The DNA Methyltransferase Inhibitor 5-Aza-4'-thio-2'-Deoxycytidine Induces C>G Transversions and Acute Lymphoid Leukemia Development.

DNA methyltransferase inhibitors (DNMTi), most commonly cytidine analogs, are compounds that decrease 5'-cytosine methylation. DNMTi are used clinically based on the hypothesis that cytosine demethylation will lead to re-expression of tumor suppressor genes. 5-Aza-4'-thio-2'-deoxycytidine (Aza-TdCyd or ATC) is a recently described thiol-substituted DNMTi that has been shown to have anti-tumor activity in solid tumor models. In this study, we investigated the therapeutic potential of ATC in a murine transplantation model of myelodysplastic syndrome. ATC treatment led to the transformation of transplanted wild-type bone marrow nucleated cells into lymphoid leukemia, and healthy mice treated with ATC also developed lymphoid leukemia. Whole-exome sequencing revealed 1,000 acquired mutations, almost all of which were C>G transversions in a specific 5'-NCG-3' context. These mutations involved dozens of genes involved in human lymphoid leukemia, such as Notch1, Pten, Pax5, Trp53, and Nf1. Human cells treated in vitro with ATC showed 1,000 acquired C>G transversions in a similar context. Deletion of Dck, the rate-limiting enzyme for the cytidine salvage pathway, eliminated C>G transversions. Taken together, these findings demonstrate a highly penetrant mutagenic and leukemogenic phenotype associated with ATC. Significance: Treatment with a DNA methyltransferase inhibitor generates a distinct mutation signature and triggers leukemic transformation, which has important implications for the research and clinical applications of these inhibitors.

Geographic EBV variants confound disease-specific variant interpretation and predict variable immune therapy responses.

ABSTRACT: Epstein-Barr virus (EBV) is a potent carcinogen linked to hematologic and solid malignancies and causes significant global morbidity and mortality. Therapy using allogeneic EBV-specific lymphocytes shows promise in certain populations, but the impact of EBV genome variation on these strategies remains unexplored. To address this, we sequenced 217 EBV genomes, including hematologic malignancies from Guatemala, Peru, Malawi, and Taiwan, and analyzed them alongside 1307 publicly available EBV genomes from cancer, nonmalignant diseases, and healthy individuals across Africa, Asia, Europe, North America, and South America. These included, to our knowledge, the first natural killer (NK)/T-cell lymphoma (NKTCL) EBV genomes reported outside of East Asia. Our findings indicate that previously proposed EBV genome variants specific to certain cancer types are more closely tied to geographic origin than to cancer histology. This included variants previously reported to be specific to NKTCL but were prevalent in EBV genomes from other cancer types and healthy individuals in East Asia. After controlling for geographic region, we did identify multiple NKTCL-specific variants associated with a 7.8-fold to 21.9-fold increased risk. We also observed frequent variations in EBV genomes that affected peptide sequences previously reported to bind common major histocompatibility complex alleles. Finally, we found several nonsynonymous variants spanning the coding sequences of current vaccine targets BALF4, BKRF2, BLLF1, BXLF2, BZLF1, and BZLF2. These results highlight the need to consider geographic variation in EBV genomes when devising strategies for exploiting adaptive immune responses against EBV-related cancers, ensuring greater global effectiveness and equity in prevention and treatment.

5-Aza-4'-thio-2'-deoxycytidine induces C>G transversions in a specific trinucleotide context and leads to acute lymphoid leukemia.

DNA methyltransferase inhibitors (DNMTi), most commonly cytidine analogs, are compounds that are used clinically to decrease 5'-cytosine methylation, with the aim of re-expression of tumor suppressor genes. We used a murine pre-clinical model of myelodysplastic syndrome based on transplantation of cells expressing a NUP98::HOXD13 transgene to investigate 5-Aza-4'-thio-2'-deoxycytidine (Aza TdCyd or ATC), a thiol substituted DNMTi, as a potential therapy. We found that ATC treatment led to lymphoid leukemia in wild-type recipient cells; further study revealed that healthy mice treated with ATC also developed lymphoid leukemia. Whole exome sequencing revealed thousands of acquired mutations, almost all of which were C > G transversions in a previously unrecognized, specific 5'-NCG-3' context. These mutations involved dozens of genes well-known to be involved in human lymphoid leukemia, such as Notch1, Pten, Pax5, Trp53 , and Nf1 . Treatment of human cells in vitro showed thousands of acquired C > G transversions in a similar context. Deletion of Dck , the rate-limiting enzyme for the cytidine salvage pathway, eliminated C > G transversions. Taken together, these findings demonstrate that DNMTi can be potent mutagens in human and mouse cells, both in vitro and in vivo .

Follicular Helper and Regulatory T Cells Drive the Development of Spontaneous Epstein-Barr Virus Lymphoproliferative Disorder.

Epstein-Barr virus (EBV) is a ubiquitous herpes virus associated with various cancers. EBV establishes latency with life-long persistence in memory B-cells and can reactivate lytic infection placing immunocompromised individuals at risk for EBV-driven lymphoproliferative disorders (EBV-LPD). Despite the ubiquity of EBV, only a small percentage of immunocompromised patients (~20%) develop EBV-LPD. Engraftment of immunodeficient mice with peripheral blood mononuclear cells (PBMCs) from healthy EBV-seropositive donors leads to spontaneous, malignant, human B-cell EBV-LPD. Only about 20% of EBV+ donors induce EBV-LPD in 100% of engrafted mice (High-Incidence, HI), while another 20% of donors never generate EBV-LPD (No-Incidence, NI). Here, we report HI donors to have significantly higher basal T follicular helper (Tfh) and regulatory T-cells (Treg), and depletion of these subsets prevents/delays EBV-LPD. Transcriptomic analysis of CD4+ T cells from ex vivo HI donor PBMC revealed amplified cytokine and inflammatory gene signatures. HI vs. NI donors showed a marked reduction in IFNγ production to EBV latent and lytic antigen stimulation. In addition, we observed abundant myeloid-derived suppressor cells in HI donor PBMC that decreased CTL proliferation in co-cultures with autologous EBV+ lymphoblasts. Our findings identify potential biomarkers that may identify individuals at risk for EBV-LPD and suggest possible strategies for prevention.