The combination of cisplatin and pemetrexed remains the gold standard chemotherapy for malignant pleural mesothelioma (MPM), although resistance and poor response pose a significant challenge. Cytidine deaminase (CDA) is a key enzyme in the nucleotide salvage pathway and is involved in the adaptive stress response to chemotherapy. The cytidine analog capecitabine and its metabolite 5\'-deoxy-5-fluorocytidine (5\'-DFCR) are converted via CDA to 5-fluorouracil, which affects DNA and RNA metabolism. This study investigated a schedule-dependent treatment strategy, proposing that initial chemotherapy induces CDA expression, sensitizing cells to subsequent capecitabine treatment. Basal CDA protein expression was low in different mesothelioma cell lines but increased in the corresponding xenografts. Standard chemotherapy increased CDA protein levels in MPM cells in vitro and in vivo in a schedule-dependent manner. This was associated with epithelial-to-mesenchymal transition and with HIF-1alpha expression at the transcriptional level. In addition, pretreatment with cisplatin and pemetrexed in combination sensitized MPM xenografts to capecitabine. Analysis of a tissue microarray (TMA) consisting of samples from 98 human MPM patients revealed that most human MPM samples had negative CDA expression. While survival curves based on CDA expression in matched samples clearly separated, significance was not reached due to the limited sample size. In non-matched samples, CDA expression before but not after neoadjuvant therapy was significantly associated with worse overall survival. In conclusion, chemotherapy increases CDA expression in xenografts, which is consistent with our in vitro results in MPM and lung cancer. A subset of matched patient samples showed increased CDA expression after therapy, suggesting that a schedule-dependent treatment strategy based on chemotherapy and capecitabine may benefit a selected MPM patient population.

Activation-induced cytidine deaminase (AID) is a B cell-specific mutator required for antibody diversification. However, it is also implicated in the etiology of several B cell malignancies. Evaluating the AID-induced mutation load in patients at-risk for certain blood cancers is critical in assessing disease severity and treatment options. We have developed a digital PCR (dPCR) assay that allows us to quantify mutations resulting from AID modification or DNA double-strand break (DSB) formation and repair at sites known to be prone to DSBs. Implementation of this assay shows that increased AID levels in immature B cells increase genome instability at loci linked to chromosomal translocation formation. This includes the CRLF2 locus that is often involved in translocations associated with a subtype of acute lymphoblastic leukemia (ALL) that disproportionately affects Hispanics, particularly those with Latin American ancestry. Using dPCR, we characterize the CRLF2 locus in B cell-derived genomic DNA from both Hispanic ALL patients and healthy Hispanic donors and found increased mutations in both, suggesting that vulnerability to DNA damage at CRLF2 may be driving this health disparity. Our ability to detect and quantify these mutations will potentiate future risk identification, early detection of cancers, and reduction of associated cancer health disparities.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has acquired multiple mutations since its emergence. Analyses of the SARS-CoV-2 genomes from infected patients exhibit a bias toward C-to-U mutations, which are suggested to be caused by the apolipoprotein B mRNA editing enzyme polypeptide-like 3 (APOBEC3, A3) cytosine deaminase proteins. However, the role of A3 enzymes in SARS-CoV-2 replication remains unclear. To address this question, we investigated the effect of A3 family proteins on SARS-CoV-2 replication in the myeloid leukemia cell line THP-1 lacking A3A to A3G genes. The Wuhan, BA.1, and BA.5 variants had comparable viral replication in parent and A3A-to-A3G-null THP-1 cells stably expressing angiotensin-converting enzyme 2 (ACE2) protein. On the other hand, the replication and infectivity of these variants were abolished in A3A-to-A3G-null THP-1-ACE2 cells in a series of passage experiments over 20 days. In contrast to previous reports, we observed no evidence of A3-induced SARS-CoV-2 mutagenesis in the passage experiments. Furthermore, our analysis of a large number of publicly available SARS-CoV-2 genomes did not reveal conclusive evidence for A3-induced mutagenesis. Our studies suggest that A3 family proteins can positively contribute to SARS-CoV-2 replication; however, this effect is deaminase-independent.

The pathway for axon regeneration in Caenorhabditis elegans is activated by SVH-1, a growth factor belonging to the HGF/plasminogen family. SVH-1 is a dual-function factor that acts as an HGF-like growth factor to promote axon regeneration and as a protease to regulate early development. It is important to understand how SVH-1 is converted from a protease to a growth factor for axon regeneration. In this study, we demonstrate that cytidine deaminase (CDD) SVH-17/CDD-2 plays a role in the functional conversion of SVH-1. We find that the codon exchange of His-755 to Tyr in the Asp-His-Ser catalytic triad of SVH-1 can suppress the cdd-2 defect in axon regeneration. Furthermore, the stem hairpin structure around the His-755 site in svh-1 mRNA is required for the activation of axon regeneration by SVH-1. These results suggest that CDD-2 promotes axon regeneration by transforming the function of SVH-1 from a protease to a growth factor through modification of svh-1 mRNA.

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.

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.

BACKGROUND: RNA-DNA hybrids or R-loops are associated with deleterious genomic instability and protective immunoglobulin class switch recombination (CSR). However, the underlying phenomenon regulating the two contrasting functions of R-loops is unknown. Notably, the underlying mechanism that protects R-loops from classic RNase H-mediated digestion thereby promoting persistence of CSR-associated R-loops during CSR remains elusive.
RESULTS: Here, we report that during CSR, R-loops formed at the immunoglobulin heavy (IgH) chain are modified by ribose 2\'-O-methylation (2\'-OMe). Moreover, we find that 2\'-O-methyltransferase fibrillarin (FBL) interacts with activation-induced cytidine deaminase (AID) associated snoRNA aSNORD1C to facilitate the 2\'-OMe. Moreover, deleting AID C-terminal tail impairs its association with aSNORD1C and FBL. Disrupting FBL, AID or aSNORD1C expression severely impairs 2\'-OMe, R-loop stability and CSR. Surprisingly, FBL, AID\'s interaction partner and aSNORD1C promoted AID targeting to the IgH locus.
CONCLUSIONS: Taken together, our results suggest that 2\'-OMe stabilizes IgH-associated R-loops to enable productive CSR. These results would shed light on AID-mediated CSR and explain the mechanism of R-loop-associated genomic instability.

Cancer initiates as a consequence of genomic mutations and its subsequent progression relies in part on increased production of ribosomes to maintain high levels of protein synthesis for unchecked cell growth. Recently, cytidine deaminases have been uncovered as sources of mutagenesis in cancer. In an attempt to form a connection between these 2 cancer driving processes, we interrogated the cytidine deaminase family of proteins for potential roles in human ribosome biogenesis. We identified and validated APOBEC3A and APOBEC4 as novel ribosome biogenesis factors through our laboratory\'s established screening platform for the discovery of regulators of nucleolar function in MCF10A cells. Through siRNA depletion experiments, we highlight APOBEC3A\'s requirement in making ribosomes and specific role within the processing and maturation steps that form the large subunit 5.8S and 28S ribosomal (r)RNAs. We demonstrate that a subset of APOBEC3A resides within the nucleolus and associates with critical ribosome biogenesis factors. Mechanistic insight was revealed by transient overexpression of both wild-type and a catalytically dead mutated APOBEC3A, which both increase cell growth and protein synthesis. Through an innovative nuclear RNA sequencing methodology, we identify only modest predicted APOBEC3A C-to-U target sites on the pre-rRNA and pre-mRNAs. Our work reveals a potential direct role for APOBEC3A in ribosome biogenesis likely independent of its editing function. More broadly, we found an additional function of APOBEC3A in cancer pathology through its function in ribosome biogenesis, expanding its relevance as a target for cancer therapeutics.

High spontaneous mutation rate is crucial for obtaining ideal phenotype and exploring the relationship between genes and phenotype. How to break the genetic stability of organisms and increase the mutation frequency has become a research hotspot. Here, we present a practical and controllable evolutionary tool (oMut-Cgts) based on dual genetic level modification engineering for Corynebacterium glutamicum. Firstly, the modification engineering of transcription and replication levels based on RNA polymerase α subunit and DNA helicase Cgl0854 as the \'dock\' of cytidine deaminase (pmCDA1) significantly increased the mutation rate, proving that the localization of pmCDA1 around transient ssDNA is necessary for genome mutation. Then, the combined modification and optimization of engineering at dual genetic level achieved 1.02 × 104-fold increased mutation rate. The genome sequencing revealed that the oMut-Cgts perform uniform and efficient C:G→T:A transitions on a genome-wide scale. Furthermore, oMut-Cgts-mediated rapid evolution of C. glutamicum with stress (acid, oxidative and ethanol) tolerance proved that the tool has powerful functions in multi-dimensional biological engineering (rapid phenotype evolution, gene function mining and protein evolution). The strategies for rapid genome evolution provided in this study are expected to be applicable to a variety of applications in all prokaryotic cells.

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.