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Targeting sickle cell root-cause pathophysiology
sor protein complexes) are logical molecular targets for therapy. Sequence-specific DNA-binding factors are par- ticular in their epigenetic co-regulator usage, e.g., even dis- tinguishing between closely similar BAF and PBAF coacti- vator complexes.45-48 Logically, the epigenetic enzymes to target for HbF induction are those that have been directly implicated in silencing of HBG2/HBG1. Multi-protein corepressor complexes directed to the HBG loci by the DNA-binding factors DRED and BCL11A have been char- acterized in great detail.49-53 Druggable epigenetic silencing enzymes contained in these recruited corepressor com- plexes include DNA methyltransferase 1 (DNMT1), vari- ous histone deacetylases (HDAC), lysine demethylase 1 (LSD1, KDM1A), and chromodomain helicase DNA bind- ing protein 4 (CHD4) and other members of the ISWI fam- ily of ATP-dependent chromatin remodelers52-55 (Table 1). Other types of biochemical studies have implicated euchromatic histone lysine methyltransferase 2 (EHMT2, G9a),56 and protein arginine methyltransferase 5 (PRMT5) in the silencing of HBG2/HBG1 57,58 (Table 1). Yet another approach to identifying candidate targets has been chem- ical screens for HbF inducers. This approach has identified histone methyltransferases EHMT1 and EHMT2 as candi- dates for inhibition59,60 (Table 1). Notably not identified by studies thus far, given that there are clinically available inhibitors for these targets, are epigenetic enzymes in polycomb repressor complex 2 (e.g., EZH2).61
Since the natural genetic experiment of HPFH provides a fundamental rationale for pursuing pharmacological induc- tion of HbF, by extension, can the genetic variants underly- ing HPFH help to identify or prioritize molecular targets for manipulation? HPFH-linked point mutations cluster in two regions 115 and 200 base-pairs upstream of the HBG2 start site, suggesting these are sites at which key repressors of HBG2/HBG1 bind.62 BCL11A and ZBTB7A have been shown to bind at these locations, and HPFH mutations have been shown to abrogate such binding.63 Moreover, some
HPFH mutations occur at BCL11A rather than β-globin gene loci.51,64 In short, the natural genetic experiment of HPFH also seems to support drugging of the corepressors recruited by BCL11A (and ZBTB7A and DRED).49-53
The candidate targets are discussed below in turn.
Histone deacetylases (HDAC)
HDAC were among the first candidate targets identified for HbF induction.65 Moreover, a number of HDAC inhibitors have already been approved by the United States Food and Drug Administration (FDA) to treat peripheral T-cell malignancies (Table 1). Unfortunately, despite exciting pre-clinical results, clinical application of marketed HDAC inhibitors for HbF induction is limited by the pleiotropic roles of HDAC outside of chromatin. That is, clinical side-effects, arising from HDAC participa- tion in multiple cellular and physiological functions, limit the achievement of an epigenetic pharmacodynamic effect in the target compartment, and thus of HbF induction in vivo.66-71 There are efforts to develop HDAC inhibitors that are more selective to specific HDAC than the broad HDAC inhibiting activity of the currently marketed drugs (Table 1), and perhaps these more selective agents will have a more suitable safety profile for HbF induction. The caution remains that even an on-target, specific drug action can generate toxicities if the molecular target of that action has pleiotropic physiological roles.
DNA methyltransferase 1 (DNMT1)
DNMT1 is well known to maintain methylation marks on DNA through cell division. In addition, DNMT1 is a corepressor that is recruited by sequence-specific DNA- binding factors, e.g., DRED (TR2/TR4) and BCL11A, which direct epigenetic silencing of HBG.43,53,72-82 The deoxycytidine analog decitabine and its pro-drug 5-azacy- tidine, FDA-approved to treat the myeloid malignancy myelodysplastic syndrome, can deplete DNMT1: a nitro- gen substituted for a carbon in the decitabine pyrimidine
Table 1. Scientifically validated molecular targets for HbF induction and candidate drugs
Target
HDAC*
DNMT1
KDM1A#
PRMT5
EHMT2
ISWI
Recruited by BCL11A
Yes
Yes
Yes
Not reported Not reported Yes
Drugs
- Depsipeptide (HDAC1,2,4,6)
- Belinostat (broad HDAC inhibitor) - Panobinostat (broad)
- Vorinostat (broad)
- Decitabine
- 5-azacytidine (decitabine pro-drug)
- ORY-1001 (related to RN-1) - GSK2879552
- 4SC-202
- INCB059872
- GSK3326595
- UNC0638
- not officially designated, patent issued
Stage
- Marketed for peripheral T-cell lymphomas - Phase I in SCD (panobinostat)
(ClinicalTrials.gov identifier: NCT01245179) - Phase II in SCD and β-thalassemia
(HQK-1001)(ClinicalTrials.gov identifiers: NCT01642758, NCT01601340)
- Marketed for myelodysplastic syndromes
- Oral forms, including in combination with inhibitors of
degradation, are in phase I/II for liquid/solid malignancies,
and SCD (ClinicalTrials.gov identifier: NCT01685515)
- Phase I/II in liquid/solid malignancies - Phase I in SCD (INCB059872)
(ClinicalTrials.gov identifier: NCT03132324)
(terminated, results not publicly available) - Phase I in liquid/solid malignancies
- Pre-clinical in vitro
- Pre-clinical in vitro
(CHD4, SMARCA5)
*Only histone deacetylase (HDAC) inhibitors approved in the USA are listed, several other HDAC inhibitors are in clinical trials. #Only KDM1A inhibitors registered in clinical trials in the USA are listed, several other compounds are in development. SCD: sickle cell disease.
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