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Heterogeneity and multi-omics features of alternative lengthening of telomeres

Hadzic, Armin

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Abstract

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

Document type: Dissertation
Supervisor: Rippe, Prof. Dr. Karsten
Place of Publication: Heidelberg
Date of thesis defense: 4 February 2022
Date Deposited: 08 Mar 2022 07:19
Date: 2023
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
DDC-classification: 570 Life sciences
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