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Karyotypic evolution in the U2-OS osteosarcoma cell line. (A) Representative, pseudo-colored, multicolor-FISH karyotypes of ten major subclones (Sc), from five U2-OS-derivative cell lines (SL) composed of 69–80 chromosomes. In U2-OS cells, every homologue of the human karyotype is affected by clonal structural or numerical aberrations. Despite the karyotypic diversity, a monoclonal origin of all side lines is evident by the common presence of six identical recombinant chromosomes (pink arrows). Based on the constitution of chromosome 5, the WT1 cells probably represent the most ancestral population. The CDT1-overexpressing, and Doxorubicin-resistant R1 and R2 cells derive from WT2. White arrows depict clone-specific rearrangements. Note that several evolutionary steps (i.e., the process from WT1 to WT2 Sc1-2 or the evolution of CDT1 and R1 cells) were accompanied by karyotypes bearing multiple duplicated copies of identical clonal recombinant chromosomes (asterisks) suggesting that leaps in karyotypic evolution were accompanied by whole genome duplication (WGD) followed by multiple chromosome losses. (B) Jumping translocations of large recombinant segments clonally present in most U2-OS-derivative cell lines and subclones. (C) Chromoanagenesis was recorded only in one recombinant chromosome, composed of multiple alternate segments of chromosomes 5 and 19, in the chemo-resistant R2 cells. (D) Identical large genome imbalances identified by aCGH are stably maintained between the U2-OS-derivative cell lines despite the karyotypic alterations produced by extensive chromosome breakage and illegitimate rejoining. Red circles indicate the presence of the same imbalance in all 5 cell lines, pink circles depict undistinguishable duplications/deletions in 4 out of 5 karyotypically-diverse U2-OS cell lines. Lower boxes include partial karyograms involving genomic material of the chromosome analyzed by aCGH, representing two major subclones per side line.
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Ongoing chromosomal instability in neoplasia (CIN) generates intratumor genomic heterogeneity and limits the efficiency of oncotherapeutics. Neoplastic human cells utilizing the alternative lengthening of telomeres (ALT)-pathway, display extensive structural and numerical CIN. To unravel patterns of genome evolution driven by oncogene-replication s...
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Telomere crisis contributes to cancer genome evolution, yet only a subset of cancers display breakage-fusion-bridge (BFB) cycles and chromothripsis, hallmarks of experimental telomere crisis identified in previous studies. We examine the spectrum of structural variants (SVs) instigated by natural telomere crisis. Eight spontaneous post-crisis clone...
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... As proximity blocks appeared to correlate with chromosomal structure, we wondered whether proximity blocks would also reflect nonreference structure in genomically abnormal cells. The U2OS line used in cpg0016 is known to be karyotypically abnormal and heterogeneous, with different clones exhibiting distinct genotypes 28 . DepMap 11 cataloged a fusion between RAD50 on chromosome 5q and ZNF536 on chromosome 19q in this cell line, and examination of the cpg0016 map shows a clear block of interchromosomal proximity bias between 5q and 19q that closely recapitulates the boundaries of the annotated fusion (Fig. 1f). ...
Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) is a powerful tool for introducing targeted mutations in DNA, but recent studies have shown that it can have unintended effects such as structural changes. However, these studies have not yet looked genome wide or across data types. Here we performed a phenotypic CRISPR–Cas9 scan targeting 17,065 genes in primary human cells, revealing a ‘proximity bias’ in which CRISPR knockouts show unexpected similarities to unrelated genes on the same chromosome arm. This bias was found to be consistent across cell types, laboratories, Cas9 delivery methods and assay modalities, and the data suggest that it is caused by telomeric truncations of chromosome arms, with cell cycle and apoptotic pathways playing a mediating role. Additionally, a simple correction is demonstrated to mitigate this pervasive bias while preserving biological relationships. This previously uncharacterized effect has implications for functional genomic studies using CRISPR–Cas9, with applications in discovery biology, drug-target identification, cell therapies and genetic therapeutics.
... Cancer cells utilizing ALT display telomere length heterogeneity and elevated chromosomal instability (CIN) 7,8 . ALT-positive (ALT+) cells are characterised by increased incidence of extrachromosomal circular telomeric DNA (C-circles) and an elevated frequency of telomeric sister chromatid exchanges (T-SCEs) 7 . ...
Telomerase-independent cancer proliferation via the alternative lengthening of telomeres (ALT) relies upon two distinct, largely uncharacterized, break-induced-replication (BIR) processes. How cancer cells initiate and regulate these terminal repair mechanisms is unknown. Here, we establish that the EXD2 nuclease is recruited to ALT telomeres to direct their maintenance. We demonstrate that EXD2 loss leads to telomere shortening, elevated telomeric sister chromatid exchanges, C-circle formation as well as BIR-mediated telomeric replication. We discover that EXD2 fork-processing activity triggers a switch between RAD52-dependent and -independent ALT-associated BIR. The latter is suppressed by EXD2 but depends specifically on the fork remodeler SMARCAL1 and the MUS81 nuclease. Thus, our findings suggest that processing of stalled replication forks orchestrates elongation pathway choice at ALT telomeres. Finally, we show that co-depletion of EXD2 with BLM, DNA2 or POLD3 confers synthetic lethality in ALT cells, identifying EXD2 as a potential druggable target for ALT-reliant cancers.
... In all clones, except for R38/1, exactly two copies/alleles of the PPOX gene were identified by sequencing. Our data thus corresponds well with previously published finding that U-2 OS cells contain 2 to 3 copies of chromosome 1, the carrier of the PPOX gene 44,45 . While the N2 clone had both PPOX encoding alleles intact, insertions, deletions, and/or mutations, resulting in disruptions to the PPOX sequence and reading frame, were identified in both alleles of remaining six clones (Table 1). ...
In humans, disruptions in the heme biosynthetic pathway are associated with various types of porphyrias, including variegate porphyria that results from the decreased activity of protoporphyrinogen oxidase IX (PPO; E.C.1.3.3.4), the enzyme catalyzing the penultimate step of the heme biosynthesis. Here we report the generation and characterization of human cell lines, in which PPO was inactivated using the CRISPR/Cas9 system. The PPO knock-out (PPO-KO) cell lines are viable with the normal proliferation rate and show massive accumulation of protoporphyrinogen IX, the PPO substrate. Observed low heme levels trigger a decrease in the amount of functional heme containing respiratory complexes III and IV and overall reduced oxygen consumption rates. Untargeted proteomics further revealed dysregulation of 22 cellular proteins, including strong upregulation of 5-aminolevulinic acid synthase, the major regulatory protein of the heme biosynthesis, as well as additional ten targets with unknown association to heme metabolism. Importantly, knock-in of PPO into PPO-KO cells rescued their wild-type phenotype, confirming the specificity of our model. Overall, our model system exploiting a non-erythroid human U-2 OS cell line reveals physiological consequences of the PPO ablation at the cellular level and can serve as a tool to study various aspects of dysregulated heme metabolism associated with variegate porphyria.
... Chromosomal duplications are commonly identified in tumors, particularly tumors that utilize alternative lengthening of telomeres for telomere maintenance or undergo chromothripsis. 187,188 Many chromosomal amplifications found in cancer are identified as double minutes (dmin), or extrachromosomal segments of DNA packaged as circular chromosomes that have been shown to independently replicate, resulting in duplications. 189,190 One hypothesis for the generation of dmin is through the breakage-fusion-bridge pathway or DNA excision and repair via NHEJ. ...
Advances in genome and tissue engineering have spurred significant progress and opportunity for innovation in cancer modeling. Human induced pluripotent stem cells (iPSCs) are an established and powerful tool to study cellular processes in the context of disease-specific genetic backgrounds; however, their application to cancer has been limited by the resistance of many transformed cells to undergo successful reprogramming. Here, we review the status of human iPSC modeling of solid tumors in the context of genetic engineering, including how base and prime editing can be incorporated into "bottom-up" cancer modeling, a term we coined for iPSC-based cancer models using genetic engineering to induce transformation. This approach circumvents the need to reprogram cancer cells while allowing for dissection of the genetic mechanisms underlying transformation, progression, and metastasis with a high degree of precision and control. We also discuss the strengths and limitations of respective engineering approaches and outline experimental considerations for establishing future models.
... In all clones, except for R38/1, exactly two copies/alleles of the PPOX gene were identified by sequencing. Our data thus corresponds well with previously published finding that U-2 OS cells contain 2 to 3 copies of chromosome 1, the carrier of the PPOX gene 44,45 . While the N2 clone had both PPOX encoding alleles intact, insertions, deletions, and/or mutations, resulting in disruptions to the PPOX sequence and reading frame, were identified in both alleles of remaining six clones (Table 1). ...
In humans, disruptions in the heme biosynthetic pathway are associated with various types of porphyrias, including variegate porphyria that results from the decreased activity of protoporphyrinogen oxidase IX (PPO; E.C.1.3.3.4), the enzyme catalyzing the penultimate step of the heme biosynthesis. Here we report the generation and characterization of human cell lines, in which PPO was inactivated using the CRISPR/Cas9 system. The PPO knock-out (PPO-KO) cell lines are viable with the normal proliferation rate and show massive accumulation of protoporphyrinogen IX, the PPO substrate. Observed low heme levels trigger a decrease in the amount of functional heme containing respiratory complexes III and IV and overall reduced oxygen consumption rates. Untargeted proteomics further revealed dysregulation of 22 cellular proteins, including strong upregulation of 5-aminolevulinic acid synthase, the major regulatory protein of the heme biosynthesis, as well as additional ten targets with unknown association to heme metabolism. Importantly, knock-in of PPO into PPO-KO cells rescued their wild-type phenotype, confirming the specificity of our model. Overall, our model system reveals physiological consequences of the PPO ablation at the cellular level and can serve as a tool to study various aspects of dysregulated heme metabolism associated with variegate porphyria.
... There is also evidence that WGD can protect normal and cancer cells from stresses in the environment, including energy depletion, oxidative stress, and chemotherapy [171,[302][303][304][305]. Polyploidy may be a major driver of treatment failure, tumor relapse, and drug-induced genomic evolution [306]. ...
Cancer is a disease of cellular evolution. For this cellular evolution to take place, a population of cells must contain functional heterogeneity and an assessment of this heterogeneity in the form of natural selection. Cancer cells from advanced malignancies are genomically and functionally very different compared to the healthy cells from which they evolved. Genomic alterations include aneuploidy (numerical and structural changes in chromosome content) and polyploidy (e.g., whole genome doubling), which can have considerable effects on cell physiology and phenotype. Likewise, conditions in the tumor microenvironment are spatially heterogeneous and vastly different than in healthy tissues, resulting in a number of environmental niches that play important roles in driving the evolution of tumor cells. While a number of studies have documented abnormal conditions of the tumor microenvironment and the cellular consequences of aneuploidy and polyploidy, a thorough overview of the interplay between karyotypically abnormal cells and the tissue and tumor microenvironments is not available. Here, we examine the evidence for how this interaction may unfold during tumor evolution. We describe a bidirectional interplay in which aneuploid and polyploid cells alter and shape the microenvironment in which they and their progeny reside; in turn, this microenvironment modulates the rate of genesis for new karyotype aberrations and selects for cells that are most fit under a given condition. We conclude by discussing the importance of this interaction for tumor evolution and the possibility of leveraging our understanding of this interplay for cancer therapy.
Argonaute 2 (AGO2) is an indispensable component of the RNA-induced silencing complex, operating at the translational or posttranscriptional level. It is compartmentalized into structures such as GW- and P-bodies, stress granules and adherens junctions as well as the midbody. Here we show using immunofluorescence, image and bioinformatic analysis and cytogenetics that AGO2 also resides in membrane protrusions such as open- and close-ended tubes. The latter are cytokinetic bridges where AGO2 colocalizes at the midbody arms with cytoskeletal components such as α-Τubulin and Aurora B, and various kinases. AGO2, phosphorylated on serine 387, is located together with Dicer at the midbody ring in a manner dependent on p38 MAPK activity. We further show that AGO2 is stress sensitive and important to ensure the proper chromosome segregation and cytokinetic fidelity. We suggest that AGO2 is part of a regulatory mechanism triggered by cytokinetic stress to generate the appropriate micro-environment for local transcript homeostasis.
