Epigenetic studies in rhabdomyosarcoma

Division Of Basic Sciences - Nci

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Abstract

To extend our studies of DNA methylation in human RMS tumors, we used Illumina HM450 and EPIC arrays to assess genome-wide DNA methylation patterns in several cohorts of RMS tumors. Unsupervised analyses confirmed our previous finding of a close association of DNA methylation pattern and fusion status. In addition to the methylation differences between the FP and FN categories, our analysis also revealed two major subsets within the FP cluster, and two major subsets within the FN cluster. In particular, these studies revealed that the P3F and P7F fusions were each enriched in one of the two FP subsets. Similarly, mutations in RAS pathway genes (HRAS, NRAS, KRAS, SOS1, NF1) were enriched or depleted in the two FN subsets. To extend these findings, we searched for genes with differential methylation between the P3F and P7F-positive categories, and between FN cases with mutant and wild-type RAS pathways. In the FP cases, one notable gene with higher promoter methylation in P3F- than P7F-positive tumors is CDKN1C, a putative tumor suppressor gene localized within a region of 11p15.5 allelic loss in RMS and other tumors. RNA and protein expression studies showed less CDKN1C expression in P3F- compared to P7F-positive tumors. In the FN cases, one notable gene with significantly lower promoter methylation in mutant RAS pathway tumors is ALDH1A3, which was previously reported as a potential marker for cancer stem cells in FN RMS. RNA expression studies revealed significantly higher ALDH1A3 mRNA expression in RAS pathway mutant versus wild-type tumors. To further investigate additional methylation-defined subsets, we compared gene mutation data with the hierarchical clustering pattern in our FN RMS cases. Of note, we determined that the small group of FN RMS cases with MYOD1 mutations were tightly clustered within the FN subset that is relatively depleted of RAS pathway mutations. Although this FN subset has statistically fewer RAS pathway mutations, the mutant MYOD1 group corresponded to many of the cases within this subset that had RAS pathway mutations. In a second analysis to extend our previous finding that the DNA methylation pattern in normal skeletal muscle is more similar to FN than FP cases, we examined the relationship of normal skeletal muscle to the two methylation-defined FN subsets. Using unsupervised bioinformatic methodologies, we determined that normal skeletal muscle was more similar to the FN subset depleted in RAS pathway mutations than the subset enriched in RAS pathway mutations. Finally, to determine which RMS model systems best recapitulate the DNA methylation patterns found in human primary RMS tumors, we compared genome-wide methylation patterns in primary RMS tumors to long-term RMS cell lines, xenografts derived from these long-term cell lines (CDXs) and patient-derived xenografts (PDXs). Unsupervised approaches demonstrate two main FP clusters and two main FN clusters. Within each pair of FP or FN clusters, one contains nearly all cell lines and CDXs and the other contains nearly all PDXs and primary tumors. Examination of the associated heat maps reveal that the vast majority of analyzed CpG sites are hypermethylated in cell lines and CDXs in contrast to the wider distribution of hypo- and hypermethylation found in primary tumors and PDXs. We did not find any statistical differences in overall methylation levels between PDXs and primary tumors in contrast to the significantly higher overall methylation levels in cell lines and CDXs. Though these findings indicate that PDXs are the optimal experimental model to study the biological significance of DNA methylation patterns in RMS tumors, we acknowledge that the PDXs cannot be manipulated as easily as cultured cell lines. Since cells from the PDXs can be cultured, we then assessed the stability of the DNA methylation patterns when short-term cultures (1-2 months) were established from the PDX tumors. Our unsupervised analysis comparing RMS PDX tumors and short-term cultures with a set of long-term RMS cell lines showed that the overall DNA methylation pattern was maintained during short-term culture for all FN short term cultures and most FP short term cultures. These findings thus provide the possibility of using a short-term culture to perform experimental manipulations of PDXs before returning these cells to the in vivo environment.