Lymphoma Driver Mutations in the Pathogenic Evolution of an Iconic Human Autoantibody.

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.

Somatic Evolution in Non-neoplastic IBD-Affected Colon.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease associated with increased risk of gastrointestinal cancers. We whole-genome sequenced 446 colonic crypts from 46 IBD patients and compared these to 412 crypts from 41 non-IBD controls from our previous publication on the mutation landscape of the normal colon. The average mutation rate of affected colonic epithelial cells is 2.4-fold that of healthy colon, and this increase is mostly driven by acceleration of mutational processes ubiquitously observed in normal colon. In contrast to the normal colon, where clonal expansions outside the confines of the crypt are rare, we observed widespread millimeter-scale clonal expansions. We discovered non-synonymous mutations in ARID1A, FBXW7, PIGR, ZC3H12A, and genes in the interleukin 17 and Toll-like receptor pathways, under positive selection in IBD. These results suggest distinct selection mechanisms in the colitis-affected colon and that somatic mutations potentially play a causal role in IBD pathogenesis.

Acquired genetic changes in human pluripotent stem cells: origins and consequences.

In the 20 years since human embryonic stem cells, and subsequently induced pluripotent stem cells, were first described, it has become apparent that during long-term culture these cells (collectively referred to as 'pluripotent stem cells' (PSCs)) can acquire genetic changes, which commonly include gains or losses of particular chromosomal regions, or mutations in certain cancer-associated genes, especially TP53. Such changes raise concerns for the safety of PSC-derived cellular therapies for regenerative medicine. Although acquired genetic changes may not be present in a cell line at the start of a research programme, the low sensitivity of current detection methods means that mutations may be difficult to detect if they arise but are present in only a small proportion of the cells. In this Review, we discuss the types of mutations acquired by human PSCs and the mechanisms that lead to their accumulation. Recent work suggests that the underlying mutation rate in PSCs is low, although they also seem to be particularly susceptible to genomic damage. This apparent contradiction can be reconciled by the observations that, in contrast to somatic cells, PSCs are programmed to die in response to genomic damage, which may reflect the requirements of early embryogenesis. Thus, the common genetic variants that are observed are probably rare events that give the cells with a selective growth advantage.

In vivo single-cell CRISPR uncovers distinct TNF programmes in tumour evolution.

The tumour evolution model posits that malignant transformation is preceded by randomly distributed driver mutations in cancer genes, which cause clonal expansions in phenotypically normal tissues. Although clonal expansions can remodel entire tissues1-3, the mechanisms that result in only a small number of clones transforming into malignant tumours remain unknown. Here we develop an in vivo single-cell CRISPR strategy to systematically investigate tissue-wide clonal dynamics of the 150 most frequently mutated squamous cell carcinoma genes. We couple ultrasound-guided in utero lentiviral microinjections, single-cell RNA sequencing and guide capture to longitudinally monitor clonal expansions and document their underlying gene programmes at single-cell transcriptomic resolution. We uncover a tumour necrosis factor (TNF) signalling module, which is dependent on TNF receptor 1 and involving macrophages, that acts as a generalizable driver of clonal expansions in epithelial tissues. Conversely, during tumorigenesis, the TNF signalling module is downregulated. Instead, we identify a subpopulation of invasive cancer cells that switch to an autocrine TNF gene programme associated with epithelial-mesenchymal transition. Finally, we provide in vivo evidence that the autocrine TNF gene programme is sufficient to mediate invasive properties and show that the TNF signature correlates with shorter overall survival of patients with squamous cell carcinoma. Collectively, our study demonstrates the power of applying in vivo single-cell CRISPR screening to mammalian tissues, unveils distinct TNF programmes in tumour evolution and highlights the importance of understanding the relationship between clonal expansions in epithelia and tumorigenesis.

Human γδ T cells are quickly reconstituted after stem-cell transplantation and show adaptive clonal expansion in response to viral infection.

To investigate how the human γδ T cell pool is shaped during ontogeny and how it is regenerated after transplantation of hematopoietic stem cells (HSCs), we applied an RNA-based next-generation sequencing approach to monitor the dynamics of the repertoires of γδ T cell antigen receptors (TCRs) before and after transplantation in a prospective cohort study. We found that repertoires of rearranged genes encoding γδ TCRs (TRG and TRD) in the peripheral blood of healthy adults were stable over time. Although a large fraction of human TRG repertoires consisted of public sequences, the TRD repertoires were private. In patients undergoing HSC transplantation, γδ T cells were quickly reconstituted; however, they had profoundly altered TCR repertoires. Notably, the clonal proliferation of individual virus-reactive γδ TCR sequences in patients with reactivation of cytomegalovirus revealed strong evidence for adaptive anti-viral γδ T cell immune responses.

T Cell Receptor Is Required for Differentiation, but Not Maintenance, of Intestinal CD4+ Intraepithelial Lymphocytes.

The gut epithelium is populated by intraepithelial lymphocytes (IELs), a heterogeneous T cell population with cytotoxic and regulatory properties, which can be acquired at the epithelial layer. However, the role of T cell receptor (TCR) in this process remains unclear. Single-cell transcriptomic analyses revealed distinct clonal expansions between cell states, with CD4+CD8αα+ IELs being one of the least diverse populations. Conditional deletion of TCR on differentiating CD4+ T cells or of major histocompatibility complex (MHC) class II on intestinal epithelial cells prevented CD4+CD8αα+ IEL differentiation. However, TCR ablation on differentiated CD4+CD8αα+ IELs or long-term cognate antigen withdraw did not affect their maintenance. TCR re-engagement of antigen-specific CD4+CD8αα+ IELs by Listeria monocytogenes did not alter their state but correlated with reduced bacterial invasion. Thus, local antigen recognition is an essential signal for differentiation of CD4+ T cells at the epithelium, yet differentiated IELs are able to preserve an effector program in the absence of TCR signaling.

Deterministic evolution and stringent selection during preneoplasia.

The earliest events during human tumour initiation, although poorly characterized, may hold clues to malignancy detection and prevention1. Here we model occult preneoplasia by biallelic inactivation of TP53, a common early event in gastric cancer, in human gastric organoids. Causal relationships between this initiating genetic lesion and resulting phenotypes were established using experimental evolution in multiple clonally derived cultures over 2 years. TP53 loss elicited progressive aneuploidy, including copy number alterations and structural variants prevalent in gastric cancers, with evident preferred orders. Longitudinal single-cell sequencing of TP53-deficient gastric organoids similarly indicates progression towards malignant transcriptional programmes. Moreover, high-throughput lineage tracing with expressed cellular barcodes demonstrates reproducible dynamics whereby initially rare subclones with shared transcriptional programmes repeatedly attain clonal dominance. This powerful platform for experimental evolution exposes stringent selection, clonal interference and a marked degree of phenotypic convergence in premalignant epithelial organoids. These data imply predictability in the earliest stages of tumorigenesis and show evolutionary constraints and barriers to malignant transformation, with implications for earlier detection and interception of aggressive, genome-instable tumours.

Spatial genomics maps the structure, nature and evolution of cancer clones.

Genome sequencing of cancers often reveals mosaics of different subclones present in the same tumour1-3. Although these are believed to arise according to the principles of somatic evolution, the exact spatial growth patterns and underlying mechanisms remain elusive4,5. Here, to address this need, we developed a workflow that generates detailed quantitative maps of genetic subclone composition across whole-tumour sections. These provide the basis for studying clonal growth patterns, and the histological characteristics, microanatomy and microenvironmental composition of each clone. The approach rests on whole-genome sequencing, followed by highly multiplexed base-specific in situ sequencing, single-cell resolved transcriptomics and dedicated algorithms to link these layers. Applying the base-specific in situ sequencing workflow to eight tissue sections from two multifocal primary breast cancers revealed intricate subclonal growth patterns that were validated by microdissection. In a case of ductal carcinoma in situ, polyclonal neoplastic expansions occurred at the macroscopic scale but segregated within microanatomical structures. Across the stages of ductal carcinoma in situ, invasive cancer and lymph node metastasis, subclone territories are shown to exhibit distinct transcriptional and histological features and cellular microenvironments. These results provide examples of the benefits afforded by spatial genomics for deciphering the mechanisms underlying cancer evolution and microenvironmental ecology.

Hematologic Cancer after Gene Therapy for Cerebral Adrenoleukodystrophy.

BACKGROUND: Gene therapy with elivaldogene autotemcel (eli-cel) consisting of autologous CD34+ cells transduced with lentiviral vector containing ABCD1 complementary DNA (Lenti-D) has shown efficacy in clinical studies for the treatment of cerebral adrenoleukodystrophy. However, the risk of oncogenesis with eli-cel is unclear. METHODS: We performed integration-site analysis, genetic studies, flow cytometry, and morphologic studies in peripheral-blood and bone marrow samples from patients who received eli-cel therapy in two completed phase 2-3 studies (ALD-102 and ALD-104) and an ongoing follow-up study (LTF-304) involving the patients in both ALD-102 and ALD-104. RESULTS: Hematologic cancer developed in 7 of 67 patients after the receipt of eli-cel (1 of 32 patients in the ALD-102 study and 6 of 35 patients in the ALD-104 study): myelodysplastic syndrome (MDS) with unilineage dysplasia in 2 patients at 14 and 26 months; MDS with excess blasts in 3 patients at 28, 42, and 92 months; MDS in 1 patient at 36 months; and acute myeloid leukemia (AML) in 1 patient at 57 months. In the 6 patients with available data, predominant clones contained lentiviral vector insertions at multiple loci, including at either MECOM-EVI1 (MDS and EVI1 complex protein EVI1 [ecotropic virus integration site 1], in 5 patients) or PRDM16 (positive regulatory domain zinc finger protein 16, in 1 patient). Several patients had cytopenias, and most had vector insertions in multiple genes within the same clone; 6 of the 7 patients also had somatic mutations (KRAS, NRAS, WT1, CDKN2A or CDKN2B, or RUNX1), and 1 of the 7 patients had monosomy 7. Of the 5 patients with MDS with excess blasts or MDS with unilineage dysplasia who underwent allogeneic hematopoietic stem-cell transplantation (HSCT), 4 patients remain free of MDS without recurrence of symptoms of cerebral adrenoleukodystrophy, and 1 patient died from presumed graft-versus-host disease 20 months after HSCT (49 months after receiving eli-cel). The patient with AML is alive and had full donor chimerism after HSCT; the patient with the most recent case of MDS is alive and awaiting HSCT. CONCLUSIONS: Hematologic cancer developed in a subgroup of patients who were treated with eli-cel; the cases are associated with clonal vector insertions within oncogenes and clonal evolution with acquisition of somatic genetic defects. (Funded by Bluebird Bio; ALD-102, ALD-104, and LTF-304 ClinicalTrials.gov numbers, NCT01896102, NCT03852498, and NCT02698579, respectively.).

Mechanisms of clonal evolution in childhood acute lymphoblastic leukemia.

Childhood acute lymphoblastic leukemia (ALL) can often be traced to a pre-leukemic clone carrying a prenatal genetic lesion. Postnatally acquired mutations then drive clonal evolution toward overt leukemia. The enzymes RAG1-RAG2 and AID, which diversify immunoglobulin-encoding genes, are strictly segregated in developing cells during B lymphopoiesis and peripheral mature B cells, respectively. Here we identified small pre-BII cells as a natural subset with increased genetic vulnerability owing to concurrent activation of these enzymes. Consistent with epidemiological findings on childhood ALL etiology, susceptibility to genetic lesions during B lymphopoiesis at the transition from the large pre-BII cell stage to the small pre-BII cell stage was exacerbated by abnormal cytokine signaling and repetitive inflammatory stimuli. We demonstrated that AID and RAG1-RAG2 drove leukemic clonal evolution with repeated exposure to inflammatory stimuli, paralleling chronic infections in childhood.

Integrating genetic and non-genetic determinants of cancer evolution by single-cell multi-omics.

Cancer represents an evolutionary process through which growing malignant populations genetically diversify, leading to tumour progression, relapse and resistance to therapy. In addition to genetic diversity, the cell-to-cell variation that fuels evolutionary selection also manifests in cellular states, epigenetic profiles, spatial distributions and interactions with the microenvironment. Therefore, the study of cancer requires the integration of multiple heritable dimensions at the resolution of the single cell - the atomic unit of somatic evolution. In this Review, we discuss emerging analytic and experimental technologies for single-cell multi-omics that enable the capture and integration of multiple data modalities to inform the study of cancer evolution. These data show that cancer results from a complex interplay between genetic and non-genetic determinants of somatic evolution.

Modeling and predicting cancer clonal evolution with reinforcement learning.

Cancer results from an evolutionary process that typically yields multiple clones with varying sets of mutations within the same tumor. Accurately modeling this process is key to understanding and predicting cancer evolution. Here, we introduce clone to mutation (CloMu), a flexible and low-parameter tree generative model of cancer evolution. CloMu uses a two-layer neural network trained via reinforcement learning to determine the probability of new mutations based on the existing mutations on a clone. CloMu supports several prediction tasks, including the determination of evolutionary trajectories, tree selection, causality and interchangeability between mutations, and mutation fitness. Importantly, previous methods support only some of these tasks, and many suffer from overfitting on data sets with a large number of mutations. Using simulations, we show that CloMu either matches or outperforms current methods on a wide variety of prediction tasks. In particular, for simulated data with interchangeable mutations, current methods are unable to uncover causal relationships as effectively as CloMu. On breast cancer and leukemia cohorts, we show that CloMu determines similarities and causal relationships between mutations as well as the fitness of mutations. We validate CloMu's inferred mutation fitness values for the leukemia cohort by comparing them to clonal proportion data not used during training, showing high concordance. In summary, CloMu's low-parameter model facilitates a wide range of prediction tasks regarding cancer evolution on increasingly available cohort-level data sets.

Somatic Mutagenesis in Mammals and Its Implications for Human Disease and Aging.

DNA mutations as a consequence of errors during DNA damage repair, replication, or mitosis are the substrate for evolution. In multicellular organisms, mutations can occur in the germline and also in somatic tissues, where they are associated with cancer and other chronic diseases and possibly with aging. Recent advances in high-throughput sequencing have made it relatively easy to study germline de novo mutations, but in somatic cells, the vast majority of mutations are low-abundant and can be detected only in clonal lineages, such as tumors, or single cells. Here we review recent results on somatic mutations in normal human and animal tissues with a focus on their possible functional consequences.

Clonal evolution dissection reveals that a high MSI2 level promotes chemoresistance in T-cell acute lymphoblastic leukemia.

ABSTRACT: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer with resistant clonal propagation in recurrence. We performed high-throughput droplet-based 5' single-cell RNA with paired T-cell receptor (TCR) sequencing of paired diagnosis-relapse (Dx_Rel) T-ALL samples to dissect the clonal diversities. Two leukemic evolutionary patterns, "clonal shift" and "clonal drift" were unveiled. Targeted single-cell DNA sequencing of paired Dx_Rel T-ALL samples further corroborated the existence of the 2 contrasting clonal evolution patterns, revealing that dynamic transcriptional variation might cause the mutationally static clones to evolve chemotherapy resistance. Analysis of commonly enriched drifted gene signatures showed expression of the RNA-binding protein MSI2 was significantly upregulated in the persistent TCR clonotypes at relapse. Integrated in vitro and in vivo functional studies suggested that MSI2 contributed to the proliferation of T-ALL and promoted chemotherapy resistance through the posttranscriptional regulation of MYC, pinpointing MSI2 as an informative biomarker and novel therapeutic target in T-ALL.

Monogenic and polygenic inheritance become instruments for clonal selection.

Clonally expanded blood cells that contain somatic mutations (clonal haematopoiesis) are commonly acquired with age and increase the risk of blood cancer1-9. The blood clones identified so far contain diverse large-scale mosaic chromosomal alterations (deletions, duplications and copy-neutral loss of heterozygosity (CN-LOH)) on all chromosomes1,2,5,6,9, but the sources of selective advantage that drive the expansion of most clones remain unknown. Here, to identify genes, mutations and biological processes that give selective advantage to mutant clones, we analysed genotyping data from the blood-derived DNA of 482,789 participants from the UK Biobank10. We identified 19,632 autosomal mosaic chromosomal alterations and analysed these for relationships to inherited genetic variation. We found 52 inherited, rare, large-effect coding or splice variants in 7 genes that were associated with greatly increased vulnerability to clonal haematopoiesis with specific acquired CN-LOH mutations. Acquired mutations systematically replaced the inherited risk alleles (at MPL) or duplicated them to the homologous chromosome (at FH, NBN, MRE11, ATM, SH2B3 and TM2D3). Three of the genes (MRE11, NBN and ATM) encode components of the MRN-ATM pathway, which limits cell division after DNA damage and telomere attrition11-13; another two (MPL and SH2B3) encode proteins that regulate the self-renewal of stem cells14-16. In addition, we found that CN-LOH mutations across the genome tended to cause chromosomal segments with alleles that promote the expansion of haematopoietic cells to replace their homologous (allelic) counterparts, increasing polygenic drive for blood-cell proliferation traits. Readily acquired mutations that replace chromosomal segments with their homologous counterparts seem to interact with pervasive inherited variation to create a challenge for lifelong cytopoiesis.

Highly parallelized laboratory evolution of wine yeasts for enhanced metabolic phenotypes.

Adaptive Laboratory Evolution (ALE) of microorganisms can improve the efficiency of sustainable industrial processes important to the global economy. However, stochasticity and genetic background effects often lead to suboptimal outcomes during laboratory evolution. Here we report an ALE platform to circumvent these shortcomings through parallelized clonal evolution at an unprecedented scale. Using this platform, we evolved 104 yeast populations in parallel from many strains for eight desired wine fermentation-related traits. Expansions of both ALE replicates and lineage numbers broadened the evolutionary search spectrum leading to improved wine yeasts unencumbered by unwanted side effects. At the genomic level, evolutionary gains in metabolic characteristics often coincided with distinct chromosome amplifications and the emergence of side-effect syndromes that were characteristic of each selection niche. Several high-performing ALE strains exhibited desired wine fermentation kinetics when tested in larger liquid cultures, supporting their suitability for application. More broadly, our high-throughput ALE platform opens opportunities for rapid optimization of microbes which otherwise could take many years to accomplish.

Homeostasis limits keratinocyte evolution.

Recent studies have revealed that normal human tissues accumulate many somatic mutations. In particular, human skin is riddled with mutations, with multiple subclones of variable sizes. Driver mutations are frequent and tend to have larger subclone sizes, suggesting selection. To begin to understand the histories encoded by these complex somatic mutations, we incorporated genomes into a simple agent-based skin-cell model whose prime directive is homeostasis. In this model, stem-cell survival is random and dependent on proximity to the basement membrane. This simple homeostatic skin model recapitulates the observed log-linear distributions of somatic mutations, where most mutations are found in increasingly smaller subclones that are typically lost with time. Hence, neutral mutations are "passengers" whose fates depend on the random survival of their stem cells, where a rarer larger subclone reflects the survival and spread of mutations acquired earlier in life. The model can also maintain homeostasis and accumulate more frequent and larger driver subclones if these mutations (NOTCH1 and TP53) confer relatively higher persistence in normal skin or during tissue damage (sunlight). Therefore, a relatively simple model of epithelial turnover indicates how observed passenger and driver somatic mutations could accumulate without violating the prime directive of homeostasis in normal human tissues.

Clonal interactions in cancer: Integrating quantitative models with experimental and clinical data.

Tumors consist of different genotypically distinct subpopulations-or subclones-of cells. These subclones can influence neighboring clones in a process called "clonal interaction." Conventionally, research on driver mutations in cancer has focused on their cell-autonomous effects that lead to an increase in fitness of the cells containing the driver. Recently, with the advent of improved experimental and computational technologies for investigating tumor heterogeneity and clonal dynamics, new studies have shown the importance of clonal interactions in cancer initiation, progression, and metastasis. In this review we provide an overview of clonal interactions in cancer, discussing key discoveries from a diverse range of approaches to cancer biology research. We discuss common types of clonal interactions, such as cooperation and competition, its mechanisms, and the overall effect on tumorigenesis, with important implications for tumor heterogeneity, resistance to treatment, and tumor suppression. Quantitative models-in coordination with cell culture and animal model experiments-have played a vital role in investigating the nature of clonal interactions and the complex clonal dynamics they generate. We present mathematical and computational models that can be used to represent clonal interactions and provide examples of the roles they have played in identifying and quantifying the strength of clonal interactions in experimental systems. Clonal interactions have proved difficult to observe in clinical data; however, several very recent quantitative approaches enable their detection. We conclude by discussing ways in which researchers can further integrate quantitative methods with experimental and clinical data to elucidate the critical-and often surprising-roles of clonal interactions in human cancers.

Single-cell transcriptome analysis reveals subtype-specific clonal evolution and microenvironmental changes in liver metastasis of pancreatic adenocarcinoma and their clinical implications.

BACKGROUND: Intratumoral heterogeneity (ITH) and tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) play important roles in tumor evolution and patient outcomes. However, the precise characterization of diverse cell populations and their crosstalk associated with PDAC progression and metastasis is still challenging. METHODS: We performed single-cell RNA sequencing (scRNA-seq) of treatment-naïve primary PDAC samples with and without paired liver metastasis samples to understand the interplay between ITH and TME in the PDAC evolution and its clinical associations. RESULTS: scRNA-seq analysis revealed that even a small proportion (22%) of basal-like malignant ductal cells could lead to poor chemotherapy response and patient survival and that epithelial-mesenchymal transition programs were largely subtype-specific. The clonal homogeneity significantly increased with more prevalent and pronounced copy number gains of oncogenes, such as KRAS and ETV1, and losses of tumor suppressor genes, such as SMAD2 and MAP2K4, along PDAC progression and metastasis. Moreover, diverse immune cell populations, including naïve SELLhi regulatory T cells (Tregs) and activated TIGIThi Tregs, contributed to shaping immunosuppressive TMEs of PDAC through cellular interactions with malignant ductal cells in PDAC evolution. Importantly, the proportion of basal-like ductal cells negatively correlated with that of immunoreactive cell populations, such as cytotoxic T cells, but positively correlated with that of immunosuppressive cell populations, such as Tregs. CONCLUSION: We uncover that the proportion of basal-like subtype is a key determinant for chemotherapy response and patient outcome, and that PDAC clonally evolves with subtype-specific dosage changes of cancer-associated genes by forming immunosuppressive microenvironments in its progression and metastasis.

Autosomal Clonal Monoallelic Expression: Natural or Artifactual?

The prevalence of mitotically heritable clonal random monoallelic expression of autosomal genes (aRME) remains controversial. Specifically, presence of clonal aRME is well supported in vitro but remains elusive in vivo. Here, we provide critical insights into this matter and discuss whether prevalent clonal aRME is natural or artifactual.