Mark DeMario

Phase I Study of RO4929097, a Gamma Secretase Inhibitor of Notch Signaling, in Patients With Refractory Metastatic or Locally Advanced Solid Tumors

PURPOSE To determine the maximum-tolerated dose (MTD) and assess safety, pharmacokinetics, pharmacodynamics, and evidence of antitumor activity of RO4929097, a gamma secretase inhibitor of Notch signaling in patients with advanced solid malignancies. PATIENTS AND METHODS Patients received escalating doses of RO4929097 orally on two schedules: (A) 3 consecutive days per week for 2 weeks every 3 weeks; (B) 7 consecutive days every 3 weeks. To assess reversible CYP3A4 autoinduction, the expanded part of the study tested three dosing schedules: (B) as above; modified A, 3 consecutive d/wk for 3 weeks; and (C) continuous daily dosing. Positron emission tomography scans with [(18)F]fluorodeoxyglucose (FDG-PET) were used to assess tumor metabolic effects. RESULTS Patients on schedule A (n = 58), B (n = 47), and C (n = 5; expanded cohort) received 302 cycles of RO4929097. Common grade 1 to 2 toxicities were fatigue, thrombocytopenia, fever, rash, chills, and anorexia. Transient grade 3 hypophosphatemia (dose-limiting toxicity, one patient) and grade 3 pruritus (two patients) were observed at 27 mg and 60 mg, respectively; transient grade 3 asthenia was observed on schedule A at 80 mg (one patient). Tumor responses included one partial response in a patient with colorectal adenocarcinoma with neuroendocrine features, one mixed response (stable disease) in a patient with sarcoma, and one nearly complete FDG-PET response in a patient with melanoma. Effect on CYP3A4 induction was observed. CONCLUSION RO4929097 was well tolerated at 270 mg on schedule A and at 135 mg on schedule B; the safety of schedule C has not been fully evaluated. Further studies are warranted on the basis of a favorable safety profile and preliminary evidence of clinical antitumor activity.

Preclinical biomarkers for a cyclin-dependent kinase inhibitor translate to candidate pharmacodynamic biomarkers in phase I patients

A genomics-based approach to identify pharmacodynamic biomarkers was used for a cyclin-dependent kinase inhibitory drug. R547 is a potent cyclin-dependent kinase inhibitor with a potent antiproliferative effect at pharmacologically relevant doses and is currently in phase I clinical trials. Using preclinical data derived from microarray experiments, we identified pharmacodynamic biomarkers to test in blood samples from patients in clinical trials. These candidate biomarkers were chosen based on several criteria: relevance to the mechanism of action of R547, dose responsiveness in preclinical models, and measurable expression in blood samples. We identified 26 potential biomarkers of R547 action and tested their clinical validity in patient blood samples by quantitative real-time PCR analysis. Based on the results, eight genes (FLJ44342, CD86, EGR1, MKI67, CCNB1, JUN, HEXIM1, and PFAAP5) were selected as dose-responsive pharmacodynamic biomarkers for phase II clinical trials. [Mol Cancer Ther 2009;8(9):2517–25]

A Phase I Monotherapy Study of RG7212, a First-in-Class Monoclonal Antibody Targeting TWEAK Signaling in Patients with Advanced Cancers

Purpose: Tumor necrosis factor (TNF)–like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible molecule 14 (Fn14) are a ligand–receptor pair frequently overexpressed in solid tumors. TWEAK:Fn14 signaling regulates multiple oncogenic processes through MAPK, AKT, and NFκB pathway activation. A phase I study of RG7212, a humanized anti-TWEAK IgG1κ monoclonal antibody, was conducted in patients with advanced solid tumors expressing Fn14. Experimental Design: Dose escalations, over a 200- to 7,200-mg range, were performed with patients enrolled in weekly (QW), bi-weekly (Q2W), or every-three-week (Q3W) schedules. Primary objectives included determination of dose and safety profile. Secondary endpoints included assessments related to inhibition of TWEAK:Fn14 signaling, tumor proliferation, tumor immune cell infiltration, and pharmacokinetics. Results: In 192 treatment cycles administered to 54 patients, RG7212 was well-tolerated with no dose-limiting toxicities observed. More than 95% of related adverse events were limited to grade 1/2. Pharmacokinetics were dose proportional for all cohorts, with a t1/2 of 11 to 12 days. Pharmacodynamic changes included clearance of free and total TWEAK ligand and reductions in tumor Ki-67 and TRAF1. A patient with BRAF wild-type melanoma who received 36 weeks of RG7212 therapy had tumor regression and pharmacodynamic changes consistent with antitumor effects. Fifteen patients (28%) received 16 or more weeks of RG7212 treatment. Conclusion: RG7212 demonstrated excellent tolerability and favorable pharmacokinetics. Pharmacodynamic endpoints were consistent with reduced TWEAK:Fn14 signaling. Tumor regression was observed and prolonged stable disease was demonstrated in multiple heavily pretreated patients with solid tumors. These encouraging results support further study of RG7212. Clin Cancer Res; 21(2); 258–66. ©2014 AACR.

RG7212 anti-TWEAK mAb inhibits tumor growth through inhibition of tumor cell proliferation and survival signaling and by enhancing the host antitumor immune response.

Purpose: To explore the role of TWEAK in tumor growth and antitumor immune response and the activity and mechanism of RG7212, an antagonistic anti-TWEAK antibody, in tumor models. Experimental Design: TWEAK-induced signaling and gene expression were explored in tumor cell lines and inhibition of these effects and antitumor efficacy with RG7212 treatment was assessed in human tumor xenograft-, patient-derived xenograft, and syngeneic tumor models and phase I patients. Genetic features correlated with antitumor activity were characterized. Results: In tumor cell lines, TWEAK induces proliferation, survival, and NF-κB signaling and gene expression that promote tumor growth and suppress antitumor immune responses. TWEAK-inducible CD274, CCL2, CXCL-10 and -11 modulate T-cell and monocyte recruitment, T-cell activation, and macrophage differentiation. These factors and TWEAK-induced signaling were decreased, and tumor, blood, and spleen immune cell composition was altered with RG7212 treatment in mice. RG7212 inhibits tumor growth in vivo in models with TWEAK receptor, Fn14, expression, and markers of pathway activation. In phase I testing, signs of tumor shrinkage and stable disease were observed without dose-limiting toxicity. In a patient with advanced, Fn14-positive, malignant melanoma with evidence of tumor regression, proliferation markers were dramatically reduced, tumor T-cell infiltration increased, and tumor macrophage content decreased. Antitumor activity, a lack of toxicity in humans and animals and no evidence of antagonism with standard of care or targeted agents in mice, suggests that RG7212 is a promising agent for use in combination therapies in patients with Fn14-positive tumors. Clin Cancer Res; 19(20); 5686–98. ©2013 AACR.

Gene expression analysis in biomarker research and early drug development using function tested reverse transcription quantitative real-time PCR assays.

The identification of new biomarkers is essential in the implementation of personalized health care strategies that offer new therapeutic approaches with optimized and individualized treatment. In support of hypothesis generation and testing in the course of our biomarker research an online portal and respective function-tested reverse transcription quantitative real-time PCR assays (RT-qPCR) facilitated the selection of relevant biomarker genes. We have established workflows applicable for convenient high throughput gene expression analysis in biomarker research with cell lines (in vitro studies) and xenograft mouse models (in vivo studies) as well as formalin-fixed paraffin-embedded tissue (FFPET) sections from various human research and clinical tumor samples. Out of 92 putative biomarker candidate genes selected in silico, 35 were shown to exhibit differential expression in various tumor cell lines. These were further analysed by in vivo xenograft mouse models, which identified 13 candidate genes including potential response prediction biomarkers and a potential pharmacodynamic biomarker. Six of these candidate genes were selected for further evaluation in FFPET samples, where optimized RNA isolation, reverse transcription and qPCR assays provided reliable determination of relative expression levels as precondition for differential gene expression analysis of FFPET samples derived from projected clinical studies. Thus, we successfully applied function tested RT-qPCR assays in our biomarker research for hypothesis generation with in vitro and in vivo models as well as for hypothesis testing with human FFPET samples. Hence, appropriate function-tested RT-qPCR assays are available in biomarker research accompanying the different stages of drug development, starting from target identification up to early clinical development. The workflow presented here supports the identification and validation of new biomarkers and may lead to advances in efforts to achieve the goal of personalized health care.

A Phase I Clinical and Pharmacokinetic Study of Ro 31-7453 Given as a 7- or 14-Day Oral Twice Daily Schedule Every 4 Weeks in Patients with Solid Tumors

Purpose: This is a dose-finding Phase I study of oral Ro 31-7453, a new class of antimitotic drug with promising preclinical activity in several chemoresistant models. Experimental Design: Two schedules of oral Ro 31-7453 (every 12 h) given for either 7 or 14 consecutive days repeated every 4 weeks were explored consecutively. Results: Thirty-seven patients with refractory cancer entered the study (14 on the 7-day schedule and 23 on the 14-day schedule). Median age was 63 years (range, 40–77 years), and median Karnofsky performance status was 80 (range, 60–100); the most frequent diagnosis was colorectal carcinoma (16 patients). Dose levels of 100, 200, 240, and 280 mg/m2 twice daily (bid) for 7 days and 70, 100, 125, and 150 mg/m2 bid for 14 days were explored. A total of 110 cycles were administered, the median number of cycles received was 3 (range, 1–7); six patients completed 6 or more cycles. Myelosuppression and mucositis were dose-limiting with both schedules. Fatigue and gastrointestinal toxicities other than mucositis were frequent but generally mild. The maximum tolerated doses were 200 mg/m2 bid and 125 mg/m2 bid for the 7- and 14-day schedules, respectively. Pharmacokinetic analysis showed rapid absorption and metabolism. The area under the concentration-time curve and trough concentrations of Ro 31-7453 and two active metabolites appeared dose proportional with a t1/2 of ∼9 h and a tmax of ∼4 h. One patient with pretreated lung cancer had a partial response. Conclusions: Both Ro 31-7453 regimens were feasible, but the 14-day schedule at the recommended dose of 125 mg/m2 bid was selected for further monotherapy Phase II evaluation because of its higher preclinical activity. This regimen is convenient, well tolerated, and has a favorable pharmacokinetic profile.

Exposure and Tumor Fn14 expression as Determinants of Pharmacodynamics of the Anti-TWEAK Monoclonal Antibody RG7212 in Patients with Fn14-positive Solid Tumors

Purpose: The TWEAK–Fn14 pathway represents a novel anticancer target that is being actively investigated. Understanding the relationship between pharmacokinetics of anti-TWEAK therapeutics and tumor pharmacodynamics is critical. We investigated exposure-response relationships of RG7212, an anti-TWEAK mAb, in patients with Fn14-expressing tumors. Experimental Design: Patients with Fn14-positive tumors (IHC≥1+) treated in a phase I first-in-human study with ascending doses of RG7212 were the basis for this analysis. Pharmacokinetics of RG7212 and dynamics of TWEAK were determined, as were changes in tumor TWEAK–Fn14 signaling in paired pre- and posttreatment tumor biopsies. The objectives of the analysis were to define exposure-response relationships and the relationship between pretreatment tumor Fn14 expression and pharmacodynamic effect. Associations between changes in TWEAK–Fn14 signaling and clinical outcome were explored. Results: Thirty-six patients were included in the analysis. RG7212 reduced plasma TWEAK to undetectable levels at all observed RG7212 exposures. In contrast, reductions in tumor Fn14 and TRAF1 protein expression were observed only at higher exposure (≥300 mg*h/mL). Significant reductions in tumor Ki-67 expression and early changes in serum concentrations of CCL-2 and MMP-9 were observed exclusively in patients with higher drug exposure who had high pretreatment tumor Fn14 expression. Pretreatment tumor Fn14 expression was not associated with outcome, but a trend toward longer time on study was observed with high versus low RG7212 exposure. Conclusions: RG7212 reduced tumor TWEAK–Fn14 signaling in a systemic exposure-dependent manner. In addition to higher exposure, relatively high Fn14 expression might be required for pharmacodynamic effect of anti-TWEAK monoclonal antibodies. Clin Cancer Res; 22(4); 858–67. ©2015 AACR.

Abstract 4708: Neuroendocrine gene transcript expression is associated with efficacy to lysine-specific demethylase-1 inhibitor RG6016 in small cell lung cancer-derived cell lines

Small cell lung cancer (SCLC), which comprises 15% of lung neoplasms, is an aggressive malignancy with limited treatment options. Survival in refractory settings is typically less than one year, exemplifying the need for more effective therapeutics. Recent published results suggest that epigenetic modulation mediated by Lysine Specific Demethylase-1 (LSD1) inhibition can impact this disease setting (Mohammed et al., Cell, 2015 28(1):57-69). RG6016 is a potent and selective inhibitor of LSD1 that promotes growth inhibition in vitro and in vivo SCLC xenograft models. Here we describe the identification of a gene expression profile associated with neuroendocrine lineages that predicts responses to RG6016 in SCLC cell lines. RG6016 was screened on a panel of 19 SCLC cell lines; potent sub-nanomolar to nanomolar activity was exhibited on a subset of these cell lines. Growth inhibitory responses were greater in classic compared to variant SCLC cell lines (p-value 0.0055). 9 of the 11 classic SCLC cell lines tested were responsive to growth inhibitory effects of RG6016, while 7 of the 8 variant cell lines were insensitive to RG6016 treatment. Differential gene expression analysis comparing classic to variant cell lines ranked neuroendocrine lineage-associated markers, such as ASCL1, amongst the highest differentially expressed genes (adj. p-value = 2.6 × 10-23). ASCL1 is a transcription factor required for proper development of pulmonary neuroendocrine cells, and was recently identified as essential for the survival of a majority of small cell lung cancers (Augustyn et al., Proc Natl Acad Sci USA 2014 111(41):14788-93). Two other established neuroendocrine markers, DDC and GRP, ranked within the top ten differentially expressed genes. We also found that a subset of insensitive SCLC cell lines harbor c-MYC amplifications, suggesting a potential pathway for resistance to LSD1 inhibition. An RG6016 response gene expression pattern that highly correlates with growth inhibition was defined using baseline expression levels of neuroendocrine lineage transcripts and c-MYC amplification. In vitro activity also correlated with in vivo responses; RG60106 treatment inhibited xenograft growth of response signature positive cell line NCI-H510A, while the response signature negative NCI-H526 xenografts were refractory to RG6016 exposure. Similar gene expression patterns were observed within SCLC cell lines and a panel of SCLC patient samples, suggesting that use of the RG6016 response gene signature may increase the likelihood of identifying patients who will clinically benefit from LSD1- based therapies. Citation Format: Francesca Milletti, Wei-Yi Cheng, Tamara Maes, Serena Lunardi, Mark DeMario, William E. Pierceall, Fiona Mack. Neuroendocrine gene transcript expression is associated with efficacy to lysine-specific demethylase-1 inhibitor RG6016 in small cell lung cancer-derived cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4708.