Targeting Aurora A kinase together with alternative survival signaling overcomes resistance to endocrine and CDK4/6 therapies in ER+ breast cancer

Background: Endocrine therapy (ET) in combination with CDK4/6 inhibitors (i) is the standard-of-care for estrogen receptor (ER)+ metastatic breast cancer (BC). Though effective, resistance is inevitable. Aurora A kinase (AURKA) drives mitotic entry and spindle assembly for proper cell division. Clinically, high AURKA levels have been linked to cancer aggressiveness, poor prognosis, and CDK4/6i resistance. A recent phase II trial (NCT02860000) reported clinically meaningful benefit of the AURKAi, alisertib (ALS), in advanced, endocrine-resistant ER+ BC. Identifying biomarkers and key signaling pathways involved in AURKA inhibition is essential to optimize ALS use and guide effective combination strategies to enhance sensitivity and overcome resistance in ER+ BC. Methods: ER+ BC cell models naïve or resistant (R) to various ET (EndoR) and/or palbociclib (PalboR, a CDK4/6i) were used. ALS-resistant (ALSR) derivatives were developed via long-term exposure to gradually increasing ALS concentrations ( > 150 nM). Molecular changes associated with growth and survival signaling, senescence, and apoptosis were assessed by Western blot upon short-term ALS treatment (72 hr) and at resistance. Signaling profiles guided the choice of specific drug combinations, which were then evaluated by cell growth assays. Results: Morphological changes, characterized by flattened, enlarged giant cells, slowed growth, senescence, and elevated p21, gammaH2AX, and c-PARP levels were observed in the ALSR derivatives of EndoR and/or PalboR models. In general, induction of various components of the HER/PI3K/MAPK pathways was seen across all ALSR models, with a specific increase in phosphorylated AKT levels in some models. Indeed, AKTi plus ALS was the most effective combination to overcome resistance in the ET-naïve/PalboR and the estrogen-deprivation (ED)R/PalboR/ALSR models. In the tamoxifenR/PalboR model and its ALSR derivative, in line with the signaling changes, EGFRi, pan-HERi (Neratinib, Nrb), or MEKi together with tamoxifen+Palbo+ALS showed strong efficacy. In the dual fulvestrantR/PalboR model, ALS+Nrb was highly effective, and adding Palbo further enhanced this effect, while in its ALSR derivative, fulvestrant+Palbo+ALS+Nrb regimen was needed to achieve substantial growth inhibition. Conclusions: Our findings highlight the potential of combining ALS with AKT, MAPK, and HER pathway inhibitors, to enhance efficacy or overcome ALS resistance in ET- and CDK4/6i-resistant ER+ BC, inferred by ALS-induced signaling changes. The presence of flattened and enlarged giant cells may reflect ongoing endoreplication, potentially indicating a dormant or persister cell state that may eventually support relapse and disease progression. Additional studies are underway to determine how these cell states can be targeted to induce cell death and overcome resistance.