Aberrant cytoplasmic localization of MLH1 characterizes a cell population that seeds breast cancer recurrence.

Estrogen receptor-positive breast cancer remains a leading cause of cancer-related death in women, with mortality largely driven by late recurrence of treatment-resistant disease. Loss of MLH1 promotes resistance to estrogen-targeting therapies by uncoupling cell cycle progression from estrogen regulation. Here, we show that even when MLH1 is abundantly expressed, aberrant cytoplasmic localization in a subset of tumor cells drives endocrine therapy resistance by enabling estrogen-independent growth. This resistance arises from failure to undergo robust cell cycle arrest in response to endocrine therapy, creating acute dependency on CDK4/6 activity. Consequently, CDK4/6 inhibitors induce strong regression in cells with cytoplasmic MLH1 compared to cells with nuclear MLH1. As cytoplasmic localization occurs in ~11% of ER+ patients, it represents a contributor to MLH1 dysregulation. Incorporating cytoplasmic MLH1 localization into diagnostics could guide the use of CDK4/6 inhibitors in this hard-to-treat subset.