Biology subjects

Manojlovic, Z.

Publications and source records attributed to Manojlovic, Z..

6 recordsLinked to original sources

bioRxiv2026

SWI/SNF Alterations Define a Chromatin-Dependent Subtype of Urothelial Carcinoma

PurposeSWI/SNF (BAF) chromatin remodeling complex alterations are common in urothelial carcinoma, yet no biomarker-directed therapeutic strategies have been established for this population. We investigated whether BAF alterations delineate a biologically distinct, therapeutically actionable urothelial carcinoma subtype. Experimental DesignWe performed integrative genomic and transcriptomic analyses of 792 urothelial carcinoma tumors from the Oncology Research Information Exchange Network (ORIEN) and validated findings in the TCGA-BLCA cohort. Mechanistic studies incorporated RNA sequencing and ATAC-seq following histone deacetylase (HDAC) inhibition. Functional dependencies were assessed using patient-derived xenograft organoids and cell line models. Clinical relevance was explored in a biomarker-enriched investigator-initiated trial. ResultsApproximately half of urothelial carcinoma tumors exhibited BAF alterations, defining a previously unrecognized chromatin-altered molecular subtype characterized by activation of proliferative programs, loss of lineage identity, and altered metabolic signaling. This subtype was enriched for transcriptomic programs associated with HDAC inhibitor sensitivity and depleted of HDAC inhibitor resistance signatures. Mechanistically, HDAC inhibition induced widespread chromatin remodeling with reduced accessibility at AP-1 and TEAD-associated regions, and downregulation of E2F- and MYC-driven transcriptional networks. Functional studies confirmed enhanced HDAC inhibition sensitivity in ARID1A-mutated cell lines and a patient-derived organoid model. Early clinical observations demonstrated a durable responder treated with HDAC inhibitors and immunotherapy. ConclusionsBAF alterations define a chromatin-dependent tumor state in urothelial carcinoma that is selectively vulnerable to HDAC inhibition. Integrating genomic, epigenomic, functional, and early clinical evidence, these findings provide a rationale for biomarker-enriched clinical trials and HDAC inhibitor-based combination strategies in urothelial carcinoma.

cancer biology↗

bioRxiv2026

Mutation count and mutation profile analyses of single-nucleotide variants in single human colon crypts

Genetic heterogeneity due to the accumulation of mutations in normal tissues can increase cancer risk and be an important factor in many age-related degenerative and chronic diseases. Somatic mutations can arise from DNA damage or replication errors and accumulate in normal tissues with age. We obtained high depth whole genome sequencing data to comprehensively profile somatic mutations in 106 single human colon crypts with matched bulk controls from 21 individuals age 10 months to 90 years old. Our analysis reveals that about half of the human crypts are polyclonal (multi-lineage) instead of entirely monoclonal as conventionally construed. Consequently, the DNA mutation count would be inflated, while the variant allele frequency for each variant would be reduced in the cell population of a multi-lineage colon crypt. Therefore, our mutation count analysis includes using single stem cell lineage colon crypts exclusively to establish the somatic mutation rate for each of the 96 trinucleotide mutation categories. In addition, the mutation profile, representing the relative presence of the 96 trinucleotide mutation categories, in each colon crypt is analyzed. Unlike mutation count, the mutation profile is not affected by crypt clonality and is very similar across all ages in individuals with no chemotherapy or radiation treatment. Importantly, combined results from mutation count and mutation profile analyses of individuals with chemotherapy or radiation suggest an intriguing impact of these treatments on cell survival. The baseline mutation rate and the normal mutation profile established in our study provide a framework for a genomic standard to assess biological age and deviations associated with factors such as lifestyle, age-related degenerative and chronic diseases, and potentially cancer treatment outcome. Future studies similar to this current study on other tissues can provide further insight into how and why different tissues age differently in humans.

genomics↗

bioRxiv2026

Complex Indel Detection: A Simulation-Based Framework and Parsing with FreeBayes

In contrast to simple deletions and simple insertions, most complex indels involve both deletions and insertions, often with base changes within a few nucleotides of the indels left and right boundaries. These complex indels often arise from double-strand breaks (DSB), which in normal somatic cells are predominantly repaired by nonhomologous DNA end joining (NHEJ). Such complex indels pose a difficult analytical problem for existing indel callers because the observed VCF representation may be locally shifted, extended with matching flanking bases, or fragmented into several closely spaced calls. To evaluate complex indel representation, we tested six variant calling approaches: FreeBayes, HaplotypeCaller, Mutect2, Strelka2, DRAGEN Germline, and DRAGEN Somatic pipelines. Among the approaches evaluated, FreeBayes most consistently represented simulated complex indels as single nearby variant records. We then developed a parsing workflow that derives effective deleted and inserted sequences from FreeBayes VCF output and enriches for candidate complex indels. This approach supports analysis of naturally occurring DSB repair events in single human colon crypts.

bioinformatics↗

bioRxiv2026

Germline VCF Annotator: a lightweight pipeline for processing germline VCFs with robust variant extraction and read evidence quality control

Raw variant calls are typically distributed as VCF files and are not well-suited for direct human review. They are intended for programmatic parsing, and spreadsheet import can distort data through automatic type conversion. Furthermore, variants in VCF are commonly annotated to add gene context and predicted functional consequences. Ensembl VEP, a widely used standard for transcript-aware variant annotation, was adapted in this study to generate standardized consequence fields across genomic features. Using a colon crypt whole-genome sequencing cohort as the motivating dataset, this study examined whether variation at DNA damage response and repair (DDR) loci could contribute to mutation-burden patterns in normal colon crypts, including patterns associated with age and potential treatment-related exposure. To make this question testable in a reproducible table-based format, the Germline VCF Annotator was developed as a two-step workflow that normalizes germline VCFs, generates VEP tabular annotations with explicit allele fields, and then extracts variants of interest and appends read-evidence metrics to assign a rules-based QC class. Within-patient concordance across technical repeats at predefined DDR loci was near-perfect after filtering for nonsilent SNVs with read depth [≥]15, with discordance concentrated among Low-QC loci. Bulk and crypt-derived samples showed no age-related trend in DDR burden. Although the demonstration centers on DDR and aging, the Germline VCF Annotator is applicable to other gene sets that require human-readable locus-level summaries with retained allele provenance and read evidence.

bioinformatics↗

bioRxiv2026

Fine Structural Features of Complex InDels and NHEJ Repair at Naturally Occurring Damage Sites in Normal Human Colon Crypts

DNA repair in biochemical and genetic experimental systems permits a precise definition of enzyme requirements and mechanistic steps. Comparing these findings to repair events at naturally occurring damage sites in multicellular organisms is essential for confirming and expanding these insights into a physiologic context. However, heterogeneity in any normal cell population increases with each cell division, and the reliable detection of replication-independent DNA damage sites and their repair has been a major barrier. Here, we examine single human colon crypts, which harbor natural cell clones, using a novel whole-genome sequencing (WGS) method to identify complex insertion-deletion (indel) in the crypt stem cells. Analysis of complex indel events likely repaired by non-homologous end joining occurring in crypt stem cells permits inferences about the in vivo repair of naturally occurring DNA damage within physiologically-relevant chromatin in normal human cells.

genomics↗

bioRxiv2025

High-depth Whole Genome Sequencing of Single Human Colon Crypts Uncovers New View on Crypt Clonality

It is generally accepted that each colon crypt is monoclonal and is populated by a single stem cell lineage after early human life. It has been impossible to profile somatic mutations genome-wide because high-depth and high-quality whole genome sequencing (WGS) of single cells is unachievable without tissue culture or whole genome amplification (WGA). Therefore, the cell-to-cell heterogeneity in each individual remains mostly unknown. Applying our novel WGA-free WGS method to obtain >30X post-alignment depth, we comprehensively profiled somatic mutations in 71 single human colon crypts with matched bulk controls from 14 individuals. Analysis reveals that colon crypts are commonly of multiple lineages in human adults. Our study is the first to demonstrate that an appropriately designed WGS approach can determine cell- to-cell heterogeneity in natural cell clones. The much higher sensitivity of WGS than the previous methods in lineage tracing can unlock the complex stem cell dynamics in the colon crypt.

genomics↗