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Mutational signatures associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC)3 cytosine deaminase activity have been found in over half of cancer types, including some therapy-resistant and metastatic tumors. Driver mutations can occur in APOBEC3-favored sequence contexts, suggesting that mutagenesis by APOBEC3 enzymes may drive cancer evolution. The APOBEC3-mediated signatures are often detected in subclonal branches of tumor phylogenies and are acquired in cancer cell lines over long periods of time, indicating that APOBEC3 mutagenesis can be ongoing in cancer. Collectively, these and other observations have led to the proposal that APOBEC3 mutagenesis represents a disease-modifying process that could be inhibited to limit tumor heterogeneity, metastasis and drug resistance. However, critical aspects of APOBEC3 biology in cancer and in healthy tissues have not been clearly defined, limiting well-grounded predictions regarding the benefits of inhibiting APOBEC3 mutagenesis in different settings in cancer. We discuss the relevant mechanistic gaps and strategies to address them to investigate whether inhibiting APOBEC3 mutagenesis may confer clinical benefits in cancer. This Perspective addresses next steps to investigate the predictions that inhibition of APOBEC3-mediated mutagenesis may limit tumor heterogeneity, metastasis and drug resistance in a broad range of cancer types by highlighting gaps in our understanding of APOBEC3 biology in cancer and in healthy tissues and strategies to address them.

While chimeric antigen receptor (CAR) T cells targeting CD19 can cure a subset of patients with B cell malignancies, most patients treated will not achieve durable remission. Identification of the mechanisms leading to failure is essential to broadening the efficacy of this promising platform. Several studies have demonstrated that disruption of CD19 genes and transcripts can lead to disease relapse after initial response; however, few other tumor-intrinsic drivers of CAR T cell failure have been reported. Here we identify expression of the Golgi-resident intramembrane protease Signal peptide peptidase-like 3 (SPPL3) in malignant B cells as a potent regulator of resistance to CAR therapy. Loss of SPPL3 results in hyperglycosylation of CD19, an alteration that directly inhibits CAR T cell effector function and suppresses anti-tumor cytotoxicity. Alternatively, over-expression of SPPL3 drives loss of CD19 protein, also enabling resistance. In this pre-clinical model these findings identify post-translational modification of CD19 as a mechanism of antigen escape from CAR T cell therapy. Loss of surface CD19 expression by leukemic cells leads to resistance and relapse to CD19-targeted CAR-T therapies. Here the authors show that loss of SPPL3 in malignant B cells results in hyperglycosylation of CD19.

BRCA2 plays a critical role in stabilizing stalled replication forks, yet critical gaps remain in understanding how BRCA2 deficiency triggers fork collapse and drives genomic instability. Here, we identify cytidine deaminase APOBEC3B as a key driver of this process. Using a unique uracil-in-DNA probe, we show that BRCA2 loss promotes APOBEC3B-mediated uracil accumulation in single-stranded DNA (U-ssDNA) at stalled forks. These lesions when processed by UNG2 and APE1, trigger fork collapse and release ssDNA fragments into the cytoplasm, activating NF-κB signaling. This in turn upregulates APOBEC3B expression, establishing a self-reinforcing loop that amplifies cytidine deamination at stalled forks and exacerbates genomic instability. Depletion of APOBEC3B, UNG2, or APE1 rescues these defects. Notably, BRCA1-deficient cells do not accumulate U-ssDNA or induce APOBEC3B under replication stress, highlighting a BRCA2-specific vulnerability. Clinically, low APE1 expression correlates with poor survival in patients with BRCA2 -mutant tumors, with high APOBEC3 levels further worsening outcomes. Together, our findings establish that replication stress, whether intrinsic or therapy induced, triggers APOBEC3B overexpression and potentially activates an APOBEC3B-driven mutagenic loop in BRCA2-deficient cells. These results position APOBEC3B, UNG2 and APE1 as critical regulators of BRCA2 -mutant tumor evolution and therapy resistance. Here the authors reveal how replication stress in BRCA2-deficient cells triggers a mutagenic cycle of APOBEC3B upregulation, uracil accumulation at stalled forks, and DNA damage, uncovering a self-reinforcing loop that fuels genomic instability.

High-grade serous ovarian cancer (HGSOC) is the most prevalent and aggressive histological subtype of ovarian cancer and often presents with metastatic disease. The drivers of metastasis in HGSOC remain enigmatic. APOBEC3A (A3A), an enzyme that generates mutations across various cancers, has been proposed as a mediator of tumor heterogeneity and disease progression. However, the role of A3A in HGSOC has not been explored. We observed an association between high levels of APOBEC3-mediated mutagenesis and poor overall survival in primary HGSOC. We experimentally addressed this correlation by modeling A3A expression in HGSOC, and this resulted in increased metastatic behavior of HGSOC cells in culture and distant metastatic spread in vivo, which was dependent on catalytic activity of A3A. A3A activity in both primary and cultured HGSOC cells yielded consistent alterations in expression of epithelial-mesenchymal transition (EMT) genes resulting in hybrid EMT and mesenchymal signatures, providing a mechanism for their increased metastatic potential. Inhibition of key EMT factors TWIST1 and IL-6 resulted in mitigation of A3A-dependent metastatic phenotypes. Our findings define the prevalence of A3A mutagenesis in HGSOC and implicate A3A as a driver of HGSOC metastasis via EMT, underscoring its clinical relevance as a potential prognostic biomarker. Our study lays the groundwork for the development of targeted therapies aimed at mitigating the deleterious effect of A3A-driven EMT in HGSOC.

DNA deaminase enzymes play key roles in immunity and have recently been harnessed for their biotechnological applications. In base editors (BEs), the combination of DNA deaminase mutator activity with CRISPR–Cas localization confers the powerful ability to directly convert one target DNA base into another. While efforts have been made to improve targeting efficiency and precision, all BEs so far use a constitutively active DNA deaminase. The absence of regulatory control over promiscuous deaminase activity remains a major limitation to accessing the widespread potential of BEs. Here, we reveal sites that permit splitting of DNA cytosine deaminases into two inactive fragments, whose reapproximation reconstitutes activity. These findings allow for the development of split-engineered BEs (seBEs), which newly enable small-molecule control over targeted mutator activity. We show that the seBE strategy facilitates robust regulated editing with BE scaffolds containing diverse deaminases, offering a generalizable solution for temporally controlling precision genome editing. The development of split-engineered base editors (seBEs) enables small-molecule control over DNA deaminase activity, decreasing off-target effects and offering a generalizable solution for temporal control over precise genome editing.

Background Children with sickle cell disease (SCD) are at risk of bone infarcts and acute osteomyelitis. The clinical differentiation between a bone infarct and acute osteomyelitis is a diagnostic challenge. Unenhanced T1-W fat-saturated MR images have been proposed as a potential tool to differentiate bone infarcts from osteomyelitis. Objective To evaluate the reliability of unenhanced T1-W fat-saturated MRI for differentiation between bone infarcts and acute osteomyelitis in children with SCD. Materials and methods We retrospectively reviewed the records of 31 children (20 boys, 11 girls; mean age 10.6 years, range 1.1–17.9 years) with SCD and acute bone pain who underwent MR imaging including unenhanced T1-W fat-saturated images from 2005 to 2010. Complete clinical charts were reviewed by a pediatric hematologist with training in infectious diseases to determine a clinical standard to define the presence or absence of osteomyelitis. A pediatric radiologist reviewed all MR imaging and was blinded to clinical information. Based on the signal intensity in T1-W fat-saturated images, the children were further classified as positive for osteomyelitis (low bone marrow signal intensity) or positive for bone infarct (high bone marrow signal intensity). Results Based on the clinical standard, 5 children were classified as positive for osteomyelitis and 26 children as positive for bone infarct (negative for osteomyelitis). The bone marrow signal intensity on T1-W fat-saturated imaging was not significant for the differentiation between bone infarct and osteomyelitis ( P  = 0.56). None of the additional evaluated imaging parameters on unenhanced MRI proved reliable in differentiating these diagnoses. Conclusion The bone marrow signal intensity on unenhanced T1-W fat-saturated MR images is not a reliable criterion to differentiate bone infarcts from osteomyelitis in children.

ATR is the master safeguard of genomic integrity during DNA replication. Acute inhibition of ATR with ATR inhibitor (ATRi) triggers a surge in origin firing, leading to increased levels of single-stranded DNA (ssDNA) that rapidly deplete all available RPA. This leaves ssDNA unprotected and susceptible to breakage, a phenomenon known as replication catastrophe. However, the mechanism by which unprotected ssDNA breaks remains unclear. Here, we reveal that APOBEC3B is the key enzyme targeting unprotected ssDNA at replication forks, triggering a reaction cascade that induces fork collapse and PARP1 hyperactivation. Mechanistically, we demonstrate that uracils generated by APOBEC3B at replication forks are removed by UNG2, creating abasic sites that are subsequently cleaved by APE1 endonuclease. Moreover, we demonstrate that APE1-mediated DNA cleavage is the critical enzymatic step for PARP1 trapping and hyperactivation in cells, regardless of how abasic sites are generated on DNA. Finally, we show that APOBEC3B-induced toxic PARP1 trapping in response to ATRi drives cell sensitivity to ATR inhibition, creating to a context of synthetic lethality when combined with PARP inhibitors. Together, these findings reveal the mechanisms that cause replication forks to break during replication catastrophe and explain why ATRi-treated cells are particularly sensitive to PARP inhibitors.

High-grade serous ovarian cancer (HGSOC) is the most prevalent and aggressive histological subtype of ovarian cancer, and often presents with metastatic disease. The drivers of metastasis in HGSOC remain enigmatic. APOBEC3A (A3A), an enzyme that generates mutations across various cancers, has been proposed as a mediator of tumor heterogeneity and disease progression. However, the role of A3A in HGSOC has not been explored. Through analysis of genome sequencing from primary HGSOC, we observed an association between high levels of APOBEC3 mutagenesis and poor overall survival. We experimentally addressed this correlation by modeling A3A activity in HGSOC cell lines and mouse models which resulted in increased metastatic behavior of HGSOC cells in culture and distant metastatic spread . A3A activity in both primary and cultured HGSOC cells yielded consistent alterations in expression of epithelial-mesenchymal-transition (EMT) genes resulting in hybrid EMT and mesenchymal signatures, and providing a mechanism for their increased metastatic potential. Our findings define the prevalence of A3A mutagenesis in HGSOC and implicate A3A as a driver of HGSOC metastasis via EMT, underscoring its clinical relevance as a potential prognostic biomarker. Our study lays the groundwork for the development of targeted therapies aimed at mitigating the deleterious impact of A3A-driven EMT in HGSOC.

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.