Cancer Mutagenesis Research Articles

FAM72A degrades UNG2 through the GID/CTLH complex to promote mutagenic repair during antibody maturation

A diverse antibody repertoire is essential for humoral immunity. Antibody diversification requires the introduction of deoxyuridine (dU) mutations within immunoglobulin genes to initiate somatic hypermutation (SHM) and class switch recombination (CSR). dUs are normally recognized and excised by the base excision repair (BER) protein uracil-DNA glycosylase 2 (UNG2). However, FAM72A downregulates UNG2 permitting dUs to persist and trigger SHM and CSR. How FAM72A promotes UNG2 degradation is unknown. Here, we show that FAM72A recruits a C-terminal to LisH (CTLH) E3 ligase complex to target UNG2 for proteasomal degradation. Deficiency in CTLH complex components result in elevated UNG2 and reduced SHM and CSR. Cryo-EM structural analysis reveals FAM72A directly binds to MKLN1 within the CTLH complex to recruit and ubiquitinate UNG2. Our study further suggests that FAM72A hijacks the CTLH complex to promote mutagenesis in cancer. These findings show that FAM72A is an E3 ligase substrate adaptor critical for humoral immunity and cancer development.

The cytidine deaminase APOBEC3A regulates nucleolar function to promote cell growth and ribosome biogenesis.

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.

ASS1 metabolically contributes to the nuclear and cytosolic p53-mediated DNA damage response

Downregulation of the urea cycle enzyme argininosuccinate synthase (ASS1) in multiple tumors is associated with a poor prognosis partly because of the metabolic diversion of cytosolic aspartate for pyrimidine synthesis, supporting proliferation and mutagenesis owing to nucleotide imbalance. Here, we find that prolonged loss of ASS1 promotes DNA damage in colon cancer cells and fibroblasts from subjects with citrullinemia type I. Following acute induction of DNA damage with doxorubicin, ASS1 expression is elevated in the cytosol and the nucleus with at least a partial dependency on p53; ASS1 metabolically restrains cell cycle progression in the cytosol by restricting nucleotide synthesis. In the nucleus, ASS1 and ASL generate fumarate for the succination of SMARCC1, destabilizing the chromatin-remodeling complex SMARCC1–SNF5 to decrease gene transcription, specifically in a subset of the p53-regulated cell cycle genes. Thus, following DNA damage, ASS1 is part of the p53 network that pauses cell cycle progression, enabling genome maintenance and survival. Loss of ASS1 contributes to DNA damage and promotes cell cycle progression, likely contributing to cancer mutagenesis and, hence, adaptability potential.

Her2 amplification, Rel-A, and Bach1 can influence APOBEC3A expression in breast cancer cells.

APOBEC-induced mutations occur in 50% of sequenced human tumors, with APOBEC3A (A3A) being a major contributor to mutagenesis in breast cancer cells. The mechanisms that cause A3A activation and mutagenesis in breast cancers are still unknown. Here, we describe factors that influence basal A3A mRNA transcript levels in breast cancer cells. We found that basal A3A mRNA correlates with A3A protein levels and predicts the amount of APOBEC signature mutations in a panel of breast cancer cell lines, indicating that increased basal transcription may be one mechanism leading to breast cancer mutagenesis. We also show that alteration of ERBB2 expression can drive A3A mRNA levels, suggesting the enrichment of the APOBEC mutation signature in Her2-enriched breast cancer could in part result from elevated A3A transcription. Hierarchical clustering of transcripts in primary breast cancers determined that A3A mRNA was co-expressed with other genes functioning in viral restriction and interferon responses. However, reduction of STAT signaling via inhibitors or shRNA in breast cancer cell lines had only minor impact on A3A abundance. Analysis of single cell RNA-seq from primary tumors indicated that A3A mRNA was highest in infiltrating immune cells within the tumor, indicating that correlations of A3A with STAT signaling in primary tumors may be result from higher immune infiltrates and are not reflective of STAT signaling controlling A3A expression in breast cancer cells. Analysis of ATAC-seq data in multiple breast cancer cell lines identified two transcription factor sites in the APOBEC3A promoter region that could promote A3A transcription. We determined that Rel-A, and Bach1, which have binding sites in these peaks, elevated basal A3A expression. Our findings highlight a complex and variable set of transcriptional activators for A3A in breast cancer cells.

A real-time biochemical assay for quantitative analyses of APOBEC-catalyzed DNA deamination

Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a need for biochemical tools that can be used to identify and characterize potential inhibitors of this enzyme family. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination (RADD) assay. This assay offers a single-step set-up and real-time fluorescent read-out, and it is capable of providing insights into enzyme kinetics and also offering a high-sensitivity and easily scalable method for identifying APOBEC3 inhibitors. This assay serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit and possesses the versatility to be readily adapted into a high-throughput format for inhibitor discovery.

Abstract 403: APOBEC3 enzymes induce chromosomal translocations in solid cancer

Abstract Introduction: Chromosomal translocations are common oncogenic drivers in human cancers, and an increasing number of translocations are being considered as crucial diagnostic and prognostic markers in clinics. In B-cell lymphoma, a number of chromosomal translocations are directed by the off-target activity of the activation-induced cytidine deaminase (AID; a B-cell specific apolipoprotein B mRNA-editing enzyme, catalytic polypeptode-like (APOBEC) family enzyme). However, in solid tumors, no such enzymes capable of directing chromosomal translocations have been identified. Given recent findings on APOBEC-mediated mutagenesis in solid cancers, APOBECs may be involved in the formation of chromosomal translocations in solid tumors, like AID does in B cells. In human, the APOBEC3 (A3) family consists of seven members (A3A-A3H) and is associated with C <t or="" c<g="" substitutions.="" recent="" evidence="" shows="" that="" a3a="" might="" be="" involved="" in="" chromosomal="" instability="" leading="" to="" drug="" resistance="" lung="" cancer.="" however,="" it="" has="" not="" been="" explored="" whether="" a3s="" generate="" dna="" dsbs,="" eventually="" translocations.="" this="" study,="" we="" investigated="" the="" role="" of="" and="" their="" mechanisms="" translocations="" solid="" cancer.Methods: We applied high-throughput genome-wide translocation sequencing (HTGTS) approach to map genome-wide chromosomal translocations induced by human A3s in mouse fibroblast and human epithelial cells. We correlated patterns of translocations induced by A3s with several other cellular features including transcription, DNA accessibility, DNA replication timing, and mutational signatures. We validated the contribution of A3 to the generation of oncogenic translocations in lung cancer. Results: Ectopic expression of A3s significantly increased chromosomal translocations in mouse fibroblasts and human epithelial cells. A3s and AID preferentially induced translocations in different parts of the genome, but some of the translocation hotspots were shared among them. Hierarchical clustering of hotspots showed that A3-mediated translocations were distinguished from AID-mediated translocations. Notably, the junction distribution of breakpoint clusters showed three distinct translocation patterns based on the locations of breakpoint clusters: (1) AID-preferred; (2) A3-preferred; and (3) AID/A3-mixed. AID-mediated translocations were heavily enriched near the transcription start site, whereas A3-mediated translocations were dispersed across the gene body and transcription end site. Furthermore, DNA replication timing and genomic architectures were linked to the formation of A3-mediated chromosomal translocations in solid cancer. Finally, A3 contributed to the formation of oncogenic rearrangements in lung cancer. Conclusions: A3 enzymes induced chromosomal translocations with distinct patterns compared to AID and facilitated chromosomal translocation formation in solid cancers. Citation Format: Taek-Chin Cheong, Qi Wang, Ahram Jang, Elif Karaca-Atabay, Roberto Chiarle. APOBEC3 enzymes induce chromosomal translocations in solid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 403.

Childhood cancer mutagenesis caused by transposase-derived PGBD5.

Genomic rearrangements are a hallmark of most childhood tumors, including medulloblastoma, one of the most common brain tumors in children, but their causes remain largely unknown. Here, we show that PiggyBac transposable element derived 5 (Pgbd5) promotes tumor development in multiple developmentally accurate mouse models of Sonic Hedgehog (SHH) medulloblastoma. Most Pgbd5-deficient mice do not develop tumors, while maintaining normal cerebellar development. Ectopic activation of SHH signaling is sufficient to enforce cerebellar granule cell progenitor-like cell states, which exhibit Pgbd5-dependent expression of distinct DNA repair and neurodevelopmental factors. Mouse medulloblastomas expressing Pgbd5 have increased numbers of somatic structural DNA rearrangements, some of which carry PGBD5-specific sequences at their breakpoints. Similar sequence breakpoints recurrently affect somatic DNA rearrangements of known tumor suppressors and oncogenes in medulloblastomas in 329 children. This identifies PGBD5 as a medulloblastoma mutator and provides a genetic mechanism for the generation of oncogenic DNA rearrangements in childhood cancer.

Abstract 3563: Multi-modal mutational signatures elucidate the comprehensive consequences of mutational processes

Abstract The development of mutational signatures defined individually in the contexts of single/double base substitutions, insertions and deletions, structural variants and copy number variation have led to improved understanding of cancer mutagenesis and potential for improved patient stratification. We hypothesized that combining these contexts into a multi-modal mutational signature framework may improve the accuracy of assigning mutational processes to samples, and provide a holistic view of the full repertoire of mutations conferred from such processes. In order to develop multi-modal signatures, bespoke feature engineering was required for each mutational modality to enhance signal, in addition to individual modality matrix normalization so that the data could be harmonized and analyzed as a whole. These methods, and subsequent signature extraction were developed on 2,438 whole genome sequenced cancers from 21 distinct cancer types from the PCAWG consortium for which all modalities of data were available, incorporating a 348 channel signature definition. Deployment of these methods identified multi-modal signatures that encompass multiple mutation types known to associate with specific processes, for example SBS, indel and rearrangement channels defining a signature of homologous recombination deficiency. In contrast, some signatures remain defined by a single mutational modality, highlighting the distinction between broadly mutational processes, and more focused mutation processes. Multi-modal signatures show promise in increasing accuracy for difficult to assign signatures, such as those for homologous recombination deficiency, and therefore in improving patient stratification for life-saving treatment. Citation Format: Christopher D. Steele, Ludmil Alexandrov, Azhar Khandekar. Multi-modal mutational signatures elucidate the comprehensive consequences of mutational processes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3563.

RADD: A real-time FRET-based biochemical assay for DNA deaminase studies.

In recent years, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become ever more apparent. This growing awareness and lack of inhibitory drugs has created a distinct need for biochemical tools that can be used to identify and characterize potential inhibitors of this family of enzymes. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination (RADD) assay. The RADD assay provides a rapid, real-time fluorescence readout of APOBEC3 DNA deamination and serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit. This method improves upon contemporary DNA deamination assays by offering a more rapid and quantifiable readout as well as providing a platform that is readily adaptable to a high-throughput format for inhibitor discovery. In this chapter we provide a detailed guide for the usage of the RADD assay for the characterization of APOBEC3 enzymes and potential inhibitors.

Constructing an APOBEC-related gene signature with predictive value in the overall survival and therapeutic sensitivity in lung adenocarcinoma

BackgroundAPOBEC family play an important role in cancer mutagenesis and tumor development. The role of APOBEC family in lung adenocarcinoma (LUAD) has not been studied comprehensively. Materials and methodsThe expression data of pan-cancer as well as LUAD was obtained from public databases. The expression level of APOBEC family genes was analyzed in different normal and cancer tissues. APOBEC mutagenesis enrichment score (AMES) was utilized to evaluate the APOBEC-induced mutations and the relation of APOBEC with genomic instability. Gene set enrichment analysis was used to identify differentially enriched pathways. Univariate Cox regression and Lasso regression were applied to screen key prognostic genes. The immune cell infiltration was estimated by CIBERSORT. RT-qPCR assay, CCK-8 and Transwell assay were conducted to explore gene expression and lung cancer cell invasion. ResultsCancer tissues had significantly altered expression of APOBEC family genes and the expression patterns of APOBEC family were different in different cancer types. APOBEC3B was the most aberrantly expressed in most cancer types. In LUAD, we observed a significantly positive correlation of AMES with intratumor heterogeneity (ITH), tumor neoantigen burden (TNB), and tumor mutation burden (TMB). High AMES group had high mutation counts of DNA damage repair pathways, and high enrichment of cell cycle and DNA repair pathways. We identified four prognostic genes (LYPD3, ANLN, MUC5B, and FOSL1) based on AMES, and constructed an AMES-related gene signature. The expressions of four genes were enhanced and accelerated the invasion ability and viability of lung cancer cells. Furthermore, we found that high group increased oxidative stress level. ConclusionsAPOBEC family was associated with genomic instability, DNA damage-related pathways, and cell cycle in LUAD. The AMES-related gene signature had a great potential to indicate the prognosis and guide immunotherapy/chemotherapy for patients suffering from LUAD.

2313P APOBEC mutagenesis and macrophage infiltration in cancer

2313P APOBEC mutagenesis and macrophage infiltration in cancer

APOBEC3B regulates R-loops and promotes transcription-associated mutagenesis in cancer

The single-stranded DNA cytosine-to-uracil deaminase APOBEC3B is an antiviral protein implicated in cancer. However, its substrates in cells are not fully delineated. Here APOBEC3B proteomics reveal interactions with a surprising number of R-loop factors. Biochemical experiments show APOBEC3B binding to R-loops in cells and in vitro. Genetic experiments demonstrate R-loop increases in cells lacking APOBEC3B and decreases in cells overexpressing APOBEC3B. Genome-wide analyses show major changes in the overall landscape of physiological and stimulus-induced R-loops with thousands of differentially altered regions, as well as binding of APOBEC3B to many of these sites. APOBEC3 mutagenesis impacts genes overexpressed in tumors and splice factor mutant tumors preferentially, and APOBEC3-attributed kataegis are enriched in RTCW motifs consistent with APOBEC3B deamination. Taken together with the fact that APOBEC3B binds single-stranded DNA and RNA and preferentially deaminates DNA, these results support a mechanism in which APOBEC3B regulates R-loops and contributes to R-loop mutagenesis in cancer.

New insights into APOBEC-mediated mutagenesis in prostate cancer.

New insights into APOBEC-mediated mutagenesis in prostate cancer.

Loss of SYNCRIP unleashes APOBEC-driven mutagenesis, tumor heterogeneity, and AR-targeted therapy resistance in prostate cancer.

Tumor mutational burden and heterogeneity has been suggested to fuel resistance to many targeted therapies. The cytosine deaminase APOBEC proteins have been implicated in the mutational signatures of more than 70% of human cancers. However, the mechanism underlying how cancer cells hijack the APOBEC mediated mutagenesis machinery to promote tumor heterogeneity, and thereby foster therapy resistance remains unclear. We identify SYNCRIP as an endogenous molecular brake which suppresses APOBEC-driven mutagenesis in prostate cancer (PCa). Overactivated APOBEC3B, in SYNCRIP-deficient PCa cells, is a key mutator, representing the molecular source of driver mutations in some frequently mutated genes in PCa, including FOXA1, EP300. Functional screening identifies eight crucial drivers for androgen receptor (AR)-targeted therapy resistance in PCa that are mutated by APOBEC3B: BRD7, CBX8, EP300, FOXA1, HDAC5, HSF4, STAT3, and AR. These results uncover a cell-intrinsic mechanism that unleashes APOBEC-driven mutagenesis, which plays a significant role in conferring AR-targeted therapy resistance in PCa.

Abstract PR001: An endogenous molecular brake preventing APOBEC-driven tumor mutational burden, heterogeneity and AR therapy resistance

Abstract Tumor mutational burden and heterogeneity fuel resistance to many targeted therapies, which inevitably limits the clinical outcome of patients. The cytosine deaminase APOBEC proteins are major drivers of mutagenesis in human cancer, with over 70% of tumors exhibiting a mutational signature that is impacted by APOBEC activity. Nevertheless, the mechanisms through which tumor cells hijack the powerful APOBEC mutagenesis machinery to promote heterogeneity and therapy resistance remain largely unknown. Through a multi-disciplinary approach integrating bulk and single cell RNA-Seq (scRNA-Seq), whole-genome exome-sequencing (WES), and CRISPR library screening, we identified a long sought-after cell-intrinsic mechanism that prevents APOBEC-driven mutagenesis in non-malignant cells. The loss of this “molecular brake” unleashes APOBEC3B-driven mutagenesis in malignant cells, which then becomes a key mutator and represents the long sought-after molecular source of driver mutations in some frequently mutated genes in cancers, including FOXA1, EP300, and AR. Functional screening identified eight crucial drivers for Androgen Receptor (AR)-targeted therapy resistance in prostate cancer that are mutated by APOBEC3B: BRD7, CBX8, EP300, FOXA1, HDAC5, HSF4, STAT3, and AR. Finally, contrary to conventional understanding, our study reveals that driver mutations in FOXA1 induced by APOBEC3B, not mutations in AR, evolutionarily outcompete other driver mutations and eventually dominate the resistant tumors. Collectively, these results uncover a cell-intrinsic mechanism that unleashes APOBEC-driven mutagenesis, which plays a significant role in conferring targeted therapy resistance and could be the potential therapeutic targets to overcome resistance. Citation Format: Ping Mu, Xiaoling Li, Yunguan Wang. An endogenous molecular brake preventing APOBEC-driven tumor mutational burden, heterogeneity and AR therapy resistance [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR001.

241P APOBEC3-induced PIK3CA mutations predict better clinical outcomes with PI3K inhibitors in patients with PIK3CA-mutated advanced breast cancer

APOBEC3 enzymes are a key source of mutagenesis in breast cancer. PI3K inhibitors are currently used in the treatment of PIK3CA-mutated advanced breast cancer. However, whether specific PIK3CA mutations (muts) are associated with better outcomes is unclear. We developed a computational framework to screen for APOBEC3-induced mutations in PIK3CA within the TCW context using whole-exome sequencing (WES) data from the breast cancer cohort of the cancer genome atlas (TCGA-BRCA) and an independent cohort of patients (pts.) with ER+/HER2- PIK3CA-mutated advanced breast cancer who were treated with PI3K and aromatase inhibitors (AI). Using the WES of 696 pts with luminal disease from the TCGA-BRCA cohort, we identified 296 pts (42.5%) with PIK3CA muts. Of these, our screen retrieved 104 pts (35.1%) with APOBEC3-induced PIK3CA muts (A3P). There was no significant difference in the median overall survival in pts with A3P compared to non-A3P mutant pts (129 vs. 114 months, p=0.49), and neither significant difference in TMB (1.3 vs 1.17 mut/Mb, p=0.75). ESR1 protein was significantly less expressed in A3P compared to non-A3P mutant samples (log2 ratio: -0.42, p=0.04). The majority of A3P muts (95%) were found in the helical domain of PIK3CA. A3P muts were numerically higher in lobular vs. ductal subtypes (43% vs. 32%, p=0.08). We validated our prioritized A3P muts using a cohort of 65 pts with PIK3CA-mutated ER+/HER2- advanced cancer. The rate of A3Pm was 45% (n=29) which was higher than the rate in pts with non-metastatic cancer in TCGA-BRCA. Pts with A3P muts were significantly associated with longer median progression-free survival on PI3K inhibitor and AI (5 vs. 2 months, hazard ratio: 0.56, 95%CI:0.31-1.00, p=0.02) and a trend toward higher objective response rate (26% vs. 5%, p=0.06). We identified a patient with the A3P E453K muts who achieved partial response for 11 months on alpelisib and AI. This mutation is not routinely included in the companion diagnostic test. Our findings indicate that using a prioritized catalog of APOBEC3-induced mutations in PIK3CA could identify driver mutations that confer oncogenic addiction, hence better response to PI3K inhibitors.

Abstract 93: An endogenous molecular brake preventing APOBEC-driven tumor mutational burden, heterogeneity and therapy resistance

Abstract Tumor mutational burden and heterogeneity fuel resistance to many targeted therapies, which inevitably limits the clinical outcome of patients. The cytosine deaminase APOBEC proteins are major drivers of mutagenesis in human cancer, with over 70% of tumors exhibiting a mutational signature that is impacted by APOBEC activity. Nevertheless, the mechanisms through which tumor cells hijack the powerful APOBEC mutagenesis machinery to promote heterogeneity and therapy resistance remain largely unknown. Through a multi-disciplinary approach integrating bulk and single cell RNA-Seq (scRNA-Seq), whole-genome exome-sequencing (WES), and CRISPR library screening, we identified a long sought-after cell-intrinsic mechanism that prevents APOBEC-driven mutagenesis in non-malignant cells. The loss of this “molecular brake”, unleashes APOBEC3B-driven mutagenesis in malignant cells, which then becomes a key mutator and represents the long sought-after molecular source of driver mutations in some frequently mutated genes in cancers, including FOXA1, EP300, and AR. Functional screening identified eight crucial drivers for Androgen Receptor (AR)-targeted therapy resistance in prostate cancer that are mutated by APOBEC3B: BRD7, CBX8, EP300, FOXA1, HDAC5, HSF4, STAT3 and AR. Finally, contrary to conventional understanding, our study reveals that driver mutations in FOXA1 induced by APOBEC3B, not mutations in AR, evolutionarily outcompete other driver mutations and eventually dominate the resistant tumors. Collectively, these results uncover a cell-intrinsic mechanism that unleashes APOBEC-driven mutagenesis, which plays a significant role in conferring targeted therapy resistance and could be the potential therapeutic targets to overcome resistance. Citation Format: Ping Mu, Xiaoling Li, Yunguan Wang, Tao Wang, Su Deng. An endogenous molecular brake preventing APOBEC-driven tumor mutational burden, heterogeneity and therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 93.

Involvement of APOBEC3A/B Deletion in Mouse Mammary Tumor Virus (MMTV)-like Positive Human Breast Cancer.

The association between mouse mammary tumor virus (MMTV)-like sequences and human breast cancer (BC) is largely documented in the literature, but further research is needed to determine how they influence carcinogenesis. APOBEC3 cytidine deaminases are viral restriction factors that have been implicated in cancer mutagenesis, and a germline deletion that results in the fusion of the APOBEC3A coding region with the APOBEC3B 3'-UTR has been linked to increased mutagenic potential, enhanced risk of BC development, and poor prognosis. However, little is known about factors influencing APOBEC3 family activation in cancer. Thus, we hypothesized that MMTV infection and APOBEC3-mediated mutagenesis may be linked in the pathogenesis of BC. We investigated APOBEC3A/B genotyping, MMTV-like positivity, and clinicopathological parameters of 209 BC patients. We show evidence for active APOBEC3-mediated mutagenesis in human-derived MMTV sequences and comparatively investigate the impact of APOBEC3A/B germline deletion in MMTV-like env positive and negative BC in a Brazilian cohort. In MMTV-like negative samples, APOBEC3A/B deletion was negatively correlated with tumor stage while being positively correlated with estrogen receptor expression. Although APOBEC3A/B was not associated with MMTV-like positivity, samples carrying both MMTV-like positivity and APOBEC3A/B deletion had the lowest age-at-diagnosis of all study groups, with all patients being less than 50 years old. These results indicate that APOBEC3 mutagenesis is active against MMTV-like sequences, and that APOBEC3A/B deletion might act along with the MMTV-like presence to predispose people to early-onset BC.

Hypoxia, a Targetable Culprit to Counter Pancreatic Cancer Resistance to Therapy

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, and it is a disease of dismal prognosis. While immunotherapy has revolutionized the treatment of various solid tumors, it has achieved little success in PDAC. Hypoxia within the stroma-rich tumor microenvironment is associated with resistance to therapies and promotes angiogenesis, giving rise to a chaotic and leaky vasculature that is inefficient at shuttling oxygen and nutrients. Hypoxia and its downstream effectors have been implicated in immune resistance and could be contributing to the lack of response to immunotherapy experienced by patients with PDAC. Paradoxically, increasing evidence has shown hypoxia to augment genomic instability and mutagenesis in cancer, suggesting that hypoxic tumor cells could have increased production of neoantigens that can potentially enable their clearance by cytotoxic immune cells. Strategies aimed at relieving this condition have been on the rise, and one such approach opts for normalizing the tumor vasculature to reverse hypoxia and its downstream support of tumor pathogenesis. An important consideration for the successful implementation of such strategies in the clinic is that not all PDACs are equally hypoxic, therefore hypoxia-detection approaches should be integrated to enable optimal patient selection for achieving improved patient outcomes.

APOBEC3A Catalytic Inactivity Mutation Induces Tertiary Structure Destabilization.

APOBEC3A (A3A)-catalyzed DNA cytosine deamination is implicated in virus and cancer mutagenesis, and A3A is a target for small molecule drug discovery. The catalytic glutamic acid (E72) is frequently mutated in biochemical studies to characterize deamination-dependent versus deamination-independent A3A functions. Additionally, catalytically active A3A is toxic in bacterial expression systems, which adversely affects yield during recombinant A3A expression. Here, we demonstrate that mutating the catalytic glutamic acid to an isosteric glutamine (E72Q) significantly decreases the thermal stability of the protein, compared to the alanine-inactivating mutation (E72A). Differential scanning fluorimetry and mass spectrometry reveal that A3A E72Q is less thermally stable than A3A E72A or wild-type A3A. Strikingly, A3A E72Q is partially denatured at 37 °C and binds single-stranded DNA with significantly poorer affinity compared to A3A E72A. This study constitutes an important cautionary note on A3A protein design and informs that A3A E72A is the preferred catalytic inactivation mutation for most applications.