Philadelphia-negative myeloproliferative neoplasms are chronic blood neoplasms. Treatments control blood counts, but disease can progress to myelofibrosis or acute myeloid leukemia. We performed longitudinal whole-genome and targeted sequencing in 30 patients, integrating clonal dynamics with 7,986 blood counts and clinical histories. Distinct evolutionary patterns distinguished stable from progressive disease, with leukemic transformation arising via TP53 loss, stepwise driver mutation acquisition within complex clones, or emergence of independent leukemic clones. In contrast, stable disease showed long-term clonal equilibrium without new drivers. Phylogenetic analysis using 203 whole-genomes of hematopoietic colonies revealed age-appropriate polyclonal hematopoiesis in triple-negative essential thrombocythemia and germline predisposition to thrombocytosis, supporting non-neoplastic origins. Therapy-associated mutagenesis was observed, including C > G mutations following azacitidine and characteristic T > A/T > G after hydroxycarbamide exposure in blood cells, although not in skin where UV damage predominated. These findings demonstrate that progression is genomically encoded years in advance and support serial monitoring and further study of treatment-related mutagenesis. SIGNIFICANCE:Longitudinal whole-genome sequencing shows MPN progression is genomically encoded years before clinical transformation, with distinct evolutionary routes to leukemia and MF. It identifies DNA mutagenesis associated with HC and 5-azacitidine, suggests some triple-negative cases are nonclonal, and supports serial clinical genomic monitoring for improved risk stratification and long-term management. See related commentary by Agarwal and Sankaran, p. 1724.

DNA suffers continual damage leaving a cell with thousands of individual DNA lesions at any given moment 1–3 . The efficiency of DNA repair means that most known classes of lesion have a half-life of minutes to hours 3,4 , but whether some DNA damage can persist for longer durations remains unknown. Here, using high-resolution phylogenetic trees from 89 donors, we identified mutations arising from 832 DNA lesions that persisted across multiple cell cycles in normal human stem cells from blood, liver and bronchial epithelium 5–12 . Persistent DNA lesions occurred at increased rates, with distinctive mutational signatures, in donors exposed to tobacco or chemotherapy, suggesting that they can arise from exogenous mutagens. In haematopoietic stem cells, persistent DNA lesions, likely from endogenous sources, generated a characteristic mutational signature, so-called SBS19 13 ; occurred steadily throughout life, including in utero ; and endured for 1.5 years on average, with 15% lasting 3+ years. We estimate that a haematopoietic stem cell has, on average, ~4-5 such lesions at any moment in time, half of which will generate a mutation with each cell cycle. Overall, 16% of mutations in blood cells are attributable to SBS19, and similar proportions of driver mutations in blood cancers exhibit this signature. These data imply the existence of a family of DNA lesions, arising from both endogenous and exogenous mutagens, present in low numbers per genome but persisting for months to years, that can generate sizable fractions of cells’ mutation burdens.

Several chemotherapeutic agents act by increasing DNA damage in cancer cells, triggering cell death. However, there is limited understanding of the extent and long-term consequences of collateral DNA damage in normal tissues. To investigate the impact of chemotherapy on mutation burdens and the cell population structure of normal tissue, we sequenced blood cell genomes from 23 individuals aged 3-80 years who were treated with a range of chemotherapy regimens. Substantial additional somatic mutation loads with characteristic mutational signatures were imposed by some chemotherapeutic agents, but the effects were dependent on the drug and blood cell types. Chemotherapy induced premature changes in the cell population structure of normal blood, similar to those caused by normal aging. The results show the long-term biological consequences of cytotoxic agents to which a substantial fraction of the population is exposed as part of disease management, raising mechanistic questions and highlighting opportunities for the mitigation of adverse effects.

Retrotransposon expression must be tightly controlled, particularly in long-lived multipotent stem cells, to prevent deleterious consequences including insertional mutations. Several mechanisms are known to repress retrotransposon transcription during development which are generally thought to persist thereafter. However, integration of retrotransposable elements into host genomes has also provided a major source of genetic variation across evolution. This has generated many sequences that have acquired advantageous host functions, but little is known about retrotransposon expression and mobility in adult stem cells or about how these processes might be dynamically regulated. Here we describe the landscape of somatic retrotransposition in haematopoietic stem cells and identify a previously unrecognised pathway which links cytokine signalling, RNA-modulating HNRNP complexes and repression of retrotransposon activity. Activation of JAK2, by thrombopoietin or gain-of-function JAK2 mutations, triggers tyrosine phosphorylation of HNRNPA1 that represses expression of ERVs, LINEs and SINEs and reduces insertional mutagenesis. This pathway coordinates retrotransposon activity with cellular context and state and provides a mechanism for protecting the haematopoietic stem cell genome. ### Competing Interest Statement ARG reports consulting for Incite; other authors declare that they have no competing interests. Wellcome Trust, https://ror.org/029chgv08, RG74909 William B Harrison Foundation, RG91681 Alborada Trust, https://ror.org/00c9bhj04, RG109433 Cancer Research UK, RG83389 Swiss National Science Foundation, P400PB_183851/1 Promedica Foundation, 1617/M

The impact of exogenous stressors, such as cancer chemotherapies, on the genomic integrity and clonal dynamics of normal hematopoiesis is not well defined. We conducted whole-genome sequencing on 1,276 single-cell-derived hematopoietic stem and progenitor cell (HSPC) colonies from ten patients with multiple myeloma treated with chemotherapies and six normal donors. Melphalan treatment significantly increased the mutational burden, producing a distinctive mutation signature, whereas other chemotherapeutic agents had minimal effects. Consequently, the clonal diversity and architecture of post-treatment HSPCs resemble those observed in normal elderly individuals, particularly through the progression of oligoclonal hematopoiesis, thereby suggesting that chemotherapy accelerates clonal aging. Integrated phylogenetic analysis of matched therapy-related myeloid neoplasm samples traced their clonal origin to a single-HSPC clone among multiple competing clones, supporting a model of oligoclonal to monoclonal transformation. These findings underscore the need for further systematic research on the long-term hematological consequences of cancer chemotherapy.

Mutation of some genes drives uncontrolled cell proliferation and cancer. The Philadelphia chromosome in chronic myeloid leukaemia (CML) provided the very first such genetic link to cancer 1,2 . However, little is known about the trajectory to CML, the rate of BCR::ABL1 clonal expansion and how this affects disease. Using whole-genome sequencing of 1,013 haematopoietic colonies from nine patients with CML aged 22 to 81 years, we reconstruct phylogenetic trees of haematopoiesis. Intronic breaks in BCR and ABL1 were not always observed, and out-of-frame exonic breakpoints in BCR , requiring exon skipping to derive BCR::ABL1 , were also noted. Apart from ASXL1 and RUNX1 mutations, extra myeloid gene mutations were mostly present in wild-type cells. We inferred explosive growth attributed to BCR::ABL1 commencing 3–14 years (confidence interval 2–16 years) before diagnosis, with annual growth rates exceeding 70,000% per year. Mutation accumulation was higher in BCR::ABL1 cells with shorter telomere lengths, reflecting their excessive cell divisions. Clonal expansion rates inversely correlated with the time to diagnosis. BCR::ABL1 in the general population mirrored CML incidence, and advanced and/or blast phase CML was characterized by subsequent genomic evolution. These data highlight the oncogenic potency of BCR::ABL1 fusion and contrast with the slow and sequential clonal trajectories of most cancers.

As we age, cells accumulate somatic mutations, undergo epigenetic alterations, and experience telomere shortening. In older individuals, normal tissues are often overtaken by expanding clones of cells that have acquired mutations promoting proliferation. Whether epigenetic changes can also provide selective advantage and induce clonal expansion in ageing is less clear. Here, we sequenced 719 whole genomes and methylomes of single-cell colonies derived from HSCs of three healthy individuals and three breast cancer patients. Using somatic mutations, we built lineage trees of the HSCs for each individual. To establish whether loss or gain of methylation was heritable, we developed a method that accurately infers, for each CpG site, its zygotic methylation state as well as the number and timing of ancestral methylation changes that explain the observed methylation state of each somatic cell. We analysed ∼24 million CpG sites per individual and find that methylation states are, in general, stably heritable over decades of life. We find that during embryogenesis, when the embryo comprises a few thousand cells, developmental cells lay down unique methylation haplotypes, spanning hundreds of base pairs in size, that are stably inherited by descendants. This phase of rewiring the methylome has striking properties: (1) thousands of regions genome-wide show heterogeneity of methylation profiles laid down during this phase; (2) these regions are often conserved across different individuals; (3) it precedes gastrulation, so that the same changes are seen in all germ layers; (4) the process happens quickly, completing within a few generations of cell division; (5) it coincides with establishment of inactive X chromosome methylation in females; and (6) the methylation affects many key regulatory regions of the genome. To assess whether embryonic methylation rewiring can contribute to cancer development, we studied women with breast cancer who had BRCA1 promoter methylation. The tumour samples did not have germline or somatic mutation of BRCA1 (or other DNA repair genes), but had BRCA1 methylation along with the typical mutational signatures of homologous recombination deficiency. Intriguingly, we found that the same methylation change was present heterozygously in a subset of HSCs; that these HSCs all derived from one embryonic cell that pre-dated gastrulation; and that the somatic mutations defining this lineage of HSCs were also present clonally in the breast cancer. Thus, aberrant methylation of the BRCA1 promoter in embryogenesis was transmitted across germ layers, in blood and breast epithelium, and (with loss-of-heterozygosity) drove homologous recombination deficiency in a breast clone that transformed to cancer 4-6 decades later. By comparing normal healthy individuals of different ages with cancer patients, we have been able to unravel the process of normal ageing from cancer development at an unprecedented resolution. Lori D. Kregar, Nicholas Williams, Joe Lee, Michael Spencer-Chapman, Oleksii Nikolaienko, Emily Mitchell, Liv B. Gansmo, Per E. Lønning, Elisa Laurenti, Lucy Yates, Stian Knappskog, Jyoti Nangalia, Peter Campbell. Rewiring of methylation during embryogenesis can seed cancer decades later [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB308.

Aplastic anemia (AA) is a life-threatening bone marrow failure driven by T-cell attack on hematopoietic stem and progenitor cells (HSPCs). Pancytopenia results in infection, bleeding, and anemia. Diagnosis remains difficult due to the lack of biomarkers, and current classification, based solely on cytopenia severity, is outdated. Most patients receive immunosuppressive therapy (IST), but 30-40% do not respond, and 10-15% of responders later develop myeloid malignancies. PIGA-mutant clones may also emerge with or without marrow failure, resulting in paroxysmal nocturnal hemoglobinuria (PNH). HSPC clones with a survival advantage emerge in AA's hostile environment. These often carry somatic mutations in genes related to myeloid malignancy, antigen presentation, or the GPI-anchor pathway. Bulk targeted sequencing – used in most studies to date – is limited to the targeted regions, and cannot identify mutation timing, clonal trajectories, or stem cell dynamics. As a result, although somatic mutations correlate with outcomes and likely reflect disease biology, they are not routinely integrated into clinical decision-making. Building somatic single-cell phylogenies from individual patients allows clonal reconstruction, mutation timing estimation, and tracking of clonal dynamics. We hypothesized that applying this approach to AA and PNH would uncover key aspects of disease pathogenesis: What is the latency between an autoimmune trigger and clinical disease? Why do specific mutations arise in some but not all patients? Can these data help predict disease course or treatment response? We used spontaneous genome-wide somatic mutations in single HSPCs to build high-resolution hematopoietic lineage trees from 10 patients with AA (n=7) or PNH (n=3). CD3-depleted peripheral blood HSPC-derived colonies were cultured in methylcellulose and underwent whole-genome sequencing (WGS) at 10-15X coverage (16-78 colonies per patient). Final dataset: 340 colonies (73 PNH, 267 AA) passed QC. Ultradeep whole-exome duplex sequencing was also performed on granulocytes from 13 patients. Using dN/dS, we identified genes under positive selection - those with more non-synonymous mutations than expected by chance. Additionally, single-cell WGS using primary template amplification (PTA) of HSPCs and lymphoid cells avoids any in vitro culture bias and enables sequencing of multiple different cell populations. Key findings:AA patients showed two distinct clonal landscapes: myeloid driver predominant (MDP) and immune escape predominant (IEP). MDP patients had parallel clonal expansions with mutations in genes such as ASXL1, U2AF1, or BCOR. IEP patients had multiple clones with inactivated HLA genes-either by 6p loss-of-heterozygosity (LOH) or nonsense mutations.Previously unreported recurrent mutations were identified in ERAP1, a gene involved in antigen processing, suggesting a novel immune escape mechanism.Immune escape variants often arose early in life. In one patient presenting with AA at age 8, the dominant 6p LOH clone was estimated to originate in utero (<15 weeks post-conception).Among PNH patients, both monoclonal (2/3) and polyclonal (1/3 with 4 independent PIGA mutations) patterns were observed. PIGA mutations were acquired prior to age 15, though clinical onset occurred decades later.Unlike in malignancy, where driver mutations and clonal expansion occur closely together, PIGA clones expanded years after mutation acquisition. Our phylogenies separate the timing of mutation acquisition from the onset of the selection pressure of autoimmunity.Ongoing analysis of PTA-based single-cell WGS and methylomes will further delineate early clonal events and epigenetic changes.Our data represent the largest WGS dataset in AA. Despite the small cohort size, our in-depth analysis of clonal architecture and dynamics provide unprecedented insights into AA evolution. Patients exhibit distinct evolutionary patterns: either immune escape or myeloid driver predominance. We also identify a novel gene under selection in AA – ERAP1. Immune escape variants typically arise early and expand gradually, whereas myeloid drivers often emerge later but expand more rapidly. These differences likely reflect underlying disease mechanisms. A revised AA classification that integrates clonal architecture could guide treatment selection, avoid ineffective IST, and identify those at increased risk of malignant transformation.

Retrotransposon expression must be tightly controlled, particularly in long-lived multipotent stem cells, to prevent deleterious consequences, including insertional mutations. Several mechanisms are known to repress retrotransposon transcription during development, which are generally thought to persist thereafter. However, integration of retrotransposable elements into host genomes has also provided a major source of genetic variation across evolution, generating many sequences that have acquired advantageous host functions. Physiologic roles for retrotransposon sequences imply the existence of regulatory mechanisms responsive to cellular states and environments, but little is known about retrotransposon expression and mobility in adult stem cells or about how these processes might be dynamically regulated.We used novel computational tools to describe the landscape of somatic retrotransposition in mouse and human hematopoietic stem cells (HSCs). Fewer retrotransposition events occur in HSCs than in intestinal stem cells, and mice unexpectedly accrue new retrotransposon insertions at a rate 350-fold higher than humans, indicating tissue-specific and evolutionary constraints on retrotransposon activity.We also identify a previously unrecognized pathway that links cytokine signaling, RNA-modulating HNRNP complexes, and repression of retrotransposon activity. Activation of JAK2, by thrombopoietin or gain-of-function JAK2 mutations, triggers tyrosine phosphorylation of HNRNPA1 that represses expression of ERV, LINE, and SINE retrotransposons and reduces insertional mutagenesis. This pathway allows dynamic regulation of retrotransposon activity in response to cellular context and coordinates maximal repression of retrotransposons with cytokine-induced proliferation, protecting the HSC genome from the increased risk of retrotransposition that exists during cell division.

Allogeneic haematopoietic cell transplantation (HCT) replaces the stem cells responsible for blood production with those from a donor1,2. Here, to quantify dynamics of long-term stem cell engraftment, we sequenced genomes from 2,824 single-cell-derived haematopoietic colonies of ten donor-recipient pairs taken 9-31 years after HLA-matched sibling HCT3. With younger donors (18-47 years at transplant), 5,000-30,000 stem cells had engrafted and were still contributing to haematopoiesis at the time of sampling; estimates were tenfold lower with older donors (50-66 years). Engrafted cells made multilineage contributions to myeloid, B lymphoid and T lymphoid populations, although individual clones often showed biases towards one or other mature cell type. Recipients had lower clonal diversity than matched donors, equivalent to around 10-15 years of additional ageing, arising from up to 25-fold greater expansion of stem cell clones. A transplant-related population bottleneck could not explain these differences; instead, phylogenetic trees evinced two distinct modes of HCT-specific selection. In pruning selection, cell divisions underpinning recipient-enriched clonal expansions had occurred in the donor, preceding transplant-their selective advantage derived from preferential mobilization, collection, survival ex vivo or initial homing. In growth selection, cell divisions underpinning clonal expansion occurred in the recipient's marrow after engraftment, most pronounced in clones with multiple driver mutations. Uprooting stem cells from their native environment and transplanting them to foreign soil exaggerates selective pressures, distorting and accelerating the loss of clonal diversity compared to the unperturbed haematopoiesis of donors.

Human aging is marked by the emergence of a tapestry of clonal expansions in dividing tissues, particularly evident in blood as clonal hematopoiesis (CH). CH, linked to cancer risk and aging-related phenotypes, often stems from somatic mutations in a set of established genes. However, the majority of clones lack known drivers. Here we infer gene-level positive selection in whole blood exomes from 200,618 individuals in UK Biobank. We identify 17 additional genes, ZBTB33, ZNF318, ZNF234, SPRED2, SH2B3, SRCAP, SIK3, SRSF1, CHEK2, CCDC115, CCL22, BAX, YLPM1, MYD88, MTA2, MAGEC3 and IGLL5, under positive selection at a population level, and validate this selection pattern in 10,837 whole genomes from single-cell-derived hematopoietic colonies. Clones with mutations in these genes grow in frequency and size with age, comparable to classical CH drivers. They correlate with heightened risk of infection, death and hematological malignancy, highlighting the significance of these additional genes in the aging process.

BACKGROUND Hematopoietic stem cells (HSCs) constantly acquire somatic mutations at a rate of ~17 per cell per year. Mutations in specific genes offer a growth advantage that may result in clonal hematopoiesis, and eventually, hematological malignancy. Understanding the processes causing these somatic mutations is fundamental to understanding the early origins of malignancy and informing interventions to reduce cancer risk. While some mutations in HSCs are from well-characterised processes, most are of unknown cause. Mutations are usually preceded by DNA damage. In fact, DNA suffers continual damage, with thousands of individual lesions at any moment. The efficiency of DNA repair means that most known classes of lesions have a half-life of minutes to hours, but might DNA damage persist longer? Understanding the dynamics of DNA lesions may help elucidate their origins and mitigate their consequences. METHODS We hypothesised that examining high-resolution phylogenetic trees of somatic cells would let us infer the persistence of DNA lesions across multiple cell cycles. A given lesion that persisted across several cell divisions would have potential to generate a mutation each time that strand was replicated, and these separate mutations could be detectable in the phylogeny. If different bases were misincorporated opposite the lesion during sequential rounds of DNA replication, closely related clones would carry two alternative mutations at the same position in the genome (‘multi-allelic’ variants; MAVs). If a persistent lesion has the same (incorrect) base misincorporated opposite during different rounds of replication, those mutations may be recognised by their contravention of the consensus phylogeny (‘phylogeny-violating’ variants; PVVs). We used seven published sets of somatic phylogenies from whole-genome sequencing of single-cell-derived colonies, organoids or laser-capture microdissections (LCM). The dataset comprised 103 phylogenies from 89 individuals, in total 11,429 genomes. Each phylogeny was generated from a single tissue type: hematopoietic stem and progenitor cells (HSPCs, n=39), bronchial epithelial cells (n=16) or liver parenchyma (n=48). The HSPC phylogenies were either from foetal and cord blood (n=4), healthy adults (n=13), stem cell transplant donor/recipient pairs (n=10), patients with myeloproliferative neoplasms (n=10) or chemotherapy-exposed patients (n=2). We examined all phylogenies to identify MAVs and PVVs caused by persistent DNA lesions. RESULTS We identified mutations arising from 816 DNA lesions that persisted across multiple cell cycles in normal human stem cells from the blood, liver and bronchial epithelium.In HSCs, persistent DNA lesions, likely from endogenous sources, generated a characteristic mutational signature, so-called SBS19.The causative lesions predominantly affected guanines in an ApG context.The persistent lesions occurred steadily throughout life, enduring 2.2 years on average, with 25% lasting 3+ years.We estimate that a single HSC has ~8 such lesions at any moment in time, half of which will generate a mutation with each cell cycle.Overall, 16% of mutations in blood cells are attributable to SBS19, and similar proportions of driver mutations in blood cancers exhibit this signature. CONCLUSION A vast array of DNA lesions emerges from the quotidian chemistry of life coupled with the rather more elective chemistry of our lifestyles. While many lesions are generated frequently and efficiently repaired, our data imply the existence of a family of DNA lesions present in low numbers, but persisting for months to years, generating sizable fractions of cells' mutation burdens. HSCs are unusual amongst normal tissues in that a substantial proportion of mutations have a pattern corresponding to SBS19. Our data explain this, as HSCs feature long-lived lesions giving rise specifically to mutations with this profile. Intriguingly, some patients with sickle cell disease have been shown to have increased rates of SBS19, suggesting there is variability in the degree of damage caused. Recognizing the unusual dynamics of this DNA damage in HSCs, future work will be well-placed to identify the causative lesion and the reason for its persistence, informing strategies to reduce the mutations it causes. In so doing, we may be able to prevent the earliest origins of blood cancer.

Therapy-related myeloid neoplasms (t-MNs) represent one of the most devastating complications associated with cancer chemotherapy. The incidence of t-MNs is notably high following autologous stem cell transplantation (ASCT) for multiple myeloma (MM) or lymphoma. Previous studies have investigated peripheral blood stem cells (PBSCs) in patients undergoing ASCT, revealing an association between the presence of clonal hematopoiesis (CH) and an increased risk of t-MNs post-ASCT. However, it remains unclear whether the CH mutations identified in PBSCs ultimately evolve into the t-MN clone, due to the absence of analyses that examine matched PBSC and t-MN samples. Furthermore, patients without CH in their PBSCs can still develop t-MNs, and the origin of t-MNs in these individuals remains unidentified. To elucidate the clonal origin and evolutionary trajectory of t-MNs post-ASCT, we studied 9 patients with MM who developed t-MNs following ASCT, with a median latency of 3 years (range: 1-8). We generated single-cell derived colonies from mobilized PBSCs of these patients and performed whole-genome sequencing (WGS) on these. A median of 86 colonies per patient was sequenced, totaling 1,032 colonies. For the matched t-MN samples, we conducted bulk WGS on bone marrow samples with a median coverage of 50x. Utilizing genome-wide somatic mutations identified in each colony, we constructed phylogenetic trees of PBSCs. Subsequently, we integrated the genome of t-MN samples within the PBSC phylogeny to identify the clonal origin (i.e., most recent common ancestor [MRCA]) of t-MNs at the single stem cell resolution. In the phylogenetic trees derived from PBSC samples, parallel evolution of distinct TP53 and PPM1D mutations was pervasive. Single-cell WGS data facilitated clone-specific analyses of mutation burden and mutation signatures. The number of somatic mutations was comparable among TP53, PPM1D, and wild-type colonies. Cells harboring TP53 or PPM1D mutations did not exhibit specific mutation signatures. Among 84 TP53-mutated cells, 82 (97.6%) exhibited normal copy number profiles, with only 2 cells displaying copy number alterations in 17p. These findings suggest that TP53-mutated cells do not yet manifest genomic instability at the clonal hematopoiesis stage. Integrated phylogenetic analysis of PBSC colonies and t-MN genomes identified the MRCA of t-MNs in 5 of 9 (56%) patients' PBSC samples. In these patients, the origin of t-MNs could be traced to a single stem cell carrying TP53 mutations, which later acquired chromosomal abnormalities or biallelic alteration of TP53 at the time of transformation. The MRCA was not identifiable in the PBSCs of 4 patients. In two of these patients, melphalan-related mutation signatures were detected in their t-MN samples. Since these patients did not receive melphalan therapy other than during ASCT conditioning, the presence of melphalan-related signatures in t-MN samples suggests that their clonal origin lies in bone marrow HSCs that were not mobilized. Our findings indicate the existence of two distinct pathways for t-MN development post-ASCT: one originating from mutant stem cells in mobilized PBSCs, and the other from bone marrow stem cells that were not mobilized. Further studies are warranted to elucidate the clinical implications of these distinct evolutionary pathways in t-MNs.

In developed countries, approximately 10% of individuals are exposed to systemic chemotherapy for cancer and other diseases. Many chemotherapeutic agents act by increasing DNA damage in cancer cells, triggering cell death. However, there is limited understanding of the extent and long-term consequences of collateral DNA damage to normal tissues. To investigate the impact of chemotherapy on mutation burdens and cell population structure of a normal tissue we sequenced blood cell genomes from 23 individuals, aged 3–80 years, treated with a range of chemotherapy regimens. Substantial additional mutation loads with characteristic mutational signatures were imposed by some chemotherapeutic agents, but there were differences in burden between different classes of agent, different agents of the same class and different blood cell types. Chemotherapy also induced premature changes in the cell population structure of normal blood, similar to those of normal ageing. The results constitute an initial survey of the long–term biological consequences of cytotoxic agents to which a substantial fraction of the population is exposed during the course of their disease management, raising mechanistic questions and highlighting opportunities for mitigation of adverse effects.### Competing Interest StatementUM and GD are GD employees of Astrazeneca.

Phylogenetic trees are a powerful means to display the evolutionary history of species, pathogens and, more recently, individual cells of the human body. Whole-genome sequencing of laser capture microdissections or expanded stem cells has allowed the discovery of somatic mutations in clones, which can be used as natural barcodes to reconstruct the developmental history of individual cells. Here we describe Sequoia, our pipeline to reconstruct lineage trees from clones of normal cells. Candidate somatic mutations are called against the human reference genome and filtered to exclude germline mutations and artifactual variants. These filtered somatic mutations form the basis for phylogeny reconstruction using a maximum parsimony framework. Lastly, we use a maximum likelihood framework to explicitly map mutations to branches in the phylogenetic tree. The resulting phylogenies can then serve as a basis for many subsequent analyses, including investigating embryonic development, tissue dynamics in health and disease, and mutational signatures. Sequoia can be readily applied to any clonal somatic mutation dataset, including single-cell DNA sequencing datasets, using the commands and scripts provided. Moreover, Sequoia is highly flexible and can be easily customized. Typically, the runtime of the core script ranges from minutes to an hour for datasets with a moderate number (50,000–150,000) of variants. Competent bioinformatic skills, including in-depth knowledge of the R programming language, are required. A high-performance computing cluster (one that is capable of running mutation-calling algorithms and other aspects of the analysis at scale) is also required, especially if handling large datasets.

Normal hematopoietic stem and progenitor cells (HSPCs) inherently accumulate somatic mutations and lose clonal diversity with age, processes implicated in the development of myeloid malignancies 1 . The impact of exogenous stressors, such as cancer chemotherapies, on the genomic integrity and clonal dynamics of normal HSPCs is not well defined. We conducted whole-genome sequencing on 1,032 single-cell-derived HSPC colonies from 10 patients with multiple myeloma (MM), who had undergone various chemotherapy regimens. Our findings reveal that melphalan treatment distinctly increases mutational burden with a unique mutation signature, whereas other MM chemotherapies do not significantly affect the normal mutation rate of HSPCs. Among these therapy-induced mutations were several oncogenic drivers such as TET2 and PPM1D . Phylogenetic analysis showed a clonal architecture in post-treatment HSPCs characterized by extensive convergent evolution of mutations in genes such as TP53 and PPM1D . Consequently, the clonal diversity and structure of post-treatment HSPCs mirror those observed in normal elderly individuals, suggesting an accelerated clonal aging due to chemotherapy. Furthermore, analysis of matched therapy-related myeloid neoplasm (t-MN) samples, which occurred 1-8 years later, enabled us to trace the clonal origin of t-MNs to a single HSPC clone among a group of clones with competing malignant potential, indicating the critical role of secondary mutations in dictating clonal dominance and malignant transformation. Our findings suggest that cancer chemotherapy promotes an oligoclonal architecture with multiple HSPC clones possessing competing leukemic potentials, setting the stage for the selective emergence of a singular clone that evolves into t-MNs after acquiring secondary mutations. These results underscore the importance of further systematic research to elucidate the long-term hematological consequences of cancer chemotherapy.

Abstract Progressive dysfunction of mitochondria, organelles responsible for energy provision to cells, is a major hallmark of ageing. How the dysfunction steadily accrues over a lifetime remains unclear, given the transient nature of free radical damage and the high turnover of mitochondria. Here, we leveraged whole-genome sequencing data from single-cell derived foetal and adult haematopoietic stem/progenitor cell colonies to study the clonal dynamics of turnover and selection in mitochondrial genomes throughout life. We found that genetic drift and independent convergence of mitochondrial mutations complicate their use as lineage marks in single-cell sequencing experiments. Point mutations accrued linearly with age at an average rate of 0.007 mutations/genome/year. Using the distribution of mtDNA allele frequencies with age, we infer that a cell’s complement of mitochondrial genomes is replicated every 4-19 weeks in adults, with faster turnover rates during foetal development. Clock-like accumulation of mutations coupled with rapid turnover induces complex evolutionary dynamics in mitochondria as individuals age. Nonsense mutations are disadvantageous regardless of their heteroplasmy level. Missense variants, however, are positively selected at low heteroplasmy but negatively selected at high heteroplasmy, suggesting that some mutations improve fitness of individual mitochondria, even though fitness of the whole cell deteriorates if their expansion proceeds too far. Thus, age-related decline in mitochondrial function can arise from preferential cellular accumulation of selfish mitochondrial clones whose superior fitness ultimately disadvantages the host cell.