Pim-1 Kinase 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.

Design and one-pot synthesis of new substituted pyrrolo[1,2-a]thieno[3,2-e]pyrimidine as potential antitumor agents: in vitro and in vivo studies

A new efficient and versatile one-pot three-component synthesis of substituted pyrrolo[1,2-a]thieno[3,2-e]pyrimidine derivatives has been developed. It is based on a multistep cascade reaction from 2-aminothiophenes and 2-hydroxy-4-oxobut-2-enoic acids, and derivatives of cyanoacetic acid catalyzed by diisopropylethylamine. As a result, novel pyrrolo[1,2-a]thieno[3,2-e]pyrimidine derivatives (21 compounds) were synthesized in a mild reaction conditions with a high yield. The structures of the developed compounds were confirmed by NMR and elemental analysis. The influence of electron-withdrawing or electron-donor substituents on the antitumor activity of the developed compounds has been identified. In vitro screening analysis of 21 compounds revealed six lead candidates (12aa, 12dc, 12hc, 12ic, 12lb, and 12mb) that demonstrated the most significant antitumor activity against B16-F10, 4T1 and CT26 cells. Necrosis/apoptosis assay showed that apoptosis was the predominant mechanism of cell death. Molecular docking analysis revealed several potential targets for tested compounds, i.e. phosphatidylinositol 5-phosphate 4-kinase (PI5P4K2C), proto-oncogene serine/threonine-protein kinase (Pim-1), nicotinamide phosphoribosyltransferase (NAMPT) and dihydrofolate reductase (DHFR). The lead compound (12aa) can effectively induce cell apoptosis, possesses a high yield (98 %) and requires low-cost starting chemicals for its synthesis. In vivo experiments with melanoma-bearing mice confirmed that 12aa compound resulted in the significant tumor inhibition on 15 d after the therapy. In particular, tumor volume was ∼0.19 cm3 for 50 mg/kg versus ∼2.39 cm3 in case of untreated mice and tumor weight was ∼71.6 mg for 50 mg/kg versus ∼452.4 mg when considered untreated mice. Thus, our results demonstrated the high potential of the 12aa compound in the treatment of melanoma and can be recommended for further preclinical studies.

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.

Regnase-1 D141N mutation induces CD4+ T cell-mediated lung granuloma formation via upregulation of Pim2.

Regnase-1 is an RNase that plays a critical role in negatively regulating immune responses by destabilizing inflammatory messenger RNAs (mRNAs). Dysfunction of Regnase-1 can be a major cause of various inflammatory diseases with tissue injury and immune cell infiltration into organs. This study focuses on the role of the RNase activity of Regnase-1 in developing inflammatory diseases. We have constructed mice with a single point mutation at the catalytic center of the Regnase-1 RNase domain, which lacks endonuclease activity. D141N mutant mice demonstrated systemic inflammation, immune cell infiltration into various organs, and progressive development of lung granuloma. CD4+ T cells, mainly affected by this mutation, upregulated the mTORC1 pathway and facilitated the autoimmune trait in the D141N mutation. Moreover, serine/threonine kinase Pim2 contributed to lung inflammation in this mutation. Inhibition of Pim2 kinase activity ameliorated granulomatous inflammation, immune cell infiltration, and proliferation in the lungs. Additionally, Pim2 inhibition reduced the expression of adhesion molecules on CD4+ T cells, suggesting a role for Pim2 in facilitating leukocyte adhesion and migration to inflamed tissues. Our findings provide new insights into the role of Regnase-1 RNase activity in controlling immune functions and underscore the therapeutic relevance of targeting Pim2 to modulate abnormal immune responses.

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.

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.

Abstract CT116: Preclinical antitumor activity and first-in-human phase I study of NB004/GDC-0570, a novel pan-PIM kinase inhibitor, in patients with advanced solid tumors

Abstract PIM proto-oncogenes encode a family of three serine/threonine kinases (PIM1, PIM2, PIM3), which are frequently overexpressed in human hematological malignancies and solid cancers. PIM kinases are promising cancer therapeutic targets for pharmacological inhibition. NB004 (aka GDC-0570) is a novel, potent, selective, orally available small molecule pan-PIM inhibitor, which exhibits antiproliferative activity as a single agent in a variety of preclinical models and can augment the activity of standard of care or targeted therapies, both in vitro and in vivo. In multiple patient-derived xenograft (PDX) models of human solid tumors, NB004 demonstrates both single agent and combination activity with agents targeting the RTK/RAS/MAPK pathways, including synergistic effects with KRAS inhibitors (KRASis). Remarkably, in NSCLC PDX models with acquired resistance to KRASis, strong combination efficacy was observed between NB004 and sotorasib, resulting in complete tumor regression. Based on the promising preclinical data, NB004 is currently being assessed in an open-label, first-in-human phase I clinical trial as monotherapy or combination therapy in patients with advanced solid tumors. The primary objective is to characterize the safety and tolerability and to recommend the phase 2 dose (RP2D). Secondary objectives include PK characterization and preliminary antitumor activity evaluation. In the phase I monotherapy dose escalation part, NB004 was administered daily in 5 dose levels, ranging from 75mg to 600mg, following a standard 3+3 design. As of December 22, 2023, a total of 18 patients were treated. Of the 13 patients (72.2%) who reported any treatment-related adverse events (TRAEs), 12 (66.7%) had Grade 1 or 2 TRAEs, 1 patient (5.6%) experienced Grade 3 neutrophil count decrease and white blood cell count decrease. No dose-limiting toxicity (DLT) or treatment-related serious adverse event (SAE) was reported, and PK exposure is in general dose proportional. These results warrant further evaluation of this drug in combination with other therapeutic agents. The combination part of the trial is currently open for enrollment (NCT05036291). Citation Format: Siqing Fu, Rachel E. Sanborn, Minxia Cai, Lijia Zhang, Yanhua Xu, Kui Lin. Preclinical antitumor activity and first-in-human phase I study of NB004/GDC-0570, a novel pan-PIM kinase inhibitor, in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT116.

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 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.

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.

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 1642: NuMA exclusive post-translational modification in epithelial cancer cells is a novel target for cell eradication

Abstract The post-translational modification of proteins implicated in mitosis in healthy and in cancer epithelial cells was measured by 2-D gels analysis. The post-translational modifications of NuMA in a variety of human epithelial cancer cells was prevented by treatment with a small phenanthridine, PJ34, while not impaired in similarly treated healthy epithelial cells. NuMA in the cancer cells was phosphorylated by pim1 kinase, and was polyADP-ribosylated by Tankyrase1. These post-translational modifications are inhibited by PJ34. Tankyrase1 is not expressed in healthy epithelial cells, and pim1 kinase was hardly expressed in these cells. Preventing the post-translational modification of NuMA in the epithelial cancer cells also suppressed the protein binding capacity of NuMA. NuMA clustering in the mitotic spindle poles, which stabilizes the mitotic spindle, and is required for the alignment of chromosomes in the spindle mid-zone was impaired. In epithelial cancer cells treated with PJ34, the mitotic spindle poles were distorted, extra-centrosomes were not clustered in multi-centrosomal cancer cells, and chromosomes were dispersed. Chromosomes dispersion violated mitosis termination. Mitosis arrested in the anaphase led to mitotic catastrophe cell death. PJ34 treated epithelial cancer cells were eradicated during 72-96 hours, regardless of their mutations, while treated healthy epithelial cells were not affected, and continued to proliferate. Citation Format: Malka Cohen-Armon. NuMA exclusive post-translational modification in epithelial cancer cells is a novel target for cell eradication [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 1642.

Abstract 650: Pim3 kinase is a poor prognostic marker and novel molecular target for triple negative breast cancer

Abstract Triple-negative Breast cancer (TNBC) is characterized by the absence of ER, PR, and HER-2 receptors and is associated with poor prognosis, early relapse, and distant metastasis. In neoadjuvant setting, most patients (~50-70%) with TNBC do not achieve complete remission. Currently, there is no effective targeted therapy for TNBC patients. Therefore, identifying novel molecular targets and developing alternative therapeutic strategies are urgently needed. PIM3 is a serine/threonine kinase and is upregulated in leukemia and various solid cancers. Although PIM1 has been shown to play a critical role in cell proliferation in cMyc-enriched TNBC tumors, the role of PIM3 in TNBC is not known. To determine the clinical significance of PIM3 in TNBC patient prognosis and progression, we performed a Kaplan-Meier analysis in the TCGA TNBC patient database. We found that PIM3 expression is significantly associated with shorter patient survival in TNBC patients (n=77, p=0.0088). PIM3 protein is highly expressed in TNBC cells compared to normal breast epithelium and other ER+ and HER2+ breast cancer subtypes. Inhibition of PIM3 by siRNA significantly suppressed TNBC cell proliferation, migration, and invasion of TNBC cells including MDA-MB-231, MDA-MB-436, and BT20 cells. Lenti-based PIM3 overexpression induced TNBC cell proliferation, invasion, and migration. Non-biased reverse phase protein array (RPPA) assay and Western blot analysis showed that PIM3 inhibition markedly reduced the expression of EF2 Kinase protein, and we found it to be associated with shorter patient survival and poor prognosis. Immunoprecipitation of PIM3 indicated that it forms a heterodimer with EF2K whose inhibition by siRNA also suppressed TNBC cell proliferation, migration, invasion, and tumor growth. Furthermore, PIM3 inhibition by siRNA significantly enhanced the antiproliferative effect of Doxorubicin, which is one of the first-line chemotherapeutics used in patients. Moreover, in vivo therapeutic delivery of PIM3 siRNA loaded albumin nanoparticles (ALNPs-PIM3 siRNA) markedly delayed tumor growth of two different TNBC tumor xenograft models (MDA-MB-231 and MDA-MB-436) in mice. Analysis of tumors by IHC after 4 weeks of treatment demonstrated that ALNP-PIM3 siRNA treatment led to inhibition of intra-tumoral proliferation and induction of apoptosis. Currently, we are conducting an in vivo study in TNBC tumor models by combining ALNP-PIM3 siRNA therapy with doxorubicin. Overall, our studies suggest that PIM3 is a marker for poor patient survival that drives TNBC tumor growth and progression and a novel potential molecular target in TNBC. Citation Format: Pinar Atalay, Goknur Kara, Rumeysa Ozyurt, Nermin Kahraman, Bulent Ozpolat. Pim3 kinase is a poor prognostic marker and novel molecular target for triple negative breast 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 650.

Abstract 665: MEN1703/SEL24 exhibits promising antitumoral activity in preclinical models of myelofibrosis both as single agent and combined with ruxolitinib

Abstract Myelofibrosis (MF) is a pathological chronic bone marrow (BM) condition characterized by abnormal growth of erythroid and megakaryocytic precursors and deposition of fibrotic tissue. The JAK/STAT pathway plays an important role in MF (Cardoso 2015), and multiple JAK1/2 inhibitors (JAKis) are approved for MF treatment. Despite initially limiting disease symptoms, JAKis often fail due to persistent JAK2 activation and survival of MF-driving cells, hence new treatment strategies are highly needed. PIM kinases also contribute to MF pathogenesis. PIM1 is highly overexpressed in granulocytes, peripheral blood mononuclear cells (PBMCs), and BM of patients with MF. PIM1/2 expression is dependent upon JAK2 signaling in myeloproliferative neoplasms (MPNs). Hence, PIM inhibitors represent potential therapeutic targets for MPNs and have been evaluated in several preclinical and clinical studies (Rampal 2021). MEN1703/SEL24 (MEN) is an oral, first-in-class, dual PIM/FLT3 kinase inhibitor in development for hematologic malignancies. This study aims to investigate the efficacy of MEN alone and in combination with the JAKi ruxolitinib (RUX) in preclinical MF models and to elucidate the underlying signaling pathways. MF cell lines (JAK2V617F and JAK2 wild type) were used in MTS assays to assess the in vitro cytotoxicity of MEN alone and in combination with RUX, compared with the PIM inhibitor TP-3654 (Dutta 2022). Cell cycle distribution and signaling pathways were analyzed via propidium iodide/FACS analysis and immunocapillary electrophoresis, respectively, upon treatment with the single agents and MEN + RUX. In vivo efficacy was assessed in a murine xenograft model treated with MEN +/- RUX. In vitro studies showed single agent MEN activity with an IC50 ranging from 0.3 to 2.3 µM. Moreover, a synergistic drug interaction was found with MEN + RUX in all tested cell lines (Combination Index [CI] range 0.41-0.80). MEN presented cytotoxic and cytostatic capacity, inducing apoptosis and arresting a proportion of cells in G2/M phase. This effect was maintained in combination with RUX. Signaling pathway analysis showed that MEN inhibits phosphorylation of STAT proteins and decreases Mcl1 protein expression. These effects were enhanced with MEN + RUX. Compared with TP-3654, MEN was more effective both as a single agent (IC50 2-3 fold lower than TP-3654) and in combination with RUX (mean CI of 0.77-0.80 compared to 0.79-0.98 with TP-3654). MEN showed anti-tumoral efficacy in MF preclinical models, moderately higher than TP-3654, exhibiting in vitro activity at clinically relevant concentrations. Importantly, MEN combined with the standard of care RUX was synergistic, and molecular analyses confirmed the role of PIM downstream target inhibition. Our results support the therapeutic potential and relevance of MEN in MF treatment strategies. Citation Format: Maria Luisa Iannitto, Diego Bisignano, Sonia Zicari, Francesco Belli, Alessio Fiascarelli, Simone Talucci, Giuseppe Merlino, Vittoria Nicolis di Robilant, Clelia Irrissuto, Mario Bigioni, Alessandro Bressan, Daniela Bellarosa, Monica Binaschi. MEN1703/SEL24 exhibits promising antitumoral activity in preclinical models of myelofibrosis both as single agent and combined with ruxolitinib [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 665.

Abstract 5944: Discovery of BH-30236: A novel macrocyclic CLK inhibitor targeting alternative splicing in cancers

Abstract Aberrant alternative splicing is a newly recognized hallmark of cancer, that has been shown to play a critical role in tumorigenesis, cancer progression, and therapeutic resistance via multiple mechanisms, including increased proliferation, decreased apoptosis, promoted migration, enhanced metastatic potential, and induced evasion of immune surveillance. Serine and arginine-rich splicing factors (SRSFs) are RNA-binding proteins (RBPs) that regulate constitutive and alternative splicing. SRSFs are often mutated or overexpressed in cancers, resulting in widespread alterations in splicing patterns. The Cdc2-like kinase (CLK) family and dual-specificity tyrosine-regulated kinase (DYRK) phosphorylate SRSFs, influencing the assembly of spliceosome machinery, exon recognition, and splicing. Therefore, targeting CLK/DYRK kinases can modulate cancer specific splicing isoforms, opening avenues for new therapeutic interventions. BH-30236 was designed as a novel orally bioavailable, ATP-competitive, macrocyclic inhibitor of CLK with IC50s of 0.134, 0.165, and 0.446 nM against CLK1, CLK2, and CLK4, respectively in enzymatic kinase assays. At clinically relevant concentrations, BH-30236 also inhibited DYRK1A/1B/2, proviral insertion site of moloney murine leukemia virus kinase 3 (PIM3), and FMS-like tyrosine kinase 3 (FLT3) with IC50s of 0.111, 0.148, 0.562, 0.115 and 0.248 nM, respectively. In cancer cells, BH-30236 impaired the phosphorylation of SRSFs, Tau and 4EBP1, the direct downstream substrates of CLK, DYRK, and PIM kinases with IC50s of 40-60 nM, ~50 nM, and ~80 nM, respectively. Furthermore, BH-30236 potently inhibited the FLT3 phosphorylation with an IC50 of 0.16 nM. Overall, BH-30236 regulated alternative splicing by primarily inducing skipped exons in favor of anti-tumor isoforms, leading to cancer cell death and growth suppression in a broad panel of cancer cell lines and in vivo efficacy studies. For example, BH-30236 potently inhibited cell proliferation with an IC50 of 0.62 nM in FLT3-ITD positive MV-4-11 cells and achieved complete tumor regression in the MV-4-11 cell-derived xenograft tumor model, even after dose cessation for 4 weeks. In MV-4-11 cells, BH-30236 increased pro-apoptosis splicing variant BCLxS, downregulated RNA expression of BCL2, MCL1, and AML stem cell markers CD33 and CD123. In addition, BH-30236 has demonstrated good human ADME and preclinical safety profiles. Collectively, the preclinical study results strongly support the clinical applications of the novel multikinase CLK inhibitor BH-30236 in hematological malignancies and solid tumors, as a single agent or in combination with other therapies. Citation Format: J. Jean Cui, Ping Jiang, Wei Deng, Dayong Zhai, Nancy Ling, Danan Li, Zhenping Wang, Yue Hu, Levan Darjania, Jeff Whitten, Evan Rogers, Eugene Rui. Discovery of BH-30236: A novel macrocyclic CLK inhibitor targeting alternative splicing in cancers [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 5944.

Abstract 4618: PIM kinases drive cell adhesion mediated drug resistance in prostate cancer

Abstract Introduction: Castration-resistant prostate cancer (CRPC) is an incurable and lethal progression of the disease that does not respond to any treatment options. Cell adhesion to the extracellular matrix (ECM) activates survival pathways, allowing cancer cells to evade anti-cancer therapy, a phenomenon known as cell adhesion-mediated drug resistance. Bone, which is the primary site of prostate cancer metastasis, is known to be enriched in laminin, a ubiquitous ECM protein. In addition, laminin binding integrins, α6β1 and α3β1, are the major integrins expressed in CRPC, suggesting that laminin-dependent adhesion provides a survival advantage. Based on these findings, activation of the laminin-binding integrin, α6β1, is an intriguing factor mediating resistance to therapy in CRPC. PIM1 is an oncogenic Ser/Thr kinase that is elevated in CRPC and is known to promote resistance to therapy. In contrast, to many pro-survival kinases, PIM kinases do not possess any obvious regulatory domains. Current evidence suggests that PIM1 is constitutively active when it is expressed, so PIM1 expression level directly correlates with its catalytic activity. Despite this fact, little is known about the internal and external mechanisms that dictate PIM1 protein levels and spatial activation in cancer cells. Here, we identify PIM1 as a key survival signal that contributes to cell adhesion-mediated drug resistance. Experimental procedures: Supporting the importance of this signaling axis in human tumors, multiplex IHC of a TMA containing prostate adenocarcinoma and CRPC bone metastases to show that integrin α6 and PIM1 are coexpressed and spatially correlated in primary and metastatic prostate cancer. To examine the spatiotemporal dynamics of PIM1 during cell adhesion, We developed a kinase activity reporter (PIMKAR) to monitor PIM catalytic activity in live cells. Because we observed PIM was highly localized to the membrane in patient tumors, we tagged PIMKAR with a myristylation sequence to specifically measure PIM activity at the membrane vs cytosol. Immunofluorescence and biochemical techniques were used to demonstrate the effect of PIM1 on mitophagy and oxidative stress. Conclusions: We demonstrate that activation of integrin α6β1 downstream of cell adhesion stabilizes PIM1 by blocking its proteasomal degradation. The resulting upregulation of PIM1 serves to reduce oxidative stress through complementary mechanisms reducing mitochondrial fragmentation and increasing the removal of damaged mitochondria by increasing mitophagy. Mechanistically, PIM1 upregulates BNIP3 to enhance mitophagy and thereby reduce oxidative stress in the cell. Together, these results expand our understanding of the mechanisms regulating PIM1 activation and provide evidence for the use of PIM inhibitors in bone mCRPC and other cancers where laminin is a major component of the ECM Citation Format: Caitlyn Flores, Shailender Chauhan, Cynthia Miranti, Anne Cress, Gregory Rogers, Noel Warfel. PIM kinases drive cell adhesion mediated drug resistance in prostate 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 4618.

Abstract 4501: First reported PIM kinase degraders: Design, profiling & optimization

Abstract The PIM serine/threonine kinases (PIM1/PIM2/PIM3) are downstream effectors of ABL, JAK2 and Flt-3 oncogenes and are required for tumorigenesis. Overexpression has been reported in hematological and solid tumors, myeloma, lymphoma, leukemia and adenocarcinoma. The first generation of PIM inhibitors to make it to the clinic, including SGI-1776, AZD1208 and PIM447, are known to be pan-PIM inhibitors, while the aim of this project was to identify novel & selective PIM3 inhibitors. To this end, several Hit-finding approaches were employed to rapidly generate novel chemical matter, which were then assessed for their activity against PIM3 using a robust ADP-Glo™ assay. In parallel to this Hit-findings activity, we used AZD1208 scaffold as a starting point to generate PIM3 bifunctional protein degraders and designed a specific screening cascade in order to profile such modalities. The PIM degrader library, synthetized using Automated Robotics Lab, was characterized using ADP-Glo assays. It was then screened on our binding and activity kinase platforms, KINOMEscan® and KinaseProfiler™, and then fully profiled including ternary complex formation with biophysics and in vitro Safety Pharmacology Profiling Panels. One example is the AZD1208 derivate degrader, i.e. EC108154-1, which showed nanomolar range affinity for PIM3 and the E3-ligase CRBN-DDB1, and its affinity range was also confirmed with the ternary complex in SPR and MST-TRIC. This comprehensive screening cascade ensures an in-depth characterization and optimization of protein degraders, with the possibility of monitoring the ternary complex formation with biophysics methods. It is a good illustration of how this combination of tests can be instrumental in selecting the best degraders to progress to the next step of drug discovery. Citation Format: Celine Legros, Paul Ratcliffe, Vanessa Porkolab, Michele Modugno, Olivier Mirguet. First reported PIM kinase degraders: Design, profiling & optimization [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 4501.