Discovery of YAP1/TAZ pathway inhibitors through phenotypic screening with potent anti-tumor activity via blockade of Rho-GTPase signaling.

This study describes the identification and target deconvolution of small molecule inhibitors of oncogenic Yes-associated protein (YAP1)/TAZ activity with potent anti-tumor activity in vivo. A high-throughput screen (HTS) of 3.8 million compounds was conducted using a cellular YAP1/TAZ reporter assay. Target deconvolution studies identified the geranylgeranyltransferase-I (GGTase-I) complex as the direct target of YAP1/TAZ pathway inhibitors. The small molecule inhibitors block the activation of Rho-GTPases, leading to subsequent inactivation of YAP1/TAZ and inhibition of cancer cell proliferation in vitro. Multi-parameter optimization resulted in BAY-593, an in vivo probe with favorable PK properties, which demonstrated anti-tumor activity and blockade of YAP1/TAZ signaling in vivo.

Safety and activity of anti-mesothelin antibody-drug conjugate anetumab ravtansine in combination with pegylated-liposomal doxorubicin in platinum-resistant ovarian cancer: multicenter, phase Ib dose escalation and expansion study.

OBJECTIVES: Anetumab ravtansine is an antibody-drug conjugate consisting of a fully human anti-mesothelin monoclonal antibody conjugated to cytotoxic maytansinoid tubulin inhibitor DM4. Mesothelin is highly expressed in ovarian cancer. This phase Ib study determines the safety, pharmacokinetics, and anti-tumor activity of anetumab ravtansine and pegylated liposomal doxorubicin in mesothelin-expressing platinum-resistant ovarian cancer. METHODS: Anetumab ravtansine (5.5 or 6.5 mg/kg) and pegylated liposomal doxorubicin (30 mg/m2) were administered intravenously every 3 weeks to 65 patients with platinum-resistant epithelial ovarian cancer. Mesothelin expression was assessed by central immunohistochemistry. Adverse events, tumor response (RECIST 1.1), and progression-free survival were determined. Biomarker samples were assessed by ELISA and next-generation sequencing. RESULTS: In dose escalation, nine patients received anetumab ravtansine across two doses (5.5 or 6.5 mg/kg). The maximum tolerated dose of anetumab ravtansine was 6.5 mg/kg every 3 weeks and no dose-limiting toxicities were observed. In dose expansion, 56 patients were treated at the maximum tolerated dose. The most common treatment-emergent adverse events of any grade were nausea (47.7%), decreased appetite (43.1%), fatigue (38.5%), diarrhea (32.3%), and corneal disorder (29.2%). In all treated patients the objective response rate was 27.7% (95% CI 17.3% to 40.2%), including one complete (1.5%) and 17 partial responses (26.2%), with median duration of response of 7.6 (95% CI 3.3 to 10.2) months and median progression-free survival of 5.0 (95% CI 3.2 to 6.0) months. In an exploratory analysis of a sub-set of patients (n=19) with high mesothelin expression who received ≤3 prior lines of systemic therapy, the objective response rate was 42.1% (95% CI 20.3% to 66.5%) with a median duration of response of 8.3 (95% CI 4.1 to 12.0) months and median progression-free survival of 8.5 (95% CI 4.0 to 11.4) months. CONCLUSIONS: Anetumab ravtansine and pegylated liposomal doxorubicin showed tolerability and promising clinical activity. These results established the dose schedule and the mesothelin-positive target population of this combination for a phase III study in platinum-resistant ovarian cancer. TRIAL REGISTRATION NUMBER: NCT02751918.

Anetumab ravtansine versus vinorelbine in patients with relapsed, mesothelin-positive malignant pleural mesothelioma (ARCS-M): a randomised, open-label phase 2 trial.

BACKGROUND: Few treatment options exist for second-line treatment of malignant pleural mesothelioma. We aimed to assess the antibody-drug conjugate anetumab ravtansine versus vinorelbine in patients with unresectable locally advanced or metastatic disease overexpressing mesothelin who had progressed on first-line platinum-pemetrexed chemotherapy with or without bevacizumab. METHODS: In this phase 2, randomised, open-label study, done at 76 hospitals in 14 countries, we enrolled adults (aged ≥18 years) with unresectable locally advanced or metastatic malignant pleural mesothelioma, an Eastern Cooperative Oncology Group performance status of 0-1, and who had progressed on first-line platinum-pemetrexed chemotherapy with or without bevacizumab. Participants were prospectively screened for mesothelin overexpression (defined as 2+ or 3+ mesothelin membrane staining intensity on at least 30% of viable tumour cells by immunohistochemistry) and were randomly assigned (2:1), using an interactive voice and web response system provided by the sponsor, to receive intravenous anetumab ravtansine (6·5 mg/kg on day 1 of each 21-day cycle) or intravenous vinorelbine (30 mg/m2 once every week) until progression, toxicity, or death. The primary endpoint was progression-free survival according to blinded central radiology review, assessed in the intention-to-treat population, with safety assessed in all participants who received any study treatment. This study is registered with ClinicalTrials.gov, NCT02610140, and is now completed. FINDINGS: Between Dec 3, 2015, and May 31, 2017, 589 patients were enrolled and 248 mesothelin-overexpressing patients were randomly allocated to the two treatment groups (166 patients were randomly assigned to receive anetumab ravtansine and 82 patients were randomly assigned to receive vinorelbine). 105 (63%) of 166 patients treated with anetumab ravtansine (median follow-up 4·0 months [IQR 1·4-5·5]) versus 43 (52%) of 82 patients treated with vinorelbine (3·9 months [1·4-5·4]) had disease progression or died (median progression-free survival 4·3 months [95% CI 4·1-5·2] vs 4·5 months [4·1-5·8]; hazard ratio 1·22 [0·85-1·74]; log-rank p=0·86). The most common grade 3 or worse adverse events were neutropenia (one [1%] of 163 patients for anetumab ravtansine vs 28 [39%] of 72 patients for vinorelbine), pneumonia (seven [4%] vs five [7%]), neutrophil count decrease (two [1%] vs 12 [17%]), and dyspnoea (nine [6%] vs three [4%]). Serious drug-related treatment-emergent adverse events occurred in 12 (7%) patients treated with anetumab ravtansine and 11 (15%) patients treated with vinorelbine. Ten (6%) treatment-emergent deaths occurred with anetumab ravtansine: pneumonia (three [2%]), dyspnoea (two [1%]), sepsis (two [1%]), atrial fibrillation (one [1%]), physical deterioration (one [1%]), hepatic failure (one [1%]), mesothelioma (one [1%]), and renal failure (one [1%]; one patient had 3 events). One (1%) treatment-emergent death occurred in the vinorelbine group (pneumonia). INTERPRETATION: Anetumab ravtansine showed a manageable safety profile and was not superior to vinorelbine. Further studies are needed to define active treatments in relapsed mesothelin-expressing malignant pleural mesothelioma. FUNDING: Bayer Healthcare Pharmaceuticals.

Prognosis of patients with malignant mesothelioma by expression of programmed cell death 1 ligand 1 and mesothelin in a contemporary cohort in Finland.

OBJECTIVES: We aimed to describe mesothelin (MSLN) and programmed cell death 1 ligand 1 (PD-L1) tumour overexpression amongst patients with malignant mesothelioma (MM), and their associations with survival, amongst a cohort of patients with MM in Finland. METHODS: Between 2004 and 2017, 91 adults with histologically confirmed MM were identified from the Auria Biobank in Finland and followed-up using linked data from electronic health records and national statistics. Biomarker content in tumour cell membranes was determined using automated Immunohistochemistry on histological sections. Stained tumour sections were scored for MSLN and PD-L1 intensity. Adjusted associations between MSLN/PD-L1 co-expression and mortality were evaluated by estimating hazard ratios (HRs) with 95% confidence intervals (CIs) using Cox regression. RESULTS: Biomarker overexpression occurred in 52 patients for MSLN and 34 patients for PD-L1 and was associated with tumour histology and certain comorbidities. Fifteen per cent of patients had a tumour that overexpressed both biomarkers; r =-0.244, p-value: 0.02. Compared with MSLN+/PD-L1+ patients, HRs (95% CIs) for death were 4.18 (1.71-10.23) for MSLN-/PD-L1+ patients, 3.03 (1.35-6.77) for MSLN-/PD-L1- patients, and 2.13 (0.97-4.67) for MSLN+/PD-L1- patients. CONCLUSIONS: Both MSLN and PD-L1 markers were independent prognostic indicators in patients with MM. Overexpression of MSLN was associated with longer survival; yet their combined expression gave a better indication of survival. The risk of death was four times higher amongst MSLN-/PD-L1+ patients than in MSLN+/PD-L1+ patients.

First-in-Human, Multicenter, Phase I Dose-Escalation and Expansion Study of Anti-Mesothelin Antibody-Drug Conjugate Anetumab Ravtansine in Advanced or Metastatic Solid Tumors.

PURPOSE: This phase I study, which to our knowledge is the first-in-human study of this kind, investigates the safety, tolerability, pharmacokinetics, and clinical activity of anetumab ravtansine, an antibody-drug conjugate of anti-mesothelin antibody linked to maytansinoid DM4, in patients with advanced, metastatic, or recurrent solid tumors known to express the tumor-differentiation antigen mesothelin. PATIENTS AND METHODS: This phase I, open-label, multicenter, dose-escalation and dose-expansion study of anetumab ravtansine enrolled 148 adult patients with multiple solid tumor types. Ten dose-escalation cohorts of patients with advanced or metastatic solid tumors (0.15-7.5 mg/kg) received anetumab ravtansine once every 3 weeks, and 6 expansion cohorts of patients with advanced, recurrent ovarian cancer or malignant mesothelioma received anetumab ravtansine at the maximum tolerated dose once every 3 weeks, 1.8 mg/kg once per week, and 2.2 mg/kg once per week. RESULTS: Forty-five patients were enrolled across the 10 dose-escalation cohorts. The maximum tolerated dose of anetumab ravtansine was 6.5 mg/kg once every 3 weeks or 2.2 mg/kg once per week. Thirty-two patients were enrolled in the 6.5 mg/kg once-every-3-weeks, 35 in the 1.8 mg/kg once-per-week, and 36 in the 2.2 mg/kg once-per-week expansion cohorts. The most common drug-related adverse events were fatigue, nausea, diarrhea, anorexia, vomiting, peripheral sensory neuropathy, and keratitis/keratopathy. There were no drug-related deaths. Anetumab ravtansine pharmacokinetics were dose proportional; the average half-life was 5.5 days. Among 148 patients with mesothelioma or ovarian, pancreatic, non-small-cell lung, and breast cancers, 1 had a complete response, 11 had partial responses, and 66 had stable disease. High levels of tumor mesothelin expression were detected in patients with clinical activity. CONCLUSION: Anetumab ravtansine exhibited a manageable safety and favorable pharmacokinetic profile with encouraging preliminary antitumor activity in heavily pretreated patients with mesothelin-expressing solid tumors. The results allowed for the determination of recommended doses, schedules, and patient populations for anetumab ravtansine in phase II studies.

First-in-human phase I study of the bromodomain and extraterminal motif inhibitor BAY 1238097: emerging pharmacokinetic/pharmacodynamic relationship and early termination due to unexpected toxicity.

BACKGROUND: Bromodomain and extraterminal motif (BET) protein inhibition is a promising cancer treatment strategy, notably for targeting MYC- or BRD4-driven diseases. A first-in-human study investigated the safety, pharmacokinetics, maximum tolerated dose and recommended phase II dose of the BET inhibitor BAY 1238097 in patients with advanced malignancies. MATERIAL AND METHODS: In this phase I, open-label, non-randomised, multicentre study, patients with cytologically or histologically confirmed advanced refractory malignancies received oral BAY 1238097 twice weekly in 21-day cycles using an adaptive dose-escalation design at a starting dose of 10 mg/week. Model-based dose-response analysis was performed to guide dose escalation. Safety, pharmacokinetics, pharmacodynamics and tumour response were evaluated. RESULTS: Eight patients were enrolled at three dose levels (10 mg/week, n = 3; 40 mg/week, n = 3; 80 mg/week, n = 2). Both patients receiving 80 mg/week had dose-limiting toxicities (DLTs) (grade 3 vomiting, grade 3 headache and grade 2/3 back pain). The most common adverse events were nausea, vomiting, headache, back pain and fatigue. Pharmacokinetic analysis indicated a linear dose response with increasing dose. Two patients displayed prolonged stable disease; no responses were observed. Biomarker evaluation of MYC and HEXIM1 expression demonstrated an emerging pharmacokinetic/pharmacodynamic relationship, with a trend towards decreased MYC and increased HEXIM1 expression in response to treatment. CONCLUSION: The study was prematurely terminated because of the occurrence of DLTs at a dose below targeted drug exposure. Pharmacokinetic modelling indicated that an alternate dosing schedule whereby DLTs could be avoided while reaching efficacious exposure was not feasible. Registration number: NCT02369029.

Phase I dose-escalation studies of roniciclib, a pan-cyclin-dependent kinase inhibitor, in advanced malignancies.

BACKGROUND: To evaluate safety, pharmacokinetics, and maximum tolerated dose of roniciclib in patients with advanced malignancies, with dose expansion to evaluate clinical benefit at the recommended phase II dose (RP2D). METHODS: Two phase I dose-escalation studies evaluated two roniciclib dosing schedules: 3 days on/4 days off or 4 weeks on/2 weeks off. The expansion phase included patients with small-cell lung cancer (SCLC), ovarian cancer, or tumour mutations involving the CDK signalling pathway. RESULTS: Ten patients were evaluable in the 4 weeks on/2 weeks off schedule (terminated following limited tolerability) and 47 in the 3 days on/4 days off schedule dose-escalation cohorts. On the 3 days on/4 days off schedule, RP2D was 5 mg twice daily in solid tumours (n=40); undetermined in lymphoid malignancies (n=7). Common roniciclib-related adverse events included nausea (76.6%), fatigue (65.8%), diarrhoea (63.1%), and vomiting (57.7%). Roniciclib demonstrated rapid absorption and dose-proportional increase in exposure. One partial response (1.0%) was observed. In RP2D expansion cohorts, the disease control rate (DCR) was 40.9% for patients with ovarian cancer (n=25), 17.4% for patients with SCLC (n=33), and 33.3% for patients with CDK-related tumour mutations (n=6). CONCLUSIONS: Roniciclib demonstrated an acceptable safety profile and moderate DCR in 3 days on/4 days off schedule.

In vitro and in vivo characterization of irreversible mutant-selective EGFR inhibitors that are wild-type sparing.

Patients with non-small cell lung carcinoma (NSCLC) with activating mutations in epidermal growth factor receptor (EGFR) initially respond well to the EGFR inhibitors erlotinib and gefitinib. However, all patients relapse because of the emergence of drug-resistant mutations, with T790M mutations accounting for approximately 60% of all resistance. Second-generation irreversible EGFR inhibitors are effective against T790M mutations in vitro, but retain affinity for wild-type EGFR (EGFR(WT)). These inhibitors have not provided compelling clinical benefit in T790M-positive patients, apparently because of dose-limiting toxicities associated with inhibition of EGFR(WT). Thus, there is an urgent clinical need for therapeutics that overcome T790M drug resistance while sparing EGFR(WT). Here, we describe a lead optimization program that led to the discovery of four potent irreversible 2,4-diaminopyrimidine compounds that are EGFR mutant (EGFR(mut)) selective and have been designed to have low affinity for EGFR(WT). Pharmacokinetic and pharmacodynamic studies in H1975 tumor-bearing mice showed that exposure was dose proportional resulting in dose-dependent EGFR modulation. Importantly, evaluation of normal lung tissue from the same animals showed no inhibition of EGFR(WT). Of all the compounds tested, compound 3 displayed the best efficacy in EGFR(L858R/T790M)-driven tumors. Compound 3, now renamed CO-1686, is currently in a phase I/II clinical trial in patients with EGFR(mut)-advanced NSCLC that have received prior EGFR-directed therapy.

Discovery of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC.

UNLABELLED: Patients with non-small cell lung cancer (NSCLC) with activating EGF receptor (EGFR) mutations initially respond to first-generation reversible EGFR tyrosine kinase inhibitors. However, clinical efficacy is limited by acquired resistance, frequently driven by the EGFR(T790M) mutation. CO-1686 is a novel, irreversible, and orally delivered kinase inhibitor that specifically targets the mutant forms of EGFR, including T790M, while exhibiting minimal activity toward the wild-type (WT) receptor. Oral administration of CO-1686 as single agent induces tumor regression in EGFR-mutated NSCLC tumor xenograft and transgenic models. Minimal activity of CO-1686 against the WT EGFR receptor was observed. In NSCLC cells with acquired resistance to CO-1686 in vitro, there was no evidence of additional mutations or amplification of the EGFR gene, but resistant cells exhibited signs of epithelial-mesenchymal transition and demonstrated increased sensitivity to AKT inhibitors. These results suggest that CO-1686 may offer a novel therapeutic option for patients with mutant EGFR NSCLC. SIGNIFICANCE: We report the preclinical development of a novel covalent inhibitor, CO-1686, that irreversibly and selectively inhibits mutant EGFR, in particular the T790M drug-resistance mutation, in NSCLC models. CO-1686 is the fi rst drug of its class in clinical development for the treatment of T790M-positive NSCLC, potentially offering potent inhibition of mutant EGFR while avoiding the on-target toxicity observed with inhibition of the WT EGFR.

The discovery of tetrahydro-beta-carbolines as inhibitors of the kinesin Eg5.

A series of tetrahydro-beta-carbolines were identified by HTS as inhibitors of the kinesin Eg5. Molecular modeling and medicinal chemistry techniques were employed to explore the SAR for this series with a focus of removing potential metabolic liabilities and improving cellular potency.

Inhibition of Cdc7/Dbf4 kinase activity affects specific phosphorylation sites on MCM2 in cancer cells.

The Cdc7/Dbf4 kinase is required for initiation of DNA replication and also plays a role in checkpoint function in response to replication stress. Exactly how Cdc7/Dbf4 mediates those activities remains to be elucidated. Cdc7/Dbf4 physically interacts with and phosphorylates the minichromosome maintenance complex (MCM), such as MCM2, MCM4 and MCM6. Cdc7/Dbf4 activity is required for association of Cdc45 followed by recruitment of DNA polymerase on the chromatin. Using high resolution mass spectrometry, we identified six phosphorylation sites on MCM2, two of them have not been described before. We provide evidence that Cdc7/Dbf4 mediates phosphorylation on serine 108 and serine 40 on human MCM2 in vitro and in vivo in cancer cells in the absence of DNA damage. Antibodies specific to pS108 or pS40 confirmed the sites and established useful read-outs for inhibition of Cdc7/Dbf4. This report demonstrates the utility of an in vitro to in vivo workflow utilizing immunoprecipitation and mass spectrometry to map phosphorylation sites on endogenous kinase substrates. The approach can be readily generalized to identify target modulation read-outs for other potential kinase cancer targets.