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.

An amino terminal phosphorylation motif regulates intranuclear compartmentalization of Olig2 in neural progenitor cells.

The bHLH transcription factor Olig2 is expressed in cycling neural progenitor cells but also in terminally differentiated, myelinating oligodendrocytes. Sustained expression of Olig2 is counterintuitive because all known functions of the protein in expansion of neural progenitors and specification of oligodendrocyte progenitors are completed with the formation of mature white matter. How are the biological functions of Olig2 suppressed in terminally differentiated oligodendrocytes? In previous studies, we have shown that a triple serine motif in the amino terminus of Olig2 is phosphorylated in cycling neural progenitors but not in their differentiated progeny. We now show that phosphorylation of the triple serine motif regulates intranuclear compartmentalization of murine Olig2. Phosphorylated Olig2 is preferentially localized to a transcriptionally active "open" chromatin compartment together with coregulator proteins essential for regulation of gene expression. Unphosphorylated Olig2, as seen in mature white matter, is localized mainly within a transcriptionally inactive, chromatin fraction characterized by condensed and inaccessible DNA. Of special note is the observation that the p53 tumor suppressor protein is confined to the open chromatin fraction. Proximity ligation assays show that phosphorylation brings Olig2 within 30 nm of p53 within the open chromatin compartment. The data thus shed light on previously noted promitogenic functions of phosphorylated Olig2, which reflect, at least in part, an oppositional relationship with p53 functions.

Structure-activity relationships of non-opioid [des-Arg(7)]-dynorphin A analogues for bradykinin receptors.

In our earlier studies, bradykinin receptors (BRs) were identified as a potential target for the neuroexcitatory effects of dynorphin A (Dyn A) in the central nervous system (CNS), and [des-Arg(7)]-Dyn A-(4-11) (6) was discovered as a lead ligand to modulate Dyn A-(2-13) induced neuroexcitatory effects in the CNS as an antagonist. In an effort to gain insights into key structural features of the Dyn A for the BRs, we pursued further structure-activity relationships (SAR) study on the [des-Arg(7)]-Dyn A analogs and confirmed that all of the [des-Arg(7)]-Dyn A analogues showed good binding affinities at the BRs.

ZFTA Translocations Constitute Ependymoma Chromatin Remodeling and Transcription Factors.

ZFTA (C11orf95)-a gene of unknown function-partners with a variety of transcriptional coactivators in translocations that drive supratentorial ependymoma, a frequently lethal brain tumor. Understanding the function of ZFTA is key to developing therapies that inhibit these fusion proteins. Here, using a combination of transcriptomics, chromatin immunoprecipitation sequencing, and proteomics, we interrogated a series of deletion-mutant genes to identify a tripartite transformation mechanism of ZFTA-containing fusions, including: spontaneous nuclear translocation, extensive chromatin binding, and SWI/SNF, SAGA, and NuA4/Tip60 HAT chromatin modifier complex recruitment. Thereby, ZFTA tethers fusion proteins across the genome, modifying chromatin to an active state and enabling its partner transcriptional coactivators to promote promiscuous expression of a transforming transcriptome. Using mouse models, we validate further those elements of ZFTA-fusion proteins that are critical for transformation-including ZFTA zinc fingers and partner gene transactivation domains-thereby unmasking vulnerabilities for therapeutic targeting. SIGNIFICANCE: Ependymomas are hard-to-treat brain tumors driven by translocations between ZFTA and a variety of transcriptional coactivators. We dissect the transforming mechanism of these fusion proteins and identify protein domains indispensable for tumorigenesis, thereby providing insights into the molecular basis of ependymoma tumorigenesis and vulnerabilities for therapeutic targeting.This article is highlighted in the In This Issue feature, p. 2113.

Cross-Species Genomics Reveals Oncogenic Dependencies in ZFTA/C11orf95 Fusion-Positive Supratentorial Ependymomas.

Molecular groups of supratentorial ependymomas comprise tumors with ZFTA-RELA or YAP1-involving fusions and fusion-negative subependymoma. However, occasionally supratentorial ependymomas cannot be readily assigned to any of these groups due to lack of detection of a typical fusion and/or ambiguous DNA methylation-based classification. An unbiased approach with a cohort of unprecedented size revealed distinct methylation clusters composed of tumors with ependymal but also various other histologic features containing alternative translocations that shared ZFTA as a partner gene. Somatic overexpression of ZFTA-associated fusion genes in the developing cerebral cortex is capable of inducing tumor formation in vivo, and cross-species comparative analyses identified GLI2 as a key downstream regulator of tumorigenesis in all tumors. Targeting GLI2 with arsenic trioxide caused extended survival of tumor-bearing animals, indicating a potential therapeutic vulnerability in ZFTA fusion-positive tumors. SIGNIFICANCE: ZFTA-RELA fusions are a hallmark feature of supratentorial ependymoma. We find that ZFTA acts as a partner for alternative transcriptional activators in oncogenic fusions of supratentorial tumors with various histologic characteristics. Establishing representative mouse models, we identify potential therapeutic targets shared by ZFTA fusion-positive tumors, such as GLI2.This article is highlighted in the In This Issue feature, p. 2113.

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

More than 60% of supratentorial 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, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin sequencing, and RNA sequencing and compared with human ZRfus-driven ependymoma. In addition to direct canonical NFκB 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 coactivators (Brd4, Ep300, Cbp, Pol2) that are amenable to pharmacologic inhibition. Downstream ZRfus target genes converge on developmental programs marked by PLAGL TF proteins, and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks. SIGNIFICANCE: Ependymomas are aggressive brain tumors. Although drivers of supratentorial ependymoma (ZFTA- and YAP1-associated gene fusions) have been discovered, their functions remain unclear. Our study investigates the biology of ZFTA-RELA-driven ependymoma, specifically mechanisms of transcriptional deregulation and direct downstream gene networks that may be leveraged for potential therapeutic testing.This article is highlighted in the In This Issue feature, p. 2113.

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.

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.

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

The 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 PDGFRα(HIGH) GSCs, a phenomenon that appears to be circumscribed in EGFR(HIGH) 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.

Various modifications of the amphipathic dynorphin A pharmacophore for rat brain bradykinin receptors.

As a unique endogenous opioid ligand, dynorphin A shows paradoxical neuroexcitatory effects at bradykinin receptors, and the effects are known to be amplified by the upregulation of dynorphin A under chronic pain and inflammatory conditions. In our earlier structure-activity relationship studies, the amphipathic dynorphin A fragment, [Des-Arg(7) ]-Dyn A-(4-11), was identified as a pharmacophore for the bradykinin receptors along with key structural features. Here, further modifications of the pharmacophore showed that the position of a Pro residue is also an important feature because of its role in making (or disrupting) a ß-turn or 310 helix structure which is crucial for receptor recognition.

Post-translational Modifications of OLIG2 Regulate Glioma Invasion through the TGF-ß Pathway.

In glioblastoma, invasion and proliferation are presumed to be mutually exclusive events; however, the molecular mechanisms that mediate this switch at the cellular level remain elusive. Previously, we have shown that phospho-OLIG2, a central-nervous-system-specific transcription factor, is essential for tumor growth and proliferation. Here, we show that the modulation of OLIG2 phosphorylation can trigger a switch between proliferation and invasion. Glioma cells with unphosphorylated OLIG2(S10, S13, S14) are highly migratory and invasive, both in vitro and in vivo. Mechanistically, unphosphorylated OLIG2 induces TGF-ß2 expression and promotes invasive mesenchymal properties in glioma cells. Inhibition of the TGF-ß2 pathway blocks this OLIG2-dependent invasion. Furthermore, ectopic expression of phosphomimetic Olig2 is sufficient to block TGF-ß2-mediated invasion and reduce expression of invasion genes (ZEB1 and CD44). Our results not only provide a mechanistic insight into how cells switch from proliferation to invasion but also offer therapeutic opportunities for inhibiting dissemination of gliomas.