Periodic Reporting for period 3 - BEAT (The functional interaction of EGFR and beta-catenin signalling in colorectal cancer: Genetics, mechanisms, and therapeutic potential.) Période du rapport: 2020-10-01 au 2022-03-31 Résumé du contexte et des objectifs généraux du projet Monoclonal antibodies against the EGF receptor (EGFR) provide substantive benefit to patients with metastatic colorectal cancer (mCRC). However, no genetic lesions that robustly predict ‘addiction’ to the EGFR pathway have been yet identified. Further, even in tumours that regress after EGFR blockade, subsets of drug-tolerant cells often linger and foster ‘minimal residual disease’ (MRD), which portends tumour relapse.Our preliminary evidence suggested that reliance on EGFR activity, as opposed to MRD persistence, could be assisted by variations in transcription factor partnerships, gene expression outputs, and biological fates related to the WNT/beta-catenin pathway. Building upon such premises, BEAT aims at elucidating the mechanisms of EGFR dependency, and escape from it, with the aim of identifying biomarkers for more efficient clinical management of CRC and developing new therapies for MRD eradication.A multidisciplinary approach is pursued spanning from integrative gene regulation analyses to functional genomics in vitro, pharmacological experiments in vivo, and clinical investigation, to address whether: (i) specific genetic alterations of the WNT pathway (and in particular TCF7L2) affect anti-EGFR sensitivity and CRC progression; (ii) combined neutralisation of EGFR and compensatory prosurvival pathways can lead to MRD deterioration; (iii) data from analysis of this synergy can lead to the discovery of clinically meaningful biomarkers with predictive and prognostic significance.This proposal capitalises on a unique proprietary platform for high-content studies based on a large biobank of viable CRC samples, which ensures strong analytical power together with unprecedented biological flexibility. By providing fresh insight into the mechanisms whereby the WNT/beta-catenin pathway influence EGFR dependency or drug tolerance, the project is expected to put forward an innovative reinterpretation of CRC molecular bases and advance the rational application of more effective therapies. Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent The most relevant results obtained in the first 30 months of BEAT focus on the characterization of the drug tolerant phenotype induced by the anti-EGFR antibody cetuximab in EGFR-dependent CRC models and on the identification of new vulnerabilities for MRD eradication (AIM 2 of the BEAT proposal).We disproved our initial hypothesis that beta-catenin directly sustains MRD in EGFR-dependent tumours treated with cetuximab. However, thanks to this initial failure, we were then able to identify the master regulator of the phenotypic switch accompanying acquisition of tolerance to cetuximab in CRC (Yes-associated protein YAP, whose inhibition is also the underlying cause of beta-catenin upregulation) and we identified alternative vulnerabilities (HER2 and HER3) which can be effectively exploited for combination therapies.We initially observed that CRC cells surviving EGFR inhibition exhibited gene expression traits reminiscent of those displayed by a quiescent subpopulation of normal intestinal secretory precursors with Paneth-cell characteristics. Mechanistically, we demonstrated that residual tumour reprogramming was mediated by inactivation of YAP – a master regulator of post-injury intestinal epithelium recovery – following EGFR neutralisation.These pseudodifferentiated remnants had reduced expression of EGFR-activating ligands, but also displayed a more pronounced activity of human epidermal growth factor 2 (HER2) and human epidermal growth factor receptor 3 (HER3) compared with untreated tumours. Through preclinical trials in PDXs, we showed that Pan-HER antibodies minimised residual disease and induced long-term tumour control after treatment discontinuation.Such results have been recently accepted for publication (Lupo et al., Science Translational Medicine, in press).Besides this, we were able to demonstrate that genetic alterations of the beta-catenin transcription partner TCF7L2 can induce addiction in CRC patient-derived models. Moreover, we set up methods to study how TCF7L2 DNA binding and transcriptional activity are affected by specific genetic alterations, as planned in the original proposal (AIM 1).Finally, we exploited biopsies from patients undergoing treatment with anti-EGFR therapy to validate the biomarkers of drug tolerance identified in context of AIM 2 (AIM 3). Moreover, by extending our collection of PDXs annotated for sensitivity to cetuximab, we prospectively validated the association between TCF7L2 genetic alterations and EGFR-dependency in CRC (AIM 3). We also observed that the extent of pro-apoptotic priming induced by cetuximab positively correlates with the extent of tumour regression in response to therapy, suggesting that therapeutic combinations with BH3 mimetics could further extend the duration of responses (AIM 2 and AIM 3). Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent) By showing that tolerance to EGFR inhibition is typified by the disengagement of an in-built lineage program that drives both regenerative signals during intestinal repair and EGFR-dependent tumorigenesis, our results shed light onto CRC lineage plasticity as an adaptive escape mechanism from therapeutic insults and suggest opportunities to target residual disease.We are currently exploring the mechanistic bases of TCF7L2 dependency in CRC and the relationship between TCF7L2 genetic alterations and EGFR autocrine activation. Morerover, we are investigating the therapeutic potential of BH3 mimetics combined with anti-EGFR and/or anti-HER2/HER3 therapies for eradication of residual disease. Schema representing the feedforward loop between EGFR and YAP in EGFR-dependent CRC.
