Sylvie Vincent

Inhibition of RhoGAP activity is sufficient for the induction of Rho-mediated actin reorganization

It is generally believed that the induction of actin cytoskeleton rearrangements by extracellular stimuli results from the activation of guanine nucleotide exchange factors for the Rho GTPases. Here, we present evidence that the inactivation of RhoGAP (GTPase activating protein) activity is an equally effective means of promoting Rho-mediated cellular processes. We observed that exposure of cultured fibroblasts to sodium fluoride (NaF) results in a rapid and potent stimulation of actin stress fiber formation. This effect is mediated by the Rho GTPase and is associated with the inactivation of cellular RhoGAP activity. Specifically, NaF promotes formation of a high-affinity complex between Rho and the two cellular p190 RhoGAPs in vivo, apparently sequestering limiting amounts of RhoGAP activity, thereby resulting in Rho activation. p190 RhoGAP activity was found to account for approximately 60% of the total RhoGAP activity detected in whole cell extracts, indicating that relatively small changes in cellular RhoGAP activity can have potent effects on Rho activation. We also found that sub-effective concentrations of NaF combined with sub-effective concentrations of the Rho pathway activator, lysophosphatidic acid, which stimulates guanine nucleotide exchange activity on the Rho GTPase, results in the rapid induction of actin stress fibers. Together, these results suggest that the Rho GTPase is regulated by a fine balance of nucleotide exchange and RhoGAP activities, and that inactivation of RhoGAP activity may be a physiologically important regulatory mechanism for activating the Rho GTPase.

Dissecting genetic requirements of human breast tumorigenesis in a tissue transgenic model of human breast cancer in mice

Breast cancer development is a complex pathobiological process involving sequential genetic alterations in normal epithelial cells that results in uncontrolled growth in a permissive microenvironment. Accordingly, physiologically relevant models of human breast cancer that recapitulate these events are needed to study cancer biology and evaluate therapeutic agents. Here, we report the generation and utilization of the human breast cancer in mouse (HIM) model, which is composed of genetically engineered primary human breast epithelial organoids and activated human breast stromal cells. By using this approach, we have defined key genetic events required to drive the development of human preneoplastic lesions as well as invasive adenocarcinomas that are histologically similar to those in patients. Tumor development in the HIM model proceeds through defined histological stages of hyperplasia, DCIS to invasive carcinoma. Moreover, HIM tumors display characteristic responses to targeted therapies, such as HER2 inhibitors, further validating the utility of these models in preclinical compound testing. The HIM model is an experimentally tractable human in vivo system that holds great potential for advancing our basic understanding of cancer biology and for the discovery and testing of targeted therapies.

Neuregulin 1 Expression Is a Predictive Biomarker for Response to AV-203, an ERBB3 Inhibitory Antibody, in Human Tumor Models

Purpose: ERBB3 is overexpressed in a broad spectrum of human cancers, and its aberrant activation is associated with tumor pathogenesis and therapeutic resistance to various anticancer agents. Neuregulin 1 (NRG1) is the predominant ligand for ERBB3 and can promote the heterodimerization of ERBB3 with other ERBB family members, resulting in activation of multiple intracellular signaling pathways. AV-203 is a humanized IgG1/κ ERBB3 inhibitory antibody that completed a first-in-human phase I clinical trial in patients with advanced solid tumors. The purpose of this preclinical study was to identify potential biomarker(s) that may predict response to AV-203 treatment in the clinic. Experimental Design: We conducted in vivo efficacy studies using a broad panel of xenograft models representing a wide variety of human cancers. To identify biomarkers that can predict response to AV-203, the relationship between tumor growth inhibition (TGI) by AV-203 and the expression levels of ERBB3 and NRG1 were evaluated in these tumor models. Results: A significant correlation was observed between the levels of NRG1 expression and TGI by AV-203. In contrast, TGI was not correlated with ERBB3 expression. The correlation between the levels of NRG1 expression in tumors and their response to ERBB3 inhibition by AV-203 was further validated using patient-derived tumor explant models. Conclusions: NRG1 is a promising biomarker that can predict response to ERBB3 inhibition by AV-203 in preclinical human cancer models. NRG1 warrants further clinical evaluation and validation as a potential predictive biomarker of response to AV-203. Clin Cancer Res; 21(5); 1106–14. ©2014 AACR.

Integrated version of reverse two-hybrid system for the postproteomic era

Publisher Summary Functional genomic databases provide information for many of the genes predicted from genome and expressed sequence tags (ESTs) sequencing projects. These databases should gradually relieve molecular biologists from the tedious and repetitive steps involved in conventional gene cloning and basic protein characterization. This in turn might help extend studies primarily focused on the function of proteins toward more precisely deciphering the function of interactions between proteins. This chapter describes an improved version of the yeast reverse two-hybrid system to generate and characterize interaction-defective alleles (IDAs) that can be used to study the function of physical interactions between proteins. Comprehensive interactome mapping projects using large-scale yeast two-hybrid systems are underway for Saccharomyces cerevisiae and Caenorhabditis elegans. Typically two-hybrid interactome maps consist of large lists of potential binary interactions made available on the World Wide Web. These maps should provide additional layers of information on various aspects of protein function.

Use of protein-interaction maps to formulate biological questions.

Protein-interaction mapping approaches generate functional information for large numbers of genes that are predicted from complete genome sequences. This information, released as databases available on the Internet, is likely to transform the way biologists formulate and then address their questions of interest.

Abstract 2509: AV-203, a humanized ERBB3 inhibitory antibody inhibits ligand-dependent and ligand-independent ERBB3 signaling in vitro and in vivo

ERBB3 is a member of the EGFR receptor tyrosine kinase (RTK) family. Agents targeting the family of EGFR RTKs have become widely used for the treatment of lung, colon, breast, gastric, and head and neck cancers. Among EGFR family members, ERBB3 is of special interest because of its ability to activate the survival pathway driven by PI3K, its essential role in HER2 mediated cancers, and its involvement in tumor progression and drug resistance. The ERBB3 receptor is expressed on cells from cancers of the head and neck, lung, breast, ovaries, prostate, colon, pancreas, and gastrointestinal tract. Its expression is often linked to poor prognosis. In addition, it has been implicated in the development of resistance to current anti-cancer agents including receptor-targeted tyrosine kinase inhibitors (TKI). ERBB3 lacks detectable tyrosine kinase activity and its activation requires the heterodimerization of ERBB3 with RTK partners, i.e., HER2, EGFR or MET. ERBB3 recruitment in heterodimer complexes takes place when RTKs are overexpressed or amplified or stimulated by ligands, i.e. Neuregulin-1 (NRG1) or epidermal growth factor (EGF). Because of the lack of ERBB3 kinase activity, antibody therapies directed against the extracellular domain of ERBB3 seem to be the most effective method to disrupt the function of ERBB3. Herein, characterization of AV-203, a humanized ERBB3 inhibitory antibody, is presented. AV-203 is a humanized immunoglobulin G1/kappa antibody that targets the ERBB3 RTK. AV-203 binds to human ERBB3 with high affinity (KD = 76 pM at 37°C) characterized by fast association and slow dissociation rates. AV-203 also binds to cynomolgus monkey ERBB3 but not to mouse ERBB3, allowing toxicological assessment of the antibody in this species. AV-203 is a potent inhibitor of ERBB3 activation and its downstream signaling molecule AKT in response to both ligands, NRG1 and EGF. In ligand-independent settings, AV-203 inhibits the steady state activation of ERBB3/AKT which depends on the presence of an overexpressed RTK such as HER2. AV-203 can prevent the ERBB3/HER2 heterodimer formation and completely inhibit the proliferation in response to NRG1 ligand in human breast cancer cell line, MCF7. AV-203 down regulates ERBB3 receptor in vitro and in vivo. Finally, AV-203 inhibits tumor growth in a broad spectrum of xenograft models in which ERRB3 is activated by its ligand NRG1 or by HER2 overexpression such as the pancreatic cancer BxPC3 or the HER2 amplified breast cancer MDA-MB-453 xenograft models, respectively. In conclusion, AV-203 demonstrated high affinity binding to ERBB3, potent inhibition of NRG1 binding and of ERBB3 activation in ligand-dependent and ligand-independent manners both in vitro and in vivo. First in human trial of AV-203 in cancer patients is planned for 2012. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2509. doi:1538-7445.AM2012-2509

Abstract P3-04-05: Identification of SAR439859, an orally bioavailable selective estrogen receptor degrader (SERD) that has strong antitumor activity in wild-type and mutant ER+ breast cancer models

Nearly 70% or more of newly diagnosed cases of breast cancer (BC) are estrogen receptor positive (ER+) where endocrine therapy is a primary treatment. However, substantial evidence describes a continued role of ER signaling in tumor progression, where approximately 40% of patients on endocrine therapy develop resistance that include mutations in the ER that drive a constitutively active receptor. Fulvestrant, an estrogen receptor degrader, is effective at shutting down ER signaling. However, fulvestrant efficacy studies report insufficient blockade of ER signaling in patients that may be a consequence of poor pharmaceutical properties. Here we describe the discovery of SAR439859, a novel, orally bioavailable SERD with potent antagonist and degradative properties against ER both in vitro and in vivo. SAR439859 has robust inhibition of ER signaling activity in multiple ER+ breast cancer cell lines including tamoxifen resistant lines harboring ER mutations. Across a large panel of ER+ cells, SAR439859 demonstrated broad and superior ER degradation activity as compared to other SERDs including improved inhibition of ER signaling and inhibition of cell growth. Similarly, in vivo treatment with SAR439859 demonstrated significant tumor regression in ER+ BC models including MCF7-ESR1 mutant-Y537S model and endocrine therapy resistant patient-derived xenograft tumor transplantation. Collectively, these results showed that SAR439859 is an oral, nonsteroidal, selective estrogen receptor antagonist and degrader that could provide therapeutic benefit to ER+ breast cancer patients. SAR439859 is currently being evaluated in a phase I clinical trial. Citation Format: Maysoun Shomali, Youssef El-Ahmad, Frank Halley, Jane Cheng, Michael Weinstein, Muchun Wang, Fangxian Sun, Natalia Malkova, Mikhail Levit, Malvika Koundinya, Zhuyan Gou, Andrew Hebert, Jessica McManus, Dietmar Hoffman, Hui Cao, Joonil Jung, Jack Pollard, Sylvie Vincent, Timothy Ackerson, Francisco Adrian, Chris Winter, Victoria Richon, Hong Chen, Karl Hsu, Joanne Lager, Albane Courjaud, Rosalia Arrebola, Laurent Besret, Pierre-Yves Abecassis, Laurent Schio, Gary McCort, Michel Tabart, Victor Certal, Fabienne Thompson, Bruno Filoche-Romme, Laurent Debussche, Patrick Cohen, Carlos Garcia-Echeverria, Monsif Bouaboula. Identification of SAR439859, an orally bioavailable selective estrogen receptor degrader (SERD) that has strong anti-tumor activity in wild-type and mutant ER+ breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5775.

Abstract 1228: AV-203, a fully humanized ERBB3 inhibitory antibody, reverses ERBB3-induced resistance to targeted therapies.

Targeted therapies have changed the treatment paradigm of cancer. Specifically, the use of tyrosine kinase inhibitors (TKIs) has improved therapeutic responses in a number of tumor types. However, initial response to TKIs is often followed by rapid relapse due to the acquisition of molecular features that lead to resistance. ERBB3, a receptor of the epidermal growth factor receptor (EGFR/ERBB1) family, has been linked to the development of resistance to multiple ERBB targeting TKIs. A proposed potential mechanism is the recruitment and activation of ERBB3 as a bypass mechanism to activate PI3K dependent pro-survival pathways. We investigated whether the inhibition of ERBB3 could reverse TKI resistance linked to ERBB3 activation. AV-203 is a humanized IgG1 antibody directed against ERBB3. AV-203 potently inhibits both ligand dependent and ligand independent activation of ERBB3. AV-203 was shown to inhibit the binding of NRG1 to ERBB3 and promote ERBB3 degradation. The tumor growth inhibitory activity of AV-203 was demonstrated in numerous xenograft models representing major human carcinomas (breast, lung, ovarian, kidney and pancreas). Here, we investigated the ability of AV-203 to restore TKI sensitivity in models with activated ERBB3. First, we demonstrated that ligand mediated (NRG1) activation of ERBB3 leads to acquired resistance to the EGFR TKI erlotinib in a lung cancer model bearing EGFR TKI sensitizing mutation, to the ERBB2/HER2 inhibitor lapatinib in a HER2 amplified breast cancer model, and to the EGFR inhibitory antibody cetuximab in head and neck cancer models. AV-203 was able to restore sensitivity to these RTK inhibitors. This data suggest that the combination of AV-203, with EGFR/HER2 targeted therapies could potentially extend the efficacy of these agents by preventing the emergence of ERBB3 mediated resistance. This hypothesis will be tested in future clinical trials. AV-203 is currently in Phase I development for the treatment of solid tumors. Citation Format: Steven M. Tyler, Sylvie Vincent, Christina Fleet, Steve Bottega, Donna McIntosh, Kristan Meetze, Jeno Gyuris. AV-203, a fully humanized ERBB3 inhibitory antibody, reverses ERBB3-induced resistance to targeted therapies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1228. doi:10.1158/1538-7445.AM2013-1228

Abstract 3787: Pharmacokinetics and pharmacodynamics of AV-203, a humanized anti-ERBB3 antibody

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL ERBB3 is widely expressed in human carcinomas, and its over expression is associated with poor prognosis in patients with various carcinomas, (i.e., breast, ovarian, prostate, colorectal, pancreatic, gastric, and head and neck cancers). The presence of ERBB3 correlates with local to distal metastasis in lung, gastric, and colorectal cancers as well as bone invasion in prostate cancer. Activation of ERBB3 is also implicated in the development of resistance to current cancer treatments. Due to its lack of kinase activity, the activation of the ERBB3 receptor is dependent on heterodimerization with active receptor tyrosine kinases (RTKs). The recruitment of ERBB3 into active, heterodimer complexes is mediated by its ligand Neuregulin-1 (NRG-1) or by amplified, over expressed RTKs in a ligand independent manner. Therefore, ERBB3 can crosstalk with most major receptors involved in cancer development and maintenance such as epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and c-MET. AV-203 is a potent, humanized anti-ERBB3 antibody that inhibits both ligand-dependent and independent activation of ERBB3 both in vitro and in vivo. Pharmacokinetics and pharmacodynamics of AV-203 were characterized in mice using the A549 non-small cell lung cancer xenograft model. AV-203 administered IV in mice exhibits acceptable pharmacokinetics supporting preclinical efficacy studies. AV-203 administered IV in A549 NSCLC xenograft tumor bearing mice had lower serum AUC than naive mice, demonstrating that the presence of human ErbB3 may alter the PK parameters of AV-203. In evaluating pharmacodynamics in vivo, AV-203 was able to down regulate total ERBB3 and ERBB3 signaling in A549 tumors in a time-dependent manner. Inhibition of ERBB3 signaling correlated with significant dose-dependent tumor growth inhibition in the A549 xenograft model. Dose scheduling studies with the constant AV-203 dose of 2.5 mg/kg revealed that the most efficacious schedule is the more frequent dosing at Q3D. In comparing the total dose of 10 mg/kg per 14 day cycle, at varying dose per injection and frequency, AV-203 resulted in significant tumor growth inhibition at all dose schedules. These data conclude that the efficacy of AV-203 is driven by total drug exposure and that AV-203 is not dependent on Cmax for its antitumor activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3787. doi:1538-7445.AM2012-3787

Abstract 642: Novel EGFR inhibitory antibodies directed against EGFR mutants with potentially reduced toxicity

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Non-small cell lung cancers (NSCLC) driven by ligand-independent activating mutations in epidermal growth factor receptor (EGFR) often respond to treatment with EGFR tyrosine kinase inhibitors (TKIs). About half of acquired resistance to EGFR-TKI therapy results from a secondary point mutation in the EGFR tyrosine kinase domain at amino acid position 790 (T790M). T790M mutants also display reduced sensitivity to Cetuximab treatment. We have investigated the molecular mechanism responsible for the reduced Cetuximab sensitivity and found that T790M mutant receptors primarily exist and signal as monomers. We have exploited this characteristic of the T790M mutant to isolate novel EGFR inhibitory antibodies with activities against all EGFR variants. In addition, as opposed to other EGFR inhibitory antibodies, these novel antibodies have reduced ligand binding inhibitory activity and minimal inhibitory effect on EGF induced human primary keratinocyte proliferation, suggesting a potentially reduced skin toxicity profile. The broad activity profile, combined with potentially reduced skin toxicity, suggests that these antibodies will have great potential for combinability with other therapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 642. doi:10.1158/1538-7445.AM2011-642