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Introduction: GATA3 is critical for the development of the mammary gland and the loss of its expression alters the estrogen receptor (ER) transcriptional program. Approximately 70%-80% of all breast cancers are ER-positive and 15-18% of them harbor GATA3 somatic mutations. Clinically, GATA3 loss defines a subset of patients with poor response to hormonal therapy and poor prognosis. As a transcription factor, however, GATA3 is not pharmacologically targetable. Synthetic lethality refers to the interaction between genetic events in two genes whereby the inactivation of either gene results in a viable phenotype, while their combined inactivation is lethal. This approach enables the indirect targeting of undruggable genes by disrupting their genetic interactors. In this study we sought to define Synthetic lethal partners for GATA3 and explore possible therapeutic targets Methods: Putative synthetic lethal partners for GATA3 were identified using the recently developed SLIdR (Synthetic Lethal Identification in R) algorithm. The synthetic lethal interaction and the anti-tumoral effect of putative partner was evaluated via genetic silencing or pharmacological inhibition using in-vitro, ex-vivo and in-vivo models. Putative mechanisms of action were investigate using RNA sequencing and confirmed using molecular biology technologies. Results: We identify MDM2 as synthetically lethal partner of GATA3 in ER-positive breast cancer. Using a siRNA approach, we first validated in-silico data by confirming that silencing MDM2 significantly reduces cell proliferation of GATA3-mutant in-vitro models by inducing apoptosis. Pharmacological inhibition of MDM2 using three different compounds (RG7388-idasanutlin, RAIN-32 and MI-733) significantly impaired tumor growth in GATA3-deficient models in vitro, in vivo and in patient-derived organoid/xenograft (PDO/PDX) harboring GATA3 somatic mutation. Additionally, we showed that the synthetic lethality between GATA3 and MDM2 is p53-dependent and acts at least partially via the PI3K/Akt/mTOR pathway. This suggests that GATA3 loss-of-function (via genetic alterations or other mechanisms) activates the PI3K/Akt/mTOR pathway and leads to resistance to apoptosis. Conclusion: Our results present MDM2 as a novel therapeutic target in the substantial cohort of ER-positive, GATA3-mutant breast cancer patients. With MDM2 inhibitors widely available, our findings can be rapidly translated into clinical trials to evaluate in-patient efficacy. Citation Format: Mattia Marinucci, Gaia Bianco, Mairene Coto-Llierena, John Gallon, Venkatesh Kancherla, Federica Panebianco, Stephanie Taha-Mehlitz, Sumana Srivatsa, Niko Beerenwinkel, Hesam Montazeri, Vijaya Tirunagaru, Marta De Menna, Caner Ercan, Ahmed Dahmani, Elodie Montaudon, Marianna Kruithof-de Julio, Luigi M. Terracciano, Rinath M. Jeselsohn, Robert C. Doebele, François-Clément Bidard, Elisabetta Marangoni, Charlotte K. Y. Ng, Salvatore Piscuoglio. GATA3 and MDM2 are synthetic lethal in estrogen receptor-positive breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3984.

Introduction: Colorectal cancer is the second cause of cancer related deaths worldwide, with the poor survival outcomes attributable to the presence of metastases. To tackle this problem, (phospho)proteomics analysis enables valuable insights into changes of protein expression and signalling in cancer that can be perturbed by drugs. To study mechanisms driving metastasis and perform subsequent drug testing, patient derived organoids (PDOs) are in development as preclinical models. PDOs are obtained by 3D culture of tumour tissue ex-vivo, which enables them to retain the heterogeneity and architecture of the tumor source. To identify putative drug targets for colon cancer metastasis, we performed comparative (phospho)proteomics analysis of metastatic colon tissues and their matched PDOs. Methods: (Phospho)proteomics profiling was performed on PDOs generated from 10 patients with colon metastases to the liver, 10 matched tumour tissues and 4 normal colon mucosa. Unsupervised analysis of the (phospho)proteomic landscape was used to identify differentially expressed genes and pathways in colon metastases and their matched PDOs, compared to normal colon mucosa. We focused on phosphosites, proteins and pathways showing consistently altered expression in tumour tissues and PDOs. Kinase-substrate enrichment analysis was used to identify aberrantly activated kinases, based on the abundance of phosphorylation on the substrates. Putative drugs were selected using the consensus expression response to multiple small molecule drugs across cell lines and conditions from the Library of Integrated Network-Based Cellular Signatures (LINCS) database. Drug treatment of drug candidates was performed using the MTT Cell Proliferation Assay. Results: Using the approach described above we identified 103 differentially expressed proteins and 236 phosphosites between tumour tissues and normals, which were also detected in PDOs, with overall high correlation of t statistics (0.6 Spearman’s rho) between tumor tissues and PDOs. MYC-targets, G2M checkpoints and E2F-targets were amongst the top positively enriched pathways in common, and the LINCS analysis identified multi-kinase inhibitor Nintedanib and NFKB pathway-targeting KIN0-260 as putative drugs for upregulated proteins. The kinase CSNK2A1 was overactivated in both groups, pointing towards the use of CK2 inhibitors for drug testing. Treatment at 5 days with Nintedanib and KIN001-260 on the PDOs resulted in significant reduction of cell viability compared with 5-FU. Conclusion: Our study provides evidence that PDOs recapitulate relevant tumor features at the proteomic and phosphoproteomic levels, supporting the utility of this ex-vivo model as a tool for drug sensitivity testing for metastatic colon cancer. Citation Format: Gina Faye Boot, Federica Panebianco, John Gallon, Charlotte Kiu Yan Ng, Salvatore Piscuoglio. (Phospho)proteomics profiling of patient-derived colon cancer organoids for the discovery of putative drug targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3074.