5-azacytidine (5-Aza) is a potent inducer of fetal hemoglobin (HbF) in people with-thalassemia and sickle cell disease. Two models have been proposed to explain this activity. The first is based on the drug's ability to inhibit global DNA methylation, including the fetal globin genes, resulting in their activation. The second is based on 5-Aza's cytotoxicity and observations that HbF production is enhanced during marrow recovery. We tested these models using human primary cells in an in vitro erythroid differentiation system. We found that doses of 5-Aza that produce near maximal induction of ␥-globin mRNA and HbF do not alter cell growth, differentiation kinetics, or cell cycle, but do cause a localized demethylation of the ␥ promoter. However, when we reduced ␥ promoter methylation to levels equivalent to those seen with 5-Aza or to the lower levels seen in primary fetal erythroid cells using DNMT1 siRNA and shRNA, we observed no induc-
IntroductionMutations which allow expression of the fetal globin genes in adults with sickle cell disease (SCD) or -thalassemia can produce a significant decrease in disease severity. 1,2 This has led to a search for pharmacologic agents that can activate fetal globin gene expression during adult erythropoiesis. Several agents have been shown to have this property when studied in human or primate systems. 3 These include the nucleoside analog DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-Aza) and 2Јdeoxy-5-azacytidine (decitabine), sodium butyrate and derivatives, histone deacetylase (HDAC) inhibitors, and cytotoxic agents including hydroxyurea, cytosine arabinoside, and vinblastine. While all of these agents are active, none exhibit the optimal combination of safety, efficacy, and convenience of use that would make them applicable to most hemoglobinopathy patients, especially those who lack access to modern medical facilities. These factors provide a strong rationale for developing new pharmacologic strategies that specifically target fetal gene activation without cytotoxicity, widespread epigenetic alterations, or difficult to manage side effects.The rational design of such targeted therapies is likely to require an in-depth knowledge of the molecular mechanisms underlying globin gene switching and pharmacologically mediated fetal gene reactivation. To this end, we have investigated the mechanism of 5-azacytidine (5-Aza). This was the first pharmacologic agent shown to have the ability to reactivate fetal gene expression in humans with -thalassemia 4 and SCD 5 and that can provide long-term clinical benefits for hemoglobinopathy patients. 6 5-Aza was originally brought to the clinic as a cytotoxic cancer chemotherapy agent. Only later was it found to be a potent inhibitor of DNA methyltransferase enzymes. 7 These 2 properties, cytotoxicity and inhibition of DNA methylation, led to the development of 2 distinct theories to explain the ability of 5-Aza to induce fetal hemoglobin (HbF) production in adult erythroid cells. The first hypothesis was that 5-Aza's abil...
A phase 1 study was conducted to determine the dose-limiting toxicities and maximum tolerated dose (MTD) for bortezomib followed by romidepsin on days 1, 8, and 15 in patients with relapsed/refractory CLL/SLL or B- or T-cell lymphoma. Eighteen treated patients were evaluable for response. The MTD was 1.3 mg/m2 bortezomib and 10 mg/m2 romidepsin; median treatment duration was 3 cycles at this dose. The dose-limiting toxicities were grade 3 fatigue, vomiting, and chills. Two patients had partial responses, one lasting >2 years, 8 had stable disease, and 8 had progressive disease. The median duration of stable disease was 3.5 cycles. Correlative studies examining expression of NF-κB, XIAP, Bcl-xL, and Bim yielded variable results. The safety profile was consistent with that reported for single-agent bortezomib and romidepsin. This regimen has modest activity in heavily pre-treated patients with relapsed/refractory CLL or B- or T-cell lymphoma. NCT00963274.
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