Abstract
TP53 (which encodes p53 protein) is the most frequently mutated gene among all human cancers. Prevalent p53 missense mutations abrogate its tumour suppressive function and lead to a ‘gain-of-function’ (GOF) that promotes cancer. Here we show that p53 GOF mutants bind to and upregulate chromatin regulatory genes, including the methyltransferases MLL1 (also known as KMT2A), MLL2 (also known as KMT2D), and acetyltransferase MOZ (also known as KAT6A or MYST3), resulting in genome-wide increases of histone methylation and acetylation. Analysis of The Cancer Genome Atlas shows specific upregulation of MLL1, MLL2, and MOZ in p53 GOF patient-derived tumours, but not in wild-type p53 or p53 null tumours. Cancer cell proliferation is markedly lowered by genetic knockdown of MLL1 or by pharmacological inhibition of the MLL1 methyltransferase complex. Our study reveals a novel chromatin mechanism underlying the progression of tumours with GOF p53, and suggests new possibilities for designing combinatorial chromatin-based therapies for treating individual cancers driven by prevalent GOF p53 mutations.
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Acknowledgements
We thank M. Tainsky for the LFS cell lines; A. Weller, J. Glover and the Stem Cell and Xenograft Core at the University of Pennsylvania for help with the tumour xenograft experiments. S.L.B. is supported by NIH grant R01 CA078831. M.A.S. is supported by a Postdoctoral Fellowship from the American Cancer Society. X.H. is supported in part by a pilot grant from ITMAT of the University of Pennsylvania. A.S. is supported by NIH grant R01 GM069905. The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from AbbVie, Bayer, Boehringer Ingelheim, Genome Canada through the Ontario Genomics Institute (OGI-055), GlaxoSmithKline, Janssen, Lilly Canada, Merck, the Novartis Research Foundation, the Ontario Ministry of Economic Development and Innovation, Pfizer, Takeda, and the Wellcome Trust (092809/Z/10/Z). Funding was also provided to C.H.A. from the Canadian Cancer Society Research Institute.
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J.Z. and S.L.B. initiated and led the project. J.Z., M.A.S., Z.D. and S.L.B. designed the experiments and interpreted the data. J.Z. and M.A.S. performed the experiments. J.Z., M.A.S. and G.D. analysed all next-generation sequencing data. M.V., M.G., D.B.-L., R.A. and C.H.A. developed OICR9429. B.W.K. and X.H. contributed to the use of menin inhibitor. A.S. and J.H. contributed to reagents used in this study. J.Z., M.A.S., C.H.A. and S.L.B. composed the manuscript. All authors reviewed and commented on the manuscript.
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Extended data figures and tables
Extended Data Figure 1 Distinct GOF p53 mutants have similar genome-wide binding patterns, but are different from that of wild-type p53.
a, Heat maps showing the enrichment of p53 peaks (±2,500 bp around peak centre) identified from each cell line (rows) in all five cell lines (columns) examined by ChIP-seq. b–e, Area under the curve, meta-peak analysis showing GOF p53(R248W) or IgG ChIP-seq signal enrichment from MDAH087 cells over TSS-proximal peaks identified in MDA-MB-468 (b), HCC70 (c), MCF7 (d) and MDA-MB-175VII (e) cells.
Extended Data Figure 2 GOF p53 genome-wide binding is in association with ETS family proteins.
a, Canonical ETS binding motif (top), and discovered motif from all TSS-proximal peaks in MDA-MB-468 predicted by MEME/TomTom (middle), or SeqPos (bottom). b, MEME/TomTom identified wild-type p53 motif from MDA-MB-175VII TSS-proximal peaks. c, GST pulldown of bacterially expressed GST or GST–ETS2 with in vitro translated wild-type p53 or p53(R175H). d, e, Co-immunoprecipitation at endogenous protein levels of ETS2 and GOF p53(R273H) (d) or wild-type p53 (e) in MDA-MB-468 (d) or MCF7 (e) cells. f, g, Box plots showing overlap of GOF p53 (f) TSS-proximal peaks from MDA-MB-468 cells or wild-type p53 (g) TSS-proximal peaks from MCF7 cells, with ETS family proteins (blue), all other transcription factors (grey) or Pol II (white) peaks from ENCODE ChIP-seq data sets. Whiskers on the box plots represent the inter-quartile range. Mann–Whitney U-tests were performed to compute significance. h, GO analysis of wild-type p53 TSS-proximal peaks (statistics are shown in Supplementary Table 1).
Extended Data Figure 3 UCSC Genome Browser views showing distinct wild-type p53 and GOF p53 binding patterns over representative canonical wild-type p53 targets and novel GOF p53 targets.
a–d, UCSC Genome Browser views of p53 occupancy over promoter regions of MLL1 (a), MLL2 (b), CDKN1A (c) and MOZ (d) in MCF7, HCC70, and BT-549 cells. e–g, Re-aligned GOF p53(R248W) and IgG ChIP-seq data from LFS MDAH087 cells, showing enrichment of GOF p53 at promoter regions of MLL1 (e), MLL2 (f), and MOZ (g). h–j, UCSC Genome Browser views of p53 occupancy over promoter regions of RBBP5 (h), MDM2 (i) and PUMA (j), in MCF7, MDA-MB-175VII, HCC70, BT-549, and MDA-MB-468 cells.
Extended Data Figure 4 ChIP–qPCR validation of GOF p53 binding at newly identified chromatin regulator genes.
a, Schematic of amplicon locations for ChIP-qPCR validations performed in this study. b, c, ChIP–qPCR showing p53 (DO-1 antibody) or IgG (mouse) enrichment (ChIP/input) over MLL1, MLL2 and MOZ peak regions, in BT-549 (b) and HCC70 (c) cells. d–f, ChIP–qPCR showing p53 (DO-1 antibody) or IgG (mouse) enrichment over OGT, PPP1CC, RBBP5, SMARCD2, and DCAF10 peak regions in BT-549 (d), HCC70 (e) and MDA-MB-468 (f) cells. g, h, ChIP–qPCR showing p53 (FL393 antibody) or IgG (rabbit) enrichment over MDM2, CDKN1A, MLL1, MLL2 and MOZ regions, in MDA-MB-468 (g) and MDA-MB-175VII (h) cells. i. ChIP–qPCR showing p53 (DO-1 antibody) or IgG (mouse) enrichment over MLL1, MLL2 and MOZ peak regions in PANC-1 cells. Error bars represent mean ± s.e.m.; n = 3; two-tailed Student’s t-test: *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Figure 5 GOF p53 regulates expression of MLL1, MLL2, and MOZ, and corresponding histone post-translational modifications in cancer cells.
a, b, RT–qPCR analysis measuring mRNA level changes upon siRNA-mediated GOF p53 knockdown in MDA-MB-468 cells (a), and shRNA-mediated wild-type p53 knockdown in MDA-MB-175VII cells (b). c, d, RT–qPCR analysis of mRNA levels (c), and western blot analysis of protein levels upon DMSO or nutlin treatment in MCF7 cells (d). e, Western blot analysis of MLL1 protein level upon shRNA-mediated wild-type p53 knockdown in MDA-MB-175VII cells. f, Western blot analysis of MOZ protein level change upon shRNA-mediated GOF p53 knockdown in MDA-MB-468 cells. g, RT–qPCR measuring mRNA levels changes upon shRNA-mediated ETS2 knockdown in MDA-MB-468 cells. h, i, RT–qPCR measuring mRNA levels (h) and western blot measuring protein levels (i) upon shRNA-mediated ETS2 knockdown in BT-549 cells. j, k, RT–qPCR measuring mRNA levels changes upon shRNA-mediated ETS1 knockdown in BT-549 (j) and MDA-MB-468 (k) cells. Numbers 89 and 91 denote two short hairpins targeting ETS1, sequences of which are shown in Supplementary Table 3. l, m, ChIP–qPCR showing p53 occupancy (l) and Pol II occupancy (m) upon shRNA-mediated ETS1 knockdown in MDA-MB-468 cells. n, o, Western blot analysis of histone methylation and acetylation level changes upon siRNA-mediated (n) or shRNA-mediated (o) knockdown of GOF p53 in MDA-MB-468 cells. p, Western blot analysis of histone methylation and acetylation level changes upon GOF p53 knockdown in PANC-1 cells. q, Western blot of H3K9ac change upon MOZ knockdown in MDA-MB-468 cells. Uncropped blots are shown in Supplementary Fig. 1. Error bars represent mean ± s.e.m.; n = 3; two-tailed Student’s t-test; *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Figure 6 GOF p53 regulates expression of Mll1, Mll2, and Moz, and corresponding histone post-translational modifications in primary MEFs.
a, RT–qPCR analysis comparing Mll1 expression levels between MEFs bearing wild-type p53, GOF p53(R172H), and p53 null. b, Western blot comparing Mll1 protein level between MEFs with wild-type p53 and GOF p53. c, RT–qPCR analysis comparing Mll2 and Moz expression levels between MEFs bearing wild-type p53, GOF p53(R172H), and p53 null. d, RT–qPCR measuring mRNA changes upon shRNA-mediated p53 knockdown in GOF p53(R172H) knock-in MEFs. e, f, RT–qPCR analysis of mRNA levels (e) and western blot of protein levels (f) upon retroviral expression of GOF p53(R172H) in MEFs with p53 knockout. g, Western blot comparing H3K4me3 and H3K9ac levels between MEFs with wild-type p53 and GOF p53(R172H). h, Western blot showing H3K4me3 and H3K9ac levels upon p53 knockdown in wild-type p53 MEFs. i, Growth curve analysis of wild-type p53 MEF proliferation upon shRNA-mediated p53 knockdown. j, k, Box plot analysis of RNA levels (left) and H3 normalized H3K4me3 levels (right) at previously discovered Mll1 target genes (j) or Hoxa cluster genes (k) compared with all genes, from RNA-seq and H3K4me3 ChIP-seq in MEFs with wild-type p53 or GOF p53 R172H. Plots are presented as ratios of GOF p53(R172H) values over wild-type p53 values. l, UCSC Genome Browser views of H3K4me3 enrichment (top) and RNA levels (bottom) of Cdkn1a, from H3K4me3 ChIP-seq and RNA-seq of MEFs with wild-type p53 or GOF p53(R172H). Tracks are presented as overlay of wild-type p53 and GOF p53 signals. Blue denotes more enriched in wild-type p53, red denotes more enriched in GOF p53(R172H), black denotes overlap. m, Box plot of H3 normalized H3K4me3 levels over all gene TSSs, from H3K4me3 ChIP-seq in MEFs with wild-type p53 or GOF p53(R172H). n, RT–qPCR analysis comparing Hox gene expression levels between MEFs bearing wild-type p53, GOF p53(R172H), and p53 null. Uncropped blots are shown in Supplementary Fig. 1. For all bar graphs, two-tailed Student’s t-test; *P <0.05; **P < 0.01; ***P < 0.001. Error bars represent mean ± s.e.m.; n = 3. For all box plots, Mann–Whitney U-test; *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Figure 7 MLL knockdown reduces proliferation and cancer phenotype specifically in GOF p53 cancer cells.
a, b, Growth curve analysis of MDA-MB-468 (a) and MDA-MB-175VII (b) cells with either non-targeting control shRNA or p53 shRNA knockdown. c, d, Growth curve analysis of MDA-MB-468 (c) and MDA-MB-175VII (d) cells with non-targeting control shRNA, MLL1 shRNA, or MLL2 shRNA knockdown. e, Growth curve analysis of MCF7 cells with non-targeting control shRNA or MLL1 shRNA knockdown. f, g, Colony-formation assay of MDA-MB-468 (f) and MCF7 (g) cells with either non-targeting control shRNA or MLL1 shRNA knockdown. Corresponding to Fig. 4a, b. h, i, Colony-formation assay of BT-549 (h) and PANC-1 (i) cells with either non-targeting control shRNA or two different MLL1 shRNA knockdown, and quantification by crystal violet staining over three biological replicates. Reduction of MLL1 protein is also shown by western blot. j, k, Anchorage-independent soft agar assay of MDA-MB-468 (j) and MCF7 (k) cells with either non-targeting control shRNA or MLL1 shRNA knockdown. Dashed boxes denote enlarged images of the selected areas. White arrows indicate visible colonies in j. Quantifications are shown as number of visible colonies. Error bars represent mean ± s.e.m.; n = 3; two-tailed Student’s t-test; **P < 0.01; ***P < 0.001.
Extended Data Figure 8 MLL knockdown reduces proliferation specifically of GOF p53 MEFs and LFS cells.
a, Growth curve analysis of GOF p53(R172H) MEFs with either non-targeting control shRNA or two different Mll1 shRNA knockdowns. b, Western blot analysis of MLL1 levels upon shRNA-mediated knockdown in LFS MDAH087 and MDAH041 cells. c, Western blot analysis of p53 protein levels in LFS MDAH087 and MDAH041 cells. d, e, Growth curve analysis of LFS MDAH087 cells upon MLL1 (d) knockdown or p53 (e) knockdown. f, Growth curve analysis of LFS MDAH041 cells upon MLL1 knockdown. g, h, Western blot analysis of MLL1 level (g) and growth curve analysis (h) of proliferation upon shRNA-mediated MLL1 knockdown in IMR90 cells. i, Growth curve analysis of LFS MDAH087 cells with non-targeting control shRNA plus empty vector, p53 shRNA plus vector, and p53 shRNA plus MLL1 expressing vector. j, k, Growth curve analysis of LFS MDAH087 (j) and LFS MDAH041 (k) cells with either non-targeting control shRNA or MLL2 shRNA knockdown.
Extended Data Figure 9 TCGA RNA expression profile analysis.
a–f, TCGA RNA expression profile of GOF p53 target genes (top), housekeeping genes (middle), and wild-type p53 target genes (bottom) in brain lower grade glioma (a), head and neck squamous cell carcinoma (b), bladder urothelial carcinoma (c), colon adenocarcinoma (d), oesophageal carcinoma (e) or pancreatic adenocarcinoma tumours (f) with wild-type p53 (blue), GOF p53 (orange), or p53 null (white). Expression values are normalized read counts (a–d, f), or RPKM values (e) from TCGA RNA-seq data sets. Mann–WhitneyU-tests were performed to compute significance.
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Zhu, J., Sammons, M., Donahue, G. et al. Gain-of-function p53 mutants co-opt chromatin pathways to drive cancer growth. Nature 525, 206–211 (2015). https://doi.org/10.1038/nature15251
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DOI: https://doi.org/10.1038/nature15251
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