Pim-1 Kinase Research Articles

Abstract 79: Crosstalk of PIM and LKB1 kinases in driving growth of prostate cancer cells

Abstract The oncogenic PIM kinases and the tumor-suppressive LKB1 kinase have been linked to regulation of cell growth and proliferation. Here we have investigated their potential interactions as well as their relative impacts on tumorigenic growth of prostate cancer cells. The cellular functions of PIM and LKB1 kinases were evaluated using either pan-PIM inhibitors or CRISPR/Cas9-based genomic editing, with which all three PIM family members and/or LKB1 were knocked out from PC3 prostate cancer cells. In addition to cell-based 2D proliferation assays, we examined tumor growth using the chick embryo chorioallantoic membrane (CAM) xenograft model. In this report, we show that inhibition of PIM expression or activity results in increased phosphorylation of AMPK at Thr172 in an LKB1-dependent fashion. With in vitro kinase assays, we demonstrate that LKB1 is a novel direct substrate for PIM kinases and that Ser334 is one of the PIM target sites in LKB1. Accordingly, wild-type LKB1, but not the phosphodeficient S334A mutant can restore PIM inhibitor-induced AMPK phosphorylation in LKB1 knock-out cells. Whereas loss of LKB1 exaggerates formation of PC3-based tumors in the CAM xenograft model, co-deletion of PIM kinases attenuates it. The impairment of cell proliferation and tumor growth in cells lacking both PIM and LKB1 kinases not only underscores the potential crosstalk in signaling between these kinases, but also suggest that PIM inhibitors could be used to restrain growth of LKB1-deficient tumors. Citation Format: Kwan Long Mung, William Eccleshall, Niina M. Santio, Adolfo Rivero-Müller, Cecilia Sahlgren, Päivi J. Koskinen. Crosstalk of PIM and LKB1 kinases in driving growth of prostate cancer cells [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 79.

Abstract 2296: Regulation of P-body dynamics and formation in tumors through EDC3 phosphorylation by PIM and AKT

Abstract Processing bodies (P-bodies) are cytoplasmic mRNA granules that form a hub to regulate mRNA translation via controlling decapping, degradation, and storage. P-bodies markedly increase as part of a stress response for example during nutrient deprivation and hypoxia. Thus, P-bodies could regulate cell fate by tuning protein levels during the stress, and thereby contribute to tumor initiation, growth, and metastasis. However, it is unclear how P-body formation is regulated by these stress and what biologic pathways P-body controls. Dysregulation of PIM and AKT kinases has been found in multiple cancers, including prostate and breast cancers. We find that the protein Enhancer of mRNA-decapping Protein 3 (EDC3), a P-body component, binds with the PIM protein kinase and is phosphorylated on serine 161 by the PIM and AKT kinases, suggesting these kinases could regulate P-body formation and function. Thus, we hypothesize that these oncogenic kinases regulate the EDC3 activity and P-body function via the phosphorylation of EDC3. Suppression of EDC3 phosphorylation by inhibiting PIM and AKT activities with drugs leads to an increase in P-body number. A knock-in mutation substituting alanine for serine 161 in PC3-LN4 prostate cancer cell line inhibited growth, migration and invasion in vitro. Prostate cancer tumor growth in these cells containing EDC3 mutation was reduced in mouse xenograft model, suggesting that this single phosphorylation is essential for regulating cell growth. These results suggest that the level of specific RNAs and its translation is being controlled by this mutation. Indeed, integrin family proteins, ITGB1 and ITGA6, are shown to be decreased in these cells consistent with the inability of these cells to attach and invade. High levels of phosphorylated EDC3 were detected in the breast cancer cell lines tested, whereas non-transformed immortalized breast epithelial cell lines showed much lower EDC3 phosphorylation. IHC staining of human breast cancer samples with phospho-specific antibodies demonstrated that EDC3 was highly phosphorylated in contrast to normal breast tissue which demonstrated low level of phosphorylation. These data indicate that in tumors, breast and prostate cancer, with activated PIM and AKT, high phosphorylation of EDC3 would increase the translation of target mRNAs via reducing their storage and destruction and affecting tumor cell growth and motility. Therefore, the phosphorylation of EDC3 by Pim and AKT kinases can be a driver of malignancy by controlling mRNA levels in breast and prostate cancers. Citation Format: Jeremiah Bearss, Sathish K. Padi, Neha Singh, Marina Cardo-Vila, Andrew S. Kraft, Koichi Okumura. Regulation of P-body dynamics and formation in tumors through EDC3 phosphorylation by PIM and AKT [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 2296.

Abstract 1428: PARP inhibitor resensitizes TKI-resistant AML to TKI

Abstract Internal tandem duplications within the juxtamembrane domain of fms-like tyrosine kinase 3 (FLT3-ITD) are present in approximately 25% of patients with acute myeloid leukemia (AML) and is an independent predictor of poor prognosis. FLT3-ITD is a constitutively active form of the receptor and aberrantly signals through signal transducer and activator of transcription (STAT) 5 which feeds-back to stabilize FLT3 through the downstream effector, Pim-1 kinase. FLT3-specific tyrosine kinase inhibitors (TKI) have clinical effects in FLT3-ITD AML but responses are limited due to the development of resistance through various means, including point mutations in the FLT3 tyrosine kinase domain commonly at D835 in the activation loop and F691 in the gate-keeper residue. Novel therapeutic strategies are therefore required for overcoming TKI-resistance. Poly (ADP-ribose) polymerase (PARP) 1 functions by poly-ADP-ribosylating (PAR) itself and other proteins primarily to catalyze DNA repair, however, PARP can also regulate the activity and function of proteins involved in other various processes. Notably, PARP inhibitors (PARPi) are novel cancer therapeutics that induce synthetic lethality in cancers with BRCA mutations. Recent studies also show that concurrent treatment of PARPi and TKI can effectively induce synthetic lethality in BRCA-proficient TKI-sensitive FLT3-ITD AML. However, the effects of PARPi in TKI-resistant disease has yet to be explored. We previously reported elevated reactive oxygen species (ROS) in FLT3-ITD AML leading to upregulated PARP1 and we now show that, compared to parental TKI-sensitive Molm14 cells, D835Y and F691L point-mutated TKI-resistant forms have significantly increased ROS production, and subsequently increased DNA damage and PARP1 protein expression. Interestingly, TKI-resistant cells also have elevated phospho-STAT5 (pSTAT5) and subsequent CRISPR-Cas9 knockout of PARP1 leads to reduction in pSTAT5 as well as the downstream targets, Pim-1 and FLT3. Significantly, STAT5 protein was found to immunoprecipitate with PAR and PARP1, suggesting for the first time that PARP1 may play a direct role in regulating STAT5. Finally, we have found that low doses (5-50nM) of the potent PARPi, Talazoparib (Tal), in combination with low dose (10nM) of the TKIs, Quizartinib (Quiz) or Gilteritinib (Gilt), is synergistically lethal in the otherwise TKI-resistant cells. Additionally, sequential treatment of TKI-resistant cells with Tal (72hr) prior to treatment with Quiz (72hr) or Gilt (72hr) was still synergistic whereas the reverse is not. This suggests a novel model whereby Tal may resensitize cells to TKI by decreasing PARP1 effects on pSTAT5 activity which feeds back to reduce FLT3 receptor activation. Therefore, this combination has potential for treatment of TKI-resistant AML and studies are underway to translate these findings with the intent of developing an effective therapy for patients with this disease. Citation Format: Anna J. Dellomo, Rena G. Lapidus, Mariusz Karbowski, Maria R. Baer, Tami J. Kingsbury, Feyruz V. Rassool. PARP inhibitor resensitizes TKI-resistant AML to TKI [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 1428.

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.

Abstract 1109: A novel strategy to cope with osimertinib resistance in non-small cell lung cancer by treatment with a PIM kinase inhibitor in combination with a MERTK-selective kinase inhibitor

Abstract Osimertinib is currently the preferred treatment for EGFR-mutated non-small cell lung cancer (NSCLC) patients due to its superior therapeutic efficacy and prolonged overall survival compared to earlier generation EGFR tyrosine kinase inhibitors, but durable responses to osimertinib treatment are rare due to acquired drug resistance. Thus, there is an urgent need for novel strategies to treat osimertinib-resistant NSCLC. Recently, we found that treatment with MRX-2843, a novel MERTK-selective kinase inhibitor currently in Phase I clinical trials, resulted in dose-dependent inhibition of cell expansion and colony formation in an osimertinib-resistant (osiR) H4006 derivative cell line. An unbiased screen of 378 kinase inhibitors was carried out to identify compounds that synergized with MRX-2843 to inhibit expansion of an osiR derivative of the EGFR-mutated H4011 cell line. Treatment with 1µM PIM kinase inhibitor SGI-1776 or 100nM MRX-2843 alone reduced cell density by 5±3% and 44±7%, respectively, while treatment with MRX-2843 and SGI-1776 combined mediated an 82±0.4% decrease. Synergy was also observed in H4006 osiR and H1650 osiR derivative cell lines. Furthermore, treatment with PIM447, a structurally distinct PIM kinase inhibitor, and MRX-2843 decreased cell expansion more effectively than either agent alone. Mechanistically, treatment with a PIM kinase inhibitor in combination with MRX-2843 decreased downstream PI3K-AKT and MAPK-ERK signaling more effectively than single agents. Additionally, combined treatment with MRX-2843 and SGI-1776 prevented colony formation, while single agents had limited effect. In all, these data indicate that combining MRX-2843 and a PIM TKI may control osimertinib resistant tumor growth, providing a potential treatment strategy for osimertinib resistant EGFR-mutated NSCLC patients for whom the choices are still limited. Citation Format: Dan Yan, Zikang Tan, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, III, Deborah DeRyckere, Douglas K. Graham. A novel strategy to cope with osimertinib resistance in non-small cell lung cancer by treatment with a PIM kinase inhibitor in combination with a MERTK-selective kinase inhibitor [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 1109.

Abstract 1338: Discovery of novel, potent USP7 inhibitors that upregulate p53 leading to anti-proliferative effects in cancer cells

Abstract Introduction: Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that regulates several proteins involved in cell cycle, DNA repair, genomic stability, and epigenetics and has been implicated in cancer progression. A key substrate of USP7 is MDM2, the oncogenic E3 ubiquitin ligase that promotes degradation of the tumor suppressor p53. USP7-mediated stabilization of MDM2 leads to the degradation of p53, preventing cell cycle arrest and the induction of apoptosis and promoting tumor cell growth. In addition to the MDM2-p53 pathway, USP7 regulates a number of other substrates involved in cancer, including PIM2 kinase, MYCN, the DNA methyltransferase DNMT1, and the PTEN tumor suppressor. Consistent with its function promoting oncogenic signaling pathways, genetic and pharmacological inhibition of USP7 has been shown to inhibit growth of a number of tumor cell lines in vitro and in vivo, and this anti-tumor activity is significantly enriched among cells expressing wild type p53. Thus, inhibition of USP7 is a promising therapeutic strategy, especially in cancers carrying wild type p53 that can be reactivated by suppression of MDM2. Results: We have discovered a new class of potent and selective USP7 inhibitors. These compounds bind to USP7 and prevent deubiquitinase activity in biochemical activity assays with picomolar potency. Our compounds induce accumulation of p53 and exhibit IC50s below 50 nM in cell viability assays in Multiple Myeloma and Acute Myeloid Leukemia (AML) cell lines. In AML cell lines, our USP7 inhibitors strongly synergize with the approved Bcl-2 inhibitor venetoclax. Unlike MDM2 antagonists currently undergoing clinical trials in AML, these USP7 inhibitors do not lead to a significant increase in MDM2 levels, and our compounds result in a more modest induction of p53, both of which could provide significant safety benefits. We have demonstrated differential sensitivity to MDM2 antagonists and our USP7 inhibitors in select cell lines and show that this effect is dependent upon p53 induction. Our compounds are orally bioavailable with a desirable PK profile in mice and induce p53 in tumor cells in CDX mouse models of Multiple Myeloma. Conclusions: We have identified novel, potent, orally bioavailable USP7 inhibitors that lead to p53 accumulation and cytotoxicity in cancer cells. We demonstrate mechanistic differences between USP7 and MDM2-p53 antagonists, which may lead to a safety advantage. We observe strong synergy between USP7 inhibitors and an approved treatment. Our lead compounds have favorable drug-like properties, promising mouse PK profiles, and can induce p53 accumulation in mouse CDX tumor models. These data demonstrate the therapeutic potential of our USP7 inhibitors and reveal specific opportunities for their use in the treatment of Multiple Myeloma and AML. Citation Format: Alan Futran, Tao Lu, Katherine Amberg-Johnson, Xiaoxiao Yang, Saidi He, Jeffrey Bell, Sarah Boyce, Markus Dahlgren, Karen Dingley, Liping Fang, Heidi Koldsoe, Zef Konst, Fang-Yu Lin, Robert Pelleltier, Heng Qian, Mats Svensson, Michael Trzoss, Jie Xu, Shuping Xu, Zhaowu Xu, Engin Yapici, Yan Zhang, Li Xing, Takao Suzuki, Xianhai Huang, Jiayi Xu, Hamish Wright, Kristian Jensen, Wayne Tang, Tao Guo, Karen Akinsanya, David Madge. Discovery of novel, potent USP7 inhibitors that upregulate p53 leading to anti-proliferative effects in cancer cells [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 1338.

Negative Regulation of Erythroid Differentiation via the CBX8-TRIM28 Axis.

Although the mechanism of chronic myeloid leukemia (CML) initiation through BCR/ABL oncogene has been well characterized, CML cell differentiation into erythroid lineage cells remains poorly understood. Using CRISPR-Cas9 screening, we identify Chromobox 8 (CBX8) as a negative regulator of K562 cell differentiation into erythrocytes. CBX8 is degraded via proteasomal pathway during K562 cell differentiation, which activates the expression of erythroid differentiation-related genes that are repressed by CBX8 in the complex of PRC1. During the differentiation process, the serine/threonine-protein kinase PIM1 phosphorylates serine 196 on CBX8, which contributes to CBX8 reduction. When CD235A expression levels are analyzed, the result reveals that the knockdown of PIM1 inhibits K562 cell differentiation. We also identify TRIM28 as another interaction partner of CBX8 by proteomic analysis. Intriguingly, TRIM28 maintains protein stability of CBX8 and TRIM28 loss significantly induces proteasomal degradation of CBX8, resulting in an acceleration of erythroid differentiation. Here, we demonstrate the involvement of the CBX8-TRIM28 axis during CML cell differentiation, suggesting that CBX8 and TRIM28 are promising novel targets for CML research.

PIM kinases inhibit AMPK activation and promote tumorigenicity by phosphorylating LKB1

BackgroundThe oncogenic PIM kinases and the tumor-suppressive LKB1 kinase have both been implicated in the regulation of cell growth and metabolism, albeit in opposite directions. Here we investigated whether these kinases interact with each other to influence AMPK activation and tumorigenic growth of prostate and breast cancer cells.MethodsWe first determined how PIM and LKB1 kinases affect AMPK phosphorylation levels. We then used in vitro kinase assays to demonstrate that LKB1 is phosphorylated by PIM kinases, and site-directed mutagenesis to identify the PIM target sites in LKB1. The cellular functions of PIM and LKB1 kinases were evaluated using either pan-PIM inhibitors or CRISPR/Cas9 genomic editing, with which all three PIM family members and/or LKB1 were knocked out from PC3 prostate and MCF7 breast cancer cell lines. In addition to cell proliferation assays, we examined the effects of PIM and/or LKB1 loss on tumor growth using the chick embryo chorioallantoic membrane (CAM) xenograft model.ResultsWe provide both genetic and pharmacological evidence to demonstrate that inhibition of PIM expression or activity increases phosphorylation of AMPK at Thr172 in both PC3 and MCF7 cells, but not in their derivatives lacking LKB1. This is explained by our observation that all three PIM family kinases can phosphorylate LKB1 at Ser334. Wild-type LKB1, but not its phosphodeficient derivative, can restore PIM inhibitor-induced AMPK phosphorylation in LKB1 knock-out cells. In the CAM model, loss of LKB1 enhances tumorigenicity of PC3 xenografts, while cells lacking both LKB1 and PIMs exhibit slower proliferation rates and form smaller tumors.ConclusionPIM kinases are novel negative regulators of LKB1 that affect AMPK activity in an LKB1-dependent fashion. The impairment of cell proliferation and tumor growth in cells lacking both LKB1 and PIMs indicates that these kinases possess a shared signaling role in the context of cancer. These data also suggest that PIM inhibitors may be a rational therapeutic option for LKB1-deficient tumors.6DjYH8_uPF8iBB8mWNNcxfVideo

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.

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 Mechanisms of PIM Kinase Inhibitor, SGI-1776 Free Base, in Treating Myeloma-Associated Bone Disease

Background and Purpose: The PIM kinase family, which includes PIM1, 2 and 3 and is expressed in a variety of solid and hematological malignancies, has an essential role in cell growth and survival. Multiple myeloma remains an incurable hematologic cancer which always companions with osteolytic bone disease. In this work, we investigated the mechanisms of a PIM kinase inhibitor, SGI-1776 free base, in anti-myeloma-associated osteolytic bone disease in vitro and in vivo.Experiment Design: CCK-8 and flow cytometry were used to assess the cytotoxicity of SGI-1776 free base on myeloma cell lines (MM.1s, CAG, ARP-1, 8226, H929, LP-1, OPM2) . The effect on RANKL-induced osteoclasts was assessed in osteoclastogenic and osteoblastic cultures. Western blot and immunofluorescence were used to investigate the mechanisms of SGI-1776 free base. SiRNAs were used to mimic the inhibition of PIM kinase by the drug. IVIS in vivo imaging system, ELISA (human Igk, CTX-I, PINP) and micro-CT (parameters such as trabecular numbers and trabecular space) were used to surveillance the tumor burden and examine the bone destruction in murine models.Results: SGI-1776 free base induced apoptosis and cell cycle arrest in the G1 phase in myeloma cells mediated by decreasing the phosphorylation of PIM kinase targets C-Myc, Bad, 4EBP1 and H3. Also, the expression of Mcl-1, C-Myc, cyclin D2 and cyclin E1 were reduced, and the inhibition of NF-kB and STAT3 pathways also observed. The IC50 varies from 2 to 6 uM in different cells. Expression of apoptosis related proteins Cleaved Caspase-3, 7, 8, 9 and PARP-1 were increased. Importantly, SGI-1776 free base could overcome the protective effect of bone marrow stromal cells and osteoclasts on myeloma cells. Knocking down PIM kinase (PIM-1, 2, and 3) by SiRNAs slowed down the proliferation rates of myeloma cells (8226 and MM.1s), indicating the pro-growth impact of PIM kinase on myeloma. Notably, SGI-1776 free base inhibited osteoclast formation and resorption though decreasing PIM-1, c-fos, NFATc1, Cathepsin K and DUSP-1. Calcium coated slide assay, TRAP staining and F-acting ring formation also proved the inhibition effect. Also, phosphorylation levels of Erk, p-38, p-65, AKT and AMPK were reduced in the cells treated with SGI-1776 free base. Knocking down PIM-1 impaired the F-acting ring formation and reduced OC numbers compared with controls, and STAT3 pathway was inhibited as well. ALP staining, ALP activity and alizarin red staining showed SGI-1776 would not suppress osteoblasts formation and its mineralization. Finally, SGI-1776 free base reduced the tumor burden in subcutaneous and disseminated myeloma murine models and prevented myeloma associated bone loss with serum PINP (bone formation) and trabecular numbers increased while serum monoclonal Igk (tumor burden), CTX-I (bone resorption) and trabecular space diminished compared with the vehicle group. Moreover, SGI-1776 free base could directly reduce the RANLK-induced rapid bone loss in non-tumor bearing mouse model.Conclusion: Our investigation reveals the mechanisms of SGI-1776 free base in treating myeloma associated bone disease. The drug has a dual effect of anti-myeloma and bone protection. These results support the clinical use of this drug in multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

Concurrent Treatment with Pim Kinase Inhibitor Downregulates Alternative Non-Homologous End-Joining Repair and Decreases Genomic Instability in FLT3-ITD Cells Treated with Topoisomerase 2 Inhibitor Chemotherapy Drugs

Internal tandem duplication of fms -like tyrosine like kinase 3 (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML) cases, causing constitutive FLT3 signaling. While FLT3-ITD AML patients generally achieve remission following combination chemotherapy including cytarabine and a topoisomerase (topo) 2 inhibitor, they relapse rapidly, and FLT3-ITD AML cells frequently have new structural chromosome changes at relapse. Genomic instability in FLT3-ITD AML appears to result from increased generation of reactive oxygen species, which cause DNA double-strand breaks (DSBs), and upregulated DNA DSB repair by an error-prone alternative non-homologous end-joining (Alt-NHEJ) pathway.Pim-1 is an oncogenic serine/threonine kinase that is transcriptionally upregulated downstream of FLT3-ITD, contributes directly to the proliferative and anti-apoptotic effects of FLT3-ITD, and also phosphorylates and stabilizes FLT3, creating a positive feedback loop in FLT3-ITD cells. Pim kinase inhibitors are in clinical trials. We previously showed that concurrent treatment with a Pim kinase inhibitor enhances apoptosis induction by topo 2 inhibitors in FLT3-ITD cells. Here, we tested the effect of concurrent Pim kinase inhibitor treatment on Alt-NHEJ repair of topo 2 inhibitor-induced DNA DSBs and on genomic instability.Functional changes in DSB repair activities induced by treatment with the topo 2 inhibitor daunorubicin (DNR) in the absence and presence of the pan-Pim kinase inhibitor AZD1208 were studied in Ba/F3-ITD cells, with FLT3-ITD, using stably integrated green fluorescent protein (GFP)-based DSB repair reporter constructs DR-GFP, EJ5-GFP and EJ2-GFP, measuring homologous recombination (HR), classical (C-)NHEJ and Alt-NHEJ, respectively. DNR, which induced DNA DSBs, did not induce HR or C-NHEJ repair activity, but induced a 50% increase in Alt-NHEJ activity at 36 hours. Concurrent treatment with AZD1208, which increased DNR-induced DNA DSBs more than two-fold, completely abrogated the DNR-induced increase in Alt-NHEJ repair activity. Treatment of Ba/F3-ITD cells with the topo 2 inhibitors mitoxantrone (MXR) or etoposide (VP-16) also induced Alt-NHEJ repair activity, which was abrogated by concurrent treatment with AZD1208.We next studied mechanisms underlying Pim kinase inhibitor downregulation of Alt-NHEJ. The effects of DNR, MXR or VP-16 on cellular and nuclear expression of the Alt-NHEJ proteins poly (ADP-ribose) polymerase (PARP) 1, DNA polymerase θ, DNA ligase 3 and co-factor X-ray repair cross-complementing protein 1 (XRCC1) were measured in the FLT3-ITD cell lines Ba/F3-ITD, 32D-ITD, MV4-11 and MOLM-14 in the absence and presence of AZD1208. Cellular and nuclear expression of XRCC1 and DNA ligase 3, but not PARP or DNA polymerase θ, decreased in cells co-treated with AZD1208 and topo 2 inhibitors, compared to topo 2 inhibitors alone. XRCC1 was identified as a potential direct Pim-1 substrate by global profiling using the reverse in-gel kinase assay, and was confirmed as a robust Pim substrate in a reverse in-gel kinase assay. This provides a potential mechanistic link between Pim-1 kinase and ALT NHEJ.Alt-NHEJ repair is associated with genomic instability characterized by increased chromosome deletions, insertions and translocations. We studied induction of chromosome changes by topo 2 inhibitor treatment with and without Pim kinase inhibitor in FLT3-ITD cells. Ba/F3-ITD and 32D-ITD cells were incubated with DNR and/or AZD1208, or DMSO control, for 36 hours, then prepared for cytogenetic analysis.For each treatment, 20 consecutive metaphases were analyzed for induction of chromatid breaks and exchanges. No chromosome changes were seen in either cell line with DMSO or AZD1208 treatment. In Ba/F3-ITD cells, chromosome abnormalities were seen in 17 of 20 metaphases after DNR treatment, but only 8 of 20 after combination treatment (p=0.008). In 32D-ITD cells, chromosome abnormalities were seen in 8 of 20 metaphases with DNR, and 0 metaphases with combination (p=0.003).Thus concurrent treatment with Pim kinase inhibitor abrogates induction of Alt-NHEJ repair by topo 2 inhibitor treatment in FLT3-ITD cells and decreases the genomic instability that may contribute to rapid relapse of FLT3-ITD AML after chemotherapy. The data support further preclinical and clinical testing of Pim kinase inhibitors with chemotherapy in AML with FLT3-ITD. DisclosuresTron:AstraZeneca: Employment, Equity Ownership.

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.

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

Inhibition of Pim-2 kinase by LT-171-861 promotes DNA damage and exhibits enhanced lethal effects with PARP inhibitor in multiple myeloma

Multiple myeloma (MM) is a malignancy of antibody-producing plasma cells with genomic instability and genetic abnormality as its two hallmarks. Therefore, DNA damage is pervasive in MM cells, which indicates irregular DNA damage response (DDR) pathway. In this study, we demonstrated that LT-171-861, a multiple kinase inhibitor, could inhibit proliferation and induce apoptosis in MM cells. LT-171-861 promoted DDR pathway and triggered DNA damage through impeding the process of homologous recombination in double strand breaks, rather than directly elevating ROS level in MM cells. Mechanism research revealed that Pim2 inhibition was responsible for LT-171-861-indcued DNA damage and cell apoptosis. LT-171-861 mainly suppressed Pim2 kinase activity and reduced the expression of its phosphorylated substrates, such as 4EBP1 and BAD. Moreover, Olaparib, a PARP inhibitor, could enhance the antitumor effect of LT-171-861 in suppressing tumor growth in MM xenografted nude mice. Taken together, our results demonstrated that LT-171-861 showed a promising therapeutic potential for MM and had an additional lethal effect with PARP inhibitors.

Novel Pan-Pim Kinase Inhibitors With Imidazopyridazine and Thiazolidinedione Structure Exert Potent Antitumor Activities.

Pim kinases are overexpressed in various types of hematological malignancies and solid carcinomas, and promote cell proliferation and survival. Here in this study, we investigated the preclinical profile of novel pan-Pim kinase inhibitors with imidazopyridazine and thiazolidinedione structure. Imidazopyridazine-thiazolidinediones inhibited activities of Pim kinases with IC50 values of tens to hundreds nanomolar. With YPC-21440 and/or YPC-21817, which exhibited especially high inhibitory activities against Pim kinases, we investigated in vitro and in vivo activities of imidazopyridazine-thiazolidinediones. In silico analysis of binding mode of YPC-21440 and Pim kinases revealed that it directly bound to ATP-binding pockets of Pim kinases. In the kinase panel tested, YPC-21440 and YPC-21817 were highly specific to Pim kinases. These compounds exerted antiproliferative activities against various cancer cell lines derived from hematological malignancies and solid carcinomas. Furthermore, they suppressed phosphorylation of Pim kinase substrates, arrested cell cycle at the G1 phase, and induced apoptosis in cultured cancer cells. In tumor xenograft models, YPC-21440 methanesulfonate and YPC-21817 methanesulfonate exerted antitumor activities. Furthermore, pharmacodynamic analysis with a xenograft model suggested that YPC-21817 methanesulfonate inhibited Pim kinases in tumors. In conclusion, our data revealed that imidazopyridazine-thiazolidinediones are novel Pim kinases inhibitors, effective on various types of cancer cell lines both in vitro and in vivo.

Suppressive neutrophils require PIM1 for metabolic fitness and survival during chronic viral infection.

SUMMARYThe immune response to a chronic viral infection is uniquely tailored to balance viral control and immunopathology. The role of myeloid cells in shaping the response to chronic viral infection, however, is poorly understood. We perform single-cell RNA sequencing of myeloid cells during acute and chronic lymphocytic choriomeningitis virus (LCMV) infection to address this question. Our analysis identifies a cluster of suppressive neutrophils that is enriched in chronic infection. Furthermore, suppressive neutrophils highly express the gene encoding Proviral integration site for Moloney murine leukemia virus-1 (PIM1), a kinase known to promote mitochondrial fitness and cell survival. Pharmacological inhibition of PIM1 selectively diminishes suppressive neutrophil-mediated immunosuppression without affecting the function of monocytic myeloid-derived suppressor cells (M-MDSCs). Decreased accumulation of suppressive neutrophils leads to increased CD8 T cell function and viral control. Mechanistically, PIM kinase activity is required for maintaining fused mitochondrial networks in suppressive neutrophils, but not in M-MDSCs, and loss of PIM kinase function causes increased suppressive neutrophil apoptosis.

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.

Co-Targeting PIM Kinase and PI3K/mTOR in NSCLC.

Simple SummaryPIM kinases interact with major oncogenic players, including the PI3K/Akt pathway, and provide an escape mechanism leading to drug resistance. This study examined PIM kinase expression in NSCLC and the potential of PIM1 as a prognostic marker. The effect on cell signaling of novel preclinical PI3K/mTOR/PIM kinase inhibitor IBL-301 was compared to PI3K/mTOR inhibition in vitro and ex vivo. PI3K-mTOR inhibitor sensitive (H1975P) and resistant (H1975GR) cells were compared for altered IL6/STAT3 pathway expression and sensitivity to IBL-301. All three PIM kinases are expressed in NSCLC and PIM1 is a marker of poor prognosis. IBL-301 inhibited c-Myc, the PI3K-Akt and JAK/STAT pathways in vitro and in NSCLC tumor tissue explants. IBL-301 also inhibited secreted pro-inflammatory cytokine MCP-1. PIM kinases were activated in H1975GR cells which were more sensitive to IBL-301 than H1975P cells. A miRNA signature of PI3K-mTOR resistance was validated. Co-targeting PIM kinase and PI3K-mTOR warrants further clinical investigation.PIM kinases are constitutively active proto-oncogenic serine/threonine kinases that play a role in cell cycle progression, metabolism, inflammation and drug resistance. PIM kinases interact with and stabilize p53, c-Myc and parallel signaling pathway PI3K/Akt. This study evaluated PIM kinase expression in NSCLC and in response to PI3K/mTOR inhibition. It investigated a novel preclinical PI3K/mTOR/PIM inhibitor (IBL-301) in vitro and in patient-derived NSCLC tumor tissues. Western blot analysis confirmed PIM1, PIM2 and PIM3 are expressed in NSCLC cell lines and PIM1 is a marker of poor prognosis in patients with NSCLC. IBL-301 decreased PIM1, c-Myc, pBAD and p4EBP1 (Thr37/46) and peIF4B (S406) protein levels in-vitro and MAP kinase, PI3K-Akt and JAK/STAT pathways in tumor tissue explants. IBL-301 significantly decreased secreted pro-inflammatory cytokine MCP-1. Altered mRNA expression, including activated PIM kinase and c-Myc, was identified in Apitolisib resistant cells (H1975GR) by an IL-6/STAT3 pathway array and validated by Western blot. H1975GR cells were more sensitive to IBL-301 than parent cells. A miRNA array identified a dysregulated miRNA signature of PI3K/mTOR drug resistance consisting of regulators of PIM kinase and c-Myc (miR17-5p, miR19b-3p, miR20a-5p, miR15b-5p, miR203a, miR-206). Our data provides a rationale for co-targeting PIM kinase and PI3K-mTOR to improve therapeutic response in NSCLC.

Effects of PIM3 in prognosis of colon cancer.

PIM kinase is called proto-oncogene, but there are less research on PIM family in colon cancer. This study was designed to explore the prognosis of PIM3 in colon cancer. In this study, we downloaded RNA-seq and clinical information of colon cancer from the Gene Expression Omnibus (GEO) database. Kaplan-Meier method was used for analyzing the impact of PIM3 on the survival of patients with colon cancer. Single-factor and multi-factor cox regression analysis were used for verifying the prognostic value of PIM3. Spearman correlation analysis was used for screening PIM3 related genes. Functional enrichment analysis was used for analyzing the biological functions and pathways in which PIM3 related genes may be involved. STRING online tools were used for building a co-expression network. Cytoscape was used for co-expression network visualization. Compared with the low expression group, the patients in the PIM3 high expression group lived longer time. Single-factor and multi-factor cox regression analysis indicated that PIM3 was an independent prognostic factor for colon cancer. Sixty-two PIM3 related genes were screened, and GO and KEGG enrichment analyses suggested that PIM3 related genes might be involved in the MAPK and WNT pathways. The co-expression network showed a strong correlation between PIM3 and MLKL, MYL5, PPP3R1 and other genes. PIM3 is an independent prognostic factor of colon cancer and may be a target for the diagnosis and treatment of colon cancer.