Abstract 1910: Advancing therapeutic interventions for NUT carcinoma: Orchestrating the precision of CRISPR/Cas9-mediated NUTM1 suppression and synergistic ADC strategies

Abstract

Abstract Purpose: NUT carcinoma (NC) is aggressive carcinoma defined by translocations involving the NUTM1 and frequent squamous differentiation. NUTM1 gene encodes a testicular nuclear protein (NUT), exclusively expressed in the testis. The fusion of NUTM1 leads to abnormal cell growth, resulting in the development of NC. This study aims to discover advanced approaches for inducing the death of cancer cells, specifically targeting those expressing NUT proteins. Methods: Spatial Transcriptome analysis and RNA sequencing were applied to formalin-fixed paraffin-embedded NUTM1 fusion-positive cancer tissues. Virus-like particles were used to introduce CRISPR/Cas9 into cells expressing the NUTM1 fusion oncoprotein. The inhibitory effect was confirmed through multiple in vitro assays. The effectiveness of Anti-Trophoblastic cell-surface antigen (TROP-2) Antibody-Drug Conjugate (ADC) was demonstrated in vivo and in vitro in cells with increased TROP2 expression subsequent to NUT suppression. Results: NCs revealed a relatively uniform pattern of gene expression, forming six unique groups. Notably, the NUT protein predominantly located in the inner regions of the tumor tissues. We employed CRISPR/Cas9 technology to specifically target cells harboring the NUTM1 fusion gene. The precise genetic editing was remarkably effective, achieving up to 90% success in the targeted area. This led to a notable decrease in both RNA and protein levels of NUT expression, effectively disrupting associated downstream pathways. The cell growth rate was reduced in several NC cell lines, and differentiation, accompanied by morphological changes, was observed in most edited cells. RNA sequencing analysis of these edited cells uncovered a substantial increase in expression of cytosolic molecules, with a marked increase in TACSTD2 expression. Interestingly, when we combined this genetic editing approach with a targeted therapy - the TROP2-ADC loaded with interferon-beta - the results were even more striking. Cells treated with this combination showed almost double the rate of growth inhibition compared to those receiving only a single form of treatment. Conclusion: This study provides valuable insights into identifying novel therapeutic targets and elucidating potential mechanisms by analyzing NUT cancer tissues using various assays. Our results support that CRISPR/Cas9-mediated targeting of NUTM1 could be a useful approach for NUT oncoprotein ablation, especially when used in combination with other therapies such as ADCs. Further studies are needed to investigate the efficacy of NUT inhibition in preclinical and clinical settings. the elimination of tumor cells facilitated by CRISPR/Cas9, along with additional tumor markers targeted by ADCs, holds potential for developing treatment strategies against incurable cancers driven by genetic alterations. Citation Format: Juyoung Choi, Hwa Lin Yi, Hee Geon Park, Misook Lee, Daesik Kim, Sung Youl Hong, Young Kee Shin, Yoon-la Choi. Advancing therapeutic interventions for NUT carcinoma: Orchestrating the precision of CRISPR/Cas9-mediated NUTM1 suppression and synergistic ADC strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1910.