Spinal Myxopapillary Ependymomas Demonstrate a Warburg Phenotype

Abstract

Purpose: Myxopapillary ependymoma (MPE) is a distinct histologic variant of ependymoma arising commonly in the spinal cord. Despite an overall favorable prognosis, distant metastases, subarachnoid dissemination, and late recurrences have been reported. Currently, the only effective treatment for MPE is gross-total resection. We characterized the genomic and transcriptional landscape of spinal ependymomas in an effort to delineate the genetic basis of this disease and identify new leads for therapy.

Experimental design: Gene expression profiling was performed on 35 spinal ependymomas, and copy number profiling was done on an overlapping cohort of 46 spinal ependymomas. Functional validation experiments were performed on tumor lysates consisting of assays measuring pyruvate kinase M activity (PKM), hexokinase activity (HK), and lactate production.

Results: At a gene expression level, we demonstrate that spinal grade II and MPE are molecularly and biologically distinct. These are supported by specific copy number alterations occurring in each histologic variant. Pathway analysis revealed that MPE are characterized by increased cellular metabolism, associated with upregulation of HIF1α. These findings were validated by Western blot analysis demonstrating increased protein expression of HIF1α, HK2, PDK1, and phosphorylation of PDHE1A. Functional assays were performed on MPE lysates, which demonstrated decreased PKM activity, increased HK activity, and elevated lactate production.

Conclusions: Our findings suggest that MPE may be driven by a Warburg metabolic phenotype. The key enzymes promoting the Warburg phenotype: HK2, PKM2, and PDK are targetable by small-molecule inhibitors/activators, and should be considered for evaluation in future clinical trials for MPE.

Figure 1. Grade II and myxopapillary spinal ependymomas are transcriptionally distinct

(A) Unsupervised consensus hierarchical clustering of 35 primary spinal ependymoma gene expression profiles generated by Affymetrix Gene 1.0ST microarrays. Shown is a rank 2 classification. Significance of clustering was measured using SigClust. (B) Unsupervised non-negative matrix factorization of 35 spinal ependymoma gene expression profiles. Shown is a rank 2 classification. Sample overlap between consensus HCL and NMF was measured using a Rand index. (C) Area under the cumulative distribution function curve indicating 2 principal subgroups of spinal ependymomas. (D) Silhouette analysis identifies ‘core’ representative samples within the 2 subgroups of spinal ependymoma

Figure 2. Grade II and myxopapillary spinal ependymomas harbor distinct copy number landscapes

(A) Bar graphs of whole chromosomal gains and losses in Grade II versus spinal myxopapillary ependymomas. Gains are show in red and losses in blue. (B) Genome-wide view of copy number alterations in 46 spinal ependymomas generated by Affymetrix SNP6.0 DNA microarrays sub-divided by Grade II and myxopapillary spinal ependymoma. Gains are show in red and losses in blue.

Figure 3. Pathway analysis identifies over-representation of metabolic gene sets in myxopapillary ependymoma

(A) Cytoscape enrichment map of significant genesets distinguishing Grade II versus myxopapillary spinal ependymomas identified by Gene Set Enrichment Analysis (GSEA) and visualized in Cytoscape. A statistical significance cutoff of p < 0.01 and FDR < 0.25 was used for the pathway analysis (B) Donut plot demonstrating significant over-representation of pathways and biological processes in involving hypoxia signaling and cellular metabolism in myxopapillary spinal ependymoma (C) Single sample GSEA demonstrating subgroup-specific over-representation of pathways and biological processes involving cellular metabolism and hypoxia signaling. Bar graph of the ages of patients is shown in the lower panel.

Figure 4. Validation of pathway analysis characterizing a metabolic signatures enriched in myxopapillary spinal ependymomas

(A) Western blot validation of up-regulation of HIF-1α, HK2, PDK1, and decreased expression of HK1. (B) Linear quantification of protein expression levels detected relating to proteins in Figure 4A. (C) Increased HK2 activity in myxopapillary spinal ependymomas measured from primary tumor lysates as compared to Grade II tumours (n = 12).

Figure 5. Spinal MPE demonstrate increased PKM2 expression and decreased overall PKM activity consistent with a 'Warburg' phenotype

(A) Protein validation of increased PKM2 protein expression relative to total PKM1 and PKM2 proteins as demonstrated by western blot analysis. (B) Linear quantification of PKM2 levels relative to total PKM1/2 demonstrated in Figure 5A. (C) Immunohistochemistry for PKM2 expression in a representative spinal Grade II and myxopapillary ependymoma (left panel). Bar graph comparing immunohistochemistry scores in a cohort of 39 non-overlapping spinal ependymomas (right panel). (D) Decreased PKM activity in myxopapillary spinal ependymomas measured from primary tumor lysates as compared to Grade II tumours (n = 12).

Figure 6. Spinal MPE exhibit increased lactate production consistent with anaerobic glycolysis and a 'Warburg' phenotype

Increased lactate production in myxopapillary spinal ependymomas measured from primary tumor lysates as compared to Grade II tumours (n = 12).