Marc J. Prindle

An in-frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy

DNA polymerase δ, whose catalytic subunit is encoded by POLD1, is responsible for lagging-strand DNA synthesis during DNA replication. It carries out this synthesis with high fidelity owing to its intrinsic 3′- to 5′-exonuclease activity, which confers proofreading ability. Missense mutations affecting the exonuclease domain of POLD1 have recently been shown to predispose to colorectal and endometrial cancers. Here we report a recurring heterozygous single-codon deletion in POLD1 affecting the polymerase active site that abolishes DNA polymerase activity but only mildly impairs 3′- to 5′-exonuclease activity. This mutation causes a distinct multisystem disorder that includes subcutaneous lipodystrophy, deafness, mandibular hypoplasia and hypogonadism in males. This discovery suggests that perturbing the function of the ubiquitously expressed POLD1 polymerase has unexpectedly tissue-specific effects in humans and argues for an important role for POLD1 function in adipose tissue homeostasis.

Sequencing small genomic targets with high efficiency and extreme accuracy

The detection of minority variants in mixed samples requires methods for enrichment and accurate sequencing of small genomic intervals. We describe an efficient approach based on sequential rounds of hybridization with biotinylated oligonucleotides that enables more than 1-million-fold enrichment of genomic regions of interest. In conjunction with error-correcting double-stranded molecular tags, our approach enables the quantification of mutations in individual DNA molecules.

DNA polymerase delta in dna replication and genome maintenance

The eukaryotic genome is in a constant state of modification and repair. Faithful transmission of the genomic information from parent to daughter cells depends upon an extensive system of surveillance, signaling, and DNA repair, as well as accurate synthesis of DNA during replication. Often, replicative synthesis occurs over regions of DNA that have not yet been repaired, presenting further challenges to genomic stability. DNA polymerase δ (pol δ) occupies a central role in all of these processes: catalyzing the accurate replication of a majority of the genome, participating in several DNA repair synthetic pathways, and contributing structurally to the accurate bypass of problematic lesions during translesion synthesis. The concerted actions of pol δ on the lagging strand, pol ϵ on the leading strand, associated replicative factors, and the mismatch repair (MMR) proteins results in a mutation rate of less than one misincorporation per genome per replication cycle. This low mutation rate provides a high level of protection against genetic defects during development and may prevent the initiation of malignancies in somatic cells. This review explores the role of pol δ in replication fidelity and genome maintenance. Environ. Mol. Mutagen. 2012.

Do mutator mutations fuel tumorigenesis

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.

Implications of genetic heterogeneity in cancer.

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.

The mutator phenotype in cancer: molecular mechanisms and targeting strategies.

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.

A substitution in the fingers domain of DNA polymerase δ reduces fidelity by altering nucleotide discrimination in the catalytic site.

Background: Amino acid substitutions near the catalytic site of DNA polymerases can affect base discrimination. Results: An A699Q substitution in the fingers domain of eukaryotic DNA polymerase δ (Pol δ) yields reduced fidelity via base misincorporation. Conclusion: Intramolecular interactions involving the Pol δ fingers and N-terminal domains can affect base selectivity. Significance: The structural determinants of fidelity intrinsic to replicative polymerases provide insight into general polymerase function. DNA polymerase δ (Pol δ) is one of the major replicative DNA polymerases in eukaryotic cells, catalyzing lagging strand synthesis as well as playing a role in many DNA repair pathways. The catalytic site for polymerization consists of a palm domain and mobile fingers domain that opens and closes each catalytic cycle. We explored the effect of amino acid substitutions in a region of the highly conserved sequence motif B in the fingers domain on replication fidelity. A novel substitution, A699Q, results in a marked increase in mutation rate at the yeast CAN1 locus, and is synthetic lethal with both proofreading deficiency and mismatch repair deficiency. Modeling the A699Q mutation onto the crystal structure of Saccharomyces cerevisiae Pol δ template reveals four potential contacts for A699Q but not for A699. We substituted alanine for each of these residues and determined that an interaction with multiple residues of the N-terminal domain is responsible for the mutator phenotype. The corresponding mutation in purified human Pol δ results in a similar 30-fold increase in mutation frequency when copying gapped DNA templates. Sequence analysis indicates that the most characteristic mutation is a guanine-to-adenine (G to A) transition. The increase in deoxythymidine 5′-triphosphate-G mispairs was confirmed by performing steady state single nucleotide addition studies. Our combined data support a model in which the Ala-to-Gln substitution in the fingers domain of Pol δ results in an interaction with the N-terminal domain that affects the base selectivity of the enzyme.

Abstract LB-283: Human cancers harbor extensive subclonal mutations

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Recent advances in next-generation DNA sequencing (NGS) have revealed greater than expected mutational heterogeneity, not only between tumors of similar cancer type, but also within individual tumors. This mutational heterogeneity could serve as a reservoir for the emergence of new phenotypes, including resistance to therapy. While mutational diversity has been reported in many human cancers, the high error-rate of conventional NGS limits its ability to confidently resolve mutations present in less than 1.0 to 5% of the cells that comprise a tumor. These low-level subclonal mutations likely contribute to the rapid emergence of resistance to radiation and chemotherapy and the ability of cancer cells to invade adjacent tissues and to metastasize. In order to study subclonal mutations, we have developed a highly accurate sequencing protocol, termed Duplex Sequencing, which takes advantage of the double-stranded nature of DNA to increase the accuracy of DNA sequencing by more than 10,000-fold; Duplex Sequencing has an unprecedented background error frequency of 1,000,000-fold, with up to 99.8% of resultant sequencing reads on target. Using these new methodologies, we have investigated the subclonal makeup of several cancers, including acute myeloid leukemia (AML), colon cancers (CRC), and glioblastoma (GBM). Upon targeting genes identified by NGS as drivers of clonal proliferation, we identified multiple subclonal mutations in each of these cancers, some with the potential to elicit drug resistance and others to drive tumor cell proliferation. We also investigated the five human replicative DNA polymerases. No reports have previously implicated mutations in these polymerases in either AML or GBM, nor in sporadic CRC with the exception of mutations in the exonuclease domain of DNA polymerase epsilon. However, our approach, combining targeted gene capture with Duplex Sequencing, revealed multiple subclonal mutations in the catalytic domains of all five replicative DNA polymerases in each of these tumors. The presence of subclonal mutations in the catalytic domains of replicative DNA polymerases supports the mutator phenotype hypothesis, which posits that an increased mutation rate is a driving force during early tumorigenesis. Furthermore, extrapolating the results on the number of subclonal mutations in target genes to the entire genome, our results indicate that sites harboring subclonal mutations are >100-fold more frequent than sites with clonal mutations. Thus, by the time a tumor is clinically diagnosed, every position in the genome could be mutated in at least one cell in the tumor; these subclones could be the reservoir for the emergence of new phenotypes and resistance to therapy. Citation Format: Michael W. Schmitt, Edward J. Fox, Marc J. Prindle, Kate S. Reid – Bayliss, Pamela S. Becker, Lawrence A. Loeb. Human cancers harbor extensive subclonal mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-283. doi:10.1158/1538-7445.AM2015-LB-283