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. 2019 Jun 1;144(11):2707-2717.
doi: 10.1002/ijc.32006. Epub 2019 Jan 15.

Relationship of DNA methylation to mutational changes and transcriptional organization in fusion-positive and fusion-negative rhabdomyosarcoma

Wenyue Sun  1 , Bishwanath Chatterjee  1 , Jack F Shern  2 , Rajesh Patidar  3 , Young Song  3 , Yonghong Wang  3 , Robert L Walker  3 , Bruce R Pawel  4 , Corinne M Linardic  5 , Peter Houghton  6 , Stephen M Hewitt  1 , Daniel C Edelman  3 , Javed Khan  3 , Paul S Meltzer  3 , Frederic G Barr  1
Affiliations

Relationship of DNA methylation to mutational changes and transcriptional organization in fusion-positive and fusion-negative rhabdomyosarcoma

Wenyue Sun et al. Int J Cancer. .

Abstract

Our previous study of DNA methylation in the pediatric soft tissue tumor rhabdomyosarcoma (RMS) demonstrated that fusion-positive (FP) and fusion-negative (FN) RMS tumors exhibit distinct DNA methylation patterns. To further examine the significance of DNA methylation differences in RMS, we investigated genome-wide DNA methylation profiles in discovery and validation cohorts. Unsupervised analysis of DNA methylation data identified novel distinct subsets associated with the specific fusion subtype in FP RMS and with RAS mutation status in FN RMS. Furthermore, the methylation pattern in normal muscle is most similar to the FN subset with wild-type RAS mutation status. Several biologically relevant genes were identified with methylation and expression differences between the two fusion subtypes of FP RMS or between the RAS wild-type and mutant subsets of FN RMS. Genomic localization studies showed that promoter and intergenic regions were hypomethylated and the 3' untranslated regions were hypermethylated in FP compared to FN tumors. There was also a significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation. Moreover, genes with PAX3-FOXO1 binding sites and promoter hypomethylation exhibited the highest frequency of overexpression in FP tumors. Finally, a comparison of RMS model systems revealed that patient-derived xenografts most closely recapitulate the DNA methylation patterns found in human RMS tumors compared to cell lines and cell line-derived xenografts. In conclusion, these findings highlight the interaction of epigenetic changes with mutational alterations and transcriptional organization in RMS tumors, and contribute to improved molecular categorization of these tumors.

Keywords: DNA methylation; Rhabdomyosarcoma; expression; fusion protein; xenograft.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest: The authors have no potential conflicts of interest to disclose.

Figures

Figure 1.
Figure 1.. DNA methylation profiling in discovery and validation cohorts identifies molecular subsets in FP and FN RMS.
Heat maps for discovery (A) and validation (B) cohorts displaying the subsets of FP and FN RMS defined by DNA methylation. These displays are based on the top 1% most varied DNA probes across RMS tumors. Differences in the top 1% most varied DNA probes between the discovery and validation cohorts may contribute to the small differences in methylation patterns displayed for the RMS subsets. Fusion status and RAS mutation status are shown in the upper panel in addition to the methylation-defined subsets. Abbreviations: FN, fusion-negative; FP, fusion-positive; WT, wild-type; MUT, mutant-type.
Figure 2.
Figure 2.. DNA methylation characteristics associated with PAX3-FOXO1- and PAX7-FOXO1-positive RMS tumors.
A, Volcano plot displaying differentially methylated probes between PAX3-FOXO1- and PAX7-FOXO1-positive RMS tumors. Red and pink dots denote hypermethylated and hypomethylated probes in PAX3-FOXO1- versus PAX7-FOXO1-positive tumors, respectively. CDKN1C promoter methylation (B) and RNA expression (C) levels in PAX3-FOXO1- and PAX7-FOXO1-positive RMS tumors. D, Correlation plot of DNA methylation versus RNA expression levels for CDKN1C in PAX3-FOXO1- and PAX7-FOXO1-positive RMS tumors. E, Representative CDKN1C immunohistochemical staining results in PAX3-FOXO1- and PAX7-FOXO1-positive tumors. Upper, CDKN1C negative staining in a PAX3-FOXO1-positive tumor; Lower, strong staining in a PAX7-FOXO1-positive tumor. Abbreviations: P3F, PAX3-FOXO1; P7F, PAX7-FOXO1.
Figure 3.
Figure 3.. DNA methylation characteristics associated with RAS mutant and wild-type FN RMS tumors.
A, Volcano plot displaying differentially methylated probes between RAS mutant and wild-type FN RMS tumors. Red and blue dots denote hypermethylated and hypomethylated probes in RAS mutant versus wild-type tumors, respectively. B, Heat map comparing DNA methylation of normal fetal and neonatal skeletal muscle with RAS mutant and wild-type FN tumors based on differentially methylated probes between RAS mutant and wild-type FN tumors. Mutation events are represented by black rectangles in the upper panel. Promoter DNA methylation (C) and RNA expression (D) levels of ALDH1A3 in RAS pathway mutant and wild-type FN RMS tumors. E, Correlation plot of DNA methylation versus RNA expression levels for ALDH1A3 in RAS pathway mutant and wild-type FN RMS tumors. Abbreviations: WT, wild-type; MUT, mutant-type; +RAS.P, FN tumors with RAS pathway gene mutations; -RAS.P, FN tumors without RAS pathway gene mutations.
Figure 4.
Figure 4.. Distribution of DNA methylation differences between FP and FN tumors across genomic features.
A, Cumulative distribution frequency plots of CpG methylation levels in FP and FN tumors based on genomic features. B, Overall DNA methylation level (mean β value) of FP and FN tumors based on genomic features. C, Frequency of differentially methylated CpG probes in FP versus FN tumors based on genomic features.
Figure 5.
Figure 5.. Comparison of methylation profiles of RMS cell lines, CDXs, PDXs and patient tumors.
Heat map (A) and PCA (B) analyses comparing DNA methylation profiles of 11 RMS cell lines, 11 CDXs, 14 PDXs and 48 patient tumors in the discovery cohort based on top 1% most varied DNA methylation probes across patient tumors. Arrowheads from left to right denote RH36 PDX and RH41 PDX. C, Overall DNA methylation levels in RMS cell lines, CDXs, PDXs and patient tumors. Data for FP samples are shown above, and data for FN samples are shown below. The plots summarize the distribution of standardized average DNA methylation levels in each sample type based on the top 1% most varied probes across RMS tumors. Adjusted P values in FP samples were calculated using one way ANOVA with Games-Howell test, and adjusted P values in FN samples were calculated using one way ANOVA with Tukey-Kramer multiple comparison post hoc test. Abbreviations: P3F, PAX3-FOXO1; P7F, PAX7-FOXO1; P3IN, PAX3-INO80D; P3NC, PAX3-NCOA1; T, tumor; CL, cell line. *, P<0.05; **, P<0.01; NS, not significant.
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