Mutational processes constantly shape the somatic genome, leading to immunity, aging, cancer, and other diseases. When cancer is the outcome, we are afforded a glimpse into these processes by the ...clonal expansion of the malignant cell. Here, we characterize a less explored layer of the mutational landscape of cancer: mutational asymmetries between the two DNA strands. Analyzing whole-genome sequences of 590 tumors from 14 different cancer types, we reveal widespread asymmetries across mutagenic processes, with transcriptional (“T-class”) asymmetry dominating UV-, smoking-, and liver-cancer-associated mutations and replicative (“R-class”) asymmetry dominating POLE-, APOBEC-, and MSI-associated mutations. We report a striking phenomenon of transcription-coupled damage (TCD) on the non-transcribed DNA strand and provide evidence that APOBEC mutagenesis occurs on the lagging-strand template during DNA replication. As more genomes are sequenced, studying and classifying their asymmetries will illuminate the underlying biological mechanisms of DNA damage and repair.
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•Replicative and transcriptional mutational asymmetries are widespread across cancer•APOBEC mutagenesis in humans primarily occurs on the lagging-strand template•Mismatch repair balances asymmetric replication errors•Transcription-coupled damage (TCD) introduces sense-strand mutations in liver cancer
Using an approach that distinguishes whether mutations in cancer genomes occurred on the transcribed or non-transcribed DNA strand with respect to transcription and on the leading or lagging strand with respect to replication, the predominant mutational mechanisms associated with different types of cancers and mutational patterns can be inferred.
How somatic mutations accumulate in normal cells is poorly understood. A comprehensive analysis of RNA sequencing data from ~6700 samples across 29 normal tissues revealed multiple somatic variants, ...demonstrating that macroscopic clones can be found in many normal tissues. We found that sun-exposed skin, esophagus, and lung have a higher mutation burden than other tested tissues, which suggests that environmental factors can promote somatic mosaicism. Mutation burden was associated with both age and tissue-specific cell proliferation rate, highlighting that mutations accumulate over both time and number of cell divisions. Finally, normal tissues were found to harbor mutations in known cancer genes and hotspots. This study provides a broad view of macroscopic clonal expansion in human tissues, thus serving as a foundation for associating clonal expansion with environmental factors, aging, and risk of disease.
Current genomics methods are designed to handle tens to thousands of samples but will need to scale to millions to match the pace of data and hypothesis generation in biomedical science. Here, we ...show that high efficiency at low cost can be achieved by leveraging general-purpose libraries for computing using graphics processing units (GPUs), such as PyTorch and TensorFlow. We demonstrate > 200-fold decreases in runtime and ~ 5-10-fold reductions in cost relative to CPUs. We anticipate that the accessibility of these libraries will lead to a widespread adoption of GPUs in computational genomics.
Chimeric antigen receptor (CAR) therapy has had a transformative effect on the treatment of haematologic malignancies
, but it has shown limited efficacy against solid tumours. Solid tumours may have ...cell-intrinsic resistance mechanisms to CAR T cell cytotoxicity. Here, to systematically identify potential resistance pathways in an unbiased manner, we conducted a genome-wide CRISPR knockout screen in glioblastoma, a disease in which CAR T cells have had limited efficacy
. We found that the loss of genes in the interferon-γ receptor (IFNγR) signalling pathway (IFNGR1, JAK1 or JAK2) rendered glioblastoma and other solid tumours more resistant to killing by CAR T cells both in vitro and in vivo. However, loss of this pathway did not render leukaemia or lymphoma cell lines insensitive to CAR T cells. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell-adhesion pathways after exposure to CAR T cells. We found that loss of IFNγR1 in glioblastoma cells reduced overall CAR T cell binding duration and avidity. The critical role of IFNγR signalling in susceptibility of solid tumours to CAR T cells is surprising, given that CAR T cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumours, IFNγR signalling was required for sufficient adhesion of CAR T cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumours differ in their interactions with CAR T cells and suggests that enhancing binding interactions between T cells and tumour cells may yield improved responses in solid tumours.
Microsatellites (MSs) are tracts of variable-length repeats of short DNA motifs that exhibit high rates of mutation in the form of insertions or deletions (indels) of the repeated motif. Despite ...their prevalence, the contribution of somatic MS indels to cancer has been largely unexplored, owing to difficulties in detecting them in short-read sequencing data. Here we present two tools: MSMuTect, for accurate detection of somatic MS indels, and MSMutSig, for identification of genes containing MS indels at a higher frequency than expected by chance. Applying MSMuTect to whole-exome data from 6,747 human tumors representing 20 tumor types, we identified >1,000 previously undescribed MS indels in cancer genes. Additionally, we demonstrate that the number and pattern of MS indels can accurately distinguish microsatellite-stable tumors from tumors with microsatellite instability, thus potentially improving classification of clinically relevant subgroups. Finally, we identified seven MS indel driver hotspots: four in known cancer genes (ACVR2A, RNF43, JAK1, and MSH3) and three in genes not previously implicated as cancer drivers (ESRP1, PRDM2, and DOCK3).
Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematologic malignancies. Approximately half of patients with refractory large B cell lymphomas achieve durable ...responses from CD19-targeting CAR-T treatment; however, failure mechanisms are identified in only a fraction of cases. To gain new insights into the basis of clinical response, we performed single-cell transcriptome sequencing of 105 pretreatment and post-treatment peripheral blood mononuclear cell samples, and infusion products collected from 32 individuals with large B cell lymphoma treated with either of two CD19 CAR-T products: axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel). Expansion of proliferative memory-like CD8 clones was a hallmark of tisa-cel response, whereas axi-cel responders displayed more heterogeneous populations. Elevations in CAR-T regulatory cells among nonresponders to axi-cel were detected, and these populations were capable of suppressing conventional CAR-T cell expansion and driving late relapses in an in vivo model. Our analyses reveal the temporal dynamics of effective responses to CAR-T therapy, the distinct molecular phenotypes of CAR-T cells with differing designs, and the capacity for even small increases in CAR-T regulatory cells to drive relapse.
Biallelic inactivation of BRCA1 or BRCA2 is associated with a pattern of genome-wide mutations known as signature 3. By analyzing ∼1,000 breast cancer samples, we confirmed this association and ...established that germline nonsense and frameshift variants in PALB2, but not in ATM or CHEK2, can also give rise to the same signature. We were able to accurately classify missense BRCA1 or BRCA2 variants known to impair homologous recombination (HR) on the basis of this signature. Finally, we show that epigenetic silencing of RAD51C and BRCA1 by promoter methylation is strongly associated with signature 3 and, in our data set, was highly enriched in basal-like breast cancers in young individuals of African descent.
Multiple myeloma is a plasma cell malignancy almost always preceded by precursor conditions, but low tumor burden of these early stages has hindered the study of their molecular programs through bulk ...sequencing technologies. Here, we generate and analyze single cell RNA-sequencing of plasma cells from 26 patients at varying disease stages and 9 healthy donors. In silico dissection and comparison of normal and transformed plasma cells from the same bone marrow biopsy enables discovery of patient-specific transcriptional changes. Using Non-Negative Matrix Factorization, we discover 15 gene expression signatures which represent transcriptional modules relevant to myeloma biology, and identify a signature that is uniformly lost in abnormal cells across disease stages. Finally, we demonstrate that tumors contain heterogeneous subpopulations expressing distinct transcriptional patterns. Our findings characterize transcriptomic alterations present at the earliest stages of myeloma, providing insight into the molecular underpinnings of disease initiation.
Nearly all prostate cancer deaths are from metastatic castration-resistant prostate cancer (mCRPC), but there have been few whole-genome sequencing (WGS) studies of this disease state. We performed ...linked-read WGS on 23 mCRPC biopsy specimens and analyzed cell-free DNA sequencing data from 86 patients with mCRPC. In addition to frequent rearrangements affecting known prostate cancer genes, we observed complex rearrangements of the AR locus in most cases. Unexpectedly, these rearrangements include highly recurrent tandem duplications involving an upstream enhancer of AR in 70%–87% of cases compared with <2% of primary prostate cancers. A subset of cases displayed AR or MYC enhancer duplication in the context of a genome-wide tandem duplicator phenotype associated with CDK12 inactivation. Our findings highlight the complex genomic structure of mCRPC, nominate alterations that may inform prostate cancer treatment, and suggest that additional recurrent events in the non-coding mCRPC genome remain to be discovered.
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•Linked-read genome sequencing of mCRPC resolves haplotypes and rearrangements•CDK12 inactivation is associated with a global tandem duplication phenotype•A majority of cases have duplications of an enhancer of the androgen receptor•Progression on androgen pathway inhibitors is associated with gains in AR and AR enhancer
Linked-read genome sequencing data from patients highlight that amplification of an enhancer upstream of the androgen receptor locus is a key feature of metastatic castration-resistant prostate cancer.
Current statistical models for assessing hotspot significance do not properly account for variation in site-specific mutability, thereby yielding many false-positives. We thus (i) detail a ...Log-normal-Poisson (LNP) background model that accounts for this variability in a manner consistent with models of mutagenesis; (ii) use it to show that passenger hotspots arise from all common mutational processes; and (iii) apply it to a ∼10,000-patient cohort to nominate driver hotspots with far fewer false-positives compared with conventional methods. Overall, we show that many cancer hotspot mutations recurring at the same genomic site across multiple tumors are actually passenger events, recurring at inherently mutable genomic sites under no positive selection.
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•Many cancer hotspots are passengers, recurring at inherently mutable genomic sites•Known genomic covariates are insufficient to fully predict inherent mutability•Our LNP model accurately infers latent variability beyond what current covariates predict•Our LNP model identifies putative driver hotspots with far fewer false-positives
Somatic hotspot mutations found in tumors are generally considered evidence for selection and are used to nominate tumor drivers. Hess et al. show that many hotspots occur at inherently mutable sites without selection and develop a model that accounts for these passenger hotspots, which can more accurately nominate true driver mutations.
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