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Mutation signature correlation graph

Seven supplementary tables. Table S1. Data yield and Duplex Sequencing statistics. Table S2. Mutation contexts significantly differ between normal stem cells and transformed cells. Table S3. Homoplasmic variants in the whole mtDNA identified using Duplex Sequencing. Table S4. Rare variants in the whole mtDNA of HBEC identified using Duplex Sequencing. Table S5. Mutations that are clonally expanded or negatively selected toward tumorigenesis. Table S6. Distribution of nonsynonymous mutations of rare variants in the whole mtDNA and within mtDNA coding regions. Table S7. The number of non-synonymous mutations of rare variants in mitochondrial protein-coding regions.

g Score sum graph

Supplementary methods

Figure legends for Supplementary figures 1, 2, and 3

Rare stochastic mutations may accumulate during dormancy of stem-like cells, but technical limitations in DNA sequencing have limited exploring this possibility. In this study, we employed a recently established deep-sequencing method termed Duplex Sequencing to conduct a genome-wide analysis of mitochondrial (mt) DNA mutations in a human breast stem cell model that recapitulates the sequential stages of breast carcinogenesis. Using this method, we found significant differences in mtDNA among normal stem cells, immortal/preneoplastic cells, and tumorigenic cells. Putative cancer stem-like cell (CSC) populations and mtDNA copy numbers increased as normal stem cells become tumorigenic cells. Transformed cells exhibited lower rare mutation frequencies of whole mtDNA than did normal stem cells. The predicted mtDNA rare mutation pathogenicity was significantly lower in tumorigenic cells than normal stem cells. Major rare mutation types in normal stem cells are C>T/G>A and T>C/A>G transitions, while only C>T/G>A are major types in transformed cells. We detected a total of 1,220 rare point mutations, 678 of which were unreported previously. With only one possible exception (m10342T>C), we did not find specific mutations characterizing mtDNA in human breast CSCs; rather, the mitochondrial genome of CSCs displayed an overall decrease in rare mutations. On the basis of our work, we suggest that this decrease (in particular T>C/A>G transitions), rather than the presence of specific mitochondrial mutations, may constitute an early biomarker for breast cancer detection. Our findings support the hypothesis that the mitochondrial genome is altered greatly as a result of the transformation of normal stem cells to CSCs, and that mtDNA mutation signatures may aid in delineating normal stem cells from CSCs. Cancer Res; 76(15); 4569–78. ©2016 AACR.

Rare and low heteroplasmic mutations frequency and mutation (%) fraction graphs

Mutations characterize diverse human cancers; there is a positive correlation between elevated mutation frequency and tumor progression. One exception is acute myeloid leukemia (AML), which has few clonal single nucleotide mutations. We used highly sensitive and accurate Duplex Sequencing (DS) to show now that AML, in addition, has an extensive repertoire of variants with low allele frequencies, 1%, which is below the accurate detection limit of most other sequencing methodologies. The subclonal variants are unique to each individual and change in composition, frequency, and sequence context from diagnosis to relapse. Their functional significance is apparent by the observation that many are known variants and cluster within functionally important protein domains. Subclones provide a reservoir of variants that could expand and contribute to the development of drug resistance and relapse. In accord, we accurately identified subclonal variants in AML driver genes NRAS and RUNX1 at allele frequencies between 0.1% and 0.3% at diagnosis, which expanded to comprise a major fraction (14-53%) of the blast population at relapse. Early and accurate detection of subclonal variants with low allele frequency thus offers the opportunity for early intervention, prior to detection of clinical relapse, to improve disease outcome and enhance patient survival.

Polyguanine tracts (PolyGs) are short guanine homopolymer repeats that are prone to accumulating mutations when cells divide. This feature makes them especially suitable for cell lineage tracing, which has been exploited to detect and characterize precancerous and cancerous somatic evolution. PolyG genotyping, however, is challenging because of the inherent biochemical difficulties in amplifying and sequencing repetitive regions. To overcome this limitation, we developed PolyG-DS, a next-generation sequencing (NGS) method that combines the error-correction capabilities of duplex sequencing (DS) with enrichment of PolyG loci using CRISPR-Cas9-targeted genomic fragmentation. PolyG-DS markedly reduces technical artifacts by comparing the sequences derived from the complementary strands of each original DNA molecule. We demonstrate that PolyG-DS genotyping is accurate, reproducible, and highly sensitive, enabling the detection of low-frequency alleles (

At least 50% of human epidermal growth factor 2 (HER2) enriched breast cancer (BC) is estrogen receptor positive (ER+). In clinical trials, patients with HER2+ BC expressing ER respond poorly to neoadjuvant anti-HER2 therapy versus those lacking ER expression. Additionally, combining anti-HER2 with endocrine therapy (ET), like aromatase inhibitor (AI), does not significantly improve pathological complete response in HER2+/ER+ patients. In advanced or de novo HER2+/ER+ metastatic BC (MBC), treatment with ET plus anti-HER2 yields the highest 5-year overall survival. Unfortunately, in the real-world setting outside of clinical trials, just 23% of patients receive this combination, and nearly 40% receive ET alone. The clinical challenge associated with treating advanced HER2+/ER+ BC demands a better understanding of ER signaling and downstream pathways in HER2-driven BC. Our goal is to identify alternative targeted therapies and/or tailor therapeutic combinatorial strategies to treat and/or delay progression of HER2+/ER+ BC. In this study, we established two long-term estrogen (E2) deprived (LTED) HER2+/ER+ cell line models from BT-474 (BT) and MDA-MB-361 (MM) to mimic endocrine therapy resistance (ETR) to AI, and characterized the response of these cell lines to anti-HER2 and other ETs. We also performed whole-genome sequencing (WGS) and single-cell RNA sequencing (scRNAseq) to identify differences between parental and derived ETR-LTEDs. The ER expression was retained in BT-LTEDs but lost in MM-LTEDs as compared to parental cells. Additionally, ER transcriptional activity was confirmed by increased expression of ER-target genes upon E2 stimulation only in BT-LTEDs. However, both LTED variants were less responsive to fulvestrant and showed upregulation of pro-survival AKT signaling. Focusing on the now ER- MM-LTED model, WGS and targeted duplex sequencing identified a significant increase in exonic missense mutations, notably C>T and C>A. Several of the mutated genes encode for transcription and chromatin regulatory factors. scRNAseq analysis showed that MM-LTEDs shift from a luminal- to more basal-like phenotype, with enrichment of genes that regulate immune response and cell motility. Thus, loss of ER expression in MM-LTEDs on the mRNA and protein levels may explain observed intrinsic resistance to fulvestrant and gain of the basal-like phenotype. In summary, we report that our ETR BT- and MM-LTED models capture distinct ER-associated phenotypes of HER2+/ER+ BC. Additional studies are necessary to understand the functional significance of these missense mutations on the development of drug resistance in HER2+/ER+ BC. Ongoing studies are testing pharmacological inhibition of key upregulated genes associated with the basal phenotype to potentially delay disease progression and provide better treatments for ETR HER2+/ER+ BC. Citation Format: Shaymaa Bahnassy, Hillary Stires, Lu Jin, Stanley Tam, Yasmin J. Dunn, Brendan F. Kohrn, Lawrence A. Loeb, Manasi Balachandran, Mircea Podar, Matthew D. McCoy, Robert A. Beckman, Rebecca B. Riggins. Loss of estrogen receptor alters drug responsiveness and supports a basal-like phenotype in endocrine-resistant HER2+/ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1776.

Significance Epimutations arising from transcriptional mutagenesis have been hypothesized to contribute to viral and bacterial evolution, drug resistance, and age-related diseases, including cancer and neurodegeneration. However, methodology limitations have inhibited progress toward elucidating the contributions of epimutations to cellular evolution and survival in vivo. Recent efforts to overcome these limitations remain constrained by artifacts arising during RNA library preparation. We present accurate RNA consensus sequencing (ARC-seq), an accurate, high-throughput RNA sequencing method that effectively eliminates errors introduced during RNA library preparation and sequencing and represents a major advance over previous methods. ARC-seq will enable investigations of the causal roles of transcriptional fidelity and epimutations in multiple fields, including viral evolution, bacterial resistance, and age-related diseases, such as cancer and neurodegeneration.

For the last 40 years the authors have collaborated on trying to understand the complexities of human cancer by formulating testable mathematical models that are based on mutation accumulation in human malignancies. We summarize the concepts encompassed by multiple mutations in human cancers in the context of source, accumulation during carcinogenesis and tumor progression, and therapeutic consequences. We conclude that the efficacious treatment of human cancer by targeted therapy will involve individualized, uniquely directed specific agents singly and in simultaneous combinations, and take into account the importance of targeting resistant subclonal mutations, particularly those subclones with alterations in DNA repair genes, DNA polymerase, and other genes required to maintain genetic stability.

Background The molecular origin of cancer drug resistance is not apparent for most cases of acute myeloid leukemia (AML). Clonal evolution appears to be associated with increasing drug resistance. We sought to determine whether the mutation patterns are associated with drug susceptibility in AML. Methods Seventy-two patient blood or marrow samples were enriched for CD34+ blasts by immunomagnetic bead selection. High throughput drug sensitivity screens were performed for 223 drugs after a 72-hour exposure to 8-12 customized drug concentrations (within the range of 5pM to 100µM) of each drug spanning 4-5 logs. Post exposure viability was determined using CellTiter Glo luminescent reagent. XLFit was used to analyze the data and generate dose response curves based on a standard 4-parameter logistic fit. Mutation analysis was performed by MyAML™ utilizing next generation sequencing (NGS) to analyze the 3' and 5' UTRs and exonic regions of 194 AML-associated genes and genomic breakpoints. Co-mutation and mutual exclusivity scores of gene aberrations were computed using three different statistical methods on three large publicly available datasets: 1) TCGA-AML, 2) BeatAML, and 3) data from 1540 patients (Moritz Gerstung, et al., Nat Genet. 2017). The scores for each co-mutation or mutually exclusive gene pair were then logarithmically transformed from the aggregated p-value using a robust rank aggregation method and used to construct a graph from which communities of co-mutated genes could be detected, resulting in higher mutual exclusivity between modules and higher co-occurrence within modules. Results We identified five main groups of co-mutations, including the following: 1) RUNX1 group, 2) CEBPA group, 3) NPM1 group, 4) TP53 group, and 5) RAS group (see Fig 1A). The co-mutation community in the RUNX1 group is featured with transcriptional dysregulation (RUNX1, ASXL1 and EZH2), and dysregulation in splicing (U2AF1, SRSF2 or SF3B1). The TP53 and CEBPA groups exhibit transcriptional dysregulation, and transcription factor alterations. The NPM1 and RAS groups exhibit signaling alteration, particularly the Ras/MAPK signaling pathway. Among each co-mutation community, the variant allele frequencies (VAFs) exhibited differences for specific mutations. For example, in the NPM1 group, FLT3 exhibits the lowest VAF, followed by NPM1 while DNMT3A shows the highest VAF. The five co-mutation groups are associated with different overall survival, including better survival for the CEBPA group and poorer survival for the TP53 group. Within each co-mutation group, specific associations with overall survival were detected, including better survival for NPM1-RAD21 co-mutated vs. wild type (WT), better survival for WT vs. ASXL1-RUNX1 co-mutated, worse survival for DNMT3A-FLT3 co-mutated vs. WT, and others. We also found that the co-mutation clusters were different between the de novo and relapse groups. Most importantly, we found significant correlations with drug sensitivity for the different co-occurring mutation groups. Cells from patients with mutations in the NPM1 co-mutation group show higher sensitivity to most of the drugs, including the tyrosine kinases inhibitors and PI3K-AKT-MTOR inhibitors. Cells from patients with mutations in the RAS cluster exhibited sensitivity to MEK inhibitors (Fig 1B), while cells from patients with mutations in the TP53 cluster show resistance to many drugs, including the MDM2 inhibitor AMG232 (Fig 1C). By mapping the mutated genes and drug targets into signaling pathways, we found that mutations in downstream signaling of the drug target exhibited resistance, while mutations in upstream signaling conferred higher sensitivity. We also constructed drug sensitivity prediction models based on the co-mutation groups. Conclusion We detected co-mutation groups through integrative analysis of large publicly available AML data sets. Patients with mutations in different groups of genes show different overall survival, while cells from patients with mutations in different co-mutation groups show different drug sensitivity. The co-mutation groups may not only reflect the clonal evolution history of AML, but also serve as important features for drug sensitivity prediction. Disclosures Carson: Invivoscribe, Inc: Current Employment. Patay:Invivoscribe, Inc: Current Employment. Becker:Cardiff Oncology: Research Funding; Novartis: Research Funding; SecuraBio: Research Funding; JW Pharmaceutical: Research Funding; Glycomimetics: Research Funding; Abbvie: Research Funding; Bristol Myers Squibb: Research Funding; Pfizer: Research Funding; Invivoscribe: Research Funding; Accordant Health Services/Caremark: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: There are ~200 drugs on the screening panel, and many would be off label for acute myeloid leukemia.

Significance Cancers evolve many mutations. Clonal driver mutations are selected early. Subsequent evolution occurs in a branching fashion, possibly without selection (“neutral evolution”). Rarer mutations occur later on smaller branches of the evolutionary tree. Using a DNA-sequencing method, duplex sequencing, with unprecedented accuracy and sensitivity, we quantified rare unique subclonal mutations in diagnostic specimens from 5 human colorectal cancers. Rarer subclones probe later evolutionary time points than previously possible. We confirm neutral evolution at later times and find many more subclonal mutations than expected. A theoretical method allowed us to extrapolate further forward in time to diagnosis. At diagnosis, every DNA base is mutated in at least one cancer cell. In particular, any therapy resistance mutation would be present., Human colorectal cancers (CRCs) contain both clonal and subclonal mutations. Clonal driver mutations are positively selected, present in most cells, and drive malignant progression. Subclonal mutations are randomly dispersed throughout the genome, providing a vast reservoir of mutant cells that can expand, repopulate the tumor, and result in the rapid emergence of resistance, as well as being a major contributor to tumor heterogeneity. Here, we apply duplex sequencing (DS) methodology to quantify subclonal mutations in CRC tumor with unprecedented depth (104) and accuracy (

The super-enhancers (SE) of lineage-specific genes in B-cells are off-target sites of somatic hypermutation. However, the inability to detect sufficient numbers of mutations in normal human B-cells has precluded the generation of a high-resolution mutational landscape of SEs. Here, we captured and sequenced 12 B-cell SEs at single-nucleotide resolution from ten healthy individuals across diverse ethnicities. We detected a total of ∼9000 subclonal mutations (allele frequencies BCL6 SE alone. Within the BCL6 SE, we identified three regions of clustered mutations where the mutation frequency is ∼7X10-4. Mutational spectra show a predominance of C>T/G>A and A>G/T>C substitutions, consistent with the activities of activation-induced-cytidine deaminase (AID) and the A-T mutator, DNA Polymerase η, respectively, in mutagenesis in normal B-cells. Analyses of mutational signatures further corroborate the participation of these factors in this process. Single base substitution signature SBS85, SBS37, and SBS39 were found in the BCL6 SE. While SBS85 is a denoted signature of AID in lymphoid cells, the etiologies of SBS37 and SBS39 are still unknown. Our analysis suggests the contribution of error-prone DNA polymerases to the latter signatures. The high-resolution mutation landscape has enabled accurate profiling of subclonal mutations in B-cell SEs in normal individuals. By virtue of the fact that subclonal SE mutations are clonally expanded in B-cell lymphomas, our studies also offer the potential for early detection of neoplastic alterations.SignificanceWe used Duplex Sequencing to detect low-frequency mutations in the BCL6 super-enhancer locus in normal human B-cells. The landscape of pre-existing mutations is remarkably conserved across different ethnicities and reveals clustered mutational hotspots that correlate with reported sites of clonal mutations and translocation breakpoints in human B-cell lymphomas. This high-resolution genomic landscape revealed by Duplex Sequencing offers accurate and thorough profiling of low frequency, pre-existing mutations in normal individuals, and the potential for early detection of neoplastic alterations.

Human colorectal cancers (CRC) contain both clonal and subclonal mutations. Clonal mutations are positively selected, present in most cells and drive malignant progression. Subclonal mutations are randomly dispersed throughout the genome; they provide a vast reservoir of mutant cells that can expand, repopulate the tumor and result in the rapid emergence of resistance, as well as being a major contributor to tumor heterogeneity. Here, we apply Duplex Sequencing (DS) methodology to quantify subclonal mutations in CRC tumor with unprecedented depth (104) and accuracy (−7). We measured mutation frequencies in genes encoding replicative DNA polymerases and in genes frequently mutated in CRC, and found an unexpectedly high effective mutation rate, 7.1 × 10−7. The curve of subclonal mutation accumulation as a function of sequencing depth, using DNA obtained from five different tumors, is in accord with a neutral model of tumor evolution. We present a new theoretical approach to model neutral evolution independent of the infinite sites assumption (which states that a particular mutation arises only in one tumor cell at any given time). Our analysis indicates that the infinite sites assumption is not applicable once the number of tumor cells exceeds the reciprocal of the mutation rate, a circumstance relevant to even the smallest clinically diagnosable tumor. Our methods allow accurate estimation of the total mutation burden in clinical cancers. Our results indicate that no DNA locus is wild type in every malignant cell within a tumor at the time of diagnosis (probability of all cells wild type = 10−308).Significance StatementCancers evolve many mutations. Clonal mutations are selected early. Subsequent evolution occurs in a branching fashion, possibly without selection (“neutral evolution”). Rarer mutations occur later on smaller branches of the evolutionary tree. Using a DNA sequencing method, duplex sequencing, with unprecedented accuracy and sensitivity, we quantified very rare unique subclonal mutations in diagnostic specimens from five human colorectal cancers. Rarer subclones probe later evolutionary timepoints than previously possible. We confirm neutral evolution at later times and find many more subclonal mutations than expected. A novel theoretical method allowed us to extrapolate further forward in time to diagnosis. At diagnosis, every base in DNA is mutated in at least one cancer cell. In particular any therapy resistance mutation would be present.

Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are human photosensitive diseases with mutations in the nucleotide excision repair (NER) pathway, which repairs DNA damage from UV exposure. CS is mutated in the transcription-coupled repair (TCR) branch of the NER pathway and exhibits developmental and neurological pathologies. The XP-C group of XP patients have mutations in the global genome repair (GGR) branch of the NER pathway and have a very high incidence of UV-induced skin cancer. Cultured cells from both diseases have similar sensitivity to UV-induced cytotoxicity, but CS patients have never been reported to develop cancer, although they often exhibit photosensitivity. Because cancers are associated with increased mutations, especially when initiated by DNA damage, we examined UV-induced mutagenesis in both XP-C and CS cells, using duplex sequencing for high-sensitivity mutation detection. Duplex sequencing detects rare mutagenic events, independent of selection and in multiple loci, enabling examination of all mutations rather than just those that confer major changes to a specific protein. We found telomerase-positive normal and CS-B cells had increased background mutation frequencies that decreased upon irradiation, purging the population of subclonal variants. Primary XP-C cells had increased UV-induced mutation frequencies compared with normal cells, consistent with their GGR deficiency. CS cells, in contrast, had normal levels of mutagenesis despite their TCR deficiency. The lack of elevated UV-induced mutagenesis in CS cells reveals that their TCR deficiency, although increasing cytotoxicity, is not mutagenic. Therefore the absence of cancer in CS patients results from the absence of UV-induced mutagenesis rather than from enhanced lethality.

Summary: High-accuracy next-generation DNA sequencing promises a paradigm shift in early cancer detection by enabling the identification of mutant cancer molecules in minimally invasive body fluid samples. We demonstrate 80% sensitivity for ovarian cancer detection using ultra-accurate Duplex Sequencing to identify TP53 mutations in uterine lavage. However, in addition to tumor DNA, we also detect low-frequency TP53 mutations in nearly all lavages from women with and without cancer. These mutations increase with age and share the selection traits of clonal TP53 mutations commonly found in human tumors. We show that low-frequency TP53 mutations exist in multiple healthy tissues, from newborn to centenarian, and progressively increase in abundance and pathogenicity with older age across tissue types. Our results illustrate that subclonal cancer evolutionary processes are a ubiquitous part of normal human aging, and great care must be taken to distinguish tumor-derived from age-associated mutations in high-sensitivity clinical cancer diagnostics. : Salk et al. demonstrate that ultra-sensitive DNA sequencing to identify TP53 mutations among cells shed into uterine fluid shows potential for minimally invasive ovarian cancer detection. Yet they also reveal ubiquitous age-related accumulations of cancer-like TP53 mutations in the normal tissues of healthy women. This highlights an important challenge of using tumor driver mutations for cancer screening. Keywords: Duplex Sequencing, next-generation sequencing, TP53, clonal evolution, early detection, somatic mutations, high-grade serous ovarian cancer, uterine lavage, aging, gynecologic oncology

High accuracy next-generation DNA sequencing promises a paradigm shift in early cancer detection by enabling the identification of mutant cancer molecules in minimally-invasive body fluid samples. We demonstrate 80% sensitivity for ovarian cancer detection using ultra-accurate Duplex Sequencing to identify TP53 mutations in uterine lavage. However, in addition to tumor DNA, we also detect low frequency TP53 mutations in nearly all lavages from women with and without cancer. These mutations increase with age and share the selection traits of clonal TP53 mutations commonly found in human tumors. We show that low frequency TP53 mutations exist in multiple healthy tissues, from newborn to centenarian, and progressively increase in abundance and pathogenicity with older age across tissue types. Our results illustrate that subclonal cancer evolutionary processes are a ubiquitous part of normal human aging and great care must be taken to distinguish tumor-derived, from age-associated mutations in high sensitivity clinical cancer diagnostics.

Purpose: Sequential treatment with targeted therapies can result in complex combinations of resistance mutations in drug targets. This mutational complexity has spurred the development of pan-target inhibitors, i.e., therapies for which no single target mutation can cause resistance. Because the propensity for on- versus off-target resistance varies across cancer types, a deeper understanding of the mutational burden in drug targets could rationalize treatment outcomes and prioritize pan-target inhibitors for indications where on-target mutations are most likely. Experimental Design: To measure and model the mutational landscape of a drug target at high resolution, we integrated single-molecule Duplex Sequencing of the ABL1 gene in Philadelphia-positive (Ph+) leukemias with computational simulations. Results: A combination of drug target mutational burden and tumor-initiating cell fraction is sufficient to predict that most patients with chronic myeloid leukemia are unlikely to harbor ABL1 resistance mutations at the time of diagnosis, rationalizing the exceptional success of targeted therapy in this setting. In contrast, our analysis predicts that many patients with Ph+ acute lymphoblastic leukemia (Ph+ ALL) harbor multiple preexisting resistant cells with single mutants. The emergence of compound mutations can be traced to initial use of an ABL1 inhibitor that is susceptible to resistance from single point mutations. Conclusions: These results argue that early use of therapies that achieve pan-inhibition of ABL1 resistance mutants might improve outcomes in Ph+ ALL. Our findings show how a deep understanding of the mutational burden in drug targets can be quantitatively coupled to phenotypic heterogeneity to rationalize clinical phenomena. Clin Cancer Res; 24(21); 5321–34. ©2018 AACR.

Despite the remarkable throughput of next-generation sequencing technologies, standard techniques are limited by the difficulty in distinguishing sequencing errors from genuine low-frequency DNA variants within heterogeneous cellular or molecular populations. This Review discusses sequencing methodologies and bioinformatic strategies that have been devised for the reliable detection of rare mutations and describes various important applications in diverse fields including cancer, ageing and metagenomics. Mutations, the fuel of evolution, are first manifested as rare DNA changes within a population of cells. Although next-generation sequencing (NGS) technologies have revolutionized the study of genomic variation between species and individual organisms, most have limited ability to accurately detect and quantify rare variants among the different genome copies in heterogeneous mixtures of cells or molecules. We describe the technical challenges in characterizing subclonal variants using conventional NGS protocols and the recent development of error correction strategies, both computational and experimental, including consensus sequencing of single DNA molecules. We also highlight major applications for low-frequency mutation detection in science and medicine, describe emerging methodologies and provide our vision for the future of DNA sequencing.

The mutator phenotype hypothesis was postulated more than 40 years ago. It was based on the multiple enzymatic steps required to precisely replicate the 6 billion bases in the human genome each time a normal cell divides. A reduction in this accuracy during tumor progression could be responsible for the striking heterogeneity of malignant cells within a tumor and for the rapidity by which cancers become resistant to therapy. Cancer Res; 76(8); 2057–9. ©2016 AACR. See related article by Loeb et al. Cancer Res. 1974;34:2311–21.

Aflatoxin B1 (AFB1) and/or hepatitis B and C viruses are risk factors for human hepatocellular carcinoma (HCC). Available evidence supports the interpretation that formation of AFB1-DNA adducts in hepatocytes seeds a population of mutations, mainly G:C→T:A, and viral processes synergize to accelerate tumorigenesis, perhaps via inflammation. Responding to a need for early-onset evidence predicting disease development, highly accurate duplex sequencing was used to monitor acquisition of high-resolution mutational spectra (HRMS) during the process of hepatocarcinogenesis. Four-day-old male mice were treated with AFB1 using a regimen that induced HCC within 72 wk. For analysis, livers were separated into tumor and adjacent cellular fractions. HRMS of cells surrounding the tumors revealed predominantly G:C→T:A mutations characteristic of AFB1 exposure. Importantly, 25% of all mutations were G→T in one trinucleotide context (CGC; the underlined G is the position of the mutation), which is also a hotspot mutation in human liver tumors whose incidence correlates with AFB1 exposure. The technology proved sufficiently sensitive that the same distinctive spectrum was detected as early as 10 wk after dosing, well before evidence of neoplasia. Additionally, analysis of tumor tissue revealed a more complex pattern than observed in surrounding hepatocytes; tumor HRMS were a composite of the 10-wk spectrum and a more heterogeneous set of mutations that emerged during tumor outgrowth. We propose that the 10-wk HRMS reflects a short-term mutational response to AFB1, and, as such, is an early detection metric for AFB1-induced liver cancer in this mouse model that will be a useful tool to reconstruct the molecular etiology of human hepatocarcinogenesis.

Loss-of-function mutations in the WRN helicase gene cause Werner syndrome- a progeroid syndrome with an elevated risk of cancer and other age-associated diseases. Large numbers of single nucleotide polymorphisms have been identified in WRN. We report here the organismal, cellular, and molecular phenotypes of variant rs3087425 (c. 2500C > T) that results in an arginine to cysteine substitution at residue 834 (R834C) and up to 90% reduction of WRN helicase activity. This variant is present at a high (5%) frequency in Mexico, where we identified 153 heterozygous and three homozygous individuals among 3,130 genotyped subjects. Family studies of probands identified ten additional TT homozygotes. Biochemical analysis of WRN protein purified from TT lymphoblast cell lines confirmed that the R834C substitution strongly and selectively reduces WRN helicase, but not exonuclease activity. Replication track analyses showed reduced replication fork progression in some homozygous cells following DNA replication stress. Among the thirteen TT homozygotes, we identified a previously unreported and statistically significant gender bias in favor of males (p = 0.0016), but none of the clinical findings associated with Werner syndrome. Our results indicate that WRN helicase activity alone is not rate-limiting for the development of clinical WS.

Human colorectal cancers (CRC) contain numerous positively selected clonal somatic mutations in 10% or more of the cells. In addition, subclonal mutations in a fraction of cells contribute to phenotypic heterogeneity. Several groups have shown either by analysis of the number of unique subclones as a function of sequencing depth, or by the evaluation of synonymous to nonsynonymous mutation ratios, that most subclonal mutations are not selected and evolve neutrally. Because of the branching nature of tumor evolution, the clonal mutations arise in the founder cell, or very early thereafter. Subclonal mutations appear next, the rare ones on progressively smaller branches of the evolutionary tree. Here, we apply Duplex Sequencing (DS) methodology to quantify subclonal mutations in 5 fresh human MSI-low CRC diagnostic samples with unprecedented depth (104) and accuracy (10-7), and confirm neutral evolution further forward in time than previously known. We find that CRCs without known DNA repair deficits harbor unexpectedly many subclonal mutations; indicating that the mutational diversity of CRCs has been greatly underestimated. The “effective mutation frequency”, or mutation frequency per new cell added to the tumor (taking cell death into account) is also unexpectedly high: 6 X 10-7per base. Given a genome length of 3 X 109, a new daughter cell (taking turnover into account) would have ca 2000 new private mutations compared to its parent. Further, the smallest clinically diagnosable tumor has ca 109 cells, leading to violation of the common modeling assumption that a given mutation arises uniquely in only one cell (“infinite sites assumption”), since at an effective mutation frequency of 6 X 10-7, a cell generation leading to the formation of 109 new cells would produce the same mutation in 600 of those cells. We have developed a new theoretical approach to model neutral evolution independent of the infinite sites assumption, and find that intratumoral heterogeneity in clinical tumors is also underestimated by previous theoretical approaches. Our novel experimental and theoretical methods show that every possible somatic point mutation is present when a tumor is clinically detectable, leading to pre-existing resistant cells to any therapy. We can more accurately predict emergence of cells simultaneously mutationally resistant to multiple non-cross resistant therapies with increasing cell number; targeting genetic instability itself may prevent this. Citation Format: Robert A. Beckman, Brendan Kohrn, Kaitlyn Loubet-Senear, Jasmin Dunn, Jacintha OSullivan, Mary Bronner, Lawrence A. Loeb. Unexpectedly high subclonal mutational diversity in human colorectal cancer and its significance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3770.

Background: Patients with ulcerative colitis (UC) are at risk of developing colorectal cancer. We have previously reported that cancer progression is associated with the presence of clonal expansions and shorter telomeres in nondysplastic mucosa. We sought to validate these findings in an independent case–control study. Methods: This study included 33 patients with UC: 14 progressors (patients with high-grade dysplasia or cancer) and 19 nonprogressors. For each patient, a mean of 5 nondysplastic biopsies from proximal, mid, and distal colon were assessed for clonal expansions, as determined by clonal length altering mutations in polyguanine tracts, and telomere length, as measured by quantitative PCR. Both parameters were compared with individual clinicopathological characteristics. Results: Clonal expansions and shorter telomeres were more frequent in nondysplastic biopsies from UC progressors than nonprogressors, but only for patients with early-onset of UC (diagnosis at younger than 50 years of age). Late-onset progressor patients had very few or no clonal expansions and longer telomeres. A few nonprogressors exhibited clonal expansions, which were associated with older age and shorter telomeres. In progressors, clonal expansions were associated with proximity to dysplasia. The mean percentage of clonally expanded mutations distinguished early-onset progressors from nonprogressors with 100% sensitivity and 80% specificity. Conclusions: Early-onset progressors develop cancer in a field of clonally expanded epithelium with shorter telomeres. The detection of these clones in a few random nondysplastic colon biopsies is a promising cancer biomarker in early-onset UC. Curiously, patients with late-onset UC seem to develop cancer without the involvement of such fields. (Inflamm Bowel Dis 2013;19:2593–2602)

The mutator phenotype hypothesis proposes that the mutation rate of normal cells is insufficient to account for the large number of mutations found in human cancers. Consequently, human tumors exhibit an elevated mutation rate that increases the likelihood of a tumor acquiring advantageous mutations. The hypothesis predicts that tumors are composed of cells harboring hundreds of thousands of mutations, as opposed to a small number of specific driver mutations, and that malignant cells within a tumor therefore constitute a highly heterogeneous population. As a result, drugs targeting specific mutated driver genes or even pathways of mutated driver genes will have only limited anticancer potential. In addition, because the tumor is composed of such a diverse cell population, tumor cells harboring drug-resistant mutations will exist prior to the administration of any chemotherapeutic agent. We present recent evidence in support of the mutator phenotype hypothesis, major arguments against this concept, and discuss the clinical consequences of tumor evolution fueled by an elevated mutation rate. We also consider the therapeutic possibility of altering the rate of mutation accumulation. Most significantly, we contend that there is a need to fundamentally reconsider current approaches to personalized cancer therapy. We propose that targeting cellular pathways that alter the rate of mutation accumulation in tumors will ultimately prove more effective than attempting to identify and target mutant driver genes or driver pathways.

[][1] On November 7, 2016, the cancer research community lost Dr. Richmond Prehn (Rich), a lifelong supporter of AACR and former member of its board, a penetrating theoretician, and a decisive experimentalist. Most scientists think in incremental steps and fear taking great leaps

Rare stochastic mutations may accumulate during dormancy of stem-like cells, but technical limitations in DNA sequencing have limited exploring this possibility. In this study, we employed a recently established deep-sequencing method termed Duplex Sequencing to conduct a genome-wide analysis of mitochondrial (mt) DNA mutations in a human breast stem cell model that recapitulates the sequential stages of breast carcinogenesis. Using this method, we found significant differences in mtDNA among normal stem cells, immortal/preneoplastic cells, and tumorigenic cells. Putative cancer stem-like cell (CSC) populations and mtDNA copy numbers increased as normal stem cells become tumorigenic cells. Transformed cells exhibited lower rare mutation frequencies of whole mtDNA than did normal stem cells. The predicted mtDNA rare mutation pathogenicity was significantly lower in tumorigenic cells than normal stem cells. Major rare mutation types in normal stem cells are C>T/G>A and T>C/A>G transitions, while only C>T/G>A are major types in transformed cells. We detected a total of 1,220 rare point mutations, 678 of which were unreported previously. With only one possible exception (m10342T>C), we did not find specific mutations characterizing mtDNA in human breast CSCs; rather, the mitochondrial genome of CSCs displayed an overall decrease in rare mutations. On the basis of our work, we suggest that this decrease (in particular T>C/A>G transitions), rather than the presence of specific mitochondrial mutations, may constitute an early biomarker for breast cancer detection. Our findings support the hypothesis that the mitochondrial genome is altered greatly as a result of the transformation of normal stem cells to CSCs, and that mtDNA mutation signatures may aid in delineating normal stem cells from CSCs. Cancer Res; 76(15); 4569–78. ©2016 AACR.

DNA sequencing studies have established that many cancers contain tens of thousands of clonal mutations throughout their genomes, which is difficult to reconcile with the very low rate of mutation in normal human cells. This observation provides strong evidence for the mutator phenotype hypothesis, which proposes that a genome-wide elevation in the spontaneous mutation rate is an early step in carcinogenesis. An elevated mutation rate implies that cancers undergo continuous evolution, generating multiple subpopulations of cells that differ from one another in DNA sequence. The extensive heterogeneity in DNA sequence and continual tumor evolution that would occur in the context of a mutator phenotype have important implications for cancer diagnosis and therapy.

Background: Leukemia stem cells (LSCs) play a critical role in AML propagation and relapse. Other investigators have also highlighted unique gene expression profiles for the leukemia stem cell population. Here we compared the results of in vitro drug sensitivity testing against a custom panel of drugs and drug combinations for blast populations vs. leukemia stem cell populations derived from the same patients, as well as mutation analysis for a panel of 194 recurrently mutated genes in AML. Patients and Methods: Patient AML samples were obtained with IRB approval. LSCs were isolated by fluorescence-activated cell sorting (FACS) and the blast population enriched to >90% using immunomagnetic beads from blood samples from 5 patients with AML. A sixth AML patient sample was used for NOD/SCID IL2R γc−/− engraftment, in order to compare characteristics of pre- and post-engraftment subclones. Our CLIA approved custom assay includes 153 drugs and targeted agents, both FDA approved and investigational, with additional drug combinations. High throughput screens (HTS) were conducted with enriched cells adherent to matrix protein in 384 well plates with 8 concentrations of each drug spanning 4 logs. Viability was assessed with CellTiter-Glo (Promega). HTS were performed on LSCs, blasts and pre- as well as post-engraftment AML subclones from the xenograft. Dose-response curves were generated to calibrate drug resistance patterns. Mutation analysis by NGS for a panel of 194 recurrently mutated genes in AML (MyAML®) including 37 translocations was also conducted for the LSC and blast populations. Results: AML blasts and LSCs exhibited divergent drug susceptibility patterns (see volcano plot in Figure). Of 11 drugs commonly used in AML, 8 were typical chemotherapy drugs. Five of these compounds were effective against blasts, but none were effective against LSCs (p-value: 0.0256), suggesting a possible mechanism for post-treatment relapse or primary refractoriness. LSCs were also resistant to mitomycin-C, an agent that induces DNA interstrand crosslinks and DNA breaks, in contrast to blasts that were variably sensitive. Of note, we identified 12 drugs from 8 classes defined by mechanism of action that may target LSCs, in some cases preferentially, when compared with blasts. Drugs effective in preferentially targeting LSCs included tyrosine kinase inhibitors, histone deacetylase inhibitors, 1 cyclin-dependent kinase inhibitor, 1 proteasome inhibitor and 1 microtubule assembly inhibitor. Several of the drugs that efficiently killed LSCs have been studied clinically in AML, while others have theoretical or established efficacy against LSCs by drug class. Only one commonly used drug in AML, sorafenib, a multikinase inhibitor used in FLT3+ disease that may improve survival in younger patients, was effective against LSCs. Blast specific drugs include romidepsin, dinaciclib, alvocidib, ganetespib, selinexor, dorsomorphin, vinblastine, cladribine, dabrafenib, selumetinib, etoposide, torkinib and those in Figure. Blast and LSC drug susceptibility patterns were distinct for each patient. Further, the engrafted xenograft subclone grew very rapidly, was resistant to standard chemotherapy, and possessed three new deleterious mutations in KMT2C (2), SF3B1 and 1 possibly damaging mutation in NUP214, suggesting possible genetic contributions to chemotherapy resistance. We also compared mutation profiles for LSCs vs. blasts in 5 patients, and identified LSC specific mutations in WNK3, WNK4 and BUB1, each in 2 of the 5, and there were also other mutations that were LSC or blast specific. Of note, Bub1 is a mitotic checkpoint serine/threonine kinase that controls mitosis in cancer stem cells (Venere et al Cancer Discov. 2013). WNK3 and WNK4 also both encode serine/threonine protein kinases. Conclusions: The distinct drug susceptibility patterns of patient-specific LSC and blast populations highlight the potential of an individualized approach to treat AML. LSCs are resistant to S-phase agents used in standard-of-care chemotherapy. Genetically distinct minority resistant LSC subclones present at diagnosis may grow rapidly under some conditions, and contribute to drug resistance and relapse. Incorporating the results of functional drug screening focused on LSC subclones may allow more individualized treatment of AML patients and identify patient-specific therapies that lead to improved outcomes. Figure Figure. Disclosures Carson: Invivoscribe Inc.: Employment. Patay:Invivoscribe Inc.: Consultancy, Equity Ownership, Patents & Royalties. Becker:Novartis: Research Funding; Trovagene: Research Funding; CVS Caremark: Consultancy; JW Pharmaceuticals: Research Funding; Rocket Pharmaceuticals: Research Funding; Pfizer: Consultancy; Amgen: Research Funding; BMS: Research Funding; Abbvie: Research Funding; GlycoMimetics: Research Funding.

DNA polymerase δ (pol δ) is one of the two main replicative polymerases in eukaryotes; it synthesizes the lagging DNA strand and also functions in DNA repair. In previous work, we demonstrated that heterozygous expression of the pol δ L604G variant in mice results in normal life span and no apparent phenotype, whereas a different substitution at the same position, L604K, is associated with shortened life span and accelerated carcinogenesis. Here, we report in vitro analysis of the homologous mutations at position Leu-606 in human pol δ. Four-subunit human pol δ variants that harbor or lack 3' → 5'-exonucleolytic proofreading activity were purified from Escherichia coli. The pol δ L606G and L606K holoenzymes retain catalytic activity and processivity similar to that of wild type pol δ. pol δ L606G is highly error prone, incorporating single noncomplementary nucleotides at a high frequency during DNA synthesis, whereas pol δ L606K is extremely accurate, with a higher fidelity of single nucleotide incorporation by the active site than that of wild type pol δ. However, pol δ L606K is impaired in the bypass of DNA adducts, and the homologous variant in mouse embryonic fibroblasts results in a decreased rate of replication fork progression in vivo. These results indicate that different substitutions at a single active site residue in a eukaryotic polymerase can either increase or decrease the accuracy of synthesis relative to wild type and suggest that enhanced fidelity of base selection by a polymerase active site can result in impaired lesion bypass and delayed replication fork progression.

Normal human cells replicate their DNA with exceptional accuracy. It has been estimated that approximately one error occurs during DNA replication for each 10(9) to 10(10) nucleotides polymerized. In contrast, malignant cells exhibit multiple chromosomal abnormalities and contain tens of thousands of alterations in the nucleotide sequence of nuclear DNA. To account for the disparity between the rarity of mutations in normal cells and the large numbers of mutations present in cancer, we have hypothesized that during tumor development, cancer cells exhibit a mutator phenotype. As a defining feature of cancer, the mutator phenotype remains an as-yet unexplored therapeutic target: by reducing the rate at which mutations accumulate it may be possible to significantly delay tumor development; conversely, the large number of mutations in cancer may make cancer cells more sensitive to cell killing by increasing the mutation rate. Here we summarize the evidence for the mutator phenotype hypothesis in cancer and explore how the increased frequency of random mutations during the evolution of human tumors provides new approaches for the design of cancer chemotherapy.

This perspective article highlights the growing evidence placing mitochondria and mitochondrial function at the center of cancer as an age-related disease. The discussion starts from the mitochondrial free radical hypothesis that predicts the involvement of endogenous mitochondrial reactive oxygen species (ROS) in cancer development and summarizes studies demonstrating the impact of the modulation of ROS levels on cancer development and metastasis. Cancer is fundamentally a complex interplay of cell growth, division, metastasis and death- processes connected to mitochondria through energy metabolism. Based on this evidence, therapeutics focused on mitochondrial function and mitochondrial ROS production are an attractive approach to modulating the progression of metastatic cancer and the general improvement of human health span.

The 40th International Symposium of the Princess Takamatsu Cancer Research Fund, entitled “DNA Repair and Human Cancers,” was held on November 10–12, 2009 at Hotel Grand Palace, Tokyo, Japan. The meeting focused on the role of DNA repair in preventing mutations by endogenous and exogenous DNA damage and increasing the efficacy of chemotherapeutic agents by interfering with DNA repair. The 14 presentations by the speakers from the United States, four from the United Kingdom, one each from Italy, The Netherlands, and France, and 13 from Japan, covered most aspects of DNA repair, spanning DNA damage, molecular structures of repair enzymes, and clinical studies on inhibition of DNA repair processes. Extensive time was reserved for discussions with the active participation of the 150 invited Japanese scientists. The choice of a symposium on DNA repair in human cancers resulted in part from the excellent basic and clinical studies that have been carried out for many years in Japan, and the general lack of recognition versus the importance of DNA repair in understanding carcinogenesis. Cancer Res; 70(11); 4269–73. ©2010 AACR.

Cancer recapitulates Darwinian evolution. Mutations acquired during life that provide cells with a growth or survival advantage will preferentially multiply to form a tumor. As a result of The Cancer Genome Atlas Project, we have gathered detailed information on the nucleotide sequence changes in a number of human cancers. The sources of mutations in cancer are diverse, and the complexity of those found to be clonally present in tumors has increasingly made it difficult to identify key rate-limiting genes for tumor growth that could serve as potential targets for directed therapies. The impact of DNA sequencing on future cancer research and personalized therapy is likely to be profound and merits critical evaluation.

Chronic inflammation predisposes to a variety of human cancers. Affected tissues slowly accumulate mutations, some of which affect growth regulation and drive successive waves of clonal evolution, whereas a far greater number are functionally neutral and serve only to passively mark expanding clones. Ulcerative colitis (UC) is an inflammatory bowel disease, in which up to 10% of patients eventually develop colon cancer. Here we have mapped mutations in hypermutable intergenic and intronic polyguanine tracts in patients with UC to delineate the extent of clonal expansions associated with carcinogenesis. We genotyped colon biopsies for length altering mutations at 28 different polyguanine markers. In eight patients without neoplasia, we detected only two mutations in a single individual from among 37 total biopsies. In contrast, for 11 UC patients with neoplasia elsewhere in the colon, we identified 63 mutations in 51 nondysplastic biopsies, and every patient possessed at least one mutant clone. A subset of clones were large and extended over many square centimeters of colon. Of these, some occurred as isolated populations in nondysplastic tissue, considerably distant from neoplastic lesions. Other large clones included regions of cancer, suggesting that the tumor arose within a preexisting clonal field. Our results demonstrate that neutral mutations in polyguanine tracts serve as a unique tool for identifying fields of clonal expansions, which may prove clinically useful for distinguishing a subset of UC patients who are at risk for developing cancer.

Thymidylate synthase (TS) inhibitors, such as 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FUdR), are amongst the most frequently used chemotherapeutic drugs available, although their efficacy is often limited by myelotoxicity. An emerging strategy for overcoming bone marrow toxicity involves ex vivo genetic transfer of drug resistance to autologous hematopoietic progenitor cells, followed by reimplantation of the transfected cells before chemotherapy. Here we establish that expression of mutant TS genes, selected from millions of engineered variants, renders human hematopoietic cells resistant to 5-FUdR, and identify the most efficacious variant for gene therapeutic rescue of drug-induced myelosuppression.

Werner syndrome is an autosomal inherited disease that is characterized by premature aging. The gene mutated in Werner syndrome (WS), WRN, encodes both a 3' --> 5' DNA helicase and a 3' --> 5' DNA exonuclease. Among the WS phenotypes is an exceptionally high incidence of sarcomas. We asked whether spontaneous sarcomas, not known to be associated with WS, also harbor mutations or unreported single nucleotide polymorphisms (SNPs) in WRN. We analyzed RNA or DNA sequences within the helicase and exonuclease domains from 51 and 69 matched sarcoma and adjacent normal tissues, respectively. Among a total of 13 nucleotide variants detected, we identified three novel nonsynonymous substitutions: c.611C>T, c.809_810insT, and c.1882C>G. We further characterized one, c.611C>T, which results in substitution of an evolutionarily conserved proline at amino acid 204 in the exonuclease domain with leucine. We show that P204L WRN exhibits a reduction of WRN exonuclease activity; the specific activity is approximately 10-fold lower than that of wild-type WRN. In contrast, the helicase activity of P204L WRN is reduced less than twofold.

Human alkyladenine-DNA glycosylase (AAG) initiates base excision repair (BER) of alkylated and deaminated bases in DNA. Here, we assessed the mutability of the AAG substrate binding pocket, and the essentiality of individual binding pocket amino acids for survival of methylation damage. We used oligonucleotide-directed mutagenesis to randomize 19 amino acids, 8 of which interact with substrate bases, and created more than 4.5 million variants. We expressed the mutant AAGs in repair-deficient Escherichia coli and selected for protection against the cytotoxicity of either methylmethane sulfonate (MMS) or methyl-lexitropsin (Me-lex), an agent that produces 3-methyladenine as the predominant base lesion. Sequence analysis of 116 methylation-resistant mutants revealed no substitutions for highly conserved Tyr(127)and His(136). In contrast, one mutation, L180F, was greatly enriched in both the MMS- and Me-lex-resistant libraries. Expression of the L180F single mutant conferred 4.4-fold enhanced survival at the high dose of MMS used for selection. The homogeneous L180F mutant enzyme exhibited 2.2-fold reduced excision of 3-methyladenine and 7.3-fold reduced excision of 7-methylguanine from methylated calf thymus DNA. Decreased excision of methylated bases by the mutant glycosylase could promote survival at high MMS concentrations, where the capacity of downstream enzymes to process toxic BER intermediates may be saturated. The mutant also displayed 6.6- and 3.0-fold reduced excision of 1,N(6)-ethenoadenine and hypoxanthine from oligonucleotide substrates, respectively, and a 1.7-fold increase in binding to abasic site-containing DNA. Our work provides in vivo evidence for the substrate binding mechanism deduced from crystal structures, illuminates the function of Leu(180) in wild-type human AAG, and is consistent with a role for balanced expression of BER enzymes in damage survival.

The American Association for Cancer Research has been the citadel for communicating research on chemical carcinogens for over a century. It therefore seems appropriate that a review of chemical carcinogenesis inaugurates a series of articles highlighting advances in understanding, treating, and

Eukaryotic DNA polymerase δ (Pol δ) is an essential, highly conserved enzyme that participates in DNA replication, DNA repair, and genetic recombination. Pol δ is believed to replicate a large portion of the genome, synthesizing most of the lagging strand and perhaps contributing to leading-strand synthesis as well (14, 22, 43) The 125-kDa catalytic subunit, encoded at the mouse Pold1 locus, contains a polymerase domain near the carboxyl terminus that catalyzes DNA synthesis and an exonuclease domain near the amino terminus that catalyzes 3′→5′ exonucleolytic proofreading (5, 18). Pol δ is highly processive in association with PCNA and synthesizes DNA with great accuracy, catalyzing about one error in 10−5 to 10−6 nucleotides polymerized (11, 46, 61). Discrimination between correct and incorrect base pairs at the polymerase active site confers most of the fidelity (error rate, ca. 1 × 10−5), while proofreading increases accuracy approximately 10- to 60-fold (11). Partitioning of the newly formed primer terminus between the polymerase and exonuclease active sites is an important determinant of accuracy as well (11, 47). In cells, mismatch repair adds an additional correction mechanism for errors that escape proofreading, reducing the overall error rate to about 1 × 10−9 bp (19, 35, 36, 55). Accurate DNA replication is essential for the maintenance of genomic stability and suppression of carcinogenesis. Mutants of prokaryotic and eukaryotic DNA polymerases that harbor amino acid substitutions in the polymerase domain or that lack proofreading exhibit increased mutation frequencies in vitro and in vivo (2, 8, 34, 42, 44, 47, 60). In the case of Pol δ, inactivation of the 3′→5′ exonuclease confers a mutator phenotype, increasing mutation rates in haploid yeast and in homozygous mouse embryo fibroblasts (MEFs) (7, 16, 17, 58). The homozygous mutant mice display elevated cancer incidence, suggesting that unrepaired Pol δ errors may contribute to tumorigenesis (16, 17). Given the essentiality of Pol δ and its central role in eukaryotic DNA replication, it is important to assess the nature and severity of the consequences of polymerase active site mutations in mammals. For this study, we created mice harboring mutations at a conserved Leu residue, L604, in motif A of the Pol δ polymerase domain. We elected to study L604 mutants because the effects on accuracy of mutation at homologous residues in other DNA polymerases have been studied extensively. For example, our laboratory has shown that substitution at the homologous Ile in both Escherichia coli Pol I and Taq Pol I creates mutator mutants (40, 41, 57). In phage T4 DNA polymerase, substitution for the homologous Leu can generate either mutator or antimutator mutants (47). In yeast and human DNA polymerase α (Pol α), substitution for the orthologous Leu generates mutators of various strengths (38). Most recently, we and others have shown that replacement of L612 in Saccharomyces cerevisiae Pol δ with each of eight different amino acids increases spontaneous mutation rates up to 37-fold (28, 62). The L612G and L612K yeast Pol δ alleles, which are homologous to the L604G and L604K mutations we studied here, elevated the mutation rate 17- and 13-fold, respectively (62). The present work represents the first description of mammals harboring a mutation in the polymerase domain of a major replicative DNA polymerase. Given the significant phenotypic defects we observed in haploid yeast (62) and the central role of Pol δ in replication, it was not certain that the murine L604G and L604K alleles would yield viable animals. However, when heterozygous, these mutator mutations are compatible with mammalian development and reproduction and do not produce a significant overall increase in end-of-life pathology, indicative of extensive compensation for the defective mutant polymerases. Interestingly, both alleles result in a mutator phenotype, and a single Pold1L604K allele, but not a Pold1L604G allele, accelerates tumorigenesis and reduces life span. We discuss the implications of our results for the ongoing search for Pol δ mutations in human cancers.