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(se abrirá en una nueva ventana)). 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).
