Kimmo Palin

L1s are repetitive sequences capable of copying themselves into new genomic loci. While L1s are typically repressed by DNA methylation in somatic tissues, they can become reactivated in cancer. Although L1 sequences are highly repetitive, ~25% of insertions carry a unique downstream sequence, transduction, that can be used to trace the source L1. Here, we apply nanopore long-read sequencing to 56 colorectal cancer samples to comprehensively detect somatic transductions and to characterize the source L1 activity. We demonstrate that earlier methods systematically miss a large proportion of mostly shorter transductions, leading to an incomplete and biased view of source L1 activity. Our analysis reveals a strong positive correlation between the number of transductions and other L1 insertions within samples and that distinct source L1s exhibit varying transduction lengths and 5’ inversion frequency. Finally, we integrate DNA methylation provided by nanopore reads and show that active elements in cancer samples have lower methylation levels in contrast to inactive L1s. Together, our results provide a more complete characterization of somatically active L1 elements in colorectal cancer and highlight the utility of long-read sequencing in retrotransposon research.

Background: The high mutational burden in microsatellite unstable colorectal cancers (MSI CRCs) results in high immunogenicity, yet response rates to immunotherapy vary, suggesting underlying heterogeneity of the tumour immune landscape. Here, our aims were (1) to characterise the immune cell infiltrate and immune evasion in MSI CRCs, (2) to correlate these with clinical and genomic features, and (3) to compare these between Lynch syndrome (LS) and sporadic MSI CRCs. Method: Immunohistochemistry was utilised to detect T cell and myeloid cell subsets. Whole-genome and RNA sequencing were utilised to analyse somatic variants, tumour clonality, neoantigen burden, antigen presentation, immune checkpoint expression, and consensus molecular subtypes. Results: Our results revealed higher immune cell scores in LS tumours, depicting higher T cell infiltration, compared to sporadic tumours. Conversely, sporadic tumours displayed increased infiltration of protumorigenic M2-like macrophages and increased expression of immune checkpoints PDCD1LG2 and CD40LG . Across our MSI CRC cohort, high neoantigen burden was associated with low tumour clonality. Conclusions: Our findings reveal differences between sporadic MSI and LS tumours in T cell and myeloid immune cell landscapes, and in immune evasion. These differences may contribute to the variable immunotherapy responses among MSI CRC patients and are targetable by emerging therapeutic approaches.

A bstract: Covalently bound DNA adducts are mutation precursors that contribute to aging and diseases such as cancer. Accurate detection of adducts in the genome will shed light on tumorigenesis. Commonly used detection methods are unable to pinpoint the exact genomic locations of adducts. Long-read nanopore sequencing has the potential to accurately detect multiple types of DNA adducts at single-nucleotide precision. In this study, we developed a novel statistical toolkit, IonStats, to profile DNA adducts in nanopore sequencing data. With IonStats, we investigated the effects of four adduct-inducing genotoxic compounds on nanopore sequencing, and found both shared and compound-specific perturbations in base quality scores, ionic current profiles, and translocation dynamics. Notably, aristolochic acid II and melphalan treatments profoundly altered nanopore readouts and led to substantial sequence-specific read interruptions. Our study shows that nanopore sequencing can be effectively employed to detect and characterize DNA adducts, paving the way for high-resolution, high-throughput profiling of DNA damage and the exposome.

Background: Transposons are DNA sequences able to move or copy themselves to other genomic locations leading to insertional mutagenesis. Although transposon-derived sequences account for half of the human genome, most elements are no longer transposition competent. Moreover, transposons are normally repressed through epigenetic silencing in healthy adult tissues but become derepressed in several human cancers, with high activity detected in colorectal cancer. Their impact on non-malignant and malignant tissue as well as the differences between somatic and germline retrotransposition remain poorly understood. With new sequencing technologies, including long read sequencing, we can access intricacies of retrotransposition, such as insertion sequence details and nested repeats, that have been previously challenging to characterize. Results: In this study, we investigate somatic and germline retrotransposition by analyzing long read sequencing from 56 colorectal cancers and 112 uterine leiomyomas. We identified 1495 somatic insertions in colorectal samples, while striking lack of insertions was detected in uterine leiomyomas. Our findings highlight differences between somatic and germline events, such as transposon type distribution, insertion length, and target site preference. Leveraging long-read sequencing, we provide an in-depth analysis of the twin-priming phenomenon, detecting it across transposable element types that remain active in humans, including Alus. Additionally, we detect an abundance of germline transposons in repetitive DNA, along with a relationship between replication timing and insertion target site. Conclusions: Our study reveals a stark contrast in somatic transposon activity between colorectal cancers and uterine leiomyomas, and highlights differences between somatic and germline transposition. This suggests potentially different conditions in malignant and non-malignant tissues, as well as in germline and somatic tissues, which could be involved in the transposition process. Long-read sequencing provided important insights into transposon behavior, allowing detailed examination of structural features such as twin priming and nested elements.

Oncogenic codon V600E mutations of the BRAF gene affect 10–15% of colorectal cancers, resulting in activation of the MAPK/ERK signaling pathway and increased cell proliferation and survival. BRAF-mutated colorectal tumors are often microsatellite unstable and characterized by high DNA methylation levels. However, the mechanistic link between BRAF mutations and hypermethylation remains controversial. Understanding this link, particularly in microsatellite stable tumors is of great interest as these often show poor survival. We characterized the metabolomic, epigenetic and transcriptomic patterns of altogether 39 microsatellite stable BRAF-mutated colorectal cancers. Metabolomic analysis of tumor tissue showed low levels of vitamin C and its metabolites in BRAF-mutated tumors. Gene expression analysis indicated dysregulation of vitamin C antioxidant activity in these lesions. As vitamin C is an important cofactor for the activity of TET DNA demethylase enzymes, low vitamin C levels could directly contribute to the high methylation levels in these tumors by decreasing enzymatic TET activity. Vitamin C transporter gene SLC23A1 expression, as well as vitamin C metabolite levels, were inversely correlated with DNA methylation levels. This work proposes a new mechanistic link between BRAF mutations and hypermethylation, inspiring further work on the role of vitamin C in the genesis of BRAF-mutated colorectal cancer.

Microsatellite unstable colorectal cancer (MSI-CRC) can arise through germline mutations in mismatch repair (MMR) genes in individuals with Lynch syndrome (LS), or sporadically through promoter methylation of the MMR gene MLH1. Despite the different origins of hereditary and sporadic MSI tumours, their genomic features have not been extensively compared. A prominent feature of MMR-deficient genomes is the occurrence of many indels in short repeat sequences, an understudied mutation type due to the technical challenges of variant calling in these regions. In this study, we performed whole genome sequencing and RNA-sequencing on 29 sporadic and 14 hereditary MSI-CRCs. We compared the tumour groups by analysing genome-wide mutation densities, microsatellite repeat indels, recurrent protein-coding variants, signatures of single base, doublet base, and indel mutations, and changes in gene expression. We show that the mutational landscapes of hereditary and sporadic MSI-CRCs, including mutational signatures and mutation densities genome-wide and in microsatellites, are highly similar. Only a low number of differentially expressed genes were found, enriched to interferon-γ regulated immune response pathways. Analysis of the variance in allelic fractions of somatic variants in each tumour group revealed higher clonal heterogeneity in sporadic MSI-CRCs. Our results suggest that the differing molecular origins of MMR deficiency in hereditary and sporadic MSI-CRCs do not result in substantial differences in the mutational landscapes of these tumours. The divergent patterns of clonal evolution between the tumour groups may have clinical implications, as high clonal heterogeneity has been associated with decreased tumour immunosurveillance and reduced responsiveness to immunotherapy.

Genome-wide association studies (GWAS) have identified more than 200 common genetic variants independently associated with colorectal cancer (CRC) risk, but the causal variants and target genes are mostly unknown. We sought to fine-map all known CRC risk loci using GWAS data from 100,204 cases and 154,587 controls of East Asian and European ancestry. Our stepwise conditional analyses revealed 238 independent association signals of CRC risk, each with a set of credible causal variants (CCVs), of which 28 signals had a single CCV. Our cis-eQTL/mQTL and colocalization analyses using colorectal tissue-specific transcriptome and methylome data separately from 1299 and 321 individuals, along with functional genomic investigation, uncovered 136 putative CRC susceptibility genes, including 56 genes not previously reported. Analyses of single-cell RNA-seq data from colorectal tissues revealed 17 putative CRC susceptibility genes with distinct expression patterns in specific cell types. Analyses of whole exome sequencing data provided additional support for several target genes identified in this study as CRC susceptibility genes. Enrichment analyses of the 136 genes uncover pathways not previously linked to CRC risk. Our study substantially expanded association signals for CRC and provided additional insight into the biological mechanisms underlying CRC development.

Transposable elements (TE) are repetitive genomic elements that harbor binding sites for human transcription factors (TF). A regulatory role for TEs has been suggested in embryonal development and diseases such as cancer but systematic investigation of their functions has been limited by their widespread silencing in the genome. Here, we utilize unbiased massively parallel reporter assay data using a whole human genome library to identify TEs with functional enhancer activity in two human cancer types of endodermal lineage, colorectal and liver cancers. We show that the identified TE enhancers are characterized by genomic features associated with active enhancers, such as epigenetic marks and TF binding. Importantly, we identify distinct TE subfamilies that function as tissue-specific enhancers, namely MER11- and LTR12-elements in colon and liver cancers, respectively. These elements are bound by distinct TFs in each cell type, and they have predicted associations to differentially expressed genes. In conclusion, these data demonstrate how different cancer types can utilize distinct TEs as tissue-specific enhancers, paving the way for comprehensive understanding of the role of TEs as bona fide enhancers in the cancer genomes.

Colorectal cancer (CRC) is a leading cause of mortality worldwide. We conducted a genome-wide association study meta-analysis of 100,204 CRC cases and 154,587 controls of European and east Asian ancestry, identifying 205 independent risk associations, of which 50 were unreported. We performed integrative genomic, transcriptomic and methylomic analyses across large bowel mucosa and other tissues. Transcriptome- and methylome-wide association studies revealed an additional 53 risk associations. We identified 155 high-confidence effector genes functionally linked to CRC risk, many of which had no previously established role in CRC. These have multiple different functions and specifically indicate that variation in normal colorectal homeostasis, proliferation, cell adhesion, migration, immunity and microbial interactions determines CRC risk. Crosstissue analyses indicated that over a third of effector genes most probably act outside the colonic mucosa. Our findings provide insights into colorectal oncogenesis and highlight potential targets across tissues for new CRC treatment and chemoprevention strategies.