A Yeast-Based Repurposing Approach for the Treatment of Mitochondrial DNA Depletion Syndromes Led to the Identification of Molecules Able to Modulate the dNTP Pool

Mitochondrial DNA depletion syndromes (MDS) are clinically heterogenous and often severe diseases, characterized by a reduction of the number of copies of mitochondrial DNA (mtDNA) in affected tissues. In the context of MDS, yeast has proved to be both an excellent model for the study of the mechanisms underlying mitochondrial pathologies and for the discovery of new therapies via high-throughput assays. Among the several genes involved in MDS, it has been shown that recessive mutations in MPV17 cause a hepatocerebral form of MDS and Navajo neurohepatopathy. MPV17 encodes a non selective channel in the inner mitochondrial membrane, but its physiological role and the nature of its cargo remains elusive. In this study we identify ten drugs active against MPV17 disorder, modelled in yeast using the homologous gene SYM1. All ten of the identified molecules cause a concomitant increase of both the mitochondrial deoxyribonucleoside triphosphate (mtdNTP) pool and mtDNA stability, which suggests that the reduced availability of DNA synthesis precursors is the cause for the mtDNA deletion and depletion associated with Sym1 deficiency. We finally evaluated the effect of these molecules on mtDNA stability in two other MDS yeast models, extending the potential use of these drugs to a wider range of MDS patients.

Nucleotide Pool Imbalance and Antibody Gene Diversification

The availability and adequate balance of deoxyribonucleoside triphosphate (dNTP) is an important determinant of both the fidelity and the processivity of DNA polymerases. Therefore, maintaining an optimal balance of the dNTP pool is critical for genomic stability in replicating and quiescent cells. Since DNA synthesis is required not only in genomic replication but also in DNA damage repair and recombination, the abnormalities in the dNTP pool affect a wide range of chromosomal activities. The generation of antibody diversity relies on antigen-independent V(D)J recombination, as well as antigen-dependent somatic hypermutation and class switch recombination. These processes involve diverse sets of DNA polymerases, which are affected by the dNTP pool imbalances. This review discusses the role of the optimal dNTP pool balance in the diversification of antibody encoding genes.

SAMHD1 in cancer: curse or cure?

Human sterile α motif and HD domain-containing protein 1 (SAMHD1), originally described as the major cellular deoxyribonucleoside triphosphate triphosphohydrolase (dNTPase) balancing the intracellular deoxynucleotide (dNTP) pool, has come recently into focus of cancer research. As outlined in this review, SAMHD1 has been reported to be mutated in a variety of cancer types and the expression of SAMHD1 is dysregulated in many cancers. Therefore, SAMHD1 is regarded as a tumor suppressor in certain tumors. Moreover, it has been proposed that SAMHD1 might fulfill the requirements of a driver gene in tumor development or might promote a so-called mutator phenotype. Besides its role as a dNTPase, several novel cellular functions of SAMHD1 have come to light only recently, including a role as negative regulator of innate immune responses and as facilitator of DNA end resection during DNA replication and repair. Therefore, SAMHD1 can be placed at the crossroads of various cellular processes. The present review summarizes the negative role of SAMHD1 in chemotherapy sensitivity, highlights reported SAMHD1 mutations found in various cancer types, and aims to discuss functional consequences as well as underlying mechanisms of SAMHD1 dysregulation potentially involved in cancer development.

SAMHD1 Is a Modulator of Nucleos(t)ide Analogues’ Efficacy

Nucleos(t)ide analogues are commonly used in the treatment of infectious disease and cancer. SAMHD1 is a deoxyribonucleotide (dNTP) triphosphohydrolase which is involved in the regulation of the intracellular dNTP pool, whose function has been linked to viral restriction, cancer development, and autoimmune disorders. Here, we evaluate SAMHD1 function on the antiviral and antiproliferative efficacy of a wide range of nucleos(t)ide analogues which are currently used to treat infections and cancer. The anti-HIV-1 and cytotoxic activity of compounds was assessed in primary and established cell lines in the presence or absence of SAMHD1. SAMHD1 effectively modified the anti-HIV-1 activity of all the nucleos(t)ide analogues tested, whereas sensitivity to a non-nucleoside inhibitor (nevirapine) or nucleoside phosphonates (cidofovir and tenofovir) was not affected. Interestingly, SAMHD1 could either enhance (gemcitabine, capecitabine, fluorouracil, and floxuridine) or inhibit (Ara-C, fludarabine, cladribine, clofarabine, and nelarabine) the antiviral potency of anticancer analogues, an effect that was not dependent on the specific nucleotide targeted. When cytotoxicity was evaluated, SAMHD1-dependent changes were less evident and were restricted to the increased efficacy of fluorouracil and floxuridine and reduced efficacy of nelarabine and ara-C in the presence of SAMHD1. In summary, our results demonstrate that SAMHD1 modifies the efficacy of a wide variety of nucleoside analogues which are used to treat infections, cancer, and other diseases. In addition, the anti-HIV activity of nucleos(t)ide analogues may represent a more sensitive measure of SAMHD1’s impact on drug efficacy. Thus, modulation of SAMHD1’s function may constitute a promising target for the improvement of multiple therapies.

Pharmacological Modulation of SAMHD1 Activity by CDK4/6 Inhibitors Improves Anticancer Therapy

Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1) is a dNTP triphosphohydrolase involved in the regulation of the intracellular dNTP pool, linked to viral restriction, cancer development and autoimmune disorders. SAMHD1 function is regulated by phosphorylation through a mechanism controlled by cyclin-dependent kinases and tightly linked to cell cycle progression. Recently, SAMHD1 has been shown to decrease the efficacy of nucleotide analogs used as chemotherapeutic drugs. Here, we demonstrate that SAMHD1 can enhance or decrease the efficacy of various classes of anticancer drug, including nucleotide analogues, but also anti-folate drugs and CDK inhibitors. Importantly, we show that selective CDK4/6 inhibitors are pharmacological activators of SAMHD1 that act by inhibiting its inactivation by phosphorylation. Combinations of a CDK4/6 inhibitor with nucleoside or folate antimetabolites potently enhanced drug efficacy, resulting in highly synergic drug combinations (CI < 0.04). Mechanistic analyses reveal that cell cycle-controlled modulation of SAMHD1 function is the central process explaining changes in anticancer drug efficacy, therefore providing functional proof of the potential of CDK4/6 inhibitors as a new class of adjuvants to boost chemotherapeutic regimens. The evaluation of SAMHD1 expression in cancer tissues allowed for the identification of cancer types that would benefit from the pharmacological modulation of SAMHD1 function. In conclusion, these results indicate that the modulation of SAMHD1 function may represent a promising strategy for the improvement of current antimetabolite-based treatments.

MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool

MutT homologue 1 (MTH1) removes oxidized nucleotides from the nucleotide pool and thereby prevents their incorporation into the genome and thereby reduces genotoxicity. We previously reported that MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis suggesting that MTH1 may also sanitize the nucleotide pool from other methylated nucleotides. We here show that MTH1 efficiently catalyzes the hydrolysis of N6-methyl-dATP to N6-methyl-dAMP and further report that N6-methylation of dATP drastically increases the MTH1 activity. We also observed MTH1 activity with N6-methyl-ATP, albeit at a lower level. We show that N6-methyl-dATP is incorporated into DNA in vivo, as indicated by increased N6-methyl-dA DNA levels in embryos developed from MTH1 knock-out zebrafish eggs microinjected with N6-methyl-dATP compared with noninjected embryos. N6-methyl-dATP activity is present in MTH1 homologues from distantly related vertebrates, suggesting evolutionary conservation and indicating that this activity is important. Of note, N6-methyl-dATP activity is unique to MTH1 among related NUDIX hydrolases. Moreover, we present the structure of N6-methyl-dAMP–bound human MTH1, revealing that the N6-methyl group is accommodated within a hydrophobic active-site subpocket explaining why N6-methyl-dATP is a good MTH1 substrate. N6-methylation of DNA and RNA has been reported to have epigenetic roles and to affect mRNA metabolism. We propose that MTH1 acts in concert with adenosine deaminase-like protein isoform 1 (ADAL1) to prevent incorporation of N6-methyl-(d)ATP into DNA and RNA. This would hinder potential dysregulation of epigenetic control and RNA metabolism via conversion of N6-methyl-(d)ATP to N6-methyl-(d)AMP, followed by ADAL1-catalyzed deamination producing (d)IMP that can enter the nucleotide salvage pathway.

Proofreading deficiency in mitochondrial DNA polymerase does not affect total dNTP pools in mouse embryos

Mitochondrial DNA (mtDNA) mutator mice express proofreading-deficient mtDNA polymerase gamma (PolgD257A) and age prematurely1,2, whereas mice with other mitochondrial defects do not show global signs of early aging. The reason for this discrepancy is not completely understood. Hämäläinen et al. recently reported that both induced pluripotent stem cells (iPSCs) and primary mutator embryonic fibroblasts harboring PolgD257A demonstrate increased mtDNA replication frequency leading to depletion of the total cellular dNTP pool3. The authors proposed that the decreased availability of cellular dNTPs for nuclear genome replication leads to compromised nuclear genome maintenance and premature aging. Here we report that total cellular dNTP pools are normal in mtDNA mutator mouse embryos (genotype: PolgD257A/D257A), which shows that a living organism exclusively expressing PolgD257A has normal total dNTP pools despite ubiquitous rapid cell division.

Viral protein X reduces the incorporation of mutagenic noncanonical rNTPs during lentivirus reverse transcription in macrophages

Unlike activated CD4+ T cells, nondividing macrophages have an extremely small dNTP pool, which restricts HIV-1 reverse transcription. However, rNTPs are equally abundant in both of these cell types and reach much higher concentrations than dNTPs. The greater difference in concentration between dNTPs and rNTPs in macrophages results in frequent misincorporation of noncanonical rNTPs during HIV-1 reverse transcription. Here, we tested whether the highly abundant SAM domain– and HD domain–containing protein 1 (SAMHD1) deoxynucleoside triphosphorylase in macrophages is responsible for frequent rNTP incorporation during HIV-1 reverse transcription. We also assessed whether Vpx (viral protein X), an accessory protein of HIV-2 and some simian immunodeficiency virus strains that targets SAMHD1 for proteolytic degradation, can counteract the rNTP incorporation. Results from biochemical simulation of HIV-1 reverse transcriptase–mediated DNA synthesis confirmed that rNTP incorporation is reduced under Vpx-mediated dNTP elevation. Using HIV-1 vector, we further demonstrated that dNTP pool elevation by Vpx or deoxynucleosides in human primary monocyte-derived macrophages reduces noncanonical rNTP incorporation during HIV-1 reverse transcription, an outcome similarly observed with the infectious HIV-1 89.6 strain. Furthermore, the simian immunodeficiency virus mac239 strain, encoding Vpx, displayed a much lower level of rNTP incorporation than its ΔVpx mutant in macrophages. Finally, the amount of rNMPs incorporated in HIV-1 proviral DNAs remained unchanged for ∼2 weeks in macrophages. These findings suggest that noncanonical rNTP incorporation is regulated by SAMHD1 in macrophages, whereas rNMPs incorporated in HIV-1 proviral DNA remain unrepaired. This suggests a potential long-term DNA damage impact of SAMHD1-mediated rNTP incorporation in macrophages.

A comparison of machine learning techniques for classification of HIV patients with antiretroviral therapy-induced mitochondrial toxicity from those without mitochondrial toxicity

Background Antiretroviral therapy (ART) has significantly reduced HIV-related morbidity and mortality. However, therapeutic benefit of ART is often limited by delayed drug-associated toxicity. Nucleoside reverse transcriptase inhibitors (NRTIs) are the backbone of ART regimens. NRTIs compete with endogenous deoxyribonucleotide triphosphates (dNTPs) in incorporation into elongating DNA chain resulting in their cytotoxic or antiviral effect. Thus, the efficacy of NRTIs could be affected by direct competition with endogenous dNTPs and/or feedback inhibition of their metabolic enzymes. In this paper, we assessed whether the levels of ribonucleotides (RN) and dNTP pool sizes can be used as biomarkers in distinguishing between HIV-infected patients with ART-induced mitochondrial toxicity and HIV-infected patients without toxicity. Methods We used data collected through a case-control study from 50 subjects. Cases were defined as HIV-infected individuals with clinical and/or laboratory evidence of mitochondrial toxicity. Each case was age, gender, and race matched with an HIV-positive without evidence of toxicity. We used a range of machine learning procedures to distinguish between patients with and without toxicity. Using resampling methods like Monte Carlo k-fold cross validation, we compared the accuracy of several machine learning algorithms applied to our data. We used the algorithm with highest classification accuracy rate in evaluating the diagnostic performance of 12 RN and 14 dNTP pool sizes as biomarkers of mitochondrial toxicity. Results We used eight classification algorithms to assess the diagnostic performance of RN and dNTP pool sizes distinguishing HIV patients with and without NRTI-associated mitochondrial toxicity. The algorithms resulted in cross-validated classification rates of 0.65–0.76 for dNTP and 0.72–0.83 for RN, following reduction of the dimensionality of the input data. The reduction of input variables improved the classification performance of the algorithms, with the most pronounced improvement for RN. Complex tree-based methods worked the best for both the deoxyribose dataset (Random Forest) and the ribose dataset (Classification Tree and AdaBoost), but it is worth noting that simple methods such as Linear Discriminant Analysis and Logistic Regression were very competitive in terms of classification performance. Conclusions Our finding of changes in RN and dNTP pools in participants with mitochondrial toxicity validates the importance of dNTP pools in mitochondrial function. Hence, levels of RN and dNTP pools can be used as biomarkers of ART-induced mitochondrial toxicity.

Effect of induced dNTP pool imbalance on HIV-1 reverse transcription in macrophages

Background Terminally differentiated/nondividing macrophages, a key target cell type of HIV-1, harbor extremely low dNTP concentrations established by a host dNTP triphosphohydrolase, SAM domain and HD domain containing protein 1 (SAMHD1). We tested whether the induction of dNTP pool imbalance can affect HIV-1 replication in macrophages. For this test, we induced a large dNTP pool imbalance by treating human primary monocyte derived macrophages with either one or three of the four deoxynucleosides (dNs), which are phosphorylated to dNTPs in cells, to establish two different dNTP imbalance conditions in macrophages. Results The transduction efficiency and 2-LTR circle copy number of HIV-1 GFP vector were greatly diminished in human primary macrophages treated with the biased dN treatments, compared to the untreated macrophages. We also observed the induced dNTP bias blocked the production of infectious dual tropic HIV-1 89.6 in macrophages. Moreover, biochemical DNA synthesis by HIV-1 reverse transcriptase was significantly inhibited by the induced dNTP pool imbalance. Third, the induced dNTP bias increased the viral mutant rate by approximately 20–30% per a single cycle infection. Finally, unlike HIV-1, the single dN treatment did not significantly affect the transduction of SIVmac239-based GFP vector encoding Vpx in macrophages. This is likely due to Vpx, which can elevate all four dNTP levels even with the single dN treatment. Conclusion Collectively, these data suggest that the elevated dNTP pool imbalance can induce kinetic block and mutation synthesis of HIV-1 in macrophages.