Patient derived organoids reveal that PI3K/AKT signaling is an escape pathway for radioresistance and a target for therapy in rectal cancer.

Partial or total resistance to preoperative chemoradiotherapy occurs in more than half of locally advanced rectal cancer patients. Several novel or repurposed drugs have been trialled to improve cancer cell sensitivity to radiotherapy, with limited success. To understand the mechanisms underlying this resistance and target them effectively, we initially compared treatment-naive transcriptomes of radiation-resistant and radiation-sensitive patient-derived organoids (PDO) to identify biological pathways involved in radiation resistance. Pathway analysis revealed that PI3K/AKT/mTOR and epithelial mesenchymal transition pathway genes were upregulated in radioresistant PDOs. Moreover, single-cell sequencing of pre & post-irradiation PDOs showed mTORC1 upregulation, which was confirmed by a genome-wide CRSIPR-Cas9 knockout screen using irradiated colorectal cancer (CRC) cell lines. Based on these findings, we evaluated cancer cell viability in vitro when treated with radiation in combination with dual PI3K/mTOR inhibitors apitolisib or dactolisib. Significant AKT phosphorylation was detected in HCT116 cells two hours post-irradiation (p=0.027). Dual PI3K/mTOR inhibitors radiosensitised HCT116 and radiation-resistant PDO lines. The PI3K/AKT/mTOR pathway upregulation contributes to radioresistance and its pharmacological inhibition leads to significant radiosensitisation in an organoid model of CRC and is a target for clinical trials.