PIM Inhibitors Research Articles

Therapeutic Targeting of PIM Protein Kinases in a Subset of T-Cell Acute Lymphoblastic Leukemia

New approaches are needed to treat and cure T-cell acute lymphoblastic leukemia (T-ALL) patients who are currently failing to achieve a remission with chemotherapy. While very intensive chemotherapy regimens result in remission in many T-ALL patients, early T-cell precursor (ETP)-ALL, defined by the absence of CD4, CD8, and CD1a and frequent expression of one or more myeloid markers, is associated with a higher rate of relapse and induction failure with a 10-year overall survival of 19% as compared with 84% for all other T-ALLs. The PIM serine/threonine protein kinase has been identified as a potential novel target for the development of new treatment regimens for the cure of hematopoietic malignancies.To explore the potential association of elevated PIM1 expression with ETP-ALL, we analyzed a publically available St. Judes gene expression dataset GSE28703. Results demonstrated that PIM1 is highly expressed in 75% of patients with ETP-ALL and in 13% of non-ETP ALL. PIM1 overexpression in immature or ETP-ALL is further validated using two other publically available independent cohorts: E-MEXP-313 and GSE62156.Based on this observation, we then tested the ability of pan-PIM inhibitors (AZD1208/LGB-321) on the growth of 6 human T-ALL cell lines. qRT-PCR and western blot analysis confirmed, PIM inhibitor sensitive (XTT assay: IC50 < 1 micromolar) cell lines (H-SB2, DU.528, and KOPT-K1) had elevated levels of PIM1 (>9 folds, p<0.01), activated JAK/STAT pathway and lower levels of MYC. Sensitive cells (H-SB2 and DU.528), had an immature or ETP-ALL phenotype (CD4-/CD8- and TdT-). Insensitive CUTLL1, SUP-T1, and HPB-ALL (XTT assay: IC50 > 10 micromolar) cell lines express significantly low PIM1, activated NOTCH mutations, activated AKT signaling, and higher levels of MYC. Immunophenotype of all the PIM inhibitor insensitive cells lines was consistent with a more mature T-ALL phenotype (CD4+/CD8+ and TdT+). When we selected NOTCH-mutant mature T-ALL cells for resistance to gamma secretase inhibitors (SUP-T1-Persister cells) they developed elevated PIM1 kinase levels and were sensitive to the growth inhibitory activities of PIM inhibition. Thus, PIM appears to be a potential target for chemotherapeutic treatment intervention in T-ALL and especially ETP-ALL tumor cells.While PIM kinase inhibitors block ETP-ALL growth they do not induce cell death. The observation that treatment with PIM inhibitors activated ERK and STAT phosphorylation, along with the knowledge that T-ALL cells have increased levels of SRC family tyrosine kinases suggested that adding a broadly active tyrosine kinase inhibitor could enhance the killing of these tumor cells. We have made the novel observation that the combination of Ponatinib, a tyrosine kinase inhibitor used in the treatment of chronic myelogenous leukemia, with a PIM kinase inhibitor is synergistically lethal to ETP-ALL cells (CI < 1.0). These agents synergize to induce PARP and Caspase cleavage, sufficient to drive apoptosis. In murine models, following engraftment of NOD/scid/IL-2Rγ null (NSG) mice with H-SB2-luc cell xenografts, three weeks of dual therapy with AZD1208 (30mg/kg/day) and Ponatinib (3mg/kg/day) significantly blocked leukemia growth as evidenced by optical scanning for luciferase producing cells (Figure 1, p<0.05), and reduced the number of leukemic cells (hCD45 staining) in the peripheral blood and bone marrow. This dual therapy significantly prolonged the survival (p<0.005) of the treated mice. This outcome was obtained at one-tenth of normal dose of Ponatinib used in animals, suggesting that in the humans the side effects induced by this agent could be avoided by using this combination therapy.Taken together, these findings highlight the important role for PIM protein kinase activity in T-ALL and especially in the ETP-ALL, phenotype. Tumor xenograft experiments provide strong preclinical rationale for a novel treatment strategy of combining PIM and tyrosine kinase inhibitors for treatment of patients with PIM overexpressing T-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Enhance T Cell Immunotherapy By Targeting PIM-2 Kinase

PIM kinases, a family of serine/threonine kinases, play an important role in regulating cell survival, cell proliferation, transcription activation and protein translation. Of the three isoforms, PIM-1, PIM-2 and PIM-3, some of them are overly expressed in several hematological malignancies as well as solid tumors. Thus, these kinases have been targeted clinically in several cancer studies. However, the roles of PIM kinases in T cells from previous studies are inconclusive and the functions of each isoform in these cells are yet to be investigated.In our study, we focused on the role of PIM-2 kinase in T cell responses to allo-antigen and tumor using pre-clinical models. Using allogeneic bone marrow transplantation (allo-BMT) models, we transferred allogeneic T cells isolated from mice deficient for Pim-2 (Pim-2-/-, FVB background) in comparison to WT and PIM1/3double deficient mice (PIM1/3-/-) into lethally irradiated Balb/C recipients. Our data revealed that upon allogeneic stimulation, PIM-2-/- T cells induced accelerated graft-versus-host-disease (GVHD) compared to WT or PIM1/3-/- T cells (Fig.1A). In addition, PIM-2-/- donor T cells were highly activated with significantly increased proliferation and IFN-γ production in recipient spleen and GVHD target organs. Donor PIM-2-/- T cells also expressed high levels of chemokine receptor, CXCR3 and a4b7 integrin (gut homing receptor) that correlated with increased capacity of donor T-cell migration to GVHD target organs such as liver and gut. In graft-versus-leukemia (GVL) study, PIM-2-/- donor T cells had roughly 4-fold higher ability to mediate GVL effect to WT and PIM1/3-/- T cells (Fig.1B). Negative regulation of T-cell responses to allo-antigens by PIM-2 was independently verified by restoring PIM-2 expression on PIM-2-/- T cells or inhibiting PIM kinases in PIM1/3-/- T cells. Altogether, the data from allo-BMT studies suggested that PIM-2 isoform functions as a negative regulator in T cell allo-response in contrast to PIM-1 and PIM-3 kinases.We then focused on evaluating T-cell immunity against syngeneic tumor. By administering tumor (TS-1 breast tumor in FVB background) into WT and PIM-2-/- mice either subcutaneously or intravenously, we observed that PIM-2-deficient mice efficiently control tumor growth in sharp contrast to WT mice (Fig. 1C). Mechanistic study revealed that tumor rejection in PIM-2-/- recipient relies on CD8+ tumor infiltrating lymphocytes (TILs) with a significant up-regulation of IFN-g, TNF-a and Fas-L expression. Lower infiltration of regulatory T cells and down regulation of PD-1 expression on T cells recovered from tumor site were also observed in PIM-2 deficient mice. Moreover, the tumor growth rate in PIM-2-/- mice after T-cell depletion with antibody was similar to that of WT mice. To further exclude the effect of other immune cells involved in tumor regression, we adoptively transferred WT and PIM-2-/- CD8+ T cells into WT mice with pre-established TS-1 tumor. CD8+ T cells deficient for PIM-2 was more efficient in controlling of tumor growth than WT or PIM1/3-/- CD8+ T cells. These data indicate that inhibition of PIM-2 in T cells display superior anti-tumor activity. In conclusion, PIM-2 kinase possesses a distinct role in anti-tumor immunity compared to the other two isoforms and it represents a potential target to enhance anti-tumor activity of adoptive T-cell immunotherapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Concurrent Inhibition of Pim-1 and FLT3 Kinases in FLT3-ITD Acute Myeloid Leukemia Post-Translationally Downregulates the Anti-Apoptotic Protein Mcl-1 through Downregulation of the Mcl-1 Deubiquitinase USP9X

Internal tandem duplication (ITD) mutations of the receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) are present in acute myeloid leukemia (AML) cells in 30% of cases and are associated with high relapse rate and short disease-free survival following both chemotherapy and allogeneic hematopoietic stem cell transplantation. Inhibitors of FLT3 signaling have shown activity in clinical trials in FLT3-ITD AML, but efficacy has generally been limited and transient. Concurrent inhibition of other targets in FLT3-ITD signaling pathways is being explored as an approach to increasing the depth and duration of responses to FLT3 inhibitors.The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD and phosphorylates and stabilizes FLT3, thereby promoting FLT3 signaling in a positive feedback loop in cells with FLT3-ITD. Pim kinase inhibitors are in clinical trials. We previously showed that combinations of clinically active Pim kinase and FLT3 inhibitors at pharmacologically relevant concentrations enhance apoptosis and decrease clonogenic growth of FLT3-ITD AML cell lines and primary patient cells in vitro and suppress growth of FLT3-ITD AML cells in vivo, in relation to treatment with FLT3 or Pim inhibitors alone. Here we studied the mechanistic effects of concurrent Pim kinase and FLT3 inhibition, demonstrating a novel mechanism of Mcl-1 downregulation in FLT3-ITD AML cells.Ba/F3-ITD cells, transfected with FLT3-ITD, were cultured with the pan-Pim kinase inhibitor AZD1208 at 1 μM, a concentration chosen based on in vitro and phase I clinical trial data, and/or the FLT3 inhibitor quizartinib at 1 nM, its IC50 concentration, and expression of the anti-apoptotic proteins Mcl-1, Bcl2 and Bcl-xL and the pro-apoptotic proteins BAD/S112 p-BAD, BAK, BAX and Bim was measured by western blot analysis. Mcl-1 expression decreased in a time-dependent manner with AZD1208 and quizartinib co-treatment, but not with treatment with either inhibitor alone, while levels of the other proteins did not change. Mcl-1 downregulation with Pim kinase and FLT3 inhibitor combination treatment was then confirmed in the human FLT3-ITD AML cell lines MV4-11 and MOLM-14.Mcl-1 expression is regulated at multiple levels, and we next sought to determine the mechanism(s) by which it is downregulated by concurrent Pim and FLT3 inhibition. While Mcl-1 protein levels decreased, Mcl-1 mRNA levels did not change, indicating post-transcriptional regulation. Additionally, levels of miR-29b, a negative regulator of Mcl-1 translation,decreased similarly in Ba/F3-ITD cells treated with AZD1208 and quizartinib, compared to quizartinib alone. Polysome profiling showed decreased total mRNA translation, but no selective reduction in Mcl-1 translation. In contrast, the progressive decrease in Mcl-1 protein expression with AZD1208 and quizartinib co-treatment was abrogated by addition of the proteasome inhibitor MG-132, demonstrating that Mcl-1 protein is downregulated by enhanced Mcl-1 proteasomal degradation. This mechanism was further confirmed by demonstration of an increase in ubiquitinated Mcl-1 prior to Mcl-1 downregulation in cells co-treated with AZD1208 and quizartinib, but not with each inhibitor alone or with DMSO control.The deubiquitinase USP9X decreases Mcl-1 ubiquitination and consequent proteasomal degradation, and we found that USP9X expression is downregulated prior to the increase in ubiquitinated Mcl-1 and the subsequent decrease in Mcl-1 protein levels during AZD1208 and quizartinib co-treatment, but was not altered by treatment with either inhibitor alone. In contrast, expression of the ubiquitin E3 ligases Mule/ARF-BP1, SCFβ-TrCP and Trim17, which mediate Mcl ubiquitination, did not change prior to Mcl-1 downregulation.Preclinical studies in our laboratory and others have shown in vitro and in vivo efficacy of combination treatment with Pim kinase and FLT3 inhibitors in FLT3-ITD AML, suggesting clinical promise of this approach. Here we show that, mechanistically, concurrent Pim kinase and FLT3 inhibition causes a post-translational decrease in expression of the anti-apoptotic protein Mcl-1 via enhanced proteasomal degradation, preceded by downregulation of the Mcl-1 deubiquitinase USP9X and an increase in ubiquitinated Mcl-1, a novel mechanism of Mcl-1 downregulation in FLT3-ITD AML cells. DisclosuresTron:AstraZeneca: Employment; AstraZeneca: Employment. Huszar:AstraZeneca: Employment.

PIM1 Inhibition Enhances and Prolongs Plerixafor-Induced Mobilization of HSCs

IntroductionThe CXCL12/CXCR4 axis was shown to be a major regulator for the interaction of hematopoietic stem cells (HSCs) with the niche and interruption of this pathway mobilizes HSCs from the bone marrow (BM).Therefore CXCR4 antagonists like AMD3100 (Plerixafor) are used in the clinic for the collection of HSCs from patients who fail to mobilize HSCs in response to G-CSF.In our previous studies, we could show that the serine/threonine kinase PIM1 is regulating the surface expression of the CXCR4 receptor on HSCs. In addition, PIM1-deficient HSCs fail to home to a wild type (WT) BM niche. Based on these results, we aimed to improve HSC mobilization by combining CXCR4 and PIM inhibition.MethodsIn order to study the mobilization efficiency in the murine model, mice were treated with AMD3100 alone or in combination with LGB321, a novel pan-PIM inhibitor. The mice were sacrificed at various timepoints and peripheral blood (PB) was isolated. The percentage of HSCs was then determined by flow cytometry. The mechanism of HSC mobilization was studied in isolated HSC and stroma populations by analyzing mRNA levels and surface expression of CXCR4, its ligand CXCL-12 and other factors, which are crucial for the interaction of HSCs and stromal cells.ResultsWe found that CXCR4 inhibition using AMD3100 leads to a compensatory upregulation of CXCR4 surface expression on total BM cells as well as HSCs. This effect can be reverted by deficiency or inhibition of PIM1 (Figure 1A). As a consequence, HSC mobilization using AMD3100 is strongly enhanced and prolonged in Pim1-deficient mice compared to WT animals (Figure 1B). Likewise, treatment of WT animals with AMD3100 in combination with LGB321 leads to increased and prolonged HSC mobilization compared to animals treated only with AMD3100 (Figure 1C). Besides the downregulation of CXCR4 on HSCs, we found that the Cxcl-12 expression as well as CXCR4 surface expression in CXCL12-abundant reticular (CAR) cells is dramatically decreased in Pim1-deficient mice, which even further increases the mobilization capacity of Pim1-deficient mice.ConclusionOur findings indicate, that PIM1 inhibition counteracts the compensatory upregulation of the CXCR4 receptor on HSCs after Plerixafor treatment and decreases CXCL12 levels within the bone marrow niche. Therefore, targeting PIM kinases in combination with CXCR4 inhibition could improve the collection of stem cells in patients at risk for poor mobilization. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

TET2 Downregulation Promotes AML Cell Proliferation Via Pim-1 Expression

Background: Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults which is still incurable although novel drugs and new combination of chemotherapies are used . With the development of genetic and molecular biology technologies, more and more genes are found to be related to leukemogenesis and drug resistance of AML. TET2, a member of the ten-eleven-translocation gene family which can modify DNA by catalyzing the conversion of 5-mehtyl-cytosine to 5-hydroxymethyl-cytosine , is often inactivated through mutation or deletion in myeloid malignancies. Recent research reported that TET2 knock-down can promote proliferation of hematopoietic stem cells and leukemic cells. Also, several clinical studies showed that patients with TET2 mutation or low levels of TET2 expression have more aggressive disease courses than those with normal levels of TET2. However, the mechanism of the phenomenon is unknown. Our aim is to uncover how TET2 protein level is negatively correlated with AML cell proliferation and to provide a better view of target therapy in AML.Methods: We determined the expression levels of TET2 and other target genes in acute leukemia cell lines, bone marrow AML specimens, and peripheral blood mononuclear cells from healthy donors by qRT-PCR and Western blot. We also determined the mutation status of TET2 in AML cell lines. CCK8 and flow cytometry were used to determine cell proliferation, cell apoptosis, and cell cycle profile. Methylation-specific PCR were used to examine the methylation status in gene promoter regions. Also, we developed TET2 knock-down lentivirus to transfect AML cell lines to examine the effect of TET2 depletion. Last, RNA-seq was used to compare gene expression level changes between TET2 knock-down cell lines and the control cell lines.Results: AML cells from AML cell lines (KG-1,U937, Kasumi, HL-60, THP-1, and MV4-11) and AML patients’ specimens expressed lower levels of TET2 than those of PBMC from the healthy donor (P<0.05). Among AML cell lines, U937 barely expressed TET2, while KG-1 expressed TET2 at a relatively higher level than those of other AML cell lines. We constructed a TET2 shRNA to transfect KG-1,THP-1,MV-4-11,Kasumi,and HL-60, and used qRT-PCR and western blot to verify the knock-down efficiency. CCK8 confirmed that knocking down TET2 could increase leukemia cell proliferation (P<0.05). Flow cytometry showed that cell cycle profile was altered in TET2 knock-down cells compared to the negative control cells. In order to identify target genes, we performed RNA-seq on wildtype and TET2 knockdown KG-1 cells and found that the expression of cell cycle related genes, DNA replication related genes, and some oncogenes were changed. We focused on Pim-1, an oncogene related to leukemogenesis, which was significantly up-regulated in the RNA-seq profile. Western blot and qPCR verified the RNA-seq results of Pim-1 expression in the transfected cells . Also, AML patients’ bone marrow samples (n=35) were tested by qPCR and 28 of them were found to express low TET2 but high Pim-1 with the other 7 being opposite. For detailed exploration in expression regulation of Pim-1 via TET2, we screened genes affecting Pim-1 expression and found SHP-1, a tumor suppress gene which is often silenced by promoter methylation in AML. Western blot band of SHP-1 was attenuated in TET2 knockdown KG-1 cells. Moreover, methylation-specific PCR showed that after knocking down TET2 in KG-1 cell line, the promoter regions were methylated much more than the control cells. These results indicated that the function of TET2 in epigenetic modulation plays an important role in regulating Pim-1 expression. Finally, using flow cytometry and CCK8 we surprisingly found that knocking down TET2 expression could lead leukemic cells (KG-1, THP-1 and MV-4-11) more sensitive to Pim-1 inhibitor (SGI-1776 free base) and decitabine (a demethylation agent treating MDS and AML) (P<0.05).Conclusion: Our study showed that knocking down TET2 promoted leukemic cell proliferation. This phenomenon may correlate to Pim-1 up-regulation. Our clinical data also showed that the expression of TET2 and Pim-1 have an inverse relationship. The mechanism of TET2 regulating Pim-1 expression may be related to the epigenetic modulation function of TET2. Finally, we found TET2 downregulation could increase leukemia vulnerability to Pim-1 inhibitor and decitbine, and provide a novel view of target therapy in AML. DisclosuresNo relevant conflicts of interest to declare.

Microenvironment-Induced Expression of PIM Kinases Supports Chronic Lymphocytic Leukemia Cells Survival and Promotes CXCR4-mTOR Pathway Dependent Migration

Lymph node microenvironment provides chronic lymphocytic leukemia (CLL) cells with pro-survival and protective signals, fostering resistance to conventional chemotherapeutics. CLL cells overexpress oncogenic PIM kinases, which modulate proteins engaged in transcription, translation, apoptosis, cell cycle and adhesion/motility (Mol Cancer Ther 2014, 13: 1231-45). Herein, we searched for the link between tumor microenvironment and PIMs expression, compared the clinical characteristics of CLL patients with high versus low expression of PIM kinases, and investigated the consequences of their inhibition with newly developed pan-PIM inhibitor, SEL24-B489 in primary CLL cells.We first evaluated the expression of PIM kinases in CD19+ cells derived from 88 newly diagnosed CLL cases. Patients with unmutated IGHV status exhibited significantly higher PIM1 transcript levels than patients with mutated IGHV genes. Subjects with advanced CLL (Binet C) exhibited higher PIM2 expression than patients in Binet A/B stage. Significantly higher PIM2 transcript abundance at the time of diagnosis was also observed in patients who relapsed after first line treatment (p=0.005). Expression of PIM2 and PIM3 kinases in lymph nodes was significantly higher than in peripheral blood, suggesting a relationship between PIM kinase expression/activity and CLL cell microenvironment. To further explore the role of microenvironment in the control of PIM expression, peripheral blood CLL cells were incubated with anti-IgM or CD40 ligand. Both stimuli induced PIM1 and PIM3 expression. Co-culture of CLL cells with stromal cell (HS5) monolayers promoted the expression of PIM3 isoform.We next assessed the consequences of PIM inhibition in CLL cells using novel pan-PIM inhibitor, SEL24-B489. Incubation with SEL24-B489 decreased phosphorylation of PIM substrates, p-FOXO1/3a(T24/T32) and p-4EBP1(S65), and induced dose-dependent apoptosis in 27 out of 28 analyzed cases, regardless of the IGHV mutation status and including relapsed patients. Of note, SEL24-B489 induced higher apoptotic response in primary CLL cells than referential pan-PIM inhibitor AZD1208. CLL cells with 17p13 deletion and obtained from chemo-refractory patients were also vulnerable to SEL24-B489, suggesting that functional p53 is not required for execution of SEL24-B489-mediated apoptosis. Importantly, SEL24-B489 was not toxic for cells derived from healthy donors.Since microenvironmental cues increase expression of PIM kinases, we hypothesized that interactions with stromal cells might hinder the in vitro activity of the PIM inhibitor. To explore this possibility, we compared apoptotic response to SEL24-B489 in CLL cells co-cultured on HS5 monolayers and CLL cells grown without the stromal support. In 6 out of 7 tested cases, SEL24-B489 overrode the protective signals from HS5 cells and induced apoptosis, although the cytotoxic effect of PIM inhibitor was stronger in the absence of stromal cells.PIM1 was shown to regulate CLL cells migration through CXCR4(S339) phosphorylation (Mol Cancer Ther 2014, 13: 1231-45). Accordingly, SEL24-B489 decreased phospho-CXCR4(S339), CXCR4 surface expression, and impaired CLL cells migration in the CXCL12 gradient. Surprisingly, decrease in the CXCR4 surface expression after SEL24-B489 was relatively modest when compared to the effect of this inhibitor on CXCL12-directed migration. We found that incubation of CLL cells with CXCL12 led to increase in the phosphorylation of mTOR(S2448) and Akt(S473). SEL24-B489 reduced the levels of p-mTOR(S2448), p-Akt(S473), p-4EBP1(T37/T46) and p-TSC2(S1798), revealing inhibitory effect on mTOR pathway. Pre-incubation of CLL cells with an mTOR inhibitor similarly restrained CXCL12-mediated mTOR activity and led to impaired CLL cells migration, uncovering the key role of mTOR axis in CXCR4-dependent migration. Thus, SEL24-B489 impairs the CLL cell migration by inhibiting CXCR4 surface expression and the CXCR4-triggered mTOR pathway.Taken together, we show that microenvironment signals increase expression of PIM kinases, supporting CLL cell survival and migration. Inhibition of PIM kinases impairs CXCR4-dependent migration and leads to CLL cells death, regardless of the p53 status. Targeting PIM kinases in CLL patients will likely release the cells from microenvironmental niches and might be a rational therapeutic strategy. DisclosuresWarzocha:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Czardybon:Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

PIM1 kinase regulates cell death, tumor growth and chemotherapy response in triple-negative breast cancer.

Triple-negative breast cancers (TNBCs) have poor prognosis and lack targeted therapies. Here we identified increased copy number and expression of the PIM1 proto-oncogene in genomic data sets of patients with TNBC. TNBC cells, but not nonmalignant mammary epithelial cells, were dependent on PIM1 for proliferation and protection from apoptosis. PIM1 knockdown reduced expression of the anti-apoptotic factor BCL2, and dynamic BH3 profiling of apoptotic priming revealed that PIM1 prevents mitochondrial-mediated apoptosis in TNBC cell lines. In TNBC tumors and their cellular models, PIM1 expression was associated with several transcriptional signatures involving the transcription factor MYC, and PIM1 depletion in TNBC cell lines decreased, in a MYC-dependent manner, cell population growth and expression of the MYC target gene MCL1. Treatment with the pan-PIM kinase inhibitor AZD1208 impaired the growth of both cell line and patient-derived xenografts and sensitized them to standard-of-care chemotherapy. This work identifies PIM1 as a malignant-cell-selective target in TNBC and the potential use of PIM1 inhibitors for sensitizing TNBC to chemotherapy-induced apoptotic cell death.

Systematic Functional Characterization of Resistance to PI3K Inhibition in Breast Cancer.

PIM kinase overexpression confers resistance to small-molecule PI3K inhibitors. Combined inhibition of PIM and PI3K may therefore be warranted in a subset of breast cancers. Cancer Discov; 6(10); 1134-47. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1069.

Discovery of imidazopyridazines as potent Pim-1/2 kinase inhibitors

High levels of Pim expression have been implicated in several hematopoietic and solid tumor cancers, suggesting that inhibition of Pim signaling could provide patients with therapeutic benefit. Herein, we describe our progress towards this goal using a screening hit (rac-1) as a starting point. Modification of the indazole ring resulted in the discovery of a series of imidazopyridazine-based Pim inhibitors exemplified by compound 22m, which was found to be a subnanomolar inhibitor of the Pim-1 and Pim-2 isoforms (IC50 values of 0.024nM and 0.095nM, respectively) and to potently inhibit the phosphorylation of BAD in a cell line that expresses high levels of all Pim isoforms, KMS-12-BM (IC50=28nM). Profiling of Pim-1 and Pim-2 expression levels in a panel of multiple myeloma cell lines and correlation of these data with the potency of compound 22m in a proliferation assay suggests that Pim-2 inhibition would be advantageous for this indication.

Control of translational activation by PIM kinase in activated B-cell diffuse large B-cell lymphoma confers sensitivity to inhibition by PIM447.

The PIM family kinases promote growth and survival of tumor cells and are expressed in a wide variety of human cancers. Their potential as therapeutic targets, however, is complicated by overlapping activities with multiple other pathways and remains poorly defined in most clinical scenarios. Here we explore activity of the new pan-PIM inhibitor PIM447 in a variety of lymphoid-derived tumors. We find strong activity in cell lines derived from the activated B-cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL). Sensitive lines show lost activation of the mTORC1 signaling complex and subsequent lost activation of cap-dependent protein translation. In addition, we characterize recurrent PIM1 protein-coding mutations found in DLBCL clinical samples and find most preserve the wild-type protein's ability to protect cells from apoptosis but do not bypass activity of PIM447. Pan-PIM inhibition therefore may have an important role to play in the therapy of selected ABC-DLBCL cases.

PIM kinases as therapeutic targets against advanced melanoma.

Therapeutic strategies for the treatment of metastatic melanoma show encouraging results in the clinic; however, not all patients respond equally and tumor resistance still poses a challenge. To identify novel therapeutic targets for melanoma, we screened a panel of structurally diverse organometallic inhibitors against human-derived normal and melanoma cells. We observed that a compound that targets PIM kinases (a family of Ser/Thr kinases) preferentially inhibited melanoma cell proliferation, invasion, and viability in adherent and three-dimensional (3D) melanoma models. Assessment of tumor tissue from melanoma patients showed that PIM kinases are expressed in pre- and post-treatment tumors, suggesting PIM kinases as promising targets in the clinic. Using knockdown studies, we showed that PIM1 contributes to melanoma cell proliferation and tumor growth in vivo; however, the presence of PIM2 and PIM3 could also influence the outcome. The inhibition of all PIM isoforms using SGI-1776 (a clinically-available PIM inhibitor) reduced melanoma proliferation and survival in preclinical models of melanoma. This was potentiated in the presence of the BRAF inhibitor PLX4720 and in the presence of PI3K inhibitors. Our findings suggest that PIM inhibitors provide promising additions to the targeted therapies available to melanoma patients.

Abstract 178: PIM1, a novel target in chemotherapy-resistant triple-negative breast cancer

Abstract Triple-Negative Breast Cancers (TNBCs) are aggressive, associated with poor prognosis and lack targeted therapies, mainly due to the absence of suitable molecular targets. The genomic region 6p21-p25 is recurrently amplified in TNBCs and encompasses the PIM1 oncogene. This study interrogated genomic breast cancer datasets and identified gene copy number-driven increase of PIM1 expression in TNBC. To understand the role of PIM1 in malignant phenotypes of TNBCs, functional studies were carried out in breast cancer and non-malignant mammary epithelial cell line models. RNA interference and rescue-of-function experiments revealed addiction to PIM1 kinase for cell population growth and apoptosis protection in TNBC cells, absent in non-malignant cells. In cells sensitive to PIM1 knockdown, we observed a subsequent reduction of the expression of the anti-apoptotic proteins BCL2 and MCL1. Moreover, exogenous expression of BCL2 prevented apoptosis caused by PIM1 knockdown. BH3-profiling analysis further confirmed that PIM1 blocks apoptosis elicited through the mitochondrial pathway in TNBC cell lines. The activity of PIM1 in other cancers has been closely linked to the regulation of c-MYC, an “un-targetable” oncogene that drives malignancy and that is frequently amplified and highly expressed in primary TNBCs and post-chemotherapy residual disease. Importantly, we found that PIM1 expression associates with several MYC-transcriptional signatures in TNBCs and mediates phosphorylation of c-MYC Ser62 and Histone-H3 Ser10, key targets for MYC-driven transcription. Exogenous expression of MYC rescued the phenotypes caused by PIM1 knockdown, providing mechanistic insights for the observed addiction. Finally, the therapeutic potential of targeting PIM1 was assessed using AZD1208, a small molecule inhibitor of the PIM kinase family. AZD1208 inhibited growth and sensitized TNBC derived cell lines, cell line xenografts and PDXs to chemotherapy by increasing apoptosis. We therefore identify PIM1 as a driver of malignancy in TNBC, illustrating relationships with MYC-activation and regulation of anti-apoptotic BCL2 and MCL1 proteins. PIM1 inhibitors could provide a potential therapeutic to abrogate chemotherapy resistance in TNBCs. Citation Format: Fara Braso-Maristany, Simone Filosto, Steven Catchpole, Rebecca Marlow, Jelmar Quist, Erika Francesch Domenech, Gianmaria Liccardi, Leila Zakka, Violeta Serra, Albert Gris, Maggie Cheang, Nirmesh Patel, Anna Perdrix Rosell, Patrycja Gazinska, Elodie Noel, Jonathan Watkins, Pierfrancesco Marra, Anita Grigoriadis, Andrew Tutt. PIM1, a novel target in chemotherapy-resistant triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 178.

Abstract 4824: Synergistic interaction of PIM kinase inhibitor, JP_11646, and cytotoxic or targeted therapies in acute leukemia models

Abstract Standard therapy for acute myeloid leukemia (AML) results in significant mortality and morbidity with an overall cure rate of only 25%. Implementation of novel therapeutic options is needed in order to significantly improve patient survival and outcome. PIM serine/threonine kinases are overexpressed in hematological malignancies and regulate cell cycle, survival and drug resistance pathways—thus providing viable therapeutic targets. We hypothesized that targeted or cytotoxic therapy will enhance anti-tumor activity in combination with JP-11646, a novel PIM2 inhibitor, in preclinical AML models. Preclinical data has shown JP-11646 exerted potent inhibition of AML cells in vitro and in vivo in subcutaneous AML xenograft models. An in vitro MTT assay was used to determine the overall anti-proliferative effects of single and combination therapies with respect to the downstream effects of PIM kinase and mechanism of action of JP-11646 in leukemia. First, the single agent efficacy in human AML (HEL, and MV(4;11)) cell lines were examined after 72 hours of exposure to JP-11646, rapamycin, daunorubicin and cytarabine. Overall potency, IC50, (0.17 μM (0.15-0.19), 0.003 μM (0.002-0.004), 0.067 μM(0.065-0.0699) and 0.31 μM (0.294-0.345), respectively) and maximal inhibition, Imax, of the various therapies was determined. Potential synergistic effects were first tested by treating AML cell lines with JP-11646 and daunorubicin, cytarabine, or rapamycin (an inhibitor of a pathway that regulates downstream molecules of the PIM kinase pathway). To quantify the synergy or interaction of JP-11646 in combination, a simple inhibitory non-competitive interaction model with an interaction parameter (Ø) as well as a three dimensional interaction surface was employed (Ø&amp;lt;1 synergism, Ø&amp;gt;1 antagonism). The intensity of the interaction (Ø) of JP-11646 with rapamycin (0.819 (0.395-0.543)), daunorubicin (0.469 (0.665-0.975)) or cytarabine (0.173 (0.118-0.229)) demonstrated synergistic effects across all combinations. Here, we demonstrate that novel drug targets and drugs for AML treatment alone, and in combination with pre-existing therapies, may lead to improved strategies for AML treatment and these findings support future development of JP-11646 in novel combinatorial regimens for AML therapy. Citation Format: Krista Pundt, Camern Baldino, Justin Caserta, Alex Adjei, Kelvin Lee, Xu Zhu, Alissa Verone-Boyle, Frank Engler, Laura Pitzonka, Gerald Fetterly. Synergistic interaction of PIM kinase inhibitor, JP_11646, and cytotoxic or targeted therapies in acute leukemia models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4824.

Abstract 394: Novel pan-PIM/pan-PI3K/mTOR inhibitors are highly active in preclinical models of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal and morbid malignancies today. Although newer chemotherapy regimens such as FOLFIRINOX and gemcitabine/nab-paclitaxel have made modest improvements in patient survival, more effective therapies are needed. With the exception of the EGFR inhibitor erlotinib, there are no approved, targeted therapies for metastatic PDA. Recent work demonstrated the importance of PIM kinases, particularly PIM1 and PIM3, in PDA. PIM kinases are involved in apoptosis, cell cycle progression, DNA damage repair mechanisms, and drug resistance mechanisms. Our work showed that hypoxic stress causes the RNA binding protein HuR to translocate to the cytoplasm where it stabilizes PIM1 mRNA leading to increased levels and activity of PIM1. Activation of PIM1 led to oxaliplatin resistance in a hypoxia specific manner and validated PIM1's upregulation upon drug exposure as a potential target for therapeutic inhibition in PDA. The KRas/PI3K/mTOR signaling pathway has been well described in PDA and PI3K as well as dual PI3K/mTOR inhibitors are currently in clinical development. Inflection Biosciences is currently developing IBL-202, a pan-PIM/pan-class I PI3K inhibitor and IBL-301, a pan-PIM/pan-class I PI3K/mTOR inhibitor. These compounds show good oral bioavailability in preclinical PK studies. Unlike prior attempts at PIM inhibition, these compounds do not interact with hERG channels or CYP enzymes, decreasing the likelihood of drug-drug interactions or prolonged QTc. Furthermore, toxicity studies in rodent models demonstrate that the compounds are well tolerated at 200mg/kg and 400mg/kg doses. Our work shows that IBL-202 and IBL-301 have significant preclinical activity in PDA cell lines with diverse genetic backgrounds, including PL5, Panc-1, and HS776t cells. Using PICOgreen assays, we show that the IC50 for IBL-202 ranges from 400nM (PL5) to 800nM (HS776t). The IC50 of IBL-301 ranges from 80nM (PL5) to 400nM (HS776t). As the PIM and PI3K pathways are important for inhibition of apoptosis, we tested the ability of these compounds to induce an apoptotic response. Cells treated for 48 hours at IC50 levels of IBL-202 showed positive cleaved caspase 3/7 staining in 31% (Panc-1) to 90% (HS776t) as examined by flow cytometry. IBL-301 elicited a positive cleaved caspase 3/7 in 28% (Panc-1) to 91% (PL5) of cells. Mechanistically, PIM and PI3K are known to lead to G1/S cell cycle progression through phosphorylation of p21 and p27. PDA cell lines dosed at IC50 levels show a potent G1 to S arrest at 48 hours in all cell lines tested. Overall, this work provides the rationale for the further preclinical development of IBL-202 and IBL-301 in PDA pre-clinical models in an effort to move these compounds towards early phase trials. Citation Format: Kevin O’Hayer, John Barbe, Avinoam Nevler, Michael O’Neill, Darren Cunningham, Jordan Winter, Jonathan Brody. Novel pan-PIM/pan-PI3K/mTOR inhibitors are highly active in preclinical models of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 394.

Abstract 3866: The clinically applicable pan-Pim kinase inhibitor PIM447 sensitizes acute myeloid leukemia cells with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitors

Abstract Introduction: Internal tandem duplication of the fms-like tyrosine kinase-3 receptor (FLT3-ITD) is present in acute myeloid leukemia (AML) cells in 30% of patients and these patients have short disease-free survival following chemotherapy. FLT3 inhibitors are clinically active, but their activity is limited and transient. The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD, and promotes FLT3 signaling in a positive feedback loop in FLT3-ITD cells. We have previously demonstrated that inhibiting Pim kinase sensitizes AML cell lines and primary AML patient blasts with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitor chemotherapy drugs. Here we studied the effects of the pan-Pim kinase inhibitor PIM447 (formerly LGH447; Novartis Pharmaceuticals), currently in clinical trials, on response of FLT3-ITD-expressing cell lines and AML patient samples to FLT3 inhibitors and to topoisomerase 2 inhibitors, with the ultimate goal of developing clinically applicable combination regimens. Methods: Cell lines and AML patient samples with FLT3-ITD were cultured with FLT3 inhibitors or topoisomerase 2 inhibitors at clinically applicable concentrations and PIM447 at a range of concentrations. Apoptosis was measured by Annexin V labeling and PARP cleavage. Mcl-1 expression was measured by immunoblotting. Reactive oxygen species (ROS) were measured with the redox-sensitive dye CM-H2DCFDA and DNA double-strand breaks (DSBs) by immunoblotting for γH2AX. Results: Transfected Ba/F3-ITD and 32D/ITD cells, MV4-11 and MOLM-14 human AML cells and primary AML patient blasts, all expressing FLT3-ITD, were treated with FLT3 inhibitors, including 100 nM midostaurin and 1 nM quizartinib, with 0, 10, 100 and 500 nM PIM447. Co-treatment with PIM447 produced a concentration-dependent increase in apoptosis induced by each FLT3 inhibitor (p&amp;lt;0.001). FLT3-ITD-expressing cells were also treated with the topoisomerase 2 inhibitors daunorubicin and etoposide at IC50 concentrations, with 0, 10, 100 and 500 nM PIM447. Co-treatment with PIM447 also produced a concentration-dependent increase in apoptosis induced by each topoisomerase 2 inhibitor (p&amp;lt;0.001). Increased apoptosis induced by PIM447 with FLT3 inhibitors was associated with Mcl-1 downregulation, while increased apoptosis induced by PIM447 with topoisomerase 2 inhibitors was associated with increased generation of ROS and increased DNA DSBs. Conclusions: The clinically applicable pan-Pim kinase inhibitor PIM447 sensitizes AML cells with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitors. Our data support in vivo testing of combination regimens and design of clinical trials aimed at improving outcomes for patients with AML with FLT3-ITD. Citation Format: Kshama A. Doshi, Patrick R. Baldwin, Shivani Kapoor, Maria R. Baer. The clinically applicable pan-Pim kinase inhibitor PIM447 sensitizes acute myeloid leukemia cells with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3866.

Abstract 4630: The pan-PIM inhibitor PIM447 enhances the antitumor activity of lenalidomide in multiple myeloma cells via synergistic inhibition of c-MYC

Abstract Introduction: Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide and pomalidomide have significantly contributed to improved overall survivals in multiple myeloma (MM) patients. However, cure remains an elusive goal for the vast majority of MM patients and novel combination regimens are needed. Recently, the mechanism of action of IMiDs was identified to be inducing degradation of two MM transcription factors, IZKF1 and IZKF3. IKZF1/IKZF3 regulates the expression of oncogene c-MYC via another transcription factor-interferon regulatory factor 4 (IRF4). This new finding provides an opportunity to study synergistic combinations of IMiDs with targeted therapies. PIM447 (formerly LGH447, Novartis Pharmaceuticals) is a pan-PIM kinase inhibitor that is in clinical development for the treatment of patients with MM and other hematological malignancies. Based on the observation that several MM lines are sensitive to both IMiD and PIM447, we evaluated the combination effects of PIM447 and lenalidomide in 9 MM cell lines. Methods: An 8×8 serial dilution dose matrix of PIM447 and lenalidomide were tested in a panel of 9 MM cell lines. RT-PCR and immunoblotting were performed in selected lines to define possible mechanisms of combination activity. Results: Combination activity (synergy score&amp;gt;2) was observed in 4 out of the 9 MM cell lines tested, with MM1-S and NCI-H929 showing the highest synergy scores. Combination activity was only noted in the presence of PIM447 single agent activity and appeared to correlate with PIM2 protein expression. In both MM1-S and NCI-H929 cells, the combination led to increased apoptosis as measured by PARP cleavage. PIM447 did not affect the levels of various targets of lenalidomide, including IKZF1, IKZF3 and IRF4, and lenalidomide did not affect PIM downstream effectors such as phospho-S6RP. Alterations involving c-MYC are amongst the most frequent genetic features found in MM, and the combination of PIM447 and lenalidomide resulted in greater reduction of c-MYC expression. In both MM1-S and NCI-H929 cells, c-MYC knockdown has previously been shown to inhibit cell proliferation, suggesting the combination activity might derive from the observed reduction of c-MYC expression. The mechanisms by which c-MYC expression were synergistically decreased by the combination may involve both enhanced downregulation of c-MYC transcripts and accelerated degradation of c-MYC protein. Conclusions: The pan-PIM kinase inhibitor PIM447 enhanced the anti-tumor activity of IMiDs in MM via synergistic inhibition of c-MYC. Our data support in vivo testing of this combination regimen and the design of future clinical trials in MM patients. Citation Format: Huaixiang Hao, Dannie Wang, Gary Vanasse, Giordano Caponigro. The pan-PIM inhibitor PIM447 enhances the antitumor activity of lenalidomide in multiple myeloma cells via synergistic inhibition of c-MYC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4630.

Abstract 4414: Insulin receptor substrate 1 is a key substrate for Pim protein kinases

Abstract [BACKGROUND] Proviral integration site for Moloney murine leukemic virus (Pim) proteins are a family of serine/threonine kinases composed of three different isoforms (Pim1, Pim2 and Pim3) that are aberrantly expressed in a wide variety of tumor types, including hematopoietic malignancies. Overexpression of Pim kinases in cells exerts a pleiotropic effect on various signal transduction pathways, including cell cycle progression, cell proliferation and apoptosis. They are now being targeted in human clinical trials by small molecules inhibitors. However, the protein substrates and the mechanism of action of the Pim protein kinases are not known. To understand how Pim is regulating this diverse set of pathways, it is essential to define the substrates for this protein kinase. [MATERIAL and METHOD] To identify novel substrates, bioinformatics analysis was carried out to identify proteins containing a consensus Pim phosphorylation site. This analysis identified the insulin receptor substrate 1 serine 1101 (IRS1-S1101) as a potential Pim substrate. Experiments were carried out in tissue culture, animals, and human samples from phase I trials to validate this observation and define the biologic readout of this phosphorylation. [RESULT] Our in vitro kinase assay showed that S1101 phosphorylation was markedly induced by incubation of active Pim1 kinase to IRS1 immunoprecipitants, demonstrating that Pim1 is capable of directly phosphorylating IRS1 protein on S1101. Mutation of this residue in IRS1 from serine to alanine (S1101A) completely abolished in vitro phosphorylation by Pim1. Similar results were obtained using the IRS2 protein that contains an identical Pim kinase phosphorylation consensus at 1149. To find whether one or all of the three Pim isoforms are regulating the phosphorylation IRS1, each isoform was transduced into Pim triple knockout (TKO) MEF cells using lentiviruses producing Pim1, Pim2 or Pim3. Each of the three isoforms was sufficient to induce the phosphorylation of IRS1 on S1101. Consistent with these results, the depletion of each Pim kinase isoform individually using siRNA in the various cancer cell lines did not decrease IRS1 phosphorylation, but the knockdown of all three isoforms abolished the phosphorylation of the IRS1 protein. In xenograft tumor experiments and in a human phase I clinical trial, a pan-Pim inhibitor administered in vivo to animals or humans decreased IRS1S1101 phosphorylation in tumor tissues. This phosphorylation was shown to have effects on the half-life of the IRS family of proteins, suggesting a role in insulin or IGF signaling. [Conclusion] These results demonstrate that IRS1-S1101 is a novel substrate for the Pim kinases and provide the possibility of a novel marker for evaluation of Pim inhibitor therapy. Citation Format: Jin H. Song, Andrew S. Kraft. Insulin receptor substrate 1 is a key substrate for Pim protein kinases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4414.

Abstract LB-024: PIM kinase inhibitors selectively kill hypoxic cancer cells by reducing Nrf2 activity and increasing reactive oxygen species

Abstract Prostate cancer (PCa) is the leading cause of cancer death in men worldwide. Patients diagnosed with metastatic prostate cancer initially respond to androgen deprivation therapy, but this response is not durable, and all patients develop hormone-refractory disease that ultimately leads to death. Current treatment options for metastatic PCa patients are limited (e.g., cytotoxic chemotherapy or radiation), and the response to these agents is invariably temporary and does not improve survival. A significant obstacle to the successful treatment of advanced PCa is tumor hypoxia. While hypoxia is toxic to normal cells, cancer cells take particular advantage of their ability to adapt to these harsh conditions by increasing the expression of proteins that promote angiogenesis and survival. Therefore, elucidating the molecular mechanisms controlling survival in hypoxia is important for the development of effective therapeutic strategies to selectively kill hypoxic tumor cells. The Proviral Integration site for Moloney murine leukemia virus (PIM) kinases represent a pro-survival pathawy that is upregulated in response to hypoxia in a HIF-1 independent manner. We demonstrate that pharmacological or genetic inhibition of PIM kinases is significantly more toxic toward cancer cells in hypoxia versus normoxia. Xenograft studies confirm that PIM kinase inhibitors impede tumor growth and selectively kill hypoxic tumor cells in vivo. Experiments show that PIM kinases enhance the ability of tumor cells to adapt to hypoxia-induced oxidative stress by increasing the nuclear localization and activity of Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), which functions to increase the expression of antioxidant genes. Small molecule PIM kinase inhibitors reduce the ability of Nrf2 to enter the nucleus and block the transcrption of cytoprotective genes, leading to the accumulation of intracellular reactive oxygen species (ROS) to toxic levels in hypoxic tumor cells. This toxic effect of PIM inhibitors can be succesfully blocked by ROS scavengers, including N-acetyl cystine and superoxide dismutase. Thus, inhibition of PIM kinases has the potential to oppose hypoxia-mediate therapeutic resistance and induce cell death in the hypoxic tumor microenvironment. Citation Format: Alva G. Sainz, Jin H. Song, Andrew S. Kraft, Noel A. Warfel. PIM kinase inhibitors selectively kill hypoxic cancer cells by reducing Nrf2 activity and increasing reactive oxygen species. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-024.

Pim1 inhibition as a novel therapeutic strategy for Alzheimer's disease.

BackgroundAlzheimer’s disease (AD) is the most prevalent neurodegenerative disorder worldwide. Clinically, AD is characterized by impairments of memory and cognitive functions. Accumulation of amyloid-β (Aβ) and neurofibrillary tangles are the prominent neuropathologies in patients with AD. Strong evidence indicates that an imbalance between production and degradation of key proteins contributes to the pathogenesis of AD. The mammalian target of rapamycin (mTOR) plays a key role in maintaining protein homeostasis as it regulates both protein synthesis and degradation. A key regulator of mTOR activity is the proline-rich AKT substrate 40 kDa (PRAS40), which directly binds to mTOR and reduces its activity. Notably, AD patients have elevated levels of phosphorylated PRAS40, which correlate with Aβ and tau pathologies as well as cognitive deficits. Physiologically, PRAS40 phosphorylation is regulated by Pim1, a protein kinase of the protoconcogene family. Here, we tested the effects of a selective Pim1 inhibitor (Pim1i), on spatial reference and working memory and AD-like pathology in 3xTg-AD mice.ResultsWe have identified a Pim1i that crosses the blood brain barrier and reduces PRAS40 phosphorylation. Pim1i-treated 3xTg-AD mice performed significantly better than their vehicle treated counterparts as well as non-transgenic mice. Additionally, 3xTg-AD Pim1i-treated mice showed a reduction in soluble and insoluble Aβ40 and Aβ42 levels, as well as a 45.2 % reduction in Aβ42 plaques within the hippocampus. Furthermore, phosphorylated tau immunoreactivity was reduced in the hippocampus of Pim1i–treated 3xTg-AD mice by 38 %. Mechanistically, these changes were linked to a significant increase in proteasome activity.ConclusionThese results suggest that reductions in phosphorylated PRAS40 levels via Pim1 inhibition reduce Aβ and Tau pathology and rescue cognitive deficits by increasing proteasome function. Given that Pim1 inhibitors are already being tested in ongoing human clinical trials for cancer, the results presented here may open a new venue of drug discovery for AD by developing more Pim1 inhibitors.

Characterization of HJ-PI01 as a novel Pim-2 inhibitor that induces apoptosis and autophagic cell death in triple-negative human breast cancer.

Pim-2 is a short-lived serine/threonine kinase, which plays a key role in metastasis of breast cancer through persistent activation of STAT3. Although the crystal structure of Pim-2 has been reported, but thus far no specific Pim-2-targeted compounds have been reported. In this study, we identified a novel Pim-2 inhibitor, HJ-PI01, by in silico analysis and experimental validation. The protein-protein interaction (PPI) network, chemical synthesis, molecular docking, and molecular dynamics (MD) simulations were used to design and discover the new Pim-2 inhibitor HJ-PI01. The anti-tumor effects of HJ-PI01 were evaluated in human breast MDA-MB-231, MDA-MB-468, MDA-MB-436, MCF-7 cells in vitro and in MDA-MB-231 xenograft mice, which were treated with HJ-PI01 (40 mg·kg(-1)·d(-1), ig) with or without lienal polypeptide (50 mg·kg(-1)·d(-1), ip) for 10 d. The apoptosis/autophage-inducing mechanisms of HJ-PI01 were elucidated using Western blots, immunoblots, flow cytometry, transmission electron microscopy and fluorescence microscopy. Based on the PrePPI network, the potential partners interacting with Pim-2 in regulating apoptosis (160 protein pairs) and autophagy (47 protein pairs) were identified. Based on the structural characteristics of Pim-2, a total of 15 compounds (HJ-PI01 to HJ-P015) were synthesized, which showed moderate or remarkable anti-proliferative potency in the human breast cancer cell lines tested. The most effective compound HJ-PI01 exerted a robust inhibition on MDA-MB-231 cells compared with chlorpromazine and the pan-Pim inhibitor PI003. Molecular dynamics (MD) simulation revealed that HJ-PI01 had a good binding score with Pim-2. Moreover, HJ-PI01 (300 nmol/L) induced death receptor-dependent and mitochondrial apoptosis as well as autophagic death in MDA-MB-231 cells. In MDA-MB-231 xenograft mice, administration of HJ-PI01 remarkably inhibited the tumor growth and induced tumor cell apoptosis in vivo. Co-administration of HJ-PI01 with lienal polypeptide could improve the anti-tumor activity of HJ-PI01 and reduce its toxicity. The newly synthesized compound, HJ-PI01, can induce death receptor/mitochondrial apoptosis and autophagic cell death by targeting Pim-2 in human breast cancer cells in vitro and in vivo.