Multi-regional genomic and transcriptomic characterization of a melanoma-associated oral cavity cancer provide evidence for CASP8 alteration-mediated field cancerization.

BACKGROUND: Precancerous and malignant tumours arise within the oral cavity from a predisposed "field" of epithelial cells upon exposure to carcinogenic stimulus. This phenomenon is known as "Field Cancerization". The molecular genomic and transcriptomic alterations that lead to field cancerization and tumour progression is unknown in Indian Oral squamous cell carcinoma (OSCC) patients. METHODS: We have performed whole exome sequencing, copy-number variation array and whole transcriptome sequencing from five tumours and dysplastic lesions (sampled from distinct anatomical subsites - one each from buccal anterior and posterior alveolus, dorsum of tongue-mucosal melanoma, lip and left buccal mucosa) and blood from a rare OSCC patient with field cancerization. RESULTS: A missense CASP8 gene mutation (p.S375F) was observed to be the initiating event in oral tumour field development. APOBEC mutation signatures, arm-level copy number alterations, depletion of CD8 + T cells and activated NK cells and enrichment of pro-inflammatory mast cells were features of early-originating tumours. Pharmacological inhibition of CASP8 protein in a CASP8-wild type OSCC cell line showed enhanced levels of cellular migration and viability. CONCLUSION: CASP8 alterations are the earliest driving events in oral field carcinogenesis, whereas additional somatic mutational, copy number and transcriptomic alterations ultimately lead to OSCC tumour formation and progression.

RNA Modifications Shape Hematopoietic Stem Cell Aging: Beyond the Code.

Hematopoietic system aging is characterized by both hematopoietic stem cell (HSC) and niche degeneration resulting in myeloid lineage-biased differentiation, reduced B cell and T cell lymphopoiesis, increased HSC mobilization, and fat deposition in the bone marrow. Both alterations in RNA splicing and editing during HSC aging contribute to increased myeloid lineage skewing and inflammation-responsive transcription factors, underscoring the importance of epitranscriptomic mechanisms in the acquisition of an age-related phenotype.

Smoldering oncolysis by foamy virus carrying CD19 as a CAR target escapes CAR T detection by genomic modification.

Chimeric antigen receptor (CAR) T cells have had limited success against solid tumors. Here, we used an oncolytic foamy virus (oFV) to display a model CAR target antigen (CD19) on tumors in combination with anti-CD19 CAR T cells. We generated oFV-Δbel2 and oFV-bel2 vectors to test the efficiency and stability of viral/CD19 spread. While both viruses conferred equal CAR T killing in vitro, the oFV-Δbel2 virus acquired G-to-A mutations, whereas oFV-bel2 virus had genome deletions. In subcutaneous tumor models in vivo, CAR T cells led to a significant decrease in oFV-specific bioluminescence, confirming clearance of oFV-infected tumor cells. However, the most effective therapy was with high-dose oFV in the absence of CAR T cells, indicating that CAR T clearance of oFV was detrimental. Moreover, in tumors that escaped CAR T cell treatment, resurgent virus contained deletions within the oFV-CD19 transgene, allowing the virus to escape CAR T elimination. Therefore, oFV represents a slow smoldering type of oncolytic virus, whose chronic spread through tumors generates anti-tumor therapy, which is abolished by CAR T therapy. These results suggest that further development of this oncolytic platform, with additional immunotherapeutic arming, may allow for an effective combination of chronic oncolysis.

APOBEC family reshapes the immune microenvironment and therapy sensitivity in clear cell renal cell carcinoma.

Emerging evidence suggests that the APOBEC family is implicated in multiple cancers and might be utilized as a new target for cancer detection and treatment. However, the dysregulation and clinical implication of the APOBEC family in clear cell renal cell cancer (ccRCC) remain elusive. TCGA multiomics data facilitated a comprehensive exploration of the APOBEC family across cancers, including ccRCC. Remodeling analysis classified ccRCC patients into two distinct subgroups: APOBEC family pattern cancer subtype 1 (APCS1) and subtype 2 (APCS2). The study investigated differences in clinical parameters, tumor immune microenvironment, therapeutic responsiveness, and genomic mutation landscapes between these subtypes. An APOBEC family-related risk model was developed and validated for predicting ccRCC patient prognosis, demonstrating good sensitivity and specificity. Finally, the overview of APOBEC3B function was investigated in multiple cancers and verified in clinical samples. APCS1 and APCS2 demonstrated considerably distinct clinical features and biological processes in ccRCC. APCS1, an aggressive subtype, has advanced clinical stage and a poor prognosis. APCS1 exhibited an oncogenic and metabolically active phenotype. APCS1 also exhibited a greater tumor mutation load and immunocompromised condition, resulting in immunological dysfunction and immune checkpoint treatment resistance. The genomic copy number variation of APCS1, including arm gain and loss, was much more than that of APCS2, which may help explain the tired immune system. Furthermore, the two subtypes have distinct drug sensitivity patterns in clinical specimens and matching cell lines. Finally, we developed a predictive risk model based on subtype biomarkers that performed well for ccRCC patients and validated the clinical impact of APOBEC3B. Aberrant APOBEC family expression patterns might modify the tumor immune microenvironment by increasing the genome mutation frequency, thus inducing an immune-exhausted phenotype. APOBEC family-based molecular subtypes could strengthen the understanding of ccRCC characterization and guide clinical treatment. Targeting APOBEC3B may be regarded as a new therapeutic target for ccRCC.

Liver-specific Coxsackievirus and adenovirus receptor deletion develop metabolic dysfunction-associated fatty liver disease.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a common liver disease associated with obesity and is caused by the accumulation of ectopic fat without alcohol consumption. Coxsackievirus and adenovirus receptor (CAR) are vital for cardiac myocyte-intercalated discs and endothelial cell-to-cell tight junctions. CAR has also been reported to be associated with obesity and high blood pressure. However, its function in the liver is still not well understood. The liver of obese mice exhibit elevated CAR mRNA and protein levels. Furthermore, in the liver of patients with non-alcoholic steatohepatitis, CAR is reduced in hepatocyte cell-cell junctions compared to normal levels. We generated liver-specific CAR knockout (KO) mice to investigate the role of CAR in the liver. Body and liver weights were not different between wild-type (WT) and KO mice fed a paired or high-fat diet (HFD). However, HFD induced significant liver damage and lipid accumulation in CAR KO mice compared with WT mice. Additionally, inflammatory cytokines transcription, hepatic permeability, and macrophage recruitment considerably increased in CAR KO mice. We identified a new interaction partner of CAR using a protein pull-down assay and mass spectrometry. Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3C (APOBEC3C) demonstrated a complex relationship with CAR, and hepatic CAR expression tightly regulated its level. Moreover, Apolipoprotein B (ApoB) and Low-density lipoprotein receptor (LDLR) levels correlated with APOBEC3C expression in the liver of CAR KO mice, suggesting that CAR may regulate lipid accumulation by controlling APOBEC3C activity. In this study, we showed that hepatic CAR deficiency increased cell-to-cell permeability. In addition, CAR deletion significantly increased hepatic lipid accumulation by inducing ApoB and LDLR expression. Although the underlying mechanism is unclear, CARs may be a target for the development of novel therapies for MAFLD.

APOBEC-1 Complementation Factor: From RNA Binding to Cancer.

BACKGROUND: APOBEC-1 complementation factor (A1CF) and Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-1 (APOBEC-1) constitute the minimal proteins necessary for the editing of apolipoprotein B (apoB) mRNA in vitro. Unlike APOBEC-1 and apoB mRNA, the ubiquitous expression of A1CF in human tissues suggests its unique biological significance, with various factors such as protein kinase C, thyroid hormones, and insulin regulating the activity and expression of A1CF. Nevertheless, few studies have provided an overview of this topic. OBJECTIVE: We conducted a literature review to describe the molecular mechanisms of A1CF and its relevance to human diseases. METHOD: In the PubMed database, we used the keywords 'A1CF' and 'APOBEC-1 complementation factor' to collect peer-reviewed articles published in English from 2000 to 2023. Two authors independently reviewed the articles and reached the consensus. RESULT: After reviewing 127 articles, a total of 61 articles that met the inclusion criteria were included in the present review. Studies revealed that A1CF is involved in epigenetic regulation of reproductive cells affecting embryonic development, and that it is closely associated with the occurrence of gout due to its editing properties on apoB. A1CF can also affect the process of epithelial-mesenchymal transition in renal tubular epithelial cells and promote liver regeneration by controlling the stability of IL-6 mRNA, but no influence on cardiac function was found. Furthermore, increasing evidence suggests that A1CF may promote the occurrence and development of breast cancer, lung cancer, renal cell carcinoma, hepatocellular carcinoma, endometrial cancer, and glioma. CONCLUSION: This review clarifies the association between A1CF and other complementary factors and their impact on the development of human diseases, aiming to provide guidance for further research on A1CF, which can help treat human diseases and promote health.

Next generation APOBEC3 inhibitors: Optimally designed for potency and nuclease stability.

APOBEC3 (or A3) enzymes have emerged as potential therapeutic targets due to their role in introducing heterogeneity in viruses and cancer, often leading to drug resistance. Inhibiting these enzymes has remained elusive as initial phosphodiester (PO) linked DNA based inhibitors lack stability and potency. We have enhanced both potency and nuclease stability, of 2'-deoxy-zebularine (dZ), substrate-based oligonucleotide inhibitors for two critical A3's: A3A and A3G. While replacing the phosphate backbone with phosphorothioate (PS) linkages increased nuclease stability, fully PS-modified inhibitors lost potency (1.4-3.7 fold) due to the structural constraints of the active site. For both enzymes, mixed PO/PS backbones enhanced potency (2.3-9.2 fold), while also vastly improving nuclease resistance. We also strategically introduced 2'-fluoro sugar modifications, creating the first nanomolar inhibitor of A3G-CTD2. With hairpin-structured inhibitors containing optimized PS patterns and LNA sugar modifications, we characterize the first single-digit nanomolar inhibitor targeting A3A. These extremely potent A3A inhibitors, were highly resistant to nuclease degradation in serum stability assays. Overall, our optimally designed A3 oligonucleotide inhibitors show improved potency and stability, compared to previous attempts to inhibit these critical enzymes, opening the door to realize the therapeutic potential of A3 inhibition.

Human and bats genome robustness under COSMIC mutational signatures.

Carcinogenesis is an evolutionary process, and mutations can fix the selected phenotypes in selective microenvironments. Both normal and neoplastic cells are robust to the mutational stressors in the microenvironment to the extent that secure their fitness. To test the robustness of genes under a range of mutagens, we developed a sequential mutation simulator, Sinabro, to simulate single base substitution under a given mutational process. Then, we developed a pipeline to measure the robustness of genes and cells under those mutagenesis processes. We discovered significant human genome robustness to the APOBEC mutational signature SBS2, which is associated with viral defense mechanisms and is implicated in cancer. Robustness evaluations across over 70,000 sequences against 41 signatures showed higher resilience under signatures predominantly causing C-to-T (G-to-A) mutations. Principal component analysis indicates the GC content at the codon's wobble position significantly influences robustness, with increased resilience noted under transition mutations compared to transversions. Then, we tested our results in bats at extremes of the lifespan-to-mass relationship and found the long-lived bat is more robust to APOBEC than the short-lived one. By revealing robustness to APOBEC ranked highest in human (and bats with much more than number of APOBEC) genome, this work bolsters the key potential role of APOBECs in aging and cancer, as well as evolved countermeasures to this innate mutagenic process. It also provides the baseline of the human and bat genome robustness under mutational processes associated with aging and cancer.

p-STAT3-elevated E3 ubiquitin ligase DTX4 confers the stability of HBV cccDNA by ubiquitinating APOBEC3B in liver.

Background: Clinically, the persistence of HBV cccDNA is the major obstacle in anti-HBV therapy. However, the underlying mechanism of HBV cccDNA is poorly understood. The transcriptional factor STAT3 is able to activate HBV replication in liver. Approach & Results: RNA-Seq analysis demonstrated that cucurbitacin I targeting STAT3 was associated with virus replication in liver. HBV-infected human liver chimeric mouse model and HBV hydrodynamic injection mouse model were established. Then, we validated that cucurbitacin I effectively limited the stability of HBV cccDNA and HBV replication in cells, in which cucurbitacin I enhanced the sensitivity of pegylated interferon α (PEG-IFN α) against HBV via combination in vitro and in vivo. Mechanistically, we identified that cucurbitacin I increased the levels of APOBEC3B to control HBV cccDNA by inhibiting p-STAT3 in cells, resulting in the inhibition of HBV replication. Moreover, RNA-Seq data showed that E3 ubiquitin ligase DTX4 might be involved in the events. Then, we observed that HBV particles could upregulate DTX4 by increasing the levels of p-STAT3 in vitro and in vivo. The p-STAT3-elevated DTX4/male-specific lethal 2 (MSL2) independently and synergistically enhanced the stability of HBV cccDNA by facilitating the ubiquitination degradation of APOBEC3B in cells, leading to the HBV replication. Conclusions: p-STAT3-elevated DTX4 confers the stability of HBV cccDNA and HBV replication by facilitating the ubiquitination degradation of APOBEC3B. Cucurbitacin Ⅰ effectively enhances the sensitivity of PEG-IFN α in anti-HBV therapy by inhibiting the p-STAT3/DTX4/MSL2/APOBEC3B signalling. Our finding provides new insights into the mechanism of HBV cccDNA. The p-STAT3 and DTX4/MSL2 might serve as the therapeutical targets of HBV cccDNA.

Genetic alteration of mRNA editing enzyme APOBEC3B in the pathogenesis of ovarian endometriosis.

RESEARCH QUESTION: What are the specific genetic alterations and associated network in endometriotic cells responsible for the disease pathogenesis? DESIGN: Case control experimental study involving 45 women with endometriosis who underwent laparoscopic surgery (case) and 45 normal samples from women undergoing total abdominal hysterectomy (control). The endometrial samples were subjected to whole exome sequencing (WES) of endometriotic tissue and copy number variation analysis. Validation of gene hits were obtained from WES using polymerase chain reaction techniques, immunological techniques, in-silico tools and transgenic cell line models. RESULTS: Germline heterozygous deletion of mRNA editing enzyme subunit APOBEC3B was identified in about 96% of endometriosis samples. The presence of germline deletion was confirmed with blood, endometrium and normal ovary samples obtained from the same patient. APOBEC3B deletions resulted in a hybrid protein that activates A1CF. APOBEC3B deletion can be a major cause of changes in the endometriotic microenvironment, and contributes to the pathogenesis and manifestation of the disease. The effect of APOBEC3B deletion was proved by in-vitro experiments in a cell line model, which displayed endometriosis-like characteristics. APOBEC3B germline deletion plays a major role in the pathogenesis of endometriosis, which is evident by the activation of A1CF, an increase in epithelial to mesenchymal transition, cellular proliferation, inflammation markers and a decrease in apoptosis markers. CONCLUSION: The deleterious effects caused by APOBEC3B deletion in endometriosis were identified and confirmed. These results might provide a base for identifying the complete pathogenetic mechanism of endometriosis, thereby moving a step closer to better diagnosis and treatment options.

RAD51 protects abasic sites to prevent replication fork breakage.

Abasic sites are DNA lesions repaired by base excision repair. Cleavage of unrepaired abasic sites in single-stranded DNA (ssDNA) can lead to chromosomal breakage during DNA replication. How rupture of abasic DNA is prevented remains poorly understood. Here, using cryoelectron microscopy (cryo-EM), Xenopus laevis egg extracts, and human cells, we show that RAD51 nucleofilaments specifically recognize and protect abasic sites, which increase RAD51 association rate to DNA. In the absence of BRCA2 or RAD51, abasic sites accumulate as a result of DNA base methylation, oxidation, and deamination, inducing abasic ssDNA gaps that make replicating DNA fibers sensitive to APE1. RAD51 assembled on abasic DNA prevents abasic site cleavage by the MRE11-RAD50 complex, suppressing replication fork breakage triggered by an excess of abasic sites or POLθ polymerase inhibition. Our study highlights the critical role of BRCA2 and RAD51 in safeguarding against unrepaired abasic sites in DNA templates stemming from base alterations, ensuring genomic stability.

ERCC2 mutations alter the genomic distribution pattern of somatic mutations and are independently prognostic in bladder cancer.

Excision repair cross-complementation group 2 (ERCC2) encodes the DNA helicase xeroderma pigmentosum group D, which functions in transcription and nucleotide excision repair. Point mutations in ERCC2 are putative drivers in around 10% of bladder cancers (BLCAs) and a potential positive biomarker for cisplatin therapy response. Nevertheless, the prognostic significance directly attributed to ERCC2 mutations and its pathogenic role in genome instability remain poorly understood. We first demonstrated that mutant ERCC2 is an independent predictor of prognosis in BLCA. We then examined its impact on the somatic mutational landscape using a cohort of ERCC2 wild-type (n = 343) and mutant (n = 39) BLCA whole genomes. The genome-wide distribution of somatic mutations is significantly altered in ERCC2 mutants, including T[C>T]N enrichment, altered replication time correlations, and CTCF-cohesin binding site mutation hotspots. We leverage these alterations to develop a machine learning model for predicting pathogenic ERCC2 mutations, which may be useful to inform treatment of patients with BLCA.

APOBEC3C-mediated NF-κB activation enhances clear cell renal cell carcinoma progression.

Renowned as the predominant form of kidney cancer, clear cell renal cell carcinoma (ccRCC) exhibits susceptibility to immunotherapies due to its specific expression profile as well as notable immune cell infiltration. Despite this, effectively treating metastatic ccRCC remains a significant challenge, necessitating a more profound comprehension of the underlying molecular mechanisms governing its progression. Here, we unveil that the enhanced expression of the RNA-binding protein DNA dC → dU-editing enzyme APOBEC-3C (APOBEC3C; also known as A3C) in ccRCC tissue and ccRCC-derived cell lines serves as a catalyst for tumor growth by amplifying nuclear factor-kappa B (NF-κB) activity. By employing RNA-sequencing and cell-based assays in ccRCC-derived cell lines, we determined that A3C is a stress-responsive factor and crucial for cell survival. Furthermore, we identified that A3C binds and potentially stabilizes messenger RNAs (mRNAs) encoding positive regulators of the NF-κB pathway. Upon A3C depletion, essential subunits of the NF-κB family are abnormally restrained in the cytoplasm, leading to deregulation of NF-κB target genes. Our study illuminates the pivotal role of A3C in promoting ccRCC tumor development, positioning it as a prospective target for future therapeutic strategies.

Hypomorphic mutation in the large subunit of replication protein A affects mutagenesis by human APOBEC cytidine deaminases in yeast.

Human APOBEC single-strand (ss) specific DNA and RNA cytidine deaminases change cytosines to uracils (U's) and function in antiviral innate immunity and RNA editing and can cause hypermutation in chromosomes. The resulting U's can be directly replicated, resulting in C to T mutations, or U-DNA glycosylase can convert the U's to abasic (AP) sites which are then fixed as C to T or C to G mutations by translesion DNA polymerases. We noticed that in yeast and in human cancers, contributions of C to T and C to G mutations depend on the origin of ssDNA mutagenized by APOBECs. Since ssDNA in eukaryotic genomes readily binds to replication protein A (RPA) we asked if RPA could affect APOBEC-induced mutation spectrum in yeast. For that purpose, we expressed human APOBECs in the wild-type (WT) yeast and in strains carrying a hypomorph mutation rfa1-t33 in the large RPA subunit. We confirmed that the rfa1-t33 allele can facilitate mutagenesis by APOBECs. We also found that the rfa1-t33 mutation changed the ratio of APOBEC3A-induced T to C and T to G mutations in replicating yeast to resemble a ratio observed in long persistent ssDNA in yeast and in cancers. We present the data suggesting that RPA may shield APOBEC formed U's in ssDNA from Ung1, thereby facilitating C to T mutagenesis through the accurate copying of U's by replicative DNA polymerases. Unexpectedly, we also found that for U's shielded from Ung1 by WT RPA, the mutagenic outcome is reduced in the presence of translesion DNA polymerase zeta.

Inhibition of Uracil DNA Glycosylase Alters Frequency and Spectrum of Action-at-a-Distance Mutations Induced by 8-Oxo-7,8-dihydroguanine.

The generation of DNA damage causes mutations and consequently cancer. Reactive oxygen species are important sources of DNA damage and some mutation signatures found in human cancers. 8-Oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) is one of the most abundant oxidized bases and induces a G→T transversion mutation at the modified site. The damaged G base also causes untargeted base substitution mutations at the G bases of 5'-GpA-3' dinucleotides (action-at-a-distance mutations) in human cells, and the cytosine deaminase apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) is involved in the mutation process. The deaminated cytosine, i.e., uracil, bases are expected to be removed by uracil DNA glycosylase. Most of the substitution mutations at the G bases of 5'-GpA-3' might be caused by abasic sites formed by the glycosylase. In this study, we expressed the uracil DNA glycosylase inhibitor from Bacillus subtilis bacteriophage PBS2 in human U2OS cells and examined the effects on the GO-induced action-at-a-distance mutations. The inhibition of uracil DNA glycosylase increased the mutation frequency, and in particular, the frequency of G→A transitions. These results indicated that uracil DNA glycosylase, in addition to APOBEC3, is involved in the untargeted mutation process induced by GO.

Highly homologous simian T-cell leukemia virus type 1 genome in Japanese macaques: a large cohort study.

BACKGROUND: Simian T-cell leukemia virus type 1 (STLV-1) is a retrovirus closely related to human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia (ATL). It has been shown that Japanese macaques (Macaca fuscata, JMs) are one of the main hosts of STLV-1 and that a high percentage of JMs (up to 60%) are infected with STLV-1; however, the molecular epidemiology of STLV-1 in JMs has not been examined. METHODS: In this study, we analyzed full-length STLV-1 genome sequences obtained from 5 independent troops including a total of 68 JMs. RESULTS: The overall nucleotide heterogeneity was 4.7%, and the heterogeneity among the troops was 2.1%, irrespective of the formation of distinct subclusters in each troop. Moreover, the heterogeneity within each troop was extremely low (>99% genome homology) compared with cases of STLV-1 in African non-human primates as well as humans. It was previously reported that frequent G-to-A single-nucleotide variants (SNVs) occur in HTLV-1 proviral genomes in both ATL patients and HTLV-1 carriers, and that a G-to-A hypermutation is associated with the cellular antiviral restriction factor, Apobec3G. Surprisingly, these SNVs were scarcely observed in the STLV-1 genomes in JMs. CONCLUSIONS: Taken together, these results indicate that STLV-1 genomes in JMs are highly homologous, at least in part due to the lack of Apobec3G-dependent G-to-A hypermutation.

Regulation, functional impact, and therapeutic targeting of APOBEC3A in cancer.

Enzymes of the apolipoprotein B mRNA editing catalytic polypeptide like (APOBEC) family are cytosine deaminases that convert cytosine to uracil in DNA and RNA. Among these proteins, APOBEC3 sub-family members, APOBEC3A (A3A) and APOBEC3B (A3B), are prominent sources of mutagenesis in cancer cells. The aberrant expression of A3A and A3B in cancer cells leads to accumulation of mutations with specific single-base substitution (SBS) signatures, characterized by C→T and C→G changes, in a number of tumor types. In addition to fueling mutagenesis, A3A and A3B, particularly A3A, induce DNA replication stress, DNA damage, and chromosomal instability through their catalytic activities, triggering a range of cellular responses. Thus, A3A/B have emerged as key drivers of genome evolution during cancer development, contributing to tumorigenesis, tumor heterogeneity, and therapeutic resistance. Yet, the expression of A3A/B in cancer cells presents a cancer vulnerability that can be exploited therapeutically. In this review, we discuss the recent studies that shed light on the mechanisms regulating A3A expression and the impact of A3A in cancer. We also review recent advances in the development of A3A inhibitors and provide perspectives on the future directions of A3A research.

Hypomorphic mutation in the large subunit of replication protein A affects mutagenesis by human APOBEC cytidine deaminases in yeast.

Human APOBEC single-strand (ss) specific DNA and RNA cytidine deaminases change cytosines to uracils and function in antiviral innate immunity, RNA editing, and can cause hypermutation in chromosomes. The resulting uracils can be directly replicated, resulting in C to T mutations, or uracil-DNA glycosylase can convert the uracils to abasic (AP) sites which are then fixed as C to T or C to G mutations by translesion DNA polymerases. We noticed that in yeast and in human cancers, contributions of C to T and C to G mutations depends on the origin of ssDNA mutagenized by APOBECs. Since ssDNA in eukaryotic genomes readily binds to replication protein A (RPA) we asked if RPA could affect APOBEC-induced mutation spectrum in yeast. For that purpose, we expressed human APOBECs in the wild-type yeast and in strains carrying a hypomorph mutation rfa1-t33 in the large RPA subunit. We confirmed that the rfa1-t33 allele can facilitate mutagenesis by APOBECs. We also found that the rfa1-t33 mutation changed the ratio of APOBEC3A-induced T to C and T to G mutations in replicating yeast to resemble a ratio observed in long-persistent ssDNA in yeast and in cancers. We present the data suggesting that RPA may shield APOBEC formed uracils in ssDNA from Ung1, thereby facilitating C to T mutagenesis through the accurate copying of uracils by replicative DNA polymerases. Unexpectedly, we also found that for uracils shielded from Ung1 by wild-type RPA the mutagenic outcome is reduced in the presence of translesion DNA polymerase zeta.

Efficient CRISPR-mediated C-to-T base editing in Komagataella phaffii.

The nonconventional methylotrophic yeast Komagataella phaffii is widely applied in the production of industrial enzymes, pharmaceutical proteins, and various high-value chemicals. The development of robust and versatile genome editing tools for K. phaffii is crucial for the design of increasingly advanced cell factories. Here, we first developed a base editing method for K. phaffii based on the CRISPR-nCas9 system. We engineered 24 different base editor constructs, using a variety of promoters and cytidine deaminases (CDAs). The optimal base editor (PAOX2*-KpA3A-nCas9-KpUGI-DAS1TT) comprised a truncated AOX2 promoter (PAOX2*), a K. phaffii codon-optimized human APOBEC3A CDA (KpA3A), human codon-optimized nCas9 (D10A), and a K. phaffii codon-optimized uracil glycosylase inhibitor (KpUGI). This optimal base editor efficiently performed C-to-T editing in K. phaffii, with single-, double-, and triple-locus editing efficiencies of up to 96.0%, 65.0%, and 5.0%, respectively, within a 7-nucleotide window from C-18 to C-12. To expand the targetable genomic region, we also replaced nCas9 in the optimal base editor with nSpG and nSpRy, and achieved 50.0%-60.0% C-to-T editing efficiency for NGN-protospacer adjacent motif (PAM) sites and 20.0%-93.2% C-to-T editing efficiency for NRN-PAM sites, respectively. Therefore, these constructed base editors have emerged as powerful tools for gene function research, metabolic engineering, genetic improvement, and functional genomics research in K. phaffii.

The role of distinct APOBEC/ADAR mRNA levels in mutational signatures linked to aging and ultraviolet radiation.

The APOBEC/AID family is known for its mutator activity, and recent evidence also supports the potential impact of ADARs. Furthermore, the mutator impacts of APOBEC/ADAR mutations have not yet been investigated. Assessment of pancancer TCGA exomes identified enriched somatic variants among exomes with nonsynonymous APOBEC1, APOBEC3B, APOBEC3C, ADAR, and ADARB1 mutations, compared to exomes with synonymous ones. Principal component (PC) analysis reduced the number of potential players to eight in cancer exomes/genomes, and to five in cancer types. Multivariate regression analysis was used to assess the impact of the PCs on each COSMIC mutational signature among pancancer exomes/genomes and particular cancers, identifying several novel links, including SBS17b, SBS18, and ID7 mainly determined by APOBEC1 mRNA levels; SBS40, ID1, and ID2 by age; SBS3 and SBS16 by APOBEC3A/APOBEC3B mRNA levels; ID5 and DBS9 by DNA repair/replication (DRR) defects; and SBS7a-d, SBS38, ID4, ID8, ID13, and DBS1 by ultraviolet (UV) radiation/ADARB1 mRNA levels. APOBEC/ADAR mutations appeared to potentiate the impact of DRR defects on several mutational signatures, and some factors seemed to inversely affect certain signatures. These findings potentially implicate certain APOBEC/ADAR mutations/mRNA levels in distinct mutational signatures, particularly APOBEC1 mRNA levels in aging-related signatures and ADARB1 mRNA levels in UV radiation-related signatures.