BackgroundMedulloblastoma, the most common malignant pediatric brain tumor, displays marked sex differences in prevalence of the four main molecular subgroups: SHH, WNT, Group 3 and Group 4. Males are more frequently diagnosed with SHH, Group 3 and 4 tumors, which have worse prognoses than WNT tumors. Little is known about sex differences in methylation profiles within subgroups.MethodsUsing publicly available methylation data (Illumina HumanMethylation450K array), we compared beta values for males versus females. Differentially methylated positions (DMP) by sex within medulloblastoma subgroups were identified on the autosomes. DMPs were mapped to genes and Reactome pathway analysis was run by subgroup. Kaplan-Meier survival curves (Log-Rank p-values) were assessed for each sex within subgroup. MethylCIBERSORT was used to investigate the tumor microenvironment using deconvolution to estimate the abundances of immune cell types using DNA methylation data.ResultsThere were statistically significant differences in sex by medulloblastoma subgroups (chi-squared p-value=0.00004): Group 3 (n=144; 65% male), Group 4 (n=326; 67% male), SHH (n=223; 57% male) and WNT (n=70; 41% male). Females had worse survival than males for SHH (p-value=0.02). DMPs by sex were identified within subgroups: SHH (n=131), Group 4 (n=29), Group 3 (n=19), and WNT (n=16) and validated in an independent dataset. Unsupervised hierarchical clustering showed that sex-DMPs in SHH did not correlate with other tumor attributes. Ten genes with sex DMPs (RFTN1, C1orf103, FKBP1B, COL25A1, NPDC1, B3GNT1, FOXN3, RNASEH2C, TLE1, and PHF17) were shared across subgroups. Significant pathways (p<0.05) associated with DMPs were identified for SHH (n=22) and Group 4 (n=4) and included signaling pathways for RET proto-oncogene, advanced glycosylation end product receptor, regulation of KIT, neurotrophic receptors, NOTCH, and TGF-β. In SHH, we identified DMPs in four genes (CDK6, COL25A1, MMP16, PRIM2) that encode proteins which are the target of therapies in clinical trials for other cancers. There were few sex differences in immune cell composition within tumor subgroups.ConclusionThere are sexually dimorphic methylation profiles for SHH medulloblastoma where survival differences were observed. Sex-specific therapies in medulloblastoma may impact outcomes.
Medulloblastoma (MB), the most common malignant brain tumor in children, has marked sex differences within molecular subtypes such that males are more frequently diagnosed with SHH, Group 3 and 4 tumors, which have worse prognoses than WNT tumors. Using publicly available data by Cavalli et al. (Cancer Cell, 2017), we identified sex differences in MB methylation and gene expression to uncover sexually dimorphic genomic profiles and biologic pathways to better understand the role of sex in MB subtype biology. There were statistically significant differences in sex by MB subtype (p-value=0.0005): Group 3 (n=144; 65% male), Group 4 (n=326; 67% male), SHH (n=223; 57% male) and WNT (n=70; 41% male). Females had statistically significantly worse survival than males for SHH (Log-Rank p-value=0.02; female 5-year survival 71%, male 85%; Hazard Ratio: 2.39; 95% Confidence Interval: 1.16-4.77). There were no sex differences in survival for other subtypes. In differential gene expression analyses using microarray data, few genes had statistically significant sexually dimorphic expression patterns (Group 3: 25 genes; Group 4: 65 genes; SHH: 37 genes; WNT: 32 genes). Ingenuity Pathway Analysis on the top 200 genes for each subtype found no sexually dimorphic pathways shared between all subtypes. Sex differences in Regulation of eIF4 and p70S6K Signaling, which has an important role in the mTOR pathway, was shared in WNT, SHH and Group 4 tumors. There were sex differences in VEGF and androgen signaling in WNT, EMT and IL-23 signaling in SHH, NF-KB and IL-8 signaling in Group 3, and mTOR and AMPK signaling in Group 4. Due to immune system sex differences, we examined MB immune cell composition, which may underlie sex differences in MB severity. We applied the LM22 signature and identified differences in most immune cell types between MB subtypes including M2 macrophages, CD8 T cells, and activated NK cells (all p<0.001). By sex, Group 3 females had higher plasma cell levels (p-value <0.01), WNT females had higher memory resting CD4 T cells (p<0.05), and WNT males had higher levels of activated NK cells (p<0.05). Concerning methylation on the autosomes, differentially methylated regions (DMR) by sex were identified in each subtype, with the largest number in SHH (n=398). Forty-four sex-specific DMR were shared across subtypes. Unsupervised hierarchical clustering showed nearly complete differentiation of samples by sex for each subtype when using DMR that differed significantly by sex. Females had more DMR with higher methylation than males in all subtypes. In pathway analysis among genes with higher methylation in females (Q-value<0.05), signaling pathways for IGF-1, HIF1α, TGF-β, ERK/MAPK and the Th1 pathway were identified. Collectively, these findings highlight sexually dimorphic genomic profiles for children with MB, particularly in SHH where survival differences were observed, which may help identify sex-specific therapeutic targets in the future. Citation Format: Rachel M. Moss, Natali Sorajja, Lauren J. Mills, Christopher L. Moertel, Logan G. Spector, David A. Largaespada, Lindsay A. Williams. Sex differences in the genomic profiles of medulloblastoma subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3026.
Ewing Sarcoma (ES) is a rare but deadly pediatric bone and soft tissue tumor, with little improvement in survival for decades despite knowing the driving fusion oncoprotein EWS-FLI1. EWS-FLI1 is notably toxic, acting as an aberrant transcription factor and leading to growth arrest and apoptosis in all but a few cell types. ES incidence in populations of European ancestry is nearly ten times that found in children with primarily African ancestry. To better understand how EWS-FLI1 transforms the transcriptome and the protective effect of the African genome, we leveraged this disparate genomic context to study ES tumorigenesis by introduction of EWS-FLI1 into cells derived from donors with a range of African ancestry followed by RNA sequencing (RNA-seq). Induced pluripotent stem cell (iPSC) lines with genome wide SNP genotyping data were obtained and local ancestry was assessed using RFMix to establish genetic ancestry. Two lines each of ~100% European and African ancestry and four lines with intermediate European/African admixture (45% to 90% African) were differentiated into neural crest cells (iNCC, a proposed cell-of-origin for ES) and transduced with a lentivirus expressing either a GFP reporter or a GFP-2A-EWS/FLI1 cassette. Interestingly, iNCC derived from European lines maintained a higher frequency of cells expressing GFP/EWS-FLI1 than the pure African lines, with admixed lines showing an intermediate frequency. RNA was extracted at 48- and 96-hour time points and sequenced for gene expression analysis. As expected, we identified pronounced changes to the transcriptional landscape in EWS-FLI1-induced cells compared to control with 13,578 differentially expressed genes (adjusted p-value < 0.05). More interestingly we identified 3,128 genes with ancestry-associated gene expression differences in response to EWS-FLI1 expression. Gene Set Enrichment Analysis (GSEA) using MSigDB 50 hallmark gene sets (v7.4) detected 24 significantly enriched gene sets with an inverse correlation with percent African ancestry, including those sets involved with oxidative phosphorylation, MYC v1 and v2, and MTORC1 signaling. To identify those expression changes that are key to ES tumorigenesis we are comparing the transcriptional profiles of our EWS-FLI1 expressing iNCC to ES tumors and ES cell lines as well as other models of ES derived from mesenchymal stem cells. Ongoing studies will include profiling of chromatin accessibility and EWS-FLI1 occupation for each ancestral iNCC line following EWS-FLI1 transduction to determine the early, ancestry-linked transitions that are permissive to oncogenic transformation. As EWS-FLI1 itself has proven elusive to direct targeting, studying its immediate downstream effects has the potential for establishing new druggable biologic pathways for treatment of ES. Citation Format: Rachel M. Moss, Kelsie L. Becklin, Lauren J. Mills, Branden S. Moriarity, Beau R. Webber, Logan G. Spector. Interaction between genetic ancestry and EWS-FLI induced transcription in Ewing sarcoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 702.
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