Targeting common denominators of DNA damage tolerance mechanisms as therapeutic strategy to cope with dynamic phenotypic changes in triple-negative breast cancer cells

Abstract Triple negative breast cancer (TNBC) is the most aggressive type of breast cancer with limited treatment options and a propensity towards chemoresistance. Given the lack of targetable receptors, treatment relies primarily on chemotherapy. While mechanisms leading to chemoresistance are vastly unknown, DNA damage tolerance (DDT) pathways have emerged as potential drivers of chemoresistance. Herein, we report a phenotypic characterization of four TNBC- and six non-TNBC cell lines, which supports the relevance of DDT in the TNBC response to chemotherapy. We unveiled an association between DDT pathway activation through PCNA-ubiquitination, augmented epithelial-to-mesenchymal transition (EMT) and stemness features, specifically in 3D spheroid culture from TNBC cells. Analysis of replication dynamics revealed a pronounced replication slowdown in 3D- versus 2D-cultured TNBC but not in non-TNBC cells. The exploration of DDT pathway usage in three TNBC cell lines (MDA-MB-231, Hs578t, MDA-MB-468) uncovers reliance on ZRANB3-, HLTF- and SMARCAL1-dependent fork reversal in 3D, while translesion DNA synthesis (TLS) polymerases (POLs) were more active under 2D conditions. Screening cytotoxicity with a panel of ten drugs targeting key DDT pathway components identified POLζ inhibition (JH-RE-06; IC 50  ≤ 7 µM/48 h) and CHK1 inhibition (PF477736; IC 50  ≤ 20 µM/48 h) as the most potent single-agent treatments in all three cell lines under both conditions. Mechanistically, both POLζ and CHK1 inhibition target several DDT pathways, in sum, causing replication slowdown, revealed consistently for CHK1. While TNBC cell lines were largely unresponsive to Olaparib mono-treatment, especially in 3D culture, all three cell lines were resensitized by combining Olaparib with low-dose POLζ-, CHK1-, or RAD51 inhibitor, except for 3D-cultured MDA-MB-231 cells, which may resist Olaparib due to their high ZRANB3 complex formation in the nucleus. These findings identify key DDT pathway components such as POLζ and CHK1 as druggable targets that alone or in combination with Olaparib may overcome chemoresistance in TNBC.