Abstract 6126: Natural killer activation drives immunotherapy efficacy in triple-negative breast cancer patients: Novel insights for therapeutic intervention

Abstract Immunotherapy in combination with neoadjuvant chemotherapy is rapidly changing the therapeutic perspective for Triple-Negative Breast Cancer (TNBC) patients, as demonstrated by the KEYNOTE522 trial. Enhancing a proficient immune response to improve neoadjuvant therapy (NAT) efficacy is an urgent clinical need. We have previously shown by applying a morphology-guided digital spatial transcriptome profiling, that Natural Killer (NK) cell infiltration in diagnostic biopsies of TNBC patients was associated with increased chemotherapy success. In this work, we aim to characterize the specific NK landscape associated with NAT response in TNBC. We prospectively enrolled 35 patients with a new diagnosis of TNBC in our Institute and underwent the KEYNOTE522 neoadjuvant schedule. We collected peripheral blood at diagnosis (T0) and after NAT completion before surgery (T1). On peripheral blood mononuclear cells (PBMCs), we performed single-cell RNA sequencing (scRNA-seq) and flow cytometry analysis to profile circulating NKs and compare patients showing either pathological complete or partial response to the KEYNOTE522 schedule. We did not observe a significant difference in NK number or sub-clustering before therapy (T0) when comparing responders to partial responders. However, after the NAT conclusion (T1), NKs from partial responders displayed a prevalent immune tolerant profile, characterized by the down-regulation of NK cytotoxicity-related genes. Besides, CD16-CD56bright NKs were significantly increased in partial responders, whereas CD16+CD56dim NKs were enhanced in complete responders, confirming a divergent activation profile among circulating NKs that was detectable only after NAT. Altogether, our results pointed out the relevance of NK activation in supporting a proficient NAT response in TNBC patients. Still, the molecular mechanisms governing NK cytotoxicity toward TNBC are largely unknown, restraining their clinical exploitation. To fill this gap, we investigated the specific transcriptional landscape associated with NK effector functions in TNBC patients. We applied a predictive bioinformatic approach to reconstruct the hierarchical organization of the regulatory transcription network associated with NK activation in TNBC, identifying a list of top-scoring Transcription Factors (TFs). Among these, we found SMAD3, VEZF1, MAZ, and RXRA as putative novel master regulators of NK cytotoxicity in anti-TNBC response. We developed a CRISPRi-based system to knock down these TFs and confirm the impact of this transcriptional reprogramming on NK effector functions. Collectively, our data unveil a driving role of NKs in triggering NAT efficacy in TNBC patients and that transcriptional reprogramming can be exploited to potentiate NK-based adoptive cell therapies and further enhance immunotherapy efficacy in TNBC patients. Citation Format: Gloria Manzotti, Elisa Salviato, Veronica Manicardi, Anna Rita Redavid, Elisa Gasparini, Moira Ragazzi, Federica Torricelli, Emanuele Vitale, Giovanna Talarico, Francesco Bertolini, Carmine Pinto, Alessia Ciarrocchi, Francesca Reggiani. Natural killer activation drives immunotherapy efficacy in triple-negative breast cancer patients: Novel insights for therapeutic intervention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6126.