Pim-1 Expression Research Articles

PIM2 kinase has a pivotal role in plasmablast generation and plasma cell survival, opening up novel treatment options in myeloma

AbstractThe differentiation of B cells into plasmablasts (PBs) and then plasma cells (PCs) is associated with extensive cell reprogramming and new cell functions. By using specific inhibition strategies (including a novel morpholino RNA antisense approach), we found that early, sustained upregulation of the proviral integrations of Moloney virus 2 (PIM2) kinase is a pivotal event during human B-cell in vitro differentiation and then continues in mature normal and malignant PCs in the bone marrow. In particular, PIM2 sustained the G1/S transition by acting on CDC25A and p27Kip1 and hindering caspase 3–driven apoptosis through BAD phosphorylation and cytoplasmic stabilization of p21Cip1. In PCs, interleukin-6 triggered PIM2 expression, resulting in antiapoptotic effects on which malignant PCs were particularly dependent. In multiple myeloma, pan-PIM and myeloid cell leukemia-1 (MCL1) inhibitors displayed synergistic activity. Our results highlight a cell-autonomous function that links kinase activity to the newly acquired secretion ability of the PBs and the adaptability observed in both normal and malignant PCs. These findings should finally prompt the reconsideration of PIM2 as a therapeutic target in multiple myeloma.

Relationship between the transcriptional expression of PIM1 and local control in patients with head and neck squamous cell carcinomas treated with radiotherapy

PurposeProviral integration site for Moloney murine leukemia virus (PIMs) are proto-oncogenes encoding serine/threonine kinases that phosphorylate a variety of substrates involved in the regulation of cellular processes. Elevated expression of PIM-1 has been associated with poor prognosis in several types of cancer. There are no studies that have analyzed the response to radiotherapy in patients with head and neck squamous cell carcinoma (HNSCC) according to the expression of PIM-1. The aim of our study was to analyze the relationship between the transcriptional expression of PIM-1 and local response to radiotherapy in HNSCC patients.MethodsWe determined the transcriptional expression of PIM-1 in 135 HNSCC patients treated with radiotherapy, including patients treated with chemoradiotherapy (n = 65) and bioradiotherapy (n = 15).ResultsDuring the follow-up, 48 patients (35.6%) had a local recurrence of the tumor. Patients with local recurrence had a higher level of PIM-1 expression than those who achieved local control of the disease (P = 0.017). Five-year local recurrence-free survival for patients with a high expression of PIM-1 (n = 43) was 44.6% (95% CI 29.2–60.0%), and for patients with low expression (n = 92) it was 71.9% (95% CI 62.5–81.3%) (P = 0.007). According to the results of multivariate analysis, patients with a high PIM-1 expression had a 2.2-fold increased risk of local recurrence (95% CI 1.22–4.10, P = 0.009).ConclusionPatients with elevated transcriptional expression levels of PIM-1 had a significantly higher risk of local recurrence after radiotherapy.

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.

Genetic ablation of Pim1 or pharmacologic inhibition with TP-3654 ameliorates myelofibrosis in murine models.

Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasm (MPN). The JAK inhibitor Ruxolitinib can reduce constitutional symptoms but it does not substantially improve bone marrow fibrosis. Pim1 expression is significantly elevated in MPN/MF hematopoietic progenitors. Here, we show that genetic ablation of Pim1 blocked the development of myelofibrosis induced by Jak2V617F and MPLW515L. Pharmacologic inhibition of Pim1 with a second-generation Pim kinase inhibitor TP-3654 significantly reduced leukocytosis and splenomegaly, and attenuated bone marrow fibrosis in Jak2V617F and MPLW515L mouse models of MF. Combined treatment of TP-3654 and Ruxolitinib resulted in greater reduction of spleen size, normalization of blood leukocyte counts and abrogation of bone marrow fibrosis in murine models of MF. TP-3654 treatment also preferentially inhibited Jak2V617F mutant hematopoietic progenitors in mice. Mechanistically, we show that TP-3654 treatment significantly inhibits mTORC1, MYC and TGF-β signaling in Jak2V617F mutant hematopoietic cells and diminishes the expression of fibrotic markers in the bone marrow. Collectively, our results suggest that Pim1 plays an important role in the pathogenesis of MF, and inhibition of Pim1 with TP-3654 might be useful for treatment of MF.

Expression of Oncogenic Molecules in Pediatric Ulcerative Colitis

Introduction: Long-term disease duration of ulcerative colitis (UC) is known to increase the risk of developing colorectal cancer in adults; however, this association has not been genetically analyzed in children with UC. Herein, we examined the expression of cancer-related genes in the colonic mucosa of pediatric UC patients and their risk of developing colorectal cancer. Methods: Microarray analysis of cancer-related gene expression was conducted on rectal mucosa biopsy specimens randomly selected from pediatric cases, including 4 active-phase UC cases, 3 remission-phase UC cases, and 3 irritable bowel syndrome control cases. The subject pool was then expanded to 10 active-phase cases, 10 remission-phase cases, and 10 controls, which were analyzed by real-time polymerase chain reaction (PCR) and immunohistochemical staining. Results: The microarray results indicated significantly higher expression levels of cancer-related genes PIM2 and SPI1 in the active group than in the remission and control groups (p < 0.05). Real-time PCR confirmed that PIM2 and SPI1 expression levels were significantly higher, whereas TP53 and APC expression levels were significantly lower, in the active-phase group than in the remission and control groups (p < 0.05). Immunohistochemical staining for PIM2, SPI1, TP53, and APC proteins supported the real-time PCR results. Conclusions: Expression levels of previously unreported cancer-related genes in adult UC patients were significantly higher in pediatric UC patients than in controls. Inflammation of the gastrointestinal mucosa increased the expression levels of cancer-related genes even in childhood-onset UC cases, suggesting that chronic inflammation from childhood may increase the risk of colorectal cancer development.

PIM1 Inhibition Affects Glioblastoma Stem Cell Behavior and Kills Glioblastoma Stem-like Cells.

Despite comprehensive therapy and extensive research, glioblastoma (GBM) still represents the most aggressive brain tumor in adults. Glioma stem cells (GSCs) are thought to play a major role in tumor progression and resistance of GBM cells to radiochemotherapy. The PIM1 kinase has become a focus in cancer research. We have previously demonstrated that PIM1 is involved in survival of GBM cells and in GBM growth in a mouse model. However, little is known about the importance of PIM1 in cancer stem cells. Here, we report on the role of PIM1 in GBM stem cell behavior and killing. PIM1 inhibition negatively regulates the protein expression of the stem cell markers CD133 and Nestin in GBM cells (LN-18, U-87 MG). In contrast, CD44 and the astrocytic differentiation marker GFAP were up-regulated. Furthermore, PIM1 expression was increased in neurospheres as a model of GBM stem-like cells. Treatment of neurospheres with PIM1 inhibitors (TCS PIM1-1, Quercetagetin, and LY294002) diminished the cell viability associated with reduced DNA synthesis rate, increased caspase 3 activity, decreased PCNA protein expression, and reduced neurosphere formation. Our results indicate that PIM1 affects the glioblastoma stem cell behavior, and its inhibition kills glioblastoma stem-like cells, pointing to PIM1 targeting as a potential anti-glioblastoma therapy.

Pim-2 kinase inhibits inflammation by suppressing the mTORC1 pathway in atherosclerosis.

Background: Inflammatory immunity theory has raised considerable concern in the pathogenesis of atherosclerosis. Proviral integration site of murine 2 (Pim-2) kinases functions in apoptosis pathways and the anti-inflammatory response. Here, we investigated whether Pim-2 kinase inhibits atherosclerotic inflammation by suppressing the mTORC1 pathway.Methods: An atherosclerosis animal model was established by feeding ApoE -/- mice a high-fat diet. THP-1-derived macrophages were subjected to ox-LDL (50 μg/ml, 24h) conditions in vitro to mimic the in vivo conditions.Result: The protein expression of Pim-2 was upregulated in ox-LDL-treated THP-1-derived macrophages and an atherosclerotic mouse model. Additionally, ox-LDL upregulated the protein expression of p-mTOR, p-S6K1 and p-4EBP1, intracellular lipid droplets, free cholesterol and cholesterylester and the mRNA expression of inflammatory cytokines, including IL-6, MCP-1, TLR-4 and TNF-α, in THP-1-derived macrophages. Functionally, overexpressed Pim-2 (Pim-2 OE) attenuated atherosclerotic inflammation associated with the mTORC1 signaling pathway in vitro and in vivo, whereas knocked down Pim-2 (Pim-2 KD) markedly promoted atherosclerotic inflammation associated with upregulation of the mTORC1 signaling pathway. The plaque areas and lesions in the whole aorta and aortic root sections were alleviated in ApoE -/- mice with Pim-2 OE, but aggravated by Pim-2 KD. Additionally, an mTOR agonist (MHY1485) counteracted the anti-inflammatory effect of Pim-2 in ox-LDL-treated THP-1-derived macrophages after Pim-2 OE, whereas rapamycin rescued atherosclerotic inflammation in ox-LDL-treated THP-1-derived macrophages after Pim-2 KD. Furthermore, si-mTOR and si-Raptor alleviated the atherosclerotic proinflammatory effect in ox-LDL-treated THP-1-derived macrophages in a the background of Pim-2 KD.Conclusions:These results indicated that Pim-2 kinase inhibits atherosclerotic inflammation by suppressing the mTORC1 pathway.

Hypoxia-activated platelets stimulate proliferation and migration of pulmonary arterial smooth muscle cells by phosphatidylserine/LOX-1 signaling-impelled intercellular communication

Continuous recruitment and inappropriate activation of platelets in pulmonary arteries contribute to pulmonary vascular remodeling in pulmonary hypertension (PH). Our previous study has demonstrated that lectin like oxidized low-density lipoprotein receptor-1 (LOX-1) regulates the proliferation of pulmonary arterial smooth muscle cells (PASMCs). Phosphatidylserine exposed on the surface of activated platelets is a ligand for LOX-1. However, whether hypoxia-activated platelets stimulate the proliferation and migration of PASMCs by phosphatidylserine/LOX-1 signaling-impelled intercellular communication remains unclear. The present study found that rats treated with hypoxia (10% O2) for 21 days revealed PH with the activation of platelets and the recruitment of platelets in pulmonary arteries, and LOX-1 knockout inhibited hypoxia-induced PH and platelets activation. Notably, co-incubation of PASMCs with hypoxic PH rats-derived platelets up-regulated LOX-1 expression in PASMCs leading to the proliferation and migration of PASMCs, which was inhibited by the phosphatidylserine inhibitor annexin V or the LOX-1 neutralizing antibody. LOX-1 knockout led to decreased proliferation and migration of PASMCs stimulated by hypoxia-activated platelets. In rats, hypoxia up-regulated the phosphorylation of signal transducer and activator of transcription 3 (Stat3) and the expression of Pim-1 in pulmonary arteries. Hypoxia-activated platelets also up-regulated the phosphorylation of Stat3 and the expression of Pim-1 in PASMCs, which was inhibited by annexin V, the LOX-1 neutralizing antibody, the protein kinase C inhibitor and LOX-1 knockout. In conclusion, we for the first time demonstrated that hypoxia-activated platelets stimulated the proliferation and migration of PASMCs by phosphatidylserine/LOX-1/PKC/Stat3/Pim-1 signaling-impelled intercellular communication, thereby potentially contributing to hypoxic pulmonary vascular remodeling.

PIM2 Promotes Epithelial–Mesenchymal Transition via Phosphorylates β-Catenin in Ovarian Endometriosis

Background: Endometriosis is a common and benign gynecological disease but has characteristics similar to the malignant tumor. The underlying pathogenesis and pathophysiology of endometriosis remain unclear. Methods: Immunohistochemistry was used to exam the expression of PIM2 and EMT related markers in tissues. Co-immunoprecipitated and western blot were used to verify the interaction between PIM2 and β-catenin in endometriosis cells and the molecular mechanism by which PIM2 phosphorylates β-catenin in regulating the protein function. PIM2 deficient endometriosis mice were used to demonstrate the role of PIM2 and EMT in vivo. Findings: Through immunohistochemistry staining, we found significant upregulation of PIM2 in ectopic endometrium compared with eutopic and normal endometrium, and associated with the expression of EMT related markers. PIM2 can directly bind and change the phosphorylation of β-catenin at Ser675 site to enhanceβ-catenin protein stability. Then increased β-catenin signaling transcriptional activity and expression of dowestream genes such as Cyclin-D1 and c-Myc. Functionally, PIM2 promoted invasion, migration and EMT process through β-catenin signaling in vitro. Moreover, in endometriosis mouse model, when uterus fragments from donor PIM2 deficient (PIM2d/d) transplanted to the abdominal cavity of recipient wide-type or PIM2d/d mice, endometriotic lesions growth and EMT process was decreased. Interpretation: PIM2/β-catenin signaling plays an important role in the pathophysiology of endometriosis. Inhibiting the PIM2 can be a therapeutic target for endometriosis. Fundings: This research was supported by funding from the National Natural Science Foundation of China (No. 81602301 and 81972489), Projects of medical and health technology development program in Shandong Province (No. 2017WS398 ). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The Human Investigation Ethical Committee of Affiliated Hospital of Weifang medical University approved this study. The experiment was approved by the institutional experimental animals review board of Weifang Medical University.

MPN-161: A Phase I Study of TP-3654, an Oral PIM Kinase Inhibitor, in Patients with Intermediate-2 or High-Risk Primary or Secondary Myelofibrosis (MF)

Context: MF is characterized by bone marrow fibrosis (BMF), ineffective hematopoiesis, splenomegaly, and debilitating symptoms. Expression of PIM-1 is significantly upregulated in MF hematopoietic cells, indicating that PIM-1 is a potential therapeutic target in MF. TP-3654, an oral small-molecule investigational PIM-1 kinase inhibitor with favorable selectivity against other kinases, has been shown to inhibit proliferation and increase apoptosis in murine and human hematopoietic cells expressing the JAK2V617F mutation. TP-3654 alone and in combination with ruxolitinib normalizes leukocyte counts and reduces spleen size and BMF in JAK2V617F and MPLW515L murine MF models. Objective: To evaluate TP-3654 monotherapy in patients with MF. Design: Phase I, open-label, dose-escalation study (NCT04176198). Setting: Multicenter, US only. Patients or Other Participants: Key eligibility criteria include primary or secondary MF (post-polycythemia vera-MF/post-essential thrombocythemia-MF); intermediate-2/high-risk MF per Dynamic International Prognostic Scoring System (DIPSS); previously failed or are ineligible to receive Janus kinase inhibitors (e.g., ruxolitinib, fedratinib); grade ≥2 BMF; platelet count ≥50x109/L; absolute neutrophil count ≥1x109/L; peripheral blood blast counts Interventions: Patients receive oral TP-3654. Dose escalation, performed using a Bayesian Logistic Regression Model with Escalation with Overdose Control, will continue until identification of the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D) is achieved. Dose expansion will follow. Main Outcomes Measures: Primary objectives: to determine MTD and/or identify the RP2D of TP-3654 monotherapy. Secondary objectives: to assess preliminary activity (spleen volume reduction, symptom improvement, and BMF reduction), determine QT interval changes, and establish the pharmacokinetic profile. Exploratory objectives: to study pharmacodynamic markers in peripheral blood and BM biopsies. This study is currently recruiting.

Pim1 knockout alleviates sarcopenia in aging mice via reducing adipogenic differentiation of PDGFRα+ mesenchymal progenitors.

BackgroundSarcopenia widely exists in elderly people and triggers numerous age‐related events. The essential pathologic change lies in the increased intramuscular adipose tissue after aging with no exception to non‐obese objects. Pim1 appears to be associated with adipogenic differentiation in recent studies, inspiring us to explore whether it regulates adipogenesis in aging muscles and affects sarcopenia.MethodsWild‐type and Pim1 knockout C57/BL6J mice were randomized into young and old groups. Histo‐pathological and molecular biological methods were applied to assess the intramuscular adipose tissue content, the atrophy and regeneration, and the expressions of Pim1 and adipogenic transcription factors. PDGFRα+ mesenchymal progenitors were separated and their replicative aging model were established. Different time of adipogenic induction and different amounts of Pim1 inhibitor were applied, after which the adipogenic potency were evaluated. The expressions of Pim1 and adipogenic transcription factors were measured through western blotting.ResultsThe aging mice demonstrated decreased forelimb grip strength (P = 0.0003), hanging impulse (P < 0.0001), exhaustive running time (P < 0.0001), tetanic force (P = 0.0298), lean mass (P = 0.0008), and percentage of gastrocnemius weight in body weight (P < 0.0001), which were improved by Pim1 knockout (P = 0.0015, P = 0.0222, P < 0.0001, P = 0.0444, P = 0.0004, and P = 0.0003, respectively). To elucidate the mechanisms, analyses showed that Pim1 knockout decreased the fat mass (P = 0.0005) and reduced the intramuscular adipose tissue content (P = 0.0008) by inhibiting the C/EBPδ pathway (P = 0.0067) in aging mice, resulting in increased cross‐sectional area of all and fast muscle fibres (P = 0.0017 and 0.0024 respectively), decreased levels of MuRF 1 and atrogin 1 (P = 0.0001 and 0.0329 respectively), and decreased content of Pax7 at the basal state (P = 0.0055). In vitro, senescent PDGFRα+ mesenchymal progenitors showed significantly increased the intracellular adipose tissue content (OD510) compared with young cells after 6 days of adipogenic induction (P < 0.0001). The Pim1 expression was elevated during adipogenic differentiation, and Pim1 inhibition significantly reduced the OD510 in senescent cells (P = 0.0040) by inhibiting the C/EBPδ pathway (P = 0.0047).ConclusionsPim1 knockout exerted protective effects in sarcopenia by inhibiting the adipogenic differentiation of PDGFRα+ mesenchymal progenitors induced by C/EBPδ activation and thus reducing the intramuscular adipose tissue content in aging mice. These results provide a potential target for the treatment of sarcopenia.

Myeloid-Derived Growth Factor Protects Against Pressure Overload–Induced Heart Failure by Preserving Sarco/Endoplasmic Reticulum Ca 2+ -ATPase Expression in Cardiomyocytes

Background: Inflammation contributes to the pathogenesis of heart failure, but there is limited understanding of inflammation’s potential benefits. Inflammatory cells secrete MYDGF (myeloid-derived growth factor) to promote tissue repair after acute myocardial infarction. We hypothesized that MYDGF has a role in cardiac adaptation to persistent pressure overload. Methods: We defined the cellular sources and function of MYDGF in wild-type (WT), Mydgf -deficient ( Mydgf −/− ), and Mydgf bone marrow–chimeric or bone marrow–conditional transgenic mice with pressure overload–induced heart failure after transverse aortic constriction surgery. We measured MYDGF plasma concentrations by targeted liquid chromatography–mass spectrometry. We identified MYDGF signaling targets by phosphoproteomics and substrate-based kinase activity inference. We recorded Ca 2+ transients and sarcomere contractions in isolated cardiomyocytes. Additionally, we explored the therapeutic potential of recombinant MYDGF. Results: MYDGF protein abundance increased in the left ventricular myocardium and in blood plasma of pressure-overloaded mice. Patients with severe aortic stenosis also had elevated MYDGF plasma concentrations, which declined after transcatheter aortic valve implantation. Monocytes and macrophages emerged as the main MYDGF sources in the pressure-overloaded murine heart. While Mydgf −/− mice had no apparent phenotype at baseline, they developed more severe left ventricular hypertrophy and contractile dysfunction during pressure overload than WT mice. Conversely, conditional transgenic overexpression of MYDGF in bone marrow–derived inflammatory cells attenuated pressure overload–induced hypertrophy and dysfunction. Mechanistically, MYDGF inhibited G protein–coupled receptor agonist–induced hypertrophy and augmented SERCA2a (sarco/endoplasmic reticulum Ca 2+ -ATPase 2a) expression in cultured neonatal rat ventricular cardiomyocytes by enhancing PIM1 (Pim-1 proto-oncogene, serine/threonine kinase) expression and activity. Along this line, cardiomyocytes from pressure-overloaded Mydgf −/− mice displayed reduced PIM1 and SERCA2a expression, greater hypertrophy, and impaired Ca 2+ cycling and sarcomere function compared with cardiomyocytes from pressure-overloaded WT mice. Transplanting Mydgf −/− mice with WT bone marrow cells augmented cardiac PIM1 and SERCA2a levels and ameliorated pressure overload–induced hypertrophy and dysfunction. Pressure-overloaded Mydgf −/− mice were similarly rescued by adenoviral Serca2a gene transfer. Treating pressure-overloaded WT mice subcutaneously with recombinant MYDGF enhanced SERCA2a expression, attenuated left ventricular hypertrophy and dysfunction, and improved survival. Conclusions: These findings establish a MYDGF-based adaptive crosstalk between inflammatory cells and cardiomyocytes that protects against pressure overload–induced heart failure.

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.

Direct phosphorylation and stabilization of HIF-1α by PIM1 kinase drives angiogenesis in solid tumors.

Angiogenesis is essential for the sustained growth of solid tumors. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of angiogenesis and constitutive activation of HIF-1 is frequently observed in human cancers. Therefore, understanding the mechanisms governing the activation of HIF-1 is critical for successful therapeutic targeting of tumor angiogenesis. Herein, we establish a new regulatory mechanism responsible for the constitutive activation of HIF-1α in cancer, irrespective of oxygen tension. PIM1 kinase directly phosphorylates HIF-1α at threonine 455, a previously uncharacterized site within its oxygen-dependent degradation domain. This phosphorylation event disrupts the ability of prolyl hydroxylases to bind and hydroxylate HIF-1α, interrupting its canonical degradation pathway and promoting constitutive transcription of HIF-1 target genes. Moreover, phosphorylation of the analogous site in HIF-2α (S435) stabilizes the protein through the same mechanism, indicating post-translational modification within the oxygen-dependent degradation domain as a mechanism of regulating the HIF-α subunits. In vitro and in vivo models demonstrate that expression of PIM1 is sufficient to stabilize HIF-1α and HIF-2α in normoxia and stimulate angiogenesis in a HIF-1-dependent manner. CRISPR mutants of HIF-1α (Thr455D) promoted increased tumor growth, proliferation and angiogenesis. Moreover, HIF-1α-T455D xenograft tumors were refractory to the anti-angiogenic and cytotoxic effects of PIM inhibitors. These data identify a new signaling axis responsible for hypoxia-independent activation of HIF-1 and expand our understanding of the tumorigenic role of PIM1 in solid tumors.

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.

Genetic Activation and Therapeutic Targeting of PIM1 in T-Cell Acute Lymphoblastic Leukemia/Lymphoma

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell acute lymphoblastic lymphoma (T-LBL) are aggressive immature T-cell malignancies that are often characterized by improper T-cell receptor (TCR) recombinations leading to aberrant activation of proto-oncogenes. Current multi-agent combination chemotherapy treatment provides an overall survival rate of ~80% in children and 50% in adults. However, the prognosis of patients with relapsed and refractory T-ALL/T-LBL remains extremely poor and the development of more effective and specific antileukemic drugs is critical. Therefore, we aim to identify new oncogenes, define their mechanism of action and establish strategies to develop targeted, highly specific antileukemic drugs.Here, we used Targeted Locus Amplification (TLA, de Vree et al., Nat Biotechnol., 2014) to identify a novel TCRβ driven t(6;7)(p21;q34) translocation in a human T-LBL patient resulting in aberrant activation of the PIM1 proto-oncogene. PIM1 is a constitutively active serine/threonine kinase involved in cell cycle progression, apoptosis, transcription and drug resistance and is overexpressed in a variety of human cancers. Its expression is controlled by the JAK/STAT signaling pathway. Further characterization of this PIM1 rearranged patient sample revealed cooperative genetic alterations that target known T-ALL/T-LBL onco- and tumor suppressor genes, including NOTCH1, IKZF1, EP300 and CDKN2A.Comparing PIM1 expression between normal T-cell subsets, T-ALL and T-LBL patient samples using qPCR revealed subsets in both T-LBL and T-ALL patients with high PIM1 levels. This PIM1 expression can be both monoallelicaly (due to translocation) or biallelicaly (due to upstream mutations in f.e. the JAK/STAT pathway). Using publically available gene expression datasets, we show that oncogenic PIM1 activation is largely restricted to immature, TLX1, TLX3, NKX2-1 or HOXA positive T-ALL and T-LBL patients, and is mostly not present in TAL1 or LMO2 rearranged tumors. This pattern of PIM1 activation is in line with the preferential occurrence of JAK-STAT pathway alterations in these genetic subtypes of human T-ALL/T-LBL.To study the oncogenic properties of PIM1 in the context of malignant T-cell transformation, we did RNA sequencing and phosphoproteomics on the T-ALL/T-LBL tumor line HSB-2 (cell line with the highest PIM1 expression) and on ex vivo treated leukemic cells harvested from the spleen of T-LBL engrafted NSG mice after PIM1 inhibition with TP-3654 (Foulks et al., Neoplasia, 2014). These data revealed that PIM1 inhibition has broad effects on transcription and phosphorylation substrates involved in cell cycle, translation and apoptosis. For example, we showed that the oncogenic properties of PIM1 are, at least in part, mediated by phosphorylation of GSK3β and subsequent stabilization of anti-apoptotic MCL1.Next, we evaluated the therapeutic potential of PIM1 inhibition. Daily TP-3654 treatment for 4 weeks of T-LBL engrafted NSG mice resulted in strong anti-leukemic effects. In vitro combination treatment with TP-3654 and glucocorticoids showed strong synergism, while the combination with other chemotherapeutics such as asparaginase and vincristine did not. Moreover, the combination of TP-3654 and the glucocorticoid dexamethasone in vivo almost completely cured the leukemic mice (<1% hCD45 positive blasts in the peripheral blood).All together, our study identifies PIM1 as a putative oncogene in T-ALL and T-LBL and suggests that inhibition of this serine/threonine kinase could serve as a novel therapeutic strategy in these aggressive T-cell neoplasms. DisclosuresNo relevant conflicts of interest to declare.

Cytokine Activation of JAK Kinases Rescues FLT3-Mutant AML from FLT3 Inhibition and Requires PIM Kinases

Mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase occur in ~30% of acute myeloid leukemias (AMLs) and are associated with a poor prognosis. Activating mutations in FLT3 predominantly occur as internal tandem duplications in the juxtamembrane region (FLT3-ITD), resulting in constitutive activation of canonical signaling pathways including STAT5, RAS/MAPK, and PI3K/mTORC1. Selective, potent 2nd and 3rd generation FLT3 inhibitors have shown significant clinical activity in patients with relapsed FLT3-ITD+ AML; however, clearance of FLT3 mutant clones does not consistently occur and most patients will eventually progress after an initial response. This challenges the model of FLT3 mutant AML being oncogene addicted and suggests that redundant signaling pathways may regulate AML cell survival after FLT3 inhibition. We hypothesized that these cells are resistant to FLT3 inhibition due to cytokines within the bone marrow microenvironment, resulting in low levels of primary resistant cells that will ultimately result in relapse. We have shown that primary FLT3-ITD+ AML cells escape FLT3 inhibition in vitro in the presence of cytokines produced normally in the bone marrow and that this effect is abrogated in patient cells by co-treatment with a JAK kinase inhibitor. To understand the mechanism of this rescue, we have studied a FLT3-ITD+ AML cell line (MV4;11). GM-CSF and IL-3 in particular are able to rescue cells from apoptosis following FLT3 inhibition. Interestingly, despite reactivating the canonical signaling pathways, GM-CSF and IL-3 maintain cell survival without rescuing proliferation, consistent with the clinical scenario. Both GM-CSF and IL-3 signal through JAK kinases. Co-treatment with the JAK1/2 inhibitor, ruxolitinib, or STAT5 knockdown re-sensitized AML cells to FLT3 inhibition when grown in the presence of cytokines. Inhibition of MEK or mTORC1 was unable to fully abrogate cytokine-mediated rescue of cell survival. These data demonstrate that JAK-dependent cytokine signaling mediate rescue from FLT3 inhibition through STAT5, but is not dependent on reactivation of MAPK or mTORC1 pathways. PIM kinases are a non-canonical target downstream of FLT3-ITD and downstream of STATs in cytokine signaling. PIM kinases are transcriptionally regulated. All three PIM isoforms were downregulated upon FLT3 inhibition. Both GM-CSF and IL-3 rescued PIM1 and PIM2 expression versus IL-6, a cytokine that is unable to protect from FLT3 inhibition. Co-treatment with a novel pan-PIM kinase inhibitor (INCB053914), was sufficient to block IL-3 rescue of cell survival. Altogether, these data demonstrate that FLT3-ITD+ AML cells utilize redundant signaling pathways activated by bone marrow cytokines through a JAK-STAT5-PIM axis to escape cell death from FLT3 inhibition. Pre-clinical testing with patient-derived xenografts is planned to validate these findings in an in vivo model and assess the feasibility of these combination approaches to improve clinical responses to FLT3 inhibitor therapy. DisclosuresSung:Radius Health Inc.: Equity Ownership. Koblish:Incyte Corporation: Employment, Equity Ownership. Perl:Seattle Genetics: Other: Advisory board; Astellas: Consultancy; Actinium Pharmaceuticals: Other: Scientific Advisory Board; Daiichi Sankyo: Consultancy; Novartis: Other: Advisory Board; Arog Pharmaceuticals: Consultancy; Pfizer: Other: Advisory Board; Asana Biosciences: Other: Scientific advisory board. Carroll:Incyte Pharmaceuticals: Research Funding; Astellas Pharmaceuticals: Research Funding.

The Pan-PIM Inhibitor INCB053914 Displays Potent Synergy at Low Doses in Combination with Ruxolitinib in Pre-Clinical Models of MPNs

Classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are hematopoietic stem cell disorders that affect about 300,000 people in the U.S. and can progress to bone marrow failure and acute myeloid leukemia. MPNs are driven by aberrant activation of the JAK2 tyrosine kinase, which leads to activation of downstream signaling molecules including STAT5, ERK, and AKT, among others. While the JAK inhibitor ruxolitinib is approved for use with patients with intermediate and high risk myelofibrosis and patients with polycythemia vera who are resistant or intolerant to hydroxyurea based on compelling clinical and/or hematologic efficacy, molecular responses to ruxolitinib therapy occur only rarely. In order to identify new MPN regimens that may enhance patient benefits, we investigated the effect of combining the JAK1/JAK2 inhibitor ruxolitinib with a new inhibitor (INCB053914) of the PIM family of Ser/Thr kinases, which are downstream targets of JAK2/STAT signaling. Members of the PIM family of kinases are proto-oncogenes that can cooperate with cMyc to induce lymphomagenesis in mice. PIM kinase activity regulates a variety of cell processes, including cell proliferation and apoptosis. For example, PIM kinases phosphorylate and inactivate the pro-apoptotic protein BAD. In addition, PIM activity can regulate translation initiation via activation of the mTORC1 complex, leading to inactivation of the translational initiation repressor 4EBP1. Triple PIM1/2/3 knockout mice demonstrate no major deleterious abnormalities suggesting specific targeting of PIM kinase activity may have limited adverse effects. However, the elimination of PIM1 in mice has suggested a role of PIM1 in the function of hematopoietic stem cells. Thus, deregulated PIM activity could affect hematopoietic stem cell disorders such as MPNs. We have recently demonstrated that PIM family members are elevated in MPN cells that persistently grow in the presence of ruxolitinib and that exogenous expression of PIM1 can induce ruxolitinib resistance. Taken together, PIMs appear to be strong candidates as therapeutic targets in MPNs.INCB053914 (PIMi) exhibits biochemical IC50s against Pim1/2/3 of 0.24 nM/30 nM/0.12 nM (at 1 mM ATP). In this study, we demonstrate that PIMi exhibits potent combination effects with ruxolitinib in pre-clinical models of MPNs. PIMi displayed synergistic effects in combination with ruxolitinib in MPN model cell lines, including BaF3/EpoR-JAK2-V617F, UKE1, and SET2. These effects include growth inhibition and, importantly, synergistic induction of apoptosis. The phosphorylation of biomarkers for Pim activity, including BAD, p70S6K, S6, and 4EBP1 was significantly diminished with combination of low dose ruxolitinib (0.1 uM) and PIMi (0.1 uM). Cells that were generated to persistently grow in ruxolitinib remained sensitive to PIMi. Importantly, neoplastic growth of erthyroid colonies from JAK2-V617F-positive MPN patients was sensitive to low dose PIMi (5 nM), with colony formation from some patient samples displaying sensitivity to as low as 1 nM of PIMi. In combination with ruxolitinib (25-50 nM), low dose PIMi displayed impressive synergy in inhibiting neoplastic colony formation of primary patient cells. Finally, in an in vivo model of JAK2-V617F-driven MPN cell growth, the combined treatment of ruxolitinib and PIMi suppressed tumor formation in CB17-SCID mice at doses that had little anti-tumor effect as single agents. The combination therapy was well tolerated in mice.Together, these data suggest that the pan-PIM inhibitor INCB053914 may provide a highly effective novel anti-MPN therapeutic, particularly in combination with ruxolitinib. INCB053914 is currently under investigation in a Phase 1/2 study in patients with advanced hematological malignancies (ClinicalTrials.gov: NCT02587598). Our findings support a clinical study investigating the combination of INCB053914 and ruxolitinib in MPN patients. DisclosuresCollins:Incyte Corporation: Employment, Equity Ownership. Koblish:Incyte Corporation: Employment, Equity Ownership. Ruggeri:Incyte Corporation: Employment, Equity Ownership. Stubbs:Incyte Corporation: Employment, Equity Ownership. Reuther:Incyte Corporation: Research Funding.

Expression and ERG regulation of PIM kinases in prostate cancer.

The three oncogenic PIM family kinases have been implicated in the development of prostate cancer (PCa). The aim of this study was to examine the mRNA and protein expression levels of PIM1, PIM2, and PIM3 in PCa and their associations with the MYC and ERG oncogenes. We utilized prostate tissue specimens of normal, benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), untreated PCa, and castration‐resistant prostate cancer (CRPC) for immunohistochemical (IHC) analysis. In addition, we analyzed data from publicly available mRNA expression and chromatin immunoprecipitation sequencing (ChIP‐Seq) datasets. Our data demonstrated that PIM expression levels are significantly elevated in PCa compared to benign samples. Strikingly, the expression of both PIM1 and PIM2 was further increased in CRPC compared to PCa. We also demonstrated a significant association between upregulated PIM family members and both the ERG and MYC oncoproteins. Interestingly, ERG directly binds to the regulatory regions of all PIM genes and upregulates their expression. Furthermore, ERG suppression with siRNA reduced the expression of PIM in PCa cells. These results provide evidence for cooperation of PIM and the MYC and ERG oncoproteins in PCa development and progression and may help to stratify suitable patients for PIM‐targeted therapies.