter of metastasis is tumor hypoxia, or low oxygen concentration. Our lab previously demonstrated that Proviral Integration site for Moloney murine leukemia virus (PIM)-1, a serine-threonine kinase, is upregulated in hypoxia and that inhibition of PIM1 reduced metastatic occurrence in orthotopic mouse models. Here, we identify a novel signaling pathway that links hypoxia and PIM kinase to the actin cytoskeleton and cell motility. An unbiased screen to discover new PIM substrates in hypoxia identified Abl-Interactor 2 (Abi2), an integral member of the actin regulating Wave Regulatory Complex (WRC), as a novel PIM kinase substrate. Biochemical analysis demonstrates that phosphorylation of Abi2 at our identified PIM sites increases Abi2 stability. Overexpression of PIM1 increases Abi2 and other WRC members at the protein level, resulting in increased actin protrusions and cell motility. CRISPR-generated Abi2 knockout cell lines showed diminished protrusive activity and negated both the necessity and sufficiency of PIM kinase to increases protrusive activity in cancer cells. Lastly, in vivo experiments show that overexpression of PIM1 increases tumor invasion into the surrounding tissue in an Abi2-dependent manner, and small molecule PIM inhibitors significantly reduce invasion. In summary, we uncovered a novel role for PIM1 in promoting cellular motility and protrusion, identified novel phosphorylation sites that control the stability of Abi2 in normoxic and hypoxic conditions, and highlight a novel PIM1/Abi2 signaling axis that regulates hypoxia-induced tumor invasion. This research provides important insight in the role of PIM signaling in prostate tumors and yields preclinical evidence for further use of PIM kinase inhibitors as a means to inhibit tumor invasion, a key driving step in the formation of lethal prostate cancer metastasis. Citation Format: Corbin C. Jensen, Noel A. Warfel. PIM kinase regulates actin dynamics in hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 263.

PIM1 is a Poor Prognostic Factor for and Potential Therapeutic Target in Serous Carcinoma of the Endometrium.

Serous carcinoma (SC) is an aggressive histologic type of endometrial carcinoma (EMC) with a poor prognosis. The development of novel therapeutics for SC is an important issue. PIM1 is a serine/threonine kinase involved in various cellular functions, such as cell cycle progression, apoptosis, and transcriptional activation via the phosphorylation of many target proteins, including MYC. PIM1 is overexpressed in several cancers and has been associated with treatment-resistance. We investigated the expression and function of PIM1 in EMC, particularly SC. Immunohistochemical analysis in 133 EMC cases [103 endometrioid carcinomas (EC) and 30 SC] revealed the significantly stronger expression of PIM1 in SC than in EC and significantly shorter survival of patients with overexpression of PIM1 in all EMC cases, as well as in only SC cases. A multivariate analysis identified overexpression of PIM1 as an independent prognostic factor. The knockdown of PIM1 by siRNA in the SC cell line, ARK1, decreased the expression of phosphorylated MYC and reduced proliferation, migration, and invasion. The PIM1 inhibitor, SGI-1776, reduced cell viability in SC cell lines (ARK1, ARK2, and SPAC1L) with IC50 between 1 and 5 µM. SGI-1776 also reduced the migration and invasion of ARK1 cells. Moreover, the oral administration of SGI-1776 significantly suppressed subcutaneous ARK1 xenograft tumor growth in nude mice without impairing health. These results indicate that PIM1 is involved in the acquisition of aggressiveness and suggest the potential of PIM1 as a novel therapeutic target and SGI-1776 as a therapeutic agent for SC.

Transcriptional analysis of lung fibroblasts identifies PIM1 signaling as a driver of aging-associated persistent fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by myofibroblast accumulation and progressive lung scarring. To identify transcriptional gene programs driving persistent lung fibrosis in aging, we performed RNA-Seq on lung fibroblasts isolated from young and aged mice during the early resolution phase after bleomycin injury. We discovered that, relative to injured young fibroblasts, injured aged fibroblasts exhibited a profibrotic state characterized by elevated expression of genes implicated in inflammation, matrix remodeling, and cell survival. We identified the proviral integration site for Moloney murine leukemia virus 1 (PIM1) and its target nuclear factor of activated T cells-1 (NFATc1) as putative drivers of the sustained profibrotic gene signatures in injured aged fibroblasts. PIM1 and NFATc1 transcripts were enriched in a pathogenic fibroblast population recently discovered in IPF lungs, and their protein expression was abundant in fibroblastic foci. Overexpression of PIM1 in normal human lung fibroblasts potentiated their fibrogenic activation, and this effect was attenuated by NFATc1 inhibition. Pharmacological inhibition of PIM1 attenuated IPF fibroblast activation and sensitized them to apoptotic stimuli. Interruption of PIM1 signaling in IPF lung explants ex vivo inhibited prosurvival gene expression and collagen secretion, suggesting that targeting this pathway may represent a therapeutic strategy to block IPF progression.

Targeting an IL-6/JAK/STAT/PIM1 pathway in renal cell carcinoma.

376 Background: Renal cell carcinoma (RCC) is the most common cancer of the kidney and accounts for over 430,000 new cases worldwide and 179,000 deaths each year. PIM1 overexpression is associated with poor clinical outcomes in patients with RCC. PIM1 is a constitutively active effector serine/threonine kinase with roles in tumor progression including cell proliferation, apoptosis, invasion, and migration. Yet, the mechanisms that underlie PIM1 expression and its function in RCC are not fully understood. Previous studies have implicated the cytokine IL-6 as an activator of STAT3 and stimulator of PIM1 expression in pancreatic cancer. The aim of the present study is to explore expression of PIM1 in RCC and identify potential signaling pathways that influence PIM1 expression. Methods: To evaluate the contribution of PIM1 expression in RCC we first established baseline PIM1 levels across four RCC cell lines (786-O, 769-P, CAKI-1, and RCC-4). Next, we quantified the presence of IL-6 in cell culture supernatants for each cell line using an ELISA assay. IL-6 signals through the JAK/STAT pathway, consequently we then used immunoblotting to determine whether ruxolitinib, a JAK1/2 inhibitor, would affect PIM1 expression. Finally, we examined cell viability post-ruxolitinib therapy using an MTS assay. Results: Our results show that 769-P, 786-O, and CAKI-1 but not RCC-4 cells overexpress PIM1 relative to normal renal proximal tubule epithelial cells. Results from the ELISA assay show that 786-O, CAKI-1, and to a lesser degree RCC-4 cells secrete IL-6 into their media. Interestingly, 769-P cells had little to no IL-6 present in their media. Based on these differences we sought to understand what effect inhibiting JAK may have on PIM1 expression. After treating cells with ruxolitinib, we found that 786-O and CAKI-1 cells show a time and dose dependent decrease in PIM1 levels whereas 769-P cells did not. To understand if these differences correlate with cell viability, we performed an MTS assay after treating cells with ruxolitinib. Preliminary results show that at 8hr post-treatment, CAKI-1 but not 769-P cells display a time and concentration dependent loss of cell viability. Conclusions: Our initial studies suggest that differential expression of PIM1 among RCC cell lines may be linked to autocrine IL-6 secretion. In CAKI-1 cells JAK inhibition decreases PIM1 expression and cell viability. In the context of 769-P cells with high PIM1 expression but low IL-6 secretion levels, JAK inhibition did not affect cell viability. These data suggest that in 769-P cells there exists a non-IL-6 dependent mechanism stimulating PIM1 expression. Further investigation is required to elucidate the contexts of these PIM1 regulatory pathways in order to develop IL-6/JAK/PIM1 targeted anti-cancer strategies in RCC.

PDGF-BB promotes vascular smooth muscle cell migration by enhancing Pim-1 expression via inhibiting miR-214.

BackgroundSeveral studies have indicated that the platelet-derived growth factor/platelet-derived growth factor receptor (PDGF/PDGFR) pathway is involved in the process of atherosclerosis. However, its underlying mechanism remains to be further elucidated. Serine/threonine-protein kinase pim-1 (Pim-1), a member of serine/threonine-specific kinases, is a pro-oncogene published to be related to cell proliferation, apoptosis, and metastasis of cancer cells. Whether Pim-1 is involved in PDGF/PDGFR pathway-mediated coronary atherosclerotic heart disease remains to be elucidated.MethodsWe established a cell model of PDGF-BB-stimulated smooth muscle cells using A7r5 cells. Transwell assay was used to detect the potential of cell migration and invasion. The targeted regulation of Pim-1 by miR-214 was confirmed by luciferase assay. Rescue experiments were performed to determine the role of the PDGF-BB/miR-214/Pim-1 axis on the cell migration of smooth muscle cells by including PDGF-BB treatment, and the overexpression of miR-214 and Pim-1. Quantitative polymerase chain reaction (qPCR) was used to examine the gene expression and western blot was performed to detect the protein expression.ResultsOur data indicated that PDGF-BB could effectively enhance smooth muscle cell migration. We also showed Pim-1 was a target of miR-214 in A7r5 cells. The expression of Pim-1 was shown to be upregulated by PDGF-BB via suppression of the expression of miR-214. Moreover, overexpression miR-214 inhibited PDGF-BB-stimulated Pim-1 expression and smooth muscle cell migration via modulating epithelial-mesenchymal transition (EMT), but no change on cell cycle. However, overexpression of Pim-1 reversed miR-214-blocked cell migration by promoting the activation of the STAT3, AKT, and ERK signaling pathways.ConclusionsOur data suggested that the PDGF/miR-214/Pim-1 axis could be a potential target for coronary atherosclerotic heart disease.

Ventilagolin Suppresses Migration, Invasion and Epithelial-Mesenchymal Transition of Hepatocellular Carcinoma Cells by Downregulating Pim-1

ObjectiveInhibition of tumor metastasis is a useful strategy to improve the efficacy of cancer therapy. Ventilagolin, a natural 1, 4-naphthoquinone derivative extracted from Ventilago leiocarpa Benth, has shown promising antitumor effects in previous studies. However, the effects and underlying mechanisms of Ventilagolin against migration, invasion and epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma (HCC) remain unclear. The present study has examined these effects and determined whether the proto-oncogene Pim-1 is involved.MethodsThe effects of Ventilagolin on migration, invasion, Pim-1 and EMT-related proteins (eg, E-cadherin, N-cadherin, Vimentin) expression were assessed by scratch wound healing, Transwell, qRT-PCR and Western blot assays, respectively. Pim-1 stably overexpressed HepG2 and SMMC-7721 cells were generated to explore whether Ventilagolin inhibited migration, invasion and EMT of HCC cells via regulating Pim-1. Subcutaneous xenograft tumor model in nude mice was established. Histopathological changes of tumor tissues were examined by H&E staining and expressions of Pim-1 and EMT-related proteins were detected by immunohistochemistry.ResultsVentilagolin significantly (P < 0.01) reduced the expression of Pim-1 levels in HepG2 and SMMC-7721 cells. Compared with the control group, the migration and invasion abilities of Pim-1-overexpressing HepG2 and SMMC-7721 cells were significantly (P < 0.05, P < 0.01) enhanced, the expression of E-cadherin was decreased (P < 0.01), and the levels of N-cadherin and Vimentin were upregulated (P < 0.05, P < 0.01). Ventilagolin treatment effectively reversed these effects of Pim-1 overexpression. In vivo experiments showed that Ventilagolin could effectively suppress HCC tumor growth, downregulate Pim-1, N-cadherin and Vimentin expression, and upregulate E-cadherin expression.ConclusionVentilagolin suppresses HCC cell proliferation, migration and invasion and reverses EMT process by downregulating Pim-1, suggesting Ventilagolin is a potential therapeutic agent for treatment of HCC.

Phytoconstituents and Medicinal Plants for Anticancer Drug Discovery: Computational Identification of Potent Inhibitors of PIM1 Kinase.

Natural products, medicinal plants, and phytoconstituents serve as important sources and accelerators for anticancer drug discovery, especially when they are combined with virtual screening and molecular simulations against molecular drug targets. Proto-oncogene serine/threonine-protein kinase Pim1 (PIM1) is involved in cell survival and proliferation, with great relevance for oncogenesis. PIM1 plays a major role in the progression of various common complex human cancers, including prostate cancer, acute myeloid leukemia, and other hematopoietic malignancies. The overexpression of PIM1 leads to cancer progression, and thus it is considered as a potential target for drug design and development purposes. Here, we report original in silico findings by employing structure-based virtual screening to discover potential phytoconstituents from the medicinal plants-based compounds, which could inhibit the PIM1 activity, using the IMPPAT (a curated database of Indian Medicinal Plants, Phytochemistry And Therapeutics) database. The initial hits were selected based on their binding affinity toward PIM1 calculated through the molecular docking approach. Subsequently, interaction analyses and molecular dynamics (MD) simulation for 100 ns was carried out to study the conformational dynamics and complex stability of PIM1 with the identified compounds. Importantly, we found that PIM1 forms stable protein-ligand complexes with the phytoconstituents Dehydrotectol and Nordracorubin in particular. Our findings suggest that identified phytoconstituents Dehydrotectol and Nordracorubin bind to PIM1 in ATP-competitive binding mode. These findings and the compounds reported herein warrant further exploration as promising scaffolds for anticancer drug design, discovery, and development.

Decreased expression of miR-3135b reduces sensitivity to 5-fluorouracil in colorectal cancer by direct repression of PIM1.

5-Fluorouracil (5-FU)-based chemotherapy is the conventional treatment approach for patients with colorectal cancer (CRC). However, de novo and acquired resistance to 5-FU are frequently observed during treatment, which eventually lead to patients succumbing to the disease. Accumulating data have revealed an association of CRC resistance to 5-FU with aberrant expression of microRNAs (miRs). In the present study, Cell Counting Kit-8 was performed to measure cell viability, flow cytometry was performed to detect cell apoptosis, reverse transcription-quantitative PCR was conducted to measure proviral integration site for Moloney murine leukemia virus 1 (PIM1) and miR-3135b expression, western blotting was conducted to measure PIM1 expression. Microarray data analysis indicated that the level of miR-3135b expression was decreased in patients with recurrent CRC that were treated with 5-FU when compared with non-recurrent cases. Overexpression of miR-3135b increased the sensitivity of CRC cells to 5-FU treatment. Moreover, PIM1 was identified as a target gene of miR-3135b using bioinformatics analysis, reverse transcription-quantitative PCR and western blotting. The direct interaction between these two targets was confirmed by luciferase reporter assays. Notably, PIM1 overexpression compensated the effect of miR-3135b in CRC cells. Furthermore, an inverse correlation between PIM1 mRNA expression levels and miR-3135b expression was observed in clinical samples. Therefore, the present study identified miR-3135b as a novel regulator of 5-FU sensitivity in CRC.

Pim1 Kinase Inhibitors Exert Anti-Cancer Activity Against HER2-Positive Breast Cancer Cells Through Downregulation of HER2.

The proviral integration site for moloney murine leukemia virus 1 (Pim1) is a serine/threonine kinase and able to promote cell proliferation, survival and drug resistance. Overexpression of Pim1 has been observed in many cancer types and is associated with the poor prognosis of breast cancer. However, it remains unclear whether Pim1 kinase is a potential therapeutic target for breast cancer patients. In this study, we found that Pim1 expression was strongly associated with HER2 expression and that HER2-overexpressing breast cancer cells were more sensitive to Pim1 inhibitor-induced inhibitions of cell viability and metastatic ability. Mechanistically, Pim1 inhibitor suppressed the expression of HER2 at least in part through transcriptional level. More importantly, Pim1 inhibitor overcame the resistance of breast cancer cells to HER2 tyrosine kinase inhibitor lapatinib. In summary, downregulation of HER2 by targeting Pim1 may be a promising and effective therapeutic approach not only for anti-cancer growth but also for circumventing lapatinib resistance in HER2-positive breast cancer patients.

Preliminary Results from an Ongoing Phase 1/2 Study of INCB053914, a Pan-Proviral Integration Sites for Moloney Virus (PIM) Kinase Inhibitor, in Patients with Advanced Hematologic Malignancies

Preliminary Results from an Ongoing Phase 1/2 Study of INCB053914, a Pan-Proviral Integration Sites for Moloney Virus (PIM) Kinase Inhibitor, in Patients with Advanced Hematologic Malignancies