(W1130-04-21) Identification of Potential Drug Combinations against BRAF V600E, p53 R273H HT-29 Colon Cancer Cells With and Without BRCA1 Knocked Down

Purpose: BRCA (BReast CAncer) gene mutations are well characterized in the context of breast and ovarian cancers but are also present in a small percentage of colorectal cancer (CRC) patients. Loss of function BRCA1 is associated with cancers with increased drug resistance and aggressiveness.  Current first-line therapies for CRC do not effectively address CRC patients with rare oncogene combinations involving loss of function BRCA1. Hence, novel drug combinations need to be investigated. Here, we screened for the cytotoxicity of a range of drug combinations against BRAF^V600E p53^R273H HT-29 CRC cells with and without BRCA1 knocked down.

Methods: HT-29 cells were genetically modified using the CRISPR/Cas9 gene editing technique to knock down the BRCA1 gene. In brief, the developed single-strand oligos and in vitro transcribed guide RNA (IVT gRNA) were co-transfected with Cas9 using an electroporation technique. The obtained BRCA1 knockdown cells (KD) were characterized by next-generation sequencing (NGS) and Sanger sequencing. The wild type (WT) and KD cell line (clones C2 and D1) were further tested for growth kinetics and wound healing. To check their drug sensitivity to a range of drug candidates including chemotherapeutics (e.g., 5-fluorouracil, oxaliplatin, paclitaxel, trifluridine-tipiracil), poly-ADP ribose polymerase-1 (PARP) inhibitors (e.g., olaparib, rucaparib, talazoparib, saruparib), kinase inhibitors (e.g., encorafenib, trametinib, nintedanib, fruquintinib, alisertib, prexasertib), zen-3694, eprenetapopt and gemcitabine were tested for cytotoxicity using the Presto blue cell viability assay. The synergistic combinations of drugs were identified using the Bliss assay model.

Results: After knocking down BRCA1, HT-29 cells changed from an epithelial morphology (characteristic of WT) to mesenchymal shape. In addition, both BRCA1 knockdown clones were observed to grow faster, with a doubling time of 13.6 h, compared to WT cells (17.58 h). In a wound healing assay the BRCA1 knockdown cells covered 55.94 ± 3.07 % of the wound area, significantly greater than 16.45 ± 5.59 % covered by WT cells (P=0.0003) at t = 42 hours, showing enhanced migration behavior. Figure 1 shows that the BRCA1 knockdown cells were more resistant to most drugs tested than WT HT-29 cells except for Zen 3694, gemcitabine and trifluridine-tipiracil. Thirty-three synergistic dual-drug combinations were identified for HT-29 WT cells (figure 2), while only 6 and 2 combinations were found for clones C2 and D1, respectively. Importantly, paclitaxel-trametinib and trametinib-prexasertib drug combinations (figure 3) were found synergistic in both HT29 WT and BRCA1 KD clones.

Conclusion: Knocking down BRCA1 in HT-29 cells resulted in CRC cells that were more aggressive (showing proliferation/migration) and chemo-resistant. The bliss assays conclude that combining the MEK1/2 inhibitor, trametinib with either paclitaxel or prexasertib (a Chk-1 inhibitor) resulted in potent synergistic cytotoxic activity against WT and BRCA1 KD HT-29 CRC cells.

Acknowledgements: This work was financially supported by Gastro-Intestinal Research Foundation (GIRF)

Figure 1. Chemosensitivity of HT-29 WT (black) and HT-29 KD C2 (red) cell lines upon 48h of exposure.

Figure 2. Heat map of synergy scores of dual drug combinations based bliss assays for HT-29 WT (upper right part) and HT-29 KD C2 (lower left part) cell lines.

Figure 3. Synergy maps depicting the behavior of paclitaxel-trametinib and trametinib-prexasertib combinations across different concentrations in HT-29 WT and BRCA1 KD clones. The square in each map represents the concentrations with the highest synergistic activity.