Clinically actionable drug synergy drives transcriptomic and metabolic reprogramming to induce ferroptosis and apoptosis in triple negative breast cancer

Introduction: The current standard of care of triple negative breast cancer (TNBC) consists of chemotherapy regimens such as anthracyclines and taxanes with an increasing role for immunotherapy, and drugs targeting DNA damage and repair. However, these regimens are not very effective in majority of patients and have serious adverse effects. Therefore, there is an unmet need for rationally designed therapies. We have developed a novel, mechanistically driven drug repurposing strategy where tamoxifen (Tam) is repurposed in combination with chemotherapeutic agent doxorubicin (Doxo) to induce efficient cell death by apoptosis and ferroptosis (iron-dependent cell death process characterized by lipid peroxidation) in molecularly stratified TNBC based on expression of estrogen receptor beta (ER beta) and mutant p53. Methods: TNBC cell lines, cell line-derived xenografts (CDXs), and patient tumor-derived xenografts (PDXs) were used to analyze protein-protein interactions, cell proliferation, cell death (apoptosis and ferroptosis), migration, invasion, and therapeutic response. Various experimental approaches including knocking down and overexpressing genes, Co-IP, ChIP, RNA-seq, bioinformatics analysis, quantitative real time PCR, reverse phase protein array (RPPA), proximity ligation assay (PLA), and PK/PD and synergy analysis were used. Results: Tam, although widely used as an effective endocrine agent to treat ER alpha-positive luminal breast cancer, has not been used to treat TNBC because of the lack of ER alpha, the canonical target of Tam, in TNBC. Our data has challenged this clinical paradigm by demonstrating that Tam synergizes with Doxo to lower IC50 of Doxo. The drug combination increased apoptosis and ferroptosis in TNBC cells and inhibited growth of TNBC CDX and PDX tumors in vivo in ER beta and mutant p53-dependet manner.Mechanistically, Tam enhances ER beta-mediated sequestration of p73-bound mutant p53, thereby disrupting p73-mutant p53 interaction resulting in reactivation of p73, and in synergy with Doxo, upregulates apoptosis and ferroptosis. Furthermore, the combination downregulated genes associated with metastatic progression and DNA damage repair (DDR) pathways. Importantly, transcriptomic reprograming and alteration of iron metabolism and lipid peroxidation led to increased ferroptosis. Ferroptosis was further augmented by lower levels of antioxidant enzyme GPX4, a consequence of downregulation of the mevalonate pathway by the drug combination. Conclusion: As large percentage of TNBCs express both mutant p53 and ER beta, successful clinical translation of our novel findings on repurposing of Tam has the potential for a relatively safe, cost-effective, and efficacious treatment option while minimizing delay for clinical availability and financial toxicity to the patients.