PIM Kinase Family Research Articles

Role of PIM Kinase Inhibitor in the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD), a neurodegenerative disorder, is the most prevalent form of senile dementia, causing progressive deterioration of cognition, behavior, and rational skills. Neuropathologically, AD is characterized by two hallmark proteinaceous aggregates: amyloid beta (Aβ) plaques and neurofibrillary tangles (NFTs) formed of hyperphosphorylated tau. A significant study has been done to understand how Aβ and/or tau accumulation can alter signaling pathways that affect neuronal function. A conserved protein kinase known as the mammalian target of rapamycin (mTOR) is essential for maintaining the proper balance between protein synthesis and degradation. Overwhelming evidence shows mTOR signaling's primary role in age-dependent cognitive decline and the pathogenesis of AD. Postmortem human AD brains consistently show an upregulation of mTOR signaling. Confocal microscopy findings demonstrated a direct connection between mTOR and intraneuronal Aβ42 through molecular processes of PRAS40 phosphorylation. By attaching to the mTORC1 complex, PRAS40 inhibits the activity of mTOR. Furthermore, inhibiting PRAS40 phosphorylation can stop the Aβ-mediated increase in mTOR activity, indicating that the accumulation of Aβ may aid in PRAS40 phosphorylation. Physiologically, PRAS40 is phosphorylated by PIM1 which is a serine/threonine kinase of proto-oncogene PIM kinase family. Pharmacological inhibition of PIM1 activity prevents the Aβ-induced mTOR hyperactivity in vivo by blocking PRAS40 phosphorylation and restores cognitive impairments by enhancing proteasome function. Recently identified small-molecule PIM1 inhibitors have been developed as potential therapeutic to reduce AD-neuropathology. This comprehensive study aims to address the activity of PIM1 inhibitor that has been tested for the treatment of AD, in addition to the pharmacological and structural aspects of PIM1.

Targeting Pim2 improves effector function and longevity of T cells in cancer immunotherapy through regulating autophagy

Abstract The Pim kinase family is critically involved in tumorigenesis, yet its role in primary T cells is not well known. We reported that Pim2, distinct from the other two isoforms, inhibits T-cell responses to alloantigen. To study how Pim2 impacts T-cell immunity against cancer, we used murine models of adoptive cell therapy (ACT) and hematopoietic cell transplantation. Pim2 deficiency enhanced the ability of antigen specific CD8 T cells and polyclonal T cells in controlling solid tumor and leukemia growth, respectively. Pim2 deficiency increased cytokine production and metabolic activities of CD8 T cells in vitro and promoted effector but constrained exhausted CD8 subsets in tumor. Pim2-/- CD8 T cells had increased Tcf1 expression and longevity after ACT. Mechanistically, Pim2 binds P62 and constrains T-cell activation through facilitating autophagy. Pim2 deficiency in T cells attenuated LC3 lipidation, P62 degradation as well as autophagosome formation. Augmentation of autophagy via Atg5 overexpression or metformin treatment reduced effector differentiation and anti-tumor function of Pim2-/- T cells. Importantly, a Pim2-specific inhibitor improved murine T-cell immunity against melanoma and leukemia and increased activity of human melanoma-specific T cells and CD19 CAR-T cells. Taken together, Pim2 restrains T-cell anti-tumor immunity through regulating autophagy, and blocking Pim2 enhances effector differentiation and persistence of T cells after cancer immunotherapy.

Abstract 4508: Defining PIM kinase protein regulation and PROTAC for pre-clinical testing

Abstract The PIM kinase family are serine/threonine kinases known to be overexpressed in castration-resistant prostate cancer (CRPC). PIM1, an isoform of PIM, is known to regulate cell survival, migration, and proliferation. Uniquely, PIM kinases are constitutively active due to the lack of a regulatory domain. Therefore, PIM protein levels are reflected in PIM activity and are regulated by protein degradation and synthesis. PIM inhibitors have been developed, targeting the PIM kinase activity and inhibiting phosphorylation of downstream targets, but have had limited efficacy. Our lab and others demonstrate that PIM inhibitor treatment increases total PIM protein levels, suggesting that PIM kinases control protein stability via autophosphorylation. To elucidate the mechanism of autophosphorylation dependent regulation of PIM1, we used phospho-proteomics to identify potential autophosphorylation sites on PIM1. We have identified overexpression of deubiquitinase USP28 inhibiting degradation of PIM1, leading to the investigation of the role of F-box protein Fbw7 in regulating PIM1. Dominant-negative expression of Fbw7 results in increased PIM1 total protein. Additionally, coimmunoprecipitation of Fbw7 knockdown cells resulted in decreased PM1 ubiquitination compared to control. Therefore, we have identified Fbw7 as a putative E3 ligase responsible for PIM1 degradation. To address the limited efficacy of PIM inhibitors, we have developed the first PIM-PROTAC (PIMTAC) with our collaborators. Proteolysis targeting chimera (PROTAC) is a promising approach to eliminating the pro-tumorigenic effects of PIM. The PIMTAC selectively targets PIM kinases for degradation and we have demonstrated in vitro efficacy. PIMTAC successfully maintains degradation of all isoforms of PIM for over 72 hours. In vitro assessment of anti-tumor efficacy demonstrated increased apoptotic death with PIMTAC alone compared to PIM inhibitor or docetaxel. Furthermore, dual treatment of PIMTAC and docetaxel had more apoptotic death compared to PIM inhibitor with docetaxel. We aim to develop the PIMTAC for pre-clinical testing. Citation Format: Hope Liou, John Brognard, Rolf Swenson, Pedro Torres-Ayuso, Noel Warfel. Defining PIM kinase protein regulation and PROTAC for pre-clinical testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4508.

Identification and assessment of new PIM2 inhibitors for treating hematologic cancers: A combined approach of energy-based virtual screening and machine learning evaluation.

PIM2, part of the PIM kinase family along with PIM1 and PIM3, is often overexpressed in hematologic cancers, fueling tumor growth. Despite its significance, there are no approved drugs targeting it. In response to this challenge, we devised a thorough virtual screening workflow for discovering novel PIM2 inhibitors. Our process includes molecular docking and diverse scoring methods likemolecular mechanics generalized born surface area, XGBOOST, and DeepDock to rank potential inhibitors by binding affinities and interaction potential. Ten compounds were selected and subjected to an adequate evaluation of their biological activity. Compound 2 emerged as the most potent inhibitor with an IC50 of approximately 135.7 nM. It also displayed significant activity against various hematological cancers, including acute myeloid leukemia, mantle cell lymphoma, and anaplastic large cell lymphoma (ALCL). Molecular dynamics simulations elucidated the binding mode of compound 2 with PIM2, offering insights for drug development. These results highlight the reliability and efficacy of our virtual screening workflow, promising new drugs for hematologic cancers, notably ALCL.

Epigenetic and Transcriptional Consequences of PIM Kinase Activity and Inhibition in Diffuse Large B-Cell Lymphoma (DLBCL)

The PIM kinase family consists of 3 proto-oncogenic proteins: PIM1, PIM2 and PIM3, expressed in numerous malignancies, including DLBCL. PIM kinases regulate crucial processes, such as proliferation, apoptosis, metabolism, or migration, therefore their inhibition is of great interest as a potential therapeutic strategy. Although recent studies have shed new light on the biological role of PIMs in lymphoid cancers, the details and mechanisms of PIM's oncogenic effects and the consequences of their inhibition in DLBCL remain insufficiently understood. Earlier studies have demonstrated a potential epigenetic role of PIM through histone H3S10 phosphorylation, however, described only at a single locus. The broad genomic role of PIM in modulating transcription in lymphoma cells remains unclear. We confirmed the effect of PIM inhibition on histone modifications using small molecule inhibitors as well as genetic silencing of PIM kinases in DLBCL cell lines. PIM kinase inhibition changed the global amounts of histone modifications (including histone H4 pan-acetylation), accompanied by lower phosphorylation of RNA polymerase II, suggesting an involvement of PIMs in the RNA polymerase II pause release/elongation phase of transcription. Local histone acetylation changes in the enhancer (H3K27ac) and promoter (H3K9ac) regions in response to treatment with the pan-PIM inhibitor were also identified, coupled with gene expression profiling in DLBCL cell lines. PIM inhibition decreased the expression of genes controlled by super-enhancers (SE), i.e. wide regulatory regions responsible for the high level of expression of the most important genes for the cell, including oncogenes. The integrated results of epigenomic and transcriptomic analyses explain the changes in expression of genes associated with many pathways, including transcription, inflammatory response, apoptosis, epigenetic mechanisms or DNA damage. The induction of the latter was further experimentally confirmed by the examination of DNA breaks using γH2AX, the activating phosphorylation of CHK2 or comet assays following PIM inhibition. The conducted studies document an epigenetic function of PIM kinases, and suggest that inhibition of PIM activity leads to alterations in the DLBCL epigenetic landscape and associated changes in transcription. They also indicate that PIM inhibitors can be a therapeutic option in DLBCL. Study supported aby Foundation For Polish Science TEAM Grant # POIR.04.04.00-00-5C84/17 and Polish National Science Centre Grant # 2018/29/B/NZ5/01471.

Targeting Pim2 improves effector function and longevity of T cells in cancer immunotherapy

Abstract The Provirus Integration sites for Moloney murine leukemia virus (Pim) kinase family has been studied extensively in tumorigenesis. We previously reported a key role of Pim2 in negatively regulating T-cell responses to alloantigen. We further investigated how Pim2 regulates T-cell mediated anti-tumor responses using murine models of adoptive cell therapy (ACT) and autologous hematopoietic cell transplantation (HCT). We found that Pim2 −/−Pmel CD8 T cells showed enhanced ability for controlling B16 melanoma growth following ACT. Pim2 −/−T cells were more potent to control C1498 leukemia growth after syngeneic HCT. Mechanistically, Pim2 −/−CD8 T cells exhibited increased effector cytokine production, metabolic activities, and unexpectedly CD62L expression than WT controls following activation. Activated Pim2 −/−CD8 T cells had less apoptosis and exhaustion during expansion in IL-2. In vivo, Pim2 −/−CD8 T cells differentiated more into effector, but less into exhausted or suppressive subsets in tumors. On the other hand, Pim2 −/−CD8 T cells exhibited increased longevity in spleen and tumor draining lymph nodes. Our data indicates that Pim2 negatively regulates CD8 T-cell anti-tumor activity by controlling their persistence in secondary lymphoid organs and effector function in tumors. Blocking Pim2 with a Pim2-specific inhibitor improved graft-versus-leukemia activity after autologous HCT and enhanced anti-melanoma effects after ACT. Pim2 inhibition also increased anti-tumor activity of human melanoma-specific T cells and CD19 CAR-T cells. Taken together, targeting Pim2 may serve as a novel strategy for improving cancer immunotherapy through enhancing differentiation and persistence of effector T cells. NIH R01 CA258440 and R21 CA263140 to Xue-Zhong Yu, MCW Center for Immunology Pilot Award to Yongxia Wu

Principles, Processes and Modalities of Interaction Between Proto-Oncoprotein PIM-1 Kinase and Cell Division Cyclin CDC25A

All malignant tumors with aberrant cell differentiation and proliferation, unchecked growth, invasion, and metastasis are referred to as cancer. Its occurrence is a multi-step, complex process with many contributing components. Proto oncoprotein and The PIM kinase family is crucial to this procedure., and proto oncoprotein PIM-1 kinase is an important member of PIM family. The expression of proto oncoprotein PIM-1 is positively correlated with the status of lymphatic metastasis, and its overexpression could promote cell cycle and inhibit apoptosis. Cell division cycle 25 (CDC25) is a bispecific phosphatase from Schizosaccharomyces cerevisiae in 1986. CDC25 can activate CDK by catalyzing the dephosphorylation of inhibitory phosphorylation sites in the cell cycle dependent protein kinases (CDK) molecule, and regulate the response to DNA damage, thus promoting cell cycle operation. The interaction between the proto oncoprotein CDC25A in the above process is one of the keys to the current mechanism research with PIM-1 kinase. In this paper, PIM-1 and CDC25 are first introduced, respectively. Then, the interaction between PIM-1 and different substrates with a focus on CDC25A is presented. Finally, their role in tumor initiation and the application of CDC25A as a therapeutic target are highlighted. Therefore, this paper will discuss the interaction between CDC25A and PIM family.

Role and mechanism of PIM family in the immune microenvironment of diffuse large B cell lymphoma

BackgroundDiffuse large B cell lymphoma (DLBCL) is a more common non-Hodgkin lymphoma (NHL). This study aims to explore the prognostic value of PIM kinase family in DLBCL and its relationship with the immune microenvironment, to provide a certain reference for the prognosis and treatment of DLBCL.MethodsThe prognostic value of PIM kinase family in DLBCL from the data set GSE10846 was verified through survival analysis and cox regression analysis. Mutations in PIM kinase family and its relationship with immune cell infiltration were explored with online cBioPortal, TIMER database, and single-gene GSEA analysis. Finally, the expression of PIM kinase family in tissues from DLBCL clinical samples was validated through immunohistochemical staining.ResultsThe proteins of PIM kinase family were highly expressed in DLBCL patients, which are good prognostic factors for DLBCL patients. Then, PIM1-3 proteins were positively correlated with the immune infiltration of B cells, whose types of mutations also showed different degrees of correlation with B cells. PIM kinase family proteins also showed a high correlation with PDL1. In addition, PIM kinase family was also associated with the commonly mutated genes in DLBCL, such as MYD88, MYC, and BTK.ConclusionPIM kinase family may be a potential therapeutic target for DLBCL patients.

PIM Kinases Regulate Super-Enhancer-Dependent Gene Expression in Diffuse Large B-Cell Lymphoma

The PIM kinase family consists of 3 proto-oncogenic proteins: PIM1, PIM2 and PIM3, expressed in numerous cancers, including diffuse large B-cell lymphoma (DLBCL). PIM kinases regulate crucial processes, such as proliferation, apoptosis, metabolism, and migration, therefore their inhibition is of great interest as a potential therapeutic strategy. However, the detailed mechanisms of PIM's oncogenic effects and the consequences of their inhibition in DLBCL remain insufficiently understood. Our earlier studies (Szydłowski et al., Blood 2017, Szydłowski et al., Cancer Res. 2021) highlighted immunomodulatory functions of PIM kinases in lymphoid malignancies, but also indicated that their inhibition has a profound impact on gene expression. Although earlier studies indicated that PIMs regulate certain transcription factors and/or regulate gene expression via epigenetic mechanisms through histone H3S10 phosphorylation, the detailed genomic functions of PIM kinases in modulating transcription in DLBCL remains unclear. In order to investigate the mechanisms and consequences of PIM kinase inhibition on gene expression in DLBCL cells, we first performed RNA sequencing-based transcriptome profiling in DLBCL cells treated with the pan-PIM and FLT3 inhibitor MEN1703, which is currently in clinical development. MEN1703 markedly downregulated the expression of transcripts controlled by super-enhancers (SE), i.e. large regulatory regions responsible for high levels of transcript expression of the key essential genes for the cell, including oncogenes. The analysis also suggested new biological processes controlled by PIMs, including the reduction of non-coding RNAs generated from the SE regions. To understand the mechanisms linking PIM inhibition with decreased SE-driven gene expression, we assessed PIM-dependent H3S10 phosphorylation in lymphoma cells. Since H3S10 phosphorylation facilitates MYST family KAT8 (MOF) acetyltransferase recruitment to certain promoters/enhancers, we evaluated the histone H3 and H4 acetylations in DLBCL cells following PIM inhibition. MEN1703 markedly reduced the H3K9Ac, H3K14Ac and H4 pan-acetylation, which was associated with reduced BRD4 binding to the SE regions and reduced RNAPII serine 5 (S5) phosphorylation, but not S3 or S7 phosphorylations. Together, these observations suggest the participation of PIM in the elongation phase of SE-dependent gene transcription via H3S10-dependent epigenetic mechanism. The effects of PIM inhibition on histone modifications and proliferation were confirmed by genetic silencing or CRISPR-Cas9 deletion of individual PIM kinases. To better characterize the function of H3S10 phosphorylation in DLBCL, non-phosphorylatable H3S10A mutants were introduced to DLBCL cells. Compared to wild type counterparts, cells with the mutated H3S10 exhibited decreased proliferation, indicating a significant biological role of this modification in DLBCL. The effect was potentiated in cells exposed to metabolic stress (starvation) conditions. Together, these studies confirm the epigenetic role of PIM kinases, especially in the regulation of SE-dependent gene transcription and suggest that inhibition of PIM activity leads to disturbances of SE integrity and DLBCL transcription. The results document new, epigenetic function of PIM kinases and present a novel cytotoxicity mechanism of their inhibition in DLBCL. They also indicate that PIM inhibition with MEN1703 can be a therapeutic option in DLBCL. Study supported aby Foundation For Polish Science TEAM Grant # POIR.04.04.00-00-5C84/17 and Polish National Science Centre Grant # 2018/29/B/NZ5/01471.

Targeting Pim2 for Improving T-Cell Effector Function and Promoting Cancer Immunotherapy

The Provirus Integration sites for Moloney murine leukemia virus (Pim) kinases are a highly conserved family of serine/threonine kinases. The Pim kinase family is composed of three different isoforms, Pim1, Pim2, and Pim3, which have been studied extensively in tumorigenesis and as a potential therapeutic target in various cancers. We previously reported an unexpected role of Pim2 in negatively regulates T-cell responses to alloantigen and tumor (JCI, 2015, PMID: 29781812). However, the mechanisms by which Pim2 modulates T-cell responses remain largely undefined. In the current study, using genetic Pim2-deficient mouse, we demonstrated a key role of Pim2 in regulating T-cell hemostatic and anti-tumor responses in aging, hematopoietic cell transplantation (HCT), and antigen-specific adoptive T-cell therapy (ACT). We observed that Pim2 was critical for T cells to retain quiescent in aged mice, as thymic Treg development was impaired while effector T-cell differentiation in lymphoid organs, including Tc1/Th1, Tc17/Th17 and follicular helper T cells, was increased in Pim2-deficient mice, but not in Pim1/Pim3-deficient mice. Furthermore, Pim2-deficient mice were capable to completely eradicate syngeneic breast cancer (NT2.5) growth (Figure A). During antigen specific anti-tumor response, adoptively transferred Pim2 -/- CD8 T cells showed enhanced ability for controlling established NT2.5 breast cancer and B16 melanoma (Figure B, C). Mechanistically, loss of Pim2 promoted G1 to S phase cell-cycle progression while reduced apoptosis in CD8 T cells. Pim2 -/- CD8 T cells exhibited elevated effector cytokine production while maintained higher levels of CD62L expression, leading to superior effector function, persistence and anti-tumor activity. Reduced differentiation of exhausted and suppressive subsets were observed in Pim2 -/- CD8 T cells after being adoptively transferred in tumor-bearing mice. In addition, Pim2 deficiency was associated with a higher metabolic potential, reflected by increased glycolysis and oxidative phosphorylation, which was at least partially attributed to a decreased level of autophagy in Pim2 -/- CD8 T cells. To further evaluate the clinical translation potential, we applied a Pim2-specific inhibitor (JP11646) and found that blocking Pim2 improved graft-versus-leukemia activity after autologous HCT and also enhanced CD8 T-cell mediated anti-melanoma effects after ACT in mice (Figure B, C). Furthermore, blocking Pim2 using JP11646 promoted human CD8 T-cell response during polyclonal stimulation and enhanced expansion, effector function and tumor killing ability of human melanoma antigen-specific CD8 T cells (data not shown) and CD19 CAR-T cells (Figure D). Our work demonstrated that Pim2 is a potent and distinct regulator of differentiation and maintenance of T effector cells through modulating metabolism and autophagy. Specifically target Pim2 can serve as a novel strategy for improving cancer immunotherapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pim Kinases as Therapeutic Targets in Early RheumatoidArthritis.

As well as being an established oncoprotein and therapeutic target in cancer, proviral integration site for Moloney murine leukemia virus 1 (Pim-1) is implicated in human autoimmunity. This study was undertaken to investigate Pim-1 and its family members as potential therapeutic targets in early rheumatoid arthritis (RA). A flow cytometry assay for PIM1 transcript measurement in peripheral blood mononuclear cells from patients with early arthritis was validated and applied as a biomarker of Pim-1 activity at the cellular level. Synovial protein expression was similarly determined by multiplex immunofluorescence in tissue samples from untreated RA patients and non-RA disease controls. Functional consequences of Pim kinase family manipulation in freshly isolated CD4+ T cells from these individuals were ascertained, along with the impact of Pim inhibition on mice with collagen-induced arthritis (CIA). The percentage of circulating CD4+ T cells positive for PIM1 transcript by flow cytometry proved a faithful surrogate for gene expression and was significantly higher in patients with early RA than in those with other diseases. Pim-1 protein levels were similarly up-regulated in synovial CD4+ T cells from patients with early RA. Ex vivo, exposure of T cell receptor-stimulated early RA CD4+ T cells to Pim kinase inhibitors restrained their activation and proliferative capacity. Diminished production of proinflammatory cytokines (interferon-γ and interleukin-17) and an expanded CD25high FoxP3+ Treg cell fraction were also observed in exposed versus unexposed cells. Finally, administration of Pim inhibitors robustly limited arthritis progression and cartilage destruction in CIA. Our findings indicate that Pim kinases are plausible therapeutic targets in a readily identifiable subgroup of patients with early RA. Repurposing of Pim inhibitors for this disease should be considered.

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.

Aminothiazolones as potent, selective and cell active inhibitors of the PIM kinase family

We have previously reported the discovery of a series of rhodanine-based inhibitors of the PIM family of serine/threonine kinases. Here we described the optimisation of those compounds to improve their physicochemical and ADME properties as well as reducing their off-targets activities against other kinases. Through molecular modeling and systematic structure activity relationship (SAR) studies, advanced molecules with high inhibitory potency, reduced off-target activity and minimal efflux were identified as new pan-PIM inhibitors. One example of an early lead, OX01401, was found to inhibit PIMs with nanomolar potency (15 nM for PIM1), inhibit proliferation of two PIM-expressing leukaemic cancer cell lines, MV4-11 and K562, and to reduce intracellular phosphorylation of a PIM substrate in a concentration dependent manner.

Insights into the Interaction Mechanisms of the Proviral Integration Site of Moloney Murine Leukemia Virus (Pim) Kinases with Pan-Pim Inhibitors PIM447 and AZD1208: A Molecular Dynamics Simulation and MM/GBSA Calculation Study.

Based on the up-regulation of the proviral integration site of the Moloney murine leukemia virus (Pim) kinase family (Pim1, 2, and 3) observed in several types of leukemias and lymphomas, the development of pan-Pim inhibitors is an attractive therapeutic strategy. While only PIM447 and AZD1208 have entered the clinical stages. To elucidate the interaction mechanisms of three Pim kinases with PIM447 and AZD1208, six Pim/ligand systems were studied by homology modeling, molecular docking, molecular dynamics (MD) simulation and molecular mechanics/generalized Born surface area (MM/GBSA) binding free energy calculation. The residues of the top group (Leu44, Val52, Ala65, Lys67, and Leu120 in Pim1) dominated the pan-Pim inhibitors binding to Pim kinases. The residues of the bottom group (Gln127, Asp128, and Leu174 in Pim1) were crucial for Pims/PIM447 systems, while the contributions of these residues were decreased sharply for Pims/AZD1208 systems. It is likely that the more potent pan-Pim inhibitors should be bound strongly to the top and bottom groups. The residues of the left, right and loop groups were located in the loop regions of the binding pocket, however, the flexibility of these regions triggered the protein interacting with diverse pan-Pim inhibitors efficiently. We hope this work can provide valuable information for the design of novel pan-Pim inhibitors in the future.

The serine-threonine kinase PIM3 is an aldosterone-regulated protein in the distal nephron.

The mineralocorticoid hormone aldosterone plays a crucial role in the control of Na+ and K+ balance, blood volume, and arterial blood pressure, by acting in the aldosterone‐sensitive distal nephron (ASDN) and stimulating a complex transcriptional, translational, and cellular program. Because the complexity of the aldosterone response is still not fully appreciated, we aimed at identifying new elements in this pathway. Here, we demonstrate that the expression of the proto‐oncogene PIM3 (Proviral Integration Site of Moloney Murine Leukemia Virus 3), a serine/threonine kinase belonging to the calcium/calmodulin‐regulated group of kinases, is stimulated by aldosterone in vitro (mCCDcl1 cells), ex vivo (mouse kidney slices), and in vivo in mice. Characterizing a germline Pim3 − / − mouse model, we found that these mice have an upregulated Renin‐Angiotensin‐Aldosterone System (RAAS), with high circulating aldosterone and plasma renin activity levels on both standard or Na+‐deficient diet. Surprisingly, we did not observe any obvious salt‐losing phenotype in Pim3 KO mice as shown by normal blood pressure, plasma and urinary electrolytes, as well as unchanged expression levels of the major Na+ transport proteins. These observations suggest that the potential effects of the loss of the Pim3 gene are physiologically compensated. Indeed, the 2 other family members of the PIM kinase family, PIM1 and PIM2 are upregulated in the kidney of Pim3 − / − mice, and may therefore be involved in such compensation. In conclusion, our data demonstrate that the PIM3 kinase is a novel aldosterone‐induced protein, but its precise role in aldosterone‐dependent renal homeostasis remains to be determined.

Discovery of novel triazolo[4,3-b]pyridazin-3-yl-quinoline derivatives as PIM inhibitors

PIM kinase family (PIM-1, PIM-2 and PIM-3) is an appealing target for the discovery and development of selective inhibitors, useful in various disease conditions in which these proteins are highly expressed, such as cancer. The significant effort put, in the recent years, towards the development of small molecules exhibiting inhibitory activity against this protein family has ended up with several molecules entering clinical trials. As part of our ongoing exploration for potential drug candidates that exhibit affinity towards this protein family, we have generated a novel chemical series of triazolo[4,3-b]pyridazine based tricycles by applying a scaffold hopping strategy over our previously reported potent pan-PIM inhibitor ETP-47453 (compound 2). The structure-activity relationship studies presented herein demonstrate a rather selective PIM-1/PIM-3 biochemical profile for this novel series of tricycles, although pan-PIM and PIM-1 inhibitors have also been identified. Selected examples show significant inhibition of the phosphorylation of BAD protein in a cell-based assay. Moreover, optimized and highly selective compounds, such as 42, did not show significant hERG inhibition at 20 μM concentration, and proved its antiproliferative activity and utility in combination with particular antitumoral agents in several tumor cell lines.

AZD1208, a Pan-Pim Kinase Inhibitor, Has Anti-Growth Effect on 93T449 Human Liposarcoma Cells via Control of the Expression and Phosphorylation of Pim-3, mTOR, 4EBP-1, S6, STAT-3 and AMPK.

Overexpression of Pim kinases has an oncogenic/pro-survival role in many hematological and solid cancers. AZD1208 is a pan-Pim kinase inhibitor that has anti-cancer and anti-adipogenic actions. Here, we investigated the effects of AZD1208 on the growth of 93T449 cells, a differentiated human liposarcoma cell line. At 20 µM, AZD1208 was cytotoxic (cytostatic) but not apoptotic, reducing cell survival without DNA fragmentation, caspase activation or increasing cells in the sub G1 phase; known apoptotic parameters. Notably, AZD1208 reduced phosphorylation of signal transducer and activator of transcription-3 (STAT-3) in 93T449 cells. STAT-3 inhibition by AG490, a JAK2/STAT-3 inhibitor similarly reduced cell survival. AZD1208 down-regulated phosphorylation of mammalian target of rapamycin (mTOR) and ribosomal S6 while up-regulated eukaryotic initiation factor-2α (eIF-2α). In addition, AZD1208 induced a LKB-1-independent AMPK activation, which was crucial for its cytostatic effect, as knock-down of AMPK greatly blocked AZD1208s ability to reduce cell survival. AZD1208 had no effect on expression of two members of Pim kinase family (Pim-1 and Pim-3) but inhibited phosphorylation of 4EBP-1, a downstream effector of Pim kinases. Importantly, a central role for Pim-3 in the actions of AZD1208 was confirmed by knock-down, which not only reduced 93T449 cell survival but also led to the inhibition of 4EBP-1, mTOR, eIF-2α and STAT-3, along with the activation of AMPK. In summary, this is the first report demonstrating that AZD1208 inhibits growth of liposarcoma cells and that this activity is mediated through Pim-3 kinase, STAT-3, mTOR, S6 and AMPK expression and phosphorylation pathways.

PIM-2 protein kinase negatively regulates T cell responses in transplantation and tumor immunity.

PIM kinase family members play a crucial role in promoting cell survival and proliferation via phosphorylation of their target substrates. In this study, we investigated the role of the PIM kinases with respect to T cell responses in transplantation and tumor immunity. We found that the PIM-2 isoform negatively regulated T cell responses to alloantigen, in contrast to the PIM-1 and PIM-3 isoforms, which acted as positive regulators. T cells deficient in PIM-2 demonstrated increased T cell differentiation toward Th1 subset, proliferation, and migration to target organs after allogeneic bone marrow transplantation, resulting in dramatically accelerated graft-versus-host disease (GVHD) severity. Restoration of PIM-2 expression markedly attenuated the pathogenicity of PIM-2-deficient T cells to induce GVHD. On the other hand, mice deficient in PIM-2 readily rejected syngeneic tumor, which was primarily dependent on CD8+ T cells. Furthermore, silencing PIM-2 in polyclonal or antigen-specific CD8+ T cells substantially enhanced their antitumor response in adoptive T cell immunotherapy. We conclude that PIM-2 kinase plays a prominent role in suppressing T cell responses, and provide a strong rationale to target PIM-2 for cancer immunotherapy.

Abstract 524: Characterizing the in vitro and in vivo effects of the PIM kinase inhibitor HS140 in triple-negative human breast cancer

Abstract Triple-negative breast cancer (TNBC) lacks expression of the estrogen receptor (ER), the progesterone receptor (PR) and the ERBB2 (also known as HER2) receptor, making it the worse subtype of all breast cancers. Currently, there are no molecular targeted therapies for this cancer and chemotherapy is only successful in a limited number of patients. Recent studies suggest that members of the oncogenic PIM kinase family, especially PIM-1, play a significant role in the growth of TNBC. However, there is very limited information on the role other PIM family members have in the growth and development of TNBCs. As an attempt to address this concern, we treated a panel of TNBC cell lines with HS140, a PIM-2 kinase inhibitor developed in the laboratory. Using cytotoxicity assays, we were able to demonstrate a decrease in anchorage-dependent growth of cell lines at different concentrations of HS140. Also, a triple negative breast cancer GEMM (C3TAg) was used for an efficacy study. Mice (FVB/N background) have C(3)SV40 T-antigen resulting in inactivation of p53 and Rb. Nine mice received HS140 treatment (80mg/kg BIW IP) and 14 left untreated for control (non-treatment [NT]). All mice were monitored for weight loss and timespan to tumor development 5 times weekly. Tumor volumes at 21 days were significantly (Mann-Whitney, p=0.0002) reduced in the treated cohort with a mean of 109mm3 (range of 0-5000) compared to untreated 1393mm3 (0-239). We observed no toxicities with body mass stable at the treatment dose of 80mg/kg. Additionally, three mice from untreated and three mice from treated cohorts were chosen for blood sampling pre- and post-treatment via a submandibular bleed; no significant differences were noted in WBC, RBC, PLT, or HGB between untreated and treated cohorts. Overall, these results suggest that other members of the PIM kinase family, including PIM-2, have an important function in the growth and development of TNBC and may serve as a potential molecular target for future therapeutics. Citation Format: Michael Cobb, Lucas Hunter, David Carlson, David Darr, Timothy Haystead, Antonio T. Baines. Characterizing the in vitro and in vivo effects of the PIM kinase inhibitor HS140 in triple-negative human breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 524. doi:10.1158/1538-7445.AM2017-524

Thiazolidine derivatives as potent and selective inhibitors of the PIM kinase family

The PIM family of serine/threonine kinases have become an attractive target for anti-cancer drug development, particularly for certain hematological malignancies. Here, we describe the discovery of a series of inhibitors of the PIM kinase family using a high throughput screening strategy. Through a combination of molecular modeling and optimization studies, the intrinsic potencies and molecular properties of this series of compounds was significantly improved. An excellent pan-PIM isoform inhibition profile was observed across the series, while optimized examples show good selectivity over other kinases. Two PIM-expressing leukemic cancer cell lines, MV4-11 and K562, were employed to evaluate the in vitro anti-proliferative effects of selected inhibitors. Encouraging activities were observed for many examples, with the best example (44) giving an IC50 of 0.75μM against the K562 cell line. These data provide a promising starting point for further development of this series as a new cancer therapy through PIM kinase inhibition.