Periodic Reporting for period 4 - BEAT (The functional interaction of EGFR and beta-catenin signalling in colorectal cancer: Genetics, mechanisms, and therapeutic potential.)
Reporting period: 2022-04-01 to 2023-09-30
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.
In the course of the project, we leveraged a multidisciplinary approach spanning from integrative gene regulation analyses to functional genomics in vitro, pharmacological experiments in vivo, and clinical investigation, to investigate EGFR dependency in colorectal tumours and the molecular based that can lead do resistance. We capitalized on a unique proprietary platform for high-content studies based on a large biobank of viable CRC samples, both in the form of patient-derived xenografts and 3D tumoroids which ensures strong analytical power together with unprecedented biological flexibility.
Through all this, we provided fresh insight into the mechanisms of drug tolerance in the context of EGFR dependency in colorectal tumours. By studying the residual disease of tumours that underwent partial regression upon EGFR inhibition, we identified several adaptive responses to treatment that allowed the development of novel rational approaches for disease eradication, which are now under consideration of the design of dedicated phase II clinical trials. Moreover, we discovered a previously overlooled heterogeneity in the rate of accumulation of new genetic variants in colorectal cancer patients. This may have major implications into the dynamics of acquisition of secondary resistance. Overall, we are convinced that the project outcomes put forward an innovative reinterpretation of CRC molecular bases and have the potential to advance the rational application of more effective therapies.
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.
In the course of the project, we leveraged a multidisciplinary approach spanning from integrative gene regulation analyses to functional genomics in vitro, pharmacological experiments in vivo, and clinical investigation, to investigate EGFR dependency in colorectal tumours and the molecular based that can lead do resistance. We capitalized on a unique proprietary platform for high-content studies based on a large biobank of viable CRC samples, both in the form of patient-derived xenografts and 3D tumoroids which ensures strong analytical power together with unprecedented biological flexibility.
Through all this, we provided fresh insight into the mechanisms of drug tolerance in the context of EGFR dependency in colorectal tumours. By studying the residual disease of tumours that underwent partial regression upon EGFR inhibition, we identified several adaptive responses to treatment that allowed the development of novel rational approaches for disease eradication, which are now under consideration of the design of dedicated phase II clinical trials. Moreover, we discovered a previously overlooled heterogeneity in the rate of accumulation of new genetic variants in colorectal cancer patients. This may have major implications into the dynamics of acquisition of secondary resistance. Overall, we are convinced that the project outcomes put forward an innovative reinterpretation of CRC molecular bases and have the potential to advance the rational application of more effective therapies.
The most relevant results obtained in the course of the BEAT are related to the characterization of the drug tolerant phenotype induced by the anti-EGFR antibody cetuximab in EGFR-dependent CRC models and on the molecular mechanisms that lead to the accumulation of new genetic variants in colorectal cancer cells.
We identified 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. (Lupo et al., Science Translational Medicine, 2020).
Moreover, to better investigate the molecular mechanisms underlying drug tolerance, starting from our PDXs we developed a platform of more than 100 patient-derived organoids (PDOs) that are amenable to genetic manipulations and in vitro studies (Leto et al, under revision in Nature Communications, https://www.biorxiv.org/content/10.1101/2023.07.10.548375v1). Through such platform, we demonstrated that the extent of response to EGFR blockade in mCRC correlates with the level of pro-apoptotic priming induced by cetuximab. Thanks to this finding, we found that BCLXL inhibitors can effectively improve the therapeutic effectiveness of EGFR blockade in terms of both extent and duration of the response (Leto et al., Clinical Cancer Research 2023).
We also exploited single-cell RNAseq to assess the functional heterogeneity of CRC cancer cells treated or not treated with cetuximab. We demonstrated that, in organoids responsive to cetuximab, treatment can induce an enrichment of slowly-cycling cells that are characterized by Paneth cell-like traits. By longitudinal tracing, based on CRISPR/Cas9 genome editing, of the Paneth cell-like state we showed that the Paneth cell-like phenotype is induced by cetuximab in a minority of CRC cells that are unevenly distributed across organoids in the same culture. Organoids containing such cells were significantly more resilient to cetuximab than their negative counterparts. This suggested that Paneth cell-like cells exerted some sort of protective effect on their neighbors. This finding was corroborated by the observation that CRISPR/Cas9-mediated knock-out of ATOH1 (the master regulator of the Paneth cell-like phenotype) not only abrogated the induction of Paneth cell-like cells upon cetuximab treatment, but also impaired the viability of the other cells in the presence of the drug. We speculate that Paneth cell-like cells produce some soluble factors that provide protective cues to their neighborhood (Catalano et al., in preparation), possibly resulting in HER2 and BCLXL activation (see above). Studies are ongoing to identify such factor(s), which represent ideal candidate targets for combination therapies.
Finally, we investigated how quiescent persistors can become overtly resistant to treatment. We showed that cells in a drug-tolerant state can increase their mutation rate (MR), thus increasing the chances to acquire resistance-causing mutations (Russo et al., 2019 and 2022). We thus decided to measure the MR in a cohort of CRC PDOs through mutation accumulation experiments. We discovered that MRs are heterogeneous across tumors. Most importantly, our data suggest that higher mutation rates are selected during metastatization, which may have major implications in terms of acquisition of resistance and response to immunotherapy (Grassi et al., submitted to Nature).
We identified 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. (Lupo et al., Science Translational Medicine, 2020).
Moreover, to better investigate the molecular mechanisms underlying drug tolerance, starting from our PDXs we developed a platform of more than 100 patient-derived organoids (PDOs) that are amenable to genetic manipulations and in vitro studies (Leto et al, under revision in Nature Communications, https://www.biorxiv.org/content/10.1101/2023.07.10.548375v1). Through such platform, we demonstrated that the extent of response to EGFR blockade in mCRC correlates with the level of pro-apoptotic priming induced by cetuximab. Thanks to this finding, we found that BCLXL inhibitors can effectively improve the therapeutic effectiveness of EGFR blockade in terms of both extent and duration of the response (Leto et al., Clinical Cancer Research 2023).
We also exploited single-cell RNAseq to assess the functional heterogeneity of CRC cancer cells treated or not treated with cetuximab. We demonstrated that, in organoids responsive to cetuximab, treatment can induce an enrichment of slowly-cycling cells that are characterized by Paneth cell-like traits. By longitudinal tracing, based on CRISPR/Cas9 genome editing, of the Paneth cell-like state we showed that the Paneth cell-like phenotype is induced by cetuximab in a minority of CRC cells that are unevenly distributed across organoids in the same culture. Organoids containing such cells were significantly more resilient to cetuximab than their negative counterparts. This suggested that Paneth cell-like cells exerted some sort of protective effect on their neighbors. This finding was corroborated by the observation that CRISPR/Cas9-mediated knock-out of ATOH1 (the master regulator of the Paneth cell-like phenotype) not only abrogated the induction of Paneth cell-like cells upon cetuximab treatment, but also impaired the viability of the other cells in the presence of the drug. We speculate that Paneth cell-like cells produce some soluble factors that provide protective cues to their neighborhood (Catalano et al., in preparation), possibly resulting in HER2 and BCLXL activation (see above). Studies are ongoing to identify such factor(s), which represent ideal candidate targets for combination therapies.
Finally, we investigated how quiescent persistors can become overtly resistant to treatment. We showed that cells in a drug-tolerant state can increase their mutation rate (MR), thus increasing the chances to acquire resistance-causing mutations (Russo et al., 2019 and 2022). We thus decided to measure the MR in a cohort of CRC PDOs through mutation accumulation experiments. We discovered that MRs are heterogeneous across tumors. Most importantly, our data suggest that higher mutation rates are selected during metastatization, which may have major implications in terms of acquisition of resistance and response to immunotherapy (Grassi et al., submitted to Nature).
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 possibility to exploit therapeutic potential of BH3 mimetics combined with anti-EGFR and/or anti-HER2/HER3 therapies for eradication of residual disease through the design of dedicated Phase II clinical trials.
We are currently exploring the possibility to exploit therapeutic potential of BH3 mimetics combined with anti-EGFR and/or anti-HER2/HER3 therapies for eradication of residual disease through the design of dedicated Phase II clinical trials.