Alain Bruno

Characterization of a Large Panel of Patient-Derived Tumor Xenografts Representing the Clinical Heterogeneity of Human Colorectal Cancer

Purpose: Patient-derived xenograft models are considered to represent the heterogeneity of human cancers and advanced preclinical models. Our consortium joins efforts to extensively develop and characterize a new collection of patient-derived colorectal cancer (CRC) models. Experimental Design: From the 85 unsupervised surgical colorectal samples collection, 54 tumors were successfully xenografted in immunodeficient mice and rats, representing 35 primary tumors, 5 peritoneal carcinoses and 14 metastases. Histologic and molecular characterization of patient tumors, first and late passages on mice includes the sequence of key genes involved in CRC (i.e., APC, KRAS, TP53), aCGH, and transcriptomic analysis. Results: This comprehensive characterization shows that our collection recapitulates the clinical situation about the histopathology and molecular diversity of CRC. Moreover, patient tumors and corresponding models are clustering together allowing comparison studies between clinical and preclinical data. Hence, we conducted pharmacologic monotherapy studies with standard of care for CRC (5-fluorouracil, oxaliplatin, irinotecan, and cetuximab). Through this extensive in vivo analysis, we have shown the loss of human stroma cells after engraftment, observed a metastatic phenotype in some models, and finally compared the molecular profile with the drug sensitivity of each tumor model. Through an experimental cetuximab phase II trial, we confirmed the key role of KRAS mutation in cetuximab resistance. Conclusions: This new collection could bring benefit to evaluate novel targeted therapeutic strategies and to better understand the basis for sensitivity or resistance of tumors from individual patients. Clin Cancer Res; 18(19); 5314–28. ©2012 AACR.

S49076 is a Novel Kinase Inhibitor of MET, AXL and FGFR with Strong Preclinical Activity Alone and in Association with Bevacizumab

Aberrant activity of the receptor tyrosine kinases MET, AXL, and FGFR1/2/3 has been associated with tumor progression in a wide variety of human malignancies, notably in instances of primary or acquired resistance to existing or emerging anticancer therapies. This study describes the preclinical characterization of S49076, a novel, potent inhibitor of MET, AXL/MER, and FGFR1/2/3. S49076 potently blocked cellular phosphorylation of MET, AXL, and FGFRs and inhibited downstream signaling in vitro and in vivo. In cell models, S49076 inhibited the proliferation of MET- and FGFR2-dependent gastric cancer cells, blocked MET-driven migration of lung carcinoma cells, and inhibited colony formation of hepatocarcinoma cells expressing FGFR1/2 and AXL. In tumor xenograft models, a good pharmacokinetic/pharmacodynamic relationship for MET and FGFR2 inhibition following oral administration of S49076 was established and correlated well with impact on tumor growth. MET, AXL, and the FGFRs have all been implicated in resistance to VEGF/VEGFR inhibitors such as bevacizumab. Accordingly, combination of S49076 with bevacizumab in colon carcinoma xenograft models led to near total inhibition of tumor growth. Moreover, S49076 alone caused tumor growth arrest in bevacizumab-resistant tumors. On the basis of these preclinical studies showing a favorable and novel pharmacologic profile of S49076, a phase I study is currently underway in patients with advanced solid tumors. Mol Cancer Ther; 12(9); 1749–62. ©2013 AACR.

Specific oncogenic activity of the Src-family tyrosine kinase c-Yes in colon carcinoma cells.

c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of β-catenin at cell membranes and a reduction of expression of β-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in β-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src.

Abstract 2782: Bcl-2 selective antagonists show antitumor activity without dose limiting platelet toxicity

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnProteins of the Bcl-2 family are central regulators of programmed cell death. Pro-survival Bcl-2 proteins, such as Bcl-2, Bcl-xL and Mcl-1 are often over-expressed in human tumours and participate in tumour initiation, progression and chemo-resistance. Therefore drugs targeting these pro-survival Bcl-2 proteins represent a promising therapeutic approach for cancer treatment. The most advanced drug targeting this protein family is ABT-263, a potent Bcl-2 and Bcl-xL inhibitor showing anti-tumour efficacy in preclinical models of leukaemia and small cell lung carcinoma. Survival of circulating platelets has been shown to be highly Bcl-xL dependent; consequently the dose-limiting toxicity of ABT-263 is an on-target peripheral thrombocytopenia. We have used a range of biophysical methods to guide the structure-based generation of a significant number of small molecules,* which bind with high affinity (MW 100 fold). In cellular assays, our lead compounds efficiently displace Bax from Bcl-2 with near complete inhibition of Bcl-2 / Bax co-immunoprecipitation at 100 nM. These compounds are strong inducers of cell death in Bcl-2 dependent cellular models such as the acute myeloblastic leukaemia (AML) cell line RS4;11, affording sub-10 nM IC50s for the most potent compounds. In vivo, in agreement with their mechanism of action, these Bcl-2 selective inhibitors, given either intravenously or orally, elicit a rapid (30 min iv, and 2 hours po) and strong apoptotic response in mouse xenografts of the RS4:11 cell line. When the most potent compounds are given orally to RS4;11 xenograft-bearing mice, apoptosis in tumor cells is induced more than 15 fold (at 25 mg/kg) and more than 20 fold (at 50 mg/kg) compared to untreated mice. Importantly, in agreement with the selectivity of the compounds for Bcl-2 versus Bcl-xL, no platelet loss was observed in mice treated with our compounds, in sharp contrast to ABT-263. Finally, we observe very robust anti-tumor activity when a lead compound is given orally at 50 mg/kg and 100 mg/kg (with complete regression at 100 mg/kg) in an RS4;11 mouse xenograft model. This anti-tumor activity was similar whether the compound was dosed daily or twice a week over two weeks. Altogether our data demonstrate that highly Bcl-2 selective antagonists show anti-tumor activity and no platelet toxicity, in contrast to Bcl-2 / Bcl-xL dual inhibitors. Such compounds represent promising drug candidates for the treatment of Bcl-2 dependent malignancies such as chronic lymphocytic leukaemia (CLL) and other leukaemias and lymphomas. * Chemical structures of compounds will not be disclosed.nnCitation 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 2782. doi:1538-7445.AM2012-2782

Abstract 848: Preclinical antitumor activity of S 49076, a novel MET / AXL / FGFR kinase inhibitor and molecular stratification for tumor sensitivity

Aberrant activity of receptor tyrosine kinases (RTKs) has been associated with tumor progression in a wide variety of human malignancies, making them promising drug targets for cancer therapy. Although, in some instances, specific inhibition of just one of these RTKs suffices for inhibition of tumor progression, in the majority of cases, targeting more than one RTK could be required for therapeutic efficacy. Moreover, increased expression or activation of RTKs is often associated with resistance to standard chemotherapy agents or signal transduction modulators. S 49076 is a novel, potent, ATP-competitive tyrosine kinase inhibitor of the RTKs MET, AXL and FGFR. S 49076 blocks autophosphorylation of these RTKs and their downstream signaling in cells with IC 50 values of less than 50 nM in the case of MET and AXL, and at below 200 nM for FGFR1/2/3. In vitro, S 49076 inhibits the proliferation of MET- and FGFR2- dependent gastric cancer cells, blocks MET-driven migration of lung carcinoma cells and inhibits colony formation of hepatocarcinoma cells overexpressing FGFR2 and AXL. In vivo, oral administration of S 49076 inhibits MET autophosphorylation and tumor growth in subcutaneous GTL-16 human gastric carcinoma and U87-MG human glioblastoma at 6 mg/kg/day. In FGFR2-dependent SNU-16 gastric tumors S 49076 inhibits FGFR2 autophosphorylation, downstream signaling and tumor growth at 25 mg/kg/day. In a panel of 53 patient-derived tumors and 14 cell lines of diverse origin growing in three-dimensional in vitro culture, twenty-five (37%) were found to be sensitive to S 49076 at 1 µM or less. In the majority of cases, analysis of the expression, activation and mutation status of MET, AXL, FGFR1/2/3 and other target kinases of S 49076 as well as that of major signaling proteins enabled hypotheses to be made concerning the sensitivity or resistance to S 49076. Moreover, in many of these resistant tumors, a rationale for association of S 49076 with other targeted therapies emerged. Examples of the efficacy of such combined therapies will be shown. Based on these preclinical studies showing a favorable and novel pharmacological profile of S 49076, a phase I study is currently underway in patients with advanced solid tumors. 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 848. doi:1538-7445.AM2012-848

Abstract A238: S 49076, a novel, potent, MET/FGFR/AXL kinase inhibitor with wide antitumor activity.

Receptor tyrosine kinases (RTKs) are key regulators of a multitude of cell processes, including survival, proliferation, migration, invasion and angiogenesis. Aberrant activity of certain of these receptor tyrosine kinases has been associated with tumor progression in a wide variety of human malignancies, making them promising drug targets for cancer therapy. Although, in some instances, specific inhibition of just one of these RTKs suffices for inhibition of tumor progression, in the majority of cases, targeting more than one RTK could be required for therapeutic efficacy. These RTKs include MET, the receptor for hepatocyte growth factor (HGF), the fibroblast growth factor receptor, FGFR, and AXL. Dysregulation of MET activity, due to its overexpression or mutation, or overexpression of its ligand, has been consistently associated with aggressive phenotype, resistance to certain anti-cancer therapies and poor outcome. The FGFRs have a well-documented role in tumor angiogenesis, and, more recently, have been implicated directly in tumor cell survival, proliferation and metastasis in a wide range of tumor types. AXL is one of a family of three tyrosine kinase receptors (TAM family) involved in the pathogenesis of several human cancers and has also recently incited interest as a cancer therapeutic target. S 49076 is a novel, potent, ATP-competitive tyrosine kinase inhibitor of MET, FGFR1/2/3 and AXL. S 49076 blocks autophosphorylation of these RTKs and their downstream signaling in cells with IC 50 values of between 1 and 200 nM depending on the target and cell line. Furthermore, in kinase binding assays, S 49076 also binds to clinically-relevant MET mutated isoforms and a number of other kinases implicated in cancer pathology at concentrations of less than 100 nM. S 49076 is not, however, a potent inhibitor of VEGFR2. Although VEGFR2 is implicated in tumor angiogenesis, it also plays a major physiological role in the maintenance of vascular tone, and its inhibition by other RTK inhibitors has limited dosing of these molecules in the clinic. The unique inhibition profile of S 49076 may allow inhibition of oncogenic RTKs potentially without toxic effects encountered by VEGFR2 inhibitors. In vitro, S 49076 inhibits the proliferation of MET- and FGFR2-dependent gastric cancer cells, blocks MET-driven migration of lung carcinoma cells and inhibits colony formation of AXL-overexpressing hepatocarcinoma cells. In vivo, oral administration of S 49076 inhibits > 80% MET autophosphorylation in subcutaneous GTL-16 human gastric carcinoma tumors at 3 mg/kg. In both this GTL-16 model and in the MET-dependent U87-MG human glioblastoma model, S 49076 inhibits > 80% tumor growth at 6 mg/kg/day. S 49076 also inhibits FGFR2 autophosphorylation, downstream signaling and tumor growth in FGFR2-dependent SNU-16 gastric tumors, and tumor growth in LS-174T colon carcinoma. Based on these preclinical studies showing a favorable and novel pharmacological profile and the potential of S 49076 as innovative anticancer agent, a phase I study is planned soon to evaluate an oral formulation of S 49076 in patients with advanced solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A238.