Oral tetrahydrouridine and decitabine for non-cytotoxic epigenetic gene regulation in sickle cell disease: A randomized phase 1 study

Abstract

BackgroundSickle cell disease (SCD), a congenital hemolytic anemia that exacts terrible global morbidity and mortality, is driven by polymerization of mutated sickle hemoglobin (HbS) in red blood cells (RBCs). Fetal hemoglobin (HbF) interferes with this polymerization, but HbF is epigenetically silenced from infancy onward by DNA methyltransferase 1 (DNMT1).Methods and findingsTo pharmacologically re-induce HbF by DNMT1 inhibition, this first-in-human clinical trial (NCT01685515) combined 2 small molecules—decitabine to deplete DNMT1 and tetrahydrouridine (THU) to inhibit cytidine deaminase (CDA), the enzyme that otherwise rapidly deaminates/inactivates decitabine, severely limiting its half-life, tissue distribution, and oral bioavailability. Oral decitabine doses, administered after oral THU 10 mg/kg, were escalated from a very low starting level (0.01, 0.02, 0.04, 0.08, or 0.16 mg/kg) to identify minimal doses active in depleting DNMT1 without cytotoxicity. Patients were SCD adults at risk of early death despite standard-of-care, randomized 3:2 to THU–decitabine versus placebo in 5 cohorts of 5 patients treated 2X/week for 8 weeks, with 4 weeks of follow-up. The primary endpoint was ≥ grade 3 non-hematologic toxicity. This endpoint was not triggered, and adverse events (AEs) were not significantly different in THU-decitabine—versus placebo-treated patients. At the decitabine 0.16 mg/kg dose, plasma concentrations peaked at approximately 50 nM (Cmax) and remained elevated for several hours. This dose decreased DNMT1 protein in peripheral blood mononuclear cells by >75% and repetitive element CpG methylation by approximately 10%, and increased HbF by 4%–9% (P < 0.001), doubling fetal hemoglobin-enriched red blood cells (F-cells) up to approximately 80% of total RBCs. Total hemoglobin increased by 1.2–1.9 g/dL (P = 0.01) as reticulocytes simultaneously decreased; that is, better quality and efficiency of HbF-enriched erythropoiesis elevated hemoglobin using fewer reticulocytes. Also indicating better RBC quality, biomarkers of hemolysis, thrombophilia, and inflammation (LDH, bilirubin, D-dimer, C-reactive protein [CRP]) improved. As expected with non-cytotoxic DNMT1-depletion, platelets increased and neutrophils concurrently decreased, but not to an extent requiring treatment holds. As an early phase study, limitations include small patient numbers at each dose level and narrow capacity to evaluate clinical benefits.ConclusionAdministration of oral THU-decitabine to patients with SCD was safe in this study and, by targeting DNMT1, upregulated HbF in RBCs. Further studies should investigate clinical benefits and potential harms not identified to date.Trial registrationClinicalTrials.gov, NCT01685515

Leukemogenic nucleophosmin mutation disrupts the transcription factor hub that regulates granulomonocytic fates

“…Immunodeficient mice were xenotransplanted with NPM1/FLT3-mutated primary AML cells (55). After bone marrow AML engraftment to at least 20% was confirmed in 3 randomly selected mice (Figure 10A), mice were randomized to treatment with (i) vehicle as control; (ii) nuclear export inhibition by 2 mg/kg selinexor by ilarly, DNMT1 was not depleted from AML cells harvested from bone marrow at time of euthanasia (Supplemental Figure 19B), consistent with previous documentation by us and others that in vitro resistance to decitabine and 5-azacytidine is by selection for malignant cells that avoid DNMT1 depletion (58)(59)(60)(61)(62)(63). Thus, resistance both in vitro and in vivo was mediated by prevention of intended molecular pharmacodynamic effects, undermonths of in vitro culture (exponential proliferation in selinexor up to 50 nM added every 3 days) demonstrated persistent cytoplasmic dislocation of mutant NPM1/PU.1, as shown both by IF ( Figure 11A) and by WB of cytoplasmic and nuclear fractions ( Figure 11B).…”

“…40); this explains meaningful clinical activity of noncytotoxic DNMT1 depletion by decitabine (or its pro-drug 5-azacytidine) in patients with myeloid malignancies containing sundry mutations and translocations (20,21,(65)(66)(67)(91)(92)(93)(94) and likely contributes to the greater efficacy of this approach compared with selinexor alone in the in vivo model containing both NPM1 and FLT3 mutations. Underscoring the importance of the targeted pathways to the malignant phenotype, resistance in vitro and in vivo was by AML cells that evaded nuclear export inhibition by selinexor and DNMT1 depletion by decitabine/5-azacytidine (58)(59)(60)(61)(62)(63).…”

“…Mice were anesthetized with isoflurane before transplantation. After confirmation of bone marrow AML engraftment to ≥20% in at least 3 randomly selected mice, remaining mice were randomized to treatment with vehicle (PBS), nuclear export inhibition by 2-5 mg/kg oral selinexor 4 times per week (n = 5 per group), DNMT1 inhibition (depletion) by 10 mg/kg tetrahydrouridine (THU) given intraperitoneally, followed by 0.1 mg/kg subcutaneous decitabine or 1 mg/ kg subcutaneous 5-azacytidine 3 times per week (the decitabine and 5-azacytidine were alternated each week; the subcutaneous route of administration of decitabine and 5-azacytidine was to produce low C max and long half-life suited to noncytotoxic DNMT1 depletion; tetrahydrouridine was used to inhibit cytidine deaminase, which otherwise rapidly deaminates decitabine and 5-azacytidine in vivo) (59)(60)(61)67). Tail vein blood samples for blood count measurement by HemaVet were obtained prior to leukemia inoculation and at intervals thereafter as indicated in the figures.…”

MIR29B mediates epigenetic mechanisms of HBG gene activation

“…Therapeutic interventions to decrease HBG gene methylation and reactivate transcription have proven beneficial in clinical studies. For example, SCD patients treated intravenously with the DNA methyltransferase (DNMT) inhibitor decitabine (Dec) demonstrated robust induction of HbF and total haemoglobin levels (Molokie et al , ; Akpan et al , ; Lavelle et al , ; Desimone et al , ). However, when administered by mouth, Dec is rapidly inactivated by cytidine deaminase.…”

“…However, when administered by mouth, Dec is rapidly inactivated by cytidine deaminase. In a recent Phase I clinical trial, where Dec was combined with tetrahydrouridine to inhibit its metabolism, DNMT1 protein levels and DNA methylation were decreased in peripheral blood mononuclear cells, while HbF levels increased in SCD patients (Molokie et al , ). Ideally, treatments for SCD will activate HBG expression without bone marrow toxicity and avoiding off target effects.…”