Abstract 2053: Hemizygous focal deletions in <i>MACROD2</i> alter tumor biology and genomic instability in colorectal cancer

Abstract Introduction: MACROD2 is a large gene located within a common fragile site (CFS), a genomic region that is vulnerable to acquiring focal deletions under replicative stress. Structural variants (SVs) in MACROD2 are highly prevalent in patients with colorectal cancer (CRC), but their putative role in tumor initiation and progression remains to be established. Aim of this study: In silico and in vitro characterization of the effects of somatic SVs in MACROD2 in CRC initiation and evolution. Methods: Five datasets were used for in silico characterization of SVs in MACROD2: HMF (metastatic CRC, n=610), PCAWG (stage I-IV CRC, n=60), PLCRC-PROVENC3 (stage III CRC, n=234), PLCRC-MEDOCC (stage II-III CRC, n=214) and IntEnd (adenomas, n=634). The system-wide impact of intragenic SVs in MACROD2 on gene expression profiles was evaluated using the tool CIBRA. SVs in MACROD2 were associated with features of genomic instability, including tumor mutational burden (TMB), fraction of genome altered (FGA), tumor break load (TBL) and length of somatic SVs. The most frequently occurring SVs in MACROD2 were mimicked in vitro by CRISPR-Cas9 genetic engineering of a colorectal cancer cell line and an adenoma organoid. Their effect on tumor biology was determined by evaluating mRNA sequencing data, cell viability, mono-ADP-ribose levels, DNA damage repair and drug testing. Results: Focal deletions in MACROD2 were absent in adenomas, and present in 56% primary non-metastatic tumors and 71% of the metastatic CRC lesions evaluated. Focal deletions affecting the MACRO domain, in particular MACROD2 exons 5 or 6, were associated with system-wide expression changes (CIBRA score 0.37; FDR &amp;lt; 0.01), increased TBL (p &amp;lt; 10-4) and an increased number of SVs genome wide, specifically deletions of approximately 100kb (p &amp;lt; 10-15). In vitro, the models containing hemizygous or homozygous focal deletions showed lower levels of mono-ADP-ribose in the nucleus, and higher levels of DNA damage accumulation in the cancer cell line. Drug testing revealed that hemizygous deletions of MACROD2 made the cells more susceptible to treatment with 5-fluorouracil compared to WT (GR50: 17.6 μM vs 62.1 μM, P= 0.004). A similar trend was observed for the homozygous deletion (GR50: 30.3 μM vs 62.1 μM, ns) Conclusions: This study evaluated the effect of the most prevalent SV in CRC. Hemizygous intragenic focal deletions in MACROD2 that affect exon 5 and/or 6 are sufficient to alter system-wide expression profiles, nuclear mADPr levels, DNA damage repair and response to 5-fluorouracil. Citation Format: Carmen Rubio Alarcon, Soufyan Lakbir, Elisabeth J. van Bree, Bram van den Broek, Amber Hondema, Petur Snaebjornsson, Steven L. Ketelaars, Marianne Tijssen, Anne Bolijn, Pien Delis-van Diemen, Margriet Lemmens, Maartje Alkemade, Mette Kalager, Magnus Løberg Løberg, Miriam Koopman, Jeanine M. Roodhart, Geraldine R. Vink, PLCRC-MEDOCC group, Ellen Stelloo, Jaap Heringa, Gerrit A. Meijer, Harma Feitsma, Beatriz Carvalho, Sanne Abeln, Remond J. Fijneman. Hemizygous focal deletions in MACROD2 alter tumor biology and genomic instability in colorectal cancer [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 2053.