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. Here, we investigated the therapeutic potential of ATC in a murine transplantation model of myelodysplastic syndrome. ATC treatment led to 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 thousands of 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 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 a highly penetrant mutagenic and leukemogenic phenotype associated with ATC.

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 .

Mcm2 hypomorph leads to acute leukemia or hematopoietic stem cell failure, dependent on genetic context.

Minichromosome maintenance proteins (Mcm2-7) form a hexameric complex that unwinds DNA ahead of a replicative fork. The deficiency of Mcm proteins leads to replicative stress and consequent genomic instability. Mice with a germline insertion of a Cre cassette into the 3'UTR of the Mcm2 gene (designated Mcm2Cre ) have decreased Mcm2 expression and invariably develop precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL), due to 100-1000 kb deletions involving important tumor suppressor genes. To determine whether mice that were protected from pre-T LBL would develop non-T-cell malignancies, we used two approaches. Mice engrafted with Mcm2Cre/Cre Lin- Sca-1+ Kit+ hematopoietic stem/progenitor cells did not develop hematologic malignancy; however, these mice died of hematopoietic stem cell failure by 6 months of age. Placing the Mcm2Cre allele onto an athymic nu/nu background completely prevented pre-T LBL and extended survival of these mice three-fold (median 296.5 vs. 80.5 days). Ultimately, most Mcm2Cre/Cre ;nu/nu mice developed B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We identified recurrent deletions of 100-1000 kb that involved genes known or suspected to be involved in BCP-ALL, including Pax5, Nf1, Ikzf3, and Bcor. Moreover, whole-exome sequencing identified recurrent mutations of genes known to be involved in BCP-ALL progression, such as Jak1/Jak3, Ptpn11, and Kras. These findings demonstrate that an Mcm2Cre/Cre hypomorph can induce hematopoietic dysfunction via hematopoietic stem cell failure as well as a "deletor" phenotype affecting known or suspected tumor suppressor genes.

The Ribbon-Helix-Helix Domain Protein CdrS Regulates the Tubulin Homolog ftsZ2 To Control Cell Division in Archaea.

Precise control of the cell cycle is central to the physiology of all cells. In prior work we demonstrated that archaeal cells maintain a constant size; however, the regulatory mechanisms underlying the cell cycle remain unexplored in this domain of life. Here, we use genetics, functional genomics, and quantitative imaging to identify and characterize the novel CdrSL gene regulatory network in a model species of archaea. We demonstrate the central role of these ribbon-helix-helix family transcription factors in the regulation of cell division through specific transcriptional control of the gene encoding FtsZ2, a putative tubulin homolog. Using time-lapse fluorescence microscopy in live cells cultivated in microfluidics devices, we further demonstrate that FtsZ2 is required for cell division but not elongation. The cdrS-ftsZ2 locus is highly conserved throughout the archaeal domain, and the central function of CdrS in regulating cell division is conserved across hypersaline adapted archaea. We propose that the CdrSL-FtsZ2 transcriptional network coordinates cell division timing with cell growth in archaea.IMPORTANCE Healthy cell growth and division are critical for individual organism survival and species long-term viability. However, it remains unknown how cells of the domain Archaea maintain a healthy cell cycle. Understanding the archaeal cell cycle is of paramount evolutionary importance given that an archaeal cell was the host of the endosymbiotic event that gave rise to eukaryotes. Here, we identify and characterize novel molecular players needed for regulating cell division in archaea. These molecules dictate the timing of cell septation but are dispensable for growth between divisions. Timing is accomplished through transcriptional control of the cell division ring. Our results shed light on mechanisms underlying the archaeal cell cycle, which has thus far remained elusive.