Abstract 7181: Gene enhancers as novel rationale therapeutic targets for impeding colorectal cancer progression

Abstract Half of of colorectal cancer (CRC) patients develop metastatic CRC (mCRC) with a low survival rate. Epigenetic abnormalities in promoters and enhancers play a significant role in CRC advancement and hold promise for diagnosis and therapy and over 5, 000 variable enhancer loci (VELs) have been shown to be gained/lost in mCRC. Here, the functional impact of these VELs on driving CRC progression was explored. We designed a CRISPRi library including 1, 524 VELs gained or lost specifically in CRC cell lines, the top 500 essential genes for the cell line as positive controls, 1, 000 non-targeting guides as negative controls and 6 sgRNAs per site, cloned into a lentiviral plasmid. CRISPR screen was applied to the HCT116-dCas9-KRAB-MeCP2 cells for NGS. Anticancer potentials of silenced VELs were evaluated by in vitro assays (i.e MTT assay, apoptosis, cell cycle assay, 3D bioprinting). Chromosomal interactions and epigenetic state were analyzed by Chromosome conformation capture (3C) coupled with sequencing (Hi-C), chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing (ChIP-seq) and RNA sequencing. We identified 12 depleted VELs causing a significant decrease in HCT116 proliferation after CRISPR-mediated silencing. VEL_1243 and VEL_1084 caused the highest level of cell growth inhibition as well as increased apoptosis and cell cycle arrest in HCT116 cells. After silencing both enhancers, 3D bioprinting showed a reduction in key markers of metastasis. HiC-Seq and transcription factor binding data showed long-range interaction of both enhancers with genes such as MYO3B, UBR3 and GINS3 and enrichment for disease-associated TF including RFX7, and ELF3. RNA seq results showed significant transcriptome-wide expression alterations regulated by VEL_1243 and VEL_1084, exemplified by their impact on disease-associated signaling pathways including PI3K-Akt and MAPK. IFNγ response pathway -which is a critical aspect of anti-tumor immunity - was downregulated following both VEL_1243 and VEL_1084 silencing. Given that immune/inflammatory cells (e.g., tumor-associated macrophages (TAMs), T-cells, natural killer (NK) and cancer-associated fibroblasts (CAFs) are the important components of the tumor microenvironment (TME), we further examined if VELs mediated regulation of TAMs or immune cells controls the TME in a way that promotes tumour growth. M1 were co-cultured with CRC cells knock-down of VELs. Both VELs repression could induce M1 polarization in HCT116 and SW620 cells. Considering M1 macrophages have the capability to promote the Th1 response and engulf and kill target tumor cells, VEL_1084 and VEL_1243 could be potential therapeutic targets for CRC by affecting the polarization of macrophages. VEL_1243 and VEL_1054 regulate CRC-associated phenotypes and warrant further exploration to determine their regulatory role and potential as a novel therapeutic target in CRC. Citation Format: Flavia Genua, Xinxin Shao, Ellen Finnegan, Christina Caragine, Sarah Maguire, Paulien Van Minsel, Grainne Kirwan, Bernard Thienpont, Neville Sanjana, Weipeng Wang, Sudipto Das. Gene enhancers as novel rationale therapeutic targets for impeding colorectal cancer progression [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 7181.