Pim-1 Kinase Research Articles

A phase Ib study targeting PIM1 and CDK4/6 kinases in metastatic renal cell carcinoma (PICKRCC).

TPS771 Background: Over the last few years there has been a rapid increase in the clinically relevant agents available to treat metastatic renal cell carcinoma (mRCC). We note, however, that the vast majority of new agents available for mRCC do not exploit new clinical targets or pathways. We believe that the identification of new, clinically relevant targets in RCC will propel the field even further forward, expand treatment options, and lead to improved survival for mRCC patients. SGI-1776, a selective PIM1 kinase inhibitor, has previously been shown to induce reduction in tumor size as monotherapy and in combination with sunitinib in mouse pre-clinical studies of RCC. In our tissue microarray studies a subset of ~26% of RCC showed high staining for PIM1 kinase while only 1% of normal adjacent tissue showed similar high staining. Wildtype PIM1 is constitutively active, thus these data suggest that PIM1 activity is increased in a subset of RCC. Abemaciclib is a potent CDK4/6 inhibitor with an IC50 of 2 and 10 nM, respectively. It is also a potent PIM1 inhibitor with an IC50 of 50 nM. We have shown that abemaciclib decreases cell viability and increases apoptosis in RCC cell lines, and does so at greatest effect in combination with sunitinib. We have also shown that abemaciclib induces regression of RCC tumors in a mouse model of RCC, with the most rapid responses observed when abemaciclib is combined with sunitinib. Based on these data we have opened a phase Ib dose escalation study to determine the safety and tolerability of abemaciclib in combination with sunitinib in patients with mRCC. The study includes an expansion cohort at the recommended phase II dose to evaluate for a signal for efficacy. Pattern and intensity of PIM1 staining in tumor tissue will be evaluated as a potential biomarker of response. We are also collecting blood and urine to evaluate additional potential biomarkers of response. Methods: Clinical trial information: NCT03905889 .[Table: see text]

Preclinical evaluation of a novel triple-acting PIM/PI3K/mTOR inhibitor, IBL-302, in breast cancer

The proviral integration of Moloney virus (PIM) family of protein kinases are overexpressed in many haematological and solid tumours. PIM kinase expression is elevated in PI3K inhibitor-treated breast cancer samples, suggesting a major resistance pathway for PI3K inhibitors in breast cancer, potentially limiting their clinical utility. IBL-302 is a novel molecule that inhibits both PIM and PI3K/AKT/mTOR signalling. We thus evaluated the preclinical activity of IBL-302, in a range of breast cancer models. Our results demonstrate in vitro efficacy of IBL-302 in a range of breast cancer cell lines, including lines with acquired resistance to trastuzumab and lapatinib. IBL-302 demonstrated single-agent, anti-tumour efficacy in suppression of pAKT, pmTOR and pBAD in the SKBR-3, BT-474 and HCC-1954 HER2+/PIK3CA-mutated cell lines. We have also shown the in vivo single-agent efficacy of IBL-302 in the subcutaneous BT-474 and HCC-1954 xenograft model in BALB/c nude mice. The combination of trastuzumab and IBL-302 significantly increased the anti-proliferative effect in HER2+ breast cancer cell line, and matched trastuzumab-resistant line, relative to testing either drug alone. We thus believe that the novel PIM and PI3K/mTOR inhibitor, IBL-302, represents an exciting new potential treatment option for breast cancer, and that it should be considered for clinical investigation.

The extracellular sulfatase SULF2 promotes liver tumorigenesis by stimulating assembly of a promoter-looping GLI1-STAT3 transcriptional complex

The expression of the extracellular sulfatase SULF2 has been associated with increased hepatocellular carcinoma (HCC) growth and poor patient survival. However, the molecular mechanisms underlying SULF2-associated tumor growth remain unclear. To address this gap, here we developed a transgenic mouse overexpressing Sulf2 in hepatocytes under the control of the transthyretin promoter. In this model, Sulf2 overexpression potentiated diethylnitrosamine-induced HCC. Further analysis indicated that the transcription factor GLI family zinc finger 1 (GLI1) mediates Sulf2 expression during HCC development. A cross of the Sulf2-overexpressing with Gli1-knockout mice revealed that Gli1 inactivation impairs SULF2-induced HCC. Transcriptomic analysis revealed that Sulf2 overexpression is associated with signal transducer and activator of transcription 3 (STAT3)-specific gene signatures. Interestingly, the Gli1 knockout abrogated SULF2-mediated induction of several STAT3 target genes, including suppressor of cytokine signaling 2/3 (Socs2/3); Pim-1 proto-oncogene, Ser/Thr kinase (Pim1); and Fms-related tyrosine kinase 4 (Flt4). Human orthologs were similarly regulated by SULF2, dependent on intact GLI1 and STAT3 functions in HCC cells. SULF2 overexpression promoted a GLI1-STAT3 interaction and increased GLI1 and STAT3 enrichment at the promoters of their target genes. Interestingly, the SULF2 overexpression resulted in GLI1 enrichment at select STAT3 consensus sites, and vice versa. siRNA-mediated STAT3 or GLI1 knockdown reduced promoter binding of GLI1 and STAT3, respectively. Finally, chromatin-capture PCR confirmed long-range co-regulation of SOCS2 and FLT3 through changes in promoter conformation. These findings define a mechanism whereby SULF2 drives HCC by stimulating formation of a GLI1-STAT3 transcriptional complex.

Structural insights for rational design of new PIM-1 kinase inhibitors based on 3,5-disubstituted indole derivatives: An integrative computational approach

Proviral integration Moloney virus (PIM) 1, 2, and 3 kinases are a family of constitutively active serine/threonine kinases that are involved in a number of signaling pathways important to cancer cells. Their overexpression in a variety of human hematopoietic malignancies and solid tumors suggest that inhibition of PIM signaling could provide patients with therapeutic benefit. In this study, a series of 3,5-disubstituted indole derivatives have been systematically studied using three-dimensional quantitative structure-activity relationship (3D−QSAR) analysis, molecular docking simulation, and partial least-squares (PLS) analysis methods to explore the influence of the structural characteristics on the inhibitory activity and use them to propose novel bioactive molecules. The comparative molecular field and comparative molecular similarity indices analyses (CoMFA and CoMSIA) models exhibited a good correlation between the predicted and experimental activities with excellent predictive capability and yielded statistically reliable value (CoMFA: Q2 = 0.535, R2 = 0.987, r2pred = 0.909; CoMSIA: Q2 = 0.785, R2 = 0.989, r2pred = 0.969). Based on the CoMFA and CoMSIA models and docking results, ten novel potent PIM-1 inhibitors (N1–N10) have been designed and the molecular models have validated their inhibitory activities. These results provided strong theoretical guidance for the development of novel PIM-1 inhibitors.

RNA-Seq analysis reveals that spring viraemia of carp virus induces a broad spectrum of PIM kinases in zebrafish kidney that promote viral entry

PIM kinases are a family of serine/threonine protein kinases that potentiate the progression of the cell cycle and inhibit apoptosis. Because of this, they are considered to be proto-oncogenes, and they represent an interesting target for the development of anticancer drugs. In mammals, three PIM kinases exist (PIM-1, PIM-2 and PIM-3), and different inhibitors have been developed to block their activity. In addition to their involvement in cancer, some publications have reported that the PIM kinases have pro-viral activity, and different mechanisms where PIM kinases favour viral infections have been proposed. Zebrafish possess more than 300 Pim kinase members in their genome, and by using RNA-Seq analysis, we found a high number of Pim kinase genes that were significantly induced after infection with spring viraemia of carp virus (SVCV). Moreover, analysis of the miRNAs modulated by this infection revealed that some of them could be involved in the post-transcriptional regulation of Pim kinase abundance. To elucidate the potential role of the 16 overexpressed Pim kinases in the infectivity of SVCV, we used three different pan-PIM kinase inhibitors (SGI-1776, INCB053914 and AZD1208), and different experiments were conducted both in vitro and in vivo. We observed that the PIM kinase inhibitors had a protective effect against SVCV, indicating that, similar to what is observed in mammals, PIM kinases are beneficial for the virus in zebrafish. Moreover, zebrafish Pim kinases seem to facilitate viral entry into the host cells because when ZF4 cells were pre-incubated with the virus and then were treated with the inhibitors, the protective effect of the inhibitors was abrogated. Although more investigation is necessary, these results show that pan-PIM kinase inhibitors could serve as a useful treatment for preventing the spread of viral diseases.

PIM kinases alter mitochondrial dynamics and chemosensitivity in lung cancer.

Resistance to chemotherapy represents a major obstacle to the successful treatment of non-small-cell lung cancer (NSCLC). The goal of this study was to determine how PIM kinases impact mitochondrial dynamics, ROS production, and response to chemotherapy in lung cancer. Live-cell imaging and microscopy were used to determine the effect of PIM loss or inhibition on mitochondrial phenotype and ROS. Inhibition of PIM kinases caused excessive mitochondrial fission and significant upregulation of mitochondrial superoxide, increasing intracellular ROS. Mechanistically, we define a signaling axis linking PIM1 to Drp1 and mitochondrial fission in lung cancer. PIM inhibition significantly increased the protein levels and mitochondrial localization of Drp1, causing marked fragmentation of mitochondria. An inverse correlation between PIM1 and Drp1 was confirmed in NSCLC patient samples. Inhibition of PIM sensitized NSCLC cells to chemotherapy and produced a synergistic antitumor response in vitro and in vivo. Immunohistochemistry and transmission electron microscopy verified that PIM inhibitors promote mitochondrial fission and apoptosis in vivo. These data improve our knowledge about how PIM1 regulates mitochondria and provide justification for combining PIM inhibition with chemotherapy in NSCLC.

Human papillomavirus insertions identify the PIM family of serine/threonine kinases as targetable driver genes in head and neck squamous cell carcinoma.

Human papillomavirus (HPV) insertions in cancer genomes have been linked to various forms of focal genomic instability and altered expression of neighboring genes. Here we tested the hypothesis that investigation of HPV insertions in a head and neck cancer squamous cell carcinoma (HNSCC) cell line would identify targetable driver genes contributing to oncogenesis of other HNSCC. In the cell line UPCI:SCC090 HPV16 integration amplified the PIM1 serine/threonine kinase gene ~16-fold, thereby increasing transcript and protein levels. We used genetic and pharmacological approaches to inhibit PIM kinases in this and other HNSCC cell lines. Knockdown of PIM1 transcripts by transfected short hairpin RNAs reduced UPCI:SCC090 viability. CRISPR/Cas9-mediated mutagenesis of PIM1 caused cell cycle arrest and apoptosis. Pharmacological inhibition of PIM family kinases decreased growth of UPCI:SCC090 and additional HNSCC cell lines in vitro and a xenograft UPCI:SCC090 model in vivo. Based on established interactions between intracellular signaling pathways and relatively high levels of gene expression in almost all HNSCC, we also evaluated combinations of PIM kinase and epidermal growth factor receptor (EGFR) inhibitors. Dual inhibition of these pathways resulted in supra-additive cell death. These data support clinical testing of PIM inhibitors alone or in combination in HNSCC.

Saccharomonosporine A inspiration; synthesis of potent analogues as potential PIM kinase inhibitors.

Saccharomonosporine A was recently reported as a natural anti-cancer agent working through inhibition of a Proviral integration site for Moloney murine leukemia virus-1 (PIM-1) kinase. Structural bioisosteres of this natural product were synthesized and tested against PIM kinase enzymes. They showed potent inhibitory activity against all the known PIM kinases (PIM-1, 2 and 3) with IC50 values ranging from 0.22 to 2.46 μM. Compound 5 was the most potent pan-inhibitor with IC50 values of 0.37, 0.41, and 0.3 μM, against PIM-1, 2, 3 respectively. Compounds 4–6 were tested for their cytotoxic activities against 3 cell lines: H1650, HT-29, and HL-60. Compound 5 exhibited significant cytotoxic activity against human colon adenocarcinoma HT-29 and the human promyelocytic leukemia HL-60, with IC50 μM values of 1.4 and 1.7 respectively. Molecular docking and homology modeling studies were carried out to confirm the affinity of these synthesized compounds to the three different PIM kinases. Additionally, a number of in silico predictions, ADME/Tox, were adopted to evaluate their drug-likeness.

Direct Phosphorylation and Stabilization of HIF-1α By PIM1 Kinase Drives Angiogenesis in Solid Tumors

Angiogenesis is essential for sustained growth of solid tumors. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of angiogenesis and constitutive activation of HIF-1 is frequently observed in human cancers. Thus, understanding mechanisms governing the activation of HIF-1 is critical for successful therapeutic targeting of tumor angiogenesis. Herein, we establish a new regulatory mechanism responsible for the constitutive activation of HIF-1α in cancer, irrespective of oxygen tension. PIM1 kinase directly phosphorylates HIF-1α at threonine 455, a previously uncharacterized site within its oxygen-dependent degradation domain. This phosphorylation event disrupts the ability of prolyl hydroxylases (PHDs) to bind and hydroxylate HIF-1α, interrupting its canonical degradation pathway and promoting constitutive transcription of HIF-1 target genes. Overexpression of PIM1 is sufficient to stabilize HIF-1α in normoxia and stimulate angiogenesis in a HIF-1-dependent manner in vivo. CRISPR mutants of HIF-1α (Thr455D) showed increased tumor growth, proliferation and angiogenesis. Moreover, T455D xenograft tumors were refractory to the anti-angiogenic and cytotoxic effects of PIM inhibitors. These data identify a new signaling axis responsible for hypoxia-independent activation of HIF-1 and expand our understanding of the tumorigenic role of PIM1 in solid tumors.

Novel therapeutics in hepatoblastoma

Hepatoblastoma is the most common primary liver malignancy in children less than 5 years of age. The incidence of this cancer is increasing but therapy for hepatoblastoma has not significantly changed and it is primarily managed with combinations of surgery and chemotherapy. Conventional chemotherapy regimens include cisplatin, as it is currently the most effective agent for this tumor. Unfortunately, with the current modalities, the prognosis for advanced-stage disease remains poor due to the high rate of recurrence and/or the development of chemoresistance. As the understanding of the pathophysiology of hepatoblastoma deepens, there is a burgeoning interest in developing novel therapeutic agents for hepatoblastoma, including targeted therapies. In the current review, we discuss the use of major signaling pathways and their effector molecules such as β-catenin, c-MET, mTOR, PIM kinases, and PLK1 as therapeutic targets. In addition, we explore various alternative pre-clinical approaches that have been described in hepatoblastoma, including immunotherapy, differentiation agents, micro-RNAs, and targeted drug delivery. Finally, we outline the therapies currently under investigation in pre-clinical and clinical settings that show promising preliminary results for hepatoblastoma treatment.

Synthesis, crystal structures, antiproliferative activities and reverse docking studies of eight novel Schiff bases derived from benzil.

Eight novel Schiff bases derived from benzil dihydrazone (BDH) or benzil monohydrazone (BMH) and four fused-ring carbonyl compounds (3-formylindole, FI; 3-acetylindole, AI; 3-formyl-1-methylindole, MFI; 1-formylnaphthalene, FN) were synthesized and characterized by elemental analysis, ESI-QTOF-MS, 1H and 13C NMR spectroscopy, as well as single-crystal X-ray diffraction. They are (1Z,2Z)-1,2-bis{(E)-[(1H-indol-3-yl)methylidene]hydrazinylidene}-1,2-diphenylethane (BDHFI), C32H24N6, (1Z,2Z)-1,2-bis{(E)-[1-(1H-indol-3-yl)ethylidene]hydrazinylidene}-1,2-diphenylethane (BDHAI), C34H28N6, (1Z,2Z)-1,2-bis{(E)-[(1-methyl-1H-indol-3-yl)methylidene]hydrazinylidene}-1,2-diphenylethane (BMHMFI) acetonitrile hemisolvate, C34H28N6·0.5CH3CN, (1Z,2Z)-1,2-bis{(E)-[(naphthalen-1-yl)methylidene]hydrazinylidene}-1,2-diphenylethane (BDHFN), C36H26N4, (Z)-2-{(E)-[(1H-indol-3-yl)methylidene]hydrazinylidene}-1,2-diphenylethanone (BMHFI), C23H17N3O, (Z)-2-{(E)-[1-(1H-indol-3-yl)ethylidene]hydrazinylidene}-1,2-diphenylethanone (BMHAI), C24H19N3O, (Z)-2-{(E)-[(1-methyl-1H-indol-3-yl)methylidene]hydrazinylidene}-1,2-diphenylethanone (BMHMFI), C24H19N3O, and (Z)-2-{(E)-[(naphthalen-1-yl)methylidene]hydrazinylidene}-1,2-diphenylethanone (BMHFN) C25H18N2O. Moreover, the in vitro cytotoxicity of the eight title compounds was evaluated against two tumour cell lines (A549 human lung cancer and 4T1 mouse breast cancer) and two normal cell lines (MRC-5 normal lung cells and NIH 3T3 fibroblasts) by MTT assay. The results indicate that four (BDHMFI, BDHFN, BMHMFI and BMHFN) are inactive and the other four (BDHFI, BDHAI, BMHFI and BMHAI) show severe toxicities against human A549 and mouse 4T1 cells, similar to the standard cisplatin. All the compounds exhibited weaker cytotoxicity against normal cells than cancer cells. The Swiss Target Prediction web server was applied for the prediction of protein targets. After analyzing the differences in frequency hits between these active and inactive Schiff bases, 18 probable targets were selected for reverse docking with the Surflex-dock function in SYBYL-X 2.0 software. Three target proteins, i.e. human ether-á-go-go-related (hERG) potassium channel, the inhibitor of apoptosis protein 3 and serine/threonine-protein kinase PIM1, were chosen as the targets. Finally, the ligand-based structure-activity relationships were analyzed based on the putative protein target (hERG) docking results, which will be used to design and synthesize novel hERG ion channel inhibitors.

Synthesis of Tetrahydrobenzo[b]thiophene-3-carbohydrazide Derivatives as Potential Anti-cancer Agents and Pim-1 Kinase Inhibitors.

Tetrahydrobenzo[b]thiophene derivatives are well known to be biologically active compounds and many of them occupy a wide range of anticancer agent drugs. One of the main aim of this work was to synthesize target molecules not only possessing anti-tumor activities but also kinase inhibitors. To achieve this goal, our strategy was to synthesize a series of 4,5,6,7- tetrahydrobenzo[b]thiophene-3-carbohydrazide derivatives using cyclohexan-1,4-dione and cyanoacetylhydrazine to give the 2-amino-6-oxo-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbohydrazide (3) as the key starting material for many heterocyclization reactions. Compound 3 was reacted with some aryldiazonium salts and the products were cyclised when reacted with either malononitrile or ethyl cyanoacetate. Thiazole derivatives were also obtained through the reaction of compound 3 with phenylisothiocyanate followed by heterocyclization with α-halocarbonyl derivatives. Pyrazole, triazole and pyran derivatives were also obtained. The compounds obtained in this work were evaluated for their in-vitro cytotoxic activity against c-Met kinase, and the six typical cancer cell lines (A549, H460, HT-29, MKN-45, U87MG, and SMMC-7721). The results of anti-proliferative evaluations and c-Met kinase, Pim-1 kinse inhibitions revealed that some compounds showed high activities. The most promising compounds 5b, 5c, 7c, 7d, 11b, 14a, 16b, 18b, 19, 21a, 23c, 23d and 23i against c-Met kinase were further investigated against the five tyrosin kinases (c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR). Compounds 5b, 5c, 7d, 7e, 11b, 11c, 16c, 16d, 18c, 19, 23e, 23k and 23m were selected to examine their Pim-1 kinase inhibitions activity where compounds 7d, 7e, 11b, 11c, 16d, 18c and 23e showed high activities. All of the synthesized compounds have no impaired effect toward the VERO normal cell line.

Current perspectives on targeting PIM kinases to overcome mechanisms of drug resistance and immune evasion in cancer.

PIM kinases are a class of serine/threonine kinases that play a role in several of the hallmarks of cancer including cell cycle progression, metabolism, inflammation and immune evasion. Their constitutively active nature and unique catalytic structure has led them to be an attractive anticancer target through the use of small molecule inhibitors. This review highlights the enhanced activity of PIM kinases in cancer that can be driven by hypoxia in the tumour microenvironment and the important role that aberrant PIM kinase activity plays in resistance mechanisms to chemotherapy, radiotherapy, anti-angiogenic therapies and targeted therapies. We highlight an interaction of PIM kinases with numerous major oncogenic players, including but not limited to, stabilisation of p53, synergism with c-Myc, and notable parallel signalling with PI3K/Akt. We provide a comprehensive overview of PIM kinase's role as an escape mechanism to targeted therapies including PI3K/mTOR inhibitors, MET inhibitors, anti-HER2/EGFR treatments and the immunosuppressant rapamycin, providing a rationale for co-targeting treatment strategies for a more durable patient response. The current status of PIM kinase inhibitors and their use as a combination therapy with other targeted agents, in addition to the development of novel multi-molecularly targeted single therapeutic agents containing a PIM kinase targeting moiety are discussed.

Bisindolemethane derivatives as highly potent anticancer agents: Synthesis, medicinal activity evaluation, cell-based compound discovery, and computational target predictions

In recent years chemical and biological interest in Turbomycin B and its various analogues have motivated researchers to develop new bioactive indole scaffolds. In this work, we focused on the development of indole alkaloids, especially bioindolemethane derivatives and their anticancer potential. Based on the excellent IC50 value against HeLa and A549 cell, cyano-substituted Turbomycin B (CN-TBM) was selected to understand the mechanism behind cancer cell death. The potential targets involved in CN-TBM mediated apoptotic cell death were predicted by comparing the results of two chemoinformatic approaches, i.e., PharmMapper and IdTarget. Four targets were predicted using these tools and the targets were further subjected to molecular docking to obtain a single target for CN-TBM. Serine/threonine-protein kinase (Pim-1) was identified as the direct target of CN-TBM with a pharmacophore model complementing well with the molecular features of CN-TBM. The interaction between CN-TBM and Pim-1 was well supported by high fit-score, Z-score, idTarget dock score and excellent binding affinity. Further, the present study also provides an insight into co-expression, shared protein domains, functional annotation and relationship within the ten putative targets of CN-TBM predicted by PharmMapper.

Apoptosis: A target for anticancer therapy with novel cyanopyridines.

One of the many methods of treating cancer is to terminate the uncontrolled growth of cancer cells. So, aiming the apoptotic pathway is an exciting approach to finding new anticancer agents. A novel series of cyanopyridines was designed and synthesized for antiproliferative evaluation. 2-Amino-6-(4-(benzyloxy)phenyl)-4-(4-(dimethylamino)phenyl) nicotinonitrile 10f was the most potent inhibitor against the growth of PC-3, and HepG-2 cancer cell lines with IC50 values of 2.04 uM (selectivity index, SI=78.63, 43, respectively). Also, 10f was safe against the growth of normal human diploid lung fibroblasts cell line (WI-38) with an IC50 value of 160.04 uM. Its analogs, 10b, 10d, 10g, and 11b, were also active against the growth of PC-3, and HepG-2 while against MCF-7 cell line, they displayed good cytotoxic activity compared to the reference standard 5-FU. Remarkably, mechanistic studies indicated that compounds 10b, 10d, 10f, 10g, and 11b stimulated the level of active caspase 3 and boosted the BAX/BCL2 ratio 20-95 folds in comparison to the control. Our results have also indicated that 10b, 10d, 10f, 10g, and 11b exhibited a very potent inhibitory activity against PIM-1 kinase enzyme, where the IC50 values unraveled very potent molecules in the micromolar range (0.47-1.27μM). Further investigations have shown that 10f, the most potent PIM-1 kinase inhibitor, induced a cell cycle arrest at the G2/M phase. Moreover, in silico evaluation of ADME properties indicated that all the cyanopyridine compounds are orally bioavailable with no permeation to the blood brain barrier.

Abstract B132: Inhibition of Haspin kinase to promote cell-intrinsic and extrinsic anticancer therapy

Abstract Oncogene-targeted therapies are initially effective in most patients with BRAF-mutated melanoma. However, patients frequently develop resistance during therapy. At the time of therapy resistance, they are also less likely to respond to immune checkpoint inhibitors (ICI) with response rates ranging from 0-12%, compared to 40-60% when used in the first line. This may be in part be due to rapid tumor growth that outpaces pharmacodynamics of ICI. New drugs that directly control tumor growth and effectively enhance ICI activity are therefore needed. We report that the small molecule CX-6258 has potent activity against both RAF/MEK inhibitor (RMS) and resistant (RMR) BRAF-mutant melanoma cell lines. CX-6258 is annotated as a pan-PIM kinase inhibitor, with an EC50 corrected for growth rate (GR50) of ~200 nM; in contrast, other PIM inhibitors had no in vitro activity, suggesting that PIMs may not be the true target in melanoma - we therefore sought to systematically study the targets of this compound. Using an unbiased kinase profiling approach, we identified Haspin Kinase (HASPIN) as the key target of CX-6258 with an IC50~10nM. Haspin kinase (Haspin) is a poorly understood mitotic kinase, whose only well-defined function is phosphorylation of Histon H3 at Thr3 (pH3T3). Treatment with CX-6258 resulted in reduced proliferation, reduced phosphorylation of pH3T3, frequent formation of micronuclei (MN), recruitment of cGAS and activation of the cGAS-STING-pathway. By using CRISPR/CAS9 knock outs of PIM kinase we show that these targets are not required for CX-6258 function. Furthermore, siRNA mediated knock-down of HASPIN phenocopied CX-6258. In murine models, CX-6258 induces a potent cGAS dependent type-I-interferon response in tumor cells, increases IFNγ-producing CD8+ T-cells, reduces Tregs, and enhances responses to ICIs in vivo. HASPIN is more strongly expressed in malignant compared to healthy tissue and its inhibition by CX-6258 has minimal toxicity in ex vivo expanded human tumor-infiltrating-lymphocytes (TILs) and in vitro differentiated human neurons, suggesting a potential therapeutic index for anti-cancer therapy. CX-6258 also shows activity in several Ewing sarcoma and multiple myeloma cell lines with. In summary, HASPIN inhibition may overcome drug resistance in melanoma, synergize with ICIs and target a vulnerability in different cancer lineages. Citation Format: Johannes C. Melms, Sreeram Vallabhaneni, Caitlin E. Mills, Kai Wucherpfennig, Peter K. Sorger, Benjamin Izar. Inhibition of Haspin kinase to promote cell-intrinsic and extrinsic anticancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B132. doi:10.1158/1535-7163.TARG-19-B132

Molecular Docking of Citronellol, Geraniol and Ester Derivatives as Pim 1 Kinase Inhibitor of Leukemia Cancer

The proviral insertion site in Moloney murine leukemia virus-1 (Pim1) kinase is overexpressed in many human cancer diseases. Inhibition of Pim1 kinase has been reported can suppress cell proliferation and induce apoptotic cell death, so that the protein could be chosen as drug target for the treatment of cancer. Citronellol and geraniol mainly contained in citronella oil that’s one of Indonesian natural products have bioactivity as antitumor agents, so that both of the compounds can be used as guiding compound to be developed as chemoprevention and chemotherapeutic agent compounds for cancer especially for leukemia cancer. In this research, sixteen citronellol and geraniol esters as an inhibitor of leukemia cancer designed were docked based on their interaction with leukemia receptor target of Pim1 kinase by using Molegro Virtual Docker (MVD). The result showed that the binding citronellol and geraniol esters to Pim1 kinase are more stable and better affinity than the binding between citronellol and geraniol, indicated by rerank score were lower than rerank score of citronellol/geraniolPim1 kinase. The docking resulted in the four top-ranked compounds, namely, citronellyl caproate, citronellyl caprylate, geranyl caproate and geranyl caprylate with rerank score value from -182.560 to -189.822 KJ/mol. These compounds have hydrogen bonding with amino acid residues of Lys 67 and Asp 186 of an active site of Pim1 kinase. Based on Lipinski’s rule of five, among of the four top-ranked compounds, citronellyl caproate and geranyl caproate were predicted have potency as new chemopreventive and chemotherapeutic agents for leukemia anticancer candidates.

The targetable kinase PIM1 drives ALK inhibitor resistance in high-risk neuroblastoma independent of MYCN status

Resistance to anaplastic lymphoma kinase (ALK)-targeted therapy in ALK-positive non-small cell lung cancer has been reported, with the majority of acquired resistance mechanisms relying on bypass signaling. To proactively identify resistance mechanisms in ALK-positive neuroblastoma (NB), we herein employ genome-wide CRISPR activation screens of NB cell lines treated with brigatinib or ceritinib, identifying PIM1 as a putative resistance gene, whose high expression is associated with high-risk disease and poor survival. Knockdown of PIM1 sensitizes cells of differing MYCN status to ALK inhibitors, and in patient-derived xenografts of high-risk NB harboring ALK mutations, the combination of the ALK inhibitor ceritinib and PIM1 inhibitor AZD1208 shows significantly enhanced anti-tumor efficacy relative to single agents. These data confirm that PIM1 overexpression decreases sensitivity to ALK inhibitors in NB, and suggests that combined front-line inhibition of ALK and PIM1 is a viable strategy for the treatment of ALK-positive NB independent of MYCN status.

Anti‑survival and pro‑apoptotic effects of meridianin C derivatives on MV4‑11 human acute myeloid leukemia cells.

Meridianin C is a marine natural product with anticancer activity. Several meridianin C derivatives (compounds7a‑j) were recently synthesized, and their inhibitory effects on pro‑viral integration site for Moloney murine leukemia virus (PIM) kinases, as well as their antiproliferative effects on human leukemia cells, were reported. However, the anti‑leukemic effects and mechanisms of action of meridianin C and its derivatives remain largely unknown. The aim of the present study was to investigate the effects of meridianin C and its derivatives on MV4‑11 human acute myeloid leukemia cell growth. The parent compound meridianin C did not markedly affect the viability and survival of MV4‑11 cells. By contrast, MV4‑11 cell viability and survival were reduced by meridianinC derivatives, with compound7a achieving the most prominent reduction. Compound7a notably inhibited the expression and activity of PIM kinases, as evidenced by reduced B‑cell lymphoma‑2 (Bcl‑2)‑associated death promoter phosphorylation at Ser112. However, meridianin C also suppressed PIM kinase expression and activity, and the pan‑PIM kinase inhibitor AZD1208 only slightly suppressed the survival of MV4‑11 cells. Thus, the anti‑survival effect of compound7a on MV4‑11 cells was unrelated to PIM kinase inhibition. Moreover, compound7a induced apoptosis, caspase‑9and‑3 activation and poly(ADP‑ribose) polymerase (PARP) cleavage, but did not affect death receptor (DR)‑4 or DR‑5 expression in MV4‑11 cells. Compound7a also induced the generation of cleaved Bcl‑2, and the downregulation of myeloid cell leukemia (Mcl)‑1 and X‑linked inhibitor of apoptosis (XIAP) in MV4‑11 cells. Furthermore, compound7a increased eukaryotic initiation factor (eIF)‑2α phosphorylation and decreased S6 phosphorylation, whereas GRP‑78 expression was unaffected. Importantly, treatment with a pan‑caspase inhibitor (z‑VAD‑fmk) significantly attenuated compound7a‑induced apoptosis, caspase‑9and‑3 activation, PARP cleavage, generation of cleaved Bcl‑2 and downregulation of Mcl‑1 and XIAP in MV4‑11 cells. Collectively, these findings demonstrated the strong anti‑survival and pro‑apoptotic effects of compound7a on MV4‑11 cells through regulation of caspase‑9and‑3, Bcl‑2, Mcl‑1, XIAP, eIF‑2α and S6 molecules.

The pan-PIM inhibitor INCB053914 displays potent synergy in combination with ruxolitinib in models of MPN

AbstractAberrant JAK2 tyrosine kinase signaling drives the development of Philadelphia chromosome–negative myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. However, JAK2 kinase inhibitors have failed to significantly reduce allele burden in MPN patients, underscoring the need for improved therapeutic strategies. Members of the PIM family of serine/threonine kinases promote cellular proliferation by regulating a variety of cellular processes, including protein synthesis and the balance of signaling that regulates apoptosis. Overexpression of PIM family members is oncogenic, exemplified by their ability to induce lymphomas in collaboration with c-Myc. Thus, PIM kinases are potential therapeutic targets for several malignancies such as solid tumors and blood cancers. We and others have shown that PIM inhibitors augment the efficacy of JAK2 inhibitors by using in vitro models of MPNs. Here we report that the recently developed pan-PIM inhibitor INCB053914 augments the efficacy of the US Food and Drug Administration–approved JAK1/2 inhibitor ruxolitinib in both in vitro and in vivo MPN models. INCB053914 synergizes with ruxolitinib to inhibit cell growth in JAK2-driven MPN models and induce apoptosis. Significantly, low nanomolar INCB053914 enhances the efficacy of ruxolitinib to inhibit the neoplastic growth of primary MPN patient cells, and INCB053914 antagonizes ruxolitinib persistent myeloproliferation in vivo. These findings support the notion that INCB053914, which is currently in clinical trials in patients with advanced hematologic malignancies, in combination with ruxolitinib may be effective in MPN patients, and they support the clinical testing of this combination in MPN patients.