Isocitrate dehydrogenase 1 and 2 mutations induce BCL-2 dependence in acute myeloid leukemia

Abstract

Mutant isocitrate dehydrogenase (IDH) 1 and 2 proteins alter the epigenetic landscape in acute myeloid leukemia (AML) cells through production of the oncometabolite (R)-2-hydroxyglutarate (2-HG). Here we performed a large-scale RNA interference (RNAi) screen to identify genes that are synthetic lethal to the IDH1(R132H) mutation in AML and identified the anti-apoptotic gene BCL-2. IDH1- and IDH2-mutant primary human AML cells were more sensitive than IDH1/2 wild-type cells to ABT-199, a highly specific BCL-2 inhibitor that is currently in clinical trials for hematologic malignancies, both ex vivo and in xenotransplant models. This sensitization effect was induced by (R)-2-HG-mediated inhibition of the activity of cytochrome c oxidase (COX) in the mitochondrial electron transport chain (ETC); suppression of COX activity lowered the mitochondrial threshold to trigger apoptosis upon BCL-2 inhibition. Our findings indicate that IDH1/2 mutation status may identify patients that are likely to respond to pharmacologic BCL-2 inhibition and form the rational basis for combining agents that disrupt ETC activity with ABT-199 in future clinical studies.

Figure 1

Identification of BCL-2 as synthetic lethal to mutant IDH1. (a) The 15 synthetic lethal gene hits. The log₁₀ of the drop-out ratio (number of barcode reads in the presence of dox to the number of reads in the absence of dox) in mutant IDH1^R132H-expressing cells (green bars) or in wild-type IDH1-expressing cells (blue bars) is shown. The red arrows highlight two antiapoptotic members of the BCL-2 family. (b) Viability of the engineered THP-1 cell lines transduced with lentiviral vectors encoding the indicated shRNAs and induced to express either wild-type or mutant IDH1^R132H for seven days. The means of three biological replicates are shown. (c) Viability of parental THP-1 cells transduced with lentiviral vectors encoding the indicated shRNAs and treated with the indicated concentrations of octyl-(R)-2-HG for two days. The means of three biological replicates are shown. NT, non-targeting. *P < 0.05. Statistical significance (P) was determined by Student’s t-test. All error bars represent sd.

Figure 2

(R)-2-HG sensitizes AML cells to pharmacologic BCL-2 inhibition. (a) Viability of the engineered THP-1 cell lines cultured in the absence or presence of doxycycline (dox) and treated with serial dilutions of ABT-199 for 5 days. (b) Viability of parental THP-1 cells treated with serial dilutions of ABT-199 in the presence of 300 μM of octyl-(R)-2-HG or PBS (vehicle control) for two days. (c) Viability of FACS-sorted blasts from the indicated primary AML samples treated with serial dilutions of ABT-199 in the presence of octyl-(R)-2-HG (250 μM for SU353 and 500 μM for SU463) or PBS (vehicle control) for 24 hours. (d) Viability of the engineered mutant IDH1^R132H–expressing THP-1 cell line treated with DMSO, ABT-199 (200 nM) and/or the mutant IDH1 inhibitor (mIDH1 inh; AGI-5198 at 10 μM) for five days either in the absence or presence of doxycycline. The data shown in this figure are the means of three biological replicates. *P < 0.05. Statistical significance (P) was determined by Student’s t-test. All error bars represent sd.

Figure 3

ABT-199 targets IDH1/2 mutant primary human AML cells. (a) ABT-199 IC₅₀ values of FACS-purified AML blasts (n = 11 for each group) and CD34⁺ enriched cord blood HSPCs (n = 3). (b) Change in AML bone marrow (BM) engraftment level relative to baseline in NSG mice transplanted with the indicated primary human AML samples and treated with either vehicle (mock) or ABT-199 daily for seven consecutive days. Each data point represents an individual mouse (n = 11 for the mock-treated WT IDH1/2 group; n = 12 for the ABT-199–treated WT IDH1/2 group; n = 17 for the mock-treated mutant IDH1 group; n = 18 for the ABT-199 treated mutant IDH1 group). (c) AML BM engraftment levels in NSG mice transplanted with the indicated primary samples and treated with vehicle or ABT-199 daily for 14 consecutive days (days 1–14). Each line represents serial measurements from an individual mouse. (d) AML BM engraftment levels in secondary NSG recipients 7 weeks after transplantation with BM cells obtained from primary NSG recipients that were engrafted with the indicated IDH1 mutant samples and treated with vehicle or ABT-199 for 7 consecutive days (SU372), 14 consecutive days (SU291F), or 21 consecutive days (SU430). Each data point represents an individual mouse. All horizontal lines represent the mean. All error bars represent sem. NS, not significant. *P ≤ 0.05, ** P < 0.001. Statistical significance (P) was determined by Student’s t-test.

Figure 4

(R)-2-HG–mediated inhibition of cytochrome c oxidase activity induces BCL-2 dependence. (a) Western blot for cytochrome C in the cytosolic fraction of THP-1 cells treated with DMSO (−) or ABT-199 (+) at 200 nM for 6 h. Left panel, parental cells were either untreated (–) or pretreated with octyl-(R)-2-HG (+) at 300 μM for 24 h. Right panel, engineered THP-1 cell lines were either untreated (–) or pretreated with doxycycline (+) for 72 h to induce expression of either WT IDH1 (WT) or mutant IDH1^R132H (M). β-actin, loading control. The data shown are representative of two experiments. (b) BH3 profiling assay of parental THP-1 cells pretreated with PBS (control) or octyl-(R)-2-HG (300 μM) for 24 h using the membrane potential–sensitive probe JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide). The data shown are representative of two experiments. (c) Time-course fluorescence of the membrane potential–sensitive probe JC-1 in purified mitochondria treated with the indicated compounds. The data shown are representative of three experiments. (d) Enzymatic activities of complex I, II, III and IV (COX) of the electron transport chain (ETC) in the absence or presence of (R)-2-HG or (S)-2-HG. (e) COX activity in the presence of (R)-2-HG or (S)-2-HG at the indicated concentrations. (f) COX activity normalized to MT-CO1 expression in FACS-sorted blasts of primary AML samples. Each data point represents an individual sample (n = 7 for each group). Horizontal line, mean. (g) MT-CO1 expression in the blast population of primary AML samples. Each data point represents an individual sample (n = 7 for each group). Horizontal line, mean. (h) Viability of parental THP-1 cells treated with DMSO (vehicle control) or ABT-199 (200nM) for 48 hours in the presence of either octyl-(S)-2-HG or octyl-(R)-2-HG at the indicated concentrations. (i) Viability of parental THP-1 cells treated with serial dilutions of ABT-199 for 48 hours in the absence or presence of sodium azide (NaN₃; 2.5 mM) or potassium cyanide (KCN; 5 mM). (j) Viability of FACS-purified blasts from the indicated wild-type IDH1/2 primary AML samples with serial dilutions of ABT-199 for 48 hours in the absence or presence of sodium azide (NaN₃; 5 mM). (k) Viability of parental THP-1 and KG-1 cells transduced with lentiviral vectors encoding the indicated shRNAs and treated with DMSO (vehicle control) or ABT-199 at 200 nM for 48 hours. (l) Viability of parental THP-1, KG-1 and Kasumi-1 cells pretreated with DMSO (vehicle control) or tigecycline (10 μM for THP-1, KG-1 and 2.5 μM for Kasumi-1) for 48 h followed by treatment with serial dilutions of ABT-199 at the indicated concentrations for 24 h. Unless otherwise indicated, the data shown in this figure are the means of three biological replicates. NS, not significant. * P < 0.05. Statistical significance (P) was determined by Student’s t-test. All error bars represent sd.