1、nature medicine VOLUME 23|NUMBER 4|APRIL 2017 493letterSDiffuse intrinsic pontine glioma(DIPG)is a highly aggressive pediatric brainstem tumor characterized by rapid and uniform patient demise1.A heterozygous point mutation of histone H3 occurs in more than 80%of these tumors and results in a lysine
2、-to-methionine substitution(H3K27M)2,3.Expression of this histone mutant is accompanied by a reduction in the levels of polycomb repressive complex 2(PRC2)-mediated H3K27 trimethylation(H3K27me3),and this is hypothesized to be a driving event of DIPG oncogenesis4,5.Despite a major loss of H3K27me3,P
3、RC2 activity is still detected in DIPG cells positive for H3K27M6,7.To investigate the functional roles of H3K27M and PRC2 in DIPG pathogenesis,we profiled the epigenome of H3K27M-mutant DIPG cells and found that H3K27M associates with increased H3K27 acetylation(H3K27ac).In accordance with previous
4、 biochemical data5,the majority of the heterotypic H3K27M-K27ac nucleosomes colocalize with bromodomain proteins at the loci of actively transcribed genes,whereas PRC2 is excluded from these regions;this suggests that H3K27M does not sequester PRC2 on chromatin.Residual PRC2 activity is required to
5、maintain DIPG proliferative potential,by repressing neuronal differentiation and function.Finally,to examine the therapeutic potential of blocking the recruitment of bromodomain proteins by heterotypic H3K27M-K27ac nucleosomes in DIPG cells,we performed treatments invivo with BET bromodomain inhibit
6、ors and demonstrate that they efficiently inhibit tumor progression,thus identifying this class of compounds as potential therapeutics in DIPG.To advance understanding of the oncogenic molecular function of the H3K27M in oncogenesis,we mapped genome-wide occupancy of the mutant histone in DIPG cell
7、lines by chromatin immunoprecipitation (ChIP)-seq,using an antibody directed against H3K27M(Supplementary Fig.1).To confirm the specificity of the H3K27M antibody,we expressed wild-type or H3K27M-mutant histone at equivalent levels in HCT116 cells,a human colorectal tumor cell line(Supplementary Fig
8、.1a)and performed ChIP-seq.ChIP-seq signal with the H3K27M antibody was detected exclusively in H3K27M-expressing cells,whereas no enrichment was observed in cells expressing the wild-type counterpart(Supplementary Fig.1bd).ChIP-seq results from SF8628 human DIPG cellswhich contain a mutant H3F3A ge
9、ne that encodes H3.3K27M8reveal a genome-wide distribution of H3.3K27M that is highly correlated with active transcription,as indicated by the colocalization of acetylated H3K27(H3K27ac)and RNA polymerase II(RNA pol II)(Fig.1ac and Supplementary Fig.2a,b).Intriguingly,this observation is recapitu-la
10、ted in SU-DIPG-IV(hereafter called DIPG-IV)cells,a DIPG cell line carrying a point mutation in the HIST1H3B gene that results in the expression of H3.1K27M9,and results obtained in SF7761 cells,another H3F3A-mutant DIPG line8,provide further support(Supplementary Figs.3 and 4).As shown by our group
11、and others,H3K27M expression is associ-ated with increased levels of H3K27ac4,5(Fig.1d).To directly dem-onstrate a functional link between the expression of H3K27M and H3K27ac accumulation,we used a doxycycline-inducible system and found that H3K27M induction of H3K27ac is a reversible process(Suppl
12、ementary Fig.5a).H3K27M localizes to H3K27ac sites that are present in wild-type H3.3-expressing cells,but importantly,it can induce a marked increase of H3K27ac at regions that show only modest H3K27ac enrichment in control cells(Supplementary Fig.5b,c).These changes in H3K27ac are reversible and r
13、eturn to distri-butions that are almost indistinguishable from those present in control H3.3-expressing cells after terminating H3K27M transgene expression(Supplementary Fig.5b,c).Importantly,H3K27ac is Therapeutic targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliom
14、asAndrea Piunti1,Rintaro Hashizume1,2,Marc A Morgan1,Elizabeth T Bartom1,Craig M Horbinski2,Stacy A Marshall1,Emily J Rendleman1,Quanhong Ma1,2,Yoh-hei Takahashi1,Ashley R Woodfin1,Alexander V Misharin3,Nebiyu A Abshiru4,Rishi R Lulla5,Amanda M Saratsis2,6,7,Neil L Kelleher4,C David James1,2,7&Ali S
15、hilatifard1,71Department of Biochemistry and Molecular Genetics,Northwestern University Feinberg School of Medicine,Chicago,Illinois,USA.2Department of Neurological Surgery,Northwestern University Feinberg School of Medicine,Chicago,Illinois,USA.3Department of Medicine,Division of Pulmonology,and De
16、partment of Pediatrics,Division of Hematology-Oncology,Northwestern University Feinberg School of Medicine,Chicago,Illinois,USA.4Department of Chemistry,Northwestern University,Evanston,Illinois,USA.5Department of Hematology,Oncology,Neuro-Oncology&Stem Cell Transplantation,Ann&Robert H.Lurie Childr
17、ens Hospital of Chicago,Chicago,Illinois,USA.6Department of Surgery,Division of Pediatric Neurosurgery,Ann&Robert H.Lurie Childrens Hospital of Chicago,Chicago,Illinois,USA.7Robert H.Lurie NCI Comprehensive Cancer Center,Northwestern University Feinberg School of Medicine,Chicago,Illinois,USA.Corres
18、pondence should be addressed to A.S.(ashnorthwestern.edu)or(C.D.J.)(charles.jamesnorthwestern.edu).Received 30 November 2016;accepted 30 January 2017;published online 27 February 2017;doi:10.1038/nm.4296 2017 Nature America,Inc.,part of Springer Nature.All rights reserved.letterS494VOLUME 23|NUMBER
19、4|APRIL 2017 nature medicinethe only substantially increased histone acetylation mark induced by H3K27M,as determined by mass spectrometry analysis(Supplementary Figs.6 and 7 and Supplementary Table 1).Thus,H3K27M expression directly increases the levels of H3K27ac at specific genomic regions.The H3
20、K27M histone mutant is suggested to have increased bind-ing affinity for EZH2(refs.4,6,10);our previous biochemical purifi-cations of mononucleosomes containing H3K27M,however,did not show any preferential enrichment for PRC2 subunits5.To determine whether PRC2 subunits colocalize with H3K27M in viv
21、o on chroma-tin,we analyzed the genome-wide distributions of PRC2,H3K27me3 and H3K27M histone in DIPG cells.Strikingly,PRC2 subunits EZH2 and SUZ12,as well as their enzymatic product,H3K27me3,are largely excluded from chromatin on sites containing H3K27M(Fig.1e,f;Supplementary Figs.2 and 8).Collecti
22、vely,these results indicate that H3K27M is not involved in PRC2 recruitment or sequestration on chromatin in vivo,but on the contrary,suggest that PRC2 binding to chromatin is excluded by locus-specific formation of H3K27M-K27ac heterotypic nucleosomes.H3K27M has been hypothesized to drive gliomagen
23、esis through PRC2 catalytic inhibition4,suggesting that a lack of PRC2 function is an important contributing factor to the development of H3K27M tumors.However,our results indicate that both PRC2 and the prod-uct of its catalytic activity,H3K27me3,are present at thousands of loci in DIPG cells6,7(Fi
24、g.1e,f and Supplementary Fig.8af).To address whether residual PRC2 activity is required by H3K27M tumor cells,we used lentiviral short hairpin RNAs(shRNAs)to suppress the expression of the essential PRC2 subunits SUZ12 and EED11.The growth and colony-forming ability of SUZ12 or EED knockdown cells a
25、re greatly reduced relative to those of control cells(Fig.2a,b and SUZ12EZH2H3K27me3H3.3H3.3K27MInputH3K27acRNA pol IISUZ12EZH2H3K27me3H3.3H3.3K27MInputH3K27acRNA pol II10 kbHISTH2BBHIST1H1AHIST1H3AHIST1H4AHIST1H4BHIST1H3BHIST1H2ABHIST1H3CHIST1H1C2-3-2-7-5-2-5-2-H3.3K27MabfcdeH3K27acH3.3RNA pol IIEZ
26、H2SUZ12H3K27me3Input5 kb0+5 kb0.00.20.40.60.8Average signal accumulation(r.p.m.)H3.3K27MH3.3RNA pol IIH3K27acSUZ12EZH2H3K27me3Input100 kbHOXA1HOTAIRM1HOXA2AK291164HOXA3AK311383BC035889HOXA4HOXA-AS3HOXA5HOXA6HOXA7HOXA9HOXA10-HOXA9HOXA-AS4MIR196BHOXA10HOXA11HOXA11-ASLOC402470HOXA13HOTTIPEVX1H3.3K27M2-
27、H3K27ac3-H3.32-RNA pol II7-EZH25-SUZ122-H3K27me35-Input2-H3K27acH3.3K27M-FLAGH3.3K27wt-FLAGInputH3FLAG+IP:FLAG00.700.7Figure 1 H3K27M correlates with H3K27ac and is excluded from PRC2 targets.(a)Representative UCSC genome-browser view of a genomic region showing chromatin immunoprecipitation(ChIP)en
28、richment for H3.3K27M and active chromatin marks(H3.3,H3K27ac,RNA pol II)and lack of PRC2 components(SUZ12,EZH2 and H3K27me3)in SF8628 cells.Input signal is included as a background control.(b)Heat-map analysis of the 5,000 most highly H3.3K27M-enriched genomic loci in SF8628 cells,ranked in descend
29、ing order from top to bottom.The center of each vertical lane represents the summit of the peak;left and right borders represent 5 kb and+5 kb,respectively.Signal for all other ChIP targets at these regions is plotted accordingly.Input signal is included as a background control.Color scale bar indic
30、ates relative intensities.(c)Metaplot showing average signal accumulation in reads per million(r.p.m.)of the top 5,000 regions bound by H3.3K27M in SF8628 cells.Each plot is centered on the summit of the average occupancy and extended 5 kb upstream and downstream(5 kb and+5 kb,respectively).Input si
31、gnal is included as a background control.(d)Western blot analysis of immunoprecipitated(IP)histone H3K27ac from mononucleosomes containing C-terminal,FLAG-tagged wild-type H3.3 or mutant H3.3K27M purified from HEK293T cells(image representative of at least two independent experiments.Uncropped image
32、s in Supplementary Fig.18).(e)Representative UCSC genome-browser view of a region enriched for PRC2 components and lacking H3.3K27M and active chromatin marks.(f)Heat-map analysis of the 5,000 most highly H3K27me3-enriched genomic loci in SF8628 cells,ranked in descending order from top to bottom,pl
33、otted as described in b.2017 Nature America,Inc.,part of Springer Nature.All rights reserved.letterSnature medicine VOLUME 23|NUMBER 4|APRIL 2017 495Supplementary Fig.9).Unexpectedly,these results demonstrate a role for PRC2 function in the maintenance of DIPG growth.To identify potential targets of
34、 PRC2 that might drive DIPG cell proliferation,we focused our attention on CDKN2A(also known as p16),a negative cell-cycle regulator and well-established target of PRC2 transcriptional repression12.SUZ12 or EED knockdown resulted in derepression of p16 expression in SF8628 cells,consistent with a ro
35、le for CDKN2A as a PRC2 target in these cells.By contrast,DIPG-IV cells do not upregulate p16 expression upon PRC2 depletion(Supplementary Figs.10a,b and 9d),despite the anti-proliferative effects of SUZ12 and EED knockdown in these cells(Fig.2a,b and Supplementary Figs.9ad and 10a,b).To further exp
36、lore the role of PRC2 in DIPG proliferation,we employed a small-molecule catalytic inhibitor of EZH2 methyltrans-ferase activity,EPZ-6438.In agreement with the SUZ12 and EED knockdown results,H3K27M-positive DIPG cells treated with EPZ-6438 show reduced proliferation as compared to vehicle-treated c
37、ells(Supplementary Fig.10c).DIPG-IV cells tolerate higher doses of EPZ-6438 than SF8628 cells(Supplementary Fig.10c),and this prob-ably reflects lower effectiveness of the chemical inhibitor in DIPG-IV,because a common PRC2 target gene,cyclin D2(CCND2)(Fig.2cf),simply requires increased doses to ach
38、ieve derepression levels simi-lar to those in SF8628 cells(Supplementary Fig.10d).Notably,the extent of target derepression is correlated with decreased proliferation in these cells(Supplementary Fig.10c,d).As for EED and SUZ12 knockdown,EZH2 catalytic inhibition does not elicit p16 upregula-tion in
39、 DIPG-IV cells(Supplementary Fig.10d).In combination,our results indicate that PRC2 pro-proliferative activity in H3K27M tumor cells can act through p16-independent mechanisms,as has been demonstrated already in other models13.To further investigate global PRC2 transcriptional regulation,we performe
40、d transcriptome analyses(RNA-sequencing)of SUZ12 shRNA and control-vector-transduced SF8628 and DIPG-IV cells.Our analysis revealed that genes highly occupied by PRC2 in SF8628 and DIPG-IV cells show a trend of derepression upon SUZ12 deple-tion(Fig.2c).As expected from our previous results,genomic
41、regions bearing PRC2 at their transcription start site(TSS)are characterized by the presence of H3K27me3(87%in SF8628 cells and 92%in DIPG-IV cells).Many PRC2 targets are shared between SF8628 and DIPG-IV cells(70%and 40%,respectively;Fig.2d,e),and are significantly derepressed upon SUZ12 knockdown
42、as com-pared to non-PRC2 target genes(P 1.5 1022 in SF8628 cells and P 1.17 1022 in DIPG-IV cells;Fig.2f,g).Thus,the activation of these genes suggests that they could functionally inhibit DIPG proliferation.Importantly,gene ontology(GO)analysis of common genes supports their importance in mediating
43、 neuronal-lineage dif-ferentiation and establishing mature neuronal functions(Fig.2h).Although still functionally active,PRC2 catalytic property toward H3K27M-containing nucleosomes is strongly inhibited4.Interestingly,upon loss of H3K27me3,these nucleosomes acquire H3K27ac,which results in the form
44、ation of H3K27M-K27ac heterotypic nucleosomes4(Fig.1d and Supplementary Fig.5b,c).These observations support the increased association of acetyl-binding bromodomain proteins to H3K27M-containing nucleosomes5,and they prompted us to investigate the role of these proteins in DIPG cells.Strikingly,bro-
45、modomain-containing protein 2(BRD2)and BRD4 show highly sig-nificant overlap with H3K27M-occupied sites P 0.01(Fig.3ad and Supplementary Figs.2 and 3ad).BRD2-or BRD4-and H3K27M-co-occupied loci represent 85%of all BRD2-and BRD4-positive regions in SF8628 cells(Fig.3d).H3K27M localizes to promoters,i
46、ntragenic regions and at H3K4me1-positive intergenic regions(Supplementary Fig.11a,b).We also found that H3K27M occupancy at active enhanc-ers(H3K4me1-and H3K27ac-positive regions)is asymmetric and characterized by high-density occupancy over a fraction of active enhancers that also show increased p
47、resence of BRD2 or BRD4 and active transcription marks(Supplementary Fig.11c,d);this suggests that H3K27M participates in the formation of super-enhancer-like elements purported to have a key role in cancer14.The strong co-occupancy between BRD2 and BRD4 proteins and H3K27M-K27ac heterotypic nucleos
48、omes suggests a potential role of BRD proteins in DIPG pathogenesis.To address this point,we treated SF8628 cells and DIPG-IV cells with JQ1,a well-known bromodomain and extra-terminal domain(BET)inhibitor15.Dose-response analysis revealed submicromolar inhibitory concentration 50%(IC50)values in th
49、e H3K27M-mutant cell lines as compared to glioblastoma cells not harboring that mutation(Supplementary Fig.12a),and the growth-inhibitory effect of single-dose JQ1 administration extended to 5 d after treatment(Fig.3e and Supplementary Fig.12b,c).This effect was fully recapitulated by treatment with
50、 another BET inhibitor(Fig.3e and Supplementary Fig.12d).Interestingly,the anti-proliferative effect of JQ1 was accompanied by a neuron-like morphological change(Supplementary Fig.12e).In accordance with the crucial function of bromodomain proteins in transcriptional elongation16,we performed RNA-se
