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Targeting sickle cell root-cause pathophysiology
DNA-binding factors direct the function of these epigenet- ic enzymes and are mandatory for gene activation.105 Stated another way, the consequences of inhibiting a par- ticular epigenetic enzyme depend very much on cellular context.105 A corollary of the above is that although inhibiting silencing epigenetic enzymes can produce cell fate or function shifts, these relate to what the cells were to begin with and are not drastic.105 This is of course criti- cal clinically, since a candidate epigenetic therapeutic for SCD will be distributed systemically.
What then about the cellular/transcription factor con- text of erythropoiesis enables inhibition of DNMT1 etc., to activate HBG2/HBG1? Several groups have found that the developmental switch from HBG2/HBG1 to HBB acti- vation is recapitulated, albeit very rapidly, during ery- throid lineage maturation (a ‘maturational switch’ during routine erythropoiesis).106-110 The maturational switch entails removal of activating and acquisition of repressive epigenetic marks at HBG2/HBG1.59,102 with physical migra- tion of the shared enhancer, the locus control region, from the HBG2/HBG1 to the HBB locus.54 These dynamics at HBG2/HBG1 and HBB during erythropoiesis creates an opportunity for pharmacological/biochemical interven- tion to prevent enhancer migration, to stall the massive gene activating machinery at HBG2/HBG1. That is, HbF induction by inhibiting epigenetic ‘off’ enzymes such as DNMT1 is not predicated on returning the enhancer from HBB back to HBG2/HBG1 (turning a gene that is ‘off’ to ‘on’), but on preventing a switch from HBG2/HBG1 to HBB in the first place (preventing a gene that is ‘on’ from being turned ‘off’). Accordingly, HbF induction by an inhibitor of the silencing epigenetic enzyme EHMT2 (UNC0638) depended on the timing of its addition to cul- tures of synchronously maturing erythroid progenitors,59 with similar observations in our hands with DNMT1- depleting drugs (personal communication).
Why are these drugs being evaluated for, or used, to treat cancer?
Some of the most recurrently mutated, deleted or ampli- fied genes in cancers encode for chromatin remodelers. Thus, another concern with epigenetic targeting is whether it might mimic some of these genetic changes and favor activation of oncogenic programs. It is reassuring to some extent, however, that the epigenetic targets and drugs discussed above have been or are being developed to treat and/or prevent cancer. We recently reviewed the bio- logical rationale for this,105 and it is briefly summarized here: cancer cells, including self-replicating cancer cells (cancer or leukemia ‘stem’ cells), contain high amounts of the lineage master transcription factors that normally acti- vate terminal lineage-fates, and depend on specific core- pressors (‘addictions’) in order to avoid these terminal fates.111 The pathway of action is activation of the terminal lineage-fates intended by cancer cell lineage master tran- scription factor content. The same chromatin-‘relaxing’ treatments that trigger terminal lineage-fates of cancer/leukemia stem cells preserve self-renewal of uncommitted tissue stem cells, since these cells express stem cell master transcription factors, not high levels of lin- eage-specifying transcription factors.92,93 This therapeutic index explains why non-cytotoxic doses and schedules of decitabine can suppress malignant clones and simultane- ously improve functional blood counts even in elderly patients with myeloid malignancies.91,105,111-115 Stated simply, several corepressor components (repressing epigenetic
enzymes), e.g., DNMT1, HDAC, KDM1A, have been bio- logically validated as molecular targets for the treatment and prevention of cancer.111
Teratogenic risks
Another concern is the potential for teratogenicity: this should be assumed for individual agents, unless shown otherwise by formal toxicological studies.
Drug metabolism is central to the clinical profile of activity
Drugs, being biologically active, are metabolized, and this too can contribute substantially to their in vivo profile of activity. For example, DNMT1-depleting decitabine is a pyrmidine nucleoside analog pro-drug that depends absolutely for its activity on the pyrimidine metabolism enzyme deoxycytidine kinase: Deoxycytidine kinase exe- cutes the initial phosphorylation of decitabine in cells, which rate-limits its conversion into the nucleotide form that actually depletes DNMT1. Serendipitously, deoxycy- tidine kinase is most highly expressed in the myeloid compartment, especially erythroid precursors. Thus, the clinical profile of decitabine activity is in major part dictat- ed by its metabolically driven tropism for the myeloid compartment.
Baseline HbF levels dictate final HbF levels
There is a wide variation in baseline HbF levels in patients with SCD and even in the general population, reflecting the influence of various genetic polymorphisms on the regulation of this locus.9 Even if a molecular target- ed therapy produces similar rates of increase in HbF% (the percentage of total hemoglobin that is HbF) in all patients, the final HbF% will be dictated by the level at which HbF% began. Moreover, in clinical trials we have conduct- ed with DNMT1-depleting decitabine, we have noticed a slightly lower slope to the rate of increase in HbF% in SCD patients with lower HbF% at baseline. Fortunately and importantly, however, HbF induced by this epigenetic strategy was well-distributed among RBC, and the rates of increase of HbF-enriched RBC (F-cells) was actually higher in patients with low F-cells at baseline.43,81,94,95
At some time-point after starting therapy, F-cells enter- ing the circulation are matched by a similar number of F- cells leaving the circulation, producing plateaus in HbF% and F-cell%.
Small molecules to chemically modify HbS to impede polymerization
The scientific foundation for efforts to chemically mod- ify HbS is the two-state allosteric Monod-Wyman- Changeux structural model which characterizes the rapid- ly reversible equilibrium between the quarternary struc- ture of hemoglobin with low oxygen affinity (fully deoxy- genated hemoglobin, ‘‘T’ quarternary structure) and the hemoglobin quarternary structure with high affinity for oxygen (oxygenated hemoglobin, ‘R’ quarternary struc- ture).116,117 The Monod-Wyman-Changeux model demon- strated incompatibility of the R conformation with poly- merization, creating a foundation to propose molecules to favor the high oxygen affinity R conformation, as a method to delay HbS polymerization.5
The basic concern with such an approach is that SCD is a disease of decreased oxygen delivery to tissues and,
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