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Epigenomic reprogramming during pancreatic cancer progression links anabolic glucose metabolism to distant metastasis


During the progression of pancreatic ductal adenocarcinoma (PDAC), heterogeneous subclonal populations emerge that drive primary tumor growth, regional spread, distant metastasis, and patient death. However, the genetics of metastases largely reflects that of the primary tumor in untreated patients, and PDAC driver mutations are shared by all subclones. This raises the possibility that an epigenetic process might operate during metastasis. Here we report large-scale reprogramming of chromatin modifications during the natural evolution of distant metastasis. Changes were targeted to thousands of large chromatin domains across the genome that collectively specified malignant traits, including euchromatin and large organized chromatin histone H3 lysine 9 (H3K9)-modified (LOCK) heterochromatin. Remarkably, distant metastases co-evolved a dependence on the oxidative branch of the pentose phosphate pathway (oxPPP), and oxPPP inhibition selectively reversed reprogrammed chromatin, malignant gene expression programs, and tumorigenesis. These findings suggest a model whereby linked metabolic–epigenetic programs are selected for enhanced tumorigenic fitness during the evolution of distant metastasis.

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Figure 1: Global epigenetic reprogramming during the evolution of distant metastasis.
Figure 2: Epigenomic reprogramming of chromatin domains during PDAC subclonal evolution.
Figure 3: Reprogrammed chromatin domains encode divergent malignant properties.
Figure 4: Hyperactive glucose metabolism and 6PG depletion in distant metastatic subclones.
Figure 5: PGD dependence in distant metastatic subclones.
Figure 6: Reversal of reprogrammed chromatin and tumorigenic potential by 6AN.
Figure 7: CDH2 and TOP2B are epigenetic targets of 6AN that selectively regulate tumorigenic potential.

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We thank J. Zacharias for technical assistance with sample processing and members of the Johns Hopkins DNA sequencing core facility for ChIP–seq on SOLiD formats. This work was supported by NIH grant CA38548 (A.P.F.), NIH grants CA140599 and CA179991 (C.A.I.-D.), the AACR Pancreatic Cancer Action Network Pathway to Leadership grant (O.G.M.), the Vanderbilt GI SPORE (O.G.M.), the Vanderbilt–Ingram Cancer Center (O.G.M.), and NIH grant CA180682 (A.M.-M.).

Author information




O.G.M., C.A.I.-D., and A.P.F. conceived the work and wrote the manuscript. A.P.F. oversaw epigenomic sequencing performed at Johns Hopkins University. C.A.I.-D. selected patient samples, performed pathological review, analyzed immunostaining data, and oversaw whole-genome sequencing studies performed at MSKCC. O.G.M. designed and performed or oversaw all experiments and data analysis conducted at Vanderbilt. X.L. and H.W. performed the bioinformatic and statistical analyses. O.G.M. and A.P.F. guided the bioinformatic analyses. T.S. performed immunohistochemical and immunofluorescence experiments. R.T. prepared sequencing libraries and performed sequencing runs, assisted by T.H.S. S.J.M., M.O.W., and J.W.L. performed the LC–HRMS measurements of metabolites. O.G.M. and A.E.W. analyzed and plotted LC–HRMS data. A.C. and K.E.W. performed and analyzed YSI glucose and lactate measurements. S.N. and K.M.S. maintained cell culture and performed NADPH assays and a subset of immunoblotting. Y.Z. performed animal injection experiments. A.M.-M. performed whole-genome sequencing. O.G.M. prepared the figures. All authors approved the final version of the paper.

Corresponding authors

Correspondence to Christine A Iacobuzio-Donahue or Andrew P Feinberg.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Global reprogramming of histone modifications during the evolution of distant metastasis.

(a) IHC staining performed on formalin-fixed, paraffin-embedded (FFPE) tissue sections revealed diffusely positive H3K9me2 and H3K9me3 staining across PDAC cells from the founder clone (top), the subclone that seeded metastasis (middle), and the peritoneal metastasis (bottom) in patient A141. Scale bars, 100 μm. (b) In contrast, heterogeneous staining for H3K9me2 and H3K9me3 was detected in FFPE tissue sections taken from the primary tumor subclone that seeded metastasis (middle) and in the matched liver metastasis itself (bottom) in patient A132. Scale bars, 100 μm. (c) FFPE tissue sections were available from primary tumor subclones that seeded both peritoneal and distant metastasis in patient A38. Similar to patients A124 (Fig. 1a) and A141, diffusely positive staining for heterochromatin modifications was observed in both the founder clone (first panel) and the subclone that seeded peritoneal metastasis (second panel). In contrast, similar to patients A125 (Fig. 1b) and A132, heterogeneous staining between PDAC cells emerged in the primary tumor subclone that seeded distant metastases (third panel). The matched lung metastasis itself was diffusely negative (fourth panel), similar to cell lines isolated from this site and a matched liver metastasis in this patient (Fig. 1c). Scale bars, 100 μm. (d) Immunoblots on cell lines showed minimal or inconsistent changes for the indicated histone modifications between regional PDAC samples, including A38Per (Per). (e) In contrast, immunoblots on cell lines isolated from a panel of distant metastatic subclones showed recurrent changes in specific modifications, as compared to A38Per. (f) Global reprogramming of H3K9me3 and H4K20me2 was also observed between cell lines isolated from primary tumor subclones (Pr1, Pr2) and a matched lung metastasis in the same patient (pA13).

Supplementary Figure 2 Specificity of reprogrammed histone modifications.

(a) Ki67 staining showed similar cell cycle rates for peritoneal (A38Per) and matched lung (A38Lg) metastases from the same patient grown in medium with 10% FBS, and serum-free medium (SFM) greatly slowed growth in both subclones. Scale bars, 100 μm. (b) Serial cell counts for the indicated times confirmed similar growth rates and growth arrest in SFM. (c) GO analysis on RNA–seq data from cells cultured in serum versus SFM further confirmed growth arrest in SFM (lung data; the peritoneal cells gave identical results). (d) Immunoblots showed persistence of reprogrammed chromatin modifications in proliferative (FBS) and growth-arrested (SFM) cells. (e) Treatment of the peritoneal subclone with PDAC chemotherapies (Gem, gemcitabine; G+FU, gemcitabine + 5-fluorouracil) did not induce loss of methylation or gain of acetylation as seen between peritoneal and distant metastases (e.g., Fig. 1c,d), supporting the notion that those changes were independent of chemotherapy treatments. As expected, treatments resulted in increased levels of γH2AX, a signature of activated DNA repair pathways.

Supplementary Figure 3 Specificity of chromatin modifications within heterochromatin domains.

(a) Plots of ChIP–seq read densities normalized to input for histone modifications (y axes) showed that heterochromatin modifications (H3K9me2, H3K9me3, H3K27me3) were enriched in regions that were called heterochromatin (0% to 100%; bottom labels) for each subclone (indicated above). In contrast, euchromatin modifications (H3K36me3, H3K27ac) were depleted from the same regions. (b) Heterochromatin domains detected using alternative thresholds for domain calls produced similar results as shown in Figure 2a, demonstrating that the domain calls and reprogramming changes within them were robust across multiple sensitivity criteria (removal of short peaks less than 10 kb in length and merging of broad peaks separated by 15 kb are shown; other sensitivity tests using alternative thresholds produced nearly identical results that even more closely matched those in Fig. 2a).

Supplementary Figure 4 Reprogramming of H3K9me3 in LOCKs during PDAC subclonal evolution.

(a) ChIP–seq data from paired peritoneal (A38Per) and lung (A38Lg) metastatic subclones detected dramatic reduction of H3K9me3 in A38Lg that overlapped with H3K9me2 in LOCK domains. (b) Similar data were found for patient A13, which also showed loss of H3K9me3 from LOCK regions in the A13Pr2 and A13Lg subclones, as compared to the A13Pr1 primary tumor subclone.

Supplementary Figure 5 Localized reprogramming of DE gene loci within LOCK domains.

(a) Mapping DE genes from RNA–seq (distant metastases and precursors versus A38Per) to LOCKs revealed reciprocal changes in H3K27me3 and H3K27ac + H3K36me3 + DNA methylation around genes downregulated in LOCKs. (b) The opposite changes in H3K27me3 and H3K27ac + H3K36me3 were detected from genes upregulated in LOCKs. DNA methylation remained high in these regions.

Supplementary Figure 6 Specificity of chromatin modifications within ECDs.

(a) Plots of ChIP–seq read densities normalized to input for histone modifications (left labels) showed that euchromatin modifications (H3K36me3, H3K27ac; H3K4me3 is not shown) were enriched in regions that were called ECDs (0% to 100%; bottom labels) for each subclone (indicated above). In contrast, heterochromatin modifications (H3K9me2, H3K9me3, H3K27me3) were depleted from ECDs. (b) Euchromatin domains detected using alternative thresholds for domain calls (removal of short peaks less than 10 kb in length and merging of broad peaks separated by 15 kb) produced similar results as shown in Figure 2d, demonstrating that the domain calls were robust across multiple criteria. The y axes represent ChIP–seq read densities normalized to input for both panels.

Supplementary Figure 7 Reprogramming of large LOCK domains during PDAC evolution.

(ac) H3K9me3 was enriched and DNA was hypomethylated within large LOCK domains. (b,c) Striking reciprocal reprogramming of H3K9me2 and H3K9me3 and DNA methylation was detected in a subset of large LOCKs in patient A38. (d) H3K9me3 was enriched across large HPDE LOCKs. (e) Examples of reprogrammed domains between samples across a 25-Mb locus on chromosome 13.

Supplementary Figure 8 Malignant heterogeneity between A38 subclones.

(a) Oxidoreductase capacity was measured with MTT assays performed on equal numbers of growth-arrested cells in the absence of serum, and MTT signals were normalized to total cell number per well. Consistent with GO results, A38Lg possessed higher oxidoreductase activity. n = 4 technical replicates; error bars, s.d. P values were calculated by two-tailed t test. (b) NADPH/NADP levels were measured on equal numbers of growth-arrested cells. More NADPH per 1 million cells was detected in A38Lg. n = 2 biological replicates; error bars, s.d. P values were calculated by two-tailed t test. (c) A38Per and A38Lg maintained well- or poorly differentiated morphology in patient tissues and across three separate in vitro culture conditions as indicated. (d) IF performed on fixed tissues from the primary tumor showed loss of E-cadherin with gain of vimentin in the precursor subclone that seeded A38Lg, consistent with EMT. (e,f) RNAi knockdown of KRAS (e) blocked 3D tumor formation in suspension assays (f) more efficiently in A38Per than A38Lg. n = 4 technical replicates; error bars, s.d. P values were calculated by two-tailed t test. Scale bars, 400 μm.

Supplementary Figure 9 Rearrangements were targeted to large LOCKs and ECDs in patient A38.

(a) Total breakpoints in the A38 genome were not significantly enriched within large LOCKs or ECDs. P values represent the results of χ2 analyses of observed breakpoints in each domain versus those expected if rearrangements were uniformly distributed across the genome. (b) Unlike typical LOCKs, rearrangements (breakpoint fusions) located within large LOCKs and ECDs were significantly enriched for joining of the breakpoints within homologous domains (e.g., ECD to ECD). χ2-test P values were calculated as above. (c) Examples of large LOCK domains where instability hotspots clustered, including both intrachromosomal (amplifications) and interchromosomal (translocations) rearrangements.

Supplementary Figure 10 Enhanced glucose metabolism with depleted 6PG across distant metastatic and precursor subclones.

(a) Extracellular glucose consumption and lactate secretion were elevated in A38Lg, A13Pr1, and A13Lg relative to A38Per. (b) Schematic of glycolytic (outside) and PPP (boxed) metabolites, with intracellular metabolite levels detected by LC–HRMS plotted for each sample. Data represent LC–HRMS signals normalized to total protein for each sample, as indicated on the y axis.

Supplementary Figure 11 6AN targets glucose metabolism and the PGD step of the oxPPP.

(a) 6AN selectively slowed rates of extracellular glucose consumption and lactate secretion in precursor (A13Pr1) and metastatic (A13Lg, A38Lg) subclones, with no effect on glutamine or glutamate. (b) 6AN reduced incorporation of intracellular 13C-labeled glucose into metabolites downstream of the oxPPP. (c) 6AN greatly increased levels of the PGD substrate (6PG) and upstream metabolites with corresponding reductions in the levels of downstream products. (d) Scatterplots with correlation coefficients confirmed that 6AN gene expression changes were targeted to DE genes detected in A38Per (versus A38Lg) and that this was a partial, quantitative effect.

Supplementary Figure 12 6AN selectively modulated the reprogrammed chromatin state of distant metastatic subclones.

(a) 6AN treatments generally increased global H3K9me2 with corresponding decreased acetylation in distant metastatic subclones. (b) Normal cells and local regional PDACs did not show such changes. (c) 6AN changes persisted after 3 d of treatment (+) followed by removal of 6AN from the medium (+–) for an additional 3 d.

Supplementary Figure 13 6AN targeted reprogrammed LOCK regions.

(a) Mapping 6AN-repressed DE genes to A38Lg LOCKs revealed that these were located in reprogrammed LOCK-EI regions. (b) ChIP–seq on DMSO- versus 6AN-treated A38Lg detected a quantitative increase in LOCK-wide H3K9me2 levels from reprogrammed regions (as aligned to A38Per LOCKs). (c) ChIP–seq also detected 6AN-induced quantitative reductions in H3K27ac specifically from genes repressed from LOCKs. (d) Scatterplots with correlation coefficients showed that the quantitative reductions in H3K27ac from 6AN-downregulated LOCK genes also partially corresponded to genes that were co-(down)-regulated in A38Per (top), relative to all LOCK genes examined together (bottom). (e) Unlike H3K27ac, no significant differences in H3K27me3 levels were detected from genes repressed in LOCKs or other genes, similar to immunoblot findings.

Supplementary Figure 14 6AN selectively blocked 3D tumor formation in distant metastatic subclones.

(a,b) 6AN did not interfere with the ability of regional PDAC samples to form tumors in 3D Matrigel assays (a) or in 3D suspension tumorsphere assays (b). n = 2–4 technical replicates; error bars, s.d. (c) In contrast, 6AN strongly blocked the ability of distant metastatic subclones to form tumors in 3D suspension tumorsphere assays. n = 4 technical replicates; error bars, s.d. *P < 0.003, two-tailed t test. Scale bars, 400 μm.

Supplementary Figure 15 Reprogramming of the TOP2B locus in response to 6AN.

(a) 6AN did not alter mRNA expression (top) or levels of H3K9me2 and H3K27ac (bottom) across the TOP2B genomic locus in the peritoneal (A38Per) subclone (ChIP–qPCR primer positions are shown with respect to the TOP2B gene). (b) In contrast, 6AN selectively repressed mRNA expression (n = 4 technical replicates; *P < 0.002, two-tailed t test) and induced enrichment of H3K9me2 across the TOP2B locus in the lung metastatic subclone matched from the same patient (A38Lg). (c,d) Representative RT–qPCR (A38Lg) verified RNAi knockdown of CDH2 and TOP2B with minimal effect on VIM (vimentin) expression. n = 3 technical replicates, each normalized to ERK; error bars, s.d.

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Supplementary Text and Figures

Supplementary Figures 1–15, Supplementary Tables 1, 5, 6 and 8–16, and Supplementary Note (PDF 4626 kb)

Supplementary Table 2

Data sets and correlation coefficients. (XLSX 30 kb)

Supplementary Table 3

Domain characteristics. (XLSX 14 kb)

Supplementary Table 4

Reprogramming within domains and sensitivity analyses. (XLSX 19 kb)

Supplementary Table 7

Gene expression changes. (XLSX 9329 kb)

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McDonald, O., Li, X., Saunders, T. et al. Epigenomic reprogramming during pancreatic cancer progression links anabolic glucose metabolism to distant metastasis. Nat Genet 49, 367–376 (2017).

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