Abstract B086: Targeting ATR to overcome Carboplatin resistance in triple-negative breast cancer patient-derived models

Abstract

Background: Triple negative breast cancer subtype is associated with poor prognosis and is mostly treated with chemotherapy, including the DNA crosslinking agent Carboplatin. Carboplatin treatment leads to DNA damage and a cell cycle inhibition allowing DNA damage repair. However, many patients are resistant or develop resistance to Carboplatin. The identification of novel treatments against chemoresistant TNBC is a major unmet clinical need.

Method: TNBC patient-derived cell lines (PDCs) from patient-derived xenografts (PDXs) generated from chemotherapy resistant tumors were used for shRNA high throughput screens to identify genetic vulnerabilities that could resensitize these cells to Carboplatin. Validation of this target was performed genetically by shRNA silencing and pharmacologically with commercially available target inhibitors both in vitro and in vivo, using Alamar Blue viability assays and calculating synergy using the Chou Talalay method. DNA damage was assessed using yH2AX expression, and apoptosis using c-PARP and Annexin V, by confocal microscopy, flow cytometry and/or western blotting.

Results: We identified ataxia telangiectasia and Rad3-related protein (ATR) as a target in one of the cell lines screened and validated it with an ATR shRNA. We tested the combination of the ATR inhibitor (ATRi), Elimusertib, with Carboplatin and found the combination to be synergistic (Average CI=0.30). DNA damage was markedly increased with combination treatment and a pan-nuclear yH2AX staining together with increased pHH3 expression were observed, suggestive of mitotic catastrophe. Cells treated with Carboplatin accumulated in S phase and a shift toward G2/M when Elimusertib was added. Apoptosis was significantly increased with combination treatment. PKMYT1 and WEE1 are two kinases regulating cell cycle entry by inhibiting CDK1, which controls entry into mitosis. Carboplatin treatment resulted in an increase in PKMYT1 expression. PKMYT1 inhibition with RP6306 re-sensitized the cells to Carboplatin more than WEE1 inhibition with Adavosertib, and this synergy was further increased with low doses of Elimusertib. Gene silencing of WEE1 and PKMYT1 both re-sensitized the cells to Carboplatin, but only WEE1 silencing alone significantly prevent proliferation, suggesting the requirement of PKMYT1 activity for resistance but not for cell proliferation without Carboplatin. In vivo validation using the PDXs from which the cell lines have been derived, demonstrated a significant delayed in tumor growth and prolonged survival when combining Carboplatin with ATRi compared to Carboplatin alone in two models.

Conclusion: ATR inhibition resensitizes drug resistant TNBC cells to Carboplatin both in vitro and in vivo. The combination of Carboplatin with ATRi led to DNA damage and mitotic catastrophe. PKMYT1 plays a role in Carboplatin-resistant TNBC, collaborating with ATR in managing the cell cycle response to DNA damage, and PKMYT1 inhibition also re-sensitizes cells to Carboplatin, an effect increased with the triple combination of adding ATR inhibition.

Citation Format: Juliet Guay, Catherine Chabot, Cédric Darini, Marguerite Buchanan, Adriana Aguilar-Mahecha, Tim Kong, Connie Yang, Mark Basik, Sidong Huang. Targeting ATR to overcome Carboplatin resistance in triple-negative breast cancer patient-derived models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B086.