Abstract A22: Deciphering natural killer transcriptional activation in triple-negative breast cancer: A novel opportunity for adoptive cell therapies?
Authors: Reggiani Francesca , Donati Benedetta , Ragazzi Moira , Manicardi Veronica , Torricelli Federica , Sauta Elisabetta , Gasparini Elisa , Piana Simonetta , Sancisi Valentina , Ciarrocchi Alessia 
Published: December 2022

Triple-Negative Breast Cancer (TNBC) patients that do not respond to neoadjuvant chemotherapy account for 60-70% of all cases and are orphans of therapeutic alternatives after chemotherapy failure. Despite the great progress that has been made in the application of immune-based strategies, clinical opportunities for TNBC patients are still lacking. We aimed to investigate the molecular mechanisms leading to the activation of the TNBC immunological microenvironment, thus providing novel clues for the design and development of immune-based therapeutic approaches and adoptive immune cell therapies for TNBC patients. We analyzed consecutive TNBC cases treated at AUSL-IRCCS Hospital of Reggio Emilia from 2011 to 2017. 24 Tru-Cut biopsies were selected, comparing 12 patients with pathological complete response (pCR) after neoadjuvant treatment and 12 patients with cancer progression. Then, we performed a morphology-guided transcriptome profiling of the immune infiltrate using our GeoMx-Digital spatial profiler (DSP) to investigate which immune populations were associated with a more favorable outcome. The immune infiltrate of patients showing pCR was significantly enriched for activated Natural Killer (NK) cells, compared to patients with disease progression. Intriguingly, both T-helper CD4- and CD8-T lymphocytes did not reach statistical significance, suggesting a secondary involvement in predicting neoadjuvant efficacy in TNBC patients. We confirmed that NK cells can significantly impair TNBC cell proliferation and viability in vitro and improved the efficacy of chemotherapeutic drugs (paclitaxel, docetaxel). To increase our knowledge of the molecular mechanisms governing NK effector functions toward TNBC, we decided to investigate the specific transcriptional landscape that triggers NK cell activation. We profiled the transcriptome of activated NK cells by RNA-sequencing, identifying several de-regulated pathways involved in NK innate anti-tumor response, including the down-regulation of Immune checkpoint (IC) molecules (CTLA4, CEACAM3, PD1, KIR2DL4, KIR3DX1, KIR3DL3, TIGIT, TIM3, LAG3, etc) and the up-regulation of vesicle-trafficking and autophagy-related proteins (STX5, STX7, BNIP3L, SNAP29, GABARAPL1, RAB4B, etc). To predict upstream transcription regulators, promoters of de-regulated genes were in silico scanned for common binding motifs with the FIMO algorithm, thus identifying a list of candidate Transcription Factors (TFs). Among top-scoring TFs, we identified PAX5 which has been already involved in NK differentiation and cytotoxicity. Other top-scoring TFs are currently under investigation as potential NK Master Regulators of their effector functions, including SMAD3, VEZF1, and MAZ. Overall, our results point out the relevance of NK cells in predicting neoadjuvant treatment response in TNBC patients. Moreover, the modulation of the NK transcriptional program may be a novel tool to potentiate their anti-tumor potential, thus proving novel clues to optimize their application in adoptive cell therapies and in the treatment of TNBC patients.

Citation Format: Francesca Reggiani, Benedetta Donati, Moira Ragazzi, Veronica Manicardi, Federica Torricelli, Elisabetta Sauta, Elisa Gasparini, Simonetta Piana, Valentina Sancisi, Alessia Ciarrocchi. Deciphering natural killer transcriptional activation in triple-negative breast cancer: A novel opportunity for adoptive cell therapies? [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A22.