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Y. Saunthararajah et al.
ring covalently binds to DNMT1 and causes its degrada- tion.83 By depleting DNMT1 protein, decitabine disrupts its scaffolding functions for other epigenetic enzymes such as KDM1A.84,85 That is, decitabine does not just inhib- it the enzyme function of DNMT1 but produces a broad corepressor disrupting effect. Because the deoxyribose moiety of decitabine is unmodified, it can incorporate into the elongating DNA strand during the S-phase without terminating chain extension or causing cytotoxicity, con- trasting with most nucleoside analogs used in the clinic to treat cancer.86,87 High concentrations of decitabine do, however, produce off-target anti-metabolite effects and cytotoxicity, in significant part via its uridine moiety degradation products that can misincorporate into DNA or inhibit thymidylate synthase.88,89 We designed decitabine dose, schedule and route-of-administration reg- imens to produce non-cytotoxic depletion of DNMT1 in vivo.43,90-93 These regimens increased HbF by >10% in SCD patients who had no HbF response (~0.3%) to hydrox- yurea in the pivotal clinical trial.43,81,94 That is, very small, non-cytotoxic doses of ~0.2 mg/kg twice weekly were sufficient to produce large increases in HbF and total hemoglobins, even in patients in whom hydroxyurea ~20 mg/kg/day, >1000-fold the molar amount of decitabine, did not induce HbF (Figure 2).43,44
Marketed decitabine, however, is a parenteral drug with trivial oral bioavailability, undermining potential for worldwide application. We have therefore combined oral decitabine with tetrahydrouridine to inhibit the enzyme that limits its oral bioavailability, cytidine deaminase.95 This combination was well-tolerated and safe in a phase I study in patients with severe SCD. The target decitabine dose of 0.16 mg/kg produced a wide decitabine concentra- tion-time profile (low Cmax, long Tmax) ideal for non-cyto- toxic DNMT1 depletion83,96-98 and decreased DNMT1 pro- tein in peripheral blood mononuclear cells by >75% and repetitive element CpG methylation by ~10%. This increased HbF by 4-9%, doubling HbF-enriched RBC (F- cells) up to ~80% of total RBC. Total hemoglobin increased by 1.2-1.9 g/dL (P=0.01) as reticulocytes simul- taneously decreased; that is, better quality and efficiency of HbF-enriched erythropoiesis elevated hemoglobin using fewer reticulocytes. Other indications of better RBC quality, biomarkers of hemolysis, thrombophilia and inflammation (lactate dehydrogenase, bilirubin, D-dimer, C-reactive protein) also improved. The side-effects were a concurrent increase in platelets and decrease in neu- trophils, expected with non-cytotoxic DNMT1 depletion. In the relatively short treatment duration of 8 weeks, these blood count shifts did not cross thresholds requiring withholding or modification of treatment, that is, neu- trophil counts and platelets remained in ranges observed in SCD patients receiving standard-of-care therapies.
The major limitation is the need for longer term studies to demonstrate durable safety and efficacy of the oral tetrahydrouridine/decitabine combination.
Lysine demethylase 1 (LSD1, KDM1A)
KDM1A, like DNMT1, is recruited by the HBG2/HBG1 repressing DNA-binding factors DRED and BCL11A, and KDM1A inhibition with either of two specific inhibitors induced HbF in vitro, in sickle mice and in non-human pri- mates.99-102 Several KDM1A inhibitors are in clinical trials for cancer indications (Table 1). At least two of the com- pounds in trials (ORY-1001, GSK2879552) are built around
a tranylcypropamine warhead that inhibits monoamine oxidases that metabolize catecholamine neurotransmit- ters in the brain. Although cancer clinical trials are ongoing and unpublished (EudraCT number: 2013-002447-29; ClinicalTrials.gov identifiers: NCT02177812, NCT02034123), there is concern regarding side-effects related to the poten- tial for inhibition of monoamine oxidases other than KDM1A. Thus, there are ongoing efforts to develop and evaluate KDM1A inhibitors with other scaffolds (e.g., ClinicalTrials.gov identifier: NCT01344707). One registered phase I clinical trial evaluated a KDM1A inhibitor for HbF induction in SCD (ClinicalTrials.gov identifier: NCT03132324). This trial has been terminated but results are not publicly available at this time.
Protein arginine methyltransferase 5 (PRMT5)
PRMT5 methylation of histone H4 arginine 3 has been implicated as a signal that recruits additional chromatin- modifying enzymes and represses HBG.57 There is a PRMT5 inhibitor in clinical trials (GSK3326595) for cancer indications. No trials of this molecule for HbF induction in SCD have been registered so far.
Chromodomain helicase DNA binding protein 4 (CHD4) and SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (SMARCA5) (ISWI family of ATP- dependent chromatin remodelers
The culmination of chromatin remodeling for gene repression or activation is nucleosome (histone octamer) repositioning around the transcription start site. This is energetically expensive work executed by SWI/SNF or ISWI family proteins containing the HELICc-DExx ATP- ase domain, with SWI/SNF moving histones away to facilitate basal transcription factor machinery access and ISWI executing the opposite.46,103,104 Since such nucleo- some repositioning is the crux of chromatin remodeling, inhibition of this action should in principle offer corre- sponding potency. CHD4 and SMARCA5 are HELICc- DExx-containing corepressors that are recruited by BCL11A and DRED to repress HBG2/HBG1.54 We have identified a first-in-class drug-like compound series that preliminarily appears to inhibit the HELICc-DExx domains of SMARCA5 and CHD4, and we are actively investigating the potential of this series to induce HbF (US20170253589A1).
Epigenetic targeting - Lessons so far and open questions
Pre-clinical and clinical experience to date provide vari- ous lessons and raise some questions regarding epigenetic targeting to induce HbF, as described below.
The consequences of inhibiting an epigenetic enzyme depend on cellular context
The baseline expression pattern of transcription factors is a key determinant of a cell’s fate or function response to epigenetic enzyme inhibition, because sequence-specific
Euchromatic histone lysine methyltransferase 2 (EHMT2, G9a)
EHMT2 has been shown to be recruited to the β-globin locus by the sequence-specific DNA binding factor NFE2, and the EHMT2 inhibitor UNC0638 has been shown to induce HbF in vitro.59,60 As of this time, there are no regis- tered clinical trials evaluating EHMT2 inhibition to induce HbF.
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haematologica | 2019; 104(9)