Periodical
Rapid and Accurate Remethylation of Dnmt3aDeficient Hematopoietic Cells with Restoration of DNMT3A Activity
| العنوان: | Rapid and Accurate Remethylation of Dnmt3aDeficient Hematopoietic Cells with Restoration of DNMT3A Activity |
|---|---|
| المؤلفون: | Li, Yang, Abel, Haley, Cai, Michelle, Lavalle, Taylor, Yin, Tiankai, Helton, Nichole, Smith, Amanda, Miller, Christopher A, Ley, Timothy J |
| المصدر: | Blood; November 2023, Vol. 142 Issue: 1, Number 1 Supplement 1 p4124-4124, 1p |
| مستخلص: | Heterozygous loss-of-function mutations in the DNMT3Agene are the most common cause of clonal hematopoiesis, and among the most common initiating events for Acute Myeloid Leukemia (AML). A reduction of DNMT3A activity causes a canonical, focal hypomethylation phenotype in hematopoietic cells, which is associated with immortalization of hematopoietic stem cells, and blockage of myeloid differentiation. The methylation defect can be partially reversed with restoration of physiologic levels of DNMT3Aexpression over a period of several months (Ketkar et al., PNAS, 2020; PMID 35313694).To identify a more efficient remethylation strategy, we first characterized the DNA methylation phenotypes of bone marrow cells from mice with hematopoietic cell deficiency of Dnmt3a, Dnmt3b(or both enzymes), or expressing the dominant negative Dnmt3aR878Hmutation (equivalent to R882H in humans; the most common mutation found in AML patients). Using these different mouse models as substrates, we then defined the patterns and completeness of DNA remethylation after “adding back” supraphysiologic levels of wild type DNMT3A1, DNMT3B1, DNMT3B3 (an inactive splice isoform of DNMT3B), or DNMT3L (a catalytically inactive “chaperone” that augments the activity of DNMT3A and DNMT3B in early embryogenesis), with MSCV-based retroviruses transduced into primary mouse hematopoietic stem/progenitor cell s. Overexpression of WT DNMT3A for 2 weeks in vitro(~3 fold) can accurately reverse the differentially methylated regions (DMRs, which are >99% hypomethylated) of Dnmt3adeficient hematopoietic cells, or cells expressing the R878H mutation. The DMRs of Dnmt3bdeficient mouse bone marrow cells can be corrected by overexpression of DNMT3A, DNMT3B1, or DNMT3B3; however, DNMT3B3 failed to remethylate the DMRs in Dnmt3a/Dnmt3bdouble knockout mouse bone marrow cells. Since DNMT3B3 is an inactive enzyme that does not cause remethylation in the absence of DNMT3A, these data suggest that DNMT3B3 functions in a DNMT3A-dependent manner in hematopoietic cells. Importantly, both DNMT3B3 and DNMT3L have been shown to facilitate DNA methylation by acting as a chaperone for DNMT3A (Duymich et al., Nat. Communication, 2016; PMID 27121154). In vitro, DNMT3L copurified with DNMT3A leads to an increase in DNMT3A methyltransferase activity that is ~5 times greater than DNMT3B3 copurified with DNMT3A. Remarkably, overexpression of DNMT3L (which is not expressed in adult hematopoietic cells, or AML cells) can completely correct the hypomethylation phenotype of Dnmt3aR878H/+bone marrow cells within 2 weeks of in vitrooverexpression, or 4 weeks of in vivooverexpression ( Figure 1A and 1B), probably by augmenting the activity of WT DNMT3A encoded by the residual WT allele in these heterozygous mutant cells. Two weeks of overexpression of DNMT3A or DNMT3L in Dnmt3aR878H/+bone marrow cells (cultured in vitrowith IL-3, SCF, FLT3L, and TPO to maintain HSPC populations) can induce a differentiation response, increasing the fraction of mature myeloid cells in the cultures by ~4-fold. This finding was recapitulated in vivowhere one month of overexpression of DNMT3Aor DNMT3Lin transplanted Dnmt3aR878H/+mouse bone marrow cells increased the fraction of mature myeloid cells by 2-fold by DNMT3L and 4-fold by DNMT3A. Together,these data show that the focal, canonical DNA hypomethylation phenotype of Dnmt3a R878H/+hematopoietic cells can be accurately and efficiently corrected by overexpressing WT DNMT3A or DNMT3L for several weeks. We have also shown that the restoration of methylation in Dnmt3a R878H/+mutant cells alters the developmental fate of progenitors, increasing the proportion of mature myeloid cells. These data suggest that DNMT3Lexpression may represent a novel approach for restoring DNMT3A activity in AMLs initiated by DNMT3Amutations, and could possibly alter AML cell fate. Additional studies are underway to determine whether the correction of DNMT3A activity in Dnmt3aR878H/+-initiated AML cells with retroviral addback of DNMT3A or DNMT3L will cause a differentiation response that alters AML cell fate. |
| قاعدة البيانات: | Supplemental Index |
Full Text Finder | تدمد: | 00064971 15280020 |
|---|---|
| DOI: | 10.1182/blood-2023-180255 |