Molecular Mechanisms of C11orf95 translocations in cancer.

Highly recurrent chromosome translocations that produce oncogenic fusions are a prime example of practical therapeutic targets translating into clinical successes. We have previously described the genetic driver of supratentorial (forebrain) ependymomas as translocations of the C11orf95 gene of unknown function and RELA, the canonical nuclear effector of NF-B signalling. Subsequent studies have reinforced our original findings on the frequency (>70%) of these C11orf95-RELA (C11-RELA) fusions in human disease, as well as their transformative potential in multiple murine models. However, the mechanisms by which C11-RELA fusions transform neural stem cells into ependymomas remain elusive. Using multi-OMICS methodologies, we show that oncogenic C11orf95 fusions co-opt spatiotemporally regulated transcriptional activators. We find that the Zinc Fingers (ZF) of C11orf95 promote nuclear translocation and a common unique transcriptional signature mediated by the trans-activation domains (TAD) of the fused c-terminal protein partner. Specifically, the ZF’s of C11orf95 direct DNA-binding (as shown with ChIP-seq) of the fused protein partner but does not appear to alter the c-terminal protein’s nuclear interactome (identified using qPLEX-RIME and IP-MS). We further show that canonical NF-kB DNA binding and dimerization, mediated by the Rel homology domain (RHD), are completely dispensable for C11-RELA fusion function in vitro and transformation of neural stem cells in vivo. We henceforth demonstrate that supratentorial ependymoma, which have been previously described as a RELA fusion diseases, are truly a C11orf95 fusion disease, with ZFs serving as a scaffold for transcriptional dysregulation. Our studies lay the architectural framework for a molecular roadmap into the heart of C11orf95 fusion disease, elucidating the biochemical mechanisms which furnish the capacity of these fusions to operate as single gene oncogenes, and henceforth providing potential avenues for therapeutic intervention in ependymoma.Cancer Research U

Lineage-Restricted OLIG2-RTK Signaling Governs the Molecular Subtype of Glioma Stem-like Cells

SummaryThe basic helix-loop-helix (bHLH) transcription factor OLIG2 is a master regulator of oligodendroglial fate decisions and tumorigenic competence of glioma stem-like cells (GSCs). However, the molecular mechanisms underlying dysregulation of OLIG2 function during gliomagenesis remains poorly understood. Here, we show that OLIG2 modulates growth factor signaling in two distinct populations of GSCs, characterized by expression of either the epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor alpha (PDGFRα). Biochemical analyses of OLIG2 function in normal and malignant neural progenitors reveal a positive feedforward loop between OLIG2 and EGFR to sustain co-expression. Furthermore, loss of OLIG2 function results in mesenchymal transformation in PDGFRαHIGH GSCs, a phenomenon that appears to be circumscribed in EGFRHIGH GSCs. Exploitation of OLIG2′s dual and antithetical, pro-mitotic (EGFR-driven), and lineage-specifying (PDGFRα-driven) functions by glioma cells appears to be critical for sustaining growth factor signaling and GSC molecular subtype

Stapled EGFR peptide reduces inflammatory breast cancer and inhibits additional HER-driven models of cancer

Background: The human epidermal growth factor receptor (HER) family of transmembrane tyrosine kinases is overexpressed and correlates with poor prognosis and decreased survival in many cancers. The receptor family has been therapeutically targeted, yet tyrosine kinase inhibitors (TKIs) do not inhibit kinase-independent functions and antibody-based targeting does not affect internalized receptors. We have previously demonstrated that a peptide mimicking the internal juxtamembrane domain of HER1 (EGFR; EJ1) promotes the formation of non-functional HER dimers that inhibit kinase-dependent and kinase-independent functions of HER1 (ERBB1/EGFR), HER2 (ERBB2) and HER3 (ERBB3). Despite inducing rapid HER-dependent cell death in vitro, EJ1 peptides are rapidly cleared in vivo, limiting their efficacy. Method: To stabilize EJ1 activity, hydrocarbon staples (SAH) were added to the active peptide (SAH-EJ1), resulting in a 7.2-fold increase in efficacy and decreased in vivo clearance. Viability assays were performed across HER1 and HER2 expressing cell lines, therapeutic-resistant breast cancer cells, clinically relevant HER1-mutated lung cancer cells, and patient-derived glioblastoma cells, in all cases demonstrating improved efficacy over standard of care pan-HER therapeutics. Tumor burden studies were also performed in lung, glioblastoma, and inflammatory breast cancer mouse models, evaluating tumor growth and overall survival. Results: When injected into mouse models of basal-like and inflammatory breast cancers, EGFRvIII-driven glioblastoma, and lung adenocarcinoma with Erlotinib resistance, tumor growth is inhibited and overall survival is extended. Studies evaluating the toxicity of SAH-EJ1 also demonstrate a broad therapeutic window. Conclusions: Taken together, these data indicate that SAH-EJ1 may be an effective therapeutic for HER-driven cancers with the potential to eliminate triple negative inflammatory breast cancer.Arizona Cancer Therapeutics; Ginny L Clements Breast Cancer Research Fund; NIH [NIH 1R41CA203353]; NCI [P30 CA023074]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

A De Novo Mouse Model of C11orf95-RELA Fusion-Driven Ependymoma Identifies Driver Functions in Addition to NF-κB.

The majority of supratentorial ependymomas (ST-ependymomas) have few mutations but frequently display chromothripsis of chromosome 11q that generates a fusion between C11orf95 and RELA (RELAFUS). Neural stem cells transduced with RELAFUSex vivo form ependymomas when implanted in the brain. These tumors display enhanced NF-κB signaling, suggesting that this aberrant signal is the principal mechanism of oncogenesis. However, it is not known whether RELAFUS is sufficient to drive de novo ependymoma tumorigenesis in the brain and, if so, whether these tumors also arise from neural stem cells. We show that RELAFUS drives ST-ependymoma formation from periventricular neural stem cells in mice and that RELAFUS-induced tumorigenesis is likely dependent on a series of cell signaling pathways in addition to NF-κB

A Glial Signature and Wnt7 Signaling Regulate Glioma-Vascular Interactions and Tumor Microenvironment.

Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. We show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. Thus, glial-encoded pathways regulate distinct glioma-vascular microenvironmental interactions

ZFTA-RELA Dictates Oncogenic Transcriptional Programs to Drive Aggressive Supratentorial Ependymoma

Over 60% of supratentorial (ST) ependymomas harbor a ZFTA-RELA (ZRfus) gene fusion (formerly C11orf95-RELA). To study the biology of ZRfus, we developed an autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain. Integrative epigenomic and transcriptomic mapping was performed on IUE driven ZRfus tumors by CUT&RUN, ChIP, ATAC, and RNA sequencing and compared to human ZRfus driven ependymoma. In addition to direct canonical NF-κB pathway activation, ZRfus dictates a neoplastic transcriptional program and binds to thousands of unique sites across the genome that are enriched with Plagl family transcription factor (TF) motifs. ZRfus activates gene expression programs through recruitment of transcriptional co-activators (Brd4, Ep300, Cbp, Pol2) that are amenable to pharmacologic inhibition. Downstream ZRfus target genes converge on developmental programs marked by Plagl transcription factor proteins, and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks

Harnessing OLIG2 function in tumorigenicity and plasticity to target malignant gliomas

Glioblastoma (GBM) is the most prevalent and malignant brain tumor, displaying notorious resistance to conventional therapy, partially due to molecular and genetic heterogeneity. Understanding the mechanisms for gliomagenesis, tumor stem/progenitor cell propagation and phenotypic diversity is critical for devising effective and targeted therapy for this lethal disease. The basic helix-loop-helix transcription factor OLIG2, which is universally expressed in gliomas, has emerged as an important player in GBM cell reprogramming, genotoxic resistance, and tumor phenotype plasticity. In an animal model of proneural GBM, elimination of mitotic OLIG2+ progenitors blocks tumor growth, suggesting that these progenitors are a seeding source for glioma propagation. OLIG2 deletion reduces tumor growth and causes an oligodendrocytic to astrocytic phenotype shift, with PDGFRα downregulation and reciprocal EGFR signaling upregulation, underlying alternative pathways in tumor recurrence. In patient-derived glioma stem cells (GSC), knockdown of OLIG2 leads to downregulation of PDGFRα, while OLIG2 silencing results in a shift from proneural-to-classical gene expression pattern or a proneural-to-mesenchymal transition in distinct GSC cell lines, where OLIG2 appears to regulate EGFR expression in a context-dependent manner. In addition, post-translational modifications such as phosphorylation by a series of protein kinases regulates OLIG2 activity in glioma cell growth and invasive behaviors. In this perspective, we will review the role of OLIG2 in tumor initiation, proliferation and phenotypic plasticity in animal models of gliomas and human GSC cell lines, and discuss the underlying mechanisms in the control of tumor growth and potential therapeutic strategies to target OLIG2 in malignant gliomas