Research progress of N1-methyladenosine RNA modification in cancer.

N1-methyladenosine (m1A) is a post-transcriptionally modified RNA molecule that plays a pivotal role in the regulation of various biological functions and activities. Especially in cancer cell invasion, proliferation and cell cycle regulation. Over recent years, there has been a burgeoning interest in investigating the m1A modification of RNA. Most studies have focused on the regulation of m1A in cancer enrichment areas and different regions. This review provides a comprehensive overview of the methodologies employed for the detection of m1A modification. Furthermore, this review delves into the key players in m1A modification, known as the "writers," "erasers," and "readers." m1A modification is modified by the m1A methyltransferases, or writers, such as TRMT6, TRMT61A, TRMT61B, TRMT10C, NML, and, removed by the demethylases, or erasers, including FTO and ALKBH1, ALKBH3. It is recognized by m1A-binding proteins YTHDF1, TYHDF2, TYHDF3, and TYHDC1, also known as "readers". Additionally, we explore the intricate relationship between m1A modification and its regulators and their implications for the development and progression of specific types of cancer, we discuss how m1A modification can potentially facilitate the discovery of novel approaches for cancer diagnosis, treatment, and prognosis. Our summary of m1A methylated adenosine modification detection methods and regulatory mechanisms in various cancers provides useful insights for cancer diagnosis, treatment, and prognosis. Video Abstract.

Histone lactylation-boosted ALKBH3 potentiates tumor progression and diminished promyelocytic leukemia protein nuclear condensates by m1A demethylation of SP100A.

Albeit N1-Methyladenosine (m1A) RNA modification represents an important regulator of RNA metabolism, the role of m1A modification in carcinogenesis remains enigmatic. Herein, we found that histone lactylation enhances ALKBH3 expression and simultaneously attenuates the formation of tumor-suppressive promyelocytic leukemia protein (PML) condensates by removing the m1A methylation of SP100A, promoting the malignant transformation of cancers. First, ALKBH3 is specifically upregulated in high-risk ocular melanoma due to excessive histone lactylation levels, referring to m1A hypomethylation status. Moreover, the multiomics analysis subsequently identified that SP100A, a core component for PML bodies, serves as a downstream candidate target for ALKBH3. Therapeutically, the silencing of ALKBH3 exhibits efficient therapeutic efficacy in melanoma both in vitro and in vivo, which could be reversed by the depletion of SP100A. Mechanistically, we found that YTHDF1 is responsible for recognition of the m1A methylated SP100A transcript, which increases its RNA stability and translational efficacy. Conclusively, we initially demonstrated that m1A modification is necessary for tumor suppressor gene expression, expanding the current understandings of dynamic m1A function during tumor progression. In addition, our results indicate that lactylation-driven ALKBH3 is essential for the formation of PML nuclear condensates, which bridges our knowledge of m1A modification, metabolic reprogramming, and phase-separation events.

Demethylation-activated light-up dual-color RNA aptamersensor for label-free detection of multiple demethylases in lung tissues.

Methylation is one of the most prevalent epigenetic modifications in natural organisms, and the processes of methylation and demethylation are closely associated with cell growth, differentiation, gene transcription and expression. Abnormal methylation may lead to various human diseases including cancers. Simultaneous analysis of multiple DNA demethylases remains a huge challenge due to the requirement of diverse substrate probes and scarcity of proper signal transduction strategies. Herein, we propose a sensitive and label-free method for simultaneous monitoring of multiple DNA demethylases on the basis of demethylation-activated light-up dual-color RNA aptamers. The presence of targets AlkB homologue-3 (ALKBH3) and fat mass and obesity-associated enzyme (FTO) erases the methyl group in DNA substrate probes, activating the ligation-mediate bidirectional transcription amplification reaction to produce enormous Spinach and Mango aptamers. The resulting RNA aptamers (i.e., Spinach and Mango aptamers) can bind with their cognate nonfluorescent fluorogens (DFHBI and TO1-biotin) to significantly improve the fluorescence signals. This aptamersensor shows high specificity and sensitivity with a limit of detection (LOD) of 8.50 × 10-14 M for ALKBH3 and 6.80 × 10-14 M for FTO, and it can apply to screen DNA demethylase inhibitors, evaluate DNA demethylase kinetic parameters, and simultaneously measure multiple endogenous DNA demethylases in a single cell. Importantly, this aptamersensor can accurately discriminate the expressions of ALKBH3 and FTO between healthy tissues and non-small cell lung cancer (NSCLC) patient tissues, offering a powerful platform for clinical diagnosis and drug discovery.

The Molecular Basis of Human ALKBH3 Mediated RNA N1 -methyladenosine (m1 A) Demethylation.

N1 -methyladenosine (m1 A) is a prevalent post-transcriptional RNA modification, and the distribution and dynamics of the modification play key epitranscriptomic roles in cell development. At present, the human AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family member ALKBH3 is the only known mRNA m1 A demethylase, but its catalytic mechanism remains unclear. Here, we present the structures of ALKBH3-oligo crosslinked complexes obtained with the assistance of a synthetic antibody crystallization chaperone. Structural and biochemical results showed that ALKBH3 utilized two ß-hairpins (ß4-loop-ß5 and ß'-loop-ß'') and the α2 helix to facilitate single-stranded substrate binding. Moreover, a bubble-like region around Asp194 and a key residue inside the active pocket (Thr133) enabled specific recognition and demethylation of m1 A- and 3-methylcytidine (m3 C)-modified substrates. Mutation of Thr133 to the corresponding residue in the AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family members FTO or ALKBH5 converted ALKBH3 substrate selectivity from m1 A to N6 -methyladenosine (m6 A), as did Asp194 deletion. Our findings provide a molecular basis for understanding the mechanisms of substrate recognition and m1 A demethylation by ALKBH3. This study is expected to aid structure-guided design of chemical probes for further functional studies and therapeutic applications.

LncRNA ALKBH3-AS1 enhances ALKBH3 mRNA stability to promote hepatocellular carcinoma cell proliferation and invasion.

Long noncoding RNAs (lncRNAs) are confirmed as the key regulators of hepatocellular carcinoma (HCC) occurrence and progression, but the role of AlkB homologue 3 antisense RNA 1 (ALKBH3-AS1) in HCC is unclear. We revealed the overexpression of ALKBH3-AS1 in HCC tissues. The upregulated levels of ALKBH3-AS1 were observed in HCC cells. ALKBH3-AS1 was expressed in the nucleus and cytoplasm of HCC cells. The high ALKBH3-AS1 expression was markedly associated with a decreased survival rate of HCC patients. ALKBH3-AS1 knockdown repressed and ALKBH3-AS1 overexpression enhanced HCC cell invasion and proliferation. ALKBH3-AS1 silencing restricted HCC growth in vivo. A significant positive correlation between ALKBH3-AS1 and ALKBH3 mRNA levels was confirmed in HCC specimens. ALKBH3-AS1 silencing reduced ALKBH3 expression by stabilizing its mRNA stability in HCC cells. Notably, the impact of ALKBH3 silencing on HCC cells was similar to that of ALKBH3-AS1 knockdown. ALKBH3 restoration prominently attenuated the suppressive effects resulting from ALKBH3-AS1 silencing in HCCLM3 cells. Hypoxia-inducible factor-1α (HIF-1α) transcriptionally activated ALKBH3-AS1 expression in hypoxic HCC cells. ALKBH3-AS1 knockdown markedly attenuated cell proliferation and invasion in hypoxic Huh7 cells. Collectively, HIF-1α-activated ALKBH3-AS1 exerted an oncogenic role by enhancing ALKBH3 mRNA stability in HCC cells.

RNA m1A methylation regulates glycolysis of cancer cells through modulating ATP5D.

Studies on biological functions of RNA modifications such as N6-methyladenosine (m6A) in mRNA have sprung up in recent years, while the roles of N1-methyladenosine (m1A) in cancer progression remain largely unknown. We find m1A demethylase ALKBH3 can regulate the glycolysis of cancer cells via a demethylation activity dependent manner. Specifically, sequencing and functional studies confirm that ATP5D, one of the most important subunit of adenosine 5'-triphosphate synthase, is involved in m1A demethylase ALKBH3-regulated glycolysis of cancer cells. The m1A modified A71 at the exon 1 of ATP5D negatively regulates its translation elongation via increasing the binding with YTHDF1/eRF1 complex, which facilitates the release of message RNA (mRNA) from ribosome complex. m1A also regulates mRNA stability of E2F1, which directly binds with ATP5D promoter to initiate its transcription. Targeted specific demethylation of ATP5D m1A by dm1ACRISPR system can significantly increase the expression of ATP5D and glycolysis of cancer cells. In vivo data confirm the roles of m1A/ATP5D in tumor growth and cancer progression. Our study reveals a crosstalk of mRNA m1A modification and cell metabolism, which expands the understanding of such interplays that are essential for cancer therapeutic application.

The ASCC2 CUE domain in the ALKBH3-ASCC DNA repair complex recognizes adjacent ubiquitins in K63-linked polyubiquitin.

Alkylation of DNA and RNA is a potentially toxic lesion that can result in mutations and even cell death. In response to alkylation damage, K63-linked polyubiquitin chains are assembled that localize the Alpha-ketoglutarate-dependent dioxygenase alkB homolog 3-Activating Signal Cointegrator 1 Complex Subunit (ASCC) repair complex to damage sites in the nucleus. The protein ASCC2, a subunit of the ASCC complex, selectively binds K63-linked polyubiquitin chains via its coupling of ubiquitin conjugation to ER degradation (CUE) domain. The basis for polyubiquitin-binding specificity was unclear, because CUE domains in other proteins typically bind a single ubiquitin and do not discriminate among different polyubiquitin linkage types. We report here that the ASCC2 CUE domain selectively binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. The ASCC2 CUE domain binds the distal ubiquitin in a manner similar to that reported for other CUE domains bound to a single ubiquitin, whereas the contacts with the proximal ubiquitin are unique to ASCC2. Residues in the N-terminal portion of the ASCC2 α1 helix contribute to the binding interaction with the proximal ubiquitin of K63-linked diubiquitin. Mutation of residues within the N-terminal portion of the ASCC2 α1 helix decreases ASCC2 recruitment in response to DNA alkylation, supporting the functional significance of these interactions during the alkylation damage response. Our study reveals the versatility of CUE domains in ubiquitin recognition.

Aberrant RNA methylation triggers recruitment of an alkylation repair complex.

Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase in vitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins.

Programmable RNA N1 -Methyladenosine Demethylation by a Cas13d-Directed Demethylase.

N1 -methyladenosine (m1 A) is a prevalent and reversible RNA modification, which plays a crucial role in the regulation of RNA fate and gene expression. However, the lack of tools to precisely manipulate m1 A sites in specific transcripts has hindered efforts to clarify the association between a specific m1 A-modified transcript and its phenotypic outcomes. Here we develop a CRISPR-Cas13d-based tool called reengineered m1 A modification valid eraser (termed "REMOVER") for targeted m1 A demethylation of a specific transcript. The catalytically inactive RfxCas13d (dCasRx) is fused to the m1 A demethylase ALKBH3, and the dCasRx-ALKBH3 fusion protein can mediate potent demethylation of m1 A-modified RNAs. We further find that REMOVER can specifically demethylate m1 A of MALAT1 and PRUNE1 RNAs, thereby significantly increasing their stability. Our study establishes REMOVER as a tool for targeted RNA demethylation of specific m1 A-modified transcripts, which enables further elucidation of the relationship between m1 A modification of specific transcripts and their phenotypic outcomes.

Single-stranded DNA damage: Protecting the single-stranded DNA from chemical attack.

Cellular processes, such as DNA replication, recombination and transcription, require DNA strands separation and single-stranded DNA is formation. The single-stranded DNA is promptly wrapped by human single-stranded DNA binding proteins, replication protein A (RPA) complex. RPA binding not only prevent nuclease degradation and annealing, but it also coordinates cell-cycle checkpoint activation and DNA repair. However, RPA binding offers little protection against the chemical modification of DNA bases. This review focuses on the type of DNA base damage that occurs in single-stranded DNA and how the damage is rectified in human cells. The discovery of DNA repair proteins, such as ALKBH3, AGT, UNG2, NEIL3, being able to repair the damaged base in the single-stranded DNA, renewed the interest to study single-stranded DNA repair. These mechanistically different proteins work independently from each other with the overarching goal of increasing fidelity of recombination and promoting error-free replication.

Human RAD51 paralogue RAD51C fosters repair of alkylated DNA by interacting with the ALKBH3 demethylase.

The integrity of our DNA is challenged daily by a variety of chemicals that cause DNA base alkylation. DNA alkylation repair is an essential cellular defence mechanism to prevent the cytotoxicity or mutagenesis from DNA alkylating chemicals. Human oxidative demethylase ALKBH3 is a central component of alkylation repair, especially from single-stranded DNA. However, the molecular mechanism of ALKBH3-mediated damage recognition and repair is less understood. We report that ALKBH3 has a direct protein-protein interaction with human RAD51 paralogue RAD51C. We also provide evidence that RAD51C-ALKBH3 interaction stimulates ALKBH3-mediated repair of methyl-adduct located within 3'-tailed DNA, which serves as a substrate for the RAD51 recombinase. We further show that the lack of RAD51C-ALKBH3 interaction affects ALKBH3 function in vitro and in vivo. Our data provide a molecular mechanism underlying upstream events of alkyl adduct recognition and repair by ALKBH3.

ALKBH overexpression in head and neck cancer: potential target for novel anticancer therapy.

The nine identified human homologues of E. coli AlkB 2-oxoglutarate (2OG) and Fe(II)-dependent dioxygenase, ALKBH1-8 and FTO, display different substrate specificities and diverse biological functions. Here we discovered the combined overexpression of members of the ALKBH family in head and neck squamous cell carcinomas (HNSCC). We found direct correlation of ALKBH3 and FTO expression with primary HNSCC tumor size. We observed unidentified thus far cytoplasmic localization of ALKBH2 and 5 in HNSCC, suggesting abnormal role(s) of ALKBH proteins in cancer. Further, high expression of ALKBHs was observed not only in HNSCC, but also in several cancerous cell lines and silencing ALKBH expression in HeLa cancer cells resulted in dramatically decreased survival. Considering the discovered impact of high expression of ALKBH proteins on HNSCC development, we screened for ALKBH blockers among newly synthetized anthraquinone derivatives and demonstrated their potential to support standard anticancer therapy.

Intersections between transcription-coupled repair and alkylation damage reversal.

The response to DNA damage intersects with many other physiological processes in the cell, such as DNA replication, chromatin remodeling, and the cell cycle. Certain damaging lesions, such as UV-induced pyrimidine dimers, also strongly block RNA polymerases, necessitating the coordination of the repair mechanism with remodeling of the elongating transcriptional machinery, in a process called transcription-coupled nucleotide excision repair (TC-NER). This pathway is typically not thought to be engaged with smaller lesions such as base alkylation. However, recent work has uncovered the potential for shared molecular components between the cellular response to alkylation and UV damage. Here, we review our current understanding of the alkylation damage response and its impacts on RNA biogenesis. We give particular attention to the Activating Signal Cointegrator Complex (ASCC), which plays important roles in the transcriptional response during UV damage as well as alkylation damage reversal, and intersects with trichothiodystrophy, an inherited disease associated with TC-NER.

DNA repair enzymes ALKBH2, ALKBH3, and AlkB oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine in vitro.

5-Methylcytosine (5mC) in DNA CpG islands is an important epigenetic biomarker for mammalian gene regulation. It is oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the ten-eleven translocation (TET) family enzymes, which are α-ketoglutarate (α-KG)/Fe(II)-dependent dioxygenases. In this work, we demonstrate that the epigenetic marker 5mC is modified to 5hmC, 5fC, and 5caC in vitro by another class of α-KG/Fe(II)-dependent proteins-the DNA repair enzymes in the AlkB family, which include ALKBH2, ALKBH3 in huamn and AlkB in Escherichia coli. Theoretical calculations indicate that these enzymes may bind 5mC in the syn-conformation, placing the methyl group comparable to 3-methylcytosine, the prototypic substrate of AlkB. This is the first demonstration of the AlkB proteins to oxidize a methyl group attached to carbon, instead of nitrogen, on a DNA base. These observations suggest a broader role in epigenetics for these DNA repair proteins.

Initial evaluation of PET/CT with 18 F-FSU-880 targeting prostate-specific membrane antigen in prostate cancer patients.

This first-in-man study was carried out to evaluate the safety, whole-body distribution, dose estimation, and lesion accumulation of 18 F-FSU-880, a newly developed probe targeting prostate-specific membrane antigen. Six prostate cancer patients with known metastatic lesions underwent serial whole-body PET/computed tomography (CT) with 18 F-FSU-880. Blood and urine were analyzed before and after PET/CT. Accumulation of 18 F-FSU-880 in organs and metastatic lesions in serial PET images were evaluated by measuring the standardized uptake values. From the biodistribution data, the organ doses and whole-body effective dose were calculated using OLINDA/EXM software was developed by Dr. Michael Stabin of Vanderbilt University, Nashville, Tennessee, USA. 18 F-FSU-880 PET/CT could be carried out without significant adverse effects. High physiological uptake was observed in the salivary/lachrymal glands and kidneys. The effective dose was calculated to be 0.921 × 10-2 mSv/MBq. Known metastatic lesions were clearly visualized with high image contrast that increased with time, except in 1 patient, whose bone metastases were well-controlled and inactive. The PET/CT with 18 F-FSU-880 could be carried out safely and could clearly visualize active metastatic lesions. The present results warrant further clinical studies with a larger number of cases to verify the clinical utility of 18 F-FSU-880 PET/CT in the management of prostate cancer patients.

Indenone derivatives as inhibitor of human DNA dealkylation repair enzyme AlkBH3.

The mammalian AlkB homologue-3 (AlkBH3) is a member of the dioxygenase family of enzymes that in humans is involved in DNA dealkylation repair. Because of its role in promoting tumor cell proliferation and metastasis of cancer, extensive efforts are being directed in developing selective inhibitors for AlkBH3. Here we report synthesis, screening and evaluation of panel of arylated indenone derivatives as new class of inhibitors of AlkBH3 DNA repair activity. An efficient synthesis of 2,3-diaryl indenones from 2,3-dibromo indenones was achieved via Suzuki-Miyaura cross-coupling. Using a robust quantitative assay, we have obtained an AlkBH3 inhibitor that display specific binding and competitive mode of inhibition against DNA substrate. Finally, we established that this compound could prevent the proliferation of lung cancer cell line and enhance sensitivity to DNA damaging alkylating agent.

A ubiquitin-dependent signalling axis specific for ALKBH-mediated DNA dealkylation repair.

DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signalling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks. Understanding the upstream signalling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation. Here we demonstrate that human cells have a previously unrecognized signalling mechanism for sensing damage induced by alkylation. We find that the alkylation repair complex ASCC (activating signal cointegrator complex) relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of the subunit ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RING finger protein 113A (RNF113A) as the E3 ligase responsible for upstream ubiquitin signalling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy, which harbour a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked trichothiodystrophy.

Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes.

Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.

CpG promoter methylation of the ALKBH3 alkylation repair gene in breast cancer.

BACKGROUND: DNA repair of alkylation damage is defective in various cancers. This occurs through somatically acquired inactivation of the MGMT gene in various cancer types, including breast cancers. In addition to MGMT, the two E. coli AlkB homologs ALKBH2 and ALKBH3 have also been linked to direct reversal of alkylation damage. However, it is currently unknown whether ALKBH2 or ALKBH3 are found inactivated in cancer. METHODS: Methylome datasets (GSE52865, GSE20713, GSE69914), available through Omnibus, were used to determine whether ALKBH2 or ALKBH3 are found inactivated by CpG promoter methylation. TCGA dataset enabled us to then assess the impact of CpG promoter methylation on mRNA expression for both ALKBH2 and ALKBH3. DNA methylation analysis for the ALKBH3 promoter region was carried out by pyrosequencing (PyroMark Q24) in 265 primary breast tumours and 30 proximal normal breast tissue samples along with 8 breast-derived cell lines. ALKBH3 mRNA and protein expression were analysed in cell lines using RT-PCR and Western blotting, respectively. DNA alkylation damage assay was carried out in cell lines based on immunofluorescence and confocal imaging. Data on clinical parameters and survival outcomes in patients were obtained and assessed in relation to ALKBH3 promoter methylation. RESULTS: The ALKBH3 gene, but not ALKBH2, undergoes CpG promoter methylation and transcriptional silencing in breast cancer. We developed a quantitative alkylation DNA damage assay based on immunofluorescence and confocal imaging revealing higher levels of alkylation damage in association with epigenetic inactivation of the ALKBH3 gene (P = 0.029). In our cohort of 265 primary breast cancer, we found 72 cases showing aberrantly high CpG promoter methylation over the ALKBH3 promoter (27%; 72 out of 265). We further show that increasingly higher degree of ALKBH3 promoter methylation is associated with reduced breast-cancer specific survival times in patients. In this analysis, ALKBH3 promoter methylation at >20% CpG methylation was found to be statistically significantly associated with reduced survival (HR = 2.3; P = 0.012). By thresholding at the clinically relevant CpG methylation level (>20%), we find the incidence of ALKBH3 promoter methylation to be 5% (13 out of 265). CONCLUSIONS: ALKBH3 is a novel addition to the catalogue of DNA repair genes found inactivated in breast cancer. Our results underscore a link between defective alkylation repair and breast cancer which, additionally, is found in association with poor disease outcome.