<em>In vivo</em> CRISPR screening identifies tada2b as a key epigenetic regulator of immune response in adrenocortical carcinoma

Kleiton S. Borges; Claudio Ribeiro; Audrey Muscato; Celeste Nobrega; Vasileios Chortis; Mesut Berber; Betul Haykir; Diana L. Carlone; Jinguy Wu; Kathleen Yates; Robert Manguso; David T. Breault

doi:10.1530/endoabs.107.011

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Endocrine Abstracts (2024) 107 011 | DOI: 10.1530/endoabs.107.011

IACS9 9th International Adrenal Cancer Symposium 2024 Abstracts (18 abstracts)

In vivo CRISPR screening identifies tada2b as a key epigenetic regulator of immune response in adrenocortical carcinoma

Kleiton S. Borges ¹ , Claudio Ribeiro ¹ , Audrey Muscato ² , Celeste Nobrega ² , Vasileios Chortis ¹ , Mesut Berber ¹ , Betul Haykir ¹ , Diana L. Carlone ¹ , Jinguy Wu ³ , Kathleen Yates ² , Robert Manguso ² & David T. Breault ¹

Author affiliations

¹Division of Endocrinology, Boston Children’s Hospital, Boston, MA, USA ²Massachusetts General Hospital/Broad Institute, Boston, MA, USA ³Dana Farber Cancer Institute/Beth Israel Deaconess Medical Center, Boston, MA, USA

Background: Adrenocortical carcinoma (ACC) is an aggressive malignancy with poor survival outcomes and resistance to immune checkpoint blockade (ICB) therapy. Progress in understanding ACC immunobiology has been hindered by the lack of suitable models. We developed a genetically engineered mouse model of ACC (BPCre) targeting Wnt/β-catenin activation and p53 loss, which closely mimics aggressive human ACC, including epigenomic alterations and poor immune cell infiltration. Additionally, we derived a syngeneic ACC cell line (BCH-ACC3) that forms subcutaneous tumors with poor response to ICB, despite increased CD8 T cell infiltration, reflecting clinical outcomes in ACC patients.

Objective and Methods: To explore the role of epigenetic dysregulation in immune suppression in aggressive ACC, we performed an in vivo CRISPR loss-of-function screen targeting 936 chromatin regulators in the BCH-ACC3 cell line. To avoid immune recognition of CRISPR components, we utilized a lentiviral system with selective CRISPR antigen removal (SCAR) to transduce the cells with a pooled sgRNA library (6 sgRNAs per gene). After gene editing and immunogenic component removal, the cells were implanted into three mouse groups: immunodeficient (NSG), immunocompetent (WT-Bl6), and WT-Bl6 treated with ICB (anti-PD-1). Genomic DNA sequencing identified sgRNAs depleted under immune pressure.

Results: Comparison of the WT-Bl6 untreated and WT-Bl6 ICB-treated groups to the NSG group revealed numerous immune-sensitizing hits, including positive control genes and genes known to regulate tumor immunity. Notably, we identified Tada2b, a transcriptional adaptor protein essential for the acetyltransferase activity of the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex, as a novel regulator of endogenous and immunotherapy-dependent antitumor immunity in ACC. Tada2b knockout (KO) tumors exhibited reduced growth compared to wild-type (WT) tumors, and ICB treatment resulted in greater tumor shrinkage and increased infiltration of activated CD8+ T cells in Tada2b-KO tumors. Additionally, analysis of the ACC-TCGA cohort revealed that TADA2B expression negatively correlates with immune signatures such as pathways of TNFA, IL6-JAK-STAT3, IL2-STAT5, and interferongamma. These findings suggest that human ACC tumors with TADA2B expression are immune excluded.

Conclusions: These findings indicate that TADA2B modulates the immune response in ACC, suggesting that targeting TADA2B could be a promising strategy to overcome resistance to immunotherapy in ACC.