Cross-species genomics matches driver mutations and cell compartments to model ependymoma

Abstract

Understanding the biology that underlies histologically similar but molecularly distinct subgroups of cancer has proven difficult because their defining genetic alterations are often numerous, and the cellular origins of most cancers remain unknown. We sought to decipher this heterogeneity by integrating matched genetic alterations and candidate cells of origin to generate accurate disease models. First, we identified subgroups of human ependymoma, a form of neural tumour that arises throughout the central nervous system (CNS). Subgroup-specific alterations included amplifications and homozygous deletions of genes not yet implicated in ependymoma. To select cellular compartments most likely to give rise to subgroups of ependymoma, we matched the transcriptomes of human tumours to those of mouse neural stem cells (NSCs), isolated from different regions of the CNS at different developmental stages, with an intact or deleted Ink4a/Arf locus (that encodes Cdkn2a and b). The transcriptome of human supratentorial ependymomas with amplified EPHB2 and deleted INK4A/ARF matched only that of embryonic cerebral Ink4a/Arf(-/-) NSCs. Notably, activation of Ephb2 signalling in these, but not other, NSCs generated the first mouse model of ependymoma, which is highly penetrant and accurately models the histology and transcriptome of one subgroup of human supratentorial tumour. Further, comparative analysis of matched mouse and human tumours revealed selective deregulation in the expression and copy number of genes that control synaptogenesis, pinpointing disruption of this pathway as a critical event in the production of this ependymoma subgroup. Our data demonstrate the power of cross-species genomics to meticulously match subgroup-specific driver mutations with cellular compartments to model and interrogate cancer subgroups.

Figure 1. The ependymoma genome

Dendrogram at top segregates 83 human ependymomas into nine subgroups (A to I) according to mRNA expression profiles. Clusters were validated by two distinct methods that confirmed agreement for location (Rand index=0.93, P<0.0001) and subgroup (Rand index=0.87, P<0.0001). Heatmap below reports in columns for each case chromosomal gains (red) and losses (blue). Each row represents a chromosome. Red ‘dots’ report focal amplifications (details right), blue ‘dots’ focal deletions (details left). Details are reported in the Supplemental Gene Cards. Site: supratentorial (pink), posterior fossa (blue), spine (yellow). Age: patients <3 years (red), 3–21 years (orange), >21 years (green).

Figure 2. Regionally, developmentally and genetically discrete NSCs match subgroups of human ependymoma

a. Co-immunofluorescence of neurospheres generated by NSC from the cerebrum (C), hindbrain (HB) and spine (SP) of embryonic (E) and adult (A) Ink4a/Arf null or WT Blbp-eGFP mice. b. Graph of percentage of multi-potent daughter neurospheres formed from each NSC type. Dendrogram segregates 177 NSC isolates into subgroups according to mRNA expression. c. Heatmap of transcriptomic (AGDEX, 14,261 orthologs) agreement between mouse NSCs and human supratentorial, posterior fossa and spinal ependymomas. NB. Supratentorial ependymomas match only embryonic cerebral Ink4a/Arf null NSCs; spinal ependymomas match mouse adult spinal NSCs.

Figure 3. EPHB2 is an ependymoma oncogene that selectively transforms embryonic cerebral Ink4a/Arf^−/− NSCs

a. SNP array profile infers focal EPHB2 amplification in human supratentorial ependymoma, validated by FISH analysis (inset) of the EPHB2 locus. b. Profiles of EPHB2 mRNA expression in the human ependymomas shown in Figure 1. c. eGFP/RFP co-immunofluorescence (top) and EphB2, RFP western blot analysis (below), of eGFP⁺ NSCs transduced with Ephb2 (Ephb2^RFP) or control (cRFP) virus. d. Tumor free survival of mice implanted with Ephb2^RFP or control transduced cells. Numbers next to each NSC type indicate the number of implanted mice (**, P<0.005; ***, P<0.0005, Log Rank Test).

Figure 4. Ephb2-driven mouse ependymomas model human subgroup ‘D’ tumors

a. Comparative histology of a representative human cerebral (left) and Ephb2-driven mouse (right) ependymoma including the neuronal marker synaptophysin and glial marker glial fibrillary acidic protein (GFAP). Scale bar=100µm, all panels. (b) Electron micrographs of EphB2-driven mouse ependymomas showing three ultrastructural hallmarks of human ependymoma. (c) Heatmap reporting the transcriptomic agreement (AGDEX) and corresponding permuted p-value in comparisons between EphB2-driven mouse ependymoma and 18 molecular subtypes of human brain tumor. NB. Mouse ependymomas matched only human subgroup D ependymomas.