Discovery of a first-in-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia

Pappalardi, Melissa B.; Keenan, Kathryn; Cockerill, Mark; Kellner, Wendy A.; Stowell, Alexandra; Sherk, Christian; Wong, Kristen; Pathuri, Sarath; Briand, Jacques; Steidel, Michael; Chapman, Phil; Groy, Arthur; Wiseman, Ashley K.; McHugh, Charles F.; Campobasso, Nino; Graves, Alan P.; Fairweather, Emma; Werner, Thilo; Raoof, Ali; Butlin, Roger J.; Rueda, Lourdes; Horton, J.R.; Fosbenner, David T.; Zhang, Cunyu; Handler, Jessica L.; Muliaditan, Morris; Mebrahtu, Makda; Jaworski, Jon Paul; McNulty, Dean E.; Burt, Charlotte; Eberl, H. Christian; Taylor, Amy; Ho, Thau; Merrihew, Susan; Foley, Shawn W.; Rutkowska, Anna; Li, Mei; Romeril, Stuart P.; Goldberg, Kristin; Zhang, Xing; Kershaw, Christopher S.; Bantscheff, Marcus; Jurewicz, Anthony J.; Minthorn, Elisabeth A.; Grandi, Paola; Patel, Mehul; Benowitz, Andrew B.; Mohammad, Helai P.; Gilmartin, Aidan G.; Prinjha, Rab K.; Ogilvie, Donald; Carpenter, Christopher L.; Heerding, Dirk A.; Baylin, Stephen B.; Jones, Peter A.; Cheng, Xiaodong; King, Bryan W.; Luengo, Juan I.; Jordan, Allan M.; Waddell, Ian D.; Kruger, Ryan G.; McCabe, Michael T.

doi:10.1038/s43018-021-00249-x

Cited by 158 publications

(153 citation statements)

References 65 publications

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“…However, gene re-expression was consistent and significant when the HD was combined with VAL, SAHA or TSA. These findings suggest that a longer incubation time or a more effective DNMT inhibitor could be used [ 63 ], or that the expression of each gene may be partially regulated by other mechanisms such as histone deacetylation, as previously discovered [ 44 , 55 , 64 ].…”

Section: Discussionmentioning

confidence: 75%

Hypermethylation-Mediated Silencing of CIDEA, MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies

Zorzan

Elgendy

Guerra

et al. 2022

IJMS

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Gene expression is controlled by epigenetic deregulation, a hallmark of cancer. The DNA methylome of canine diffuse large B-cell lymphoma (cDLBCL), the most frequent malignancy of B-lymphocytes in dog, has recently been investigated, suggesting that aberrant hypermethylation of CpG loci is associated with gene silencing. Here, we used a multi-omics approach (DNA methylome, transcriptome and copy number variations) combined with functional in vitro assays, to identify putative tumour suppressor genes subjected to DNA methylation in cDLBCL. Using four cDLBCL primary cell cultures and CLBL-1 cells, we found that CiDEA, MAL and PCDH17, which were significantly suppressed in DLBCL samples, were hypermethylated and also responsive (at the DNA, mRNA and protein level) to pharmacological unmasking with hypomethylating drugs and histone deacetylase inhibitors. The regulatory mechanism underneath the methylation-dependent inhibition of those target genes expression was then investigated through luciferase and in vitro methylation assays. In the most responsive CpG-rich regions, an in silico analysis allowed the prediction of putative transcription factor binding sites influenced by DNA methylation. Interestingly, regulatory elements for AP2, MZF1, NF-kB, PAX5 and SP1 were commonly identified in all three genes. This study provides a foundation for characterisation and experimental validation of novel epigenetically-dysregulated pathways in cDLBCL.

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Section: Discussionmentioning

confidence: 75%

Hypermethylation-Mediated Silencing of CIDEA, MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies

Zorzan

Elgendy

Guerra

et al. 2022

IJMS

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“…The recent development of a specific DNMT1 inhibitor, which does not require incorporation into DNA for its activity 17 , provided us with a useful tool to probe the role of the enzyme in the maintenance of DNA methylation patterns in cancer cell lines. Long term treatment of the HCT116 cells resulted in the derivation of cells which showed impaired growth rates and were markedly resistant to the compound.…”

Section: Discussionmentioning

confidence: 99%

Chromosome-specific retention of cancer-associated DNA hypermethylation following pharmacological inhibition of DNMT1

et al. 2022

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The DNA methylation status of the X-chromosome in cancer cells is often overlooked because of computational difficulties. Most of the CpG islands on the X-chromosome are mono-allelically methylated in normal female cells and only present as a single copy in male cells. We treated two colorectal cancer cell lines from a male (HCT116) and a female (RKO) with increasing doses of a DNA methyltransferase 1 (DNMT1)-specific inhibitor (GSK3685032/GSK5032) over several months to remove as much non-essential CpG methylation as possible. Profiling of the remaining DNA methylome revealed an unexpected, enriched retention of DNA methylation on the X-chromosome. Strikingly, the identified retained X-chromosome DNA methylation patterns accurately predicted de novo DNA hypermethylation in colon cancer patient methylomes in the TCGA COAD/READ cohort. These results suggest that a re-examination of tumors for X-linked DNA methylation changes may enable greater understanding of the importance of epigenetic silencing of cancer related genes.

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“…This shift may reflect the fact that mESCs depend heavily on TRIM28 to silence transposons, but upon differentiation of mESCs or uterine implantation of embryos, DNA methylation gains importance for transposon repression [ 7 , 32 , 36 , 37 ]. Thus, researchers working with other cell types may well observe specific toxicity at lower doses, and indeed GSK-3484862 and related compounds show a striking effect in leukemias [ 26 ]. We also cannot rule out that non-specific toxicity may occur in some cell types, a result suggested by the GSK-3484862’s ability to halt mouse development at relatively low concentrations [ 25 ].…”

Section: Discussionmentioning

confidence: 99%

“…At this low concentration, only a 34% global drop in DNA methylation is observed relative to untreated Dnmt3a/3b KO embryos; however, the treatment is sufficient to induce a marked increase in IAP-Ez transposon expression, consistent with demethylating activity of the compound. Most recently, Pappalardi and colleagues described the discovery of GSK-3484862 in a screen for DNMT1 inhibitors and the characterization of two related compounds, GSK-3685032 and GSK-3830052 [ 26 ]. They demonstrated that these inhibitors make simultaneous contact with DNA and the DNMT1 active-site loop and block activity of the enzyme.…”

Section: Introductionmentioning

confidence: 99%

The DNMT1 inhibitor GSK-3484862 mediates global demethylation in murine embryonic stem cells

Portilho

Saini

Hossain

et al. 2021

Epigenetics & Chromatin

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Background DNA methylation plays an important role in regulating gene expression in mammals. The covalent DNMT1 inhibitors 5-azacytidine and decitabine are widely used in research to reduce DNA methylation levels, but they impart severe cytotoxicity which limits their demethylation capability and confounds interpretation of experiments. Recently, a non-covalent inhibitor of DNMT1 called GSK-3484862 was developed by GlaxoSmithKline. We sought to determine whether GSK-3484862 can induce demethylation more effectively than 5-azanucleosides. Murine embryonic stem cells (mESCs) are an ideal cell type in which to conduct such experiments, as they have a high degree of DNA methylation but tolerate dramatic methylation loss. Results We determined the cytotoxicity and optimal concentration of GSK-3484862 by treating wild-type (WT) or Dnmt1/3a/3b triple knockout (TKO) mESC with different concentrations of the compound, which was obtained from two commercial sources. Concentrations of 10 µM or below were readily tolerated for 14 days of culture. Known DNA methylation targets such as germline genes and GLN-family transposons were upregulated within 2 days of the start of GSK-3484862 treatment. By contrast, 5-azacytidine and decitabine induced weaker upregulation of methylated genes and extensive cell death. Whole-genome bisulfite sequencing showed that treatment with GSK-3484862 induced dramatic DNA methylation loss, with global CpG methylation levels falling from near 70% in WT mESC to less than 18% after 6 days of treatment with GSK-3484862. The treated cells showed a methylation level and pattern similar to that observed in Dnmt1-deficient mESCs. Conclusions GSK-3484862 mediates striking demethylation in mESCs with minimal non-specific toxicity.

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Discovery of a first-in-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia

Cited by 158 publications

References 65 publications

Hypermethylation-Mediated Silencing of CIDEA, MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies

Hypermethylation-Mediated Silencing of CIDEA, MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies

Chromosome-specific retention of cancer-associated DNA hypermethylation following pharmacological inhibition of DNMT1

The DNMT1 inhibitor GSK-3484862 mediates global demethylation in murine embryonic stem cells

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