Page 210 - 2021_07-Haematologica-web

A.G. Gilmartin et al.
hemolysis. These changes in the sickle erythrocytes pro- duce a cascade of effects that result in anemia, impaired blood flow, and painful vaso-occlusive events that ultimate- ly cause tissue ischemia and long-term damage.2
; HbF) that is 2
sites in the promoters of HBG1 and HBG2, resulting in increased γ-globin expression and elevated HbF levels. While they are effective inducers of HbF, the decitabine and 5-aza mechanism of action relies on incorporation into DNA, and they both carry drug label warnings for genotox- icity and cytotoxicity. Decitabine and 5-aza are currently approved only for use in myelodysplastic syndromes and acute myeloid leukemia, and they are not currently approved to treat β-hemoglobinopathies.
In the current work, we describe the identification of a novel class of orally-dosed, reversible DNMT1-selective inhibitors, exemplified by GSK3482364. This molecule caused decreased DNA methylation in cultured human EPC, resulting in increased γ-globin gene expression and increased HbF. In a murine model of SCD, orally dosed GSK3482364 decreased DNA methylation in bone marrow, and increased HbF expression in erythrocytes. Notably, although GSK3482364 and decitabine were comparable in their maximal effects on DNA methylation in cells, GSK3482364 treatment resulted in lower cytotoxicity in cultured cells as well as improved in vivo tolerability in pre- clinical models. These results indicate that selective, reversible inhibition of DNMT1 is sufficient for the induc- tion of HbF, is well-tolerated in vivo, and that neither irre- versible DNMT1 inhibition nor inhibition of DNMT3A or DNMT3B is required for this effect.
Methods
Erythroid progenitor cell culture
Cryopreserved human bone marrow CD34+ cells (AllCells) were confirmed to be sourced ethically, and their research use was in accord with the terms of the informed consents under an Institutional Review Board/Ethics Commity approved protocol. Cells were cultured according to previously described methods23 for 7 days to generate EPC. For compound treatment studies, cell culture plates were typically incubated for 3-5 days unless other- wise indicated. In order to investigate potential drug effects on erythroid maturation to reticulocytes, CD34+ cells were cultured for 19 days in a three stage protocol that models maturation into reticulocytes with continuous compound treatment. Details for cell culture and methods for cellular assays can be found in the Online Supplemental Appendix.
Methylation-sensitive restriction endonuclease assays
Genomic DNA was extracted from EPC or bone marrow using Zymo Quick-gDNA kits (Zymo Research). Total DNA was meas- ured on a NanoDrop (ThermoFisher), diluted, and split into tubes containing reaction buffer +/- methylation-sensitive HpaII (New England Biolabs) for a 1-hour reaction. Reaction products were then quantitated in a 50 mL SYBR Green quantitative polymerase chain reaction (qPCR) (Applied Biosystems). -53 base pair: Primer 1: 5ˈ-GAACTGCTGAAGGGTGCT-3ˈ, Primer 2: 5ˈ-GACAAG- GCAAACTTGACCAATAG-3ˈ.
In vivo studies
All studies were conducted in accordance with the
GlaxoSmithKline (GSK) Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Male and female human hemoglobin transgenic mice [B6;129-HBAtm1(HBA)Tow/HBBtm2(HBG1,HBB*)Tow/J Mice] (Jackson Laboratories) were 6-8 weeks of age and grouped into
During fetal development and until shortly after birth, erythrocytes preferentially express an alternγative hemoglo-
bin tetramer termed fetal hemoglobin (a 2
composed of two γ-globin chains paired with a-globin chains rather than β-globin chains. The genes encoding for γ-globin, HBG1 and HBG2, lack the mutation that causes SCD. Consequently, symptoms of SCD first manifest sev- eral months after birth following the “hemoglobin switch”, the transition from HbF to HbA, or to HbS in the case of SCD patients.3 During the transition from HbF to HbA/HbS, the genes encoding for γ-globin, HBG1 and HBG2, are repressed by transcriptional complexes that include GATA1, TR2/TR4, MYB, KLF1, Sox6, BCL11A, LRF, DNMT1, and HDAC1/2.4-6 The repressor complexes cause significant chromatin remodeling, controlled in part through increased DNA methylation of HBG1 and HBG2 gene promoters and demethylation of the HBB gene pro- moter. 7, 8 Although HbF typically decreases to a few percent of total hemoglobin shortly after birth, HbF levels can remain elevated in a rare condition called hereditary persist- ence of HbF (HPFH) in which mutations prevent the normal repression of γ-globin.9 When HPFH co-occurs with the mutations that cause SCD, elevated levels of HbF can pre- vent the aggregation of HbS and protect erythrocytes from sickling, significantly ameliorating the disease.10
To date, the most important pharmacological agent for the management of SCD remains the ribonucleotide reduc- tase inhibitor hydroxyurea (HU), which benefits patients through increasing HbF expression and reducing the inci- dence of vaso-occlusive crises. Although HU mitigates the clinical severity of disease for many SCD patients, there are important limitations to the clinical utility of HU. Importantly, there is typically a narrow therapeutic win- dow between the efficacious dose of HU for beneficial HbF induction and the maximum tolerated dose typically defined by acceptable myelosuppression. As a conse- quence, there are variable pharmacological responses to HU in many patients.11-13 There is therefore a desire to identify alternative agents that safely and consistently induce HbF to therapeutic levels for the treatment of SCD.
The hypomethylating agent (HMA) 5-azacytidine (5-aza) is a cytidine analog that was first demonstrated to induce HbF in an anemic baboon model.14 It was subsequently con- firmed to increase HbF in investigational studies of patients with SCD and β-thalassemia15-18 as well as in patients with myelodysplastic syndrome and acute myeloid leukemia.19, 20 Low doses of decitabine were also confirmed to increase HbF levels in SCD patients, in some cases exceeding the maximal HbF levels observed with HU.18 Decitabine and 5- aza are inhibitors of DNA methyltransferases (DNMT), enzymes that establish and maintain the epigenetic pattern of DNA methylation that functions in chromatin condensa- tion and gene silencing. The catalytically active members of the DNMT family are DNMT3A, DNMT3B, and DNMT1. DNMT3A and DNMT3B establish the de novo pattern of DNA methylation, while DNMT1 is the primary mainte- nance methyltransferase that propagates the pattern of DNA methylation to daughter cells during cell division.21 In cultured human erythroid progenitor cells (EPC)22-24 and in vivo models with monkeys,25, 26 treatments with either decitabine or 5-aza decreased methylation of multiple CpG
1980
haematologica | 2021; 106(7)