Purpose: In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. Experimental Design: Leukemia cell lines, genetic mouse models, and isogenic cells with and without DNMT3A(mut) were used to evaluate sensitivity to nucleoside analogues such as cytarabine in vitro and in vivo, followed by analysis of DNA damage and signaling, replication restart, and cell-cycle progression on treatment and after drug removal. Transcriptome profiling identified pathways deregulated by DNMT3A(mut) expression. Results: We found increased sensitivity to pharmacologically induced replication stress in cells expressing DNMT3A(R882)-mutant, with persistent intra–S-phase checkpoint activation, impaired PARP1 recruitment, and elevated DNA damage, which was incompletely resolved after drug removal and carried through mitosis. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine washout demonstrated a higher rate of fork collapse in DNMT3A(mut)-expressing cells. RNA-seq studies supported deregulated cell-cycle progression and p53 activation, along with splicing, ribosome biogenesis, and metabolism. Conclusions: Together, our studies show that DNMT3A mutations underlie a defect in recovery from replication fork arrest with subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during replication stress. See related commentary by Viny, p. 573

Diffuse large B-cell lymphoma (DLBCL), when associated with Epstein–Barr virus (EBV) in immunocompromised individuals such as AIDS patients, presents a significant treatment challenge. Lytic induction therapy, which reactivates latent EBV to directly kill tumor cells and sensitize them to nucleoside analogs that block viral replication and immune clearance, offers promise. However, little is known about EBV reactivation in DLBCL. Here, we examined four EBV-positive DLBCL cell lines and found variable, cell-line-specific responses to lytic stimuli, with most showing an abortive response—either before or after genome replication, without virus release. This is in contrast to commonly studied lymphoma cells in which EBV reactivation typically leads to a full lytic cycle. Mechanistically, we show that the unfolded protein response (UPR), via a splice variant of the transcription factor XBP1, upregulates TXNIP and NLRP3, activating the inflammasome and removing a barrier to transcription of the EBV latent-to-lytic switch gene BZLF1. Combining lytic induction with the nucleoside analog ganciclovir enhanced oncolytic cell death. This study identifies a pivotal link between two danger sensing pathways, the UPR and the inflammasome, in reactivating the virus resident in DLBCL and suggests that controlled lytic reactivation could provide a basis for EBV-targeted therapies to improve outcomes in this malignancy.

Lactate, primarily produced by the gut microbiota, performs as a necessary “information transmission carrier” between the gut and the microbiota. To investigate the role of lactate in the gut epithelium cell–microbiota interactions as a metabolic signal, we performed a combinatory, global, and unbiased analysis of metabolomic and transcriptional profiling in human colon epithelial cells (Caco-2), using a lactate treatment at the physiological concentration (8 mM). The data demonstrated that most of the genes in oxidative phosphorylation were significantly downregulated in the Caco-2 cells due to lactate treatment. Consistently, the levels of fumarate, adenosine triphosphate (ATP), and creatine significantly decreased, and these are the metabolic markers of OXPHOS inhibition by mitochondria dysfunction. The one-carbon metabolism was affected and the polyol pathway was activated at the levels of gene expression and metabolic alternation. In addition, lactate significantly upregulated the expressions of genes related to self-protection against apoptosis. In conclusion, lactate participates in gut–gut microbiota communications by remodeling the metabolomic and transcriptional signatures, especially for the regulation of mitochondrial function. This work contributes comprehensive information to disclose the molecular mechanisms of lactate-mediated functions in human colon epithelial cells that can help us understand how the microbiota communicates with the intestines through the signaling molecule, lactate.

Epstein-Barr virus (EBV), a common gamma-herpesvirus linked to various malignancies, exploits host cellular mechanisms to promote oncogenesis. Our previous research identified the zinc finger protein ZC3H18 as a novel component of the cellular DNA replication machinery in the context of EBV-driven tumorigenesis. We now demonstrate that ZC3H18 expression is upregulated in EBV-transformed and cancer cell lines, as well as in EBV-positive diffuse large B-cell lymphomas from AIDS patients, compared to their EBV-negative counterparts, supporting its activation by EBV. Our experiments show that ZC3H18 expression is regulated by the key oncogenic factors STAT3 and MYC, as well as the essential viral protein EBNA1. Using inhibitors and genetic knockdown, we find that suppressing STAT3, MYC, or EBNA1 leads to decreased ZC3H18 levels, reduced cell viability, and increased apoptosis in EBV-positive B lymphoma cells. Furthermore, ZC3H18, STAT3, MYC, and EBNA1 mutually support each other’s expression through a complex transcriptional network. Notably also, ZC3H18 transcriptionally enhances components of the NF-κB pathway, contributing to NF-κB signaling even in the absence of the EBV oncoprotein LMP1, which is crucial for cell proliferation and survival of several EBV-associated malignancies. Our findings reveal a novel regulatory axis in EBV-positive cancer cells involving STAT3, MYC, EBNA1, & ZC3H18, also linking ZC3H18 to the NF-κB pathway independently of LMP1. The involvement of EBNA1 in this network may explain, at least in part, the preferential upregulation of ZC3H18 in EBV-associated tumors and highlights predictive and therapeutic possibilities for such cancers.

Supplementary Data from DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks

Supplementary Data from DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks