Therapeutic targeting of ependymoma as informed by oncogenic enhancer profiling

Genomic sequencing has driven precision-based oncology therapy; however, the genetic drivers of many malignancies remain unknown or non-targetable, so alternative approaches to the identification of therapeutic leads are necessary. Ependymomas are chemotherapy-resistant brain tumours, which, despite genomic sequencing, lack effective molecular targets. Intracranial ependymomas are segregated on the basis of anatomical location (supratentorial region or posterior fossa) and further divided into distinct molecular subgroups that reflect differences in the age of onset, gender predominance and response to therapy1,2,3. The most common and aggressive subgroup, posterior fossa ependymoma group A (PF-EPN-A), occurs in young children and appears to lack recurrent somatic mutations2. Conversely, posterior fossa ependymoma group B (PF-EPN-B) tumours display frequent large-scale copy number gains and losses but have favourable clinical outcomes1,3. More than 70% of supratentorial ependymomas are defined by highly recurrent gene fusions in the NF-κB subunit gene RELA (ST-EPN-RELA), and a smaller number involve fusion of the gene encoding the transcriptional activator YAP1 (ST-EPN-YAP1)1,3,4. Subependymomas, a distinct histologic variant, can also be found within the supratetorial and posterior fossa compartments, and account for the majority of tumours in the molecular subgroups ST-EPN-SE and PF-EPN-SE. Here we describe mapping of active chromatin landscapes in 42 primary ependymomas in two non-overlapping primary ependymoma cohorts, with the goal of identifying essential super-enhancer-associated genes on which tumour cells depend. Enhancer regions revealed putative oncogenes, molecular targets and pathways; inhibition of these targets with small molecule inhibitors or short hairpin RNA diminished the proliferation of patient-derived neurospheres and increased survival in mouse models of ependymomas. Through profiling of transcriptional enhancers, our study provides a framework for target and drug discovery in other cancers that lack known genetic drivers and are therefore difficult to treat.This work was supported by an Alex's Lemonade Stand Young Investigator Award (S.C.M.), The CIHR Banting Fellowship (S.C.M.), The Cancer Prevention Research Institute of Texas (S.C.M., RR170023), Sibylle Assmus Award for Neurooncology (K.W.P.), the DKFZ-MOST (Ministry of Science, Technology & Space, Israel) program in cancer research (H.W.), James S. McDonnell Foundation (J.N.R.) and NIH grants: CA154130 (J.N.R.), R01 CA169117 (J.N.R.), R01 CA171652 (J.N.R.), R01 NS087913 (J.N.R.) and R01 NS089272 (J.N.R.). R.C.G. is supported by NIH grants T32GM00725 and F30CA217065. M.D.T. is supported by The Garron Family Chair in Childhood Cancer Research, and grants from the Pediatric Brain Tumour Foundation, Grand Challenge Award from CureSearch for Children’s Cancer, the National Institutes of Health (R01CA148699, R01CA159859), The Terry Fox Research Institute and Brainchild. M.D.T. is also supported by a Stand Up To Cancer St. Baldrick’s Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113)

ADAR1-mediated RNA editing links ganglioside catabolism to glioblastoma stem cell maintenance.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor, containing GBM stem cells (GSCs) that contribute to therapeutic resistance and relapse. Exposing potential GSC vulnerabilities may provide therapeutic strategies against GBM. Here, we interrogated the role of adenosine-to-inosine (A-to-I) RNA editing mediated by adenosine deaminase acting on RNA 1 (ADAR1) in GSCs and found that both ADAR1 and global RNA editomes were elevated in GSCs compared with normal neural stem cells. ADAR1 inactivation or blocking of the upstream JAK/STAT pathway through TYK2 inhibition impaired GSC self-renewal and stemness. Downstream of ADAR1, RNA editing of the 3'-UTR of GM2A, a key ganglioside catabolism activator, proved to be critical, as interference with ganglioside catabolism and disruption of ADAR1 showed a similar functional impact on GSCs. These findings reveal that RNA editing links ganglioside catabolism to GSC self-renewal and stemness, exposing a potential vulnerability of GBM for therapeutic intervention

FBXO44 promotes DNA replication-coupled repetitive element silencing in cancer cells.

Repetitive elements (REs) compose ∼50% of the human genome and are normally transcriptionally silenced, although the mechanism has remained elusive. Through an RNAi screen, we identified FBXO44 as an essential repressor of REs in cancer cells. FBXO44 bound H3K9me3-modified nucleosomes at the replication fork and recruited SUV39H1, CRL4, and Mi-2/NuRD to transcriptionally silence REs post-DNA replication. FBXO44/SUV39H1 inhibition reactivated REs, leading to DNA replication stress and stimulation of MAVS/STING antiviral pathways and interferon (IFN) signaling in cancer cells to promote decreased tumorigenicity, increased immunogenicity, and enhanced immunotherapy response. FBXO44 expression inversely correlated with replication stress, antiviral pathways, IFN signaling, and cytotoxic T cell infiltration in human cancers, while a FBXO44-immune gene signature correlated with improved immunotherapy response in cancer patients. FBXO44/SUV39H1 were dispensable in normal cells. Collectively, FBXO44/SUV39H1 are crucial repressors of RE transcription, and their inhibition selectively induces DNA replication stress and viral mimicry in cancer cells

Non-metabolic functions of phosphofructokinase-1 orchestrate tumor cellular invasion and genome maintenance under bevacizumab therapy

Background Glioblastoma (GBM) is a highly lethal malignancy for which neoangiogenesis serves as a defining hallmark. The anti-VEGF antibody, bevacizumab, has been approved for the treatment of recurrent GBM, but resistance is universal. Methods We analyzed expression data of GBM patients treated with bevacizumab to discover potential resistance mechanisms. Patient-derived xenografts (PDXs) and cultures were interrogated for effects of phosphofructokinase-1, muscle isoform (PFKM) loss on tumor cell motility, migration, and invasion through genetic and pharmacologic targeting. Results We identified PFKM as a driver of bevacizumab resistance. PFKM functions dichotomize based on subcellular location: cytosolic PFKM interacted with KIF11, a tubular motor protein, to promote tumor invasion, whereas nuclear PFKM safeguarded genomic stability of tumor cells through interaction with NBS1. Leveraging differential transcriptional profiling, bupivacaine phenocopied genetic targeting of PFKM, and enhanced efficacy of bevacizumab in preclinical GBM models in vivo. Conclusion PFKM drives novel molecular pathways in GBM, offering a translational path to a novel therapeutic paradigm

The Meningioma Enhancer Landscape Delineates Novel Subgroups and Drives Druggable Dependencies

Meningiomas are the most common primary intracranial tumor with current classification offering limited therapeutic guidance. Here, we interrogated meningioma enhancer landscapes from 33 tumors to stratify patients based upon prognosis and identify novel meningioma-specific dependencies. Enhancers robustly stratified meningiomas into three biologically distinct groups (adipogenesis/cholesterol, mesodermal, and neural crest) distinguished by distinct hormonal lineage transcriptional regulators. Meningioma landscapes clustered with intrinsic brain tumors and hormonally responsive systemic cancers with meningioma subgroups, reflecting progesterone or androgen hormonal signaling. Enhancer classification identified a subset of tumors with poor prognosis, irrespective of histologic grading. Superenhancer signatures predicted drug dependencies with superior in vitro efficacy to treatment based upon the NF2 genomic profile. Inhibition of DUSP1, a novel and druggable meningioma target, impaired tumor growth in vivo. Collectively, epigenetic landscapes empower meningioma classification and identification of novel therapies. SIGNIFICANCE: Enhancer landscapes inform prognostic classification of aggressive meningiomas, identifying tumors at high risk of recurrence, and reveal previously unknown therapeutic targets. Druggable dependencies discovered through epigenetic profiling potentially guide treatment of intractable meningiomas.This article is highlighted in the In This Issue feature, p. 1611

Targeting EYA2 tyrosine phosphatase activity in glioblastoma stem cells induces mitotic catastrophe

Glioblastoma ranks among the most lethal of primary brain malignancies, with glioblastoma stem cells (GSCs) at the apex of tumor cellular hierarchies. Here, to discover novel therapeutic GSC targets, we interrogated gene expression profiles from GSCs, differentiated glioblastoma cells (DGCs), and neural stem cells (NSCs), revealing EYA2 as preferentially expressed by GSCs. Targeting EYA2 impaired GSC maintenance and induced cell cycle arrest, apoptosis, and loss of self-renewal. EYA2 displayed novel localization to centrosomes in GSCs, and EYA2 tyrosine (Tyr) phosphatase activity was essential for proper mitotic spindle assembly and survival of GSCs. Inhibition of the EYA2 Tyr phosphatase activity, via genetic or pharmacological means, mimicked EYA2 loss in GSCs in vitro and extended the survival of tumor-bearing mice. Supporting the clinical relevance of these findings, EYA2 portends poor patient prognosis in glioblastoma. Collectively, our data indicate that EYA2 phosphatase function plays selective critical roles in the growth and survival of GSCs, potentially offering a high therapeutic index for EYA2 inhibitors

Three-dimensional bioprinted glioblastoma microenvironments model cellular dependencies and immune interactions.

Brain tumors are dynamic complex ecosystems with multiple cell types. To model the brain tumor microenvironment in a reproducible and scalable system, we developed a rapid three-dimensional (3D) bioprinting method to construct clinically relevant biomimetic tissue models. In recurrent glioblastoma, macrophages/microglia prominently contribute to the tumor mass. To parse the function of macrophages in 3D, we compared the growth of glioblastoma stem cells (GSCs) alone or with astrocytes and neural precursor cells in a hyaluronic acid-rich hydrogel, with or without macrophage. Bioprinted constructs integrating macrophage recapitulate patient-derived transcriptional profiles predictive of patient survival, maintenance of stemness, invasion, and drug resistance. Whole-genome CRISPR screening with bioprinted complex systems identified unique molecular dependencies in GSCs, relative to sphere culture. Multicellular bioprinted models serve as a scalable and physiologic platform to interrogate drug sensitivity, cellular crosstalk, invasion, context-specific functional dependencies, as well as immunologic interactions in a species-matched neural environment