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Hormone or endocrine therapy is the primary treatment for estrogen receptor-positive (ER+) breast cancer. Selective Estrogen Receptor Modulators (SERMs) have been used for women with ER+ invasive cancer in the adjuvant setting. However, they do not fully inhibit ER transcriptional activity. Therefore, selective estrogen receptor degraders (SERDs) such as fulvestrant have been developed to overcome the partial modulation of ER transcriptional activity by SERMs. However, the clinical benefit of fulvestrant is limited by its pharmaceutical properties, and burden of intramuscular administration. We developed amcenestrant (SAR439859), a novel, orally bioavailable SERD to improve drug properties and is under clinical investigation.To assess the relative ER modulating activity, we developed a gene signature by transcriptional profiling of multiple ER+ cell lines. We identified 87 genes that were either up- or down-regulated by estradiol and reversed by SERM and SERD compounds or differentially expressed between a SERM/SERD compound and estradiol. We assessed ER activity scores by applying Gene Set Variation Analysis (GSVA) method to the ER gene signature and evaluated the effect of 6 SERD compounds. Amcenestrant and fulvestrant illuminated a deeper inhibition of ER activity compared to the other compounds. In addition, these ER activity scores could potentially be utilized in clinics as pharmacodynamic (PD) biomarker to assess target engagement. We applied this ER signature to RNA-seq data from paired pre- and post-treatment tumor biopsies from patients of NCT03284957 study (AMEERA-1), an open-label, phase 1/2 study of amcenestrant in postmenopausal women with ER+/HER2- metastatic breast cancer. The results of our analysis showed that ER activity is down-regulated by amcenestrant (oral SERD) for 3 out of 5 patients, in parallel with clinical benefit. Validation in a larger patient cohort is needed. Citation Format: Joon Sang Lee, Maysoun Shomali, Monsif Bouaboula, Emma Wang, Wilson Dos-Santos-Bele, Colette Dib, Eric Boitier, Vasiliki Pelekanou, Nils Ternes, Alice Gosselin, Patrick Cohen, Marina Celanovic, Christopher Soria, Alexei Protopopov, Jack Pollard. Development of a gene signature assessing ER modulation by SERMs and SERDs as a target engagement biomarker for endocrine therapy in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 406.

Over the past years, the HIPPO tumor suppressor pathway and its downstream effector Yes1-associated transcriptional regulator (YAP1) present increasing interest in cancer research. The YAP1-TEAD complex regulates transcription for cell proliferation, survival, plasticity, and migration. Aberrant activation of YAP1 has been reported for multiple different cancer types. To estimate the proportion of cases where YAP1/TEAD-dependent transcription may be responsible for tumor development or evolution and evaluate the pharmacodynamics of novel TEAD-inhibitors, we have identified a transcriptional signature of YAP1 activity. The signature comprises 500 genes, positive and negative downstream effectors at about equal proportions, selected by differential expression analyses from 10 published datasets reporting, in total, 19 YAP1-activation and 18 YAP1-inhibition experiments. YAP1/TEAD-dependent transcription was scored in single samples as the difference Rp - Rn, where Rp is the mean fractional rank of the positive YAP1-effectors, and Rn, that of the negative effectors. We so scored, and called, YAP1 activation in normal tissues (GTEx collection; N~9000), in primary, recurrent, or metastatic tumors (TCGA; N~10000), and in cell lines (CCLE; N~1000) for the selection of models. We further applied the signature to follow the degree of YAP1 inhibition in cell and tumor models either after genetic knockdown of HIPPO-YAP1 pathway components or by pharmacologic inhibition using small-molecule TEAD-inhibitors. Our data confirm the robustness of the YAP1-transcription signature as a biomarker for both, tumor indication selection and pharmacodynamics of YAP1-TEAD activity. Citation Format: Emmanuel Spanakis, Loreley Calvet, Odette Dos Santos, Colette Dib, Sukhvinder Sidhu, Jurgen Moll, Laurent Debussche, Jack Pollard, Iris Valtingojer. A transcriptomic signature for measuring YAP1 activity in patient samples and tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2161.

Yes associated protein YAP1 (YAP1) is a cofactor of gene transcription and exerts its action through association with transcription factors from the TEAD family. Activity of YAP1-TEAD is tightly regulated by the HIPPO pathway and upon activation the expression of pro-survival and anti-apoptotic genes is induced. The HIPPO pathway consists of a series of Ser/Thr kinases, which lead to the phosphorylation of YAP1, and hence to its inactivation by either sequestration in the cytoplasm, or degradation. HIPPO pathway deletions and YAP1 activation are particularly prevalent (> 60 %) in patients with malignant pleural mesothelioma. We use a malignant mesothelioma tumor cell line with a HIPPO deleted genetic background (MSTO-211H, LATS1 loss) to demonstrate, that downregulation of YAP1 leads to the inhibition of YAP1-dependent gene expression, the induction of apoptosis and the inhibition of cell growth in vitro. When these cell lines are grown as an in vivo subcutaneous xenograft in mice, downregulation of YAP1 leads to the inhibition of YAP1-dependent gene expression and eventually results in the regression of established tumors. This effect is specific to YAP1 activated mesothelioma models and is not observed in the HIPPO pathway independent HCT116 colon cancer model. We show here, that YAP1 activity alone is required for tumor maintenance in vivo in the context of HIPPO pathway genetic alterations, and thus confirm the relevance of this target for a drug discovery approach. Citation Format: Loreley Calvet, Odette Dos Santos, Véronique Jean-Baptiste, Emmanuel Spanakis, Yvette Ruffin, Isabelle Sanchez, Jessica Mestadier, Stéphane Soubigou, Sylvie Feteanu, Pascale Picard, Chagri Boumaya, Jack Pollard, Colette Dib, Sukhvinder Sidhu, Laurent Debussche, Iris Valtingojer. Oncogenic HIPPO-YAP1: in vivo target validation of YAP1 in malignant mesothelioma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4858.

The field of liquid biopsies is of enormous interest in cancer using a noninvasive process. There are currently three major approaches: Circulating Cell Tumors (CTCs), circulating cell free DNA (cfDNA) and Extracellular Vesicles (EVs) such as exosomes, microvesicles and apoptotic bodies. The EVs are cell-derived small membrane vesicles secreted by cells that convey biological messages, either by surface-to-surface interaction or by shuttling bioactive molecules to a recipient cell's cytoplasm. The EVs released by cells harbor the cargos of their original cells and could be a good tool to follow drug activity. We are developing anti-TGFβ antibody SAR439459 for the treatment of cancer (NCT03192345), and in this vitro study we explore the effect of anti-TGFβ antibody in EVs carrying TGFβ. The cargo of EVs were analyzed by an Elisa test for both latent and active free TGFβ and by miRNA expression. Cell lines were selected based on their expression of TGFβ (A549, DU45, PC3, MCF7 and Caco2), treated for 72h and compared with respective untreated controls. The EVs were isolated from the supernatant and purified. Nanoparticle Tracking Analysis was used to quantify and determine size distribution of EVs. The expression of active free and latent TGFβ was done using Elisa tests. TGFβ antibody-coated magnetic beads were used to capture EVs, and miRNA expression sequencing was done using the Illumina NextSeq. Anti-TGFβ treatment reduced notably the expression of latent TGFβ in the supernatant of cultured tumor cells; the active free TGFβ was very low expressed or not detected in the supernatants. EVs express both latent and active free TGFβ; the anti-TGFβ antibody treatment significantly reduced the active free TGFβ and altered miRNA expression in EVs. Finally, using TGFβ Antibody-coated magnetic beads, we demonstrate that intact EVs express TGFβ at their surface. In conclusion, these results suggest that EVs express TGFβ at their surface and carry the active free TGFβ. Finally, the expression of active free TGFβ in EVs is decreased with anti-TGFβ antibody, suggesting that it might be a biomarker for monitoring anti-TGFβ antibody activity in patients by liquid biopsies. Citation Format: Manoel Nunes, Camille Bernard, Gaelle Muzard, Wilson Dos-Santos-Bele, Jack Pollard, Alexei Protopopov, Colette Dib. Tumor-derived Extracellular Vesicles (EVs) expressing TGFb as potential biomarkers for anti-TGFb antibody activity and drug activity in liquid biopsy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 773.

GPR88, coding for a G protein-coupled orphan receptor that is highly represented in the striatum, is a strong functional candidate gene for neuropsychiatric disorders and is located at 1p22-p21, a chromosomal region that we have previously linked to bipolar disorder (BD) in the Sardinian population. In order to ascertain the relevance of GPR88 as a risk factor for psychiatric diseases, we performed a genetic association analysis between GPR88 and BD in a sample of triads (patient and both parents) recruited in the Sardinian and the Palestinian population as well as between GPR88 and schizophrenia (SZ) in triads from the Xhosa population in South Africa. We found a positive association between GPR88 and BD in the Sardinian and Palestinian triads. Moreover, we found a positive association between GPR88 and SZ in triads from the Xhosa population in South Africa. When these results were corrected for multiple testing, the association between GPR88 and BD was maintained in the Palestinian population. Thus, these results suggest that GPR88 deserves consideration as a candidate gene for psychiatric diseases and requires to be further investigated in other populations.

Alzheimer's disease is a genetically complex disorder, for which new putative susceptibility genes are constantly proposed in the literature. We selected 16 candidate genes involved in biological pathways closely related to the pathology, and for which a genetic association with Alzheimer's disease was previously detected: ACE, BACE1, BDNF, ECE1, HSPG2, IDE, IL1a, IL6, IL10, MAPT, PLAU, PrnP, PSEN1, SORL1, TFCP2 and TGFb1. The variants originally associated with the disease were genotyped in a French Caucasian sample including 428 cases and 475 controls and tested for association in order to replicate the initial results. Despite a careful replication study design, we failed to validate the initial findings for any of these variants, with the possible exception of MAPT, SORL1 and TFCP2 for which some nominal but inconsistent evidence of association was observed.

Development of targeted therapeutics required translationally relevant preclinical models with well-characterized cancer genome alterations. Here, by studying 52 colorectal patient-derived tumor xenografts (PDX), we examined key molecular alterations of the IGF2–PI3K and ERBB–RAS pathways and response to cetuximab. PDX molecular data were compared with that published for patient colorectal tumors in The Cancer Genome Atlas. We demonstrated a significant pattern of mutual exclusivity of genomic abnormalities in the IGF2–PI3K and ERBB–RAS pathways. The genomic anomaly frequencies observed in microsatellite stable PDX reproduce those detected in nonhypermutated patient tumors. We found frequent IGF2 upregulation (16%), which was mutually exclusive with IRS2, PIK3CA, PTEN, and INPP4B alterations, supporting IGF2 as a potential drug target. In addition to maintaining the genomic and histologic diversity, correct preclinical models need to reproduce drug response observed in patients. Responses of PDXs to cetuximab recapitulate also clinical data in patients, with partial or complete response in 15% (8 of 52) of PDXs and response strictly restricted to KRAS wild-type models. The response rate reaches 53% (8 of 15) when KRAS, BRAF, and NRAS mutations are concomitantly excluded, proving a functional cross-validation of predictive biomarkers obtained retrospectively in patients. Collectively, these results show that, because of their clinical relevance, colorectal PDXs are appropriate tools to identify both new targets, like IGF2, and predictive biomarkers of response/resistance to targeted therapies. Cancer Res; 75(8); 1560–6. ©2015 AACR.

The discovery of the genetic factors implicated in the predisposition to complex diseases may greatly profit from genetic studies in isolated populations. In this perspective, we performed a genome-wide scan using 507 microsatellite markers, with an average interval size of 7.6 cM, on a sample of 88 nuclear families with at least two affected sibs with bipolar disorder recruited in the Sardinian population. An initial analysis yielded non-parametric linkage exceeding 3.4 with P-values

SummaryCeliac disease (CD), a malabsorption disorder of the small intestine, results from ingestion of gluten. The HLA risk factors involved in CD are well known but do not explain the entire genetic susceptibility. To determine the localization of other genetic risk factors, a systematic screening of the genome has been undertaken. The typing information of 281 markers on 110 affected sib pairs and their parents was used to test linkage. Systematic linkage analysis was first performed on 39 pairs in which both sibs had a symptomatic form of CD. Replication of the regions of interest was then carried out on 71 pairs in which one sib had a symptomatic form and the other a silent form of CD. In addition to the HLA loci, our study suggests that a risk factor in 5qter is involved in both forms of CD (symptomatic and silent). Furthermore, a factor on 11qter possibly differentiates the two forms. In contrast, none of the regions recently published was confirmed by the present screening.

Aflibercept (also called VEGF Trap) is a novel anti-angiogenic agent currently tested in combination with standard chemotherapy in several phase 3 clinical trials including second-line metastatic colorectal cancer. It is a recombinant fusion protein consisting of Vascular Endothelial Growth Factor (VEGF)-binding portions from the extracellular domains of human VEGF Receptors 1 and 2 fused to the Fc portion of the human IgG1. Aflibercept acts as a soluble decoy receptor that binds to VEGF-A, with high affinity, as well as the related ligands PIGF and VEGF-B. It neutralizes all isoforms of VEGF-A, blocks angiogenesis and effectively suppresses tumor growth in vivo. Aflibercept also binds Placental Growth Factor (P1GF), which has been involved in pathological angiogenesis and tumor progression. Preclinical studies were performed to assess the influence of somatic mutations on the activity of aflibercept against the growth of 37 human tumor xenografts in mice covering 17 different indications. These models were previously documented for KRAS, BRAF, and PIK3CA activating mutations and PTEN protein deficiency according to the Catalogue of Somatic Mutations in Cancer (COSMIC) database, literature, and internal studies. Aflibercept was administered twice weekly at 40 mg/kg. Aflibercept was found highly active in 6 models with tumor growth inhibition (ΔT/ΔC) ≤ 0%, very active in 9 models (ΔT/ΔC 0–10%), displayed some activity in 17 models (ΔT/ΔC 10–40%), and had no effect in 5 xenograft models (ΔT/ΔC > 40%). We stratified the models into two groups according to the tumor response: the “partially reponsive and non-responsive models” with detectable tumor growth under treatment (22/37 models), and the “responsive models” with no detectable tumor growth or regression under treatment (15/37 models). Mutations in KRAS (G12D/V/S, G13D, Q61H) were present in both responsive (3 models) and partially responsive/non-responsive models (4 models). Ten of 28 (36%) KRAS wild-type tumors and 3 of 7 (43%) KRAS mutated tumors were responsive to aflibercept indicating that KRAS status has no significant influence on aflibercept activity on tumors (p=1; Fisher exact test). Similarly, tumor models were also found sensitive to the anti-tumor effects of aflibercept whatever the BRAF and PIK3CA genotype status, and PTEN protein expression status. In summary, the status of KRAS, BRAF, PIK3CA and PTEN in these preclinical tumor models did not significantly influence the response to aflibercept. The impact of these genetic alterations on aflibercept activity will be further explored in clinical trials. 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 C162.