Sorana Morrissy

Hotspot Mutations in H3F3A and IDH1 Define Distinct Epigenetic and Biological Subgroups of Glioblastoma

Glioblastoma (GBM) is a brain tumor that carries a dismal prognosis and displays considerable heterogeneity. We have recently identified recurrent H3F3A mutations affecting two critical amino acids (K27 and G34) of histone H3.3 in one-third of pediatric GBM. Here, we show that each H3F3A mutation defines an epigenetic subgroup of GBM with a distinct global methylation pattern, and that they are mutually exclusive with IDH1 mutations, which characterize a third mutation-defined subgroup. Three further epigenetic subgroups were enriched for hallmark genetic events of adult GBM and/or established transcriptomic signatures. We also demonstrate that the two H3F3A mutations give rise to GBMs in separate anatomic compartments, with differential regulation of transcription factors OLIG1, OLIG2, and FOXG1, possibly reflecting different cellular origins.

Quiescent Sox2+ Cells Drive Hierarchical Growth and Relapse in Sonic Hedgehog Subgroup Medulloblastoma

Functional heterogeneity within tumors presents a significant therapeutic challenge. Here we show that quiescent, therapy-resistant Sox2(+) cells propagate sonic hedgehog subgroup medulloblastoma by a mechanism that mirrors a neurogenic program. Rare Sox2(+) cells produce rapidly cycling doublecortin(+) progenitors that, together with their postmitotic progeny expressing NeuN, comprise tumor bulk. Sox2(+) cells are enriched following anti-mitotic chemotherapy and Smoothened inhibition, creating a reservoir for tumor regrowth. Lineage traces from Sox2(+) cells increase following treatment, suggesting that this population is responsible for relapse. Targeting Sox2(+) cells with the antineoplastic mithramycin abrogated tumor growth. Addressing functional heterogeneity and eliminating Sox2(+) cells presents a promising therapeutic paradigm for treatment of sonic hedgehog subgroup medulloblastoma.

HDAC and PI3K Antagonists Cooperate to Inhibit Growth of MYC-Driven Medulloblastoma

Medulloblastoma (MB) is a highly malignant pediatric brain tumor. Despite aggressive therapy, many patients succumb to the disease, and survivors experience severe side effects from treatment. MYC-driven MB has a particularly poor prognosis and would greatly benefit from more effective therapies. We used an animal model of MYC-driven MB to screen for drugs that decrease viability of tumor cells. Among the most effective compounds were histone deacetylase inhibitors (HDACIs). HDACIs potently inhibit survival of MYC-driven MB cells in vitro, in part by inducing expression of the FOXO1 tumor suppressor gene. HDACIs also synergize with phosphatidylinositol 3-kinase inhibitors to inhibit tumor growth in vivo. These studies identify an effective combination therapy for the most aggressive form of MB.

Digital Gene Expression by Tag Sequencing on the Illumina Genome Analyzer

This unit provides a protocol for performing digital gene expression profiling on the Illumina Genome Analyzer sequencing platform. Tag sequencing (Tag‐seq) is an implementation of the LongSAGE protocol on the Illumina sequencing platform that increases utility while reducing both the cost and time required to generate gene expression profiles. The ultra‐high‐throughput sequencing capability of the Illumina platform allows the cost‐effective generation of libraries containing an average of 20 million tags, a 200‐fold improvement over classical LongSAGE. Tag‐seq has less sequence composition bias, leading to a better representation of AT‐rich tag sequences, and allows a more accurate profiling of a subset of the transcriptome characterized by AT‐rich genes expressed at levels below the threshold of detection of LongSAGE (Morrissy et al., 2009). Curr. Protoc. Hum. Genet. 65:11.11.1‐11.11.36

Identification of GPC2 as an Oncoprotein and Candidate Immunotherapeutic Target in High-Risk Neuroblastoma

We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target.

Medulloblastoma-associated DDX3 variant selectively alters the translational response to stress

DDX3X encodes a DEAD-box family RNA helicase (DDX3) commonly mutated in medulloblastoma, a highly aggressive cerebellar tumor affecting both children and adults. Despite being implicated in several facets of RNA metabolism, the nature and scope of DDX3′s interactions with RNA remain unclear. Here, we show DDX3 collaborates extensively with the translation initiation machinery through direct binding to 5′UTRs of nearly all coding RNAs, specific sites on the 18S rRNA, and multiple components of the translation initiation complex. Impairment of translation initiation is also evident in primary medulloblastomas harboring mutations in DDX3X, further highlighting DDX3′s role in this process. Arsenite-induced stress shifts DDX3 binding from the 5′UTR into the coding region of mRNAs concomitant with a general reduction of translation, and both the shift of DDX3 on mRNA and decreased translation are blunted by expression of a catalytically-impaired, medulloblastoma-associated DDX3R534H variant. Furthermore, despite the global repression of translation induced by arsenite, translation is preserved on select genes involved in chromatin organization in DDX3R534H-expressing cells. Thus, DDX3 interacts extensively with RNA and ribosomal machinery to help remodel the translation landscape in response to stress, while cancer-related DDX3 variants adapt this response to selectively preserve translation.

Opposing Effects of CREBBP Mutations Govern the Phenotype of Rubinstein-Taybi Syndrome and Adult SHH Medulloblastoma

Recurrent mutations in chromatin modifiers are specifically prevalent in adolescent or adult patients with Sonic hedgehog-associated medulloblastoma (SHH MB). Here, we report that mutations in the acetyltransferase CREBBP have opposing effects during the development of the cerebellum, the primary site of origin of SHH MB. Our data reveal that loss of Crebbp in cerebellar granule neuron progenitors (GNPs) during embryonic development of mice compromises GNP development, in part by downregulation of brain-derived neurotrophic factor (Bdnf). Interestingly, concomitant cerebellar hypoplasia was also observed in patients with Rubinstein-Taybi syndrome, a congenital disorder caused by germline mutations of CREBBP. By contrast, loss of Crebbp in GNPs during postnatal development synergizes with oncogenic activation of SHH signaling to drive MB growth, thereby explaining the enrichment of somatic CREBBP mutations in SHH MB of adult patients. Together, our data provide insights into time-sensitive consequences of CREBBP mutations and corresponding associations with human diseases.

BMI1 is a therapeutic target in recurrent medulloblastoma

Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor, representing 20% of newly diagnosed childhood central nervous system malignancies. Although advances in multimodal therapy yielded a 5-year survivorship of 80%, MB still accounts for the leading cause of childhood cancer mortality. In this work, we describe the epigenetic regulator BMI1 as a novel therapeutic target for the treatment of recurrent human Group 3 MB, a childhood brain tumor for which there is virtually no treatment option beyond palliation. Current clinical trials for recurrent MB patients based on genomic profiles of primary, treatment-naive tumors will provide limited clinical benefit since recurrent metastatic MBs are highly genetically divergent from their primary tumor. Using a small molecule inhibitor against BMI1, PTC-028, we were able to demonstrate complete ablation of self-renewal of MB stem cells in vitro. When administered to mice xenografted with patient tumors, we observed significant reduction in tumor burden in both local and metastatic compartments and subsequent increased survival, without neurotoxicity. Strikingly, serial in vivo re-transplantation assays demonstrated a marked reduction in tumor initiation ability of recurrent MB cells upon re-transplantation of PTC-028-treated cells into secondary recipient mouse brains. As Group 3 MB is often metastatic and uniformly fatal at recurrence, with no current or planned trials of targeted therapy, an efficacious targeted agent would be rapidly transitioned to clinical trials.

Abstract 685: GPC2 is an oncogene and immunotherapeutic target in high-risk neuroblastoma

Background: GD2-directed immunotherapeutic strategies have improved outcomes in neuroblastoma; however, the majority of patients treated suffer relapse and GD2 expression on pain fibers causes dose-limiting toxicities. Methods: To identify alternative cell surface immunotherapeutic targets, we compared high-risk neuroblastoma (n=126 tumors) and normal tissue RNA sequencing data (GTEx; n=7859 samples from 31 normal tissues) and prioritized genes by differential and absolute expression and cell surface prediction. Genes were further surveyed for somatic copy number gain and correlative expression with MYCN amplification. Differential protein expression and localization were confirmed in neuroblastoma primary tumors (n=98), patient-derived xenografts (n=32; PDXs), cell lines (n=23), and normal pediatric tissues (n=36). Cell lines were subjected to candidate gene gain and loss of function studies (n=11). Additional pediatric tumor RNA sequencing data was surveyed followed by confirmatory immunohistochemistry (IHC). Finally, candidate specific antibodies were isolated from a human Fab phage library and utilized for antibody-drug conjugate (ADC) engineering followed by cytotoxicity studies. Results: We identified 33 differentially expressed cell surface molecules from which we prioritized glypican-2 (GPC2) for validation given GPC2’s robust differential expression (log-fold change tumor vs. normal tissue = 1.71-9.22; p=1.99 x 10-9-1.88 x10-300), high-level absolute RNA expression (median FPKM=60), and frequent DNA copy number gain associated with higher GPC2 expression (35%, n=182 tumors; p Conclusions: GPC2 is an oncogene and immunotherapeutic target in neuroblastoma and potentially other cancers. Citation Format: Kristopher R. Bosse, Pichai Raman, Maria Lane, Robyn T. Sussman, Jo Lynne Harenza, Daniel Martinez, Sabine Heitzeneder, Zhongyu Zhu, Komal Rathi, Michael Randall, Laura Donovan, Sorana Morrissy, Doncho V. Zhelev, Yang Feng, Jennifer Hwang, Yanping Wang, Bruce Pawel, Tricia Bhatti, Mariarita Santi, Javed Khan, Michael Taylor, Dimiter S. Dimitrov, Crystal Mackall, John M. Maris. GPC2 is an oncogene and immunotherapeutic target in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 685. doi:10.1158/1538-7445.AM2017-685