PO-381 Response project: chromatin regulators as biomarkers and drug targets in colorectal cancer

Abstract

IntroductionColorectal cancer (CRC) is the third most common cancer worldwide. The majority of patients diagnosed at advanced disease develop primary/acquired resistance. Chromatin-regulatory factors modulate accessibility to DNA and showed some alterations in tumours at variable frequencies.Our aim is to identify which chromatin factors could be novel targets for overcoming resistance to chemotherapy, and if they can be considered as novel biomarkers to predict treatment response. We are using a potent genetic loss-of- function screen that will reveal which chromatin factors lead to sensitisation or resistance in non-treated vs. treated cells with drugs that mimic therapeutic regimes in patients.Material and methodsRESPONSE project is an explorative approach based on a pooled retroviral library containing 7300 short hairpin RNAs (shRNA) targeting 912 chromatin factor genes in an optimised miRNA-backbone that allow maximal knockdown by single copy integration. The experiments were performed using HT-29 CRC cell line, modified with a murine ecotropic receptor (EcoR).Co-infection assays were performed with labelled GFP and mCherry vectors using several viral dilutions to confirm the percentage of virus required for single copy integration. Kill-assays were performed to monitor by flow cytometry the shRNA lethality caused by the knockdown of essential MYC and RPA3 genes compared with neutral shRNA Renilla gene. The half maximal inhibitory concentration (IC50) of 5-Fluoroacil (5-FU), Oxaliplatin (Oxa) and Irinotecan (SN-38) drugs were calculated.FOLFOX (5-FU+Oxa) and FOLFIRI (5-FU+SN-38) like treatments were tested in dose-response three-week experiments to determine the suitable drug concentration for the screening. The experiments were analysed by XTT and DiOC/DAPI staining.Results and discussionsEcoR receptor was correctly expressed in transformed HT-29 cells and functional when infected with GFP and mCherry labelled vectors. Infection rates were 40% and 12%, respectively. The suitable viral dilution ranged from 2.5% to 5%. Kill assays confirmed shRNA inhibition efficiency leading HT-29 EcoR infected cells with MYC and RPA3 shRNA to death. The IC50 of 5-FU, Oxa and SN-38 were 10 µM, 2 µM and 5.5 nM respectively and the FOLFOX and FOLFIRI dilution that allowed 70%–80% of cell survival after a long-term treatment were determined.ConclusionWe have successfully set up the conditions to perform the loss-of-function screening. Currently, we are working on the effect of each relevant shRNA to validate the results of the screening.