Abstract PO-069: Characterizing cell-to-cell heterogeneity and chromosome instability induced by USP22 deficiency in colorectal cancer

Abstract

Abstract Chromosome instability (CIN) is an aberrant phenotype characterized by an increased rate of gains and losses of chromosomes. CIN is observed in most cancer types, including 85% of colorectal cancer (CRC) cases. Although CIN is a driver of tumor heterogeneity and associated with drug resistance and poor patient prognosis, the genetic defects underlying CIN remain poorly understood. Ubiquitin Specific Peptidase 22 (USP22) is a deubiquitinating enzyme that targets mono-ubiquitin on lysine 120 of histone H2B (H2Bub1). H2Bub1 is a dynamic post-translational modification that modulates chromatin compaction. Importantly, homozygous or heterozygous USP22 deletion is observed in 45% of CRC cases, suggesting this may be a pathogenic event promoting CRC progression. Accordingly, we sought to characterize the impact reduced USP22 expression has on CIN. To reduce USP22 expression in karyotypically stable CRC (HCT116) and immortalized fibroblast (hTERT) cell lines, we employed transient siRNA silencing and stable CRISPR-Cas9 knockout (KO) models. Single-cell quantitative image-based analyses were utilized to monitor CIN-associated phenotypes, including changes in nuclear areas, micronucleus formation and chromosome numbers. In HCT116, USP22 silencing induced CIN phenotypes and an increase in H2Bub1 abundance in mitotic chromosomes. Subsequent mechanistic studies determined that this is accompanied by an increase in the recruitment of the spindle assembly checkpoint kinase BUB1 at metaphase centromeres and an increased frequency of lagging chromosomes in anaphase. Collectively, these data suggest that reduced USP22 expression alters H2Bub1 regulation in mitosis, which impairs mitotic fidelity, thereby inducing CIN. In addition, to assess the relevance of reduced USP22 expression in a distinct cellular context, the impact of USP22 silencing was assessed in hTERT, revealing similar CIN phenotypes, including changes in nuclear area and chromosome numbers. To assess the long-term impact USP22 deletions have on CIN, novel homozygous and heterozygous USP22-KO HCT116 clones were generated and monitored for three months, which revealed dynamic CIN phenotypes and ongoing changes in chromosome complements relative to controls. Collectively, these data identify USP22 as a novel CIN gene and indicate that USP22 deletions in cancer may drive intra-tumor genetic heterogeneity and thereby promote oncogenesis. Citation Format: Lucile M. Jeusset, Zelda D. Lichtensztejn, Kirk J. McManus. Characterizing cell-to-cell heterogeneity and chromosome instability induced by USP22 deficiency in colorectal cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-069.