Targeting RUNX transcriptional activity reduces tumor heterogeneity and aggressiveness in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a highly aggressive disease, characterized by early relapse and enriched with cancer stem cells (CSCs), contributing to chemoresistance and metastasis. Our research focuses on RUNXs role in TNBC aggressiveness, cell migration promotion and regulation of gene expression. We reported that RUNX1 influences chemoresistance in androgen-responsive TNBC cell lines and Ferrari (2016) showed its correlation with poor prognosis in TNBC patients. Lv (2024) showed that RUNX2 is also implicated in chemoresistance, and Halperin (2022) established that RUNX1 is upregulated in cancer-associated fibroblasts of breast cancer patients and predicts poor disease outcomes. Our study explores how inhibiting RUNX transcriptional activity impacts TNBC cell lines aggressiveness and drug response. Using RUNX-CBFbeta commercial inhibitors (AI-10-104 and AI-10-49) we observed significant reduction in viability and migration, and increased apoptosis in MDA-MB-231 and -468 cell lines. In a forced-suspended cell model, inhibiting RUNX transcriptional activity decreased mRNA levels of crucial genes for CSC maintenance. In MDA-MB-231 cells, RUNX inhibition completely hindered mammosphere formation. RUNX1 mRNA and protein expression increased in doxorubicin (Doxo) and paclitaxel (Px) treated cells. RUNX inhibition enhances drug toxicity, reducing viability and boosting apoptosis. Combined AI-10-104 and Doxo significantly reduced the number of mammospheres even more than Doxo alone, while AI-10-104 restored Px sensitivity in Px-resistant cells. By flow cytometry we observed that RUNX inhibition significantly downregulates PD-L1 expression, suggesting an important role in tumor immune escape. Furthermore, RUNX1 inhibition alters mitophagy-related gene expression and induces mitochondrial fragmentation in SUM-159PT cells. Finally, by transcriptomic analysis (cDNA-seq) using long-read oxford nanopore technology, we compare the expression profiles of adherent with forced-suspension growing MDA-MB-468 cells, treated with AI-10-104, and gain insights into the underlying mechanisms. Analyses have identified 10 genes including metabolic, stress, drug resistance' genes, among others, whose expression is differentially affected by RUNX transcriptional inhibition depending on the culture model. These findings point to potentially distinct RUNX-dependent regulatory mechanisms across tumor subpopulations, which are currently under our investigation. In summary, our results suggest that RUNXs play a critical role in generating chemoresistant TNBC cells by influencing intracellular heterogeneity and immune surveillance cancer hallmarks.