Serine/Threonine Protein Kinases as Attractive Targets for Anti-Cancer Drugs-An Innovative Approach to Ligand Tuning Using Combined Quantum Chemical Calculations, Molecular Docking, Molecular Dynamic Simulations, and Network-like Similarity Graphs.

Serine/threonine protein kinases (CK2, PIM-1, RIO1) are constitutively active, highly conserved, pleiotropic, and multifunctional kinases, which control several signaling pathways and regulate many cellular functions, such as cell activity, survival, proliferation, and apoptosis. Over the past decades, they have gained increasing attention as potential therapeutic targets, ranging from various cancers and neurological, inflammation, and autoimmune disorders to viral diseases, including COVID-19. Despite the accumulation of a vast amount of experimental data, there is still no "recipe" that would facilitate the search for new effective kinase inhibitors. The aim of our study was to develop an effective screening method that would be useful for this purpose. A combination of Density Functional Theory calculations and molecular docking, supplemented with newly developed quantitative methods for the comparison of the binding modes, provided deep insight into the set of desirable properties responsible for their inhibition. The mathematical metrics helped assess the distance between the binding modes, while heatmaps revealed the locations in the ligand that should be modified according to binding site requirements. The Structure-Binding Affinity Index and Structural-Binding Affinity Landscape proposed in this paper helped to measure the extent to which binding affinity is gained or lost in response to a relatively small change in the ligand's structure. The combination of the physico-chemical profile with the aforementioned factors enabled the identification of both "dead" and "promising" search directions. Tests carried out on experimental data have validated and demonstrated the high efficiency of the proposed innovative approach. Our method for quantifying differences between the ligands and their binding capabilities holds promise for guiding future research on new anti-cancer agents.

PIM kinase inhibitors: an updated patent review (2016-present).

INTRODUCTION: PIM Kinases (PIM-1, PIM-2, and PIM-3) have been reported to play crucial role in signaling cascades that govern cell survival, proliferation, and differentiation. Over-expression of these kinases leads to hematological malignancies such as diffuse large B cell lymphomas (DLBCL), multiple myeloma, leukemia, lymphoma and prostate cancer etc. PIM kinases as biomarkers and potential therapeutic targets have shown promise toward precision cancer therapy. The selective PIM-1, PIM-2, and/or PIM-3 isoform inhibitors have shown significant results in patients with advanced stages of cancer including relapsed/refractory cancer. AREAS COVERED: A comprehensive literature review of PIM Kinases (PIM-1, PIM-2, and PIM-3) in oncogenesis, the patented PIM kinase inhibitors (2016-Present), and their pharmacological and structural insights have been highlighted. EXPERT OPINION: Recently, PIM kinases viz. PIM-1, PIM-2, and PIM-3 (members of the serine/threonine protein kinase family) as therapeutic targets have attracted considerable interest in oncology especially in hematological malignancies. The patented PIM kinase inhibitors comprised of heterocyclic (fused)ring structure(s) like indole, pyridine, pyrazine, pyrazole, pyridazine, piperazine, thiazole, oxadiazole, quinoline, triazolo-pyridine, pyrazolo-pyridine, imidazo-pyridazine, oxadiazole-thione, pyrazolo-pyrimidine, triazolo-pyridazine, imidazo-pyridazine, pyrazolo-quinazoline and pyrazolo-pyridine etc. showed promising results in cancer chemotherapy.

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.

A review on structure-function mechanism and signaling pathway of serine/threonine protein PIM kinases as a therapeutic target.

The proviral integration for the Moloney murine leukemia virus (PIM) kinases, belonging to serine/threonine kinase family, have been found to be overexpressed in various types of cancers, such as prostate, breast, colon, endometrial, gastric, and pancreatic cancer. The three isoforms PIM kinases i.e., PIM1, PIM2, and PIM3 share a high degree of sequence and structural similarity and phosphorylate substrates controlling tumorigenic phenotypes like proliferation and cell survival. Targeting short-lived PIM kinases presents an intriguing strategy as in vivo knock-down studies result in non-lethal phenotypes, indicating that clinical inhibition of PIM might have fewer adverse effects. The ATP binding site (hinge region) possesses distinctive attributes, which led to the development of novel small molecule scaffolds that target either one or all three PIM isoforms. Machine learning and structure-based approaches have been at the forefront of developing novel and effective chemical therapeutics against PIM in preclinical and clinical settings, and none have yet received approval for cancer treatment. The stability of PIM isoforms is maintained by PIM kinase activity, which leads to resistance against PIM inhibitors and chemotherapy; thus, to overcome such effects, PIM proteolysis targeting chimeras (PROTACs) are now being developed that specifically degrade PIM proteins. In this review, we recapitulate an overview of the oncogenic functions of PIM kinases, their structure, function, and crucial signaling network in different types of cancer, and the potential of pharmacological small-molecule inhibitors. Further, our comprehensive review also provides valuable insights for developing novel antitumor drugs that specifically target PIM kinases in the future. In conclusion, we provide insights into the benefits of degrading PIM kinases as opposed to blocking their catalytic activity to address the oncogenic potential of PIM kinases.

PIM Kinase Inhibition Sensitizes Neuroblastoma to Doxorubicin.

BACKGROUND: Chemoresistance 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. METHODS: Kocak 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. RESULTS: Kocak 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. CONCLUSIONS: The 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 STUDY: Basic Science Research. LEVEL OF EVIDENCE: NA.

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.

Combined BRAF and PIM1 inhibitory therapy for papillary thyroid carcinoma based on BRAFV600E regulation of PIM1: Synergistic effect and metabolic mechanisms.

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, and its incidence has increased rapidly in recent years. The BRAF inhibitor vemurafenib is effective against BRAFV600E-positive PTC; however, acquired resistance to single agent therapy frequently leads to tumor recurrence and metastasis, underscoring the need to develop tailored treatment strategies. We previously showed that the oncogenic kinase PIM1 was associated with the malignant phenotype and prognosis of PTC. In this study, we showed that sustained expression of the PIM1 protein in PTC was affected by the BRAFV600E mutation. Based on this regulatory mechanism, we tested the synergistic effects of inhibitors of BRAF (BRAFi) and PIM1 in BRAFV600E-positive PTC cell lines and xenograft tumors. LC-MS metabolomics analyses suggested that BRAFi/PIMi therapy acted by restricting the amounts of critical amino acids and nucleotides required by cancer cells as well as modulating DNA methylation. This study elucidates the role of BRAFV600E in the regulation of PIM1 in PTC and demonstrates the synergistic effect of a novel combination, BRAFi/PIMi, for the treatment of PTC. This discovery, along with the pathways that may be involved in the powerful efficacy of BRAFi/PIMi strategy from the perspective of cell metabolism, provides insight into the molecular basis of PTC progression and offers new perspectives for BRAF-resistant PTC treatment.

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.

Targeting Host PIM Protein Kinases Reduces Mayaro Virus Replication.

Mayaro virus (MAYV) manipulates cell machinery to successfully replicate. Thus, identifying host proteins implicated in MAYV replication represents an opportunity to discover potential antiviral targets. PIM kinases are enzymes that regulate essential cell functions and also appear to be critical factors in the replication of certain viruses. In this study we explored the consequences of PIM kinase inhibition in the replication of MAYV and other arboviruses. Cytopathic effects or viral titers in samples from MAYV-, Chikungunya-, Una- or Zika-infected cells treated with PIM kinase inhibitors were evaluated using an inverted microscope or plaque-forming assays. The expression of viral proteins E1 and nsP1 in MAYV-infected cells was assessed using an immunofluorescence confocal microscope or Western blot. Our results revealed that PIM kinase inhibition partially prevented MAYV-induced cell damage and also promoted a decrease in viral titers for MAYV, UNAV and ZIKV. The inhibitory effect of PIM kinase blocking was observed for each of the MAYV strains tested and also occurred as late as 8 h post infection (hpi). Finally, PIM kinase inhibition suppressed the expression of MAYV E1 and nsP1 proteins. Taken together, these findings suggest that PIM kinases could represent an antiviral target for MAYV and other arboviruses.

Design, Synthesis, Molecular Docking, and Biological Studies of New Heterocyclic Compounds Derived from ß-Diketones as Novel EGFR and Pim-1 Inhibitors Endowed with AntitumorActivity.

BACKGROUND: Cancer is a disease illustrated by a shift in the controlled mechanisms that control both cell proliferation and differentiation. It is regarded as a prime health problem worldwide and a leading cause of human death rate exceeded only by cardiovascular diseases. Many reported works are concerned with discovering new antitumor compounds, encouraging us to synthesize new anticancer agents. OBJECTIVE: In this work, we aimed to synthesize target molecules from 1,3-dicarbonyl compounds through heterocyclization reactions. METHODS: The reaction of either 4-methylaniline (1a) or 1-naphthylamine (1b) with diethyl malonate (2) gave the anilide derivatives 3a and 3b, respectively. The latter underwent a series of heterocyclization reactions to give the pyridine, pyran, and thiazole derivatives confirmed by the required spectral data. RESULTS: The in-vitro antitumor evaluation of the newly synthesized products against three cancer cell lines, MCF-7, NCI-H460, SF-268, and WI 38, which were used as the normal cell lines, was conducted, and the data revealed that compounds 11a, 18b, 18c, and 20d showed high antitumor activity and 20d individualized with potential antitumor activity towards cell lines with lowest cytotoxicity effect. Both EGFR and PIM-1 enzymes inhibition were investigated for the compound 20d, and it was found that the inhibition effect of compound 20d was promising for each enzyme, showing IC50 = 45.67 ng and 553.3 ng for EGFR and PIM-1, respectively. CONCLUSION: Molecular docking results of compound 20d showed strong binding interactions with both the enzymes, where good binding modes were obtained in the case of EGFR, which was closely similar to the binding mode of standard Erlotinib.While 20d showed complete superimposition binding interactions with VRV-cocrystallized ligand of PIM-1 that may expound the in-vitro antitumor activity.

Pd and photoredox dual catalysis assisted decarboxylative ortho-benzoylation of N-phenyl-7-azaindoles.

The directing group assisted decarboxylative ortho-benzoylation of N-aryl-7-azaindoles with α-keto acids has been achieved by synergistic visible light promoted photoredox and palladium catalysis. The approach tenders rapid entry to aryl ketone architectures from simple α-keto acid precursors via the in situ generation of a benzoyl radical intermediate. The transformation provides a range of ortho-benzoylated N-aryl-7-azaindoles, with excellent site-selectivity and good functional group compatibility under mild reaction conditions. Biological target predictions indicate that these molecules may serve as potential anti-cancer and anti-viral agents.

Discovery of Dual CDK6/PIM1 Inhibitors with a Novel Structure, High Potency, and Favorable Druggability for the Treatment of Acute Myeloid Leukemia.

Nowadays, the simultaneous inhibition of two or more pathways plays an increasingly important role in cancer treatment due to the complex and diverse pathogenesis of cancer, and the combination of the cyclin-dependent kinase 6 (CDK6) inhibitor and PIM1 inhibitor was found to generate synergistic effects in acute myeloid leukemia (AML) treatment. Therefore, we discovered a novel lead 1 targeting CDK6/PIM1 via pharmacophore-based and structure-based virtual screening, synthesized five different series of new derivates, and obtained a potent and balanced dual CDK6/PIM1 inhibitor 51, which showed high kinase selectivity. Meanwhile, 51 displayed an excellent safety profile and great pharmacokinetic properties. Furthermore, 51 displayed stronger potency in reducing the burden of AML than palbociclib and SMI-4a in vivo. In summary, we offered a new direction for AML treatment and provided a great lead compound for AML preclinical studies.

Genetic ablation of Pim1 or pharmacologic inhibition with TP-3654 ameliorates myelofibrosis in murine models.

Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasm (MPN). The JAK inhibitor Ruxolitinib can reduce constitutional symptoms but it does not substantially improve bone marrow fibrosis. Pim1 expression is significantly elevated in MPN/MF hematopoietic progenitors. Here, we show that genetic ablation of Pim1 blocked the development of myelofibrosis induced by Jak2V617F and MPLW515L. Pharmacologic inhibition of Pim1 with a second-generation Pim kinase inhibitor TP-3654 significantly reduced leukocytosis and splenomegaly, and attenuated bone marrow fibrosis in Jak2V617F and MPLW515L mouse models of MF. Combined treatment of TP-3654 and Ruxolitinib resulted in greater reduction of spleen size, normalization of blood leukocyte counts and abrogation of bone marrow fibrosis in murine models of MF. TP-3654 treatment also preferentially inhibited Jak2V617F mutant hematopoietic progenitors in mice. Mechanistically, we show that TP-3654 treatment significantly inhibits mTORC1, MYC and TGF-ß signaling in Jak2V617F mutant hematopoietic cells and diminishes the expression of fibrotic markers in the bone marrow. Collectively, our results suggest that Pim1 plays an important role in the pathogenesis of MF, and inhibition of Pim1 with TP-3654 might be useful for treatment of MF.

Utilization of cyanopyridine in design and synthesis of first-in-class anticancer dual acting PIM-1 kinase/HDAC inhibitors.

Herein, we report design and synthesis of twenty-one dual PIM-1/HDAC inhibitors utilizing 3-cyanopyridines as a novel cap moiety linked with aliphatic /aromatic linker bearing carboxylic acid 3a-g, hydroxamic acid 4a-g or 2-aminoanilide moieties 5a-g as zinc-binding group. Most of the target hybrids revealed promising growth inhibition according to one dose NCI protocol against 60 cancer cell lines. Meanwhile, hydroxamic acids 4b, 4d and 4e displayed strong and broad-spectrum activity against nine tumor subpanels tested (GI50 0.176-8.87 µM); 4d displayed strong antiproliferative activity with GI50 ≤ 3 µM against different cancer cell lines (GI50 range from 0.325 to 2.9 µM). Furthermore, 4a, 4d-4g and 5f manifested a high inhibitory activity against HDACs 1 and 6 isozymes; 4g, displayed potent HDAC 1 and 6 inhibitory activity (45.01 ± 2.1 and 19.78 ± 1.1 nM) more than the reference SAHA (51.54 ± 2.4 and 21.38 ± 1.2 nM, respectively), while 4f was more potent (30.09 ± 1.4 nM) than SAHA against HDAC 1 and less potent (30.29 ± 1.7 nM) than SAHA against HDAC 6. Hybrids 4b, 4d, 4e and 4f exhibited potent PIM-1 inhibitory activity; 4d showed comparable activity to quercetin (IC50 of 343.87 ± 16.6 and 353.76 ± 17.1 nM, respectively); it exhibited pre G1 apoptosis and arrest cell cycle at G2/M phase. Moreover, it revealed good binding into pocket of HDACs 1,6 and PIM-1 kinase enzymes with good correlation with biological results. Moreover, 4b, 4d and 4e had reasonable drug-likeness properties according to Lipinski's rule. However, multitarget inhibitor of PIM-1/HDAC is a promising strategy in anticancer drug discovery; the most potent hybrids require further in vivo and clinical investigations.

Ventilagolin Suppresses Migration, Invasion and Epithelial-Mesenchymal Transition of Hepatocellular Carcinoma Cells by Downregulating Pim-1.

OBJECTIVE: Inhibition of tumor metastasis is a useful strategy to improve the efficacy of cancer therapy. Ventilagolin, a natural 1, 4-naphthoquinone derivative extracted from Ventilago leiocarpa Benth, has shown promising antitumor effects in previous studies. However, the effects and underlying mechanisms of Ventilagolin against migration, invasion and epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma (HCC) remain unclear. The present study has examined these effects and determined whether the proto-oncogene Pim-1 is involved. METHODS: The effects of Ventilagolin on migration, invasion, Pim-1 and EMT-related proteins (eg, E-cadherin, N-cadherin, Vimentin) expression were assessed by scratch wound healing, Transwell, qRT-PCR and Western blot assays, respectively. Pim-1 stably overexpressed HepG2 and SMMC-7721 cells were generated to explore whether Ventilagolin inhibited migration, invasion and EMT of HCC cells via regulating Pim-1. Subcutaneous xenograft tumor model in nude mice was established. Histopathological changes of tumor tissues were examined by H&E staining and expressions of Pim-1 and EMT-related proteins were detected by immunohistochemistry. RESULTS: Ventilagolin significantly (P < 0.01) reduced the expression of Pim-1 levels in HepG2 and SMMC-7721 cells. Compared with the control group, the migration and invasion abilities of Pim-1-overexpressing HepG2 and SMMC-7721 cells were significantly (P < 0.05, P < 0.01) enhanced, the expression of E-cadherin was decreased (P < 0.01), and the levels of N-cadherin and Vimentin were upregulated (P < 0.05, P < 0.01). Ventilagolin treatment effectively reversed these effects of Pim-1 overexpression. In vivo experiments showed that Ventilagolin could effectively suppress HCC tumor growth, downregulate Pim-1, N-cadherin and Vimentin expression, and upregulate E-cadherin expression. CONCLUSION: Ventilagolin suppresses HCC cell proliferation, migration and invasion and reverses EMT process by downregulating Pim-1, suggesting Ventilagolin is a potential therapeutic agent for treatment of HCC.

Dibenzofuran Derivatives Inspired from Cercosporamide as Dual Inhibitors of Pim and CLK1 Kinases.

Pim kinases (proviral integration site for Moloney murine leukemia virus kinases) are overexpressed in various types of hematological malignancies and solid carcinomas, and promote cell proliferation and survival. Thus, Pim kinases are validated as targets for antitumor therapy. In this context, our combined efforts in natural product-inspired library generation and screening furnished very promising dibenzo[b,d]furan derivatives derived from cercosporamide. Among them, lead compound 44 was highlighted as a potent Pim-1/2 kinases inhibitor with an additional nanomolar IC50 value against CLK1 (cdc2-like kinases 1) and displayed a low micromolar anticancer potency towards the MV4-11 (AML) cell line, expressing high endogenous levels of Pim-1/2 kinases. The design, synthesis, structure-activity relationship, and docking studies are reported herein and supported by enzyme, cellular assays, and Galleria mellonella larvae testing for acute toxicity.

PIM1 Inhibition Affects Glioblastoma Stem Cell Behavior and Kills Glioblastoma Stem-like Cells.

Despite comprehensive therapy and extensive research, glioblastoma (GBM) still represents the most aggressive brain tumor in adults. Glioma stem cells (GSCs) are thought to play a major role in tumor progression and resistance of GBM cells to radiochemotherapy. The PIM1 kinase has become a focus in cancer research. We have previously demonstrated that PIM1 is involved in survival of GBM cells and in GBM growth in a mouse model. However, little is known about the importance of PIM1 in cancer stem cells. Here, we report on the role of PIM1 in GBM stem cell behavior and killing. PIM1 inhibition negatively regulates the protein expression of the stem cell markers CD133 and Nestin in GBM cells (LN-18, U-87 MG). In contrast, CD44 and the astrocytic differentiation marker GFAP were up-regulated. Furthermore, PIM1 expression was increased in neurospheres as a model of GBM stem-like cells. Treatment of neurospheres with PIM1 inhibitors (TCS PIM1-1, Quercetagetin, and LY294002) diminished the cell viability associated with reduced DNA synthesis rate, increased caspase 3 activity, decreased PCNA protein expression, and reduced neurosphere formation. Our results indicate that PIM1 affects the glioblastoma stem cell behavior, and its inhibition kills glioblastoma stem-like cells, pointing to PIM1 targeting as a potential anti-glioblastoma therapy.

Inhibition of PIM Kinases in DLBCL Targets MYC Transcriptional Program and Augments the Efficacy of Anti-CD20 Antibodies.

The family of PIM serine/threonine kinases includes three highly conserved oncogenes, PIM1, PIM2, and PIM3, which regulate multiple prosurvival pathways and cooperate with other oncogenes such as MYC. Recent genomic CRISPR-Cas9 screens further highlighted oncogenic functions of PIMs in diffuse large B-cell lymphoma (DLBCL) cells, justifying the development of small-molecule PIM inhibitors and therapeutic targeting of PIM kinases in lymphomas. However, detailed consequences of PIM inhibition in DLBCL remain undefined. Using chemical and genetic PIM blockade, we comprehensively characterized PIM kinase-associated prosurvival functions in DLBCL and the mechanisms of PIM inhibition-induced toxicity. Treatment of DLBCL cells with SEL24/MEN1703, a pan-PIM inhibitor in clinical development, decreased BAD phosphorylation and cap-dependent protein translation, reduced MCL1 expression, and induced apoptosis. PIM kinases were tightly coexpressed with MYC in diagnostic DLBCL biopsies, and PIM inhibition in cell lines and patient-derived primary lymphoma cells decreased MYC levels as well as expression of multiple MYC-dependent genes, including PLK1. Chemical and genetic PIM inhibition upregulated surface CD20 levels in an MYC-dependent fashion. Consistently, MEN1703 and other clinically available pan-PIM inhibitors synergized with the anti-CD20 monoclonal antibody rituximab in vitro, increasing complement-dependent cytotoxicity and antibody-mediated phagocytosis. Combined treatment with PIM inhibitor and rituximab suppressed tumor growth in lymphoma xenografts more efficiently than either drug alone. Taken together, these results show that targeting PIM in DLBCL exhibits pleiotropic effects that combine direct cytotoxicity with potentiated susceptibility to anti-CD20 antibodies, justifying further clinical development of such combinatorial strategies. SIGNIFICANCE: These findings demonstrate that inhibition of PIM induces DLBCL cell death via MYC-dependent and -independent mechanisms and enhances the therapeutic response to anti-CD20 antibodies by increasing CD20 expression.

Optimization of 4,6-Disubstituted Pyrido[3,2-d]pyrimidines as Dual MNK/PIM Inhibitors to Inhibit Leukemia Cell Growth.

Mitogen-activated protein kinase-interacting kinases (MNKs) and provirus integration in maloney murine leukemia virus kinases (PIMs) are downstream enzymes of cell proliferation signaling pathways associated with the resistance of tyrosine kinase inhibitors. MNKs and PIMs have complementary effects to regulate cap-dependent translation of oncoproteins. Dual inhibitors of MNKs and PIMs have not been developed. We developed a novel 4,6-disubstituted pyrido[3,2-d]pyrimidine compound 21o with selective inhibition of MNKs and PIMs. The IC50's of 21o to inhibit MNK1 and MNK2 are 1 and 7 nM and those to inhibit PIM1, PIM2, and PIM3 are 43, 232, and 774 nM, respectively. 21o inhibits the growth of myeloid leukemia K562 and MOLM-13 cells with GI50's of 2.1 and 1.2 µM, respectively. 21o decreases the levels of p-eIF4E and p-4EBP1, the downstream products of MNKs and PIMs, as well as cap-dependent proteins c-myc, cyclin D1, and Mcl-1. 21o inhibits the growth of MOLM-13 cell xenografts without causing evident toxicity. 21o represents an innovative dual MNK/PIM inhibitor with a good pharmacokinetic profile.

2-Thioxothiazolidin-4-one Analogs as Pan-PIM Kinase Inhibitors.

Proviral integration site for Moloney murine leukemia virus (PIM) kinases are proto-oncogenic kinases involved in the regulation of several cellular processes. PIM kinases are promising targets for new drug development because they play a major role in many cancer-specific pathways, such as survival, apoptosis, proliferation, cell cycle regulation, and migration. Here, 2-thioxothiazolidin-4-one derivatives were synthesized and evaluated as potent pan-PIM kinase inhibitors. Optimized compounds showed single-digit nanomolar IC50 values against all three PIM kinases with high selectivity over 14 other kinases. Compound 17 inhibited the growth of Molm-16 cell lines (EC50 = 14 nM) and modulated the expression of pBAD and p4EBP1 in a dose-dependent manner.