Abstract B071: Unveiling natural killer transcriptional plasticity in triple-negative breast cancer patients: a novel dynamic biomarker of immune checkpoint blockade response?

Abstract Immunotherapy in combination with neoadjuvant chemotherapy is rapidly changing the therapeutic perspective for Triple-Negative Breast Cancer (TNBC) patients, underscoring the relevance of immune response in defining patients’ outcomes. The use of immune checkpoint inhibitors (ICIs) drastically enhanced the pathological complete response rate achieved by these patients, confirming that the proper stimulation of the immune system improves chemotherapy efficacy. We previously showed that higher Natural Killer cell (NK) tumor infiltration at patient diagnosis was associated with increased neoadjuvant chemotherapy efficacy. To further investigate the role of NKs in predicting immunotherapy response in TNBC patients, a single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) was applied to compare patients showing either a complete or partial pathological response to the neoadjuvant schedule with chemotherapy and pembrolizumab (KEYNOTE522). Results indicated that, even though the NK cell number at diagnosis was highly similar in all patients, NKs from partial responders were significantly impaired after therapy completion compared to complete responders. Besides, NKs displayed a prevalent immune-tolerant phenotype associated with partial/low therapy responders. Trajectory analysis confirmed that, in these patients, NKs were predominantly stacked at immature stages, displaying a significant impairment in their cytotoxicity. Altogether, these results highlight the relevance of a proper NK activation to reach a proficient immunotherapy response in TNBC patients. NKs are characterized by high transcriptional plasticity associated with dynamic changes in their anti-tumor effector properties. Still, the molecular mechanisms driving NK activation toward TNBC are largely unknown. To fill this gap, we built a 300-gene NK-specific signature. A predictive bioinformatic approach was then applied to reconstruct the hierarchical organization of the regulatory transcriptional landscape associated with the signature. We identified top-scoring upstream Transcription Factors (TFs), including VEZF1, RXRA, and MAZ, whose specific role in NKs was poorly investigated. ChIP-sequencing analysis revealed that each TF controls a distinct transcriptional network, but with mutual regulation. We characterized for the first time the role of VEZF1, which controls vesicle trafficking and lytic granule exocytosis, both crucial processes in triggering NK effector functions. VEZF1 and MAZ were identified as transcriptional co-operators promoting NK cytotoxicity. Conversely, the retinoic acid and its receptor, RXRA, constituted a novel transcriptional axis of repression of NK anti-tumor response. The transcriptional program controlled by these TFs was validated in an independent scRNA-seq dataset and correlated with TNBC patient immunotherapy response. Altogether, these data support that NKs and their transcriptional activation may constitute a dynamic biomarker predicting TNBC patient response to neoadjuvant treatment with ICIs. Citation Format: MARTINA BIGLIARDI, ELISA SALVIATO, VERONICA MANICARDI, GLORIA MANZOTTI, ELISA GASPARINI, MOIRA RAGAZZI, FEDERICA SILVESTRI, EMANUELE VITALE, FEDERICA TORRICELLI, CARMINE PINTO, ALESSIA CIARROCCHI, FRANCESCA REGGIANI. Unveiling natural killer transcriptional plasticity in triple-negative breast cancer patients: a novel dynamic biomarker of immune checkpoint blockade response? [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B071.