ABSTRACT:Telomere length shortening has been associated with genomic instability and acquisition of molecular lesions, but these processes have not been systematically studied across large cohorts of myeloid neoplasia (MN). As proof of concept for a novel, cross-validated whole-genome sequencing-based method of telomere content (TC) determination combined with mutations, transcriptomics, and functional assays, we studied TC in correlation with specific molecular features of a large cohort (N = 1804) of patients with MN, including acute myeloid leukemia (AML) and myelodysplastic syndrome. When compared with healthy participants and patients with nonclonal diseases such as persistent polyclonal B-cell lymphocytosis, both MN and nonmalignant controls with clonal disease, such as paroxysmal nocturnal hemoglobinuria and aplastic anemia, exhibited decreased TC. Furthermore, we show that TC is lowered in adult MN abrogating correlation with age with considerable TC diversification among certain morphologic and molecular subtypes. For instance, AML harbored the lowest TC. Furthermore, MN originating from a more mature cell of origin (eg, acute promyelocytic leukemia) or characterized by hyperproliferative driver mutations (eg, RAS pathway genes) had lower TC, possibly indicating a loss of telomere maintenance capacity. In contrast, compared with other mutations, MN subtypes arising in a context of profound genetic alterations, such as TP53 mutations and complex karyotype, exhibited a relatively higher/preserved TC. This phenomenon did not involve alternative lengthening processes but was rather consistent with an increased TC due to preserved activity of the telomerase complex. Our results describe a common and genotype-specific telomeric makeup of a large cohort of patients with MN providing a molecular benchmark for future therapeutic targeting of the telomere machinery.

Motivation Telomere biology plays a critical role in multiple biological processes including carcinogenesis, aging, and genome stability. With increasing availability of DNA-sequence datasets, telomere length and composition are more frequently directly inferred in silico. The TelomereHunter software is used in genome research and precision oncology to study telomere maintenance mechanisms from routine sequencing data. However, bioinformatics tools face constant challenges such as increasing the number and size of genomic datasets, novel file formats and deprecating software components.Results We developed TelomereHunter2 (TH2) to create a sustainable framework for telomere analysis. By containerizing our software and improving the runtime by up to 74%, we simplify the integration of TH2 into diverse precision oncology workflows and computational environments. We also extended TH2 to support non-human genomes and single-cell sequencing approaches, broadening its applications across species and methodologies. We demonstrate TH2 improvements on a pilot dataset.Availability and implementation TelomereHunter2 is an open-source Python package released under the GPL-3.0 license. It is distributed via PyPI and the source code, documentation, and wiki are available at: https://github.com/ferdinand-popp/TelomereHunter2.

Abstract Complex karyotype sarcomas (CKS) are heterogeneous mesenchymal malignancies that typically lack recurrent actionable oncogenic drivers and remain therapeutically challenging. Loss of ATRX is a recurrent feature of CKS and defines a particularly high-risk subgroup. ATRX loss is also associated with activation of the alternative lengthening of telomeres (ALT) pathway, and ALT-positive sarcomas have been linked to poor clinical outcomes. However, the molecular underpinnings underlying ALT-status-dependent differences in CKS, as well as the therapeutic vulnerabilities associated with ALT, remain poorly defined. By integrating C-circle-based ALT detection across 776 sarcoma samples with multi-modal sequencing of five CKS subtypes, we find that ALT activity is associated with enriched hallmarks of genomic instability. ALT-positive transcriptomes are dominated by a coordinated DNA damage response and mitotic program, in contrast to oncogenic signaling pathways that drive TERT activation in ALT-negative tumors. Long-read sequencing reveals telomere repeat clusters and telomere-mediated healing at structural breakpoints in ALT-positive tumors. These events also occur on extrachromosomal DNA (ecDNA), linking ALT activity to ecDNA biology. Together, our findings position ALT status as an important stratifying feature of CKS and identify ALT-associated transcriptional programs as potential therapeutic targets.

The lysine-specific demethylase 1 (LSD1) regulates hematopoietic stem cell differentiation and has been identified as a therapeutic target in hematological disorders. LSD1 demethylates mono and dimethylated histones 3 at lysine 4 and 9. In addition, it acts as a scaffold for the formation of chromatin-modifying complexes that regulates the transcription of myeloid-lineage-specific genes in complex with GFI1, a transcriptional repressor. While both enzymatic and non-enzymatic functions of LSD1 have been well defined, the relative importance of these two functions in hematopoiesis remains incompletely understood. Here, we investigated the contribution of enzymatic and non-enzymatic functions of LSD1 to myelopoiesis. We show that myeloid differentiation is independent of the enzymatic functions of LSD1 but requires the non-enzymatic, scaffolding function, which directs GFI1 binding to target sequences. In the absence of the LSD1 protein, GFI1 DNA binding is diminished, and myeloid cell differentiation arrests at an immature, myelomonocytic-like cell stage, which overexpresses Prtn3. We provide functional data implicating Prtn3 as an effector of the stem cell expansion and myeloid maturation block caused by the loss of LSD1.

Telomere length shortening has been associated with genomic instability and acquisition of molecular lesions, but these processes have not been systematically studied across large cohorts of myeloid neoplasia (MN).As proof of concept for a novel, cross-validated WGS-based method of telomere content (TC) determination combined with mutations, transcriptomics, and functional assays, we studied TC in correlation with specific molecular features of a large cohort (n=1804) of MN patients including acute myeloid leukemia (AML) and myelodysplastic syndrome. When compared to healthy subjects and patients with non-clonal diseases such as persistent polyclonal B cell lymphocytosis, both MN and non-malignant controls with clonal disease, such as paroxysmal nocturnal hemoglobinuria and aplastic anemia, exhibited decreased TC. Furthermore, we show that TC is lowered in adult MN abrogating correlation with age with considerable TC diversification among certain morphologic and molecular subtypes. For instance, AML harbored the lowest TC. Furthermore, MN originating from a more mature cell of origin (e.g., APL), and those characterized by hyperproliferative driver mutations (e.g., RAS pathway genes) had lower TC, possibly indicating a loss of telomere maintenance capacity. In contrast, MN subtypes arising in a context of profound genetic alterations, such as TP53 mutations and complex karyotype, exhibited a relatively higher/preserved TC compared to other mutations. This phenomenon did not involve alternative lengthening processes but was rather consistent with an increased TC due to preserved activity of the telomerase complex. Our results describe a common and genotype-specific telomeric make-up of a large cohort of patients with MN providing a molecular benchmark for future therapeutic targeting of the telomere machinery.

Telomeres are repetitive nucleoprotein structures that cap the ends of linear chromosomes and are essential for maintaining genomic stability. While individual chromosome ends maintain distinct telomere lengths, the extent of this conservation across tissues and populations remains unclear due to the difficulty of analyzing repetitive telomeric sequences. Here, we show that high-coverage whole-genome Nanopore sequencing enables robust measurement of telomere length at the level of individual chromosomes. Nanopore reads yield reproducible telomere length estimates across replicates, in contrast to PacBio HiFi reads. Across > 250 individuals from 1000 Genomes and the SMaHT projects, chromosome- specific telomere length patterns are conserved across individuals and tissues, with tissues from the same individual showing highly similar patterns. This conserved landscape suggests coordinated regulation, whose disruption may contribute to genomic instability. Nanopore sequencing also allows simultaneous detection of structural variants, including disruption of TERT and NHP2 that drive global telomere shortening. Furthermore, our quantification of telomere variant repeats in positional context indicates active telomerase-mediated elongation. Our integrated profiling of telomere length and structural variation enables inference of variant effects on chromosome-specific telomere dynamics and may uncover risk factors for short telomere syndromes and cancer. Importantly, positionally fully resolved telomeric variant repeat patterns may predict activated telomere maintenance mechanisms with high accuracy. ### Competing Interest Statement The authors have declared no competing interest.

The recent FDA approval of Imetelstat, a new class of antineoplastic drugs for the treatment of MDS1 opens the question on whether telomere content (TC) and/or telomerase function might constitute therapeutic targets and diagnostic biomarkers. Early studies have not specifically delved in elucidating such possibility. In general, cancer relies on excessive shortening of telomeres, and one could envision that systematic and comprehensive genomic and functional studies of TC in myeloid neoplasia (MN) might reveal the “Achilles' heel” e.g., the group with a TC likely responding to Imetelstat. We have adopted WGS-based pipelines to measure TC in MN. Taking advantage of a large, well-annotated cohort (n=1804) of patients with MN including AML (n=730), MDS (n=702), MPN (n=372) and 163 non-malignant controls (PNH/AA [n=102], persistent polyclonal B lymphocytosis (n=50) and healthy subjects [n=11]) we studied TC in correlation with pathomorphologic, cytogenetic and molecular subtypes of the disease. First, we cross-validated different bioinformatic WGS-based pipelines23 and established consistency with PCR-based TC measurements. Thus, we observed a general decrease in TC in MN patients irrespective of age. In both AML and MDS cohorts (for subsequent analysis referred together as MN), blast percentage correlated with telomere shortening (p<.0001, r2=0.0231 and p=.0346, r2=0.0063, respectively). Comparing TC among karyotypic abnormalities in MN, we detected higher TC in patients with del(5q) (p<.0001) and lower TC in patients with t(6;9) (p=.0002), t(8;21) (p<.0001), inv(16) (p=.0003), t(9;11) (p<.0001), t(15;17) (p<.0001) when compared to patients with normal karyotype. The analysis of TC according to the genetic landscape revealed higher TC in patients harboring ETV6 (p=.0172), SF3B1 (p=.0015), SRSF2 (p=.0005) and TP53 (p=.0025) mutations and lower TC in patients carrying FLT3 (p<.0001), JAK2 (p=.0191), KRAS (p=.0001), NPM1 (p<.0001), NRAS (p=.0234) and WT1 (p=.0002) mutations when compared to the overall MN population. We then explored specific TC signatures and we observed that high TC MN (above 90th percentile) were mainly represented by MDS and were enriched in -7/del(7q), inv(3)/t(3;3) and complex karyotype abnormalities as well as ASXL1, SRSF2 and TP53 mutations. Low TC MN were mainly represented by AML and were enriched in t(15;17) and t(8;21) as well as KRAS, NRAS, NPM1 and WT1 mutations. In a multivariate analysis nor the diagnosis (AML vs MDS), the age or the karyotype (abnormal vs normal) correlated to TC, whereas we detected an independent role of blast percentage (p<.0001) and TP53 mutations (positively correlating with TC, p=.001). To corroborate our findings, we investigated germline mutations in genes involved in telomere machinery (18 variants identified, 2 co-occurring with TP53 mutation [ATRX, c.6332G>C; TERT, c.1807del]) and excluded an impact of them on TC in this context. RNASeq supported these findings as mRNA expression of genes involved in telomere maintenance (RAP1A, TERC, TINF2, TPP1, CTC1) were only increased in patients with MN and high TC. Functional studies, including singletons composition of telomeres investigation, C-circle analysis and transcriptome-based scores to assess telomerase activity (EXTENDScore), showed that among all mutations, the ones with profound genomic alterations (e.g., TP53 mutations) maintain telomere elongation activity. In conclusion, our findings demonstrate that telomere shortening is common in MN irrespective of telomere machinery hypomorphic variants. In addition, mutations driving hyperproliferation (e.g., NRAS, KRAS) might exceed the compensatory capacity of normal telomere lengthening as also suggested by the negative correlation between TL and blast percentage. These results open the question as to whether TC might be used as a marker of response to Imetelstat and whether can identify disease phenotypes more likely to respond to the drug (MN relying on high TC (e.g., TP53 mutants) vs MN reaching a critical level of telomeres shortening (e.g., NRAS/KRAS mutated).

Cancer is a heterogeneous disease that arises due to mutations that drive cancer progression. However, the identification of these functional mutations has typically focused only on protein-coding DNA. Among non-coding mutations, only a few have been clearly associated with cancer. We hypothesize that this gap in discovery is partly due to the limitations of current methods requiring high recurrence of mutations. To support candidate selection for experimental validation of lowly recurrent and singleton promoter mutations, new computational approaches for the integrated analysis of multi-omics data are required. To address this challenge, the REMIND-Cancer Pipeline leverages whole-genome sequencing and RNA-Seq data to extract and prioritize functional promoter mutations, regardless of their recurrence status. Subsequently, pSNV Hunter aggregates and visualizes comprehensive information for each candidate. We demonstrate the functionality of both tools by applying it to the PCAWG dataset. This workflow successfully identified and prioritized known highly-recurrent mutations, as well as, novel singletons and lowly recurrent candidates. Hence, the output of our workflow directly supports hypothesis generation for subsequent experimental validation to overcome limitations of recurrence-based approaches. ### Competing Interest Statement The authors have declared no competing interest.

The characterization of somatic genomic variation associated with the biology of tumors is fundamental for cancer research and personalized medicine, as it guides the reliability and impact of cancer studies and genomic-based decisions in clinical oncology. However, the quality and scope of tumor genome analysis across cancer research centers and hospitals are currently highly heterogeneous, limiting the consistency of tumor diagnoses across hospitals and the possibilities of data sharing and data integration across studies. With the aim of providing users with actionable and personalized recommendations for the overall enhancement and harmonization of somatic variant identification across research and clinical environments, we have developed ONCOLINER. Using specifically designed mosaic and tumorized genomes for the analysis of recall and precision across somatic SNVs, insertions or deletions (indels), and structural variants (SVs), we demonstrate that ONCOLINER is capable of improving and harmonizing genome analysis across three state-of-the-art variant discovery pipelines in genomic oncology.

Cancer is a heterogeneous disease caused by genetic alterations. Computational analysis of cancer genomes led to the expansion of the catalog of driver mutations. While individual high-impact mutations have been discovered also in gene promoters, frequency-based approaches have only characterized a few novel candidates. To investigate the promoter mutation paucity in cancer, we developed the REMIND-Cancer workflow to predict activating promoter mutations in silico , irrespective of their recurrence frequency, and applied it to the PCAWG dataset. We positively validated 7 candidates by luciferase assay including mutations within the promoters of ANKRD53 and MYB . Our analysis indicates that particular mutational signatures and necessary co-alterations constrain the creation and positive selection of functional promoter mutations. We conclude that activating promoter mutations are more frequent in the PCAWG dataset than previously observed, which has potential implications for personalized oncology. ### Competing Interest Statement The authors have declared no competing interest.

Introduction: Biological processes such as aging, carcinogenesis, and immune response rely on the ability to maintain or rapidly expand cell populations. The fitness of the involved cells is constrained by their replicative potential, which is reflected in the cellular telomere content. Method: We apply TelomereHunter to scATAC-seq data to determine telomere content on single-cell level, in a hematopoietic dataset consisting of 35,139 cells from samples of basal cell carcinoma patients receiving programmed cell death protein 1 (PD1) blockade treatment. Integrating information from open-chromatin-based signatures to assess cell identity, we characterize the heterogeneity of telomere length for individual cell populations pre- and post-immunotherapy. Results: The extracted telomeric reads reflect the expected telomereome-to-genome fraction. Telomere content distributions differ significantly between cell populations, and the median telomere content in intermediate and terminal exhausted CD8+ T-cells pre-treatment is significantly correlated to response to PD-1 checkpoint blockade. Likewise, telomere content correlates with post-treatment cell proliferation in terminally exhausted and T follicular helper cells from responding patients. Conclusion: Telomere content measurement from scATAC-seq data has a sufficiently high signal-to-noise ratio to detect significant differences between cell types. Furthermore, the telomere content of CD8+ exhausted T-cells pre-treatment is a putative biomarker for successful PD-1-based immunotherapy. ### Competing Interest Statement The authors have declared no competing interest.

Supplementary Table S2 shows sequencing coverage metrics.

Supplementary Figure S1 shows circos plots including 17 PDACs of the patient cohort and a PDCM.

Supplementary Figure S3 illustrates genomic breakpoints of fusions in KRASwt tumors.

Supplementary Figure S2 shows experimental data demonstrating the proliferative capacity of the ATP1B1-NRG1 fusion.

Supplementary Table S1 lists potentially clinically relevant somatic alterations in the PDAC patient cohort.

Supplementary patient data provides a summary on the clinical course of patient 16.

The prognosis of AML patients with adverse genetics, such as a complex, monosomal karyotype and TP53 lesions, is still dismal even with standard chemotherapy. DNA-hypomethylating agent monotherapy induces an encouraging response rate in these patients. When combined with decitabine (DAC), all-trans retinoic acid (ATRA) resulted in an improved response rate and longer overall survival in a randomized phase II trial (DECIDER; NCT00867672). The molecular mechanisms governing this in vivo synergism are unclear. We now demonstrate cooperative antileukemic effects of DAC and ATRA on AML cell lines U937 and MOLM-13. By RNA-sequencing, derepression of >1200 commonly regulated transcripts following the dual treatment was observed. Overall chromatin accessibility (interrogated by ATAC-seq) and, in particular, at motifs of retinoic acid response elements were affected by both single-agent DAC and ATRA, and enhanced by the dual treatment. Cooperativity regarding transcriptional induction and chromatin remodeling was demonstrated by interrogating the HIC1, CYP26A1, GBP4, and LYZ genes, in vivo gene derepression by expression studies on peripheral blood blasts from AML patients receiving DAC + ATRA. The two drugs also cooperated in derepression of transposable elements, more effectively in U937 (mutated TP53) than MOLM-13 (intact TP53), resulting in a "viral mimicry" response. In conclusion, we demonstrate that in vitro and in vivo, the antileukemic and gene-derepressive epigenetic activity of DAC is enhanced by ATRA.