Kinase Profiling Research Articles

A tightly controlled Src-YAP signaling axis determines therapeutic response to dasatinib in renal cell carcinoma.

Over the past decade, therapies targeting the VEGF/VEGFR and mTOR pathways have served as the standard of care for the clinical management of renal cell carcinoma (RCC) patients. Albeit promising, these targeted drugs have attained only modest clinical benefits with limited prolonged progression-free survival. Therefore, alternative reasonable and applicable therapeutic approaches should be introduced to improve the clinical outcome of RCC patients.Methods: FDA approved kinase inhibitors were screened to evaluate their abilities to suppress the proliferation of RCC cells. Then, the downstream effector, therapeutic target and signaling pathway of the selected drug were identified by gene expression array, RNAi, kinase profile and rescue verification. Finally, the in vivo effectiveness of the drug was assessed in cell line-based xenograft models and patient-derived xenograft models.Results: In this study, we discovered that dasatinib is a potent agent that can impair RCC cell viability in vitro and decrease tumor growth in vivo. Mechanistically, we improved the understanding of the precise mechanistic role of YAP as a pivotal effector of dasatinib-induced anti-proliferation through Src-JNK-LIMD1-LATS signaling cascade in RCC cells. Meanwhile, our results indicated that the alteration of p-YAP is closely correlated to the growth inhibition caused by dasatinib in sensitive RCC models.Conclusion: Our findings provide evidence that dasatinib may serve as a powerful drug candidate to treat subgroups of RCC patients with hyper-activated Src-YAP signaling axis, and the alteration of p-YAP could serve as a functional response biomarker of dasatinib in RCC.

Design, synthesis and evaluation of (R)-3-(7-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-5-azaspiro[2.4]heptan-5-yl)-3-oxopropanenitrile as a JAK1-selective inhibitor.

Based on (R)-N-methyl-N-(5-azaspiro[2.4]heptan-7-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine as a core scaffold, we identified (R)-3-(7-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-5-azaspiro[2.4]heptan-5-yl)-3-oxopropanenitrile [(R)-6c] as a JAK1 selective inhibitor. The structural design was based on the combination of tofacitinib's 7-deazapurine and 5-azaspiro[2.4]heptan-7-amine. Compound (R)-6c exhibited an IC50 value of 8.5 nM against JAK1 with a selectivity index of 48 over JAK2. To optimize (R)-6c as a lead compound, we performed in vitro ADME, hERG, kinase profiling, and pharmacokinetic tests. Mouse and rat in vivo studies verified that (R)-6c exhibited desired efficacies in CIA and AIA models.

Subtyping Of Triple Negative Breast Carcinoma On The Basis Of RTK Expression.

Background: "Triple-negative breast cancers" (TNBC) comprise a heterogeneous group of about 15% of invasive BCs lacking the expression of estrogen and progesterone receptors (ER, PR) and the expression of HER2 (ERBB2) and are therefore no established candidates for targeted treatment options in BC, i.e., endocrine and anti-HER2 therapy. The aim of the present study was to use gene expression profiling and immunohistochemical (IHC) characterization to identify receptor tyrosine kinase (RTK) profiles that would allow patient stratification for the purposes of target-oriented personalized tumor therapy in TNBC.Methods: Twenty-nine cases of TNBC selected according to routine diagnostic IHC/cytogenetic criteria were examined by reverse transcription polymerase chain reaction (RT-PCR). RTK mRNA expression profiles were generated for a total of 31 tumor-relevant biomarkers, mainly belonging to the IGF- and EGF-receptor families but also including biomarkers related to downstream signaling. Protein expression of selected biomarkers was investigated by IHC.Results: Hierarchical cluster analysis revealed a dichotomous differentiation pattern amongst TNBCs. A significant difference in gene expression was observed for 16 of the 31 RTK-associated tumor relevant biomarkers between the two newly identified TNBC subgroups. The findings were verified at the posttranslational level by the IHC data. The RTKs HER4, IGF-1R and IGF-2R and the hormone receptors ER and PR below the IHC detection limit play a central role in the differentiation of the two TNBC subgroups. Observed survival was reported as Kaplan-Meier estimates and point towards an improved survival of patients with RTK-high with superior three-year survival rate of 100% compared to RTK-low gene signatures with superior three-year survival rate of 60% (log-rank test, p-value = 0.022).Conclusion: Gene-expression and IHC analysis of the EGF and IGF receptor families and biomarkers associated with downstream signaling point to the existence of two distinct TNBC subtypes. The RTKs HER4, IGF-1R, IGF-2R and the hormone receptors ER and PR appear to be of particular importance here. Based on survival analysis the differentiation of TNBC with RTK-high and RTK-low gene signatures seems to be of prognostic relevance. Additionally, correlation analysis of the relationship between RTKs and ER suggests co-regulatory mechanisms that may have potential significance in new therapeutic approaches.

Discovery and characterization of an iminocoumarin scaffold as an inhibitor of MEKK2 (MAP3K2)

The kinase MEKK2 (MAP3K2) activates the MEK5/ERK5 cell signaling pathway and may play an important role in tumor growth and metastasis. Thus, MEKK2 may represent a novel kinase target for cancer. In order to identify inhibitors of MEKK2, we screened a library of compounds using a high throughput MEKK2 intrinsic ATPase enzyme assay. We identified two hits with validated structures and confirmed activity in the primary assay (IC50 values = 322 nM and 7.7 μM) and two orthogonal MEKK2 biochemical assays. Compound 1, the more potent hit, was the subject of further investigation. Limited structure-activity relationship (SAR) studies were performed on this iminocoumarin hit which resulted in ≥20-fold more potent analogs (e.g. 8 and 16 nM IC50). Two analogs had improved selectivity in a 50-member kinase profiling panel compared to the hit. These studies suggested that substitutions around the phenoxy ring of this scaffold can impart improved potency and selectivity for MEKK2. Analog Compound 1s (16 nM IC50) was further verified by external testing to inhibit MEKK2 and MEKK3 with similar potencies. Compound 1s displayed activity in cell-based assays in which it inhibited ERK5 pathway activation in cells and inhibited cell migration in a scratch assay. Thus, we have identified a scaffold that has promising potential to be developed into a highly selective and potent inhibitor of MEKK2. Information from these SAR studies provides specific guidance for the future design of MEKK2 inhibitor probes.

Abstract B182: The novel tyrosine kinase inhibitors UJ26 and UJ30 are active in solid tumor and hematologic cancer cell lines

Abstract Background: Tyrosine kinase (TK) inhibitors represent the class of targeted antitumoral drugs that has been so far more successful. Here, we present two novel TK inhibitors, UJ26 and UJ30, with antitumor activity in preclinical models of solid tumors and hematologic cancers. Materials and Methods: Biochemical kinase profiling was performed (DiscoverX, CA, USA). Cell lines included in the NCI60 panel and lymphoma cell lines were exposed to increasing doses of UJ26, UJ30, and, as control, to the SRC inhibitor dasatinib for 72 hours. Cell proliferation was evaluated by using MTT assay. In vitro synergy was assessed with the Chou-Talalay combination index (CI): synergism (<0.9), additive (0.9-1.1), antagonism/no benefit (> 1.1). DLBCL RI-1 cells (15x106) were s.c. inoculated in NOD-Scid (NOD.CB17-Prkdcscid/NCrHsd) mice. Tumor size was measured on regular basis and drug treatments with UJ26 and UJ30 both given i.p, at the dose of 100mg/kg started when tumors reached 5 mm in diameter (100 mm3). Results: The two novel kinase inhibitors UJ26 and UJ30 presented broad specificity comparable to that of dasatinib, able to inhibit clinically relevant kinases ABL1, EGFR, FLT3, KIT, and MET. UJ26 and UJ30 presented antitumor activity in the NCI60 cell line panel against models of hematologic malignancies and solid tumors, in particular in the K562 leukemia and KM12 colon cancer cell lines (GI50 < 50 nM). Antiproliferative activity of UJ26 and UJ30 was then assessed in 8 B-cell lymphoma cell lines derived from mantle cell lymphoma (MCL) (Jeko1, MAVER1), chronic lymphocytic leukemia (MEC-1, PCL-12), and diffuse large B cell lymphoma (DLBCL) of the activated B cell like (ABC) (RI-1, SU-DHL-2) or of the germinal center B cell (GCB) (VAL, SU-DHL-4). Compounds were active with median IC50s of 600nM (95%C.I. 300-1065nM) and 250nM (95%C.I. 100-1049nM), respectively. Their antitumor activity appeared more potent than dasatinib (median IC50=1325nM, 95%C.I. 100-16625nM), in particular in 3 of the cell lines (MAVER1, SU-DHL-2 and MEC1). UJ26 and UJ30 were evaluated in combination with the BET inhibitor OTX015, exposing the four most sensitive lymphoma cell lines to increasing concentrations of the single compound or of both compounds for 72h. Both the UJ26/OTX015 combination, as well as the UJ30/OTX015 combination, were synergistic in all the cell lines. In vivo experiments showed that UJ26 was very toxic already after the first dose with swelling of the injection area. UJ26-treated mice started to recover after 2 weeks and were not treated any more, and there was no antitumor effect. UJ30 given once every 5 days reduced tumor volume compared to control group (D5, D11, D15; P < 0.05) and was well tolerated. UJ30-treated xenografts were three times smaller than both control and UJ26 groups at the end of the experiment (D15, UJ30 median tumor volume=867, 95%C.I. 600-1267.5). Body weight loss was observed after treatment with compound UJ26 whereas UJ30 showed similar weight growth as in the control group. Experiment was terminated after 2 weeks for vehicle- and UJ26-treated groups (D15) while UJ30-treated mice were sacrificed at day 20. Conclusions: The novel TK inhibitors UJ26 and UJ30 showed preclinical antitumor activity and in particular UJ30 is worth further investigations. CN, AC, FB: co-senior authors. Citation Format: Chiara Tarantelli, Eugenio Gaudio, Andrea Unzue, Pawel Sledz, Hillarie Ekeh, Elena Bernasconi, Filippo Spriano, Cristina Nevado, Amedeo Caflisch, Francesco Bertoni. The novel tyrosine kinase inhibitors UJ26 and UJ30 are active in solid tumor and hematologic cancer cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B182.

Structure-Based Kinase Profiling To Understand the Polypharmacological Behavior of Therapeutic Molecules.

Several drugs elicit their therapeutic efficacy by modulating multiple cellular targets and possess varied polypharmacological actions. The identification of the molecular targets of a potent bioactive molecule is essential in determining its overall polypharmacological profile. Experimental procedures are expensive and time-consuming. Therefore, computational approaches are actively implemented in rational drug discovery. Here, we demonstrate a computational pipeline, based on reverse virtual screening technique using several consensus scoring strategies, and perform structure-based kinase profiling of 12 FDA-approved drugs. This target prediction showed an overall good performance, with an average AU-ROC greater than 0.85 for most drugs, and identified the true targets even at the top 2% cutoff. In contrast, 10 non-kinase binder drugs exhibited lower binding efficiency and appeared in the bottom of ranking list. Subsequently, we validated this pipeline on a potent therapeutic molecule, mahanine, whose polypharmacological profile related to targeting kinases is unknown. Our target-prediction method identified different kinases. Furthermore, we have experimentally validated that mahanine is able to modulate multiple kinases that are involved in cross-talk with different signaling molecules, which thereby exhibits its polypharmacological action. More importantly, in vitro kinase assay exhibited the inhibitory effect of mahanine on two such predicted kinases' (mTOR and VEGFR2) activity, with IC50 values being ∼12 and ∼22 μM, respectively. Next, we generated a comprehensive drug-protein interaction fingerprint that explained the basis of their target selectivity. We observed that it is controlled by variations in kinase conformations followed by significant differences in crucial hydrogen-bond and van der Waals interactions. Such structure-based kinase profiling could provide useful information in revealing the unknown targets of therapeutic molecules from their polypharmacological behavior and would assist in drug discovery.

PF-114 Mesylate, a Novel Third Generation ATP-Competitive BCR-ABL Tyrosine Kinase Inhibitor: First Safety and Efficacy Data from a Phase I Study in Patients with CML with Failure of Prior TKI Therapy

Background: PF-114 mesylate is a novel third generation oral tyrosine kinase inhibitor (TKI) that blocks native and mutated Bcr-Abl isoforms, including the gatekeeper mutant T315I, which is uniformly resistant to all approved first- and second generation TKIs. At the present time extensive pre-clinical studies of PF-114, including mechanism of action, kinase profiling, in vitro and in vivo efficacy, safety pharmacology, ADME, and toxicology studies in mice, rats and dogs have been completed and supported initiation of phase I clinical trial of PF-114 mesylate in patients with chronic or accelerated phase Ph+ CML who are resistant to at least one of the second generation TKIs or intolerant to previous treatment with TKIs or who have T315I mutation in the BCR-ABL gene (NCT02885766).Methods: The trial represents a classical 3+3 design of dose escalation till the maximum tolerated dose (MTD) followed by the expanded cohorts planned for dose(s) below the MTD. The total expected enrollment is 44 patients. Escalating doses of single-agent PF-114 mesylate were administered orally on a continuous once daily (QD) dosing schedule. Treatment continued until disease progression, unacceptable toxicity, consent withdrawal, or death. The primary objective was to study the dose-limiting toxicities (DLTs) occurring in cycle 1 of treatment and determine the MTD. Secondary objectives included safety, anti-CML activity (based on hematologic, cytogenetic, and molecular assessments), pharmacodynamic and pharmacogenetic properties. Adverse events (AEs) were assessed and graded according to NCI-CTCAE v4.03.Results: At data cutoff, 4-dose cohorts - 50 mg, 100 mg, 200 mg and 400 mg have completed cycle 1 and 500 mg cohort has started cycle 1 of treatment. Overall 18 patients (8 males) had been treated at the following QD doses: 50 mg (n = 1), 200 mg (n = 5), 400 mg (n = 11), 500 mg (n = 1), and the MTD has not yet been reached. A total of 7 patients with T315I mutation were included into the trial thus far. Median age was 50.5 years (range, 32-66 years). Median time from diagnostics to treatment was 11.5 years (range, 16-1 years) All patients had baseline Eastern Cooperative Oncology Group performance status 0-1. Patients were heavily pretreated; 9 (50%) had received ≥ 3 prior TKIs, 6 (33%) had received 2 prior TKIs and 3 (17%) patients with T315I had received 1 prior TKI. In 11 patients, treatment is ongoing at doses 200-500 mg QD, with median duration of exposure of 8 months (range, 2-12 months), and 7 patients discontinued (disease progression [n = 5], AEs [n = 2]). Preliminary data suggest PF-114 pharmacokinetics is dose-proportional. At the end of cycle 1 of study therapy, no drug-related adverse events greater than grade 1 in severity were observed in patients treated at the 50 mg, 100 mg and 200 mg dose levels. At the dose of 400 mg QD, a single DLT case of grade 3 erythematous rash was observed. Most common grade 2/3 AEs on 400 mg QD were dermatologic toxicity (4/11), neutropenia (1/11). No deterioration of the ankle-brachial index (ABI), which is being prospectively measured, or vascular occlusive events were observed so far. The patient with recurrent pleural effusions on previous treatment with dasatinib did not reveal effusions after 11 cycle of therapy; the patient with ischemic stroke on previous treatment with nilotinib did not reveal cardio-vascular events after 9 cycles. To date, no SAEs have been reported. Overall, during the period of 3 cycles, major cytogenetic response (CyR) have been obtained in 4 of 11 patients who completed 3 cycles (2 cases of complete CyR in and 2 cases of partial CyR). Of those patients who revealed cytogenetic response two patients have T315I mutation. Pharmacodynamic and pharmacogenetic assessments are underway.Conclusion: PF-114 mesylate exhibits antitumor activity in a heavily pretreated subgroup of patients with resistant forms of CML including cases with T315I mutation. The evaluation of the safety profile continues. The dose escalation stage of the current phase 1 study continues, while the including of patients into the expanded cohorts with doses below the MTD has already started. DisclosuresChelysheva:Fusion Pharma LLC: Consultancy. Nemchenko:Fusion Pharma LLC: Consultancy. Zaritskey:Janssen: Consultancy; Novartis: Consultancy, Speakers Bureau. Shuvaev:Fusion Pharma LLC: Consultancy. Novikov:Fusion Pharma LLC: Employment. Shulgina:Fusion Pharma LLC: Employment. Chilov:Fusion Pharma LLC: Employment, Patents & Royalties.

Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement

SummaryFor kinase inhibitors, intracellular target selectivity is fundamental to pharmacological mechanism. Although a number of acellular techniques have been developed to measure kinase binding or enzymatic inhibition, such approaches can fail to accurately predict engagement in cells. Here we report the application of an energy transfer technique that enabled the first broad-spectrum, equilibrium-based approach to quantitatively profile target occupancy and compound affinity in live cells. Using this method, we performed a selectivity profiling for clinically relevant kinase inhibitors against 178 full-length kinases, and a mechanistic interrogation of the potency offsets observed between cellular and biochemical analysis. For the multikinase inhibitor crizotinib, our approach accurately predicted cellular potency and revealed improved target selectivity compared with biochemical measurements. Due to cellular ATP, a number of putative crizotinib targets are unexpectedly disengaged in live cells at a clinically relevant drug dose.

Kinase profiling of liposarcomas using RNAi and drug screening assays identified druggable targets

BackgroundLiposarcoma, the most common soft tissue tumor, is understudied cancer, and limited progress has been made in the treatment of metastatic disease. The Achilles heel of cancer often is their kinases that are excellent therapeutic targets. However, very limited knowledge exists of therapeutic critical kinase targets in liposarcoma that could be potentially used in disease management.MethodsLarge RNAi and small-molecule tyrosine kinase inhibitor screens were performed against the proliferative capacity of liposarcoma cell lines of different subtypes. Each small molecule inhibitor was either FDA approved or in a clinical trial.ResultsScreening assays identified several previously unrecognized targets including PTK2 and KIT in liposarcoma. We also observed that ponatinib, multi-targeted tyrosine kinase inhibitor, was the most effective drug with anti-growth effects against all cell lines. In vitro assays showed that ponatinib inhibited the clonogenic proliferation of liposarcoma, and this anti-growth effect was associated with apoptosis and cell cycle arrest at the G0/G1 phase as well as a decrease in the KIT signaling pathway. In addition, ponatinib inhibited in vivo growth of liposarcoma in a xenograft model.ConclusionsTwo large-scale kinase screenings identified novel liposarcoma targets and a FDA-approved inhibitor, ponatinib with clear anti-liposarcoma activity highlighting its potential therapy for treatment of this deadly tumor.

Genome-wide analysis and expression profiling of calcium-dependent protein kinases in potato (Solanum tuberosum)

Calcium-dependent protein kinases (CDPKs or CPKs), unique to plants and some protists, are involved in growth and developmental processes as well as in defence against diverse environmental stresses. CDPKs are encoded by multi-gene families. Despite extensive studies of the CDPKs in many species, information about the evolutionary history and expression patterns of the CDPK family in the staple crop potato (Solanum tuberosum) remains poorly known. In this study, we performed bioinformatics analysis of the potato whole genome sequence and identified 23 potential CDPK genes. These genes are located in 11, of 12, potato chromosomes. Based on the phylogenetic tree and gene structures, the CDPKs were divided into four subfamilies. To determine their expression, reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis was carried out for the CDPK genes in different organs of potato such as young and mature leaves, stems, young shoots, roots, stolons, swollen stolons, flowers and tubers. The CDPKs were expressed in all the organs analysed, but their expression patterns varied greatly. The expression of some CDPKs was strongly organ specific, for example StCPK13 and StCPK18 was found only/mostly in flowers. In Solanum genotypes differing in resistance to Phytophthora infestans, the expression and activity of CDPKs increased in response to a P. infestans elicitor with different kinetics and intensity. The expression levels and activity of the CDPKs correlated positively with the level of the resistance. Our results support earlier suggestion that CDPKs are involved in potato organ development and defence against stresses. We provide new information about the CDPK gene family in the potato and a perspective on its evolutionary history and biological roles of the individual kinases.

Coumestrol Epigenetically Suppresses Cancer Cell Proliferation: Coumestrol Is a Natural Haspin Kinase Inhibitor.

Targeting epigenetic changes in gene expression in cancer cells may offer new strategies for the development of selective cancer therapies. In the present study, we investigated coumestrol, a natural compound exhibiting broad anti-cancer effects against skin melanoma, lung cancer and colon cancer cell growth. Haspin kinase was identified as a direct target protein of coumestrol using kinase profiling analysis. Histone H3 is a direct substrate of haspin kinase. We observed haspin kinase overexpression as well as greater phosphorylation of histone H3 at threonine 3 (Thr-3) in the cancer cells compared to normal cells. Computer modeling using the Schrödinger Suite program identified the binding interface within the ATP binding site. These findings suggest that the anti-cancer effect of coumestrol is due to the direct targeting of haspin kinase. Coumestrol has considerable potential for further development as a novel anti-cancer agent.

Identification of 4-phenylquinolin-2(1H)-one as a specific allosteric inhibitor of Akt

Akt plays a major role in tumorigenesis and the development of specific Akt inhibitors as effective cancer therapeutics has been challenging. Here, we report the identification of a highly specific allosteric inhibitor of Akt through a FRET-based high-throughput screening, and characterization of its inhibitory mechanism. Out of 373,868 compounds screened, 4-phenylquinolin-2(1H)-one specifically decreased Akt phosphorylation at both T308 and S473, and inhibited Akt kinase activity (IC50 = 6 µM) and downstream signaling. 4-Phenylquinolin-2(1H)-one did not alter the activity of upstream kinases including PI3K, PDK1, and mTORC2 as well as closely related kinases that affect cell proliferation and survival such as SGK1, PKA, PKC, or ERK1/2. This compound inhibited the proliferation of cancer cells but displayed less toxicity compared to inhibitors of PI3K or mTOR. Kinase profiling efforts revealed that 4-phenylquinolin-2(1H)-one does not bind to the kinase active site of over 380 human kinases including Akt. However, 4-phenylquinolin-2(1H)-one interacted with the PH domain of Akt, apparently inducing a conformation that hinders S473 and T308 phosphorylation by mTORC2 and PDK1. In conclusion, we demonstrate that 4-phenylquinolin-2(1H)-one is an exquisitely selective Akt inhibitor with a distinctive molecular mechanism, and a promising lead compound for further optimization toward the development of novel cancer therapeutics.

Synthesis and biological activity of imidazo[4,5-c]quinoline derivatives as PI3K/mTOR inhibitors

A series of imidazo[4,5-c]quinoline derivatives(12a—12m) was synthesized with 2-amino-5-bromobenzoic acid and 4-nitrophenylacetonitrile as starting materials, 6-bromo-4-chloro-3-nitroquinoline as intermediate and Suzuki reaction and closure of the imidazolinone ring with triphosgene as key steps. The structures of the key intermediate and target compounds were confirmed by means of 1H NMR, 13C NMR and HRMS. These compounds show an interesting kinase profile as dual PI3K/mTOR tool compounds.

SKLB188 inhibits the growth of head and neck squamous cell carcinoma by suppressing EGFR signalling

Background:Overexpression of epidermal growth factor receptor (EGFR) occurs in approximately 90% of head and neck squamous cell carcinoma (HNSCC), and is correlated with poor prognosis. Thus, targeting EGFR is a promising strategy for treatment of HNSCC. Several small molecule EGFR inhibitors have been tested in clinical trials for treatment of HNSCC, but none of them are more effective than the current chemotherapeutic drugs. Thus, it is urgently needed to develop novel EGFR inhibitors for HNSCC treatment.Methods:By screening an in-house focused library containing approximately 650 000 known kinase inhibitors and kinase inhibitor-like compounds containing common kinase inhibitor core scaffolds, we identified SKLB188 as a lead compound for inhibition of EGFR. The anticancer effects of SKLB188 on HNSCC cells were investigated by in vitro cell growth, cell cycle and apoptosis assays, as well as in vivo FaDu xenograft mouse model. Molecular docking, in vitro kinase profiling and western blotting were performed to characterise EGFR as the molecular target.Results:SKLB188 inhibited HNSCC cell proliferation by inducing G1 cell cycle arrest, which was associated with downregulating the expression of Cdc25A, cyclins D1/A and cyclin-dependent kinases (CDK2/4), and upregulating the expression of cyclin-dependent kinase (CDK) inhibitors (p21Cip1 and p27Kip1), leading to decreased phosphorylation of Rb. SKLB188 also induced caspase-dependent apoptosis of HNSCC cells by downregulating the expression of Mcl-1 and survivin. Molecular docking revealed that SKLB188 could bind to the kinase domain of EGFR through hydrogen bonds and hydrophobic interactions. In vitro kinase assay showed that SKLB188 inhibited the activity of a recombinant human EGFR very potently (IC50=5 nM). Western blot analysis demonstrated that SKLB188 inhibited the phosphorylation of EGFR and its downstream targets, extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) and Akt in the cells. In addition, SKLB188 dose-dependently inhibited FaDu xenograft growth in nude mice, and concurrently inhibited the phosphorylation of Erk1/2 and Akt in the tumours.Conclusions:SKLB188 potently inhibits the growth of HNSCC cells in vitro and in vivo by targeting EGFR signalling. The results provide a basis for further clinical investigation of SKLB188 as a targeted therapy for HNSCC. Our findings may open a new avenue for development of novel EGFR inhibitors for treatment of HNSCC and other cancers.

Abstract 7: Ceftriaxone, an FDA-approved cephalosporin antibiotic, suppresses lung cancer growth by targeting Aurora B

Abstract Ceftriaxone, an FDA-approved third-generation cephalosporin antibiotic, has antimicrobial activity against both gram-positive and gram-negative organisms. Generally, ceftriaxone is used for a variety of infections such as community-acquired pneumonia, meningitis and gonorrhea. Its primary molecular targets are the penicillin-binding proteins. However, other activities of ceftriaxone remain unknown. Herein, we report for the first time that ceftriaxone has antitumor activity in vitro and in vivo. Kinase profiling results predicted that Aurora B might be a potential ‘off’ target of ceftriaxone. Pull-down assay data confirmed that ceftriaxone could bind with Aurora B in vitro and in A549 cells. Furthermore, ceftriaxone (500 µM) suppressed anchorage-independent cell growth by targeting Aurora B in A549, H520 and H1650 lung cancer cells. Importantly, in vivo xenograft animal model results showed that ceftriaxone effectively suppressed A549 and H520 lung tumor growth by inhibiting Aurora B. These data suggest the anticancer efficacy of ceftriaxone for the treatment of lung cancers through its inhibition of Aurora B. Citation Format: Xiang Li, Haitao Li, Shengqing Li, Feng Zhu, Dong Joon Kim, Hua Xie, Yan Li, Janos Nadas, Naomi Oi, Tatyana Zykova, Dong Hoon Yu, Mee-Hyun Lee, Myoung Ok Kim, Lei Wang, Wei Ya Ma, Ronald A. Lubet, Ann M. Bode, Ziming Dong, Zigang Dong. Ceftriaxone, an FDA-approved cephalosporin antibiotic, suppresses lung cancer growth by targeting Aurora B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 7. doi:10.1158/1538-7445.AM2017-7

Abstract 221: Integrated functional proteomics of MET/VEGFR inhibitors reveals complex mechanism of action of foretinib in NSCLC

Abstract BACKGROUND: Foretinib (FORE) and cabozantinib (CABO) are two MET/VEGFR inhibitors with similar chemical structures. CABO is FDA-approved for medullary thyroid and renal cancer; in addition, it is in clinical trials for treatment of non-small cell lung cancer (NSCLC). Through an unbiased viability screen we have observed potent cellular activity of FORE, but not CABO, in several NSCLC cell lines. We have previously shown that most NSCLC cell lines are insensitive to MET or VEGFR inhibition, suggesting off-target activity of FORE in these cells. The aim of this project is to identify the mechanism of action of FORE in NSCLC and design an optimized combination therapy. METHODS: Cellular viability assays were done using CellTiter-Glo, cell cycle analysis by flow cytometry. Western blotting was performed to evaluate the induction of apoptosis through PARP1 and caspase cleavage, as well changes in signaling. We synthesized FORE and CABO analogues and performed differential quantitative chemical and phosphoproteomics to determine the target kinase profile and pathway effects in NSCLC cells. Changes in gene expression upon drug treatment were measured by RNA-seq. RNAi in combination with pharmacological inhibitors was performed to interrogate targets and pathways. RESULTS: FORE showed greater potency in NSCLC cell lines than CABO with regard to inhibition of viability and induction of apoptosis. FORE decreased phosphorylation of AKT and ERK. Chemical and phosphoproteomics revealed several kinases, such as MEK and MAP4K5, to bind preferentially by FORE over CABO that differentially affect the adherens junction and MAPK signaling pathways. Target validation showed differential inhibition of MEK1/2 and MAP4K5. Cellular validation with RNAi in combination with pharmacological inhibitors suggested that MEK1/2, MAP4K5 and IGF1R are involved in the mechanism of action of foretinib in NSCLC cells. RNA-seq pathway analysis furthermore suggested regulation of chromatin organization and Wnt pathway signaling by foretinib. CONCLUSION: Our results suggest that the difference in the efficacy between FORE and CABO is related to polypharmacology of FORE, which simultaneously targets IGF1R, MEK1/2 and MAP4K5. This difference results in divergence in signaling pathway inhibition and induces distinct effects in NSCLC. The establishment of FORE targets and signaling pathways can lead to optimized combination therapy for NSCLC and identification of new actionable kinases in lung cancer cells. Citation Format: Natalia J. Sumi, Bin Fang, Lily L. Rix, Muhammad Ayaz, Fumi Kinose, Eric A. Welsh, Steven A. Eschrich, Harshani R. Lawrence, John M. Koomen, Eric B. Haura, Uwe Rix. Integrated functional proteomics of MET/VEGFR inhibitors reveals complex mechanism of action of foretinib in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 221. doi:10.1158/1538-7445.AM2017-221

Abstract 4141: Phosphoproteome networks display consistent hyperactive kinase activity in pancreatic cancer: evidence for new therapeutic options

Abstract Introduction Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease due to its aggressive nature. Patients typically present with distant metastases, at which point cytotoxic agents can extend life expectancy by several months at most. Large-scale phosphoproteomics complements our knowledge obtained from genomics and transcriptomics as it provides information on which proteins and kinases are phosphorylated, thereby implicating pathways that are activated. This approach in cancer research may lead to improved patient selection for treatment with tyrosine kinase inhibitors (TKI). This study is the first to employ phosphotyrosine-based phosphoproteomics on three different preclinical PDAC models as well as patient tumor tissues to understand the aggressive nature of this disease and identify new drug targets. Approach We performed phosphoproteomics on a panel of 11 PDAC cell lines, 7 primary cell cultures, 10 patient-derived xenografts (PDX) and 16 fresh frozen human tumor tissues. Tyrosine phosphopeptides were enriched via immunoprecipitation and phosphopeptides were analyzed by high-resolution nano-LC mass spectrometry. Results Using phosphotyrosine-based phosphoproteomics, we identified a total of 1723 tyrosine phosphorylated proteins and 138 phosphorylated kinases, representing 27% of the kinome. The reproducibility of our workflow was very high, with Pearson correlation coefficients of r = 0.937 for technical replicates of cell lines and r = 0.876 for biological replicates of tumors. In our cell line panels, multiple kinases were commonly highly phosphorylated (e.g. PTK2, EPHA2, EGFR and MET). Functional testing of PTK2 by using TKI defactinib in primary cell lines with high phosphorylation resulted in inhibition of proliferation and migration in vitro. Inhibition of EPHA2 by shRNAs resulted in reduced proliferation in vitro. To validate the relevance of these candidate target proteins in vivo, the tyrosine phosphoproteome of PDXs and human tumors was analyzed. In these tumors, kinase activity analysis based on kinase phosphorylation levels and kinase-substrate networks validated these common active nodes in the majority of these tumors. Conclusion Our extensive tyrosine phosphoproteome analysis spanning a wide range of PDAC models revealed high phosphorylation levels of multiple kinases. Interestingly, the phosphorylated kinase profiles of tumors and cell lines did not show as much heterogeneity as expected, taken into account the existence of biological subtypes in PDAC identified by others via transcriptomics. The aggressive biology of this disease may be correlated with the consistent activation of multiple pathways, some of which we have shown to be targetable in vitro. This study prompts further validation and prognostic evaluation of the identified active kinases to improve treatment of PDAC. Citation Format: Tessa Y. Le Large, Maarten F. Bijlsma, Btissame El Hassouni, Nicolla Funel, Nicole C. van Grieken, Helene Damhofer, Jaco C. Knol, Sander R. Piersma, Thang V. Pham, Henk M. Verheul, Hanneke W. van Laarhoven, Geert Kazemier, Elisa Giovannetti, Connie R. Jimenez. Phosphoproteome networks display consistent hyperactive kinase activity in pancreatic cancer: evidence for new therapeutic options [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4141. doi:10.1158/1538-7445.AM2017-4141

Kinome-Wide Profiling Prediction of Small Molecules.

Extensive kinase profiling data, covering more than half of the human kinome, are available nowadays and allow the construction of activity prediction models of high practical utility. Proteochemometric (PCM) approaches use compound and protein descriptors, which enables the extrapolation of bioactivity values to thus far unexplored kinases. In this study, the potential of PCM to make large-scale predictions on the entire kinome is explored, considering the applicability on novel compounds and kinases, including clinically relevant mutants. A rigorous validation indicates high predictive power on left-out kinases and superiority over individual kinase QSAR models for new compounds. Furthermore, external validation on clinically relevant mutant kinases reveals an excellent predictive power for mutations spread across the ATP binding site.

Human Papillomavirus Regulates HER3 Expression in Head and Neck Cancer: Implications for Targeted HER3 Therapy in HPV+ Patients.

Purpose: Human papillomavirus (HPV) 16 plays an etiologic role in a growing subset of head and neck squamous cell carcinomas (HNSCC), where viral expression of the E6 and E7 oncoproteins is necessary for tumor growth and maintenance. Although patients with HPV+ tumors have a more favorable prognosis, there are currently no HPV-selective therapies. Recent studies identified differential receptor tyrosine kinase (RTK) profiles in HPV+ versus HPV- tumors. One such RTK, HER3, is overexpressed and interacts with phosphoinositide-3-kinase (PI3K) in HPV+ tumors. Therefore, we investigated the role of HPV oncoproteins in regulating HER3-mediated signaling and determined whether HER3 could be a molecular target in HPV+ HNSCC.Experimental Design: HER3 was investigated as a molecular target in HPV+ HNSCC using established cell lines, patient-derived xenografts (PDX), and human tumor specimens. A mechanistic link between HPV and HER3 was examined by augmenting E6 and E7 expression levels in HNSCC cell lines. The dependency of HPV+ and HPV- HNSCC models on HER3 was evaluated with anti-HER3 siRNAs and the clinical stage anti-HER3 monoclonal antibody KTN3379.Results: HER3 was overexpressed in HPV+ HNSCC, where it was associated with worse overall survival in patients with pharyngeal cancer. Further investigation indicated that E6 and E7 regulated HER3 protein expression and downstream PI3K pathway signaling. Targeting HER3 with siRNAs or KTN3379 significantly inhibited the growth of HPV+ cell lines and PDXs.Conclusions: This study uncovers a direct relationship between HPV infection and HER3 in HNSCC and provides a rationale for the clinical evaluation of targeted HER3 therapy for the treatment of HPV+ patients. Clin Cancer Res; 23(12); 3072-83. ©2016 AACR.

Tetrandrine, an Activator of Autophagy, Induces Autophagic Cell Death via PKC-α Inhibition and mTOR-Dependent Mechanisms.

Emerging evidence suggests the therapeutic role of autophagic modulators in cancer therapy. This study aims to identify novel traditional Chinese medicinal herbs as potential anti-tumor agents through autophagic induction, which finally lead to autophagy mediated-cell death in apoptosis-resistant cancer cells. Using bioactivity-guided purification, we identified tetrandrine (Tet) from herbal plant, Radix stephaniae tetrandrae, as an inducer of autophagy. Across a number of cancer cell lines, we found that breast cancer cells treated with tetrandrine show an increase autophagic flux and formation of autophagosomes. In addition, tetrandrine induces cell death in a panel of apoptosis-resistant cell lines that are deficient for caspase 3, caspase 7, caspase 3 and 7, or Bax-Bak respectively. We also showed that tetrandrine-induced cell death is independent of necrotic cell death. Mechanistically, tetrandrine induces autophagy that depends on mTOR inactivation. Furthermore, tetrandrine induces autophagy in a calcium/calmodulin-dependent protein kinase kinase-β (CaMKK-β), 5′ AMP-activated protein kinase (AMPK) independent manner. Finally, by kinase profiling against 300 WT kinases and computational molecular docking analysis, we showed that tetrandrine is a novel PKC-α inhibitor, which lead to autophagic induction through PKC-α inactivation. This study provides detailed insights into the novel cytotoxic mechanism of an anti-tumor compound originated from the herbal plant, which may be useful in promoting autophagy mediated- cell death in cancer cell that is resistant to apoptosis.