Use of CRISPR-modified human stem cell organoids to study the origin of mutational signatures in cancer.

Mutational processes underlie cancer initiation and progression. Signatures of these processes in cancer genomes may explain cancer etiology and could hold diagnostic and prognostic value. We developed a strategy that can be used to explore the origin of cancer-associated mutational signatures. We used CRISPR-Cas9 technology to delete key DNA repair genes in human colon organoids, followed by delayed subcloning and whole-genome sequencing. We found that mutation accumulation in organoids deficient in the mismatch repair gene MLH1 is driven by replication errors and accurately models the mutation profiles observed in mismatch repair-deficient colorectal cancers. Application of this strategy to the cancer predisposition gene NTHL1, which encodes a base excision repair protein, revealed a mutational footprint (signature 30) previously observed in a breast cancer cohort. We show that signature 30 can arise from germline NTHL1 mutations

VIBE: an R-package for VIsualization of Bulk RNA Expression data for therapeutic targeting and disease stratification

BackgroundDevelopment of cancer treatments such as antibody-based therapy relies on several factors across the drug-target axis, including the specificity of target expression and characterization of downstream signaling pathways. While existing tools for analyzing and visualizing transcriptomic data offer evaluation of individual gene-level expression, they lack a comprehensive assessment of pathway-guided analysis, relevant for single- and dual-targeting therapeutics. Here, we introduce VIBE (VIsualization of Bulk RNA Expression data), an R package which provides a thorough exploration of both individual and combined gene expression, supplemented by pathway-guided analyses. VIBE’s versatility proves pivotal for disease stratification and therapeutic targeting in cancer and other diseases.ResultsVIBE offers a wide array of functions that streamline the visualization and analysis of transcriptomic data for single- and dual-targeting therapies. Its intuitive interface allows users to evaluate the expression of target genes and their associated pathways across various cancer indications, aiding in target and disease prioritization. Metadata, such as treatment or number of prior lines of therapy, can be easily incorporated to refine the identification of patient cohorts hypothesized to derive benefit from a given drug. We demonstrate how VIBE can be used to assist in indication selection and target identification in three user case studies using both simulated and real-world data. VIBE integrates statistics in all graphics, enabling data-informed decision-making.ConclusionsVIBE facilitates detailed visualization of individual and cohort-level summaries such as concordant or discordant expression of two genes or pathways. Such analyses can help to prioritize disease indications that are amenable to treatment strategies such as bispecific or monoclonal antibody therapies. With this tool, researchers can enhance indication selection and potentially accelerate the development of novel targeted therapies with the goal of precision, personalization, and ensuring treatments align with an individual patient’s disease state across a spectrum of disorders. Explore VIBE’s full capabilities using the vignettes on the GitLab repository (https://gitlab.com/genmab-public/vibe)

Genomic landscape of rat strain and substrain variation

Background: Since the completion of the rat reference genome in 2003, whole-genome sequencing data from more than 40 rat strains have become available. These data represent the broad range of strains that are used in rat research including commonly used substrains. Currently, this wealth of information cannot be used to its full extent, because the variety of different variant calling algorithms employed by different groups impairs comparison between strains. In addition, all rat whole genome sequencing studies to date used an outdated reference genome for analysis (RGSC3.4 released in 2004). Results: Here we present a comprehensive, multi-sample and uniformly called set of genetic variants in 40 rat strains, including 19 substrains. We reanalyzed all primary data using a recent version of the rat reference assembly (RGSC5.0 released in 2012) and identified over 12 million genomic variants (SNVs, indels and structural variants) among the 40 strains. 28,318 SNVs are specific to individual substrains, which may be explained by introgression from other unsequenced strains and ongoing evolution by genetic drift. Substrain SNVs may have a larger predicted functional impact compared to older shared SNVs. Conclusions: In summary we present a comprehensive catalog of uniformly analyzed genetic variants among 40 widely used rat inbred strains based on the RGSC5.0 assembly. This represents a valuable resource, which will facilitate rat functional genomic research. In line with previous observations, our genome-wide analyses do not show evidence for contribution of multiple ancestral founder rat subspecies to the currently used rat inbred strains, as is the case for mouse. In addition, we find that the degree of substrain variation is highly variable between strains, which is of importance for the correct interpretation of experimental data from different labs

Deficiency of nucleotide excision repair is associated with mutational signature observed in cancer

Nucleotide excision repair (NER) is one of the main DNA repair pathways that protect cells against genomic damage. Disruption of this pathway can contribute to the development of cancer and accelerate aging. Mutational characteristics of NER-deficiency may reveal important diagnostic opportunities, as tumors deficient in NER are more sensitive to certain treatments. Here, we analyzed the genome-wide somatic mutational profiles of adult stem cells (ASCs) from NER-deficient Ercc1−/Δ mice. Our results indicate that NER-deficiency increases the base substitution load twofold in liver but not in small intestinal ASCs, which coincides with the tissue-specific aging pathology observed in these mice. Moreover, NER-deficient ASCs of both tissues show an increased contribution of Signature 8 mutations, which is a mutational pattern with unknown etiology that is recurrently observed in various cancer types. The scattered genomic distribution of the base substitutions indicates that deficiency of global-genome NER (GG-NER) underlies the observed mutational consequences. In line with this, we observe increased Signature 8 mutations in a GG-NER-deficient human organoid culture, in which XPC was deleted using CRISPR-Cas9 gene-editing. Furthermore, genomes of NER-deficient breast tumors show an increased contribution of Signature 8 mutations compared with NER-proficient tumors. Elevated levels of Signature 8 mutations could therefore contribute to a predictor of NER-deficiency based on a patient's mutational profile

Long-term culture of genome-stable bipotent stem cells from adult human liver

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy

Decoding the mutational history of human stem cells : From patterns to mechanisms

The extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of adult stem cell (ASC) divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal human ASCs remain unknown. In this thesis, a new method is presented for cataloging mutations in individual human ASCs. Single ASCs are expanded in vitro into clonal organoid cultures to generate sufficient DNA for whole-genome sequencing (WGS) analysis, which allows detection of the mutations that accumulated in vivo in the original ACS. Using this method, we determined the genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. However, mutational signature analysis suggests that different mutational processes contribute to mutation accumulation in liver ASCs compared to colon and small intestinal ASCs. In the intestinal ASCs the majority of the mutations are a result of spontaneous deamination of methylated cytosine residues. In liver, a mutational signature with an as-yet-unknown underlying mechanism is predominant. In addition to adult stem cells, we directly measured the base substitutions that have accumulated in single stem cells of second-trimester human fetuses in the liver and intestine. We detected on average ~48 somatic base substitutions per fetal stem cell, indicating that the mutation accumulation rate is at least 5-fold higher during fetal development than during postnatal life. Furthermore, stem cells of the fetal liver and intestine show distinct mutational profiles, suggesting that different DNA damage and/or repair mechanisms are at play in these tissues during development. Intriguingly, the mutational landscape of the fetal intestinal stem cells closely resembled that observed in intestinal ASCs from postnatal tissue. We used the CRISPR-Cas9 gene-editing technology to delete key DNA repair genes in human colon organoids and asses the mutational consequences in vitro. We found that organoids deficient in base excision repair harbor a mutational footprint similar to signature 30, which is previously observed in a breast cancer cohort. Furthermore, nucleotide excision repair (NER) deficient ASCs show an increased contribution of Signature 8 mutations, which is a pattern with unknown etiology that is recurrently observed in human cancers. The presence of these signatures in cancer genomes could hold diagnostic and prognostic value, and may improve personalized cancer treatment strategies.. For example, tumors deficient in NER are more sensitive to cisplatin treatment

Decoding the mutational history of human stem cells : From patterns to mechanisms

The extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of adult stem cell (ASC) divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal human ASCs remain unknown. In this thesis, a new method is presented for cataloging mutations in individual human ASCs. Single ASCs are expanded in vitro into clonal organoid cultures to generate sufficient DNA for whole-genome sequencing (WGS) analysis, which allows detection of the mutations that accumulated in vivo in the original ACS. Using this method, we determined the genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. However, mutational signature analysis suggests that different mutational processes contribute to mutation accumulation in liver ASCs compared to colon and small intestinal ASCs. In the intestinal ASCs the majority of the mutations are a result of spontaneous deamination of methylated cytosine residues. In liver, a mutational signature with an as-yet-unknown underlying mechanism is predominant. In addition to adult stem cells, we directly measured the base substitutions that have accumulated in single stem cells of second-trimester human fetuses in the liver and intestine. We detected on average ~48 somatic base substitutions per fetal stem cell, indicating that the mutation accumulation rate is at least 5-fold higher during fetal development than during postnatal life. Furthermore, stem cells of the fetal liver and intestine show distinct mutational profiles, suggesting that different DNA damage and/or repair mechanisms are at play in these tissues during development. Intriguingly, the mutational landscape of the fetal intestinal stem cells closely resembled that observed in intestinal ASCs from postnatal tissue. We used the CRISPR-Cas9 gene-editing technology to delete key DNA repair genes in human colon organoids and asses the mutational consequences in vitro. We found that organoids deficient in base excision repair harbor a mutational footprint similar to signature 30, which is previously observed in a breast cancer cohort. Furthermore, nucleotide excision repair (NER) deficient ASCs show an increased contribution of Signature 8 mutations, which is a pattern with unknown etiology that is recurrently observed in human cancers. The presence of these signatures in cancer genomes could hold diagnostic and prognostic value, and may improve personalized cancer treatment strategies.. For example, tumors deficient in NER are more sensitive to cisplatin treatment

NERdeficiency vcfs VAF < 0.3

NERdeficiency vcfs VAF < 0.3</p

NERdeficiency vcfs

NER-deficiency filtered VCF files</p

MutationalPatterns: comprehensive genome-wide analysis of mutational processes

AbstractBase substitution catalogs represent historical records of mutational processes that have been active in a system. Such processes can be distinguished by typical characteristics, like mutation type, sequence context, transcriptional and replicative strand bias, and distribution throughout the genome. MutationalPatterns is an R/Bioconductor package that characterizes this broad range of mutational patterns and potential relations with (epi-)genomic features. Furthermore, it offers an efficient method to quantify the contribution of known mutational signatures. Such analyses can be used to determine whether certain DNA repair mechanisms are perturbed and to further characterize the processes underlying known mutational signatures.</jats:p