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A.G. Gilmartin et al.
Discussion
As potential agents for the management of SCD, the DNMT inhibitors 5-aza and decitabine have demonstrat- ed significant induction of HbF levels in preclinical and clinical studies.14, 18, 23, 41-45 The robust HbF induction caused by these compounds is explained both by the high prolif- erative index of bone marrow progenitor cells and by transitional changes in DNA methylation that they under- go during erythropoiesis. Changes in DNA methylation occur during key determining events in hematopoiesis.46 During erythropoiesis a rapid decrease in global DNA methylation marks a commitment toward erythropoietin dependence and the expression of erythroid specific mas- ter regulators GATA1 and KLF1.8, 47 As hematopoietic stem cells commit to erythropoiesis, DNMT1 becomes the dominantly expressed DNMT with the primary role of maintaining DNA methylation and regulating globin gene transcription.8, 31, 48 For EPC in adult bone marrow, key cytosine residues in the promoter region of HBG1 and HBG2 become highly methylated while the β-globin HBB gene promoter is largely unmethylated, correspon- ding to the increased expression of β-globin and the repression of γ-globin expression.31, 49
Although the known HMA decitabine and 5-aza have proven to be valuable probes to study the biology of DNMT methylation, and partial and complete DNMT1 knockout studies in animals have established a critical role for DNMT1 in cellular differentiation and stem cell maintenance, it has not been previously possible to study the in vitro and in vivo cellular and hematopoietic effects of reversible and selective DNMT1 inhibition. GSK3482364 represents a novel class of DNMT1-selective inhibitors that are mechanistically distinct from other HMA. In cel- lular studies, GSK3482364 treatment caused DNA hypomethylation and HbF induction with maximal effects that were approximately equivalent to decitabine treatment. However, at concentrations of GSK3482364 and of decitabine that produced equivalent HbF induc- tion, decitabine was observed to consistently cause more cell growth inhibition. In an in vitro model of erythro- poiesis, expanding and differentiating from CD34+ cells to enucleated reticulocytes, GSK3482364 and decitabine caused equivalent increases in HbF-cells, but GSK3482364 treatment resulted in a larger proportion of cells maturing into HbF expressing reticulocytes. In a transgenic mouse model of SCD, the effects of orally- dosed GSK3482364 on bone marrow DNA methylation and erythrocyte HbF elevation exceeded the correspon- ding effects of decitabine at tolerated doses over a 12-day period. Examination of complete blood counts and bone marrow cellularity from in vivo studies with GSK3482364 suggests that the effects of this compound on DNA methylation in the bone marrow were well-tolerated without evidence of other adverse hematological effects.
Chronic intermittent pharmacologic inhibition of
DNMT1 does not appear to fully phenocopy the effects of permanent DNMT1 deletion. In our mouse studies, the limited impact on bone marrow cellularity or blood cell populations appears to indicate that repeat daily doses of GSK3482364 were tolerated by hematopoietic stem and progenitor cells. Notably, in our in vivo studies we did not observe the significant increase in platelets that has been reported in clinical studies with low dose decitabine and that is attributed to effects of hypomethylation in promoting megakaryocyte matura- tion.18, 45, 50 Since the increase in platelets does not appear to be captured in our mouse model, we cannot currently draw any conclusions about potential differentiating effects on platelets for GSK3482364 or related com- pounds. Future studies in non-human primate models, where multiple HbF inducers were initially character- ized, are warranted to address this and other questions about the optimal dosing regimens for this class of DNMT1 selective inhibitors. The differential cellular biology and in vivo pharmacology observed with GSK3482364 as compared to decitabine suggest that this may be a useful tool molecule to study the selective, reversible inhibition of DNMT1 in hematopoiesis and for the elevation of HbF in erythrocytes.
Disclosures
AGG, AG, EG, CA, EL, MM, ZW, WH, DM, DE, LR, MP, MTM, RGK, JL, and ABB are current or former employees of GlaxoSmithKline; AR, RB, AS, MC, DO, IW, and AJ are cur- rent or former employees of CRUK Manchester Institute; patent US20190194166 pertains to compounds discussed in this man- uscript.
Contributions
AGG, AG, EG, CA, EL, MNM, ZW, WH, DM, DE, AS, MP, and ABB designed or performed experiments; AR, RB, MC, AJ, LR, DO,IW, MTM, RGK, and JL were involved in the iden- tification and synthesis of the lead compound; AGG wrote the manuscript and all authors read and approved the final version of the manuscript.
Acknowledgments
The authors wish to thank Karen Evans, Rakesh Nagilla, Elizabeth Rivera, Karen Lynch, Denise Depagnier, Chris Traini, Leonard Azzarano, and Shanker K. Sundaram for con- tributions to this work. Additionally, we wish to thank Jerry Adams, Dirk Heerding, Chris Carpenter, Steve Pessagno, Philip Chapman, Charlotte Burt, Martyn Bottomley, Kristin Goldberg, and Chris Kershaw for their contributions to the iden- tification of GSK3482364. Work conducted at the Cancer Research UK Manchester Institute was wholly funded by Cancer Research UK (Grant numbers C480/A1141 and C5759/A17098).
Funding
Work conducted at the Cancer Research UK Manchester Institute was wholly funded by Cancer Research UK (Grant numbers C480/A1141 and C5759/A17098).
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