Kumarasamypet M. Mohankumar

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

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.

C11orf95 – RELA fusions drive oncogenic NF-κB signalling in ependymoma

Members of the nuclear factor-κB (NF-κB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-κB signalling is present in most human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-κB activity in cancer. Here we show that more than two-thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-κB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95–RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95–RELA fusion proteins translocated spontaneously to the nucleus to activate NF-κB target genes, and rapidly transformed neural stem cells—the cell of origin of ependymoma—to form these tumours in mice. Our data identify a highly recurrent genetic alteration of RELA in human cancer, and the C11orf95–RELA fusion protein as a potential therapeutic target in supratentorial ependymoma.

An in vivo screen identifies ependymoma oncogenes and tumor-suppressor genes

Cancers are characterized by non-random chromosome copy number alterations that presumably contain oncogenes and tumor-suppressor genes (TSGs). The affected loci are often large, making it difficult to pinpoint which genes are driving the cancer. Here we report a cross-species in vivo screen of 84 candidate oncogenes and 39 candidate TSGs, located within 28 recurrent chromosomal alterations in ependymoma. Through a series of mouse models, we validate eight new ependymoma oncogenes and ten new ependymoma TSGs that converge on a small number of cell functions, including vesicle trafficking, DNA modification and cholesterol biosynthesis, identifying these as potential new therapeutic targets.

Abstract 4759: Integrated in vitro and in vivo screening of tumor and normal neural stem cells identifies potential new treatments of ependymoma

Ependymomas are rare brain tumors that are incurable unless completely excised. The low incidence of the disease and lack of pre-clinical models has limited research efforts to advance understanding of biology and treatment. Recently, we used interspecies genomics to match specific driver mutations with distinct types of mouse neural stem cell (NSC) to accurately model human ependymoma. Here we report the use of these models for high throughput drug screening (HTS). Stem-like mouse ependymoma cells (mEPCs), non-ependymoma mouse brain tumor cells (mBTCs) and control transduced NSCs (mNSCs) were cultured in neurosphere conditions adapted for use in an automated small molecule HTS. We first performed replicate primary screens of 7,579 agents drawn from a bioactive library, FDA approved drug library and kinase library. Primary screens were conducted in a single concentration format (10µM). The primary screen was highly reproducible and ROC analysis of primary screen data was used to assess predictive power of the screen (ROC AUC>0.89 (0.85-0.92 95% CI)). A total of 602 compounds representing diverse drug classes progressed from primary to secondary screening. These included full 10-point dose response assays that identified a total of 181 agents with activity in at least one cell population. In all, 2.3% of compounds displayed anti-mEPC activity and were enriched for anti-cancer drugs (Fishers Exact P=1.9 × 10-7: Bonferroni correction threshold, p=0.0016). Since our HTS strategy included non-ependymoma tumor cells and mNSCs, we were able to refine our classification of activity to define compounds more active against mEPCs than other cells (0.08%); equally active against mEPCs and mBTCs relative to mNSCs (0.04%); equally active against all four cell types (2.2%); more active against mBTCs relative to all other cells (0.2%); more active against mNSCs relative to tumor cells (0.8%); inactive against mEPCs relative to all other cells (0.1%); and inactive against all four cell types (96%). Interestingly, anticancer compounds displayed patterns of cell-selective activity that varied according to their mechanism of action with some drug classes appearing significantly more toxic to mNSCs than either mEPCs or mBTCs. These HTS studies identified treatments for ependymoma including drugs that were relatively non-toxic to normal NSCs. Five agents were selected for assessment of in vivo against the originating ependymoma mouse models. Using the xenogen system to monitor tumor growth and assessing animal survival, we identified FDA-approved agents with activity in ependymoma which have not previously been implicated in the disease, and may be translated directly into the clinic. In summary, this approach identified a number of potential new treatments with potent activity against ependymoma relative to normal NSCs, and could be used to develop effective therapies for other rare cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4759. doi:10.1158/1538-7445.AM2011-4759

Abstract A52: An interspecies genomics based high‐throughput screen for novel treatments of ependymoma

Brain tumors are often chemoresistant and these diseases are heterogeneous complicating efforts to discover effective new therapies. We describe a powerful interspecies genomics approach that meticulously matches subgroup specific driver mutations with cellular compartments to model cancer subgroups for drug screening. First, we performed a comprehensive genomics analysis to identify disease subgroups among >200 ependymomas. Subgroup specific alterations included amplifications and homozygous deletions of genes not yet implicated in ependymoma. We then identified CNS cell compartments most likely to give rise to ependymoma subgroups by matching the transcriptomes of human tumors to those of distinct types of mouse neural stem cell (NSC). Remarkably, activation of oncogenes in appropriate NSCs generated ependymomas that modelled the histology and transcriptome of the human disease. Stem‐like tumor cells isolated from these mouse ependymomas were then cultured under conditions that promote stem cell growth as neurospheres. These conditions were adapted for use in an automated high‐throughput screening system. Control transduced NSCs were also included to identify ependymoma‐selective agents. Tumor and control spheres were seeded in 384 well plates and drug treated using pin tool transfer 24 hours after plating. Cells were exposed to drug for 96 hours before compound cytotoxicity measured using the cell‐titre glo luciferase based assay. We first performed replicate primary screens of a large ‘bioactive library’ including natural products, bioactive alkaloids and marketed drugs (5760 compounds [3460 unique in structure]) in a single concentration format (10uM). Active compound hit rate varied from 0.9% to 4.8% in spheres derived from mouse tumors and 3.1% in control NSCs. The primary screen was highly reproducible (263 and 261 hits in replicate assays, of which 226 of were common in an example tumor line). ROC analysis of primary screen data was used to assess predictive power of the screen. For all cells the ROC AUC was >0.89 (0.85–0.92 95% CI). We next performed secondary screens of all primary screen hits. These included full 10‐point dose response assays that identified a total of 292 active agents with activity in at least one cell population. Following analysis of the ‘bioactive library, we screened a collection of 320 FDA approved active pharmaceutical ingredients representing all drug classes and all approved chemotherapy agents in a 10‐point dose response format. The hit rate of this drug collection in all cell types was between 10.36% to 15.4% and hit compounds represented a variety of drug classes including chemotherapeutics, NSAIDs, antibiotics and dopamine‐like agents. These compounds and the related families and structures will be described in detail as well as the results of ongoing in vivo activity against the originating ependymoma mouse models. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A52.