5-Aza-4'-thio-2'-deoxycytidine (ATC) is an azanucleoside cytidine analog under investigation in preclinical studies for solid tumors as a promising DNA methyltransferase 1 (DNMT1) inhibitor. Repeated treatment with ATC has previously been shown to induce acute lymphoblastic leukemia (ALL) of both B-cell and T-cell origin in mice. Herein, RAG-1 deficient or "knockout" (KO) mice (B6.129S7-RAG-1tm1Mom/J) were treated with ATC to determine if ATC could be oncogenic in nonlymphoid cells. However, ATC treatment targeted early B progenitors and invariably led to B-lineage ALL, with a gene expression signature similar to human B-cell precursor (BCP)-ALL. Whole-exome sequencing revealed numerous single base substitutions of cytosine, primarily C>G transversions at CpG dinucleotides, within genes important for BCP-ALL. Bisulfite sequencing and treatment with a noncovalent DNMT1 inhibitor indicated that methylated cytosines were preferred targets for mutagenesis. This study reveals that ATC exposure leads to both DNMT1-dependent and -independent mutagenesis and provides a direct link between ATC exposure, a complex mutational signature, and malignant transformation.

Abstract 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.

PDF file, 208K, Identfication of miR-652*.

<p>Supplementary Figure S1 PDF file - 1274K, Schematic presentation of the workflow of experiments and analytical procedures</p>

Supplementary Figure S3 PDF file - 2243K, RNAi analysis and measurement of cell viability of amplified and overexpressed chromosome 13 candidate genes

XLS file, 56K, miRNAs that are differentially expressed between rectal cancer tissue and normal adjacent mucosa.

<p>Supplementary Figure S9 PDF file - 1478K, Expression levels of the LNX2 module</p>

Supplementary Table S1 PDF file - 435K, ist of RT-PCR primers used for validation

Supplementary Figure 1 from Chromosomal Breakpoints in Primary Colon Cancer Cluster at Sites of Structural Variants in the Genome

Supplementary Table S4 PDF file - 358K, List of filtered genes used in our experimental setup

Supplementary Table S2 PDF file - 589K, List of siRNAs

Supplementary Tables 1-5 from Gene Expression Profiling Reveals a Massive, Aneuploidy-Dependent Transcriptional Deregulation and Distinct Differences between Lymph Node–Negative and Lymph Node–Positive Colon Carcinomas

Supplementary Figure S2 PDF file - 1800K, Corroboration of gene expression levels by qRT-PCR

PDF file, 165K, Little overlap between prediction algorithms.

Supplementary Figure S8 PDF file - 391K, Reduction of CTNNB1 after siRNA against LNX2

XLS file, 32K, Target sequences for the miRNA PCR reactions.

Supplementary Figure S4 PDF file - 1138K, Cell viability and corresponding mRNA reduction of eight candidate genes selected for further experimentation

Supplementary Tables 1-9 from Aneuploidy-Dependent Massive Deregulation of the Cellular Transcriptome and Apparent Divergence of the Wnt/β-catenin Signaling Pathway in Human Rectal Carcinomas