Epigenetic studies in rhabdomyosarcoma

为了扩展我们对人类RMS肿瘤中DNA甲基化的研究，我们使用Illumina HM 450和EPIC阵列来评估几个RMS肿瘤队列中的全基因组DNA甲基化模式。无监督分析证实了我们先前的发现，即DNA甲基化模式与融合状态密切相关。除了FP和FN类别之间的甲基化差异之外，我们的分析还揭示了FP簇内的两个主要子集和FN簇内的两个主要子集。特别地，这些研究揭示了P3 F和P7 F融合体各自富集两个FP亚群中的一个。类似地，RAS途径基因（HRAS、NRAS、KRAS、SOS 1、NF 1）的突变在两个FN亚组中富集或耗尽。为了扩展这些发现，我们寻找P3 F和P7 F阳性类别之间以及突变型和野生型RAS通路的FN病例之间具有差异甲基化的基因。在FP病例中，一个值得注意的基因是CDKN 1C，其在P3 F阳性肿瘤中的启动子甲基化高于P7 F阳性肿瘤，这是一种位于RMS和其他肿瘤中11p15.5等位基因丢失区域内的假定肿瘤抑制基因。RNA和蛋白质表达研究显示，与P7 F阳性肿瘤相比，P3 F中的CDKN 1C表达较少。在FN病例中，突变型RAS通路肿瘤中具有显著较低启动子甲基化的一个值得注意的基因是ALDH 1A 3，其先前被报道为FN RMS中癌症干细胞的潜在标志物。RNA表达研究显示RAS通路突变体中ALDH 1A 3 mRNA表达显著高于野生型肿瘤。为了进一步研究额外的甲基化定义的子集，我们比较了基因突变数据与我们的FN RMS病例的分层聚类模式。值得注意的是，我们确定了一小组MYOD 1突变的FN RMS病例紧密聚集在RAS途径突变相对缺失的FN亚组中。尽管该FN亚群具有统计学上较少的RAS通路突变，但突变MYOD 1组对应于该亚群中具有RAS通路突变的许多病例。在第二项分析中，我们扩展了我们先前的发现，即正常骨骼肌中的DNA甲基化模式与FN比FP病例更相似，我们研究了正常骨骼肌与两个甲基化定义的FN子集的关系。使用无监督的生物信息学方法，我们确定正常骨骼肌与RAS途径突变缺失的FN亚组比RAS途径突变富集的亚组更相似。最后，为了确定哪些RMS模型系统最好地概括了人类原发性RMS肿瘤中发现的DNA甲基化模式，我们将原发性RMS肿瘤中的全基因组甲基化模式与长期RMS细胞系、来源于这些长期细胞系的异种移植物（CDX）和患者来源的异种移植物（PDX）进行了比较。无监督方法展示了两个主要的FP聚类和两个主要的FN聚类。在每对FP或FN簇中，一个包含几乎所有的细胞系和CDX，另一个包含几乎所有的PDX和原发性肿瘤。相关热图的检查显示，绝大多数分析的CpG位点在细胞系和CDX中是高甲基化的，而在原发性肿瘤和PDX中发现的低甲基化和高甲基化的分布更广。我们没有发现PDX和原发性肿瘤之间的总体甲基化水平存在任何统计学差异，而细胞系和CDX中的总体甲基化水平显著较高。虽然这些发现表明PDX是研究RMS肿瘤中DNA甲基化模式生物学意义的最佳实验模型，但我们承认PDX不能像培养的细胞系那样容易操作。由于来自PDX的细胞可以培养，因此我们随后评估了当从PDX肿瘤建立短期培养物（1-2个月）时DNA甲基化模式的稳定性。我们将RMS PDX肿瘤和短期培养物与一组长期RMS细胞系进行比较的无监督分析显示，对于所有FN短期培养物和大多数FP短期培养物，在短期培养期间维持了总体DNA甲基化模式。因此，这些发现提供了在将这些细胞返回到体内环境之前使用短期培养来进行PDX实验操作的可能性。

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.