Biology subjects

Durfee, C.

Publications and source records attributed to Durfee, C..

9 recordsLinked to original sources

bioRxiv2025

Mus Musculus papillomavirus MmuPV1 resists restriction by human APOBEC3B

The single-stranded DNA deaminase APOBEC3B (A3B) is capable of potently restricting the replication of a range of viruses including retroviruses (cDNA) and herpesviruses (genomic DNA). However, these and likely other DNA virus families have evolved host species-specific counter-defenses that are equally potent and serve to protect viral DNA from restriction. Although high-risk human papillomavirus (HPV) infection triggers A3B upregulation, potentially as part of an antiviral response, the impact of this restriction factor on papillomavirus replication and pathogenesis has yet to be assessed. To study human A3B antiviral function in the absence of a species-specific counter-defense, here we ask whether human A3B is capable of restricting Mus musculus papillomavirus (MmuPV1) in cellulo and in vivo. First, we created human A3B and catalytic mutant A3B-E255A expressing FVB/N mice. Second, MmuPV1 gene expression and replication was quantified in primary keratinocytes from these animals and, surprisingly, enzymatically active human A3B caused no measurable impairment in viral transcript or DNA accumulation. Third, A3B, catalytic mutant A3B-E255A, and nontransgenic FVB/N animals were infected with MmuPV1 and similar pathologies were found regardless of A3B functionality. Thus, despite likely never being exposed to human A3B during evolution, MmuPV1 appears to be unaffected by this potent, primate-specific antiviral factor. These results suggest that MmuPV1 and perhaps papillomaviruses more broadly possess a conserved mechanism to efficiently escape restriction by human A3B and related DNA deaminases. IMPORTANCEHuman papillomaviruses (HPVs) are nearly ubiquitous, and persistent infection with high-risk types causes approximately 5% of cancers worldwide. Although HPV vaccination is effective for preventing infection, insufficient global coverage and a rising incidence of HPV-associated malignancies, such as oropharyngeal carcinoma, highlight the need to understand innate virus clearance mechanisms. APOBEC3 enzymes are a central component of the mammalian innate immune system and are hypothesized to restrict papillomavirus infection, particularly between species. Here, we establish mice that express the human antiviral enzyme APOBEC3B (A3B). Surprisingly, we find that human A3B is incapable of blocking the replication of a murine papillomavirus (Mus musculus papillomavirus 1, MmuPV1) in relevant primary cells from these animals or in infected tissues in vivo. These findings highlight the complexity of teasing apart host-pathogen interactions and suggest that papillomaviruses may have a general mechanism for escaping restriction by antiviral enzymes such as A3B.

microbiology↗

bioRxiv2025

Murine models for triple-negative breast cancer with differential responsiveness to immunotherapy

Breast cancer is the most common cancer diagnosis in women. Clinical studies with triple-negative breast cancer (TNBC) are encouraging for immunotherapy combined with chemotherapy (anti-PD-1 with paclitaxel and/or carboplatin). However, additional clinical advances may be pursued more rapidly with assistance from preclinical TNBC models including syngeneic mammary tumor cell lines. Here, we report two mammary tumor cell lines that exhibit differential responsiveness to immunotherapy in vivo. Spontaneous mammary tumors from C57BL/6J MMTV-Cre Trp53fl/+ animals were passaged serially in cell culture and in vivo in the mammary fat pad of fully wildtype animals. The resulting lines, MM001i and MM008i, lost Trp53 and formed 1000 mm3 tumors in the mammary fat pad within 21-28 days. Despite originating from the same genetic background, these lines exhibit differential responses to immunotherapy. For anti-PD-1 therapy, MM001i is poorly responsive and MM008i is strongly responsive with near-complete tumor regression. In comparison, both MM001i and MM008i respond rapidly to anti-CTLA-4 therapy. Both models express unique tumor antigens as evidenced by immunity to subsequent engraftments. Primary MM008i tumors exhibit greater T cell infiltration, and CD8-positive T lymphocytes are required for anti-PD-1 responses. These TNBC models are promising for further mechanistic studies and testing future single and combinatorial therapies.

cancer biology↗

bioRxiv2025

Tobacco smoke carcinogens exacerbate APOBEC mutagenesis and carcinogenesis

Mutational processes are thought to act independently and additively. We challenge this view by demonstrating that DNA-adducting agents sensitize the genome to mutagenesis by APOBEC enzymes. A model tobacco carcinogen (NQO) triggers a 100-fold increase in APOBEC3B- catalyzed signature mutations and elevates oral carcinoma levels in vivo. A hallmark of this unidirectional synergy is strand-coordinated pairs of APOBEC signature mutations within 32nt of each other (didyma). Biochemical experiments show that APOBEC3B can catalyze didyma formation, and genetic studies demonstrate requirements for APOBEC3B and functional nucleotide excision repair (XPA in human cells and Rad14 in yeast). Analyses of lung and head & neck cancers show that APOBEC mutagenesis and didyma are elevated in tumors from smokers compared to non-smokers. Additional tumor types with links to DNA-adducting agents also exhibit didyma. These studies support a model in which DNA-adducting carcinogens activate nucleotide excision repair and amplify mutagenesis by APOBEC enzymes in cancer.

cancer biology↗

bioRxiv2023

Mesoscale DNA Features Impact APOBEC3A and APOBEC3B Deaminase Activity and Shape Tumor Mutational Landscapes

Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets singlestranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have been fully established, and the specific influence of the DNA sequence on APOBEC3A APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B selectively targets DNA stem-loop structures, and they are distinct from those subjected deamination by APOBEC3A. We develop Oligo-seq, a novel in vitro sequencing-based to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A an APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate mutation landscapes in cancer genomes, driven by their unique substrate selectivity.

biochemistry↗

bioRxiv2023

Human APOBEC3B promotes tumor heterogeneity in vivo including signature mutations and metastases

The antiviral DNA cytosine deaminase APOBEC3B has been implicated as a source of mutation in many different cancers. Despite over 10 years of work, a causal relationship has yet to be established between APOBEC3B and any stage of carcinogenesis. Here we report a murine model that expresses tumor-like levels of human APOBEC3B after Cre-mediated recombination. Animals appear to develop normally with full-body expression of APOBEC3B. However, adult males manifest infertility and older animals of both sexes show accelerated rates of tumorigenesis (mostly lymphomas or hepatocellular carcinomas). Interestingly, primary tumors also show overt heterogeneity, and a subset spreads to secondary sites. Both primary and metastatic tumors exhibit increased frequencies of C-to-T mutations in TC dinucleotide motifs consistent with the established biochemical activity of APOBEC3B. Elevated levels of structural variation and insertion-deletion mutations also accumulate in these tumors. Together, these studies provide the first cause-and-effect demonstration that human APOBEC3B is an oncoprotein capable of causing a wide range of genetic changes and driving tumor formation in vivo.

cancer biology↗

bioRxiv2021

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in non-structural protein 14 (nsp14), which excises nucleotides including antiviral drugs mis-incorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here we determined a 1.6-[A] resolution crystal structure of SARS-CoV-2 ExoN in complex with its essential co-factor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. Molecular dynamics simulations further show remarkable flexibility of multi-domain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA-binding to support its exoribonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anti-coronaviral drugs or strategies to attenuate the viral virulence.

biochemistry↗

bioRxiv2020

APOBEC3B expression generates an immunogenic model of Kras mutant lung cancer

Mutations in oncogenes such as KRAS and EGFR cause a high proportion of lung cancers. Drugs targeting these proteins cause tumour regression but ultimately fail to cure these cancers, leading to intense interest in how best to combine them with other treatments, such as immunotherapies. However, preclinical systems for studying the interaction of lung tumours with the host immune system are inadequate, in part due to the low tumour mutational burden in genetically engineered mouse models. Here we set out to develop mouse models of mutant KRAS-driven lung cancer with an elevated tumour mutational burden by expressing the human DNA cytosine deaminase, APOBEC3B, to mimic the mutational signature seen in human lung cancer. This failed to substantially increase clonal tumour mutational burden and autochthonous tumours remained refractory to immunotherapy. However, by establishing clonal cell lines from these tumours we generated an immunogenic syngeneic transplantation model of KRAS mutant lung adenocarcinoma that was sensitive to immunotherapy. Unexpectedly, we found that anti-tumour immune responses were not directed against neoantigens but instead targeted derepressed endogenous retroviral antigens. The ability of KRASG12C inhibitors to cause regression of KRASG12C-expressing versions of these tumours was markedly potentiated by the adaptive immune system, providing a unique opportunity for the study of combinations of targeted and immunotherapies in immune-hot lung cancer.

cancer biology↗

bioRxiv2020

Targeted cancer therapy induces APOBEC fuelling the evolution of drug resistance

Introductory paragraphThe clinical success of targeted cancer therapy is limited by drug resistance that renders cancers lethal in patients1-4. Human tumours can evolve therapy resistance by acquiring de novo genetic alterations and increased heterogeneity via mechanisms that remain incompletely understood1. Here, through parallel analysis of human clinical samples, tumour xenograft and cell line models and murine model systems, we uncover an unanticipated mechanism of therapy-induced adaptation that fuels the evolution of drug resistance. Targeted therapy directed against EGFR and ALK oncoproteins in lung cancer induced adaptations favoring apolipoprotein B mRNA-editing enzyme, catalytic polypeptide (APOBEC)-mediated genome mutagenesis. In human oncogenic EGFR-driven and ALK-driven lung cancers and preclinical models, EGFR or ALK inhibitor treatment induced the expression and DNA mutagenic activity of APOBEC3B via therapy-mediated activation of NF-{kappa}B signaling. Moreover, targeted therapy also mediated downregulation of certain DNA repair enzymes such as UNG2, which normally counteracts APOBEC-catalyzed DNA deamination events. In mutant EGFR-driven lung cancer mouse models, APOBEC3B was detrimental to tumour initiation and yet advantageous to tumour progression during EGFR targeted therapy, consistent with TRACERx data demonstrating subclonal enrichment of APOBEC-mediated mutagenesis. This study reveals how cancers adapt and drive genetic diversity in response to targeted therapy and identifies APOBEC deaminases as future targets for eliciting more durable clinical benefit to targeted cancer therapy.

cancer biology↗

bioRxiv2017

Quantification of cellular distribution as Poisson process in 3D matrix using a multiview light-sheet microscope

The absence of quantitative in vitro cell-extracellular matrix models represents an important bottleneck for basic research and human health. Randomness of cellular distributions provides an opportunity for the development of a quantitative in vitro model. However, quantification of the randomness of random cell distributions is still lacking. In this paper, we have imaged cellular distributions in an alginate matrix using a multiview light-sheet microscope and developed quantification metrics of randomness by modeling it as a Poisson process, a process that has constant probability of occurring in space or time. Our light-sheet microscope can image more than 5 mm thick optically clear samples with 2.9 {+/-}0.4 m depth-resolution. We applied our method to image fluorescently labeled human mesenchymal stem cells (hMSCs) embedded in an alginate matrix. Simulated randomness agrees well with the experiments. Quantification of distributions and validation by simulations will enable quantitative study of cell-matrix interactions in tissue models.

biophysics↗