2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity

Abstract

2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This "BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.

Figure 1. Mutant IDH cells are deficient in DNA DSB break repair

(A) Western blot analysis of IDH1-R132H expression in IDH1-mutant HCT116 and HeLa cells. SMC1 is used as loading control. (B) Quantification of (R)-2HG by LC-MS in WT and R132/+ HeLa and HCT116 cells (n=3, mean +/− SD). (C–D) Western blot analysis of doxycycline-inducible, FLAG-tagged (C) WT or mutant IDH1 and (D) WT or mutant IDH2 expression in HEL cells. (E) Representative images and quantification of neutral comet assays performed 24 h after 5 Gy IR in wild type, IDH1 R123H/+ (Mut), and DNA-PK (PRKDC) knockout HCT116 cells (PK). Scale bar = 400 μm. Statistical analysis by t-test (n=3). (F) Representative images and quantification of neutral comet assay performed 24 h after 5 Gy IR in wild type and IDH1 R123H/+ HeLa cells. Scale bar = 400 μm. Statistical analysis by t-test (n=3). (G) Quantification of neutral comet assay performed in log-phase, wild-type or IDH1 R132H/+ HCT116 and HeLa cells. (H and I) Representative images and quantification of neutral comet assay in HEL cells infected with (H) pSLIK IDH1-WT or pSLIK IDH1 R132H and (I) pSLIK IDH2 WT or pSLIK IDH2-R172K with or without doxycycline induction of the target protein, 7 days after addition of doxycycline or vehicle control. Scale bar = 400 μm (n=3). (J) Quantification of γH2AX and phospho-53BP1foci per nucleus in log-phase, untreated IDH1-WT and IDH1 R132H matched-pair HeLa and HCT116 cells (n=3). For E, F, G, H, I, and J, bars represent mean +/− SEM.

Figure 2. The IDH1 R132H-dependent homologous recombination deficiency confers synthetic lethality with PARP inhibition

(A) Schematic representation of the luciferase-based reporter assays for HR and NHEJ. Note the inactivating I-SceI gene in the luciferase open reading frame of the HR reporter, allowing restoration of functional luciferase only by homologous recombination at the I-SceI induced double strand break. Pathway-specific, luciferase-based HR and NHEJ reporter assays in the (B) WT and R132H heterozygous HCT116 cells, (C) WT or IDH1 R132H HeLa cells, and (D) doxycycline inducible HEL + WT IDH1, IDH1 R132H, WT IDH2, or IDH2 R172K. Statistical analysis by t-test (n=3). (E) Targeted small molecule screen in the WT and R132H heterozygous HeLa cells identifies a synthetic lethal interaction between IDH1 R132H and the PARP inhibitor BMN-673. (F) Clonogenic survival assays in the IDH1-WT and R132H HCT116 cells or HeLa cells treated with indicated doses of BMN-673 (n=6). (G) Clonogenic survival assays in the IDH1-WT or R132H HCT116 cells or HeLa cells treated with indicated doses of olaparib. Dashed lines indicate surviving fraction at 10 nM BMN-673 in F and 12.5 μM olaparib in G (n=6). (H and I) Quantification of neutral comet assay performed in WT or R132H/+ HeLa cells treated with indicated doses of (H) BMN-673 or (I) olaparib. (J) Synergy surface plots in WT or IDH1 R132H/+ HeLa cells show a mutant specific increase in synergy between BMN-673 and cisplatin (n=3). For B, C, D, H and I, bars represent mean +/− SEM.

Figure 3. 2-Hydroxyglutarate is sufficient to induce homologous recombination deficiency and PARP inhibitor synthetic lethality

(A) Quantification of neutral comet assay performed in WT HeLa cells after 24 h exposure to indicated amounts of (R)-2-hydroxyglutarate and (S)-2-hydroxyglutarate (n=3). (B) Quantification of neutral comet assay performed in immortalized astrocytes after 24 h exposure to indicated amounts of (R)-2-hydroxyglutarate (n=3). (C) Quantification of neutral comet assay performed in primary melanoma cultures treated with 300 μM (R)-2-hydroxyglutarate for 24 h (n=3). (D) U2OS DR-GFP show a dose response of HR suppression after 24 h exposure to both (2R)-octyl-2-HG and(2S)-octyl-2-HG but not the octyl- α-ketoglutarate control (n=3). (E) Schematic and representative dot plot of the DR-GFP assay results that are quantified in F. (F) Quantification of U2OS DR-GFP assay performed after 7 day culture in 2HG or DMSO control or after siRNA knockdown of the indicated targets, with western blot analysis of siRNA target knockdown. Statistical analysis by t-test (n=3). (G) Relative HR capacity determined by quantification of luciferase-based plasmid HR reporter assay normalized to DMSO-treated WT for each respective cell type in HeLa, HCT116, DLD1, and PEO1 C4-2 cells (WT BRCA2) treated with 900 μM and 300 μM (2R)-octyl-2-HG, assayed in parallel with DMSO-treated WT and DMSO-treated BRCA2 homozygous knockout DLD1 cells and BRCA2 functional-null PEO1 cells (n=3). (H) Short-term growth delay assays in HCT116 WT cells after 4-day culture in 900 μM (2R)-octyl-2-HG or DMSO treated with indicated concentrations of BMN-673 (n=3). (I) Short-term growth delay assay in wild-type HCT116 cells after 4-day culture in 300 nM BMN-673 or DMSO control, treated with increasing concentrations of (2R)-octyl-2-HG, as indicated (n=3). Fractional survival is normalized to survival in either DMSO or 300 nM BMN-673 without (2R)-octyl-2-HG. (J) Quantification of neutral comet assay performed in WT U2OS cells after 24 h exposure to indicated amounts of the α-ketoglutarate-dependent dioxygenase inhibitor, DMOG. (n=3). (K) Quantification of U2OS DR-GFP assay performed after 24 h pretreatment with indicated concentrations of DMOG or α-ketoglutarate (n=3). (L) Quantification and representative images of neutral comet assay performed in IDH1-mutant HeLa cells treated with exogenous octyl-α-ketoglutarate and WT HeLa cells. Scale bar = 400 μm (n=3). (M) Robust Z-Score of normalized HR from the ligand-inducible DR-GFP of 64 smart pool siRNAS targeting α-ketoglutarate dependent dioxygenases compared to the effects of 1 mM (R)-2HG, (S)-2HG, and DMOG, as well as control siRNAs targeting known core DNA repair proteins (n=3). (N-O) Quantitation of (N) ligand-inducible DR-GFP and (O) neutral comet assay (n=3) with deconvoluted siRNAs against KDM4A and KDM4B, each performed 96 h after siRNA transfection in U2OS inducible DR-GFP cells. (P and Q) Quantification of neutral comet assay performed in WT and R132H/+ HeLa cells (n=3) (P) 72 h after transfection with indicated deconvoluted siRNAs and (Q) 24 h after transfection with expression vectors for indicated ORFs. (n=3) Note two independent KDM4A expression constructs KDM4A #1 (pCMV-HA-KDM4A) and KDM4A #2 (pCMV-FLAG-KDM4A). For A, B, C, D, F, G, J, K, O, P, and Q, bars represent mean +/− SEM.

Figure 4. Inhibition of mutant IDH1 reverses the DSB repair defect and PARP inhibitor vulnerability

(A) Quantification of neutral comet assay performed in R132H/+ HeLa cells treated with indicated doses of AG1-5198 or WT HeLa cells treated with indicated doses of (R)-2HG (n=3). (B) Quantification of neutral comet assay performed in R132H/+ HCT116 cells treated with indicated doses of AG1-5198 (n=3). (C) Representative images from quantification shown in (A–B). Scale bar = 400 μm. (D–E) Quantification of neutral comet assays performed in (D) HCT116 IDH1 R132H/+ cells (n=3) and (E) HeLa IDH1 R132H/+ cells (n=3) treated with indicated amounts of the mutant IDH1 inhibitors AG120 and IDH1-C227. (F) Quantification of neutral comet assay in HEL cells, performed after the indicated conditions of induction of IDH1 R132H and treatment with 1.2 μM AGI-5198 and 1 mM (R)-2HG (n=3). (G) Quantification of neutral comet assay. WT or R132H/+ Hela cells were cultured in 1.2 μM AGI-5198 or DMSO control for 96 h, then supplemented with 300 μM (2R)-octyl-2-HG or DMSO control for 24 h before being split and treated with 10 nM BMN-673 for 48 h with continued exposure to 1.2 μM AGI-5198, 300 μM (2R)-octyl-2-HG, or DMSO control, then collected for analysis (n=3). (H) Quantification of neutral comet assay in HT1080 cells harboring an endogenous IDH1 R132C mutation treated with 1 μM AGI-5198 or DMSO control. Statistical analysis by t-test (n=3). (I) Quantification and representative images of clonogenic survival assays of WT and R132H/+ (Mut) HeLa Cells treated with the indicated concentrations of BMN-673 in the presence or absence of AGI-5198. HeLa cells were cultured in 1.2 μM AGI-5198 or DMSO control for 96 h before seeding for clonogenic survival, and exposure to AGI-5198 was maintained throughout the clonogenic survival assay. Representative images are HeLa seeded at 2400 cells per well and treated with 10 nM BMN and 1.2 μM AGI-5198 or DMSO control (n=6). (J) Short term viability assay in HEL cells with dox inducible IDH1 R132H treated with either 1 μM AGI-5198 or DMSO control and indicated doses of BMN-673 (n=4). For A, B, D, E, F, G, and H, bars represent mean +/− SEM.

Figure 5. IDH1 mutations confer repair deficiency and PARP inhibitor sensitivity in human glioma cells and mouse tumor models

(A) Patient information and clinical characteristics for the primary, patient-derived glioma culture collection. (B) Quantification of (R)-2HG production in IDH mutant primary glioma cultures (n=3). (C) Representative images and (D) quantification of neutral comet assays performed on primary, patient-derived glioma cultures (n=3). Scale bar = 400 μm. For comparison, primary cultures 80 and 97 were grown in media supplemented or not with 300 μM (2R)-octyl-2-HG for 7 days as indicated. (E) Quantification of γH2AX foci in primary glioma cultures treated the same way as in (C–D) (n=3). (F) Host cell reactivation assay for HR in primary glioma cultures. (G) Quantification and representative images of clonogenic survival assays in response to BMN-673 performed in primary glioma cultures. Gliomas 80 and 97 in black are IDH1 WT. Gliomas 129 and 2 in brown are IDH1 mutant. WT gliomas 80 and 97 were also cultured in 300 μM (2R)-octyl-2-HG for 7 days before seeding for the clonogenic survival assay and were maintained in 2HG throughout the assay; these are in blue (n=4). (H) Tumor volume growth curves for IDH1 R132C/+ HT1080 xenografts treated daily with either 50 mg/kg olaparib or vehicle control (ANOVA p=0.026) starting 4 days after implantation (n=9 per group). (I and J) Murine tumor volume growth curves for (I) IDH1 R132H heterozygous (ANOVA P = 0.001) (n=9 per group) and (J) IDH1 WT HCT116 xenografts (ANOVA P = 0.30) (n=9 per group) after treatment with 50 mg/kg olaparib once daily five days per week versus vehicle control. Treatment was initiated on day 4 after tumor implantation. (K) Representative images of in vivo luciferase activity in HCT116 IDH1 R132H heterozygous xenografts before and after olaparib treatment. For B, D, E, and F, bars represent mean +/− SEM.

Figure 6. Patient-derived primary acute myeloid leukemia samples show a 2HG-dependent DSB repair defect

(A) Patient information and clinical characteristics for the matched pairs of primary AML samples. (B) Cell cycle analysis of the IDH1 WT and R132H primary AML samples. (C) Quantification and (D) representative images of neutral comet assays performed on primary AML patient cells (n=3). Scale bar = 400 μm. (E) Radiation survival of primary AML cultures assayed by a short term viability assay 48 h after 5 Gy IR (n=3). (F) Representative images of neutral comet assay performed 24 h after 5 Gy IR on the primary AML cells WT-1 and Mut-1 (IDH1 R132H), indicating differential persistence of DNA DSBs after IR. Scale bar = 400 μm. (G) Proposed mechanism by which the (R)-2-hydroxyglutarate produced by IDH1 R132H (or (S)-2HG produced by hypoxia) induces an HR-deficient “BRCAness” phenotype and subsequent vulnerability to PARP inhibition. For C and E, bars represent mean +/− SEM.