A high-resolution landscape of mutations in the BCL6 super-enhancer in normal human B cells.

The super-enhancers (SEs) 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 10 healthy individuals across diverse ethnicities. We detected a total of approximately 9,000 subclonal mutations (allele frequencies <0.1%); of these, approximately 8,000 are present in the BCL6 SE alone. Within the BCL6 SE, we identified 3 regions of clustered mutations in which the mutation frequency is ∼7 × 10-4 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 signatures 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 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.

Tumor necrosis factor, tumor necrosis factor inhibition, and cancer risk.

OBJECTIVE: Tumor necrosis factor (TNF) is a highly pleiotropic cytokine with multiple activities other than its originally discovered role of tumor necrosis in rodents. TNF is now understood to play a contextual role in driving either tumor elimination or promotion. Using both animal and human data, this review examines the role of TNF in cancer development and the effect of TNF and TNF inhibitors (TNFis) on malignancy risk. RESEARCH DESIGN: A literature review was performed using relevant search terms for TNF and malignancy. RESULTS: Although administration of TNF can cause tumor regression in specific rodent tumor models, human expression polymorphisms suggest that TNF can be a tumor-promoting cytokine, whereas blocking the TNF pathway in a variety of tumor models inhibits tumor growth. In addition to direct effects of TNF on tumors, TNF can variously affect immunity and the tumor microenvironment. Whereas TNF can promote immune surveillance designed to eliminate tumors, it can also drive chronic inflammation, autoimmunity, angiogenesis, and other processes that promote tumor initiation, growth, and spread. Key players in TNF signaling that shape this response include NF-κB and JNK, and malignant-inflammatory cell interactions, each of which may have different responses to TNF signaling. Focusing on rheumatoid arthritis (RA) patients, where clinical experience is most extensive, a review of the clinical literature shows no increased risk of overall malignancy or solid tumors such as breast and lung cancers with exposure to TNFis. Lymphoma rates are not increased with use of TNFis. Conflicting data exist regarding the risks of melanoma and nonmelanoma skin cancer. Data regarding the risk of recurrent malignancy are limited. CONCLUSIONS: Overall, the available data indicate that elevated TNF is a risk factor for cancer, whereas its inhibition in RA patients is not generally associated with an increased cancer risk. In particular, TNF inhibition is not associated with cancers linked to immune suppression. A better understanding of the tumor microenvironment, molecular events underlying specific tumors, and epidemiologic studies of malignancies within specific disease indications should enable more focused pharmacovigilance studies and a better understanding of the potential risks of TNFis.

DNA replication error-induced extinction of diploid yeast.

Genetic defects in DNA polymerase accuracy, proofreading, or mismatch repair (MMR) induce mutator phenotypes that accelerate adaptation of microbes and tumor cells. Certain combinations of mutator alleles synergistically increase mutation rates to levels that drive extinction of haploid cells. The maximum tolerated mutation rate of diploid cells is unknown. Here, we define the threshold for replication error-induced extinction (EEX) of diploid Saccharomyces cerevisiae. Double-mutant pol3 alleles that carry mutations for defective DNA polymerase-δ proofreading (pol3-01) and accuracy (pol3-L612M or pol3-L612G) induce strong mutator phenotypes in heterozygous diploids (POL3/pol3-01,L612M or POL3/pol3-01,L612G). Both pol3-01,L612M and pol3-01,L612G alleles are lethal in the homozygous state; cells with pol3-01,L612M divide up to 10 times before arresting at random stages in the cell cycle. Antimutator eex mutations in the pol3 alleles suppress this lethality (pol3-01,L612M,eex or pol3-01,L612G,eex). MMR defects synergize with pol3-01,L612M,eex and pol3-01,L612G,eex alleles, increasing mutation rates and impairing growth. Conversely, inactivation of the Dun1 S-phase checkpoint kinase suppresses strong pol3-01,L612M,eex and pol3-01,L612G,eex mutator phenotypes as well as the lethal pol3-01,L612M phenotype. Our results reveal that the lethal error threshold in diploids is 10 times higher than in haploids and likely determined by homozygous inactivation of essential genes. Pronounced loss of fitness occurs at mutation rates well below the lethal threshold, suggesting that mutator-driven cancers may be susceptible to drugs that exacerbate replication errors.

Mutator suppression and escape from replication error-induced extinction in yeast.

Cells rely on a network of conserved pathways to govern DNA replication fidelity. Loss of polymerase proofreading or mismatch repair elevates spontaneous mutation and facilitates cellular adaptation. However, double mutants are inviable, suggesting that extreme mutation rates exceed an error threshold. Here we combine alleles that affect DNA polymerase δ (Pol δ) proofreading and mismatch repair to define the maximal error rate in haploid yeast and to characterize genetic suppressors of mutator phenotypes. We show that populations tolerate mutation rates 1,000-fold above wild-type levels but collapse when the rate exceeds 10⁻³ inactivating mutations per gene per cell division. Variants that escape this error-induced extinction (eex) rapidly emerge from mutator clones. One-third of the escape mutants result from second-site changes in Pol δ that suppress the proofreading-deficient phenotype, while two-thirds are extragenic. The structural locations of the Pol δ changes suggest multiple antimutator mechanisms. Our studies reveal the transient nature of eukaryotic mutators and show that mutator phenotypes are readily suppressed by genetic adaptation. This has implications for the role of mutator phenotypes in cancer.

DNA replication fidelity and cancer.

Cancer is fueled by mutations and driven by adaptive selection. Normal cells avoid deleterious mutations by replicating their genomes with extraordinary accuracy. Here we review the pathways governing DNA replication fidelity and discuss evidence implicating replication errors (point mutation instability or PIN) in carcinogenesis.

Case series: acute tumor lysis syndrome in mutator mice with disseminated lymphoblastic lymphoma.

Acute tumor lysis syndrome (ATLS) is characterized by severe metabolic abnormalities and organ dysfunction resulting from rapid destruction of neoplastic cells. Metabolic disturbances are thought to be the primary cause of clinical ATLS symptoms, which include renal dysfunction, seizures, and cardiac arrhythmias. The histopathologic lesions associated with organ dysfunction are largely unknown because of the low rate of mortality of ATLS in humans and the few cases of ATLS identified in laboratory animals. Here, we describe histologic, immunohistochemical, and electron microscopic analyses of thirty-one ATLS cases from a cohort of 499 mice that are prone to spontaneous lymphoblastic lymphoma owing to genetic defects in DNA replication fidelity. Seventy-three percent of our cohort died with lymphoblastic lymphoma, and 8% of affected mice died with diffuse microthromboemboli consistent with ATLS. Mice with ATLS had a high spontaneous mortality rate (>50%), a large tumor burden with disseminated disease, and evidence of leukemia. Blood vessels in the lung, kidney, and other organs were occluded by microthromboemboli composed of chromatin, cellular debris, fibrin, platelets, and entrapped erythrocytes and malignant cells. This case series suggests that ATLS can occur at high frequency in mice with disseminated lymphoblastic lymphoma and leads to a high rate of spontaneous death from microthromboemboli.

Divergent cellular phenotypes of human and mouse cells lacking the Werner syndrome RecQ helicase.

Werner syndrome (WS) is a human autosomal recessive genetic instability and cancer predisposition syndrome with features of premature aging. Several genetically determined mouse models of WS have been generated, however, none develops features of premature aging or an elevated risk of neoplasia unless additional genetic perturbations are introduced. In order to determine whether differences in cellular phenotype could explain the discrepant phenotypes of Wrn-/- mice and WRN-deficient humans, we compared the cellular phenotype of newly derived Wrn-/- mouse primary fibroblasts with previous analyses of primary and transformed fibroblasts from WS patients and with newly derived, WRN-depleted human primary fibroblasts. These analyses confirmed previously reported cellular phenotypes of WRN-mutant and WRN-deficient human fibroblasts, and demonstrated that the human WRN-deficient cellular phenotype can be detected in cells grown in 5% or in 20% oxygen. In contrast, we did not identify prominent cellular phenotypes present in WRN-deficient human cells in Wrn-/- mouse fibroblasts. Our results indicate that human and mouse fibroblasts have different functional requirements for WRN protein, and that the absence of a strong cellular phenotype may in part explain the failure of Wrn-/- mice to develop an organismal phenotype resembling Werner syndrome.

DNA polymerase epsilon and delta proofreading suppress discrete mutator and cancer phenotypes in mice.

Organisms require faithful DNA replication to avoid deleterious mutations. In yeast, replicative leading- and lagging-strand DNA polymerases (Pols epsilon and delta, respectively) have intrinsic proofreading exonucleases that cooperate with each other and mismatch repair to limit spontaneous mutation to less than 1 per genome per cell division. The relationship of these pathways in mammals and their functions in vivo are unknown. Here we show that mouse Pol epsilon and delta proofreading suppress discrete mutator and cancer phenotypes. We found that inactivation of Pol epsilon proofreading elevates base-substitution mutations and accelerates a unique spectrum of spontaneous cancers; the types of tumors are entirely different from those triggered by loss of Pol delta proofreading. Intercrosses of Pol epsilon-, Pol delta-, and mismatch repair-mutant mice show that Pol epsilon and delta proofreading act in parallel pathways to prevent spontaneous mutation and cancer. These findings distinguish Pol epsilon and delta functions in vivo and reveal tissue-specific requirements for DNA replication fidelity.

Mutation at the polymerase active site of mouse DNA polymerase delta increases genomic instability and accelerates tumorigenesis.

Mammalian DNA polymerase delta (Pol delta) is believed to replicate a large portion of the genome and to synthesize DNA in DNA repair and genetic recombination pathways. The effects of mutation in the polymerase domain of this essential enzyme are unknown. Here, we generated mice harboring an L604G or L604K substitution in highly conserved motif A in the polymerase active site of Pol delta. Homozygous Pold1(L604G/L604G) and Pold1(L604K/L604K) mice died in utero. However, heterozygous animals were viable and displayed no overall increase in disease incidence, indicative of efficient compensation for the defective mutant polymerase. The life spans of wild-type and heterozygous Pold1(+/L604G) mice did not differ, while that of Pold1(+/L604K) mice was reduced by 18%. Cultured embryonic fibroblasts from the heterozygous strains exhibited comparable increases in both spontaneous mutation rate and chromosome aberrations. We observed no significant increase in cancer incidence; however, Pold1(+/L604K) mice bearing histologically diagnosed tumors died at a younger median age than wild-type mice. Our results indicate that heterozygous mutation at L604 in the polymerase active site of DNA polymerase delta reduces life span, increases genomic instability, and accelerates tumorigenesis in an allele-specific manner, novel findings that have implications for human cancer.

Expression of a human cytochrome p450 in yeast permits analysis of pathways for response to and repair of aflatoxin-induced DNA damage.

Aflatoxin B1 (AFB1) is a human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In humans, AFB1 is primarily bioactivated by cytochrome P450 1A2 (CYP1A2) and 3A4 to a genotoxic epoxide that forms N7-guanine DNA adducts. A series of yeast haploid mutants defective in DNA repair and cell cycle checkpoints were transformed with human CYP1A2 to investigate how these DNA adducts are repaired. Cell survival and mutagenesis following aflatoxin B1 treatment was assayed in strains defective in nucleotide excision repair (NER) (rad14), postreplication repair (PRR) (rad6, rad18, mms2, and rad5), homologous recombinational repair (HRR) (rad51 and rad54), base excision repair (BER) (apn1 apn2), nonhomologous end-joining (NHEJ) (yku70), mismatch repair (MMR) (pms1), translesion synthesis (TLS) (rev3), and checkpoints (mec1-1, mec1-1 rad53, rad9, and rad17). Together our data suggest the involvement of homologous recombination and nucleotide excision repair, postreplication repair, and checkpoints in the repair and/or tolerance of AFB1-induced DNA damage in the yeast model. Rev3 appears to mediate AFB1-induced mutagenesis when error-free pathways are compromised. The results further suggest unique roles for Rad5 and abasic endonuclease-dependent DNA intermediates in regulating AFB1-induced mutagenicity.

Incorporation of uracil into minus strand DNA affects the specificity of plus strand synthesis initiation during lentiviral reverse transcription.

Many retroviruses either encode dUTP pyrophosphatase (dUTPase) or package host-derived uracil DNA glycosylase as a means to limit the accumulation of uracil in DNA strands, suggesting that uracil is detrimental to one or more steps in the viral life cycle. In the present study, the effects of DNA uracilation on (-) strand DNA synthesis, RNase H activity, and (+) strand DNA synthesis were investigated in a cell-free system. This system uses the activities of purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase to convert single-stranded RNA to double-stranded DNA in a single reaction mixture. Substitution of dUTP for dTTP had no effect on (-) strand synthesis but significantly decreased yields of (+) strand DNA. Mapping of nascent (+) strand 5' ends revealed that this was due to decreased initiation from polypurine tracts with a concomitant increase in initiation at non-polypurine tract sites. Aberrant initiation correlated with a change in RNase H cleavage specificity when assayed on preformed RNA-DNA duplexes containing uracilated DNA, suggesting that appropriate "selection" of the (+) strand primer is affected. Collectively, these data suggest that accumulation of uracil in retroviral DNA may disrupt the viral life cycle by altering the specificity of (+) strand DNA synthesis initiation during reverse transcription.

High incidence of epithelial cancers in mice deficient for DNA polymerase delta proofreading.

Mutations are a hallmark of cancer. Normal cells minimize spontaneous mutations through the combined actions of polymerase base selectivity, 3' --> 5' exonucleolytic proofreading, mismatch correction, and DNA damage repair. To determine the consequences of defective proofreading in mammals, we created mice with a point mutation (D400A) in the proofreading domain of DNA polymerase delta (poldelta, encoded by the Pold1 gene). We show that this mutation inactivates the 3' --> 5' exonuclease of poldelta and causes a mutator and cancer phenotype in a recessive manner. By 18 months of age, 94% of homozygous Pold1(D400A/D400A) mice developed cancer and died (median survival = 10 months). In contrast, only 3-4% of Pold1(+/D400A) and Pold1(+/+) mice developed cancer in this time frame. Of the 66 tumors arising in 49 Pold1(D400A/D400A) mice, 40 were epithelial in origin (carcinomas), 24 were mesenchymal (lymphomas and sarcomas), and two were composite (teratomas); one-third of these animals developed tumors in more than one tissue. Skin squamous cell carcinoma was the most common tumor type, occurring in 60% of all Pold1(D400A/D400A) mice and in 90% of those surviving beyond 8 months of age. These data show that poldelta proofreading suppresses spontaneous tumor development and strongly suggest that unrepaired DNA polymerase errors contribute to carcinogenesis. Mice deficient in poldelta proofreading provide a tractable model to study mechanisms of epithelial tumorigenesis initiated by a mutator phenotype.

Defects in homologous recombination repair in mismatch-repair-deficient tumour cell lines.

Loss of mismatch repair (MMR) leads to a complex mutator phenotype that appears to drive the development of a subset of colon cancers. Here we show that MMR-deficient tumour cell lines are highly sensitive to the toxic effects of thymidine relative to MMR-proficient lines. This sensitivity was not a direct consequence of MMR deficiency or alterations of DNA precursor metabolism. Instead, MMR-defective tumour cell lines are also defective in homologous recombination repair (HRR) induced by DNA double-strand breaks. Furthermore, a frameshift mutation of the human RAD51 paralog XRCC2 found in the MMR-deficient uterine tumour cell line SKUT-1 can confer thymidine sensitivity when introduced into a MMR-proficient line. Like other cells with defective XRCC2, SKUT-1 is sensitive to mitomycin C, and MMR-proficient cells expressing the mutant XRCC2 allele become more sensitive to this agent. These data suggest that the thymidine sensitivity of MMR-deficient tumour cell lines may be a consequence of defects in the HRR pathway. The increased thymidine sensitivity and the loss of an important pathway for the repair of DNA double-strand breaks create new opportunities for therapies directed specifically against this subset of tumours.