Supplemental Methods. DNA constructs, Gel electrophoresis and western blotting, Metabolite extraction and analysis

Allosteric inhibitors of mutant IDH1 or IDH2 induce terminal differentiation of the mutant leukemic blasts and provide durable clinical responses in approximately 40% of acute myeloid leukemia (AML) patients with the mutations. However, primary resistance and acquired resistance to the drugs are major clinical issues. To understand the molecular underpinnings of clinical resistance to IDH inhibitors (IDHi), we perform multipronged genomic analyses (DNA sequencing, RNA sequencing and cytosine methylation profiling) in longitudinally collected specimens from 60 IDH1- or IDH2-mutant AML patients treated with the inhibitors. The analysis reveals that leukemia stemness is a major driver of primary resistance to IDHi, whereas selection of mutations in RUNX1 / CEBPA or RAS - RTK pathway genes is the main driver of acquired resistance to IDHi, along with BCOR , homologous IDH gene, and TET2 . These data suggest that targeting stemness and certain high-risk co-occurring mutations may overcome resistance to IDHi in AML.

Azacitidine and enasidenib are two therapies available for treatment of acute myelogenous leukemia (AML), and the mechanisms of action of these drugs involve alteration of aberrant DNA methylation. We hypothesized that a combination of these agents could have interactive effects on DNA methylation and enhance differentiation in mIDH2 cells. Combination treatment enhanced cellular differentiation in TF-1 cells overexpressing IDJ2R140Q through increased hemoglobinization and increased hemoglobin g RNA expression compared with the effects of single agents. Furthermore, in primary AML samples (IDH2R140Q or R172K), combination treatment reduced CD34+ cells and increased CD15+ cells to a greater extent than attained with single agents. To explore the mechanism of enhanced differentiation with combination treatment, the TF-1 epigenome was analyzed by profiling 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) DNA methylation changes. Enasidenib treatment alone increased 5hmC, consistent with reactivation of ten-eleven-translocation (TET) enzyme activity. Compared with treatment with azacitidine alone, combination treatment reduced 5mC levels at greater numbers of sites and these loci were significantly enriched in regions with increased 5hMC (25.8% vs. 7.4%). Results are consistent with a model in which enasidenib-mediated reactivation of ten-eleven-translocation enzymes cooperates with azacitidine-mediated inhibition of DNA methyltransferase enzymes, leading to greater reductions in DNA methylation and enhanced erythroid differentiation. (c) 2021 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Introduction: LR-MDS are characterized by ineffective erythropoiesis that leads to anemia and red blood cell (RBC) transfusion dependence. Luspatercept is a first-in-class erythroid maturation agent that binds to select TGF-β superfamily ligands and enhances late-stage erythropoiesis. MEDALIST is a phase 3, randomized, double-blind, placebo-controlled trial to evaluate the safety and efficacy of luspatercept in pts with LR-MDS (IPSS-R-defined Very low-, Low-, and Intermediate-risk) with ring sideroblasts who required RBC transfusions and were ineligible for, intolerant of, or refractory to erythropoiesis-stimulating agents. Clinical benefit (CB; defined as RBC transfusion independence [RBC-TI] ≥ 8 weeks and/or modified hematologic improvement-erythroid [mHI-E] per IWG 2006 criteria) in the primary MEDALIST treatment phase (Weeks 1-24) was achieved by 58.2% of pts in the luspatercept arm and 21.1% in the placebo arm (P < 0.0001). The objective of the study was to investigate the effect of luspatercept treatment on erythropoiesis biomarkers and their relationship to CB in the primary MEDALIST treatment phase (Weeks 1-24). Methods: In the MEDALIST trial, 229 pts were randomized to receive either luspatercept (N = 153) or placebo (N = 76). Reticulocyte count was determined in blood samples collected at baseline and during the primary treatment phase. Serum biomarkers (soluble transferrin receptor 1 [sTfR1], erythroferrone [ERFE], and erythropoietin [EPO]) were measured by ELISA. Bone marrow (BM) erythroid precursors (EP) were determined by cytomorphology from BM aspirates. Biomarker levels were compared between baseline and Week 25 within treatment arms and between pts with CB and without CB in the luspatercept arm using a paired 2-tailed t-test and unpaired t-test (parametric method). Results: In the luspatercept arm, mean reticulocyte count increased from baseline, starting at 8 days after first dose (55.1 vs 34.5 × 109/L at baseline, P < 0.0001), and remained elevated throughout the evaluation period (Figure). Mean EPO levels increased significantly within 6 weeks after first dose (440.1 vs 220.4 IU/L at baseline, P < 0.0001) and remained elevated up to Week 25. Similarly, levels of sTfR1 (P < 0.0001), ERFE (P < 0.0001), and EP (P = 0.0010) were elevated at Week 25 relative to baseline (Table). The mean transfusion burden (within 16 weeks) was significantly reduced at Week 25 compared with baseline (7.2 vs 11.0 units, P < 0.0001). In contrast, in the placebo arm, reticulocyte count, EPO levels, and 16-week transfusion burden remained largely unchanged, while levels of sTfR1 (P < 0.0001), ERFE (P = 0.0431), and EP (P = 0.0010) were significantly lower at Week 25 relative to baseline. In the luspatercept arm, mean baseline EP were higher in 87 pts with CB (31.3%) compared with 63 pts without CB (26.5%; P = 0.0298). No statistically significant differences in baseline EPO, ERFE, sTfR1, reticulocyte count, and 16-week transfusion burden were observed in either group. At Week 25, pts with luspatercept and CB had a significantly greater increase of reticulocyte count (2.7 vs 1.8 mean fold increase from baseline, P = 0.0017), but not EPO levels (2.9 vs 4.3 mean fold increase from baseline, P = 0.1370) compared with pts without CB. Changes in erythropoiesis-related biomarkers (EP, ERFE, and sTfR1) did not differ significantly between pts with and without CB. To investigate whether luspatercept affects erythroid maturation, the ratio of reticulocyte/sTfR1 was calculated. This ratio was reasoned to be an approximation of the ratio of late-stage erythropoiesis (reticulocytes) within total erythropoiesis (sTfR1). Luspatercept increased the mean ratio of reticulocyte/sTfR1 in pts with CB (2.2 in Week 25 vs 1.5 at baseline, P < 0.0001) and no CB (1.9 in week 25 vs 1.3 at baseline, P = 0.0071). Conclusions: Luspatercept-treated pts in the MEDALIST trial had an increase of erythropoiesis-associated biomarkers. Luspatercept-mediated CB (RBC-TI ≥ 8 weeks and/or mHI-E) was associated with increased blood reticulocyte counts and was higher in pts with expanded BM erythropoiesis (as measured by EP) at baseline. Together with the observation that the ratio of reticulocytes/sTfR1 increased during luspatercept treatment, this suggests that the luspatercept mechanism of efficacy in pts with LR-MDS is associated with an increase of erythroid maturation and reticulocytes. Disclosures Platzbecker: Novartis: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Geron: Consultancy, Honoraria; Amgen: Honoraria, Research Funding. Zhu:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Ha:Bristol Myers Squibb: Current Employment. Risueño:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: Named in BMS (before Celgene) patent filings related to predictive patient response biomarkers in hematological malignancies. Chan:Bristol Myers Squibb: Current Employment. Zhang:BMS: Current Employment. Dunshee:Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Genentech Inc.: Current Employment, Current equity holder in publicly-traded company. Acar:Bristol Myers Squibb: Ended employment in the past 24 months. Shetty:BMS: Current Employment, Current equity holder in publicly-traded company. Ito:BMS: Current Employment, Current equity holder in publicly-traded company. MacBeth:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Santini:Menarini: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Acceleron: Consultancy; Novartis: Consultancy, Honoraria; Johnson & Johnson: Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Garbowski:Imara: Consultancy; Vifor Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees. Fenaux:BMS: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Schwickart:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

Luspatercept is a recombinant fusion protein that binds and sequesters several endogenous transforming growth factor-beta superfamily ligands, including growth differentiation factor 11, thereby diminishing Smad2/3 signaling in target cells involved in erythropoiesis. Luspatercept, and its murine analog RAP-536, have been shown to act as erythroid maturation agents via their effects on late-stage erythropoiesis by inducing erythroblast maturation, leading to increases in red blood cells (RBCs) and hemoglobin (Hb). This study demonstrated that thalassemic (th3/+) reticulocytes are unstable, and that RAP-536, in addition to its function as an erythroid maturation agent, modulates the maturation of wild-type (WT) and th3/+ reticulocytes. Furthermore, RAP-536 treatment increased RBCs and decreased bilirubin in a mouse model of alpha-thalassemia (129S-Hba-a1tm1Led/J). To examine whether acute RAP-536 treatment acts on reticulocytes and alters reticulocyte levels in blood, the blood of WT mice was analyzed 3, 12, and 24 hours, and 2, 3, 4, and 7 days after a single dose of RAP-536 (10 or 30 mg/kg) or vehicle. RAP-536 treatment increased RBCs, Hb, and hematocrit significantly at all time points, compared with vehicle. However, in mice treated with RAP-536, reticulocytes in blood decreased significantly on Days 2, 3, and 4 and returned to normal baseline levels on Day 7. Analysis of reticulocyte subpopulations in blood 3 days after RAP-536 treatment showed that the relative percentages of immature reticulocytes (CD71+ or high RNA content) within the blood reticulocyte population decreased, suggesting that reticulocytes released from the bone marrow (BM) were more mature and/or reticulocytes matured faster in blood. A quantitative pharmacology (QP) model was developed to explore which RAP-536-induced modulations of erythropoiesis in WT mice can simulate the experimental observations. The model represents erythroblast, reticulocyte, and RBC (erythrocyte) maturation stages in BM, peripheral blood, and spleen, in the presence or absence of a RAP-536 effect. The QP model consists of a system of ordinary differential equations, with homeostatic parameter values assigned from literature or experimental measures, and RAP-536-perturbed parameter values regressed by fitting the model to erythropoiesis data of RAP-536-treated WT mice. Comparison of model parameters for homeostatic versus RAP-536-perturbed states indicated that RAP-536 leads to an increase in the erythroblast-to-reticulocyte and reticulocyte-to-RBC conversion rates, the transfer of BM reticulocytes to blood, and a delayed increase in erythroblast production. To directly test whether RAP-536 treatment affects reticulocyte development in blood, comparative blood transfusion experiments were performed. Biotinylated GFP+ blood from WT mice (C57BL/6-Tg(UBC-GFP)30Scha/J) and biotinylated GFP− blood from th3/+ beta-thalassemic mice (B6.129P2-Hbb-b1tm1Unc Hbb-b2tm1Unc/J) were co-transfused into GFP− WT recipient mice (C57BL/6J), which were subsequently treated with RAP-536 or vehicle. In the donor reticulocyte population, th3/+ reticulocyte percentage decreased continuously up to 3 days after transfusion, suggesting that many of the th3/+ reticulocytes were eliminated before they could form RBCs. However, compared with vehicle, RAP-536 treatment led to increased persistence of the relative percentages of th3/+ reticulocytes (Figure A). Consequently, 7 days after transfusion, when most reticulocytes have matured to RBCs, the percentage of th3/+ RBC among donor RBCs was higher with RAP-536 (Figure B). Finally, treatment of an alpha-thalassemia mouse model (129S-Hba-a1tm1Led/J) with RAP-536 10 mg/kg for 8 weeks increased RBCs and hematocrit and reduced serum bilirubin, compared with vehicle. These results suggest that RAP-536 is, as previously shown, an erythroid maturation agent, which also modulates reticulocyte maturation in blood. In WT mice, RAP-536 modulated blood reticulocyte dynamics consistent with faster maturation. RAP-536 also prolonged the persistence of th3/+ reticulocytes and maintained a higher frequency of th3/+ RBCs. These data, together with the finding that RAP-536 reduces hemolysis in an experimental alpha-thalassemia disease model, suggest that luspatercept has the potential to improve anemias associated with hemolysis and/or reticulocytosis. Disclosures Acar: Bristol Myers Squibb: Ended employment in the past 24 months. Jupelli:Bristol Myers Squibb: Current Employment. Abbiati:Bristol Myers Squibb: Current Employment. Ramanathan:Acceleron Pharma: Current Employment, Current equity holder in publicly-traded company. Santini:Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Ratushny:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Dunshee:Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Genentech Inc.: Current Employment, Current equity holder in publicly-traded company. Lopes de Menezes:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. MacBeth:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Suragani:Acceleron Pharma: Current Employment, Current equity holder in publicly-traded company. Loos:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Schwickart:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

Background Mutations in isocitrate dehydrogenase-2 (IDH2) occur in around 5% of patients with myelodysplastic syndromes. Neomorphic activity of mutant IDH2 proteins results in hypermethylation of DNA arid histones, leading to blocked haemopoietic differentiation. Enasidenib, an inhibitor of mutated IDH2 proteins, induces responses in patients with IDH2-inutated, relapsed or refractory acute myeloid leukaemia. We aimed to establish the clinical outcomes of enasidenib monotherapy in a subgroup of patients with myelodysplastic syndromes harbouring mutations in IDH2 from the AG221-C-001 trial. Methods The multicentre, open-label, phase 1-2 AG221-C-001 trial enrolled patients with advanced haematological malignancies (2008 WHO criteria) harbouring an IDH2 mutation. The present study is a subgroup analysis of patients with IDH2-mutated myelodysplastic syndromes in the phase 1 dose-escalation and expansion portions of the trial. Patients with myelodysplastic syndromes were aged 18 years or older with an ECOG performance status score of 2 or lower, and were relapsed or refractory to, or ineligible for, standard treatments. Patients received oral doses of enasidenib at 60-300 mg per day in repeated 28-day treatment cycles. In this subgroup analysis, we focused on the safety and activity of enasidenib as main outcomes. Overall response rate, duration of response, and overall and event-free survival analyses were by intention-to-treat. Safety was assessed in all participants who received at least one dose of study drug in terms of treatment-emergent adverse events. The AG221-C-001 trial is registered on ClinicalTrials.gov, NCT01915498, status ongoing but closed to recruitment. Findings 17 patients with myelodysplastic syndromes harbouring an IDH2 mutation (median age, 67.0 years [IQR 60.5-73.0]) were enrolled between Feb 18,2014, and Sept 1,2015. At data cutoff (Oct 1, 2018), after a median followup of 11.0 months (IQR 6.8-23.0), all patients had discontinued enasidenib, with a median of 3 treatment cycles (2-15) for all patients (five 129%1 received a12 cycles). At entry, three (18%) patients had relapsed after allogeneic stem-cell transplants, 13 (76%) had previously received therapy with hypomethylating agents, and ten (59%) had received at least two previous therapies. No dose-limiting toxicities were reported. The most common treatment-emergent adverse events were diarrhoea and nausea (in nine 153%] patients each). Most common grade 3-4 treatment-emergent adverse events were indirect hyperbilirubinaernia (in six [35%] patients), pneumonia (in five [29%] patients), and thrombocytopaenia (in four [24%] patients). Serious treatment-emergent adverse events in more than one patient were pneumonia (in five [29% patients); tumor lysis syndrome (in three [18%] patients); and sepsis, atrial flutter, indirect hyperbilirubinaemia, cerebral hemorrhage, and mental status change (in two [12%1 patients each). No treatment-related deaths occurred. An overall response was achieved in 9 patients (53% [95% CI 28-771), with a median duration of response of 9.2 months (95% CI 1.0-not reached). Six (46%) of 13 patients previously treated with hypomethylating agents responded. Median overall survival was 16.9 months (95% CI 1.5-32.3), and median event-free survival was 11.0 months (1.5-16.7). Interpretation Enasidenib is generally well tolerated and can induce responses in patients with mutant IDH2 myelodysplastic syndromes, including in those who have had previous therapy with hypomethylating agents. Testing for IDH2 mutations in myelodysplastic syndromes is essential for identifying patients who might benefit from enasidenib therapy, including those patients in whom conventional treatments have been unsuccessful. Copyright (C) 2020 Elsevier Ltd. All rights reserved.

BACKGROUND: CC-486, a DNA hypomethylating agent and epigenetic modifier, is an oral formulation of azacitidine (AZA) that is administered at lower exposures for extended durations (300 mg/day [d] for 14 or 21d/28d cycle) compared with the injectable formulation of AZA, which is given in a high exposure, limited duration regimen of 75mg/m2 for 7d/28d cycle. AZA induces DNA damage and cytotoxicity, and promotes changes in gene expression leading to cellular differentiation. As DNA incorporation of AZA is S-phase-dependent, it has been hypothesized that extended dosing with CC-486 prolongs drug exposure and DNA incorporation to enhance epigenetic activity. The mechanism of action imparted by extended dosing schedules of CC-486 is not fully understood. In patients with myeloid malignancies, DNA hypomethylation in blood is sustained throughout the 28d Tx cycle with extended CC-486 dosing regimens (Laille, 2015; Garcia-Manero, 2016). To better understand the mechanism of CC-486, we assessed the kinetics of expression of myeloid markers of cellular differentiation and cytotoxicity with various AZA dosing schedules in in vitro and in vivo models of AML. METHODS: AML cell lines (AML-193, KG1a, and MV4-11) were treated in vitro with AZA (0.05 - 5 µM daily for 5d or 15d), and at cumulative concentrations of 1 or 3 µM administered once or fractionated over 2-5d to experimentally model CC-486 extended exposures: 1 µM cumulative dose (1 µM × 1d, 0.5 µM × 2d, 0.33 µM × 3d, 0.25 µM × 4d, or 0.2 µM × 5d); 3 µM cumulative dose (3 µM × 1d, 1.5 µM × 2d, 1 µM × 3d, 0.75 µM × 4d, or 0.6 µM × 5d). AZA- or vehicle-treated cells were analyzed by flow cytometry, DNA methylation (Illumina Infinium EPIC assay), and RNA-Seq. Temporal expression of CD11b was assessed as a surface marker of myeloid differentiation, and Annexin-V staining was used to determine the extent of apoptosis and cell death. In efficacy studies, mouse models of AML (syngeneic, cell line-derived xenografts) were treated intraperitoneally with AZA regimens at 1 mg/kg/d × 15d (extended) or 3 mg/kg/d x 5d. RESULTS: Tx of AML-193 cells with 0.05 - 5 µM daily AZA led to upregulation of markers of myeloid differentiation (including CD11b) at lower doses, and a dose-dependent increase in apoptosis up to 7d after Tx initiation. Following Tx with 1 µM AZA for 1d, maximal cellular differentiation (ie, CD11b expression) occurred at d3 in 30% of AML-193 cells; conversely, cells treated with 0.2 µM/d AZA for 5d showed greater differentiation (40%) peaking on d7 (Fig A). CD11b expression was increased upon each subsequent cell division; after 5 cell divisions, CD11b upregulation was 4-fold higher in cells treated with multiple, lower AZA doses than with 1 µM AZA administered for 1d (Fig B). CD11b upregulation was not observed in the absence of cell division under serum starvation conditions for 3d (to induce cell cycle arrest), further suggesting that cell division is a requirement for AZA-induced CD11b changes (Fig C). Similarly, AML-193 cells treated with a 3 µM cumulative AZA dose over 1, 2, 3, 4, or 5d showed greater changes in myeloid differentiation marker expression, with peak apoptosis at d7 with extended dosing regimens (Fig D). In KG1a and MV4-11 cells, Tx with 1 µM AZA QD for 5d led to induction of myeloid differentiation by d7, and cell death (followed by recovery of undifferentiated cells) by d28. In contrast, daily Tx with 0.3 µM AZA for 15d led to slower, more robust upregulation of differentiation markers, peaking at d21 and accompanied by a gradual loss of cell viability. Extended AZA exposure to cells led to pronounced changes in gene expression (Fig E) and DNA methylation (Fig F) at both d7 (immune response gene signature) and d28 (cell adhesion gene signature) compared with limited duration exposure AZA. In mice, low exposure, extended regimens of AZA exhibited higher DNA and RNA incorporation into peripheral blood mononuclear cells (PBMCs) and bone marrow cells when compared with higher exposure, limited duration regimens. Extended AZA dosing led to significant efficacy in murine AML models. CONCLUSIONS: In AML cell lines, low exposure, extended duration AZA schedules modeling CC-486 induced robust changes in differentiation. These results suggest that CC-486-mediated effects using extended exposure regimens preferentially promote a differentiation effect and cell death of AML tumor cells. These mechanistic insights may help inform rational CC-486 combination Tx strategies. Disclosures Dunshee: Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Genentech Inc.: Current Employment, Current equity holder in publicly-traded company. Dai:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Jang:Bristol Myers Squibb: Ended employment in the past 24 months. Risueño:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: Named in BMS (before Celgene) patent filings related to predictive patient response biomarkers in hematological malignancies. Jeyaraju:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hagner:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. See:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. MacBeth:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Wang:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. La Torre:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Skikne:Bristol Myers Squibb: Current Employment. Beach:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Kumar:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Thakurta:Oxford University: Other: visiting professor; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Lopes de Menezes:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

Luspatercept, a fusion protein composed of a modified activin receptor type-2B (ActRIIB) and IgG1 Fc, is a novel erythroid maturation agent that has been shown to increase red blood cell (RBC) and hemoglobin (Hb) levels. Luspatercept binds to and sequesters several endogenous transforming growth factor-beta superfamily ligands, thereby diminishing Smad2/3 signaling. RAP-536, the murine analog of luspatercept, has been shown to induce erythroblast maturation and increase RBC, Hb, and hematocrit levels in wild-type (WT) mice and in murine disease models of beta-thalassemia and myelodysplastic syndromes. Previous studies have shown that rapamycin (sirolimus), an mTOR inhibitor, improved anemia in a mouse model of beta-thalassemia by activating the autophagy pathway. In this study, rapamycin was tested in combination with RAP-536 by treating WT mice and th3/+ mice (a beta-thalassemia disease model; B6.129P2-Hbb-b1tm1Unc Hbb-b2tm1Unc/J) with vehicle, RAP-536 10 mg/kg twice weekly, rapamycin 4 mg/kg daily, or RAP-536 plus rapamycin for 2 weeks. Single-agent dosing with either RAP-536 or rapamycin increased RBC, Hb, and hematocrit levels significantly compared with vehicle treatment in both WT and th3/+ mice (Figure). The percentage increase in RBC, Hb, and hematocrit levels upon RAP-536 or rapamycin administration was substantially higher in th3/+ mice than in WT mice. Interestingly, much higher and significant increases in RBC and Hb levels in both WT and th3/+ mice, and in hematocrit levels in th3/+ mice, were observed with RAP-536 plus rapamycin, when compared with the single agents. For example, in th3/+ mice, the increase in Hb levels compared with vehicle control was 19.2% with RAP-536 alone, 13.0% with rapamycin alone, and 44.7% with RAP-536 plus rapamycin (Figure). In th3/+ mice, RAP-536 plus rapamycin treatment also significantly reduced spleen enlargement compared with vehicle (P < 0.001). RAP-536 increased and rapamycin decreased overall reticulocyte levels and immature reticulocyte relative percentages in whole reticulocyte populations in the blood of WT mice, suggesting that RAP-536 and rapamycin have different mechanisms of action through which they increase RBC and Hb levels. Altogether, these preclinical results provide a rationale for combining rapamycin, and potentially other mTOR inhibitors, with luspatercept to treat anemia associated with beta-thalassemia. Disclosures Acar: Bristol Myers Squibb: Ended employment in the past 24 months. Jupelli:Bristol Myers Squibb: Current Employment. MacBeth:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Schwickart:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

Somatic gene mutations are key determinants of outcome in patients with myelodysplastic syndromes (MDS) and secondary AML (sAML). In particular, patients with TP53 mutations represent a distinct molecular cohort with uniformly poor prognosis. The precise pathogenetic mechanisms underlying these inferior outcomes have not been delineated. In this study, we characterized the immunological features of the malignant clone and alterations in the immune microenvironment in patients with TP53-mutant and wild-type MDS or sAML. Notably, PDL1 expression is significantly increased in hematopoietic stem cells of patients with TP53 mutations, which is associated with MYC upregulation and marked downregulation of MYC's negative regulator miR-34a, a p53 transcription target. Notably, patients with TP53 mutations display significantly reduced numbers of bone marrow-infiltrating OX40+ cytotoxic T cells and helper T cells, as well as decreased ICOS+ and 4-1BB+ natural killer cells. Further, highly immunosuppressive regulatory T cells (Tregs) (ie, ICOShigh/PD-1-) and myeloid-derived suppressor cells (PD-1low) are expanded in cases with TP53 mutations. Finally, a higher proportion of bone marrow-infiltrating ICOShigh/PD-1- Treg cells is a highly significant independent predictor of overall survival. We conclude that the microenvironment of TP53 mutant MDS and sAML has an immune-privileged, evasive phenotype that may be a primary driver of poor outcomes and submit that immunomodulatory therapeutic strategies may offer a benefit for this molecularly defined subpopulation.

Allosteric inhibitors of mutant IDH1 or IDH2 induce terminal differentiation in mutant leukemic blasts and may provide durable clinical responses in approximately 40% of AML patients with the mutations. However, most responders eventually relapse.

Background: Luspatercept is a first-in-class erythroid maturation agent that binds TGF-β superfamily ligands to reduce aberrant Smad2/3 signaling and enhance late-stage erythropoiesis. The phase 3 MEDALIST trial evaluated luspatercept in pts with RBC transfusion-dependent, IPSS-R-defined very low-, low-, and intermediate-risk MDS with ring sideroblasts (RS+) who were refractory, intolerant, or ineligible to receive erythropoiesis-stimulating agents. This study explored associations of gene mutations, as analyzed by next-generation sequencing (NGS), with response to luspatercept, as well as dynamics of gene mutations on therapy in MEDALIST pts. Methods: DNA was isolated from bone marrow (BM) mononuclear cells from 222 of 229 pts enrolled in the study (148 luspatercept, 74 placebo) at screening and, when available, following treatment. NGS of 23 MDS-relevant genes was performed at screening and every 24 weeks; mean coverage was 1,000-fold and the variant allele frequency (VAF) cutoff was ≥ 1%. BM cell populations were analyzed by cytomorphology. Response criterion of RBC transfusion independence (RBC-TI) of ≥ 8 weeks within the first 24 weeks of treatment was used for correlative analyses. Results: Mutations in SF3B1 were found in 91.0% of pts analyzed at screening (median VAF 42%, range 6-71%), consistent with the study population being RS+. Overall, a median of 2 (range 0-5) of the 23 MDS-relevant genes analyzed were mutated per pt. In addition to SF3B1, the most frequently mutated genes were TET2 (41.9%), DNMT3A (18.9%), ASXL1 (13.1%), and SRSF2 (8.1%). Mutation profiles were similar to those found in previous studies of refractory anemia with RS (RARS; Malcovati L, et al. Blood. 2015;126:233-41) and balanced between luspatercept and placebo arms. Numbers of mutated genes at baseline were distributed similarly in luspatercept responders (R) and non-responders (NR) (Figure A), and comparable response rates were achieved irrespective of number of mutations, with response rates of 36.4%, 34.9%, 42.4%, and 33.3% for pts with 1 mutation, 2 mutations, 3 mutations, and 4 or 5 mutations in the 23 MDS-relevant genes analyzed, respectively. Response to luspatercept was independent of the presence of mutations in any of the genes analyzed individually (Figure B) or when grouped by functional categories (e.g. spliceosome, epigenetic regulation, transcription factor, etc.) (Figure C). Circos plots of co-occurring mutations showed similar mutation profiles in R and NR (Figure D). Response rates were also similar regardless of baseline SF3B1 allelic burden (R: 43%, NR: 42%; P = 0.11). At baseline, BM erythroid precursors were higher in R (R: 32.8%, NR: 26%; P = 0.008; while R and NR had similar levels of RS+ cells [R: 80%, NR: 84%; P = 0.25], Figure E), consistent with the postulated activity of luspatercept on the erythroid lineage. When comparing the frequency of mutation changes in luspatercept- vs placebo-treated pts at week 24 of the study, no statistically significant differences were observed in the frequency of newly acquired mutations (13/126 [10.3%] pts in luspatercept vs 8/64 [12.5%] pts in placebo, P = 0.63) or mutation losses (4/126 [3.2%] in luspatercept vs 5/64 [7.8%] in placebo, P = 0.17). Evaluation of changes in allele burden (median VAF at week 24 vs baseline) for mutations in genes associated with adverse prognosis (ASXL1, SRSF2, U2AF1, NRAS, IDH2, GATA2, TP53, RUNX1, and EZH2; Bejar R. Curr Opin Hematol. 2017;24:73-8) showed no change between luspatercept- or placebo-treated pts (1.01-fold, n = 58 and 0.95-fold, n = 19, respectively, P = 0.69). Conclusions: Pts enrolled in the MEDALIST study had mutations consistent with RS+, lower-risk MDS with a preponderance of SF3B1 mutations; genes associated with poor prognosis (and other genes) were balanced between study arms. RBC-TI responses with luspatercept were achieved regardless of SF3B1 allelic burden, number of baseline mutations, and presence of individual mutations, including adverse mutations, or co-mutations. Disclosures Platzbecker: Abbvie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Dunshee:Celgene Corporation: Employment, Equity Ownership. Komrokji:DSI: Consultancy; pfizer: Consultancy; Agios: Consultancy; JAZZ: Consultancy; Novartis: Speakers Bureau; Incyte: Consultancy; celgene: Consultancy; JAZZ: Speakers Bureau. Mufti:Cellectis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Garcia-Manero:Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding; Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding. Buckstein:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Research Funding. Santini:Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Honoraria; Acceleron: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees. Díez-Campelo:Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. See:Celgene Corporation: Other: Contractor. Tsai:Celgene Corporation: Employment. Risueño:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties: Named in Celgene patent filings related to predictive patient response biomarkers in hematological malignancies. Ma:Celgene Corporation: Employment, Equity Ownership. Schwickart:Celgene Corporation: Employment, Equity Ownership. Rampersad:Celgene Corp: Employment, Equity Ownership. Zhang:Celgene Corporation: Employment, Equity Ownership. Laadem:Celgene Corporation: Employment, Equity Ownership. Menezes:Celgene Corporation: Employment, Equity Ownership. MacBeth:Celgene Corporation: Employment, Equity Ownership. Linde:Acceleron Pharma: Employment, Equity Ownership; Abbott Laboratories, Inc.: Equity Ownership; Fibrogen, Inc.: Equity Ownership. Reynolds:Acceleron Pharma: Employment, Equity Ownership. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Fenaux:Celgene Corporation: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Aprea: Research Funding. OffLabel Disclosure: Luspatercept is an investigational therapy that is not approved for any use in any country. Luspatercept is currently being evaluated for potential use in patients with anemia due to myelodysplastic syndromes, beta-thalassemia, or myelofibrosis.

Background: Mutations in IDH2 occur in 8-19% of patients (pts) with AML. Enasidenib (ENA; AG-221) is an oral, small-molecule inhibitor of mutant IDH2 (mIDH2) that promotes myeloid cell differentiation. ENA is approved in the US for use in adult pts with relapsed/refractory mIDH2 AML. Azacitidine (AZA) is a hypomethylating agent that prolongs survival vs. conventional care regimens in older unfit pts with newly diagnosed (ND) AML. AZA promotes DNA hypomethylation by inhibiting DNA methyltransferases. ENA indirectly reduces DNA methylation by suppressing the oncometabolite, 2-hydroxyglutarate (2-HG), thereby restoring function to α-ketoglutarate-dependent TET family enzymes, among other substrates. In vitro, combination ENA + AZA enhances cell differentiation. This is the first report of interim outcomes from the randomized, phase II portion of an ongoing, open-label, phase I/II study of ENA + AZA vs. AZA monotherapy (AZA-only) in pts with mIDH2 ND-AML who are not candidates for intensive chemotherapy (IC) (NCT02677922). Methods: Adult pts with mIDH2 ND-AML who were ineligible to receive IC and had ECOG PS scores ≤2 were randomized in a 2:1 ratio to receive ENA + AZA or AZA-only in repeated 28-day cycles. All pts receive SC AZA 75 mg/m2/day for the first 7 days of each treatment (Tx) cycle; pts randomized to ENA + AZA also receive continuous ENA 100 mg QD. The primary endpoint is overall response rate (ORR), which includes complete remission (CR), CR with incomplete blood or platelet count recovery (CRi/CRp), partial remission (PR), and morphologic leukemia-free state (MLFS), per modified IWG 2003 AML response criteria. P values for response comparisons between Tx arms were derived using chi-square test. Duration of response (DOR) was estimated by Kaplan-Meier method. mIDH2 variant allele frequencies (VAF) in bone marrow mononuclear cells (BMMCs) were assessed by digital PCR. Results: Between Oct. 2016 and Aug. 2018, 101 pts were randomized to receive ENA + AZA (n=68) or AZA-only (n=33). Median ages were 74 years (range 62-85) in the ENA + AZA arm and 75 years (57-85) in the AZA-only arm. Among pts with available data, 78% in the ENA + AZA arm (43/55) and 90% (19/21) in the AZA-only arm had intermediate-risk cytogenetics, respectively, and 18% and 10% had poor-risk cytogenetics. At data cutoff (Feb. 2019), 39 pts were still receiving their randomized Tx. Most common reasons for study discontinuation in the ENA + AZA and AZA-only arms were death (31% and 27%, respectively) and pt decision (4% and 12%). Two pts in the ENA + AZA arm and 1 pt in the AZA-only arm proceeded to transplant. Median number of Tx cycles was 8 (range 1-24) in the ENA + AZA arm and 6 (1-22) in the AZA-only arm; 27% and 19% of pts, respectively, received ≥12 Tx cycles. Response rates were significantly higher with combination treatment vs. AZA alone: ORRs were 68% vs. 42%, respectively (P=0.0155), and CR rates were 50% vs. 12% (P=0.0002) (Table). Median DOR was not reached with ENA + AZA and was 10.2 months in the AZA-only arm (P=0.13). Maximal mIDH2 VAF suppression from baseline was significantly greater with ENA + AZA vs. AZA-only (median -69.3% vs. -14.1%, respectively; P=0.0004). Tx-related grade 3-4 adverse events occurring in ≥10% of pts in the combination arm were neutropenia (34%), thrombocytopenia (34%), anemia (21%), febrile neutropenia (12%), and IDH differentiation syndrome (IDH-DS; 10%); these events occurred in 19%, 19%, 22%, 13%, and 0% of pts in the AZA-only arm. Rate of Tx-related grade 3-4 infections was 16% in the ENA + AZA arm and 31% in the AZA-only arm. In all, 12 pts (18%) in the ENA + AZA arm experienced IDH-DS (any grade) at a median of 37 days. In the first 60 days, 5 deaths (7%) were reported in the ENA + AZA arm (3 due to infectious complications and 2 due to possible IDH-DS) and 1 death (3%) was reported in the AZA-only arm due to progressive disease. Conclusions: ENA + AZA was associated with significantly improved complete remission and overall response rates and significant mIDH2 VAF reductions compared with AZA-only. Combination Tx was generally well tolerated, with a safety profile similar to that reported for either monotherapy. An updated data cutoff that includes at least 1 year of follow-up for all pts will be presented at the conference. Updated data will include overall survival, event-free survival, and comprehensive analyses of 2-HG and co-mutation dynamics. Disclosures DiNardo: syros: Honoraria; jazz: Honoraria; medimmune: Honoraria; agios: Consultancy, Honoraria; abbvie: Consultancy, Honoraria; celgene: Consultancy, Honoraria; daiichi sankyo: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees. Schuh:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Teva Canada Innovation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria; Teva Canada Innovation: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Stein:Genentech: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Fernandez:Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Wei:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Honoraria, Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: AHW is a former employee of the Walter and Eliza Hall Institute and receives a fraction of its royalty stream related to venetoclax, Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Macrogenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. De Botton:Novartis: Consultancy; Astellas: Consultancy; AbbVie: Consultancy; Pfizer: Consultancy; Pierre Fabre: Consultancy; Daiichi: Consultancy; Celgene Corporation: Consultancy, Speakers Bureau; Servier: Consultancy; Syros: Consultancy; Janssen: Consultancy; Forma: Consultancy, Research Funding; Bayer: Consultancy; Agios: Consultancy, Research Funding. Zeidan:ADC Therapeutics: Research Funding; Jazz: Honoraria; Ariad: Honoraria; Agios: Honoraria; Novartis: Honoraria; Astellas: Honoraria; Daiichi Sankyo: Honoraria; Cardinal Health: Honoraria; Seattle Genetics: Honoraria; BeyondSpring: Honoraria; Medimmune/AstraZeneca: Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Otsuka: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Acceleron Pharma: Consultancy, Honoraria, Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding. Fathi:Amphivena, Kite, Jazz, NewLink Genetics,: Honoraria; Agios, Astellas, Celgene, Daiichi Sankyo, Novartis, Takeda, Amphivena, Kite, Forty Seven,Trovagene, NewLink genetics, Jazz, Abbvie, and PTC Therapeutics: Consultancy. Quek:Celgene: Research Funding, Speakers Bureau; Agios: Research Funding. Kantarjian:Immunogen: Research Funding; Cyclacel: Research Funding; Agios: Honoraria, Research Funding; Astex: Research Funding; BMS: Research Funding; Novartis: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Research Funding; Ariad: Research Funding; Daiichi-Sankyo: Research Funding; Amgen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding. Frattini:Celgene Corporation: Employment, Equity Ownership. Lersch:Celgene: Employment, Equity Ownership. Gong:Celgene: Employment, Equity Ownership. Franovic:Celgene: Employment, Equity Ownership. MacBeth:Celgene Corporation: Employment, Equity Ownership. Vyas:Celgene: Research Funding, Speakers Bureau; Daiichi Sankyo: Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Forty Seven, Inc.: Research Funding; Abbvie: Speakers Bureau; Pfizer: Speakers Bureau; Astellas: Speakers Bureau. Döhner:Celgene, Novartis, Sunesis: Honoraria, Research Funding; AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding.

Background:The β‐thalassemias are a group of inherited disorders characterized by absent or reduced production of the β‐globin chains of hemoglobin (Hb), leading to ineffective erythropoiesis, chronic anemia, and multiple morbidities, and often require lifelong RBC transfusions. Increasing HbF is a therapeutic approach to rescuing the defect imposed by reduced/absent β‐globin in β‐thalassemias. Luspatercept is a first‐in‐class erythroid maturation agent that binds to select TGF‐β superfamily ligands and enhances late‐stage erythropoiesis by a mechanism that is still not fully understood. The phase 3, randomized, double‐blind, placebo‐controlled BELIEVE study evaluated the efficacy and safety of luspatercept in adult β‐thalassemia patients requiring regular RBC transfusions. In the BELIEVE study, treatment with luspatercept resulted in significant reductions in RBC transfusion burden compared with placebo.Aims:To explore the effects of luspatercept treatment on HbF levels in patients with RBC transfusion‐dependent (TD) β‐thalassemia, and to explore the relationship of HbF changes with transfusion burden reduction.Methods:In the BELIEVE study, 336 adults with TD β‐thalassemia were randomized to receive either luspatercept or placebo, administered subcutaneously every 21 days for 48 weeks, plus best supportive care. Hb variants were analyzed by HPLC and HbF was reported as a percentage of total Hb from whole blood samples collected at baseline and throughout the treatment period. P values were derived by 2‐tailed t‐test (parametric method) and Wilcoxon test (non‐parametric method). Differences in HbF changes during treatment were compared between responders and non‐responders (as defined by transfusion burden decrease of ≥33% over any 12 weeks), patients with baseline HbF≤1% and HbF>1%, and luspatercept‐ and placebo‐treated patients.Results:HbF levels increased by a mean 1.2‐fold in luspatercept‐treated patients beginning at Dose2 Day1, increasing to 2.5‐fold by the end of the evaluation period (Dose16 Day1). Mean change in HbF levels remained relatively unchanged in placebo‐treated patients through Dose16 Day1, ranging from 0.9‐ to 1.2‐fold of baseline levels.Among luspatercept‐treated patients starting at Dose5 until Dose16 Day1, HbF fold increase was greater in responders vs non‐responders (Dose5 Day1: 2.1 vs 1.8 mean fold increase, P < 0.024, Dose16 Day1: 2.7 vs 2.1 mean fold increase, P = 0.012). To determine whether changes in HbF were secondary to RBC transfusion reduction or a direct effect of luspatercept, a subgroup analysis was performed in patients with normal HbF (HbF≤1%) at baseline. In luspatercept‐treated patients with normal HbF at baseline, HbF increased by a mean 1.4‐fold in responders and 1.5‐fold in non‐responders, beginning at Dose2 Day1, and continued to increase to 3.8‐fold in responders and 2.9‐fold in non‐responders by Dose16 Day1, with no statistically significant difference between HbF changes in responders and non‐responders (Figure). HbF levels were not modulated in placebo‐treated patients. In luspatercept‐treated patients with elevated (>1%) levels of HbF at baseline, HbF levels increased more in responders compared with non‐responders (2.8 vs 2.0 mean fold increase, P = 0.009).Summary/Conclusion:Luspatercept treatment was associated with increased HbF in patients with RBC TD β‐thalassemia. This luspatercept treatment effect on increasing HbF levels was observed in both responders and non‐responders. Luspatercept‐mediated increases in HbF were observed early and maintained throughout the treatment period.image

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.

Older adults with acute myeloid leukemia (AML) who are not fit for standard chemotherapy historically have poor outcomes. Approximately 12-15% of older patients with AML harbor isocitrate dehydrogenase 2 (IDH2) gene mutations. Enasidenib is an oral inhibitor of mutant IDH2 proteins. Among 39 patients with newly diagnosed mutant-IDH2 AML who received enasidenib monotherapy in this phase I/II trial, median age was 77 years (range 58-87) and 23 patients (59%) had had an antecedent hematologic disorder. The median number of enasidenib treatment cycles was 6.0 (range 1-35). The most common treatment-related adverse events were indirect hyperbilirubinemia (31%), nausea (23%), and fatigue, decreased appetite, and rash (18% each). Treatment-related grade 3-4 cytopenias were reported for eight patients (21%); there was no treatment-related grade 3-4 infections. Twelve patients achieved a response (overall response rate 30.8% [95% CI 17.0%, 47.6%]), including seven patients (18%) who attained complete remission. At a median follow-up of 8.4 months, the median duration of any response was not reached (NR). Median overall survival for all patients was 11.3 months (95% CI 5.7, 15.1), and was NR for responders. Oral, outpatient targeted treatment with enasidenib may benefit older adults with newly diagnosed mutant-IDH2 AML who are not candidates for cytotoxic regimens.

Introduction: Preclinical and early clinical studies suggest that combining epigenetic agents with checkpoint inhibitors can potentially improve outcomes in patients with previously treated advanced non-small cell lung cancer (NSCLC). This phase 2 trial examined second-line pembrolizumab thorn CC-486 (oral azacitidine) in patients with advanced NSCLC. Methods: Patients with one prior line of platinum-containing therapy were randomised in a ratio of 1:1 to CC-486 or placebo, on days 1-14, in combination with pembrolizumab on day 1 of a 21-day cycle. The primary end-point was progression-free survival (PFS). Key secondary end-points included overall survival (OS), overall response rate (ORR) and safety. Results: Among 100 patients randomised (pembrolizumab thorn CC-486: 51; pembrolizumab thorn placebo: 49), most were male (57.0%), were white (87.0%) and had Eastern Cooperative Oncology Group performance status 1 (68.0%). No significant difference in PFS was observed between the pembrolizumab thorn CC-486 and pembrolizumab thorn placebo arms (median, 2.9 and 4.0 months, respectively; hazard ratio [HR], 1.374; 90% confidence interval [CI], 0.926-2.038; P = 0.1789). Median OS was 11.9 months versus not estimable (HR, 1.375; 90% CI, 0.830-2.276; P = 0.2968); ORR was 20% versus 14%. Median treatment duration was shorter (15.0 versus 24.1 weeks), and the number of cycles was lower (5.0 versus 7.0) with pembrolizumab thorn CC-486 versus pembrolizumab thorn placebo. No new safety signals for CC-486 or pembrolizumab were detected. Treatment-emergent adverse events were more common in the pembrolizumab thorn CC-486 arm, particularly gastrointestinal, potentially impacting treatment feasibility. Conclusions: No improvement in PFS was observed with pembrolizumab thorn CC-486 versus pembrolizumab thorn placebo. Decreased treatment exposure due to adverse events may have impacted efficacy with pembrolizumab thorn CC-486. (C) 2018 Elsevier Ltd. All rights reserved.

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.

Abstract Somatic mutations in cytosolic or mitochondrial isoforms of isocitrate dehydrogenase (IDH1 or IDH2, respectively) contribute to oncogenesis via production of the metabolite 2-hydroxyglutarate (2HG). Isoform-selective IDH inhibitors suppress 2HG production and induce clinical responses in patients with IDH1- and IDH2-mutant malignancies. Despite the promising activity of IDH inhibitors, the mechanisms that mediate resistance to IDH inhibition are poorly understood. Here, we describe four clinical cases that identify mutant IDH isoform switching, either from mutant IDH1 to mutant IDH2 or vice versa, as a mechanism of acquired clinical resistance to IDH inhibition in solid and liquid tumors. Significance: IDH-mutant cancers can develop resistance to isoform-selective IDH inhibition by “isoform switching” from mutant IDH1 to mutant IDH2 or vice versa, thereby restoring 2HG production by the tumor. These findings underscore a role for continued 2HG production in tumor progression and suggest therapeutic strategies to prevent or overcome resistance. This article is highlighted in the In This Issue feature, p. 1494