ABSTRACT Estrogen receptor-positive (ER+)/HER2-negative breast cancer represents 70% of all breast cancer cases. Surgery and adjuvant/neo-adjuvant endocrine therapy (ET) are mainstays of treatment in early stage disease. However, some patients receiving ET for early stage ER-positive breast cancer only have a partial reduction in their risk of recurrence and mortality, and those with advanced breast cancer (ABC) either progress shortly after initiating therapy (intrinsic resistance), or ultimately experience progression over time (acquired resistance). CDK4/6 inhibitors (CDK4/6is) with ET are currently considered standard of care for patients with advanced ER +/HER2 negative breast cancer. A key feature of CDK4/6 inhibition is the cell cycle inhibitory response it elicits through induction of senescence, which can be escaped resulting in cells readily re-entering the cell cycle as soon as drug is withdrawn. Senescent cells secrete interleukins, inflammatory cytokines, and growth factors, which comprise the senescence-associated secretory phenotype (SASP) that affects surrounding cells and promotes tumor growth. The most prominent SASP cytokine is interleukin-6 (IL-6), which is associated with metastasis, tumor aggressiveness and decreased survival. IL-6 activates STAT3, which is associated with a more aggressive phenotype and resistance to many therapies [chemotherapy, targeted therapy, and immune checkpoint inhibitor therapy]. We developed CDK4/6i (i.e. palbociclib) resistant breast tumor cell line models and their molecular analysis showed that resistant cells adapt to palbociclib treatment by upregulation of IL-6 and activation of STAT3 (phosphorylation of STAT3 on Y705, pY-STAT3). Treatment of the resistant cells with an oral, small-molecule inhibitor of STAT3 (TTI-101) decreased cell viability by >25-fold and resulted in decreased levels of pY-STAT3 with concomitant decreases in (i) stem-like (CD44high/CD24low) population, (ii) primary and secondary mammosphere formation, (iii) the EMT pathway. Furthermore, TTI-101 treatment of mice bearing patient derived xenografts (PDX) that express a similar gene expression signature as palbociclib-resistant cell lines resulted in a marked decrease in tumor volume, prolonged tumor-free survival and downregulation of serum IL-6 levels. We hypothesize that inhibition of IL-6 and/or STAT3 can reverse acquired CDK4/6i resistance in vivo transgenic and PDX models and in patients who have progressed on CDK4/6i based therapy. We propose a coordinated mechanistic, preclinical and early phase clinical testing strategy to develop biomarker-qualified therapy for the clinical need to overcome CDK4/6i resistance. To address these goals we will 1): Determine the mechanism of IL-6 induction by long term CDK4/6 inhibition in vivo and the impact of IL-6 on tumorigenesis in transgenic mouse models; 2) Conduct pre-clinical trials in palbociclib resistant PDX and transgenic mouse models to determine if inhibition of STAT3 a...