586 | Nature | Vol 582 | 25 June 2020
Article
Oncometabolites suppress DNA repair by
disrupting local chromatin signalling
Parker L. Sulkowski1,2, Sebastian Oeck1,3, Jonathan Dow1,2, Nicholas G. Economos1,2,
Lily Mirfakhraie^4 , Yanfeng Liu^1 , Katelyn Noronha1,5, Xun Bao^6 , Jing Li^6 , Brian M. Shuch^7 ,
Megan C. King^4 , Ranjit S. Bindra1,8,9 ✉ & Peter M. Glazer1,2,9 ✉Deregulation of metabolism and disruption of genome integrity are hallmarks of
cancer^1. Increased levels of the metabolites 2-hydroxyglutarate, succinate and
fumarate occur in human malignancies owing to somatic mutations in the isocitrate
dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate
hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD),
respectively^2 –^4. Recent work has made an unexpected connection between these
metabolites and DNA repair by showing that they suppress the pathway of
homology-dependent repair (HDR)^5 ,^6 and confer an exquisite sensitivity to inhibitors
of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials.
However, the mechanism by which these oncometabolites inhibit HDR remains
poorly understood. Here we determine the pathway by which these metabolites
disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine
demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9
(H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal
that is essential for the proper execution of HDR. Consequently, recruitment of TIP60
and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with
reduced end resection and diminished recruitment of downstream repair factors.
These findings provide a mechanistic basis for oncometabolite-induced HDR
suppression and may guide effective strategies to exploit these defects for
therapeutic gain.The oncometabolites 2-hydroxyglutarate (2HG), succinate and fuma-
rate inhibit α-ketoglutarate (αKG)-dependent dioxygenases^7 ,^8 including
histone lysine demethylases and other epigenetic modifiers^9 –^11. We
initially identified two lysine demethylases, KDM4A and KDM4B, as
potential targets for oncometabolite suppression of HDR in an initial
screen^5. To further investigate this, we assembled a series of human
cancer cell lines with endogenous and engineered mutations in IDH1
or IDH2, FH and SDH, short hairpin RNA (shRNA) knockdowns, and
CRISPR modifications and confirmed the expected levels of oncome-
tabolites and the corresponding hypermethylation of H3K9—a target
for demethylation by KDM4A and KDM4B (Extended Data Fig. 1a–h).
On the basis of the capacity of KDM4A or KDM4B to regulate gene
expression^11 ,^12 , we considered that 2HG, fumarate and succinate might
suppress HDR via gene downregulation. Transcriptome analyses com-
paring IDH1R132H/+ cells to wild-type (IDH1+/+) cells showed broad changes
in gene expression (Extended Data Fig. 1i), but no correlation of IDH1
status with HDR genes (Extended Data Fig. 1j). Gene expression pat-
terns in human gliomas in The Cancer Genome Atlas (TCGA) lower
grade glioma cohort indicated that HDR genes are not suppressed in
mutant IDH tumours (Extended Data Fig. 1k). Western blot analyses
of isogenic cell lines with or without R132H mutant IDH1 or FH or SDH
shRNA knockdown showed no differences in the HDR factors RAD51,
BRCA2, ATM, TIP60, MRE11 or RPA (Extended Data Fig. 1l).
Alternatively, we tested for a direct functional effect on HDR. We
observed increased levels of double-strand breaks (DSBs), which are
characteristic of HDR-deficient cells^5 ,^6 , as early as 2 h after the addition
of 2HG, fumarate or succinate to cells (Fig. 1a, Extended Data Fig. 1m, n).
We confirmed that intracellular levels of the metabolites and H3K9me3
were increased at the 2-h time point, indicating inhibition of KDM4A
or KDM4B (Extended Data Fig. 1o–t). Such rapid kinetics pointed to a
direct effect of the metabolites on HDR rather than on gene expression.
We next examined the formation of DNA repair foci by RAD51 and
BRCA1 in response to ionizing radiation. In control cells, RAD51 foci are
detectable at 2 h after ionizing radiation, peaking at 4–6 h (Fig. 1b, c,
Extended Data Fig. 2a,b), consistent with previous studies^13. But RAD51
foci were attenuated in cells treated with fumarate or succinate and in
cells with shFH or shSDH knockdown (Fig. 1b, c, Extended Data Fig. 2c–
e). We also compared RAD51 foci in cells with or without mutant IDH1 or
IDH2 (including CRISPR–Cas9-mediated knockout of the endogenous
IDH1R132C allele in HT1080 fibrosarcoma cells), with or without 2HGhttps://doi.org/10.1038/s41586-020-2363-0
Received: 1 February 2019
Accepted: 28 April 2020
Published online: 3 June 2020
Check for updates(^1) Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA. (^2) Department of Genetics, Yale University School of Medicine, New Haven, CT, USA.
(^3) Department of Medical Oncology, University of Duisburg-Essen, Essen, Germany. (^4) Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA. (^5) Department of
Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.^6 Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA.^7 Department of Urology, University of
California at Los Angeles, Los Angeles, CA, USA.^8 Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.^9 These authors jointly supervised this work: Ranjit S.
Bindra, Peter M. Glazer. ✉e-mail: [email protected]; [email protected]