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Y. Saunthararajah et al.
bone marrow from severe insults or stress.24-29 One way of creating such stress is to administer cytotoxic (cell killing) drugs, leading to clinical evaluation in SCD of the oral ribonucleotide reductase inhibitor hydroxyurea.28-30 In the pivotal trial, hydroxyurea (15-35 mg/kg) increased HbF for
30,31
2 years in ~50% of the adult SCD patients treated. As
predicted, HbF increases with hydroxyurea correlated strongly with longer RBC half-life,32,33 fewer pain crises,31 and better quality of life34 (the benefits of hydroxyurea ther- apy in sickle cell mice also depended on HbF induction).35 Trial patients with HbF levels >0.5 g/dL also survived longer15 although a caveat to these analyses was that it was not known whether the higher HbF levels were intrinsic to the patients or a result of the hydroxyurea therapy.
There were, however, noteworthy limitations to the induction of HbF by hydroxyurea: (i) average HbF increas- es at 2 years were modest (3.6%);28-31,36 (ii) HbF increases were particularly unlikely in patients with the lowest baseline HbF levels and thus at highest risk of morbidity and mortality,31,33,37,38 and (iii) HbF increases diminished over time, even in the ~50% of patients with excellent ini- tial HbF induction.31,39
A shared basis for these several limitations was suggest- ed by the correlation between lower HbF increases and fewer reticulocytes (<300,000x109/L) and neutrophils (<7.5x109/L) at baseline: this correlation underscored that HbF induction by cytotoxicity requires sufficient reserves of hematopoietic precursors to mount repeated recoveries from the stress that destroys their counterparts.29,31 Such reserves are circumscribed, subject to attrition via vaso- occlusion in the marrow and kidneys, and decline with aging.31,33,37,38 A declining capacity to compensate for hemolytic anemia is a problem even separate from consid- erations of sustainable HbF induction via cytotoxicity: SCD patients require erythropoiesis at >10-fold the nor- mal rate simply to sustain hemoglobin levels compatible with life, and dwindling compensatory reticulocytosis is a
major cause of early death.2,15,31,40 Therefore, alternative, non-cytotoxic, durable, and more potent methods of inducing HbF are needed.
Directly targeting the enzymes that silence the γ-globin gene
DNA in nuclei is packaged together with RNA and structural proteins – histones - to form chromatin. Chromatin regulates gene transcription by determining accessibility of genes to the massive machinery (~150 pro- teins) that transcribes genes. Reorganization (‘remodel- ing’) of chromatin, to facilitate or hinder this machinery, is signaled via post-translational modifications to histones - methylation and acetylation of lysine residues, phospho- rylation of threonines and serines – and by modifications to DNA, mainly, methylation of cytosines that precede guanines (CpG). These signals determine whether ATP- dependent chromatin remodelers shift histones towards or away from gene transcription start sites, repositioning these physical barriers to either welcome or obstruct the gene transcribing basal transcription factor machinery.
Thus, induction of HbF, even when it is indirectly via bone marrow stress, implies remodeling γ-globin and β-glo- bin gene loci, to activate one and not the other.41 Specifically, persistent HbF expression requires: (i) decreased operation at HBG2/HBG1 of epigenetic enzymes that create ‘off’ marks and that reposition histones to obstruct transcription start sites, and (ii) increased function of the epigenetic enzymes that create epigenetic ‘on’ marks and that reposition histones away from transcription start sites, with vice versa at HBB. Cytotoxic methods of inducing HbF achieve such chromatin remodeling crudely and indi- rectly, via bone marrow stress29,41,42 (Figure 2).
So why not identify repressing epigenetic enzymes and inhibit them directly43,44 (Figure 2)? Cells contain dozens of epigenetic enzymes mediating gene activation and repres- sion, and not all repressing epigenetic enzymes (corepres-
Figure 2. Induction of fetal hemoglobin (HbF) requires chromatin remodeling, including DNA hypomethylation, of the HbF gene locus. Bone marrow stress, e.g., from cytotoxic drugs such as hydroxyurea, can create chromatin remodeling during the recovery phase of surviving erythroid precursors. An alternative approach is to remodel the hemoglobin F (HbF) gene locus (HBG) directly, e.g., by directly inhibiting/repressing epigenetic enzymes. Enzymes shown are those known to be recruited by BCL11A, TR2 or TR4 (EHMT2 and PRMT5 are not reported participants in the BCL11A/TR2/TR4 hub). The relative efficiencies of these approaches are illustrated by the greater HbF increases produced in the same non-human primates or patients by decitabine ~0.2 mg/kg twice weekly versus hydroxyurea ~20 mg/kg daily. That is, the molar amount of decitabine administered per week is <1/1000th the amount of hydroxyurea administered per week. We published variations of this figure in Molokie et al.95 and Lavelle et al.23
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haematologica | 2019; 104(9)