Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Apr 1;24(7):1705-1715.
doi: 10.1158/1078-0432.CCR-17-2796. Epub 2018 Jan 16.

IDH1/2 Mutations Sensitize Acute Myeloid Leukemia to PARP Inhibition and This Is Reversed by IDH1/2-Mutant Inhibitors

Remco J Molenaar  1   2   3   4 , Tomas Radivoyevitch  5 , Yasunobu Nagata  1 , Mohammed Khurshed  2   4 , Bartolomiej Przychodzen  1 , Hideki Makishima  1 , Mingjiang Xu  6 , Fonnet E Bleeker  7   8 , Johanna W Wilmink  3   4 , Hetty E Carraway  9 , Sudipto Mukherjee  9 , Mikkael A Sekeres  1   9 , Cornelis J F van Noorden  2   4 , Jaroslaw P Maciejewski  10
Affiliations

IDH1/2 Mutations Sensitize Acute Myeloid Leukemia to PARP Inhibition and This Is Reversed by IDH1/2-Mutant Inhibitors

Remco J Molenaar et al. Clin Cancer Res. .

Abstract

Purpose: Somatic mutations in IDH1/2 occur in approximately 20% of patients with myeloid neoplasms, including acute myeloid leukemia (AML). IDH1/2MUT enzymes produce D-2-hydroxyglutarate (D2HG), which associates with increased DNA damage and improved responses to chemo/radiotherapy and PARP inhibitors in solid tumor cells. Whether this also holds true for IDH1/2MUT AML is not known.Experimental Design: Well-characterized primary IDH1MUT, IDH2MUT, and IDH1/2WT AML cells were analyzed for DNA damage and responses to daunorubicin, ionizing radiation, and PARP inhibitors.Results:IDH1/2MUT caused increased DNA damage and sensitization to daunorubicin, irradiation, and the PARP inhibitors olaparib and talazoparib in AML cells. IDH1/2MUT inhibitors protected against these treatments. Combined treatment with a PARP inhibitor and daunorubicin had an additive effect on the killing of IDH1/2MUT AML cells. We provide evidence that the therapy sensitivity of IDH1/2MUT cells was caused by D2HG-mediated downregulation of expression of the DNA damage response gene ATM and not by altered redox responses due to metabolic alterations in IDH1/2MUT cells.Conclusions:IDH1/2MUT AML cells are sensitive to PARP inhibitors as monotherapy but especially when combined with a DNA-damaging agent, such as daunorubicin, whereas concomitant administration of IDH1/2MUT inhibitors during cytotoxic therapy decrease the efficacy of both agents in IDH1/2MUT AML. These results advocate in favor of clinical trials of PARP inhibitors either or not in combination with daunorubicin in IDH1/2MUT AML. Clin Cancer Res; 24(7); 1705-15. ©2018 AACR.

PubMed Disclaimer

Conflict of interest statement

Conflict of Interest disclosure: Dr. Maciejewski has received honoraria, has performed consultancy and has served as a speaker on behalf of Celgene. Dr. Sekeres has served on an advisory committee of Celgene. Dr. Carraway has received research funding and has served as a speaker on behalf of Celgene, and has served on an advisory committee of Novartis. These companies have IDH1/2-mutant inhibitors in development.

Figures

Figure 1
Figure 1. IDH1/2MUT increase DNA DSBs and sensitize AML cells to PARP inhibitors
(A) IDH1MUT, IDH2MUT, IDH1/2WT, and TET2−/− primary AML cells (n=5 for each group) were incubated in the presence or absence of 1 μM AGI-5198 (IDH1MUT inhibitor) or AGI-6780 (IDH2MUT inhibitor) for 7 days, harvested, and analyzed for ATM protein expression by immunoblotting. β-Actin served as loading control. Lanes were reordered horizontally for clarity. ATM mRNA expression by qRT-PCR was also measured in these cells. (B) D-2HG levels as determined by enantiomer-specific mass spectrometry in cell lysates of 106 cells. Values were normalized to the D-2HG concentration of untreated IDH1/2WT cells and are shown as arbitrary units. (C) ATM mRNA expression data was taken from The Cancer Genome Atlas (TCGA) database and plotted on the basis of the IDH1, IDH2 and TET2 mutational status. Statistical comparisons were made using the one-way ANOVA test, comparing each group with the IDH1/2WT group, with Dunnett correction for multiple comparisons. (D) IDH1/2MUT and IDH1/2WT primary human AML cells (n=5 for each group) were incubated in the presence or absence of 1 μM IDH1/2MUT inhibitor for 3 days or 7 days and pretreated with either 2 Gy IR or 25 nM daunorubicin. Cells were immunocytochemically stained for ɣH2AX/DSBs and DAPI/DNA content. Numbers of ɣH2AX+ foci per cell are shown (20 cells per patient sample). P values were obtained using one-way ANOVA on the difference between patient samples, using Tukey’s correction for multiple comparisons.
Figure 2
Figure 2. IDH1/2MUT sensitize AML cells to the PARP inhibitors olaparib and talazoparib
(A-B) Colony-forming assays with IDH1MUT, IDH2MUT, IDH1/2WT or TET2−/− primary AML cells (n=5 for each group) after 48 h pretreatment with, and during 7 days after plating with (A) 0-25 μM olaparib or (B) 0-25 nM talazoparib. (C) Pretreatment schedules for the IDH1MUT inhibitor (AGI-5198) or the IDH2MUT inhibitor (AGI-6780) for 3 or 7 days shown in panels D-I. Cells were exposed to daunorubicin or IR on day 7 and subsequently pretreated with daunorubicin for 48 h or irradiated and plated for colony-forming assays. Throughout, squares are for control conditions, upward triangles for 3 days inhibition and downward triangles for 7 days inhibition. (C-H) Same as in (A-B), but after pretreatment or not with an IDH1/2MUT inhibitor for the indicated period. Data are mean±SD from 3 independent experiments. The clonogenic fraction is the number of colonies counted, divided by the number of cells plated and corrected for the plating efficiency. Y-axes are on a logarithmic scale. Data obtained in control conditions are from the same experiments. Black significance indicators compare the indicated group with IDH1/2WT AML cells. Colored significance indicators compare the indicated group with its IDH1/2MUT inhibitor-untreated counterpart. P values are indicated as * <.05; ** <.01; ***<0.005; ****<0.001.
Figure 3
Figure 3. IDH1/2MUT sensitize AML cells to irradiation and daunorubicin
Colony-forming assays with IDH1MUT, IDH2MUT, IDH1/2WT or TET2−/− primary AML cells (n=5 for each group) after treatment with (A) 0-50 nM daunorubicin for 48 h or (B) 0-6 Gy ionizing radiation (IR). (C-H) Same as in (A-B), but after pretreatment or not with an IDH1/2MUT inhibitor for the indicated period according to the pretreatment schedule shown in Figure 2C. Data are mean±SD from 3 independent experiments. The clonogenic fraction is the number of colonies counted, divided by the number of cells plated and corrected for the plating efficiency. Y-axes are on a logarithmic scale. Data obtained in control conditions are from the same experiments. Black significance indicators compare the indicated group with IDH1/2WT AML cells. Colored significance indicators compare the indicated group with its IDH1/2MUT inhibitor-untreated counterpart. P values are indicated as * <.05; ** <.01.
Figure 4
Figure 4. PARP inhibitors and daunorubicin have additive effects in IDH1/2MUT AML cells
Colony-forming assays with IDH1MUT, IDH2MUT and IDH1/2WT primary AML cells (n=5 for each group) after 48 h pretreatment before plating and during 7 days after plating with 0-25 μM olaparib or 0-25 nM talazoparib and with 0-15 μM daunorubicin. Data are mean±SD from 3 independent experiments. The clonogenic fraction is the number of colonies counted, divided by the number of cells plated and corrected for the plating efficiency. Y-axes are on a logarithmic scale. Black significance indicators compare the indicated group with IDH1/2WT AML cells. Colored significance indicators compare the indicated group with its IDH1/2MUT inhibitor-treated counterpart. P values are indicated as * <.05; ** <.01; *** <.005; **** <.001.
Figure 5
Figure 5. ATM knockdown sensitizes IDH1/2WT AML cells to cytotoxic treatment, but not in the presence of D-2HG or IDH1/2MUT
(A-H) Colony-forming assays with IDH1MUT, IDH2MUT or IDH1/2WT AML cells (n=5 for each group) after treatment with 0-50 nM daunorubicin for 48 h or 0-6 Gy IR in the presence or absence of siRNA against ATM and/or 10 mM D2HG and/or 1 μM AGI-5198 or 1 μM AGI-6780. ATM siRNA was controlled for using a negative control siRNA. Data are mean±SD from 3 independent experiments. The clonogenic fraction is the number of colonies counted divided by the number of cells plated and corrected for the plating efficiency. Y-axes are on a logarithmic scale. Significance indicators compare the adjacent group with untreated IDH1/2WT AML cells. P values are indicated as * <.05; ** <.01. (I) Model of IDH1MUT-mediated therapy sensitization in AML cells and glioma cells, based on findings in this study and in a previous study on IDH1MUT in solid tumor cells (shaded part) (30).
Figure 6
Figure 6. IDH1/2MUT AML cells have decreased IDH1/2 activity but similar redox states as IDH1/2WT AML cells
(A) NADP+-dependent IDH1/2 activity, NAD+-dependent IDH3 activity and NADP+-dependent G6PD activity of IDH1MUT, IDH2MUT and IDH1/2WT primary AML cells was determined using image analysis as absorbance of blue formazan produced from nitroBT per cell as a readout of NADPH production. Values were normalized to the NADP+-dependent IDH activity of IDH1/2WT cells and are shown as arbitrary units. (B) Representative photomicrographs of NADP+-dependent IDH and G6PD activity in IDH1MUT and IDH1WT AML cells. Scale bars = 50 μm. (C) NADP+-dependent IDH1/2 activity after pretreatment in the presence or absence of 1 μM AGI-5198, 1 μM AGI-6780 or 10 mM exogenous D2HG; units are arbitrary and relative to IDH1/2WT rates under control conditions. (D) IDH1MUT, IDH2MUT and IDH1/2WT AML cells (n=5 for each group) were pretreated with 0-50 nM daunorubicin (DAU) for 48 h or 0-2 Gy IR and were harvested, prepared, and analyzed, colorimetrically for NADP+:NADPH ratios and fluorometrically for GSH:GSSG ratios and for ROS levels. Data are mean±SD from 3 independent experiments. P values are indicated as * <.05; ** <.01.
See this image and copyright information in PMC

References

    1. Makishima H, Yoshizato T, Yoshida K, Sekeres MA, Radivoyevitch T, Suzuki H, et al. Dynamics of clonal evolution in myelodysplastic syndromes. Nat Genet. 2017;49(2):204–12. doi: 10.1038/ng.3742. - DOI - PMC - PubMed
    1. Molenaar RJ, Radivoyevitch T, Maciejewski JP, van Noorden CJ, Bleeker FE. The driver and passenger effects of isocitrate dehydrogenase 1 and 2 mutations in oncogenesis and survival prolongation. Biochim Biophys Acta. 2014;1846(2):326–41. doi: 10.1016/j.bbcan.2014.05.004. - DOI - PubMed
    1. Tefferi A, Lasho TL, Abdel-Wahab O, Guglielmelli P, Patel J, Caramazza D, et al. IDH1 and IDH2 mutation studies in 1473 patients with chronic-, fibrotic- or blast-phase essential thrombocythemia, polycythemia vera or myelofibrosis. Leukemia. 2010;24(7):1302–9. doi: 10.1038/leu.2010.113. - DOI - PMC - PubMed
    1. Molenaar RJ, Thota S, Nagata Y, Patel B, Clemente M, Przychodzen B, et al. Clinical and biological implications of ancestral and non-ancestral IDH1 and IDH2 mutations in myeloid neoplasms. Leukemia. 2015;29(11):2134–42. doi: 10.1038/leu.2015.91. - DOI - PMC - PubMed
    1. Haferlach T, Nagata Y, Grossmann V, Okuno Y, Bacher U, Nagae G, et al. Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia. 2014;28(2):241–7. doi: 10.1038/leu.2013.336. - DOI - PMC - PubMed

Publication types

MeSH terms