Single-cell chromatin profiling reveals demethylation-dependent metabolic vulnerabilities of breast cancer epigenome

Abstract

17Metabolic reprogramming in cancer cells not only sustains bioenergetic and biosynthetic needs 18 but also influences transcriptional programs, yet how chromatin regulatory networks are rewired 19 by altered metabolism remains elusive. Here we investigate genome-scale chromatin remodeling 20 in response to 2-hydroxyglutarate (2HG) oncometabolite using single-cell assay for transposase 21 accessible chromatin with sequencing (scATAC-seq). We find that 2HG enantiomers differentially 22 disrupt exquisite control of epigenome integrity by limiting α-ketoglutarate (αKG)-dependent DNA 23 and histone demethylation, while enhanced cell-to-cell variability in the chromatin regulatory 24 landscape is most evident upon exposure to L2HG enantiomer. Despite the highly heterogeneous 25 responses, 2HG largely recapitulates two prominent hallmarks of the breast cancer epigenome, 26 i.e., global loss of 5-hydroxymethylcytosine (5hmC) and promoter hypermethylation, particularly 27 at tumor suppressor genes involved in DNA damage repair and checkpoint control. Single-cell 28 mass cytometry further demonstrates downregulation of BRCA1, MSH2 and MLH1 in 2HG-29 responsive subpopulations, along with acute reversal of chromatin remodeling upon withdrawal. 30 Collectively, this study provides a molecular basis for metabolism-epigenome coupling and 31 identifies metabolic vulnerabilities imposed on the breast cancer epigenome. 33 As a dynamic system, cancer cells continuously adapt to the fluctuating microenvironment by 34 rerouting metabolic fluxes and evolve from early initiation through progression and 35 dissemination 1,2 . Metabolic reprogramming in cancer cells facilitates energy production and 36 macromolecular synthesis to fuel cell proliferation 3,4 . In addition to supporting bioenergetic and 37 biosynthetic needs, altered metabolism involves promiscuous production of non-canonical 38 metabolic intermediates, which have been described as metabolic waste products or metabolite 39 damage 5,6 . Recent studies suggest that these previously uncharacterized metabolites including 40 oncometabolites are linked to 'non-metabolic' signaling mechanisms in cell-type-specific fate 41 decisions 7 . 42 The oncometabolite 2-hydroxyglutarate (2HG) occurs as two enantiomers and 43 accumulates up to millimolar concentrations in a broad range of hematological and solid 44 malignancies 8,9 . Somatic mutations in isocitrate dehydrogenase genes, IDH1 and IDH2, found in 45 glioma and acute myeloid leukemia (AML) result in stereospecific production of the D-enantiomer 46 (D2HG) 10,11 , while breast tumors frequently exhibit elevated levels of 2HG despite the lack of IDH 47 mutations 12,13 . Recent studies indicate that the L-enantiomer (L2HG) can accumulate under 48 acidic 14,15 and hypoxic conditions 16,17 that often coexist in the tumor microenvironment, yet the 49 potential sources and functions of L2HG are less well established. Both enantiomers structurally 50 resemble α-ketoglutarate (αKG), a key intermediate ...