Kinase Profiling Research Articles

Acetylshikonin suppressed growth of colorectal tumour tissue and cells by inhibiting the intracellular kinase, T-lymphokine-activated killer cell-originated protein kinase.

Background and PurposeOverexpression or aberrant activation of the T‐lymphokine‐activated killer cell‐originated protein kinase (TOPK) promotes gene expression and growth of solid tumours, implying that TOPK would be a rational target in developing novel anticancer drugs. Acetylshikonin, a diterpenoid compound isolated from Lithospermum erythrorhizon root, exerts a range of biological activities. Here we have investigated whether acetylshikonin, by acting as an inhibitor of TOPK, can attenuate the proliferation of colorectal cancer cells and the growth of patient‐derived tumours, in vitro and in vivo.Experimental ApproachTargets of acetylshikonin, were identified using kinase profiling analysis, kinetic/binding assay, and computational docking analysis and knock‐down techniques. Effects of acetylshikonin on colorectal cancer growth and the underlying mechanisms were evaluated in cell proliferation assays, propidium iodide and annexin‐V staining analyses and western blots. Patient‐derived tumour xenografts in mice (PDX) and immunohistochemistry were used to assess anti‐tumour effects of acetylshikonin.Key ResultsAcetylshikonin directly inhibited TOPK activity, interacting with the ATP‐binding pocket of TOPK. Acetylshikonin suppressed cell proliferation by inducing cell cycle arrest at the G1 phase, stimulated apoptosis, and increased the expression of apoptotic biomarkers in colorectal cancer cell lines. Mechanistically, acetylshikonin diminished the phosphorylation and activation of TOPK signalling. Furthermore, acetylshikonin decreased the volume of PDX tumours and reduced the expression of TOPK signalling pathway in xenograft tumours.Conclusion and ImplicationsAcetylshikonin suppressed growth of colorectal cancer cells by attenuating TOPK signalling. Targeted inhibition of TOPK by acetylshikonin might be a promising new approach to the treatment of colorectal cancer.

PROFIL PROTEIN TIROSIN KINASE DALAM SEMINAL PLASMA DOMBA MERINO DENGAN TEKNIK SODIUM DODECYL SULPHATE POLYACRILAMIDE GEL ELECTROPHORESIS

The aim of this study was to find out protein tyrosine kinase profile in Merino Sheep seminal plasma. This study consist of collecting semen of Merino Sheep containing plasma and spermatozoa, the separation between seminal plasma and spermatozoa, then tyrosine kinase analysis using Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis (SDS-PAGE). Semen was collected by artificial vagina and then centrifugated for 40 minutes at 4000 rpm to separated seminal plasma and spermatozoa. Protein was analyzed using SDS-PAGE, to separate each protein based on their molecular weight. The result showed that there were 13 protein bands in 3 seminal plasma samples with an average 149,63 kDa, 139,7 kDa, 114,97 kDa, 109,3 kDa, 97,33 kDa, 93,83 kDa, 86,23 kDa, 77,6 kDa, 64,6 kDa, 52,3 kDa, 41,93 kDa, 38,13 kDa, and 34,5 kDa. In conclusion, it is believed that tyrosine kinase located on the sixth band with molecular weight 93,83 kDa.

P48 A case of myasthenia gravis presenting to the rheumatology clinic

Abstract Background A 32-year-old Lithuanian lady, with a 6-year history of undifferentiated inflammatory arthritis and autoimmune neutropenia, contacted her rheumatologist reporting a constellation of new symptoms. This began with a month’s history of facial weakness and slurred speech. She progressively experienced nasal regurgitation on drinking fluids, reduced exercise tolerance, as well as difficulty chewing, swallowing, and coughing. Her symptoms initially came on in the evening but subsequently appeared earlier in the day. There were no symptoms suggestive of infection. Her arthritis had been well-controlled on hydroxychloroquine and sulfasalazine. She was previously on methotrexate, but this was discontinued due to plans to conceive. Methods Clinical examination during an urgent outpatient review yielded dysphonic, mildly dysarthric speech. Extraocular movements were normal with no ophthalmoplegia. There was mild lower motor neurone facial weakness and an absent gag reflex. She demonstrated fatiguability on repetitive tongue movements and arm abduction. There were no upper motor neurone or cerebellar signs. Blood tests showed normal inflammatory markers, full blood count, creatine kinase, immunoglobulins, liver, and renal profile. Her anti-nuclear antibody titre, previously weakly positive at time of diagnosis of inflammatory arthritis, was 1:1280 (homogenous). Anti-cyclic citrullinated peptide, rheumatoid factor, and extractable nuclear antigens were negative. Virology screen was unremarkable. Neurology input was sought. Anti-acetylcholine receptor antibody was positive at 5.2nmol/L (normal range 0-0.45nmol/L), anti-muscle specific receptor tyrosine kinase antibody was negative. Nerve conduction studies showed decremental response on repetitive stimulation of her left nasalis, consistent with a post-synaptic neuromuscular junction transmission disorder. There was no evidence of myopathy. Results The clinical symptoms and the investigations led to the diagnosis of myasthenia gravis with predominant bulbar features. Computed tomography showed no evidence of thymoma or thymic hyperplasia. Magnetic resonance imaging of the brain showed no evidence of space-occupying lesion or brainstem abnormality. She initially received pyridostigmine 30mg three times daily with propantheline cover to which she reported a good response. A viral illness led to deterioration of her symptoms of breathlessness and worsening dysphagia resulting in a hospital admission. She received intravenous immunoglobulins 20g daily for three days. Hydroxychloroquine and sulfasalazine were replaced with methotrexate 15mg weekly. She now remains on pyridostigmine 90mg five times daily and 10mg prednisolone daily with good effect. Conclusion Myasthenia gravis is a rare condition (prevalence of 10-20 per 100,000 in the United Kingdom). However, this occurs at a higher prevalence in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. This case illustrates the importance of remaining vigilant about symptoms which patients may disclose in the rheumatology clinic which may not fit with their known diagnosis. Her previous use of methotrexate for her arthritis could have masked her myasthenic symptoms which became apparent after the drug was stopped for a prolonged period. Disclosures S. Yeoh None. S. Salam None. G. Christofi None. V. Morris None.

Discovery of 1-Pyrimidinyl-2-Aryl-4,6-Dihydropyrrolo [3,4-d]Imidazole-5(1H)-Carboxamide as a Novel JNK Inhibitor.

We designed and synthesized 1-pyrimidinyl-2-aryl-4, 6-dihydropyrrolo [3,4-d] imidazole-5(1H)-carboxamide derivatives as selective inhibitors of c-Jun-N-terminal Kinase 3 (JNK3), a target for the treatment of neurodegenerative diseases. Based on the compounds found in previous studies, a novel scaffold was designed to improve pharmacokinetic characters and activity, and compound 18a, (R)-1-(2-((1-(cyclopropanecarbonyl)pyrrolidin-3-yl)amino)pyrimidin-4-yl)-2-(3,4-dichlorophenyl)-4,6-dihydro pyrrolo [3,4-d]imidazole-5(1H)-carboxamide, showed the highest IC50 value of 2.69 nM. Kinase profiling results also showed high selectivity for JNK3 among 38 kinases, having mild activity against JNK2, RIPK3, and GSK3β, which also known to involve in neuronal apoptosis.

Abstract A78: Preclinical evaluation of 3D185, a novel potent inhibitor of FGFR1/2/3 and CSF-1R, in FGFR-dependent and macrophage-dominant cancer models

Abstract The interaction between tumor cells and their immunosuppressive microenvironment promotes tumor progression and drug resistance. Thus, simultaneously targeting tumor cells and stromal cells is expected to have synergistic antitumor effects. Herein, we present the preclinical antitumor investigation of 3D185, a novel dual inhibitor targeting FGFRs, which are oncogenic drivers, and CSF-1R, which is the major survival factor for protumor macrophages. The antitumor characteristics of 3D185 were assessed by a range of assays, including kinase profiling, cell viability, cell migration, immunoblotting, CD8+ T-cell suppression, and in vivo antitumor efficacy, followed by flow cytometric and immunohistochemical analyses of tumor-infiltrating immune cells and endothelial cells in nude mice and immune-competent mice. We found that 3D185 significantly inhibited the kinase activity of FGFR1/2/3 and CSF-1R, with equal potency and high selectivity over other kinases. 3D185 suppressed FGFR signaling and tumor cell growth in FGFR-driven models both in vitro and in vivo. In addition, 3D185 could inhibit the survival and M2-like polarization of protumor macrophages, reversing the immunosuppressive effect of macrophages on CD8+ T cells as well as CSF1-differentiated macrophage induced-FGFR3-aberrant cancer cell migration. Furthermore, 3D185 inhibited tumor growth via remodeling the tumor microenvironment in TAM-dominated tumor models. In summary, 3D185 is a dual inhibitor against FGFR1/2/3 and CSF-1R that exhibits potent antitumor activities. This compound is promising because it simultaneously targets tumor cells per se and the immunosuppressive tumor microenvironment to synergistically antagonize tumors. Our study provides a solid foundation for the investigation of 3D185 in cancer patients, particularly in patients with aberrant FGFR and abundant macrophages, who respond poorly to classic pan-FGFRi treatment. Now 3D185 is in phase I clinical trials. Citation Format: Yang Dai, Yichun Ji, Yanyan Shen, Yi Su, Bo Liu, Yueliang Wang, Deqiao Sun, Yuchen Jiang, Chuantao Zha, Zuoquan Xie, Jian Ding, Meiyu Geng, Jing Ai, Xia Peng, Pengcong Hou, Yi Chen. Preclinical evaluation of 3D185, a novel potent inhibitor of FGFR1/2/3 and CSF-1R, in FGFR-dependent and macrophage-dominant cancer models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A78.

PO-322 exerts potent immunosuppressive effects in vitro and in vivo by selectively inhibiting SGK1 activity.

Immunosuppressive drugs have shown great promise in treating autoimmune diseases in recent years. A series of novel oxazole derivatives were screened for their immunosuppressive activity. PO-322 [1H-indole-2,3-dione 3-(1,3-benzoxazol-2-ylhydrazone)] was identified as the most effective of these compounds. Here, we have investigated the mechanism(s) underlying the inhibition of T-cell proliferation in vitro by PO-322, as well as its effects on the delayed-type hypersensitivity (DTH) response and imiquimod-induced dermatitis in vivo. T-cell proliferation and apoptosis were analysed with flow cytometry. Cell viability was assessed with a CCK-8 assay. Protein kinase activity was assessed by SelectScreen Kinase Profiling Services. The phosphorylation of signal-regulated molecules was measured by Western blot. Cytokine levels were determined by elisa. The effect of PO-322 on DTH and imiquimod-induced dermatitis was evaluated in BALB/c mice. PO-322 inhibited human T-cell proliferation with anti-CD3/anti-CD28 mAbs or alloantigen without significant cytotoxicity. Importantly, PO-322 was a selective inhibitor of the serum- and glucocorticoid-regulated kinase 1 (SGK1) and decreased NDRG1 phosphorylation but not p70S6K, STAT5, Akt, or ERK1/2 phosphorylation. Furthermore, PO-322 inhibited IFN-γ, IL-6, and IL-17 expression but not IL-10 expression. Finally, treatment with PO-322 was safe and effective for ameliorating the DTH response and imiquimod-induced dermatitis in mice. PO-322 exerted immunosuppressive activity in vitro and in vivo by selectively inhibiting SGK1 activity. PO-322 represents a potential lead compound for the design and development of new drugs for the treatment of autoimmune diseases.

Effects of Glycyrrhetic Acid on Human Chronic Myelogenous Leukemia Cells.

Chronic myelogenous leukemia (CML) is a type of blood cancer that is initially treated with imatinib (first Abl kinase inhibitor). However, some patients with CML develop imatinib resistance. Several new generation drugs have been developed, but do not overcome this problem. Glycyrrhetic acid (GA) is a plant-derived pentacyclic triterpenoid that exhibits multiple pharmacological properties for the treatment of cancers. The current study aimed to investigate the effects of GA on the K562 cell line (Bcr-Abl positive leukemia). The MTT cell proliferation assay was employed to evaluate the cytotoxic effect of GA compared with imatinib (positive control) against leukemia and normal blood cells. For detection of cell death, an apoptotic/necrotic/healthy assay was performed against the K562 cell line. To investigate the kinase inhibitory activity of GA, the Abl1 kinase profiling assay and a molecular docking study were performed. GA showed Abl kinase inhibitory activity with an IC50 value of 29.2 μM and induced apoptosis in the K562 cell line after 6 h of treatment. The current findings indicate that this class of plant extract could be a potential candidate for treatment of CML.

Effects of the multi-kinase inhibitor midostaurin in combination with chemotherapy in models of acute myeloid leukaemia.

Recently, several targeted agents have been developed for specific subsets of patients with acute myeloid leukaemia (AML), including midostaurin, the first FDA‐approved FLT3 inhibitor for newly diagnosed patients with FLT3 mutations. However, in the initial Phase I/II clinical trials, some patients without FLT3 mutations had transient responses to midostaurin, suggesting that this multi‐targeted kinase inhibitor might benefit AML patients more broadly. Here, we demonstrate submicromolar efficacy of midostaurin in vitro and efficacy in vivo against wild‐type (wt) FLT3‐expressing AML cell lines and primary cells, and we compare its effectiveness with that of other FLT3 inhibitors currently in clinical trials. Midostaurin was found to synergize with standard chemotherapeutic drugs and some targeted agents against AML cells without mutations in FLT3. The mechanism may involve, in part, the unique kinase profile of midostaurin that includes proteins implicated in AML transformation, such as SYK or KIT, or inhibition of ERK pathway or proviability signalling. Our findings support further investigation of midostaurin as a chemosensitizing agent in AML patients without FLT3 mutations.

Early high-frequency spinal cord stimulation treatment inhibited the activation of spinal mitogen-activated protein kinases and ameliorated spared nerve injury-induced neuropathic pain in rats

BackgroundNeuromodulation therapies offer a treatment option that has minimal side effects and is relatively safe and potentially reversible. Spinal cord stimulation (SCS) has been used to treat various pain conditions for many decades. High-frequency SCS (HFSCS) involves the application of a single waveform at 10,000 Hz at a subthreshold level, therefore providing pain relief without any paresthesia. MethodsWe tested whether early HFSCS treatment attenuated spared nerve injury (SNI)-induced neuropathic pain. The phosphorylation profile of mitogen-activated protein kinases (MAPKs), i.e., extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38, was evaluated to elucidate the potential underlying mechanism. ResultsSNI of rat unilateral sciatic nerves induced mechanical hyperalgesia in the ipsilateral hind paws. Rats were assigned to SCS sessions with HFSCS (frequency 10 kHz; pulse width 30 μs; pulse shape of charge-balanced, current controlled; delivered continuously for 72 h), or sham stimulation immediately after SNI. Tissue samples were examined at 1, 3, 7, and 14 days after SNI. Behavioral studies showed that HFSCS applied to the T10/T11 spinal cord significantly attenuated SNI-induced mechanical hyperalgesia compared with the sham stimulation group. Moreover, western blotting revealed a significant attenuation of the activation of ERK1, ERK2, JNK1, and p38 in the dorsal root ganglia and the spinal dorsal horn. ConclusionApplication of HFSCS provides an effective treatment for SNI-induced persistent mechanical hyperalgesia by attenuating ERK, JNK, and p38 activation in the dorsal root ganglia and the spinal dorsal horn.

P019 Colonic mucosal kinase activity, cytokine and chemokine profiles in inflammatory bowel disease

Abstract Background With the approval of tofacitinib, an oral Janus Kinase (JAK) inhibitor, modulation of kinase activity has been added to the therapeutic armamentarium of inflammatory bowel disease (IBD). Despite its established efficacy, at least a third of patients will not respond to this or other therapeutic options such as anti-tumour necrosis factor (TNF), anti-interleukin (IL)23/IL12 compounds or vedolizumab. A better understanding of the inflammatory profile could aid in tailoring drugs to individual patients. We therefore explored mucosal cytokine, chemokine and kinase activity profiles in IBD. Methods Colonic mucosal biopsies were collected from (1) patients with Crohn’s disease (CD, N = 8), (2) patients with ulcerative colitis (UC, N = 8) and (3) healthy controls (N = 4). IBD samples were collected both from inflamed and non-inflamed tissue from the same patients. All IBD patients were biological-naïve and had not used corticosteroids in the past 3 months. Biopsies were snap frozen for later kinase activity determination or directly used in a 24-h explant culture. Whole biopsy kinase activity (tyrosine, serine and threonine kinases) was assessed using the Pamgene platform. A 64-analyte panel was examined in the supernatant of the cultured biopsies employing a multiplex assay (Luminex). Results Whole-biopsy kinase activity differed between inflamed and non-inflamed mucosa of IBD patients, with more overall tyrosine kinase activity in inflamed mucosa in UC, and serine/threonine kinase activity in inflamed mucosa in CD as compared with non-inflamed mucosa (Figure 1). The kinase activity profile of non-inflamed mucosa of CD and UC patients was similarly different from mucosa of healthy control participants (Figure 2). The cytokine and chemokine profile of inflamed biopsies differed from non-inflamed IBD biopsies and healthy control biopsies, with higher levels of S100A8, TNFα, IL-6, oncostatin M (OSM) and triggering receptor expressed on myeloid cells-1 (TREM-1), amongst others (Figure 3). Conclusion In IBD, inflammation in the mucosa can be characterised both by explant-culture and kinase activity assessment. The difference in kinase activity between non-inflamed IBD mucosa and healthy control mucosa suggests the presence of sub-clinical alterations in cell signalling. The observed differences in the kinase, cytokine and chemokine profiles underscore the importance of this approach in the elucidation of the pathophysiology in IBD.

PamgeneAnalyzeR: open and reproducible pipeline for kinase profiling.

Protein phosphorylation––catalyzed by protein kinases–is the most common post-translational modification. It increases the functional diversity of the proteome and influences various aspects of normal physiology and can be altered in disease states. High throughput profiling of kinases is becoming an essential experimental approach to investigate their activity and this can be achieved using technologies such as PamChip® arrays provided by PamGene for kinase activity measurement. Here, we present ‘pamgeneAnalyzeR’, an R package developed as an alternative to the manual steps necessary to extract the data from PamChip® peptide microarrays images in a reproducible and robust manner. The extracted data can be directly used for downstream analysis.Availability and implementationPamgeneAnalyzeR is implemented in R and can be obtained from https://github.com/amelbek/pamgeneAnalyzeR.

Circulating Neutrophils Predict Poor Survival for HCC and Promote HCC Progression Through p53 and STAT3 Signaling Pathway.

Background: Tumor-associated neutrophils (TANs) contribute to tumor progression, invasion, and angiogenesis. However, most studies focus on tumor infiltration neutrophils while the roles of circulating neutrophils in tumor progression remain unclear. This study was aimed to verify the pro-tumor effects of circulating neutrophils and its' mechanism in HCC.Methods: We collected clinical data of 127 HCC patients underwent TACE. The prognostic factors for overall survival (OS) were analyzed by Kaplan-Meier curve and Cox models. Circulating neutrophils of HCC patients were sorted and co-cultured with human HCC cell lines MHCC-97H and SMMC-7721. Then we detected tumor cells' proliferation, migration, and invasion. Phosphokinase array was used to determine the kinase profile on MHCC-97H and SMMC-7721 cultured with or without circulating neutrophils.Results: The result of multivariate analyses of 127 patients showed that increased circulating neutrophils was an independent poor prognostic factor for OS of HCC patients underwent TACE. Circulating neutrophils promoted migration and invasion of HCC cell lines but had no impact on proliferation. The kinase profile on HCC cell lines showed that p-p53S46 and p-STAT3Y705 were up-regulated after co-cultured with circulating neutrophils. Repeated scratch tests and transwell tests showed a reversed impact on migration and invasion of circulating neutrophils after we treated HCC cell lines with inhibitors of p53 or STAT3.Conclusion: Circulating neutrophils was an independent poor prognostic factor for OS of HCC patients underwent TACE. It had pro-tumor effect on HCC through p53 and STAT3 signaling pathway.

Quantitative High-Throughput Screening Using an Organotypic Model Identifies Compounds that Inhibit Ovarian Cancer Metastasis.

The tumor microenvironment (TME) is a key determinant of metastatic efficiency. We performed a quantitative high-throughput screen (qHTS) of diverse medicinal chemistry tractable scaffolds (44,420 compounds) and pharmacologically active small molecules (386 compounds) using a layered organotypic, robust assay representing the ovarian cancer metastatic TME. This 3D model contains primary human mesothelial cells, fibroblasts, and extracellular matrix, to which fluorescently labeled ovarian cancer cells are added. Initially, 100 compounds inhibiting ovarian cancer adhesion/invasion to the 3D model in a dose-dependent manner were identified. Of those, eight compounds were confirmed active in five high-grade serous ovarian cancer cell lines and were further validated in secondary in vitro and in vivo biological assays. Two tyrosine kinase inhibitors, PP-121 and milciclib, and a previously unreported compound, NCGC00117362, were selected because they had potency at 1 μmol/L in vitro Specifically, NCGC00117362 and PP-121 inhibited ovarian cancer adhesion, invasion, and proliferation, whereas milciclib inhibited ovarian cancer invasion and proliferation. Using in situ kinase profiling and immunoblotting, we found that milciclib targeted Cdk2 and Cdk6, and PP-121 targeted mTOR. In vivo, all three compounds prevented ovarian cancer adhesion/invasion and metastasis, prolonged survival, and reduced omental tumor growth in an intervention study. To evaluate the clinical potential of NCGC00117362, structure-activity relationship studies were performed. Four close analogues of NCGC00117362 efficiently inhibited cancer aggressiveness in vitro and metastasis in vivo Collectively, these data show that a complex 3D culture of the TME is effective in qHTS. The three compounds identified have promise as therapeutics for prevention and treatment of ovarian cancer metastasis.

Promiscuity analysis of a kinase panel screen with designated p38 alpha inhibitors

Protein phosphorylation by kinases is of critical importance for the regulation of many cellular functions. When kinases are deregulated numerous biological processes are affected, which may cause a variety of diseases. Therefore, kinase inhibition plays an important role for therapeutic intervention. A number of kinase inhibitors have been approved as drugs, initially in oncology where promiscuous (multi-kinase) inhibitors were most efficacious. Exploring kinase inhibitor selectivity and promiscuity for therapy is among the most challenging aspects of kinase drug discovery. Herein, we thoroughly analyze a kinase profiling experiment in which 637 designated inhibitors of p38α MAP kinase (p38α) were tested against a panel of 60 kinases distributed across the human kinome. In this experiment, only 19% of the inhibitors were found to be promiscuous when the median p38α inhibition level was applied as an activity threshold. Promiscuous inhibitors had a median value of two targets per compound, and many of these inhibitors were only active against the p38α and closely related JNK3 enzymes. Promiscuity cliffs were identified and analyzed in a network representation revealing structural modifications that were implicated in triggering compound promiscuity. Taken together, the findings revealed a high degree of selectivity of designated p38α directed inhibitors although they target the ATP binding site that is largely conserved across the human kinome.

Discovery of 3-alkyl-5-aryl-1-pyrimidyl-1H-pyrazole derivatives as a novel selective inhibitor scaffold of JNK3

3-alkyl-5-aryl-1-pyrimidyl-1H-pyrazole derivatives were designed and synthesised as selective inhibitors of JNK3, a target for the treatment of neurodegenerative diseases. Following previous studies, we have designed JNK3 inhibitors to reduce the molecular weight and successfully identified a lead compound that exhibits equipotent activity towards JNK3. Kinase profiling results also showed high selectivity for JNK3 among 38 kinases. Among the derivatives, the IC50 value of 8a, (R)-2-(1-(2-((1-(cyclopropanecarbonyl)pyrrolidin-3-yl)amino)pyrimidin-4-yl)-5-(3,4-dichlorophenyl)-1H-pyrazol-3-yl)acetonitrile exhibited 227 nM, showing the highest inhibitory activity against JNK3.

Flavone-based arylamides as potential anticancers: Design, synthesis and in vitro cell-based/cell-free evaluations

Several arylamide-based antiproliferative agents are known and some of them are currently FDA-approved anticancer drugs. Provoked by the need to fill the existing room with new drugs, 31 compounds constituting a series of flavone-based arylamide derivatives were synthesized and biologically evaluated. Towards extensive evaluation, sixty diverse cancer cell lines representing nine cancer diseases of various origins have been used for evaluation of their efficacy, spectrum and potency. Two compounds 2aw and 2ax emerged as effective, broad-spectrum and potent anticancer agents that outperformed masitinibt and imatininb, which are FDA-approved anticancer drugs. Kinases profiling as possible targets for the potent compound 2aw showed that it might be a hit compound offering a starting point to develop inhibitors of STE20/GCK-IV kinase family members including HGK, TNIK and MINK1 kinases. Mechanistic study showed that compounds 2aw triggers cell cycle arrest in HT29 colon cancer cells. In conclusion, this work presents compound 2aw as a new broad-spectrum anticancer agent for further development of promising treatment of diverse cancers.

Discovery and Evaluation of Pyrazolo[3,4-d]pyridazinone as a Potent and Orally Active Irreversible BTK Inhibitor

The identification and lead optimization of a series of pyrazolo[3,4-d]pyridazinone derivatives are described as a novel class of potent irreversible BTK inhibitors, resulting in the discovery of compound 8. Compound 8 exhibited high potency against BTK kinase and acceptable PK profile. Furthermore, compound 8 demonstrated significant in vivo efficacy in a mouse-collagen-induced arthritis (CIA) model.

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

Abstract LB-C12: Derazantinib (DZB): A dual FGFR/CSF1R-inhibitor active in PDX-models of urothelial cancer

Abstract Background: DZB is an oral small-molecule Fibroblast Growth Factor Receptor 1/2/3 inhibitor (FGFRi) with clinically relevant activity in FGFR2-fusion cholangiocarcinoma. Extensive kinase profiling identified Colony-Stimulating Factor 1 receptor (CSF1R) as an additional anti-cancer target for DZB. CSF1R plays a role in the maintenance of tumor-promoting M2-macrophages; inhibition facilitates repolarization to M1-type thus restoring tumor T cell activity. Screening of urothelial cancer (UC) models both in vitro and in vivo has provided information on potential response biomarkers additional to FGFR genetic aberrations. Methods: Kinase assays used a radiometric assay and anti-proliferative activity was assessed using crystal-violet (72h incubation). Bone-marrow derived mouse macrophages were CSF1 starved (12h), pre-incubated with DZB/BLZ945 (30/10m) and stimulated with 0.3 μM CSF1 (3m). CSF1R phosphorylation (pCSF1R) was analyzed by immunoblotting. Compound docking experiments used MOE software and public X-ray structures. DZB was tested at MTD (75 mg/kg, po, qd) in UC-CDX (8 mice/group) and -PDX (3 mice/group) models with FGFR-mutations and/or differing FGFR copy-number (CN)/RNA-seq expression levels. Efficacy and tolerability were quantified at the 3-week endpoint as a dT/C (treated/control). Results: Comparative kinase IC50s showed that DZB had 1:1 nM activity against FGFR1/2/3 and CSF1R, a potency not observed for other clinically relevant FGFRi’s. Structural analyses suggested a different size of the inhibitor binding-site of FGFR- and CSF1R-structures, with DZB efficiently occupying the smaller CSF1R kinase sub-pocket. Indeed, DZB reduced ligand-stimulated pCSF1R in mouse macrophages in a concentration-dependent manner, with a maximal effect similar to the selective CSF1R inhibitor BLZ945. Based on an in vitro anti-proliferative screen across 14 UC-lines, DZB had a mean GI50 of 1.7±0.2 μM (range 0.4-3.4 μM). The most sensitive lines were RT4 and RT112/84, both of which had FGFR3-TACC3 fusions, a known oncogenic-driver. In mice bearing s.c. RT4 tumors, DZB induced tumor-stasis (dT/C<0.1) and was well tolerated (dT/C>1.0) but no response was observed in the RT112/84 model suggesting that not only FGFR mutations contribute to DZB response. An unbiased UC-PDX screen indicated efficacy in 4/17 models (dT/C≤0.4; median=0.81) with DZB-response significantly positively-associated with high FGFR expression. The most sensitive tumor had high FGFR2 RNA-expression yet low CN. Data will be presented from confirmatory efficacy experiments and bioinformatic analyses. Conclusion: DZB is a potent FGFRi and CSF1R inhibitor. Screens in UC models indicate that DZB efficacy is driven by FGFR mutation and expression and, potentially, CSF1R modulation. A clinical trial is ongoing in UC patients (NCT04045613) to assess DZB monotherapy, and combination with the PD-L1 antibody atezolizumab. Citation Format: Paul McSheehy, Felix Bachmann, Nicole Forster-Gross, Marc Lecoultre, Mahmoud El Shemerly, Mila Roceri, Stefan Reinelt, Laurenz Kellenberger, Paul R Walker, Heidi Lane. Derazantinib (DZB): A dual FGFR/CSF1R-inhibitor active in PDX-models of urothelial cancer [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 LB-C12. doi:10.1158/1535-7163.TARG-19-LB-C12

Abstract B002: Discovery of selective and potent EGFR kinase inhibitors for exon20 insertion mutations

Abstract The third generation EGFR tyrosine kinase inhibitors (TKIs), like Osimertinib, target classical activating EGFR mutations containing T790M resistance mutation. However, the exon 20 insertion (Ex20Ins), the third most common EGFR mutations in entire NSCLC, still lacks its therapeutic option. Recently, Robichaux JP et al., showed that Poziotinib, which had been developed as a second-generation EGFR TKI, had efficacy for the EGFR and Her2 Ex20Ins compared to EGFR T790M. Here, we developed inhibitors, structurally distinct from both Poziotinib and Osimertinib, targeting for the EGFR Ex20Ins mutations as well as EGFR T790M. According to kinase profiling, the inhibitors are highly selective to EGFR, ERBB2 and ERBB4 family. The candidate inhibitors demonstrated great activity (GI50 < 20 nM) in cell growth inhibition of Ba/F3 cells containing EGFR Ex20Ins (D770_N771insSVD, V769_D770insASV, D770_N771insNPG, A763_Y764insFQEA, and H773_V774insNPH), Her2 Ex20Ins (A775_G776insYVMA), or cancer cells containing activating EGFR mutations and T790M resistance mutation (L858R/T790M and Del19/T790M). Finally, in several Ex20Ins patient-derived xenograft models, the candidate inhibitors induced tumor regression as strong as Poziotinib and increased survival rates along with minimal weight loss. These novel small molecule compounds inhibiting EGFR exon 20 insertion mutations will provide future therapeutic options for patients with this molecular subtype of NSCLC. Citation Format: Sunghwan Kim, Younho Lee, Hwan Kim, Juhee Kang, Jiyoon Seok, Kyung-Ah Seo, Jaeyoung Ahn, Hee-Bum Kang, Sun-Hwa Lee, Jung Beom Son, Hwan Geun Choi, Nam Doo Kim. Discovery of selective and potent EGFR kinase inhibitors for exon20 insertion mutations [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 B002. doi:10.1158/1535-7163.TARG-19-B002