Stabilization of mutant BRCA1 protein confers PARP inhibitor and platinum resistance

Breast Cancer Type 1 Susceptibility Protein (BRCA1)-deficient cells have compromised DNA repair and are sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Despite initial responses, the development of resistance limits clinical efficacy. Mutations in the BRCA C-terminal (BRCT) domain of BRCA1 frequently create protein products unable to fold that are subject to protease-mediated degradation. Here, we show HSP90-mediated stabilization of a BRCT domain mutant BRCA1 protein under PARP inhibitor selection pressure. The stabilized mutant BRCA1 protein interacted with PALB2-BRCA2-RAD51, was essential for RAD51 focus formation, and conferred PARP inhibitor as well as cisplatin resistance. Treatment of resistant cells with the HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin reduced mutant BRCA1 protein levels and restored their sensitivity to PARP inhibition. Resistant cells also acquired a TP53BP1 mutation that facilitated DNA end resection in the absence of a BRCA1 protein capable of binding CtIP. Finally, concomitant increased mutant BRCA1 and decreased 53BP1 protein expression occur in clinical samples of BRCA1-mutated recurrent ovarian carcinomas that have developed resistance to platinum. These results provide evidence for a two-event mechanism by which BRCA1-mutant tumors acquire anticancer therapy resistance.The breast cancer 1, early onset (BRCA1) gene is commonly mutated in hereditary breast and ovarian cancers. The BRCA1 protein has multiple domains that mediate protein interactions; BRCA1 gene mutations may produce truncated proteins that lose the ability to interact with associated proteins. Additionally, mutations in the BRCA C-terminal (BRCT) domain of BRCA1 create protein folding defects that result in protease-mediated degradation (1–3).Cells that contain dysfunctional BRCA1 proteins are hypersensitive to DNA damaging agents (4). In particular, BRCA1-deficient cell lines are exquisitely sensitive to poly(ADP-ribose) polymerase (PARP) inhibition (5). Despite initial responses of BRCA1-mutant cancers to PARP inhibitor treatment (6), acquired resistance universally develops. Resistance may result from secondary mutations in the BRCA1 gene that restore the reading frame and produce a functional BRCA1 protein (7, 8). In Brca1-mutated mouse mammary tumors, activation of p-glycoprotein or loss of p53 binding protein 1 (53BP1) expression resulting from truncating TP53BP1 mutations confers PARP inhibitor resistance (9). Loss of 53BP1 in BRCA1-deficient cells provides the C-terminal binding protein interacting protein (CtIP) with unrestricted access to DNA breaks, facilitating DNA end resection, an early step in homologous recombination (HR) (9–11).Following BRCA1-CtIP–mediated activation of DNA end resection, eventual BRCA2-mediated assembly of the RAD51 recombinase in nucleoprotein filaments is a critical step in HR. A role for BRCA1 in RAD51 loading and the mechanisms by which it participates have not been fully clarified. Of note, in PARP inhibitor-resistant BRCA1- and 53BP1-deficient tumors and derived cell lines, RAD51 γ-irradiation–induced foci were detected, although at a lower level than in BRCA1- and 53BP1-proficient cells (9). Previous studies demonstrated that RAD51 foci were partially reduced in BRCA1- or partner and localizer of BRCA2 (PALB2)-deficient cells reconstituted with BRCA1 or PALB2 constructs carrying mutations that disrupt the BRCA1–PALB2 interaction (12, 13), suggesting that BRCA1 may enlist PALB2, which in turn organizes the recruitment of BRCA2 and RAD51.To date, the described mechanisms of PARP inhibitor resistance occur in only a fraction of the BRCA1 mutant patient population or in PARP inhibitor-resistant Brca1-mutated mouse mammary tumors (8, 10). Here, we used a human breast cancer cell line that contains a BRCT domain BRCA1 mutation to identify additional mechanisms of acquired PARP inhibitor resistance, and demonstrate that stabilization of the mutant BRCA1 protein is critical for the restoration of RAD51 focus formation.     

Selective resistance to the PARP inhibitor olaparib in a mouse model for BRCA1-deficient metaplastic breast cancer

Metaplastic breast carcinoma (MBC) is a rare histological breast cancer subtype characterized by mesenchymal elements and poor clinical outcome. A large fraction of MBCs harbor defects in breast cancer 1 (BRCA1). As BRCA1 deficiency sensitizes tumors to DNA cross-linking agents and poly(ADP-ribose) polymerase (PARP) inhibitors, we sought to investigate the response of BRCA1-deficient MBCs to the PARP inhibitor olaparib. To this end, we established a genetically engineered mouse model (GEMM) for BRCA1-deficient MBC by introducing the MET proto-oncogene into a BRCA1-associated breast cancer model, using our novel female GEMM ES cell (ESC) pipeline. In contrast to carcinomas, BRCA1-deficient mouse carcinosarcomas resembling MBC show intrinsic resistance to olaparib caused by increased P-glycoprotein (Pgp) drug efflux transporter expression. Indeed, resistance could be circumvented by using another PARP inhibitor, AZD2461, which is a poor Pgp substrate. These preclinical findings suggest that patients with BRCA1-associated MBC may show poor response to olaparib and illustrate the value of GEMM-ESC models of human cancer for evaluation of novel therapeutics.Poly(ADP-ribose) polymerase (PARP) inhibition provides a promising therapeutic strategy for targeting homologous recombination (HR)-deficient tumors, such as breast cancer 1 (BRCA1)-mutated cancers (1). Indeed, clinical phase I and phase II trials have shown potent anticancer activity of small molecule inhibitors of PARP, such as olaparib, in patients with BRCA1-associated breast cancer (2, 3). However, it remains to be established whether different breast cancer subtypes in BRCA1 mutation carriers respond equally to PARP inhibition. Reduced sensitivity of breast cancers to anticancer drugs has frequently been associated with an epithelial-to-mesenchymal transition (EMT) (4–7). Metaplastic breast carcinomas (MBCs) are a subset of triple-negative breast cancers (TNBCs) characterized by a claudin-low and EMT-like phenotype (8) and a poor prognosis compared with other TNBCs (9). More than 60% of MBCs have BRCA1 promoter methylation, raising the question whether these tumors can be effectively targeted by using PARP inhibitors (10). To address this issue in an experimentally controlled setting, we set out to generate a genetically engineered mouse model (GEMM) of BRCA1-deficient MBC by inducing EMT via MET overexpression in a previously established GEMM of BRCA1-mutated breast cancer. We report that EMT is associated with olaparib resistance and can be effectively bypassed by administration of AZD2461, a PARP inhibitor with low affinity for the P-glycoprotein (Pgp) drug efflux transporter.     

BRCA2 regulates DMC1-mediated recombination through the BRC repeats

In somatic cells, BRCA2 is needed for RAD51-mediated homologous recombination. The meiosis-specific DNA strand exchange protein, DMC1, promotes the formation of DNA strand invasion products (joint molecules) between homologous molecules in a fashion similar to RAD51. BRCA2 interacts directly with both human RAD51 and DMC1; in the case of RAD51, this interaction results in stimulation of RAD51-promoted DNA strand exchange. However, for DMC1, little is known regarding the basis and functional consequences of its interaction with BRCA2. Here we report that human DMC1 interacts directly with each of the BRC repeats of BRCA2, albeit most tightly with repeats 1–3 and 6–8. However, BRC1–3 bind with higher affinity to RAD51 than to DMC1, whereas BRC6–8 bind with higher affinity to DMC1, providing potential spatial organization to nascent filament formation. With the exception of BRC4, each BRC repeat stimulates joint molecule formation by DMC1. The basis for this stimulation is an enhancement of DMC1–ssDNA complex formation by the stimulatory BRC repeats. Lastly, we demonstrate that full-length BRCA2 protein stimulates DMC1-mediated DNA strand exchange between RPA–ssDNA complexes and duplex DNA, thus identifying BRCA2 as a mediator of DMC1 recombination function. Collectively, our results suggest unique and specialized functions for the BRC motifs of BRCA2 in promoting homologous recombination in meiotic and mitotic cells.The breast cancer susceptibility protein 2, BRCA2, regulates RAD51-mediated homologous recombination (HR) (1–3). Both RAD51, a DNA strand exchange protein, and its meiotic counterpart, DMC1 (disrupted meiotic cDNA 1 or DNA meiotic recombinase 1), promote HR through the formation of a nucleoprotein filament on ssDNA (4). This filament finds and invades a homologous template, resulting in a DNA strand invasion product called a joint molecule or a displacement-loop (D-loop). The joint molecule provides a primer template for the new DNA synthesis required to repair the DNA double strand break (DSB).The first evidence implicating BRCA2 in meiosis came from studies in Ustilago maydis, where strains lacking the BRCA2 ortholog, Brh2, resulted in absence of meiotic products (5). Shortly thereafter, mouse BRCA2 was inferred to coordinate the activities of RAD51 and DMC1 (6). The first direct interaction between BRCA2 and DMC1 was observed in plants (7) and later in humans (8). In the plant, Arabidopsis thaliana, the interaction between Brca2 and Dmc1 was mapped to the BRC repeats (9), a highly conserved motif comprising a sequence of ∼35 amino acids that is present at least once in all BRCA2-like proteins (10). In humans, BRCA2 contains eight BRC repeats that bind with different affinities to RAD51, and they segregate into two functional classes (11). Within a BRC repeat, two motifs that bind RAD51 have been identified: one comprising the consensus sequence FxxA that mimics the oligomerization interface (12) and contacts the catalytic domain of RAD51; the other binding module comprises the alpha-helical region of the BRC repeat, contains the consensus sequence LFDE, and binds to RAD51 through a different hydrophobic pocket (13).Importantly, loss of Brca2 in plants causes chromosomal aberrations during meiosis (14). In humans, GST-pull down assays using peptide fragments of BRCA2 mapped a unique DMC1 interacting site to residues 2386–2411 (8). However, in mouse, mutation of a key residue (Phe-2406) within this site, which had been shown to disrupt the interaction of BRCA2 with DMC1 by peptide array analysis, had no effect in meiosis (15), suggesting that another site or sites in BRCA2 provide the functions needed during meiosis in this organism (6). A direct physical interaction was indeed established for purified full-length human BRCA2 and DMC1 (2), but the functional relevance of this interaction was not elaborated.We have previously shown that the BRC repeats of BRCA2 modulate the DNA binding selectivity of RAD51 to stimulate the assembly on ssDNA by inhibiting its ATP hydrolysis and preventing its association with dsDNA (2, 11, 16); as a result, BRCA2 catalyzes the recombination activity of RAD51 (17).A comprehensive analysis of aligned sequences of RAD51 orthologs and human RAD51 paralogues suggested that most eukaryotic RAD51 proteins, including DMC1, could interact with the BRC repeats, at least in principle (10). Here we investigate whether and how BRCA2 modulates DMC1-mediated recombination.     

Exome sequencing identifies FANCM as a susceptibility gene for triple-negative breast cancer

Inherited predisposition to breast cancer is known to be caused by loss-of-function mutations in BRCA1, BRCA2, PALB2, CHEK2, and other genes involved in DNA repair. However, most families severely affected by breast cancer do not harbor mutations in any of these genes. In Finland, founder mutations have been observed in each of these genes, suggesting that the Finnish population may be an excellent resource for the identification of other such genes. To this end, we carried out exome sequencing of constitutional genomic DNA from 24 breast cancer patients from 11 Finnish breast cancer families. From all rare damaging variants, 22 variants in 21 DNA repair genes were genotyped in 3,166 breast cancer patients, 569 ovarian cancer patients, and 2,090 controls, all from the Helsinki or Tampere regions of Finland. In Fanconi anemia complementation gene M (FANCM), nonsense mutation c.5101C>T (p.Q1701X) was significantly more frequent among breast cancer patients than among controls [odds ratio (OR) = 1.86, 95% CI = 1.26–2.75; P = 0.0018], with particular enrichment among patients with triple-negative breast cancer (TNBC; OR = 3.56, 95% CI = 1.81–6.98, P = 0.0002). In the Helsinki and Tampere regions, respectively, carrier frequencies of FANCM p.Q1701X were 2.9% and 4.0% of breast cancer patients, 5.6% and 6.6% of TNBC patients, 2.2% of ovarian cancer patients (from Helsinki), and 1.4% and 2.5% of controls. These findings identify FANCM as a breast cancer susceptibility gene, mutations in which confer a particularly strong predisposition for TNBC.Breast cancer is the most common cancer affecting women worldwide. It is also the principal cause of death from cancer among women globally, accounting for 14% of all cancer deaths (1). The etiology of breast cancer is multifactorial, and the risk depends on various factors like age, family history, and reproductive, hormonal, or dietary factors. The majority of breast cancers are sporadic, but approximately 15% of cases show familial aggregation (2, 3). Since the identification of the first breast and ovarian cancer susceptibility genes breast cancer 1 and 2 (BRCA1 and BRCA2, respectively) by linkage analysis and positional cloning, several breast cancer susceptibility genes and alleles with different levels of risk and prevalence in the population have been recognized. BRCA1 and BRCA2 mutation carriers have more than 10-fold increased risk of breast cancer compared with women in the general population, and mutations in TP53, PTEN, STK11, and CDH1 have also been associated with a high lifetime risk of breast cancer in the context of rare inherited cancer syndromes (4). In addition, rare variants in genes such as checkpoint kinase 2 (CHEK2), ataxia telangiectasia mutated (ATM), and BRCA1 interacting helicase BRIP1, that confer a two- to fourfold increased risk, and in partner and localizer of BRCA2 (PALB2), with even higher risk estimates, have been found with candidate gene approaches (5, 6), and an increasing number of common low-risk loci with modest odds ratios (ORs; as much as 1.26-fold increased risk for heterozygous carriers) have been identified by genome-wide association studies (7).However, the major portion of hereditary breast cancer still remains unexplained, and many susceptibility loci are yet to be found. Exome sequencing combined with genotyping of the identified variants in case-control analysis is an effective method to recognize novel risk alleles, based on the assumption that disease-causing variants are rare and often accumulate in the protein-coding areas of the genome (8–10).Since the discovery that proteins encoded by the BRCA1 and BRCA2 breast/ovarian cancer susceptibility genes are directly involved in homologous recombination repair of DNA double-strand breaks, it has been evident that other genes involved in DNA repair are attractive breast cancer susceptibility candidates (4). Biallelic mutations in ATM gene cause rare ataxia telangiectasia disease and are associated with an increased risk for breast cancer as a result of improper DNA damage response (11). Fanconi anemia (FA) is a rare genetic disorder caused by biallelic mutations in FA genes that also participate in DNA repair. At least 15 FA genes have been identified (12). Patients with heterozygous mutations in certain FA genes have an elevated risk for various cancers, and monoallelic mutations in at least four of these genes [BRCA2, BRIP1, PALB2, and RAD51 paralog C (RAD51C)] are associated with an increased risk of breast or ovarian cancer (12, 13). Recurrent founder mutations in several cancer susceptibility genes, including the BRCA2, PALB2, and RAD51C FA genes, have been identified in the Finnish population (14–16). The PALB2 and RAD51C founder mutations have been detected at 2% frequency in Finnish breast or ovarian cancer families (15–17), whereas, in other populations, mutations in these genes are rare and often unique for each family. Founder effects in the isolated populations such as Finland or Iceland may enrich certain mutations and thus explain a significant proportion of all mutations in certain genes (18, 19). This provides an advantage in the search for novel susceptibility genes and alleles.In this study, we used exome sequencing to uncover previously unidentified recurrent breast or ovarian cancer predisposing variants in the Finnish population with a focus on DNA repair genes. Selected variants were further genotyped in a large case-control sample set. Our investigation revealed an association of a nonsense mutation (rs147021911) in an FA complementation gene, FANCM, with breast cancer, especially with triple-negative (TN) breast cancer (TNBC).     

Synthetic lethality between CCNE1 amplification and loss of BRCA1

High-grade serous ovarian cancers (HGSCs) are characterized by a high frequency of TP53 mutations, BRCA1/2 inactivation, homologous recombination dysfunction, and widespread copy number changes. Cyclin E1 (CCNE1) gene amplification has been reported to occur independently of BRCA1/2 mutation, and it is associated with primary treatment failure and reduced patient survival. Insensitivity of CCNE1-amplified tumors to platinum cross-linking agents may be partly because of an intact BRCA1/2 pathway. Both BRCA1/2 dysfunction and CCNE1 amplification are known to promote genomic instability and tumor progression. These events may be mutually exclusive, because either change provides a path to tumor development, with no selective advantage to having both mutations. Using data from a genome-wide shRNA synthetic lethal screen, we show that BRCA1 and members of the ubiquitin pathway are selectively required in cancers that harbor CCNE1 amplification. Furthermore, we show specific sensitivity of CCNE1-amplified tumor cells to the proteasome inhibitor bortezomib. These findings provide an explanation for the observed mutual exclusivity of CCNE1 amplification and BRCA1/2 loss in HGSC and suggest a unique therapeutic approach for treatment-resistant CCNE1-amplified tumors.Epithelial ovarian cancer is complex and histologically diverse but still largely treated as a single disease with limited stratification based on histological or molecular characteristics. High-grade serous ovarian cancer (HGSC) accounts for the majority of epithelial ovarian cancer-related deaths (>60%), and almost no improvement in survival has been observed in the last 20 y (1). Widespread copy number changes are a hallmark of HGSC, including focal amplification of Cyclin E1 (encoded by CCNE1), which is associated with primary treatment failure (2) and reduced survival (3). Amplification of CCNE1 is one of very few well-defined molecular targets in HGSC.Cyclin E1 forms a complex with cyclin-dependent kinase 2 (CDK2) to regulate G1/S transition as well as having kinase-independent functions, including in DNA replication (4). Ovarian cell lines with CCNE1 amplification show a specific dependency for maintenance of CCNE1 expression (5, 6). We have validated CDK2 as a therapeutic target by showing selective sensitivity to suppression either by gene knockdown or using small molecule inhibitors (7), consistent with findings in breast cancer (8).Recent genomic studies have revealed a high frequency of BRCA1/2 (Breast cancer 1/2, early onset) inactivation and homologous recombination (HR) dysfunction in HGSC (9). Alterations of genes in the HR pathway include germ-line and somatic mutations of BRCA1 or BRCA2 (∼20% of patients) and epigenetic silencing of BRCA1 by hypermethylation (∼10%). Other genes inactivated by deletion, mutation, or hypermethylation include ATM, ATR, RAD51C, and PTEN (∼10%), key Fanconi anemia members (∼5%), and amplification or mutation of EMSY (∼8%). Collectively, at least 50% of HGSCs are thought to have HR pathway defects (9).Approximately 30% of HGSC tumors have alterations in the Rb pathway or genes involved in Rb-mediated DNA repair and cell cycle control, including amplification of CCNE1 (∼20%), loss of RB1 (∼10%), or gain of RBBP8 (∼4%) (10). Strikingly, activation of the RB1/CCNE1 pathway is largely exclusive of BRCA1/2 mutation for reasons that are unclear (9, 10). Both BRCA1/2 dysfunction and CCNE1 amplification are known to promote genomic instability and tumor progression (4, 11); therefore, they may be mutually exclusive, because either change provides a path to tumor development, with no selective advantage to having both mutations (10). Insensitivity of CCNE1-amplified tumors to platinum cross-linking agents may be partly because of an intact BRCA1/2 pathway, suggesting that these patients are unlikely to respond to poly-ADP-ribose polymerase (PARP) inhibitors.Here, we show that BRCA1 and members of the ubiquitin pathway are selectively required in cancers that harbor CCNE1 amplifications. Furthermore, we show specific sensitivity of CCNE1-amplified tumor cells to the proteasome inhibitor bortezomib. These findings provide an explanation for the observed mutual exclusivity of CCNE1 amplification and BRCA1/2 loss in HGSCs and suggest a unique therapeutic approach for treatment-resistant CCNE1-amplified tumors.     

Targeting RPL39 and MLF2 reduces tumor initiation and metastasis in breast cancer by inhibiting nitric oxide synthase signaling

We previously described a gene signature for breast cancer stem cells (BCSCs) derived from patient biopsies. Selective shRNA knockdown identified ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2) as the top candidates that affect BCSC self-renewal. Knockdown of RPL39 and MLF2 by specific siRNA nanoparticles in patient-derived and human cancer xenografts reduced tumor volume and lung metastases with a concomitant decrease in BCSCs. RNA deep sequencing identified damaging mutations in both genes. These mutations were confirmed in patient lung metastases (n = 53) and were statistically associated with shorter median time to pulmonary metastasis. Both genes affect the nitric oxide synthase pathway and are altered by hypoxia. These findings support that extensive tumor heterogeneity exists within primary cancers; distinct subpopulations associated with stem-like properties have increased metastatic potential.Large-scale sequencing analyses of solid cancers have identified extensive tumor heterogeneity within individual primary cancers (1). Recent studies indicate that such tumoral heterogeneity is associated with heterogeneous protein function, which fosters tumor adaptation, treatment resistance, and failure through Darwinian selection (2–4). Cancer stem cells are a subpopulation of cells within the primary tumor responsible for tumor initiation and metastases (5–9). Three groups have recently independently provided functional evidence for the presence of cancer stem cells by lineage-tracing experiments (10–12). These observations suggest that these subpopulations of cancer stem cells (CSCs) within the bulk primary tumor are resistant to conventional therapies through different adaptive mechanisms with the potential for self-renewal and metastases (7, 13, 14). However, few studies have determined the genetic profile of the cells that escape the primary cancer and evolve in distant metastatic sites (1). Additionally, no large-scale sequencing studies of metastases have been conducted because the majority of patients are treated with systemic therapies and not surgery.Tumor clonal heterogeneity within a primary tumor may in part be explained by hypoxic regions within the bulk tumor that have been correlated with invasiveness, therapeutic resistance, and metastasis (15–18). Cancer stem cells have been found to reside near hypoxic regions in some solid cancers (19–21). We have previously published a 477-gene tumorigenic signature by isolating breast cancer stem cells (BCSCs) derived from patient biopsies (22). Here, we have identified two previously unidentified cancer genes, ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2), by selective shRNA knockdown of genes from this tumorigenic signature, that impact breast cancer stem cell self-renewal and lung metastases. Analysis of 53 patient lung metastases confirmed damaging mutations in RPL39 and MLF2 in a significant number of samples, which conferred a gain-of-function phenotype. These mutations were statistically associated with shorter median time to distant relapse. We further describe a common mechanism of action through nitric oxide synthase signaling that is regulated by hypoxia.     

ECHIDNA-mediated post-Golgi trafficking of auxin carriers for differential cell elongation

The plant hormone indole-acetic acid (auxin) is essential for many aspects of plant development. Auxin-mediated growth regulation typically involves the establishment of an auxin concentration gradient mediated by polarly localized auxin transporters. The localization of auxin carriers and their amount at the plasma membrane are controlled by membrane trafficking processes such as secretion, endocytosis, and recycling. In contrast to endocytosis or recycling, how the secretory pathway mediates the localization of auxin carriers is not well understood. In this study we have used the differential cell elongation process during apical hook development to elucidate the mechanisms underlying the post-Golgi trafficking of auxin carriers in Arabidopsis. We show that differential cell elongation during apical hook development is defective in Arabidopsis mutant echidna (ech). ECH protein is required for the trans-Golgi network (TGN)–mediated trafficking of the auxin influx carrier AUX1 to the plasma membrane. In contrast, ech mutation only marginally perturbs the trafficking of the highly related auxin influx carrier LIKE-AUX1-3 or the auxin efflux carrier PIN-FORMED-3, both also involved in hook development. Electron tomography reveals that the trafficking defects in ech mutant are associated with the perturbation of secretory vesicle genesis from the TGN. Our results identify differential mechanisms for the post-Golgi trafficking of de novo-synthesized auxin carriers to plasma membrane from the TGN and reveal how trafficking of auxin influx carriers mediates the control of differential cell elongation in apical hook development.Polar auxin transport (PAT) plays a key role in plant development (1–5). PAT is mediated by plasma membrane localized auxin influx and efflux carriers of the auxin-resistant (AUX)/like-AUX (LAX), pin-formed (PIN), and ABCB families (6–12). Highly regulated tissue, cellular localization, and amount of auxin carriers at the plasma membrane (PM) provide directionality to the auxin transport and underlies the creation of auxin concentration gradient that is essential for controlling several aspects of plant development (13–18). One of the developmental programs in which auxin concentration gradient plays a central role is the formation of apical hook, a bending in the embryonic stem during early seedling germination (19). Hook formation involves differential elongation of cells on the two opposite sides of the hypocotyl. This process is mediated by the formation of an auxin maximum at the concave side of the hook, leading to the inhibition of cell elongation (20–25). A model based on mutational analysis shows that auxin carriers including polarly localized auxin efflux and influx facilitators PIN3 and AUX1/LAX3, respectively, are important for hook development (23, 24). The amount of auxin carriers at the PM is important for the regulation of auxin concentration, and this depends on the balance between secretion, endocytosis, and recycling. The analysis of PIN efflux carriers has revealed how cell wall anchoring, endocytosis, targeted degradation, and also posttranslational modifications strongly influence the location and amount of these carriers at the PM (15, 17, 26–29). In contrast, little is known about the mechanisms and molecular components underlying the deposition of auxin carriers at the PM. Post-Golgi secretion to the PM occurs via the trans-Golgi network (TGN), a post-Golgi compartment (30). The TGN is a complex tubulo-vesicular membrane network maturing from the trans-most cisternae of the Golgi apparatus to become a highly dynamic independent structure from which secretory vesicles (SVs) and CLATHRIN-coated vesicles (CCVs) originate (31–34). Although auxin carriers traffic via TGN, components and mechanisms specifically involved in trafficking to the PM of de novo-synthesized auxin carriers remain largely undefined (35, 36). Importantly, it is not known whether auxin carriers traffic through SV or CCV sites of the TGN on their way to the PM. We have used apical hook development as a model system to investigate the mechanisms that link post-Golgi trafficking of auxin carriers to the PM with control of differential cell elongation. We previously identified the transmembrane TGN-localized protein ECHIDNA (ECH) that is required for cell elongation (37). We discovered that the ech mutant is defective in hook development and is insensitive to ethylene like the aux1 mutant. These data prompted us to investigate the role of ECH and the TGN in post-Golgi trafficking of auxin carriers during hook development. Using genetic, pharmacological, and cell biological approaches, we show that distinct mechanisms/components underlie post-Golgi trafficking of influx and efflux carriers. We show that post-Golgi trafficking of de novo-synthesized AUX1 occurs via an ECH-dependent SV-based pathway, whereas that of PIN3 and LAX3 are largely independent of ECH at the TGN. Thus, these results reveal the complexity of trafficking from the TGN to PM as shown by the differential trafficking of influx carriers AUX1 versus LAX3 and the efflux carrier PIN3. Hence, our results reveal an additional layer of regulatory control to auxin transport.     

Novel recurrently mutated genes in African American colon cancers

We used whole-exome and targeted sequencing to characterize somatic mutations in 103 colorectal cancers (CRC) from African Americans, identifying 20 new genes as significantly mutated in CRC. Resequencing 129 Caucasian derived CRCs confirmed a 15-gene set as a preferential target for mutations in African American CRCs. Two predominant genes, ephrin type A receptor 6 (EPHA6) and folliculin (FLCN), with mutations exclusive to African American CRCs, are by genetic and biological criteria highly likely African American CRC driver genes. These previously unsuspected differences in the mutational landscapes of CRCs arising among individuals of different ethnicities have potential to impact on broader disparities in cancer behaviors.Colorectal cancer (CRC) is a leading cause of cancer mortality world-wide. CRC incidence and mortality rates are both increased in African Americans (AA) compared with Caucasians Americans (1–3). Although several factors likely play a role, the contribution of potential differences in tumor genetics to this disparity have yet to be fully explored (1, 3). In particular, AA CRCs were notably underrepresented in the four major published CRC sequencing studies (4–7), accounting for only two annotated AA cases of the 333 total CRCs studied (4–7). Accordingly, we initiated this study to compare the mutational landscapes of CRCs from AA individuals versus Caucasians.     

UbcH7 regulates 53BP1 stability and DSB repair

DNA double-strand break (DSB) repair is not only key to genome stability but is also an important anticancer target. Through an shRNA library-based screening, we identified ubiquitin-conjugating enzyme H7 (UbcH7, also known as Ube2L3), a ubiquitin E2 enzyme, as a critical player in DSB repair. UbcH7 regulates both the steady-state and replicative stress-induced ubiquitination and proteasome-dependent degradation of the tumor suppressor p53-binding protein 1 (53BP1). Phosphorylation of 53BP1 at the N terminus is involved in the replicative stress-induced 53BP1 degradation. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end resection. Therefore, UbcH7-depleted cells display increased nonhomologous end-joining and reduced homologous recombination for DSB repair. Accordingly, UbcH7-depleted cells are sensitive to DNA damage likely because they mainly used the error-prone nonhomologous end-joining pathway to repair DSBs. Our studies reveal a novel layer of regulation of the DSB repair choice and propose an innovative approach to enhance the effect of radiotherapy or chemotherapy through stabilizing 53BP1.Prompt response to double-strand breaks (DSBs) caused by, for example, ionization radiation (IR), requires sequential and coordinated assembly of DNA damage response (DDR) proteins at damage sites (1). Recent research findings reveal key roles of the tumor suppressor p53-binding protein 1 (53BP1) and BRCA1 in the decision making of DSB repair. 53BP1, together with Rif1, suppress BRCA1-dependent homologous recombination (HR), thereby promoting nonhomologous end-joining (NHEJ) in G1 phase (2–6). Conversely, BRCA1 antagonizes 53BP1/Rif1, favoring HR in S and G2 phases (7, 8). In the absence of BRCA1 or with enhanced retention of 53BP1 at DSB sites, cells primarily use the error-prone NHEJ to repair DSBs throughout the cell cycle, which leads to gene rearrangement, cell death, and increased sensitivity to anticancer therapies (9–11). Consistently, BRCA1-null mice are early embryonic lethal (12, 13) and codepletion of TP53BP1 rescued the lethality phenotype of BRCA1-null mice (12–14).Low expression level of 53BP1 was found to be associated with poor clinical outcome in triple negative breast cancer patients with BRCA1 mutation (12, 15), as well as resistance to genotoxins and poly(ADP-ribose) polymerase inhibitors (12, 16, 17). This finding is probably because loss of 53BP1 restored HR and promoted cell survival (12–14). Reduced expression of 53BP1 was also observed in tumors from the brain (18), lymph node (19), and pancreas (20). These data indicate that loss of 53BP1 might be a common mechanism for advanced tumors to evade from radiotherapy or chemotherapy. However, molecular mechanisms controlling the protein level of 53BP1 remain less well understood.Here we show that UbcH7, an E2 enzyme involved in the ubiquitin (Ub) pathway, controls the protein stability of 53BP1, thereby determining the DSB repair choice. Loss of UbcH7 stabilizes 53BP1, forcing cells to choose NHEJ, but not HR, to repair DSBs, which poses a significant threat to cells treated with DNA damage, especially S-phase genotoxins, such as camptothecin (CPT), a topoisomerase 1 (Top1) inhibitor. The ternary CPT-Top1-DNA complex places a roadblock in the path of advancing DNA replication forks, leading to replication fork collapse and generation of one-ended DSBs. Such one-ended DSBs require HR, but not NHEJ, to repair (8). In contrast, repair of one-ended DSBs by NHEJ leads to radial chromosomes and cell death (12–14). Therefore, stabilization of 53BP1 by UbcH7 depletion increased the sensitivity of cancers cells to CPT and other DNA damaging agents. Our data suggest a novel strategy in enhancing the anticancer effect of radiotherapy or chemotherapy through stabilizing or increasing 53BP1.     

Double-strand break repair by homologous recombination in primary mouse somatic cells requires BRCA1 but not the ATM kinase

Homology-directed repair (HDR) is a critical pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. Efficient HDR is thought to be crucial for maintenance of genomic integrity during organismal development and tumor suppression. However, most mammalian HDR studies have focused on transformed and immortalized cell lines. We report here the generation of a Direct Repeat (DR)-GFP reporter-based mouse model to study HDR in primary cell types derived from diverse lineages. Embryonic and adult fibroblasts from these mice as well as cells derived from mammary epithelium, ovary, and neonatal brain were observed to undergo HDR at I-SceI endonuclease-induced DSBs at similar frequencies. When the DR-GFP reporter was crossed into mice carrying a hypomorphic mutation in the breast cancer susceptibility gene Brca1, a significant reduction in HDR was detected, showing that BRCA1 is critical for HDR in somatic cell types. Consistent with an HDR defect, Brca1 mutant mice are highly sensitive to the cross-linking agent mitomycin C. By contrast, loss of the DSB signaling ataxia telangiectasia-mutated (ATM) kinase did not significantly alter HDR levels, indicating that ATM is dispensable for HDR. Notably, chemical inhibition of ATM interfered with HDR. The DR-GFP mouse provides a powerful tool for dissecting the genetic requirements of HDR in a diverse array of somatic cell types in a normal, nontransformed cellular milieu.DNA damage poses a threat to genomic integrity and must be repaired in an accurate and timely manner for the health and survival of the organism. A particularly cytotoxic lesion is a chromosomal double-strand break (DSB), which can arise from endogenous sources, including DNA replication and antigen receptor rearrangements in lymphocytes, as well as exogenous sources, such as ionizing radiation (IR) (1, 2). DSBs activate an elaborate cellular signaling network of proteins, a key component of which is the ataxia telangiectasia-mutated (ATM) protein kinase (3, 4). There are three major pathways for repairing DSBs in mammalian cells: (i) homology-directed repair (HDR), (ii) nonhomologous end joining (NHEJ), and (iii) single-strand annealing (SSA) (2, 5, 6). HDR, which is considered the most precise of the three repair pathways, uses a homologous donor sequence as a template for the repair event. Template preference is biased to the sister chromatid in mammalian cells, thus restoring the original sequence before damage. NHEJ refers to joining of ends without the use of extensive homology, and it is often accompanied by modification of the sequence surrounding the break site (1). SSA occurs when complementary strands from sequence repeats flanking the DSB anneal to each other, resulting in repair of the DSB but deletion of the intervening sequence (5). Consequently, both NHEJ and SSA are considered to be more error-prone than HDR. The importance of HDR to the organism is emphasized by the requirement for HDR factors for development, tumor suppression, and fertility (5).HDR was established as a DSB repair pathway in mammalian cells using genomic reporters into which a DSB is introduced by the I-SceI endonuclease (7–9). Repair of the DSB from the homologous template on the sister chromatid or the same chromatid leads to expression of a scoreable marker, such as GFP from the DR-GFP reporter (10). Use of such reporters has led to direct evidence for the role of several proteins in HDR, including the breast cancer suppressors BRCA1 and BRCA2 (11, 12). However, these studies have been performed in transformed cell lines and immortalized ES cells, raising questions as to their relevance to normal somatic cells. Some studies have suggested that DSB repair pathways are developmentally regulated, with HDR being critical during embryonic cell cycles and NHEJ in differentiated tissues (13). Furthermore, a critical protein in HDR, the RAD51 recombinase, has been reported to be overexpressed in tumor cell lines, suggesting that HDR levels are altered in transformed cells (14, 15).In the present study, we describe a mouse model to analyze the efficiency of HDR in primary somatic cell types. To this end, the DR-GFP reporter was targeted to a defined chromosomal locus in the mouse genome. We found that primary somatic cell types derived from different lineages from DR-GFP mice, including fibroblasts, brain (glial) cells, and mammary epithelial cells, undergo HDR at I-SceI–induced DSBs at similar frequencies. The frequency of HDR is lower in the tested somatic cell types compared with ES cells, which correlates with the lower fraction of cells in S phase in the more differentiated cell types. To examine the genetic requirements for HDR in somatic cells, we crossed the DR-GFP reporter into mice carrying mutations in the Brca1 and Atm genes. We found that BRCA1 is necessary for efficient HDR in somatic cells. By contrast, ATM is not required for HDR in primary fibroblasts, although chemical inhibition of ATM can interfere with HDR.     

Contingency and entrenchment in protein evolution under purifying selection

The phenotypic effect of an allele at one genetic site may depend on alleles at other sites, a phenomenon known as epistasis. Epistasis can profoundly influence the process of evolution in populations and shape the patterns of protein divergence across species. Whereas epistasis between adaptive substitutions has been studied extensively, relatively little is known about epistasis under purifying selection. Here we use computational models of thermodynamic stability in a ligand-binding protein to explore the structure of epistasis in simulations of protein sequence evolution. Even though the predicted effects on stability of random mutations are almost completely additive, the mutations that fix under purifying selection are enriched for epistasis. In particular, the mutations that fix are contingent on previous substitutions: Although nearly neutral at their time of fixation, these mutations would be deleterious in the absence of preceding substitutions. Conversely, substitutions under purifying selection are subsequently entrenched by epistasis with later substitutions: They become increasingly deleterious to revert over time. Our results imply that, even under purifying selection, protein sequence evolution is often contingent on history and so it cannot be predicted by the phenotypic effects of mutations assayed in the ancestral background.Whether a heritable mutation is advantageous or deleterious to an organism often depends on the evolutionary history of the population. A mutation that is beneficial at the time of its introduction may confer its beneficial effect only in the presence of other potentiating or permissive mutations (1–9). Thus, the fate of a mutation arising in a population may be contingent on previous mutations (10–13). Conversely, once a mutation has fixed in a population, the mutation becomes part of the genetic background onto which subsequent modifications are introduced. Because the beneficial effects of the subsequent modifications may depend on the focal mutation, as time passes reversion of the focal mutation may become increasingly deleterious, leading to a type of evolutionary conservatism, or entrenchment (14–18).In the context of protein evolution, the effects of contingency and entrenchment are most easily studied by considering a sequence of single amino acid changes (19) that extends both forward and backward in time from some focal substitution. To assess the roles of contingency and entrenchment we can study the degree to which each focal substitution was facilitated by previous substitutions, and the degree to which the focal substitution influences the subsequent course of evolution (Fig. 1A).Open in a separate windowFig. 1.(A) A schematic model indicating how a focal substitution may be contingent on prior substitutions and may constrain future substitutions along an evolutionary trajectory, owing to epistasis. (B) A model of protein evolution under weak mutation and purifying selection for thermodynamic stability. Starting from the wild-type sequence of argT we propose 10 random 1-aa point mutations. For each of the proposed mutants we compute its predicted stability (ΔG) using FoldX, and its associated fitness. The fitness function is assumed to be either Gaussian or semi-Gaussian, with a maximum at the wild-type stability. One of the proposed mutants fixes in the population, based on its relative fixation probability under the Moran model with effective population size N_e. This process is iterated for 30 consecutive substitutions to produce an evolutionary trajectory. We simulate 100 replicate trajectories, each initiated at the wild-type argT sequence.Dependencies within a sequence of substitutions are closely connected to the concept of epistasis—that is, the idea that the phenotypic effect of a mutation at a particular genetic site may depend on the genetic background in which it arises (20–24). In the absence of epistasis, a mutation has the same effect regardless of its context and therefore regardless of any prior history or subsequent evolution. By contrast, in the presence of epistasis, each substitution may be contingent on the entire prior history of the protein, and it may constrain all subsequent evolution.The potential for epistasis to play an important role in evolution, including protein evolution, has not been overlooked by researchers (1, 8, 25–34), nor have the concepts of contingency (3, 4, 9, 12, 35–38) and, more recently, entrenchment (18, 39, 40). However, most studies have addressed the role of epistasis in the context of adaptive evolution (1–9, 27, 30, 31, 36, 38), whereas the consequences of epistasis under purifying selection have received less attention (18, 41–44). Indeed, although some more sophisticated models have been proposed (e.g., refs. 45–50), all commonly used phylogenetic models of long-term protein evolution assume that epistasis is absent so that sites evolve independently (51–56).Here we explore the relationships between epistasis, contingency, and entrenchment under long-term purifying selection on protein stability. Our analysis combines computational models for protein structures with population-genetic models for evolutionary dynamics. We use a force-field-based model, FoldX (57), to characterize the effects of point mutations on a protein’s stability and fitness. This approach allows us to simulate evolutionary trajectories of protein sequences under purifying selection, by the sequential fixation of nearly neutral mutations. We can then dissect the epistatic relationships between these substitutions by systematically inserting or reverting particular substitutions at various time points along the evolutionary trajectory.Our analysis considers epistasis both at the level of protein stability and at the level of fitness. Whereas empirical studies in diverse proteins have demonstrated that the stability effects of point mutations are typically additive across sites (58, 59), in this study we are specifically interested in epistasis for stability among the mutations that fix during evolution. Even if most random mutations are virtually additive in their effects on stability, the mutations that fix under purifying selection are highly nonrandom, and so there is reason to suspect that epistasis for stability may be enriched among such mutations. Moreover, because the mapping from stability to fitness is itself nonlinear (18, 26, 60, 61) and because selection is sensitive to selection coefficients as small as the inverse of the population size (62), even slight variation in the stability effects of mutations across different genetic backgrounds may be sufficient to influence the course of evolution.Using the computational approach summarized above, we will demonstrate that the nearly neutral mutations that fix under purifying selection are, indeed, often epistatic with each other for both stability and fitness. In particular, we find that each mutation that fixes is typically permitted to fix by the presence of preceding substitutions—that is, most substitutions would be too deleterious to fix were it not for epistasis with preceding substitutions. Conversely, we also find that mutations that fix typically become entrenched over time by epistasis—so that a substitution that was nearly neutral when it fixed becomes increasingly deleterious to revert as subsequent substitutions accumulate (18, 39). These results imply an important role for epistasis in shaping the course of sequence evolution in a protein under selection to maintain thermodynamic stability.     

CHK2–BRCA1 tumor-suppressor axis restrains oncogenic Aurora-A kinase to ensure proper mitotic microtubule assembly

BRCA1 (breast cancer type 1 susceptibility protein) is a multifunctional tumor suppressor involved in DNA damage response, DNA repair, chromatin regulation, and mitotic chromosome segregation. Although the nuclear functions of BRCA1 have been investigated in detail, its role during mitosis is little understood. It is clear, however, that loss of BRCA1 in human cancer cells leads to chromosomal instability (CIN), which is defined as a perpetual gain or loss of whole chromosomes during mitosis. Moreover, our recent work has revealed that the mitotic function of BRCA1 depends on its phosphorylation by the tumor-suppressor kinase Chk2 (checkpoint kinase 2) and that this regulation is required to ensure normal microtubule plus end assembly rates within mitotic spindles. Intriguingly, loss of the positive regulation of BRCA1 leads to increased oncogenic Aurora-A activity, which acts as a mediator for abnormal mitotic microtubule assembly resulting in chromosome missegregation and CIN. However, how the CHK2–BRCA1 tumor suppressor axis restrains oncogenic Aurora-A during mitosis to ensure karyotype stability remained an open question. Here we uncover a dual molecular mechanism by which the CHK2–BRCA1 axis restrains oncogenic Aurora-A activity during mitosis and identify BRCA1 itself as a target for Aurora-A relevant for CIN. In fact, Chk2-mediated phosphorylation of BRCA1 is required to recruit the PP6C–SAPS3 phosphatase, which acts as a T-loop phosphatase inhibiting Aurora-A bound to BRCA1. Consequently, loss of CHK2 or PP6C-SAPS3 promotes Aurora-A activity associated with BRCA1 in mitosis. Aurora-A, in turn, then phosphorylates BRCA1 itself, thereby inhibiting the mitotic function of BRCA1 and promoting mitotic microtubule assembly, chromosome missegregation, and CIN.Breast cancer type 1 susceptibility protein (BRCA1) is a major and multifunctional tumor-suppressor protein involved in the regulation of chromatin organization, gene expression, DNA damage response, and DNA repair (1–3). In addition to its functions in interphase, BRCA1 also is required for faithful execution of mitosis. Consequently, loss of BRCA1 results in abnormal mitotic progression, chromosome missegregation, and chromosomal instability (CIN), hallmark phenotypes of human cancer (4–6). However, the mitotic function of BRCA1 and its regulation during mitosis is little understood. BRCA1 localizes to centrosomes throughout the cell cycle and is phosphorylated during mitosis on S988 by the checkpoint kinase 2 (Chk2). This positive regulation is essential to ensure proper mitotic spindle assembly and chromosomal stability (5, 7, 8). Importantly, we recently have found that the loss of CHK2 or of Chk2-mediated phosphorylation on BRCA1 causes an increase in microtubule plus-end assembly rates, which results in transient spindle geometry defects facilitating the generation of erroneous microtubule–kinetochore attachments. These defects finally lead to chromosome missegregation and the induction of aneuploidy, thus constituting CIN (6).Interestingly, BRCA1 associates with the Aurora-A kinase during mitosis (6). Aurora-A is a key mitotic kinase encoded by the AURKA oncogene and is involved in various functions during mitosis, including centrosome separation and spindle assembly (9, 10). Aurora-A activation occurs at mitotic centrosomes and requires its autophosphorylation within the T-loop at threonine-288. Subsequent inactivation of Aurora-A can be mediated by the serine/threonine protein phosphatase 6 (PP6), which acts as the T-loop phosphatase for Aurora-A, and involves the catalytic subunit (PP6C) as well as regulatory subunits referred to as “Sit4-associated proteins” (SAPS1-3) and ankyrin repeat proteins (11–13). Significantly, in human cancer, AURKA frequently is overexpressed, and increased activity of AURKA is sufficient to induce enhanced microtubule plus end assembly rates, chromosome missegregation, and CIN (6). Thus, overexpression of AURKA and loss of the CHK2–BRCA1 axis result in the same mitotic abnormalities triggering CIN. Moreover, loss of the Chk2-mediated phosphorylation of BRCA1 causes an increase in BRCA1-bound Aurora-A kinase activity at mitotic centrosomes, which mediates the induction of abnormal microtubule plus end dynamics and CIN (6). However, the molecular mechanism by which the loss of the CHK2–BRCA1 tumor-suppressor axis unleashes oncogenic Aurora-A activity during mitosis and the mitotic target for Aurora-A relevant for the induction of increased microtubule dynamics and CIN remain unknown.     

Heterozygous colon cancer-associated mutations of SAMHD1 have functional significance

Even small variations in dNTP concentrations decrease DNA replication fidelity, and this observation prompted us to analyze genomic cancer data for mutations in enzymes involved in dNTP metabolism. We found that sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), a deoxyribonucleoside triphosphate triphosphohydrolase that decreases dNTP pools, is frequently mutated in colon cancers, that these mutations negatively affect SAMHD1 activity, and that several SAMHD1 mutations are found in tumors with defective mismatch repair. We show that minor changes in dNTP pools in combination with inactivated mismatch repair dramatically increase mutation rates. Determination of dNTP pools in mouse embryos revealed that inactivation of one SAMHD1 allele is sufficient to elevate dNTP pools. These observations suggest that heterozygous cancer-associated SAMHD1 mutations increase mutation rates in cancer cells.Recent advances in whole-genome sequencing have revealed that human cancers often contain thousands of subclonal mutations (1–3), lending support to the mutator phenotype hypothesis that postulates that an elevation in spontaneous mutation rate is an early step in cancer evolution (4, 5). The three major determinants of DNA replication fidelity that control the spontaneous mutation rate are nucleotide selectivity by DNA polymerases, proofreading by replicative DNA polymerases, and the mismatch repair (MMR) system (6). Failures in the two latter determinants have now been firmly associated with the development of cancer (7), but they cannot account for the increased spontaneous mutation rates in most cancers (5).The first determinant, nucleotide selectivity by DNA polymerases, can be affected by changes in the absolute and relative concentrations of the four deoxyribonucleoside triphosphates (dNTPs). We have previously demonstrated that severely imbalanced dNTP pools strongly decrease DNA replication fidelity in Saccharomyces cerevisiae (8, 9) without affecting cell proliferation, as long as none of the dNTPs is limiting for DNA replication (10). An equimolar elevation in dNTP pools also decreases DNA replication fidelity, both in yeast and bacteria, presumably by suppressing the proofreading activity of replicative DNA polymerases and by stimulating lesion bypass by both replicative and translesion DNA polymerases (11–16). Recently, we showed in yeast that even a small elevation of the dNTP pool dramatically decreases the replication fidelity of exonuclease-deficient DNA polymerase ε (Pol ε) and DNA polymerase δ (Pol δ) harboring the cancer-associated R696W mutation (17–19). Based on these observations, we hypothesized that decreased nucleotide selectivity caused by changes in the absolute or relative concentrations of dNTPs could be one of the reasons for the increased mutation rates in cancers.The absolute and relative concentrations of dNTPs are controlled by several dozen proteins (20), and mutations or a change in abundance in any of these could in principle result in a distortion of the dNTP pool. Ribonucleotide reductase (RNR), dCMP deaminase, dUTPase, dTMP synthase, dTMP kinase, and NDP kinases control dNTP biosynthesis. Purine and pyrimidine de novo synthesis pathways provide substrates for RNR, and multiple (deoxy)nucleoside kinases and 5′ nucleotidases control cellular and mitochondrial dNTP salvage. We analyzed the mutation status of the genes involved in dNTP metabolism in colon cancers using a public dataset from The Cancer Genome Atlas (TCGA) and identified SAMHD1 (sterile alpha motif and histidine-aspartate domain-containing protein 1) as one of the frequently mutated genes.SAMHD1 is a dual-function enzyme with both nuclease and deoxyribonucleoside triphosphate triphosphohydrolase (dNTPase) activities (21, 22). Germ-line mutations in SAMHD1 have been associated with Aicardi–Goutieres syndrome, a congenital autoimmune disease (23), and more recently SAMHD1 was shown to be an HIV-1 restriction factor operating in nondividing blood cells (24, 25). Initially, the restriction function of SAMHD1 was attributed to its dNTPase activity, which was presumed to decrease the intracellular dNTP concentrations to levels incompatible with viral replication (26). Later, it was suggested that restriction of HIV-1 was primarily caused by the nuclease activity of SAMHD1 degrading viral RNA (27). However, more recently it was proposed that SAMHD1 lacks nuclease activity altogether and that the restriction of HIV-1 is caused by alternating ssRNA-binding and dNTPase activities (28). Franzolin et al. showed that SAMHD1 is expressed in a cell cycle-regulated manner and that loss of SAMHD1 has large effects on dNTP pool composition in vitro in both quiescent and cycling cells (29). SAMHD1 has also been identified as a potential driver gene in chronic lymphatic leukemia, where it is recurrently mutated in early stages of tumor development (30–32). In solid tumors, lower protein and RNA expression of SAMHD1 has been observed, presumably caused by promoter methylations (30, 33, 34). However, it is unknown whether SAMHD1 somatic mutations found in cancers affect its dNTPase activity (35).Here, we show that colon cancer-associated mutations in SAMHD1 either abolish its dNTPase activity or change its specificity, leading to unequal degradation of individual dNTPs. Importantly, even a hemizygous deletion of SAMHD1 leads to an increase of dNTP pools in mouse embryos, which, similar to tumors, contain actively dividing cells. This result suggests that, although the SAMHD1 mutations identified in cancers are heterozygous, they would still result in an alteration of dNTP pools. Interestingly, several of the identified SAMHD1 mutations were present in MMR-deficient hypermutated cancers. Analysis of MMR-deficient yeast strains and human colorectal carcinoma cells demonstrated that even a small alteration of dNTP pools results in a multiplicative increase of mutation rates. Together, these findings suggest that mutations affecting the activity of SAMHD1 are likely to result in increased mutation rates in cancer.