Hélène Goulaouic

A highly potent and selective Vps34 inhibitor alters vesicle trafficking and autophagy

Vps34 is a phosphoinositide 3-kinase (PI3K) class III isoform that has attracted major attention over the recent years because of its role in autophagy. Herein we describe the biological characterization of SAR405, which is a low-molecular-mass kinase inhibitor of Vps34 (KD 1.5 nM). This compound has an exquisite protein and lipid kinase selectivity profile that is explained by its unique binding mode and molecular interactions within the ATP binding cleft of human Vps34. To the best of our knowledge, this is the first potent and specific Vps34 inhibitor described so far. Our results demonstrate that inhibition of Vps34 kinase activity by SAR405 affects both late endosome-lysosome compartments and prevents autophagy. Moreover, we show that the concomitant inhibition of Vps34 and mTOR, with SAR405 and the US Food and Drug Administration-approved mTOR inhibitor everolimus, results in synergistic antiproliferative activity in renal tumor cell lines, indicating a potential clinical application in cancer.

MET Genetic Abnormalities Unreliable for Patient Selection for Therapeutic Intervention in Oropharyngeal Squamous Cell Carcinoma

Background Identification of MET genetic alteration, mutation, or amplification in oropharyngeal squamous cell carcinoma (OPSCC) could lead to development of MET selective kinase inhibitors. The aim of this study was to assess the frequency and prognostic value of MET gene mutation, amplification, and protein expression in primary OPSCC. Methods A retrospective chart review was conducted of patients treated for single primary OPSCC between January 2007 and December 2009. Pre-treatment OPSCC tissue samples were analyzed for MET mutations, gene amplification, and overexpression using Sanger sequencing, FISH analysis, and immunohistochemistry respectively. Univariate and multivariate analyses were used to analyze correlations between molecular abnormalities and patient survival. Results 143 patients were included in this study. Six cases (4%) were identified that had a genetic variation, but previously described mutations such as p.Tyr1235Asp (Y1235D) or p.Tyr1230Cys (Y1230C) were not detected. There were 15 high polysomy cases, and only 3 cases met the criteria for true MET amplification, with ≥10% amplified cells per case. Immunohistochemistry evaluation showed 43% of cases were c-MET negative and in 57% c-MET was observed at the tumor cell level. Multivariate analysis showed no significant association between MET mutation, amplification, or expression and survival. Conclusions Our study shows a low frequency of MET mutations and amplification in this cohort of OPSCC. There was no significant correlation between MET mutations, amplification, or expression and patient survival. These results suggest that patient selection based on these MET genetic abnormalities may not be a reliable strategy for therapeutic intervention in OPSCC.

Discovery and Pharmacokinetic and Pharmacological Properties of the Potent and Selective MET Kinase Inhibitor 1-{6-[6-(4-Fluorophenyl)-[1,2,4]triazolo[4,3-b]pyridazin-3-ylsulfanyl]benzothiazol-2-yl}-3-(2-morpholin-4-ylethyl)urea (SAR125844).

The HGF/MET pathway is frequently activated in a variety of cancer types. Several selective small molecule inhibitors of the MET kinase are currently in clinical evaluation, in particular for NSCLC, liver, and gastric cancer patients. We report herein the discovery of a series of triazolopyridazines that are selective inhibitors of wild-type (WT) MET kinase and several clinically relevant mutants. We provide insight into their mode of binding and report unprecedented crystal structures of the Y1230H variant. A multiparametric chemical optimization approach allowed the identification of compound 12 (SAR125844) as a development candidate. In this chemical series, absence of CYP3A4 inhibition was obtained at the expense of satisfactory oral absorption. Compound 12, a promising parenteral agent for the treatment of MET-dependent cancers, promoted sustained target engagement at tolerated doses in a human xenograft tumor model. Preclinical pharmacokinetics conducted in several species were predictive for the observed pharmacokinetic behavior of 12 in cancer patients.

The Selective Intravenous Inhibitor of the MET Tyrosine Kinase SAR125844 Inhibits Tumor Growth in MET-Amplified Cancer

Activation of the MET/HGF pathway is common in human cancer and is thought to promote tumor initiation, metastasis, angiogenesis, and resistance to diverse therapies. We report here the pharmacologic characterization of the triazolopyridazine derivative SAR125844, a potent and highly selective inhibitor of the MET receptor tyrosine kinase (RTK), for intravenous administration. SAR125844 displayed nanomolar activity against the wild-type kinase (IC50 value of 4.2 nmol/L) and the M1250T and Y1235D mutants. Broad biochemical profiling revealed that SAR125844 was highly selective for MET kinase. SAR125844 inhibits MET autophosphorylation in cell-based assays in the nanomolar range, and promotes low nanomolar proapoptotic and antiproliferative activities selectively in cell lines with MET gene amplification or pathway addiction. In two MET-amplified human gastric tumor xenograft models, SNU-5 and Hs 746T, intravenous treatment with SAR125844 leads to potent, dose- and time-dependent inhibition of the MET kinase and to significant impact on downstream PI3K/AKT and RAS/MAPK pathways. Long duration of MET kinase inhibition up to 7 days was achieved with a nanosuspension formulation of SAR125844. Daily or every-2-days intravenous treatment of SAR125844 promoted a dose-dependent tumor regression in MET-amplified human gastric cancer models at tolerated doses without treatment-related body weight loss. Our data demonstrated that SAR125844 is a potent and selective MET kinase inhibitor with a favorable preclinical toxicity profile, supporting its clinical development in patients with MET-amplified and MET pathway–addicted tumors. Mol Cancer Ther; 14(2); 384–94. ©2014 AACR.

Abstract 2911: SAR125844: a potent and selective ATP-competitive inhibitor of MET kinase

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The tyrosine kinase MET is a membrane receptor that is essential for embryonic development and wound healing in normal cells. Stimulation of MET by its natural ligand, the hepatocyte growth factor (HGF), induces cell proliferation, migration, and invasion. Abnormal MET activation (over-expression of MET protein, amplification or mutations of the MET gene) has been observed in multiple human cancer types. We report herein the discovery of a potent and selective small molecule inhibitor (SAR125844) with potential therapeutic application in cancer patients with deregulated MET-dependent malignancies. Our initial hit identification approach was based on the biochemical screen of an in-house kinase inhibitor biased library where a series of benzimidazole sulfonate derivatives were identified with sub-micromolar MET inhibition. In particular, the initial hit exhibited an IC50 of 140 nM vs MET but it also had strong affinity for CDK9 (IC50= 6 nM), a CDK isoform involved in gene transcription. Chemical modifications of the series to dial out CDK9 affinity and remove potential normal cell cytotoxicity led to a more selective derivative with IC50s of 80nM and 1355nM vs MET and CDK9 respectively. Further sub-structural exploration allowed us to identify a heteorocyclic moiety which was shown by X-ray data to specifically interact with Tyr1230 in a non active conformation of the protein. The resulting highly favourable U-shape mode of binding in MET of representative examples from these series (e.g. IC50= 1nM) was not tolerated in CDK9 (IC50 > 10µM). Final multi-parametric medicinal chemistry optimisation led to SAR125844 with single digit nanomolar antiproliferative activity on MET-amplified cell lines. SAR125844 is highly selective for MET kinase in a panel of 275 kinases tested, with only 5 other protein kinases inhibited at IC50 values below 300 nM. This compound exhibits also satisfactory eADMET in vitro properties and has shown moderate total plasma clearance, large volume of distribution and moderate to long terminal elimination half-life in rats. 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 2911. doi:1538-7445.AM2012-2911

Abstract 3550: Inhibition of either LIN28A or ZCCHC11 (TUT4) provides distinct effects on the expression of the let-7 miRNA family and tumor cell proliferation

The LIN28A and LIN28B oncogenes are overexpressed in about 15% of human cancers, and they selectively block the expression of the tumor suppressor miRNA let-7 family, comprising twelve members (let-7a-1, -2, -3, let-7b, let-7c, let-7d, let-7e, let-7f-1, -2, let-7g, let-7i and miR-98) expressed from eight distinct loci. Upon binding to pre-let-7, LIN28A recruits ZCCHC11, a 3′ terminal uridylyl transferase, responsible for let-7 poly-uridylation and subsequent targeting of poly-uridylated let-7 miRNA for degradation. Expression of several let-7 miRNA family members was measured using quantitative RT-PCR in LIN28-positive or -negative tumor cell lines. In agreement with the inverse relationship between LIN28A/B and let-7 expression already observed by others (1), the lowest expression of let-7 miRNA was observed in LIN28A (e.g., IGROV-1 and T-47D) or LIN28B (e.g., NCI-H838, Hep-G2 and NCI-H1299) positive cell lines, and the degree of let-7 miRNA repression in LIN28A or LIN28B positive cell lines was particularly prominent for miR-98, let-7i and let-7b family members. Although the proliferation of LIN28B-positive tumor cell lines was sensitive to the 3 LIN28B siRNAs tested, the expression of LIN28A was not required for the proliferation and survival of LIN28A-expressing tumor cell lines, as demonstrated with one LIN28A siRNA that displayed an efficient knock-down at low concentrations with minimal impact on the proliferation of the T-47D tumor cell line. This LIN28A siRNA was further demonstrated to significantly upregulate the expression of several let-7 miRNAs such as miR-98 or let-7i, supporting an on-target modulation of the pathway. We also tested several siRNAs against ZCCHC11 uridylyl transferase, and found that the growth of either LIN28A- or LIN28B-positive tumor cell lines was inhibited by ZCCHC11 knockdown. However, ZCCHC11 protein knock-down was not able to restore let-7 miRNA expression, as it was seen for the LIN28A knock-down. These findings suggest that the role of ZCCHC11 in tumor cell lines might be more complex than just targeting poly-uridylated let-7 miRNA for degradation. Overall, our results seems to indicate that in LIN28A-positive tumor cell lines, LIN28A knock-down impacts let-7 miRNA expression, but has not a significant antiproliferative effect, whereas ZCCHC11 knock-down inhibits cell proliferation and this effect seems to be disconnected from let-7 miRNAs modulation. This lack of apparent effect on the expression of the miRNA let-7 expression might be related to the dual role of uridylyl transferases recently described for group II let-7 miRNAs, the mono- and poly-uridylation that leads to let-7 biogenesis and let-7 degradation respectively (2). 1- Viswanathan SR, Powers JT, Einhorn W, et al. Nat Genet 2009; 41: 843-848. 2- Heo I, Ha M, Lim J et al. Cell 2012; 151: 521-532. Citation Format: Laurent VIDARD, Claire MARIET, Eric BOITIER, Veronique SIERRA, Elisabeth CAVROIS, Carlos GARCIA-ECHEVERRIA, Helene GOULAOUIC. Inhibition of either LIN28A or ZCCHC11 (TUT4) provides distinct effects on the expression of the let-7 miRNA family and tumor cell proliferation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3550. doi:10.1158/1538-7445.AM2014-3550

Abstract 563: A prospective biomarker study to demonstrate high MET expression and gene amplification in a subset of patients with advanced gastric cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Aim: To characterize molecular features of HGF-MET axis in patients (pts) with advanced gastric cancer (GC). Methods: Pts with advanced GC undergoing routine surgery for tumor staging or resection were studied. Tumor specimens were fixed in 10% neutral buffered formalin immediately. Total (t)- and phosphorylated (p)-MET protein were assayed by IHC, and MET gene copy number by FISH. Circulating tumor cells were enumerated by CellSearch. Blood hepatocyte growth factor (HGF) and shedMET levels were measured at pre-, and post-operatively at 2 and 7 days, and 4 weeks. Biomarkers were correlated with disease stage, tumor size, histopathology, metastasis status, and overall-survival. Results: Forty pts were enrolled from East Asian. The median age was 67 years (range, 31-86 years); 78% were male; 37 and 3 pts had stage III or IV disease, respectively; tumor histopathology was poorly, moderately, or well differentiated in 18, 17, and 4 samples, respectively (1 sample with unknown status). Biomarker results were summarized in Table 1. Five out of 32 evaluable pts (15.6%) had MET gene amplification with amplified tumor cells ranged from 14-100%. Pts who were p-MET positive tended to have moderately differentiated tumors, larger tumor size (>5 cm), and distant metastasis compared with pts who were negative. Pts with MET gene amplification appeared to have distant metastasis, poor survival, and elevated blood CEA levels. Elevated circulating HGF levels were associated with larger tumor size (>5cm). Conclusions: In pts with advanced GC, positive p-MET and MET gene amplification was associated with larger tumor size, distant metastasis, and poor survival. These results support using a molecular basis to identify a subset of pts suitable for anti-MET therapy. This study is sponsored by Sanofi. Table 1: View this table: 1Among 100 nuclei counted, >10% tumor cells with MET gene copy number>4 and MET/CEP7≥2; 2Pts having ≥50% tumor cells with membranous stain level ≥2; 3H-score ≥15 on membrane; 4HGF > 787 pg/mL. Citation Format: Yang Han-Kwang, Hiroya Takeuchi, Seong-Ho Kong, Hyuk-Joon Lee, Elias Shamiyeh, Nikki Daskalakis, Helene Goulaouic, Christelle Castell, Marie-Laure Ozoux, Ju OK Lim, Kaida Wu. A prospective biomarker study to demonstrate high MET expression and gene amplification in a subset of patients with advanced gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 563. doi:10.1158/1538-7445.AM2014-563

Abstract 845: In vitro and in vivo pharmacology of SAR125844, a potent and selective intravenous MET kinase inhibitor undergoing Phase I clinical trial

SAR125844 is a potent MET kinase inhibitor for intravenous (IV) administration, with nanomolar activity against the wild-type enzyme (IC50 = 4.2 nM) and some kinase domain mutants, such as M1250T and Y1235D. It is highly selective for MET kinase in a panel of 275 kinases tested, with only 5 other protein kinases inhibited at IC50 values below 300 nM. In cellular assays, SAR125844 inhibits MET autophosphorylation at the nanomolar range (IC50 from 1.4 to 5.1 nM), translating into antiproliferative activity selectively in MET-driven cell lines such as MET-amplified cell lines, with IC50 values in the low nanomolar range and induction of apoptosis. SAR125844 induces a G1 block in the two MET-amplified tumor cell lines tested. Moreover, the compound is also able to inhibit HGF-induced cell migration in PC-3 prostate tumor cell line. In two MET-amplified human gastric tumor xenograft models tested, SNU-5 and Hs 746T, SAR125844 intravenous treatment leads to potent impact on the MET signaling pathway in a dose and time dependent manner, with potent inhibition of MET autophosphorylation, as well as a significant effect on downstream PI3K/AKT and RAS/MAPK pathways. As a single agent, this translates into a dose-dependent antitumor activity in these two xenograft models, with tumor stasis at the lowest active dose and tumor regression at the highly active doses. High loading single dose administration of SAR125844 using a nanosuspension formulation allowed a sustained P-MET inhibition in tumors up to 7 days. In all models, antitumor activity was achieved at well tolerated doses without significant effect on body weight. The observed correlation between P-MET inhibition in the tumor and antitumor activity in preclinical settings suggests that P-MET evaluation in tumor biopsies could be a direct pharmacodynamic biomarker of SAR125844 activity in patients. In order to investigate whether a non-invasive biomarker could also be used as pharmacodynamic read-out, we measured shed-MET (soluble extracellular domain of the MET receptor) in the plasma of tumor-bearing mice and showed that its level correlates with tumor burden in 2 MET-amplified xenograft models. 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 845. doi:1538-7445.AM2012-845