SummaryLenalidomide is a highly effective treatment for myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Here, we demonstrate that lenalidomide induces the ubiquitination of casein kinase 1A1 (CK1α) by the CRL4CRBN E3 ubiquitin ligase, resulting in CK1α degradation. CK1α is encoded by a gene within the common deleted region for del(5q) MDS and haploinsufficient expression sensitizes cells to lenalidomide therapy, providing a mechanistic basis for lenalidomide's therapeutic window in del(5q) MDS. We found that mouse cells are resistant to lenalidomide but that changing a single amino acid in mouse Crbn to the corresponding human residue enables lenalidomide-dependent degradation of CK1α. We further demonstrate that minor side chain modifications in thalidomide and a novel analogue, CC-122, can modulate the spectrum of substrates targeted by CRL4CRBN. These findings have implications for the clinical activity of lenalidomide and related compounds and demonstrate the therapeutic potential of novel modulators of E3 ubiquitin ligases.

BackgroundThe cytidine nucleoside analogs azacitidine (AZA) and decitabine (DAC) are used for the treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Few non-clinical studies have directly compared the mechanisms of action of these agents in a head-to-head fashion, and the agents are often viewed as mechanistically similar DNA hypomethylating agents. To better understand the similarities and differences in mechanisms of these drugs, we compared their in vitro effects on several end points in human AML cell lines.Methodology/Principal FindingsBoth drugs effected DNA methyltransferase 1 depletion, DNA hypomethylation, and DNA damage induction, with DAC showing equivalent activity at concentrations 2- to 10-fold lower than AZA. At concentrations above 1 µM, AZA had a greater effect than DAC on reducing cell viability. Both drugs increased the sub-G1 fraction and apoptosis markers, with AZA decreasing all cell cycle phases and DAC causing an increase in G2-M. Total protein synthesis was reduced only by AZA, and drug-modulated gene expression profiles were largely non-overlapping.Conclusions/SignificanceThese data demonstrate shared mechanisms of action of AZA and DAC on DNA-mediated markers of activity, but distinctly different effects in their actions on cell viability, protein synthesis, cell cycle, and gene expression. The differential effects of AZA may be mediated by RNA incorporation, as the distribution of AZA in nucleic acid of KG-1a cells was 65∶35, RNA∶DNA.

Approximately 8% to 19% of patients with acute myeloid leukemia (AML) have isocitrate dehydrogenase-2 (IDH2) mutations, which occur at active site arginine residues R140 and R172. IDH2 mutations produce an oncometabolite, 2-hydroxyglutarate (2-HG), which leads to DNA and histone hypermethylation and impaired hematopoietic differentiation. Enasidenib is an oral inhibitor of mutant-IDH2 proteins. This first-in-human phase 1/2 study evaluated enasidenib doses of 50 to 650 mg/d, administered in continuous 28-day cycles, in patients with mutant-IDH2 hematologic malignancies. Overall, 214 of 345 patients (62%) with relapsed or refractory (R/R) AML received enasidenib, 100 mg/d. Median age was 68 years. Forty-two patients (19.6%) attained complete remission (CR), 19 patients (10.3%) proceeded to an allogeneic bone marrow transplant, and the overall response rate was 38.8% (95% confidence interval [CI], 32.2-45.7). Median overall survival was 8.8 months (95% CI, 7.7-9.6). Response and survival were comparable among patients with IDH2-R140 or IDH2-R172 mutations. Response rates were similar among patients who, at study entry, were in relapse (37.7%) or were refractory to intensive (37.5%) or nonintensive (43.2%) therapies. Sixty-six (43.1%) red blood cell transfusion–dependent and 53 (40.2%) platelet transfusion–dependent patients achieved transfusion independence. The magnitude of 2-HG reduction on study was associated with CR in IDH2-R172 patients. Clearance of mutant-IDH2 clones was also associated with achievement of CR. Among all 345 patients, the most common grade 3 or 4 treatment-related adverse events were hyperbilirubinemia (10%), thrombocytopenia (7%), and IDH differentiation syndrome (6%). Enasidenib was well tolerated and induced molecular remissions and hematologic responses in patients with AML for whom prior treatments had failed. The study is registered at www.clinicaltrials.gov as #NCT01915498.

A B S T R A C T PurposeTo determine the maximum-tolerated dose (MTD), safety, pharmacokinetic and pharmacodynamic profiles, and clinical activity of an oral formulation of azacitidine in patients with myelodysplastic syndromes (MDSs), chronic myelomonocytic leukemia (CMML), or acute myeloid leukemia (AML). Patients and MethodsPatients received 1 cycle of subcutaneous (SC) azacitidine (75 mg/m 2 ) on the first 7 days of cycle 1, followed by oral azacitidine daily (120 to 600 mg) on the first 7 days of each additional 28-day cycle. Pharmacokinetic and pharmacodynamic profiles were evaluated during cycles 1 and 2. Adverse events and hematologic responses were recorded. Cross-over to SC azacitidine was permitted for nonresponders who received Ն 6 cycles of oral azacitidine. ResultsOverall, 41 patients received SC and oral azacitidine (MDSs, n ϭ 29; CMML, n ϭ 4; AML, n ϭ 8). Dose-limiting toxicity (grade 3/4 diarrhea) occurred at the 600-mg dose and MTD was 480 mg. Most common grade 3/4 adverse events were diarrhea (12.2%), nausea (7.3%), vomiting (7.3%), febrile neutropenia (19.5%), and fatigue (9.8%). Azacitidine exposure increased with escalating oral doses. Mean relative oral bioavailability ranged from 6.3% to 20%. Oral and SC azacitidine decreased DNA methylation in blood, with maximum effect at day 15 of each cycle. Hematologic responses occurred in patients with MDSs and CMML. Overall response rate (ie, complete remission, hematologic improvement, or RBC or platelet transfusion independence) was 35% in previously treated patients and 73% in previously untreated patients. ConclusionOral azacitidine was bioavailable and demonstrated biologic and clinical activity in patients with MDSs and CMML.

Mutations in the gene encoding isocitrate dehydrogenase 2 (IDH2) occur in several types of cancer, including acute myeloid leukemia (AML). In model systems, mutant IDH2 causes hematopoietic differentiation arrest. Enasidenib, a selective small-molecule inhibitor of mutant IDH2, produces a clinical response in 40% of treated patients with relapsed/refractory AML by promoting leukemic cell differentiation. Here, we studied the clonal basis of response and acquired resistance to enasidenib treatment. Using sequential patient samples, we determined the clonal structure of hematopoietic cell populations at different stages of differentiation. Before therapy, IDH2-mutant clones showed variable differentiation arrest. Enasidenib treatment promoted hematopoietic differentiation from either terminal or ancestral mutant clones; less frequently, treatment promoted differentiation of nonmutant cells. Analysis of paired diagnosis/relapse samples did not identify second-site mutations in IDH2 at relapse. Instead, relapse arose by clonal evolution or selection of terminal or ancestral clones, thus highlighting multiple bypass pathways that could potentially be targeted to restore differentiation arrest. These results show how mapping of clonal structure in cell populations at different stages of differentiation can reveal the response and evolution of clones during treatment response and relapse.