Overcoming drug resistance in Luminal Breast Cancer harboring PIK3CA and TP53 alterations

Breast Cancer (BC) is the most common malignancy and the second leading cause of cancer-related death in women worldwide. It is a highly heterogeneous disease, differing in histology, clinical behavior, and therapeutic response. Among main molecular subtypes, Luminal Breast Cancer (LBC; ER+, PR+/−, HER2−) is the most prevalent and generally associated with a more favorable prognosis. PIK3CA and TP53 represent the two most commonly mutated genes. Our previous data on a large cohort of LBC patients (>200 LBC) highlighted that a not negligible percentage of tumors carries both gene alterations (12-17% of all LBC). Endocrine and radio-therapy, combined with surgery and chemotherapy, represent cornerstones of LBC treatment. However, therapeutic efficacy is often limited by the emergence of resistant clones, leading to recurrence and progression. Key determinants of resistance are genetic alterations, such as those in PIK3CA and TP53 genes. PIK3CA mutations activate the PI3K/AKT/mTOR pathway, promoting proliferation and survival, while TP53 mutations impair cell-cycle control and DNA repair. Together, these alterations are critical to drive progression and influence therapy response. We wondered whether LBC carrying both TP53 and PIK3CA alterations may represent a particularly aggressive biological entity, leading to resistance to endocrine and radio-therapy. We thus generated and characterized two different cellular models carrying both TP53 and PIK3CA gene alterations (p53/PI3Kco-mut) and analyzed their response to endocrine and radio-therapy. Then, we used an high throughput (HTP) drug screening to identify novel therapeutic vulnerabilities. To investigate endocrine resistance, we used the luminal Breast Primary Epithelial (BPE) cell line and introduced PI3KH1047R and p53R175H mutations. p53/PI3Kco-mut cells were then fully characterized for most prominent hallmarks of cancer. We observed that p53/PI3Kco-mut cells acquired stem-like features, enhanced proliferation and tumorigenicity in both 2D- and 3D-assays. As hypothesized, they also displayed resistance to Tamoxifen treatment, one of the most common endocrine therapy. The HTP drug screening identified COX inhibitors as the most promising agents able to re-sensitize p53/PI3Kco-mut cells. To dissect the mechanisms underlying resistance to radiation therapy, we used the LBC cell line T47D, naturally mutated for both TP53 and PIK3CA. From T47D, we generated the resistance to radiotherapy, by exposing cells to incremental doses of γ-irradiation. The resistant isogenic cell line (T47D RR), confirmed by increased clonogenic survival and reduced sensitivity to Bleomycin, a radiomimetic agent, also exhibited cross-resistance to CDK4/6, ATM, DNA-PK, and PI3K inhibitors, together with enhanced proliferation and tumorigenicity in both 2D- and 3D-assays. To elucidate the mechanisms underlying radio-resistance, we analyzed DNA damage response (DDR) and survival pathways in T47D RR and T47D parental cells. Alteration in DDR and homologous recombination pathway activation were evident in radio-resistant cells, suggesting reliance on alternative repair mechanisms, such as NHEJ. To identify therapeutic vulnerabilities associated with radio-resistance, we performed a HTP drug screening, identifying microtubule inhibitors and DNA/RNA synthesis interfering compounds as synthetically lethal with the alterations accumulated with radio-resistance. Together, our study provides new insights into the molecular mechanisms associated with endocrine and radio-therapy resistance. Our HTP drug screenings open new windows of opportunity to overcome endocrine and radiation resistance and improve responses in patients with p53+PI3Kco-mut LBC.