Heterogeneity and multi-omics features of alternative lengthening of telomeres

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Telomere maintenance mechanisms (TMM) are crucial for cancer cells as they are required \nfor their unlimited proliferation capacity. While most cancers reactivate the reverse transcriptase \ntelomerase, a significant fraction of tumors maintains telomere length without it. These cancers \nemploy the alternative lengthening of telomeres (ALT) pathway, which relies on DNA repair and \nrecombination to extend telomere repeats. ALT presence is primarily confirmed with the goldstandard C-circle assay, which quantifies extrachromosomal telomere repeats that are only found in \nALT-positive cells. Mutations within the repeat repressor ATRX/DAXX/H3.3 are overrepresented in ALT \ncancers, which are believed to further telomere dysfunction and gene expression programs in ALT. \nALT presence has an impact on long-term survival in cancer, and detection of ALT in omics data is \ncurrently lacking. Although much progress has been made in the past few decades in elucidating the \nALT mechanism, the details concerning ALT are still unknown. This thesis addresses this question from \nthree angles: (i) Describing ALT activity heterogeneity in primary tumor samples; (ii) Using sequencing \nreadouts to define ALT and extract a characteristic signature; (iii) Inhibiting epigenetic modifiers with \ndrugs and observing their effect on viability in ALT cells. \nFirstly, I quantified C-circle levels in 687 primary tumor biopsies from sarcomas. The \nheterogeneous distribution indicates that ALT-activity can vary about tenfold within the same tumor \nentity. Next, I conducted ATAC-seq and RNA-seq of long and short RNAs in ALT positive and negative \ncell lines from pediatric glioblastoma and osteosarcoma to find shared ALT features. Information on \nopen chromatin regions, transcriptome, miRNA, transposable elements, and piRNA was extracted \nfrom these data. From the ATAC-seq data, it was found that ALT+ cell lines had predominantly \nincreased chromatin accessibility in non-coding regions. This change may be driven by AP-1 and RUNX \ntranscription factors (TF), whilst downregulated accessible regions result from reduced SOX TFs. From \ntranscriptomic data, it was revealed that immune TFs were enriched in upregulated ALT genes. This \nled to the identification of NFATC2 as a potential ALT biomarker, as it was found in differential \nexpression and transcriptome TF motif analysis. The immune TFs may be induced by genetic instability, \nyet the multi-omics ALT signature indicated that the cell lines have a reduced response to oxidative \nstress as well. These factors may cooperate in inducing a heightened inflammatory state that drives \nchromatin accessibility and gene expression. Differential miRNAs were extracted and could explain \nboth TERT and SOX downregulation and RUNX upregulation, indicating another gene regulatory \nmechanism employed by ALT cell lines. Furthermore, an integrative multi-omics analysis was \nperformed to extract an ALT signature. ALT-positive cells displayed a characteristic signature that was \ninfluenced by transcriptome, miRNA, and chromatin accessibility. As more upregulated open regions \nin ATAC data were observed, epigenetic drugs for repressive marks were applied to assess the \nrelationship between ALT activity and cell viability. I found that inhibition of repressive H3K27me3 and \nDNA methylation was correlated with an ALT-specific lethality and survival, respectively. Another \naberrant epigenetic feature found in ALT, an H3.3S31p chromosome-wide signal during mitosis, was \nstudied with different inhibitors to elucidate which kinase is responsible for its establishment. The \nHASPIN kinase was identified to reduce H3.3S31 phosphorylation upon treatment with a \ncorresponding inhibitor. This kinase is involved in chromosomal segregation and links ALT genetic \ninstability to DNA damage signaling during mitosis.