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Proteogenomic analysis reveals adaptive strategies to alleviate the consequences of aneuploidy in cancer. Dataset 3

ABSTRACT: Aneuploidy is prevalent in cancer, conferring fitness advantage, multidrug resistance, and poor prognosis. In contrast, experimentally induced aneuploidy often results in adverse effects and impaired proliferation. This paradox underscores the necessity of cancer cells to adapt to abnormal chromosome numbers. To identify molecular mechanisms of adaptation to aneuploidy, we initiated in vitro evolution of cells with extra chromosomes added via microcell-mediated chromosome transfer. To this end, we cultured cells in a nutrient-rich medium for 50 passages or plated the cells at a low density and selectively collected the largest colonies originating from a single cell (colony selection). One of the striking observations following evolution of cells with extra chromosome 5 in HCT116 cell line was the frequent loss of the 5q, while maintaining the 5p arm after in vitro evolution. Chromosome 5 is a frequent target of large copy number alterations in several malignancies, such as ovarian, gastric, and oesophageal cancer, and malignant myeloid diseases. Moreover, analysis of chromosome arm level events in the TCGA dataset clearly shows that loss of chromosome arm 5q and gain of 5p are among the most frequent events. We asked whether these specific changes in copy numbers of chromosome 5 – loss of 5q with simultaneous retention of 5p – could affect the proliferation of the evolved cells. To this end, we used the recently developed technique ReDACT-TR (Restoring Disomy in Aneuploid cells using CRISPR Targeting with Telomere Replacement (Girish et al., 2023)), and transfected cells of a separate clone of Htr5 (before evolution) with a gRNA that cuts near the centromere of chromosome 5, simultaneously with a cassette encoding ~100 repeats of the human telomere seed sequence. Targeting the q-arm generated two independent cell lines with 5p trisomy (Htr5p_1 and Htr5p_2), which were confirmed by FISH with probes specific for 5p and 5q arms and by shallow WGS. No cell lines with trisomy of the q-arm were generated, but we have obtained two diploid cell lines (Hdi5_1 and Hdi5_2), most likely due to the CRISPR/Cas9 induced loss of chromosome 5 (Papathanasiou, Markoulaki et al., 2021, Tsuchida, Brandes et al., 2023). The anaylsis of global proteomes of the ReDACT cell lines was then carried out using a TMT quantification strategy.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Hct-116 Cell, Cell Culture

DISEASE(S): Colon Cancer

SUBMITTER: Markus Räschle

LAB HEAD: Markus Räschle

PROVIDER: PXD056432 | Pride | 2025-01-14

REPOSITORIES: pride

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			Action	DRS
	20240622_HF1_MaRa_SA_P0229B_R1_01.raw	Raw
	20240622_HF1_MaRa_SA_P0229B_R1_02.raw	Raw
	20240622_HF1_MaRa_SA_P0229B_R1_03.raw	Raw
	20240622_HF1_MaRa_SA_P0229B_R1_04.raw	Raw
	20240622_HF1_MaRa_SA_P0229B_R1_05.raw	Raw

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Project description:Chromosomal instability (CIN), defined as an increased occurrence of chromosome segregation errors during cell division, is a prominent form of genomic instability (Bakhoum and Avi Landau, 2017). It is the major cause of aneuploidy, an imbalanced complement of whole chromosomes or chromosome arms, which is the most prevalent genetic alteration in human cancers (Vasudevan et al., 2021; Santaguida and Amon, 2015). Importantly, aneuploidy is commonly associated with ongoing CIN through consecutive cell divisions (Shelzer et al. 2011; Passerini et al., 2016), resulting in intratumor genetic heterogeneity, a central driver of cancer evolution and therapeutic resistance (Sansregret et al., 2018; Ben-David and Amon, 2019). Indeed, aneuploidy has been shown to act both as a tumor suppressor and as a tumor initiator (Weaver et al., 2007; Silk et al., 2013; Vasudevan et al., 2020), most likely depending on the specific chromosomes that are gained or lost (Ben-David et al., 2011; Sack et al., 2018; Adell et al., 2023). Despite the ubiquitous presence of CIN in several aneuploid cancer types and its clinical relevance, its presence in B-cell acute lymphoblastic leukemia (B-ALL) remains largely unexplored owing to the impaired proliferation of leukemic cells in vitro and the lack of reliable experimental models to comprehensively assess chromosome segregation in vivo. B-ALL is the most frequent childhood cancer, with 75% of cases occurring in children under 6 years of age, and it is characterized by the accumulation of highly proliferative immature B-cell precursors in the bone marrow (BM) (Hunger and Mullighan, 2015). The presence of CIN and its contribution to aneuploid cB-ALL progression is largely unknown due to the lack of preclinical models to study actively dividing cells. Accordingly, studies of CIN in cB-ALL are limited to the characterization of chromosomal copy-number heterogeneity (chr-CNH) in primary cB-ALL samples, but there is controversy over its presence due to the different techniques used to assess karyotype variability (Raimondi et al., 1996; Talamo et al., 2010; Alpar et al., 2014; Heerema et al.; 2007; Ramos-Muntada et al., 2022). Here, we explored the presence and the levels of CIN in different clinically-relevant aneuploid subtypes of cB-ALL using single-cell whole-genome sequencing (WGS) of primary samples to reliably assess chr-CNH, and by generating a large cohort of PDX models from primary cB-ALL samples (cB-ALL-PDX). Our results in cB-ALL-PDX models revealed variable levels of CIN in aneuploid cB-ALL subtypes, which significantly correlate with intraclonal karyotype heterogeneity and with disease progression. Additionally, mass-spectrometry analyses of cB-ALL-PDX samples revealed a CIN “signature” enriched in mitosis and chromosome segregation regulatory pathways. We speculate that this signature identifies adaptive mechanisms to ongoing CIN in aneuploid cB-ALL cells, which displayed a transcriptional signature characterized by an impaired mitotic spindle as observed by RNA-sequencing (RNA-Seq) analyses of a large cohort of primary cB-ALL patient samples. Our work might help to improve stratification of patients with cB-ALL with different levels of CIN who could benefit in the future from new therapeutic approaches aiming to target ongoing CIN.

Project description:Aneuploidy, an imbalance in chromosome copy numbers, causes genetic disorders, and drives cancer progression, drug tolerance, and antimicrobial resistance. While aneuploidy can confer stress resistance, it is not well understood how cells overcome the fitness burden caused by aberrant chromosomal copy numbers. Studies using both systematically generated and natural aneuploid yeasts triggered an intense debate about the role of dosage compensation, concluding that aneuploidy is transmitted to the transcriptome and proteome without significant buffering at the chromosome-wide level, and is, at least in lab strains, associated with significant fitness costs. Conversely, systematic sequencing and phenotyping of large collections of natural isolates revealed that aneuploidy is frequent and has few, if any, fitness costs in nature. To address these discrepant findings, we developed a platform that yields highly precise proteomic measurements across large numbers of genetically diverse samples and applied it to natural isolates collected as part of the 1011 genomes project (Peter, J. et al, 2018). For 613 of the isolates, we were able to match the proteomes to their corresponding transcriptomes and genomes, subsequently quantifying the effect of aneuploidy on gene expression by comparing 95 aneuploid with 518 euploid strains. We find, as in previous studies, that aneuploid gene dosage is not buffered chromosome-wide at the transcriptome level. Importantly, in the proteome, we detect an attenuation of aneuploidy by about 25% below the aneuploid gene dosage in natural yeast isolates. Furthermore, this chromosome-wide dosage compensation is associated with the ubiquitin-proteasome system (UPS), which is expressed at higher levels and has increased activity across natural aneuploid strains. Thus, through systematic exploration of the species-wide diversity of the yeast proteome, we shed light on a long-standing debate about the biology of aneuploids, revealing that aneuploidy tolerance is mediated through chromosome-wide dosage compensation at the proteome level.

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Proteogenomic analysis reveals adaptive strategies to alleviate the consequences of aneuploidy in cancer. Dataset 3

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