Pim Kinase Inhibitors Research Articles

PIM Kinase Inhibitors as Novel Promising Therapeutic Scaffolds in Cancer Therapy

: Cancer involves the uncontrolled, abnormal growth of cells and affects other tissues. Kinase has an impact on proliferating the cells and causing cancer. For the purpose of treating cancer, PIM kinase is a potential target. The pro-viral Integration site for moloney murine leukaemia virus (PIM) kinases is responsible for the tumorigenesis, by phosphorylating the proteins that control the cell cycle and cell proliferation. PIM-1, PIM-2, and PIM-3 are the three distinct isoforms of PIM kinases. The JAK/STAT pathway is essential for controlling how PIM genes are expressed. PIM kinase is also linked withPI3K/AKT/mTOR pathway in various types of cancers. The overexpression of PIM kinase will cause cancer. Currently, there are significant efforts being made in medication design and development to target its inhibition. A few small chemical inhibitors (E.g., SGI-1776, AZD1208, LGH447) that specifically target the PIM proteins' adenosine triphosphate (ATP)-binding domain have been identified. PIM kinase antagonists have a remarkable effect on different types of cancer. Despite conducting clinical trials on SGI-1776, the first PIM inhibitory agent, was prematurely withdrawn, making it unable to generate concept evidence. On the other hand, in recent years, it has aided in hastening the identification of multiple new PIM inhibitors. Cyanopyridines and Pyrazolo[1,5-a]pyrimidinecan act as potent PIM kinase inhibitors for cancer therapy. We explore the involvement of oncogenic transcription factor c-Mycandmi-RNA in relation to PIM kinase. In this article, we highlight the oncogenic effects, and structural insights into PIM kinase inhibitors for the treatment of cancer.

Pim1 inactivating induces RUNX3 upregulation that improves/alleviates airway inflammation and mucus hypersecretion in vitro and in vivo

PurposeOur research aimed to evaluate whether proto-oncogene serine/threonine-protein kinase Pim-1 (Pim1) inactivation could attenuate asthma by promoting runt-related transcription factor 3 (Runx3) expression and explore the underlying molecular mechanism.MethodPhorbol 12-myristate...

Inhibition of PIM kinase in tumor associated macrophages suppresses inflammasome activation and sensitizes prostate cancer to immunotherapy.

Immunotherapy has changed the treatment paradigm for many types of cancer, but immune checkpoint inhibitors (ICIs) have not shown benefit in prostate cancer (PCa). Chronic inflammation contributes to the immunosuppressive prostate tumor microenvironment (TME) and is associated with poor response to ICIs. The primary source of inflammatory cytokine production is the inflammasome. Here, we identify PIM kinases as important regulators of inflammasome activation in tumor associated macrophages (TAMs). Analysis of clinical data from a cohort of treatment naïve, hormone responsive PCa patients revealed that tumors from patients with high PIM1/2/3 display an immunosuppressive TME characterized by high inflammation (IL-1β and TNFα) and a high density of repressive immune cells, most notably TAMs. Strikingly, macrophage-specific knockout of PIM reduced tumor growth in syngeneic models of prostate cancer. Transcriptional analyses indicate that eliminating PIM from macrophages enhanced the adaptive immune response and increased cytotoxic immune cells. Combined treatment with PIM inhibitors and ICIs synergistically reduced tumor growth. Immune profiling revealed that PIM inhibitors sensitized PCa tumors to ICIs by increasing tumor suppressive TAMs and increasing the activation of cytotoxic T cells. Collectively, our data implicate macrophage PIM as a driver of inflammation that limits the potency of ICIs and provides preclinical evidence that PIM inhibitors are an effective strategy to improve the efficacy of immunotherapy in prostate cancer.

Molecular insights and inhibitory dynamics of flavonoids in targeting Pim-1 kinase for cancer therapy.

Pim-1 kinase, a serine/threonine kinase, is often overexpressed in various cancers, contributing to disease progression and poor prognosis. In this study, we explored the potential of flavonoids as inhibitors of Pim-1 kinase using a combination of molecular docking and steered molecular dynamics (SMD) simulations. Our docking studies revealed two main binding orientations for the flavonoid molecules. The SMD simulations showed that the binding mode with higher pulling forces was linked to stronger inhibitory activity, with a strong positive correlation (R 2 ≈ 0.92) between pulling forces and IC50 values. Quercetin stood out as the most potent inhibitor, showing a pulling force of about 820pN and an IC_(5) 0 of less than 6µM. Further dynamic simulations indicated that quercetin's hydroxyl groups at the C3, C-5 and C-7 positions formed stable hydrogen bonds with key residues GLU-121, Leu-44 and Val-126, respectively enhancing its binding stability and effectiveness. Our results emphasized the critical role of the hydroxyl group at the C-3 position, which plays a pivotal function in effectively anchoring these molecules in the active site of Pim-1 kinase. Principal component analysis (PCA) of Pim-1 kinase's conformational changes revealed that potent inhibitors like quercetin, galangin, and kaempferol significantly restricted the enzyme's flexibility, suggesting potential inhibitory effect. These findings provide insights into the structural interactions between flavonoids and Pim-1 kinase, offering a foundation for future experimental investigations. However, further studies, including in vitro and in vivo validation, are necessary to assess the pharmacological relevance and specificity of flavonoids in cancer therapy.

Pim Kinase Inhibition Disrupts CXCR4 Signalling in Megakaryocytes and Platelets by Reducing Receptor Availability at the Surface.

A key step in platelet production is the migration of megakaryocytes to the vascular sinusoids within the bone marrow. This homing is mediated by the chemokine CXCL12 and its receptor CXCR4. CXCR4 is also a positive regulator of platelet activation and thrombosis. Pim-1 kinase has been shown to regulate CXCR4 signalling in other cell types, and we have previously described how Pim kinase inhibitors attenuate platelet aggregation to CXCL12. However, the mechanism by which Pim-1 regulates CXCR4 signalling in platelets and megakaryocytes has yet to be elucidated. Using human platelets, murine bone marrow-derived megakaryocytes, and the megakaryocyte cell line MEG-01, we demonstrate that pharmacological Pim kinase inhibition leads to reduced megakaryocyte and platelet function responses to CXCL12, including reduced megakaryocyte migration and platelet granule secretion. Attenuation of CXCL12 signalling was found to be attributed to the reduced surface expression of CXCR4. The decrease in CXCR4 surface levels was found to be mediated by rapid receptor internalisation, in the absence of agonist stimulation. We demonstrate that pharmacological Pim kinase inhibition disrupts megakaryocyte and platelet function by reducing constitutive CXCR4 surface expression, decreasing the number of receptors available for agonist stimulation and signalling. These findings have implications for the development and use of Pim kinase inhibitors for the treatment of conditions associated with elevated circulating levels of CXCL12/SDF1α and increased thrombotic risk.

Evaluating the Physicochemical Properties-Activity Relationship and Discovering New 1,2-Dihydropyridine Derivatives as Promising Inhibitors for PIM1-Kinase: Evidence from Principal Component Analysis, Molecular Docking, and Molecular Dynamics Studies.

In this study, we evaluated the physicochemical properties related to the previously reported anticancer activity of a dataset comprising thirty 1,2-dihydropyridine derivatives. We utilized Principal Component Analysis (PCA) to identify the most significant influencing factors. The PCA analysis showed that the first two principal components accounted for 59.91% of the total variance, indicating a strong correlation between the molecules and specific descriptors. Among the 239 descriptors analyzed, 18 were positively correlated with anticancer activity, clustering with the 12 most active compounds based on their IC50 values. Six of these variables-LogP, Csp3, b_1rotN, LogS, TPSA, and lip_don-are related to drug-likeness potential. Thus, we then ranked the 12 compounds according to these six variables and excluded those violating the drug-likeness criteria, resulting in a shortlist of nine compounds. Next, we investigated the binding affinity of these nine shortlisted compounds with the use of molecular docking towards the PIM-1 Kinase enzyme (PDB: 2OBJ), which is overexpressed in various cancer cells. Compound 6 exhibited the best docking score among the docked compounds, with a docking score of -11.77 kcal/mol, compared to -12.08 kcal/mol for the reference PIM-1 kinase inhibitor, 6-(5-bromo-2-hydroxyphenyl)-2-oxo-4-phenyl-1,2-dihydropyridine-3-carbonitrile. To discover new PIM-1 kinase inhibitors, we designed nine novel compounds featuring hybrid structures of compound 6 and the reference inhibitor. Among these, compound 31 displayed the best binding affinity, with a docking score of -13.11 kcal/mol. Additionally, we performed PubChem database mining using the structure of compound 6 and the similarity search tool, identifying 16 structurally related compounds with various reported biological properties. Among these, compound 52 exhibited the best binding affinity, with a docking score of -13.03 kcal/mol. Finally, molecular dynamics (MD) studies were conducted to confirm the stability of the protein-ligand complexes obtained from docking the studied compounds to PIM-1 kinase, validating the potential of these compounds as PIM-1 kinase inhibitors.

Topoisomerase Inhibitors and PIM1 Kinase Inhibitors Improve Gene Editing Efficiency Mediated by CRISPR-Cas9 and Homology-Directed Repair.

The CRISPR-Cas9 system has emerged as the most prevalent gene editing technology due to its simplicity, high efficiency, and low cost. However, the homology-directed repair (HDR)-mediated gene knock-in in this system suffers from low efficiency, which limits its application in animal model preparation, gene therapy, and agricultural genetic improvement. Here, we report the design and optimization of a simple and efficient reporter-based assay to visualize and quantify HDR efficiency. Through random screening of a small molecule compound library, two groups of compounds, including the topoisomerase inhibitors and PIM1 kinase inhibitors, have been identified to promote HDR. Two representative compounds, etoposide and quercetagetin, also significantly enhance the efficiency of CRISPR-Cas9 and HDR-mediated gene knock-in in mouse embryos. Our study not only provides an assay to screen compounds that may facilitate HDR but also identifies useful tool compounds to facilitate the construction of genetically modified animal models with the CRISPR-Cas9 system.

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.

Deep PIM kinase substrate profiling reveals new rational cotherapeutic strategies for acute myeloid leukemia

AbstractProvirus integration site for Moloney murine leukemia virus (PIM) family serine/threonine kinases perform protumorigenic functions in hematologic malignancies and solid tumors by phosphorylating substrates involved in tumor metabolism, cell survival, metastasis, inflammation, and immune cell invasion. However, a comprehensive understanding of PIM kinase functions is currently lacking. Multiple small-molecule PIM kinase inhibitors are currently being evaluated as cotherapeutics in patients with cancer. To further illuminate PIM kinase functions in cancer, we deeply profiled PIM1 substrates using the reverse in-gel kinase assay to identify downstream cellular processes targetable with small molecules. Pathway analyses of putative PIM substrates nominated RNA splicing and ribosomal RNA (rRNA) processing as PIM-regulated cellular processes. PIM inhibition elicited reproducible splicing changes in PIM-inhibitor–responsive acute myeloid leukemia (AML) cell lines. PIM inhibitors synergized with splicing modulators targeting splicing factor 3b subunit 1 (SF3B1) and serine-arginine protein kinase 1 (SRPK1) to kill AML cells. PIM inhibition also altered rRNA processing, and PIM inhibitors synergized with an RNA polymerase I inhibitor to kill AML cells and block AML tumor growth. These data demonstrate that deep kinase substrate knowledge can illuminate unappreciated kinase functions, nominating synergistic cotherapeutic strategies. This approach may expand the cotherapeutic armamentarium to overcome kinase inhibitor–resistant disease that limits durable responses in malignant disease.

Assessment of structural and activity-related contributions of various PIM-1 kinase inhibitors in the treatment of leukemia and prostate cancer.

One of the most perilous illnesses in the world is cancer. The cancer may be associated with the mutation of different genes inside the body. The PIM kinase, also known as the serine/threonine kinase, plays a critical role in the biology of different kinds of cancer. They are widely distributed and associated with several biological processes, including cell division, proliferation, and death. Aberration of PIM-1 kinase is found in varieties of cancer. Prostate cancer and leukemia can both be effectively treated with PIM-1 kinase inhibitors. There are several potent compounds that have been explored in this review based on heterocyclic compounds for the treatment of prostate cancer and leukemia that have strong effects on the suppression of PIM-1 kinase. The present review summarizes the PIM-1 kinase pathway, their inhibitors under clinical trial, related patents, and SAR studies of several monocyclic, bicyclic, and polycyclic compounds. The study related to their molecular interactions with receptors is also included in the present manuscript. The study may be beneficial to scientists for the development of novel compounds as PIM-1 inhibitors in the treatment of prostate cancer and leukemia.

Inhibition of Pim kinases triggers a broad antiviral activity by affecting innate immunity and via the PI3K-Akt-mTOR axis the endolysosomal system

Zoonoses such as ZIKV and SARS-CoV-2 pose a severe risk to global health. There is urgent need for broad antiviral strategies based on host-targets filling gaps between pathogen emergence and availability of therapeutic or preventive strategies. Significant reduction of pathogen titers decreases spread of infections and thereby ensures health systems not being overloaded and public life to continue. Based on previously observed interference with FGFR1/2-signaling dependent impact on interferon stimulated gene (ISG)-expression, we identified Pim kinases as promising druggable cellular target. We therefore focused on analyzing the potential of pan-Pim kinase inhibition to trigger a broad antiviral response.The pan-Pim kinase inhibitor AZD1208 exerted an extraordinarily high antiviral effect against various ZIKV isolates, SARS-CoV-2 and HBV. This was reflected by strong reduction in viral RNA, proteins and released infectious particles. Especially in case of SARS-CoV-2, AZD1208 led to a complete removal of viral traces in cells. Kinome-analysis revealed vast changes in kinase landscape upon AZD1208 treatment, especially for inflammation and the PI3K/Akt-pathway. For ZIKV, a clear correlation between antiviral effect and increase in ISG-expression was observed. Based on a cell culture model with impaired ISG-induction, activation of the PI3K-Akt-mTOR axis, leading to major changes in the endolysosomal equilibrium, was identified as second pillar of the antiviral effect triggered by AZD1208-dependent Pim kinase inhibition, also against HBV.We identified Pim-kinases as cellular target for a broad antiviral activity. The antiviral effect exerted by inhibition of Pim kinases is based on at least two pillars: innate immunity and modulation of the endolysosomal system.

What doesn't fit is made to fit: Pim-1 kinase adapts to the configuration of stilbene-based inhibitors.

Recently, we have developed novel Pim-1 kinase inhibitors starting from a dihydrobenzofuran core structure using a computational approach. Here, we report the design and synthesis of stilbene-based Pim-1 kinase inhibitors obtained by formal elimination of the dihydrofuran ring. These inhibitors of the first design cycle, which were obtained as inseparable cis/trans mixtures, showed affinities in the low single-digit micromolar range. To be able to further optimize these compounds in a structure-based fashion, we determined the X-ray structures of the protein-ligand-complexes. Surprisingly, only the cis-isomer binds upon crystallization of the cis/trans-mixture of the ligands with Pim-1 kinase and the substrate PIMTIDE, the binding mode being largely consistent with that predicted by docking. After crystallization of the exclusively trans-configured derivatives, a markedly different binding mode for the inhibitor and a concomitant rearrangement of the glycine-rich loop is observed, resulting in the ligand being deeply buried in the binding pocket.

PIM1 inhibitor SMI-4a attenuated concanavalin A-induced acute hepatitis through suppressing inflammatory responses.

Serine/threonine kinase 1 (PIM1) plays a crucial role in cell growth, differentiation, and apoptosis. However, its role in the pathogenesis of concanavalin A (ConA)-induced acute hepatitis is not well understood. PIM1 kinase inhibitor can reduce the expression of PIM1. This study aims to investigate the effects of PIM1 kinase inhibitor and its protective mechanism in ConA-induced acute hepatitis. C57/BL six mice were injected with ConA (20, 15, and 12 mg/kg) to induce acute hepatitis, and PIM1 kinase inhibitor SMI-4a (60 mg/kg) was administered orally 24 h before ConA injection. The survival rate of the mice was observed after ConA injection. The levels of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. Serum inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was performed on liver tissue collected at different time points. The major cytokines expression in liver tissue was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The number of macrophages, T-cell and neutrophils in liver tissue were detected by flow cytometry (FCM). PIM1 in liver tissue was detected by western blot (WB) and qRT-PCR. SMI-4a (80 µM) was pretreated for 24 h and ConA (400 µg/mL) was stimulated for 12 h in RAW264.7 cell model. Phosphorylated p65 (p-p65) and cleaved caspase-3 (c-caspase-3) in liver tissue and macrophages were detected by WB. Different concentrations of ConA caused different acute hepatitis mortality, 12 mg/kg concentration within 24 h of the mortality showed a gradient increase. The levels of AST and ALT increased significantly at 12 h after ConA injection. PIM1 expression was upregulated at 12 h. SMI-4a can suppress the PIM1 expression. SMI-4a suppressed cytokines production, AST, and ALT in ConA-treated serum. SMI-4a suppressed the major cytokines in liver tissue. Tests in liver tissue showed that SMI-4a reduced the number of T cells, neutrophils, and macrophages. SMI-4a inhibited the inflammatory response by downregulating the expression of p-p65. Meanwhile, apoptosis was decreased by decreasing the expression of c-caspase-3. In conclusion, the protective effect of SMI-4a against acute hepatitis is by reducing the inflammatory response and apoptosis. These findings suggest that SMI-4a may have therapeutic potential in the treatment of autoimmune hepatitis.

Abstract 6875: PIM kinases alter the prostate tumor immune microenvironment

Abstract Prostate cancer is the second leading cause of cancer-related deaths in American men. While prostate cancer patients typically respond to androgen-deprivation therapy, androgen receptor signaling inhibitor (ARSI), and/or taxane chemotherapy, patients inevitably develop resistance, and the disease progresses to an untreatable and lethal form known as castrate resistant prostate cancer (CRPC). Immunotherapy has changed the treatment paradigm for many types of cancer; however, immune checkpoint inhibitors have not shown clinical benefits in CRPC. Clinical and preclinical evidence indicates that this lack of efficacy can largely be attributed to the immunosuppressive tumor microenvironment. Tumor associated macrophages (TAMs) secrete factors that promote tumor progression and suppress antitumor immunity, making them a promising therapeutic target. The Proviral Integration site for Moloney murine leukemia virus (PIM) kinases are serine/threonine kinases that are overexpressed in prostate cancer. PIM regulates many signaling pathways that promote cell proliferation and survival. However, how PIM kinase alters the prostate tumor immune microenvironment and impacts immunotherapy resistance is not well understood. We analyzed primary prostate and metastatic lymph node samples from treatment-naive metastatic hormone-sensitive prostate cancer patients. Prostate cancer samples with PIM1/PIM2/PIM3-high and -low expression were classified by top and bottom quartile, respectively. PIM high tumors had higher expression of immunostimulatory genes (IL1β, TNF, and TNFSF13), increased infiltration of M2 macrophages, B cells, and NK Cells, and a higher T cell inflamed score compared to PIM-low tumors. Our results demonstrate that PIM overexpressing prostate tumors display increased inflammation and infiltration of immunosuppressive immune cells, including M2 macrophages. Due to the changes observed in inflammation, we hypothesized that PIM kinase may regulate macrophage inflammatory signaling. We treated bone marrow derived macrophages with a PIM kinase inhibitor and showed that PIM inhibition suppresses inflammasome signaling and the release of the pro-inflammatory cytokine, IL-1β. Chronic inflammation can lead to the recruitment of TAMs, further promoting resistance to immune checkpoint inhibitors. Utilizing a syngeneic mouse model of prostate cancer, we demonstrated that PIM inhibition in combination with immune checkpoint blockade synergistically decreased tumor growth. Furthermore, immunoprofiling demonstrated that combination treatment enhances T cell activity. Overall, our results suggest that PIM kinase plays an important role in regulating the prostate tumor immune microenvironment and that PIM kinase may be a potential target to enhance the efficacy of immunotherapy for the treatment of prostate cancer. Citation Format: Amber N. Clements, Kai Sutterby, Sachin Kumar Deshmukh, Sharon Wu, Joanne Xiu, Alex Farrell, Milan Radovich, Chadi Nabhan, Elisabeth I. Heath, Rana R. McKay, Alejandro Recio-Boiles, Noel A. Warfel. PIM kinases alter the prostate tumor immune microenvironment [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 6875.

Abstract 4746: PIM kinase inhibition synergizes with a MERTK inhibitor to treat osimertinib resistant EGFR-mutant non-small cell lung cancer

Abstract There is an unmet need for effective treatment in the setting of acquired osimertinib resistance in EGFR-mutant non-small cell lung cancer (NSCLC) patients and MERTK tyrosine kinase is a potential therapeutic target in this context. Treatment with the MERTK inhibitor MRX-2843 was sufficient to abrogate downstream PI3K-AKT and MAPK-ERK signaling, cell expansion, and colony formation in osimertinib-resistant EGFR-mutant NSCLC cell lines, suggesting dependence on MERTK signaling for tumor cell proliferation/survival. Further, two structurally distinct PIM kinase inhibitors, SGI-1776 and PIM447, provided synergistic inhibition of cell expansion and colony formation in osimertinib-resistant cell line cultures when combined with MRX-2843 treatment. Mechanistically, treatment with MRX-2843 in combination with either PIM kinase inhibitor decreased downstream PI3K-AKT and MAPK-ERK signaling more effectively than single agents. Furthermore, knockdown of PIM kinases (PIM1, 2, 3) using nine sets of siRNAs led to various changes in expression of TAM receptor family members (TYRO3, AXL, MERTK), indicating a role for PIM kinases in regulation of TAM kinase expression. These studies suggest a novel treatment strategy for osimertinib-resistant EGFR-mutant NSCLC using a first-in-class MERTK kinase inhibitor that is currently under evaluation in multiple Phase I clinical trials. Citation Format: Dan Yan, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. PIM kinase inhibition synergizes with a MERTK inhibitor to treat osimertinib resistant EGFR-mutant non-small cell lung cancer [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 4746.

Abstract 4587: Development of highly selective, potent, orally available PIM1 inhibitor BLX0631 shows a therapeutics potential in multiple myeloma models

Abstract Background: The Proto-oncogenic PIMs family are nuclear and cytoplasmic Ser/Thr kinases (PIM1, PIM2, and PIM3) that are constitutively active and play a vital role in proliferation and survival in Multiple Myeloma (MM). Activated PIM1 kinase can induce progression of the cell cycle, inhibition of apoptosis, and modulation of other signal transduction pathways. Methods: BLX-0631 and a few analogs evaluated against a panel of MM cell lines (EJM, IM-9, L-363, LP-1, MM-1R, MM.1S, MOLP-2, NCI-H929, OPM-2, RPMI8226 and U-266) and primary MM patient samples by CellTiter-Glo® (CTG) assays. Cellular apoptosis, Cell cycle analysis, Flow Cytometry, and bone marrow microenvironment experiments on the most sensitive cells were performed. PIM1 and downstream targets by western blot, cell migration, and invasion on our lead PIM1 inhibitor BLX-0631 along with, kinome selectivity, ADME-Tox, PK, and in vivo MM.1S efficacy experiments were conducted. Results: The application of our empirical MolecuLernTM fragment library, PIM1 crystal structure, scaffold hopping, and the enumeration features of MolecuLernTM in combination with 3, 6, and 7-group analysis on pyridazine, lead to a synthesizable potential new lead series, representing Med Chem tractability and developable characteristics. Further, the consensus scoring, binding energies, and MolecuLern tractability filters were applied and led to the discovery of the BLX-0631 series that inhibits the PIM1 kinase. The active compounds, with their binding modes and lead optimization strategies, lead to the synthesis and testing of over 60 novel entities as PIM1 kinase inhibitors. Thus, based on our previous chemotype series and scaffold hopping method, the impact of changing the 3, 6, and 7 positions, while retaining the pyridazine core led to the discovery of BLX-0631 and its series of compounds exhibiting <5 nM potency. BLX-0631 in CTG assay demonstrated 100% cell growth inhibition across all MM cell lines tested. The lead compound BLX-0631 is a very potent inhibitor with an IC50 of 5.0/17/5.0 nM when tested against PIM1, PIM2, and PIM3 kinases respectively, and selective against a panel of 468 kinase panels. Dosing through IV and PO delivery routes, formulations, salt forms of BLX-0631 in mice, PK properties along with ADME-Tox, P450, and hERG results will be presented. In vivo MM.1S mouse xenograft models of BLX-0631 at 15, 30 and 45 mg/Kg, p.o, QD demonstrated TGI of 16.28%, 31.81% & 35.16% dose-dependently. Conclusions: The present study demonstrates that PIM1 plays a vital role in the proliferation and survival of Multiple Myeloma. BLX-0631 induces full inhibition of all MM cell lines, suppresses cancer cell proliferation in vitro, and exhibits promising efficacy in MM tumor models. BLX-0631, a nominated IND candidate can serve as a novel targeted agent for treating Multiple Myeloma patients. Citation Format: Zhaoang li, Chenyu Lin, David J. Bearss, Hariprasad Vankayalapati. Development of highly selective, potent, orally available PIM1 inhibitor BLX0631 shows a therapeutics potential in multiple myeloma models [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 4587.

Discovery of Benzoxazepines as a New Class of PIM1 Kinase Inhibitors Through Structure Based Virtual Screening, Biochemical Evaluation and Cytotoxicity Studies

AbstractPIM kinase is a serine/threonine protein kinase implicated in human cancers because it promotes cell proliferation while inhibiting apoptosis and is a crucial target for malignancy. In the present study, we used 700000 commercially available Chembridge small molecules library for structure‐based drug discovery to identify novel potent PIM1 kinase inhibitors. Based on the virtual screening results, ten compounds were chosen and screened enzymatically at 10 μM. Out of ten compounds, only two compounds, 22876894 (61 %) and 58626268 (63 %), inhibited PIM1 kinase. Subsequently, the enzymatic IC50 value for compounds 22876894 and 58626268 was calculated and found to be 0.8 μM and 3.9 μM. Cell viability was evaluated on human malignant cell lines HCT‐116 (colorectal carcinoma), U‐2OS (osteosarcoma), and non‐cancerous hTERT‐RPE‐1. It was found that compound 22876894 possessed significant inhibitory activity against HCT‐116 (IC50=12.1 μM), U‐2OS (IC50=23.2 μM), and hTERT‐RPE‐1 (IC50=12.7 μM) cell lines. The compound 58626268 possessed significant inhibitory activities against HCT‐116 (IC50=28.1 μM), U‐2OS (IC50=23.5 μM), and hTERT‐RPE‐1 (IC50=21 μM) cell lines. Further, the molecular dynamics simulation of identified compounds (22876894 and 58626268) was performed. Based on kinase profiling, cytotoxicity studies, and molecular dynamics simulation results, we claim 5‐{[7‐(1‐benzothien‐3‐yl)‐9‐hydroxy‐2,3‐dihydro‐1,4‐benzoxazepin‐4(5H)‐yl]carbonyl}pyridin‐2(1H)‐one (22876894) and 4‐(2‐aminoisonicotinoyl)‐7‐(1‐benzothien‐3‐yl)‐2,3,4,5‐tetrahydro‐1,4‐benzoxazepin‐9‐ol (58626268) as a selective and potential PIM1 kinase inhibitors.

PIM Kinase Inhibition Sensitizes Neuroblastoma to Doxorubicin

BackgroundChemoresistance contributes to relapse in high-risk neuroblastoma. Cancer cells acquire resistance through multiple mechanisms, including drug efflux pumps. In neuroblastoma, multidrug resistance-associated protein-1 (MRP1/ABCC1) efflux pump expression correlates with worse outcomes. These pumps are regulated by PIM kinases, a family of serine–threonine kinases, overexpressed in neuroblastoma. We hypothesized PIM kinase inhibition would sensitize neuroblastoma cells by modulating MRP1. MethodsKocak database query evaluated ABCC1, PIM1, PIM2, and PIM3 expression in neuroblastoma patients. SK-N-AS and SK-N-BE(2) cells were treated with doxorubicin or the pan-PIM kinase inhibitor, AZD1208. Flow cytometry assessed intracellular doxorubicin accumulation. AlamarBlue assay measured viability. The lethal dose 50% (LD50) of each drug and combination indices (CI) were calculated and isobolograms constructed to determine synergy. ResultsKocak database query demonstrated positive correlation between PIM genes and ABCC1. PIM kinase inhibition increased intracellular doxorubicin accumulation in both cell lines, suggesting PIM kinase regulation of MRP1. Isobolograms showed synergy between AZD1208 and doxorubicin. ConclusionsThe correlation between PIM and ABCC1 gene expression suggests PIM kinases may contribute to neuroblastoma chemotherapeutic resistance. PIM kinase inhibition increased intracellular doxorubicin accumulation. Combination treatment with AZD1208 and doxorubicin decreased neuroblastoma cell viability in a synergistic fashion. These findings support further investigations of PIM kinase inhibition in neuroblastoma. Type of StudyBasic Science Research. Level of EvidenceNA.

S100A8/A9 predicts response to PIM kinase and PD-1/PD-L1 inhibition in triple-negative breast cancer mouse models

BackgroundUnderstanding why some triple-negative breast cancer (TNBC) patients respond poorly to existing therapies while others respond well remains a challenge. This study aims to understand the potential underlying mechanisms distinguishing early-stage TNBC tumors that respond to clinical intervention from non-responders, as well as to identify clinically viable therapeutic strategies, specifically for TNBC patients who may not benefit from existing therapies.MethodsWe conducted retrospective bioinformatics analysis of historical gene expression datasets to identify a group of genes whose expression levels in early-stage tumors predict poor clinical outcomes in TNBC. In vitro small-molecule screening, genetic manipulation, and drug treatment in syngeneic mouse models of TNBC were utilized to investigate potential therapeutic strategies and elucidate mechanisms of drug action.ResultsOur bioinformatics analysis reveals a robust association between increased expression of immunosuppressive cytokine S100A8/A9 in early-stage tumors and subsequent disease progression in TNBC. A targeted small-molecule screen identifies PIM kinase inhibitors as capable of decreasing S100A8/A9 expression in multiple cell types, including TNBC and immunosuppressive myeloid cells. Combining PIM inhibition and immune checkpoint blockade induces significant antitumor responses, especially in otherwise resistant S100A8/A9-high PD-1/PD-L1-positive tumors. Notably, serum S100A8/A9 levels mirror those of tumor S100A8/A9 in a syngeneic mouse model of TNBC.ConclusionsOur data propose S100A8/A9 as a potential predictive and pharmacodynamic biomarker in clinical trials evaluating combination therapy targeting PIM and immune checkpoints in TNBC. This work encourages the development of S100A8/A9-based liquid biopsy tests for treatment guidance.

Pim Kinase Inhibitors Increase Gilteritinib Cytotoxicity in FLT3-ITD Acute Myeloid Leukemia Through GSK-3β Activation and c-Myc and Mcl-1 Proteasomal Degradation.

FLT3-ITD is present in 25% of in AML, with continued poor outcomes. Combining Pim kinase inhibitors with the FDA-approved FLT3 inhibitor gilteritinib increases cytotoxicity in vitro and in vivo through activation of GSK-3β, which phosphorylates and posttranslationally downregulates c-Myc and Mcl-1. The data support efficacy of GSK-3βactivation in FLT3-ITD AML, and also support development of a clinical trial combining the Pim inhibitor TP-3654 with gilteritinib.