The Hidden Genomic and Transcriptomic Plasticity of Giant Marker Chromosomes in Cancer

Abstract

Genome amplification in the form of rings or giant rod-shaped marker chromosomes (RGMs) is a common genetic alteration in soft tissue tumors. The mitotic stability of these structures is often rescued by perfectly functioning analphoid neocentromeres, which therefore significantly contribute to cancer progression. Here, we disentangled the genomic architecture of many neocentromeres stabilizing marker chromosomes in well-differentiated liposarcoma and lung sarcomatoid carcinoma samples. In cells carrying heavily rearranged RGMs, these structures were assembled as patchworks of multiple short amplified sequences, disclosing an extremely high level of complexity and definitely ruling out the existence of regions prone to neocentromere seeding. Moreover, by studying two well-differentiated liposarcoma samples derived from the onset and the recurrence of the same tumor, we documented an expansion of the neocentromeric domain that occurred during tumor progression, which reflects a strong selective pressure acting toward the improvement of the neocentromeric functionality in cancer. In lung sarcomatoid carcinoma cells we documented, extensive "centromere sliding" phenomena giving rise to multiple, closely mapping neocentromeric epialleles on separate coexisting markers occur, likely due to the instability of neocentromeres arising in cancer cells. Finally, by investigating the transcriptional activity of neocentromeres, we came across a burst of chimeric transcripts, both by extremely complex genomic rearrangements, and cis/trans-splicing events. Post-transcriptional editing events have been reported to expand and variegate the genetic repertoire of higher eukaryotes, so they might have a determining role in cancer. The increased incidence of fusion transcripts, might act as a driving force for the genomic amplification process, together with the increased transcription of oncogenes. KEYWORDS neocentromere; fusion transcript; WDLPS; LSC; gene amplification G ENOME amplification is a frequent genetic alteration in cancer, with variable cytogenetic manifestations including double minutes, homogeneously-staining regions, and/or ring and giant rod-shaped marker chromosomes (RGMs)

Promoter G-quadruplexes and transcription factors cooperate to shape the cell type-specific transcriptome

“…A recent work by Hänsel-Hertsch and colleagues 22 supported by previous literature 50, [68][69][70] showed that cancer-related copy number variants (CNVs), i.e., amplifications, as well as single nucleotide variants, are enriched in breast cancer-specific G4s. To test whether WDLPS-specific G4s were associated with CNVs and also pan-cancer somatic variants (SVs), we calculated the overlap between 93T449 cell-specific CNVs or SVs 37 , and G4 peaks found in 93T449 or HaCaT cells. We found that 73% of 93T449 CNVs (299 out of 411 detected CNVs) overlapped with folded G4s peaks, and 47% of all CNVs (193 out of 411) were in genomic regions that harbor 93T449 cell line-specific G4s (i.e., G4s that are not folded in the HaCaT cell line).…”

“…The R package "ChIPpeakAnno" 34,94 was employed to calculate overlapping of 93T449 CNVs and SVs regions with G4-ChIP-seq peaks. CNVs data obtained by BICseq and SVs obtained by WGS (Crest) were retrieved from Macchia et al 37 . Genomic regions coordinates were converted from GRCh37/hg19 to GRCh38/hg38 by mean of UCSC Lift Genome Annotations tool (https://genome.ucsc.edu/cgi-bin/hgLiftOver).…”

“…In the present work, we performed ChIP-seq analysis with a G4 antibody to map the folded G4s in cells of well-differentiated liposarcoma (WDLPS), a malignant neoplasia affecting the general human population, including young adults and children. WDLPS is resistant to current chemotherapies and has a largely unexplored biological background [35][36][37] . The integration of RNA-seq and assay for transposase-accessible chromatin followed by high-throughput sequencing (ATAC-seq) data allowed us to build a relationship between folded G4s, chromatin state, and transcriptional output.…”

The MDM2 inducible promoter folds into four-tetrad antiparallel G-quadruplexes targetable to fight malignant liposarcoma

“…MDM2 amplification in WDLPS is associated to the formation of giant ring marker chromosomes containing multiple copies of the gene. Chimeric and fusion transcripts arising from such unstable regions are hypothesized to push the genomic amplification process during tumour progression ( 70 ). The deficiency of one or more MDM2 G4 binding/unwinding proteins may cause replicative stress that worsens or is at the root of the pathological genome amplification, as recently proposed for ATRX-deficient malignant glioma ( 71 ).…”

Small ring has big potential: insights into extrachromosomal DNA in cancer

“…In ring-chromosomes, the ends of the DNA sequence are fused together to form a ring shape [ 12 ]. Neochromosomes contain centromere and telomere sequences, with a typical sequence length of 30–600 Mb [ 7 , 8 ]. Neochromosomes have been shown to contain high copy numbers of oncogenes and can be created through chromothripsis [ 9 ].…”