Oncometabolite D-2-Hydroxyglutarate Inhibits ALKBH DNA Repair Enzymes and Sensitizes IDH Mutant Cells to Alkylating Agents

Abstract

Chemotherapy of a combination of DNA alkylating agents, procarbazine and lomustine (CCNU), and a microtubule poison, vincristine, offers a significant benefit to a subset of glioma patients. The benefit of this regimen, known as PCV, was recently linked to IDH mutation that occurs frequently in glioma and produces D-2-hydroxyglutarate (D-2-HG), a competitive inhibitor of α-ketoglutarate (α-KG). We report here that D-2-HG inhibits the α-KG-dependent alkB homolog (ALKBH) DNA repair enzymes. Cells expressing mutant IDH display reduced repair kinetics, accumulate more DNA damages, and are sensitized to alkylating agents. The observed sensitization to alkylating agents requires the catalytic activity of mutant IDH to produce D-2-HG and can be reversed by the deletion of mutant IDH allele or overexpression of ALKBH2 or AKLBH3. Our results suggest that impairment of DNA repair may contribute to tumorigenesis driven by IDH mutations and that alkylating agents may merit exploration for treating IDH-mutated cancer patients.

Figure 1. 2-HG inhibits ALKBH2 and ALKBH3 and accumulates DNA damages

(A) D-2-HG inhibits the activity of ALKBH3 in vitro, with IC₅₀ values being 3.09 mM. Shown are average values of triplicated results with standard deviation (S.D.). (B) 2-HG accumulation causes decreased DNA adduct repair after MMS treatment. U87-MG cells stably expressing the indicated proteins was treated with 2 mM MMS for 1 hr. After the treatment, the cells were cultured in fresh medium containing no MMS for the indicated time. Genomic DNA was hydrolyzed to nucleotide by enzyme digestion, and was then subjected to LC-MS/MS to determine the concentrations of deoxyadenosine (dA) and 1-methyldeoxyadenosine (1MedA). Shown are average values of triplicated results with standard deviation (S.D.). (C, D, E) 2-HG accumulation causes increased double strand breaks after MMS treatment. U87-MG and U-373 cells stably expressing the indicated proteins were exposed to increasing concentrations of MMS for 1 hr. After the treatment, the cells were cultured in fresh medium containing no MMS for another 10 hours, and the level of phosphorylated histone variant H2A.X (γ-H2AX) was determined by western blot (C) and immunofluorescence (D). Scale bar:100 μm. The number of γ-H2AX foci was counted from 25 randomly selected cells (E). Shown are average values of triplicated results with standard error of means (SEM).

Figure 2. Expression of tumor-derived mutant IDH1 sensitizes cells to alkylating agents

U87-MG and U373-MG cells stably expressing the indicated proteins were exposed to increasing concentrations of MNNG or MMS for 1 hour. The cells were cultured in fresh medium containing no alkylating agents for another 47 hours. After that, cell death was assessed by performing flow cytometry analysis (A), MTT assay (B), and trypan blue staining for viable cell counting (C). Shown are average values of triplicated results with standard deviation (S.D.). *denotes the p < 0.05 for cells expressing mutant IDH1 versus wild-type IDH1; n.s.= not significant. (D). Knockdown ALKBH2 sensitize U87-MG cells expressing wild-type, but not mutant IDH1 to MMS treatment. Cell death was assessed by flow cytometry analysis (right panel).

Figure 3. Alkylating agents sensitizing effect of mutant IDH1 is dependent on 2-HG

(A, B) Characterization of 2-HG producing activity of IDH1 double mutants. The activity of various IDH1 double mutants in producing 2-HG was determined by both the rate of NADPH oxidation (A) and GC-MS analysis (B). Peaks at 19.6 min were identified as bis-TBDMSD derived 2-HG by the mass fragment spectra. The bis-TBDMS 2-HG-specific 433-m/z fragment is shown in the insert. Moreover, the peaks at 18.9 min and 20.3 min were identified as the derivatives of aspartate (Asp) and glutamate (Glu), respectively. ** indicates p < 0.01 by student’s t-test between IDH1^R132H and IDH1. (C) U87-MG cells stably expressing the indicated proteins were exposed to increasing concentrations of MNNG or MMS. Cell death was assessed by performing flow cytometry analysis and trypan blue staining for viable cell counting. (D) The mRNA expression of selected DNA repair genes was tested in U87-MG cells stably expressing the indicated proteins, as determined by qRT-PCR analysis. (E) HT1080 (IDH1+/−) cell line was generated by knocking-out the R132C allele in parental HT1080 (IDH1+/R132C) cells using TALEN technique. Metabolites extracted from HT1080 (IDH1+/−) and parental HT1080 (IDH1+/R132C) cells were subjected to GC-MS analysis, showing the loss of 2-HG accumulation in HT1080 (IDH1+/−) cells. (F) HT1080 (IDH1+/−) and parental HT1080 (IDH1+/R132C) cells were exposed to increased concentrations of MNNG or MMS. Cell death was assessed by performing flow cytometry analysis and trypan blue staining for viable cell counting. Shown are average values of triplicated results with standard deviation (S.D.). *denotes the p < 0.05 for HT1080 (IDH1+/−) versus parental HT1080 (IDH1+/R132C) cells. (G, H) Parental HT1080 (IDH1^+/R132C) and TALEN-edited HT1080 (IDH1^+/−) cells stably expressing FLAG tagged ALKBH2 or ALKBH3 were established by retrovirus transduction. Expression of ALKBH was verified by western blot (G). The sensitivity of both IDH1^+/R132C and IDH1^+/− cells to MMS was determined by MTT assay (H).

Figure 4. Expression of tumor-derived mutant IDH1 sensitizes cells to clinical alkylating agents

(A, B) U87-MG cells stably expressing the indicated proteins were exposed to different concentrations of busulfan(A), procabazine, CCNU or vincristine (B) for 48 hrs. Cell viability was assessed by flow cytometry analysis (left), and cells were also stained with trypan blue for viable cell counting (right). (C) U87-MG cells stably expressing the indicated proteins were treated with different concentrations of CCNU, along with 1 mM procarbazine or 500 nM vincristine for 48 hours. Cell viability was assessed by flow cytometry analysis (upper) and trypan blue exclusion (lower). Shown are average values of triplicated results with standard deviation (S.D.). *denotes the p < 0.05 for cells expressing mutant IDH1 versus wild-type IDH1; n.s.= not significant. (D) Parental HT1080 (IDH1^+/R132C) and TALEN-edited HT1080 (IDH1^+/−) cells stably expressing ALKBH2 or ALKBH3 were exposed to different concentrations of CCNU for 72 hr. Cell viability was determined by MTT assay. *denotes the p < 0.05 for HT1080 (IDH1+/−) versus parental HT1080 (IDH1+/R132C) cells.