Translation of circHGF RNA encodes an HGF protein variant promoting glioblastoma growth through stimulation of c-MET.

INTRODUCTION: HGF/c-MET signaling is a significant driver of glioblastoma (GBM) growth and disease progression. Unfortunately, c-MET targeted therapies have been found to be largely ineffective suggesting additional redundant mechanisms of c-MET activation. METHODS: Utilizing RNA-sequencing (RNA-seq) and ribosome profiling analyses of circular RNAs, circ-HGF (hsa_circ_0080914) was identified as markedly upregulated in primary GBM and found to potentially encode an HGF protein variant (C-HGF) 119 amino acids in length. This candidate HGF variant was characterized and evaluated for its ability to mediate c-MET activation and regulate PDX GBM cell growth, motility and invasive potential in vitro and tumor burden in intracranial xenografts in mice. RESULTS: An internal ribosome entry site (IRES) was identified within the circ-HGF RNA which mediated translation of the cross-junctional ORF encoding C-HGF and was observed to be highly expressed in GBM relative to normal brain tissue. C-HGF was also found to be secreted from GBM cells and concentrated cell culture supernatants or recombinant C-HGF activated known signaling cascades downstream of c-MET. C-HGF was shown to interact directly with the c-MET receptor resulting in its autophosphorylation and activation in PDX GBM lines. Knockdown of C-HGF resulted in suppression of c-MET signaling and marked inhibition of cell growth, motility and invasiveness, whereas overexpression of C-HGF displayed the opposite effects. Additionally, modulation of C-HGF expression regulated tumor growth in intracranial xenografted PDX GBM models. CONCLUSIONS: These results reveal an alternative mechanism of c-MET activation via a circular RNA encoded HGF protein variant which is relevant in GBM biology. Targeting C-HGF may offer a promising approach for GBM clinical management.

Targeting the YAP-TEAD interaction interface for therapeutic intervention in glioblastoma.

INTRODUCTION: Recent studies have suggested that dysregulated Hippo pathway signaling may contribute to glioblastoma proliferation and invasive characteristics. The downstream effector of the pathway, the Yes-associated protein (YAP) oncoprotein, has emerged as a promising target in glioblastoma multiforme (GBM). METHODS: Utilizing a high-throughput yeast two-hybrid based screen, a small molecule was identified which inhibits the association of the co-transcriptional activator YAP1 and the TEA domain family member 1 (TEAD1) transcription factor protein-protein interaction interface. This candidate inhibitor, NSC682769, a novel benzazepine compound, was evaluated for its ability to affect Hippo/YAP axis signaling and potential anti-glioblastoma properties. RESULTS: NSC682769 potently blocked association of YAP and TEAD in vitro and in GBM cells treated with submicromolar concentrations. Moreover, inhibitor-coupled bead pull down and surface plasmon resonance analyses demonstrate that NSC682769 binds to YAP. NSC682769 treatment of GBM lines and patient derived cells resulted in downregulation of YAP expression levels resulting in curtailed YAP-TEAD transcriptional activity. In GBM cell models, NSC682769 inhibited proliferation, colony formation, migration, invasiveness and enhanced apoptosis. In tumor xenograft and genetically engineered mouse models, NSC682769 exhibited marked anti-tumor responses and resulted in increased overall survival and displayed significant blood-brain barrier penetration. CONCLUSIONS: These results demonstrate that blockade of YAP-TEAD association is a viable therapeutic strategy for glioblastoma. On the basis of these favorable preclinical studies further clinical studies are warranted.

Repurposing Potential of Riluzole as an ITAF Inhibitor in mTOR Therapy Resistant Glioblastoma.

Internal ribosome entry site (IRES)-mediated protein synthesis has been demonstrated to play an important role in resistance to mechanistic target of rapamycin (mTOR) targeted therapies. Previously, we have demonstrated that the IRES trans-acting factor (ITAF), hnRNP A1 is required to promote IRES activity and small molecule inhibitors which bind specifically to this ITAF and curtail IRES activity, leading to mTOR inhibitor sensitivity. Here we report the identification of riluzole (Rilutek®), an FDA-approved drug for amyotrophic lateral sclerosis (ALS), via an in silico docking analysis of FDA-approved compounds, as an inhibitor of hnRNP A1. In a riluzole-bead coupled binding assay and in surface plasmon resonance imaging analyses, riluzole was found to directly bind to hnRNP A1 and inhibited IRES activity via effects on ITAF/RNA-binding. Riluzole also demonstrated synergistic anti-glioblastoma (GBM) affects with mTOR inhibitors in vitro and in GBM xenografts in mice. These data suggest that repurposing riluzole, used in conjunction with mTOR inhibitors, may serve as an effective therapeutic option in glioblastoma.

The protein arginine methyltransferase PRMT5 confers therapeutic resistance to mTOR inhibition in glioblastoma.

INTRODUCTION: Clinical trials directed at mechanistic target of rapamycin (mTOR) inhibition have yielded disappointing results in glioblastoma (GBM). A major mechanism of resistance involves the activation of a salvage pathway stimulating internal ribosome entry site (IRES)-mediated protein synthesis. PRMT5 activity has been implicated in the enhancement of IRES activity. METHODS: We analyzed the expression and activity of PRMT5 in response to mTOR inhibition in GBM cell lines and short-term patient cultures. To determine whether PRMT5 conferred resistance we used genetic and pharmacological approaches to ablate PRMT5 activity and assessed the effects on in vitro and in vivo sensitivity. Mutational analyses of the requisite IRES-trans-acting factor (ITAF), hnRNP A1 determined whether PRMT5-mediated methylation was necessary for ITAF RNA binding and IRES activity. RESULTS: PRMT5 activity is stimulated in response to mTOR inhibitors. Knockdown or treatment with a PRMT5 inhibitor blocked IRES activity and sensitizes GBM cells. Ectopic expression of non-methylatable hnRNP A1 mutants demonstrated that methylation of either arginine residues 218 or 225 was sufficient to maintain IRES binding and hnRNP A1-dependent cyclin D1 or c-MYC IRES activity, however a double R218K/R225K mutant was unable to do so. The PRMT5 inhibitor EPZ015666 displayed synergistic anti-GBM effects in vitro and in a xenograft mouse model in combination with PP242. CONCLUSIONS: These results demonstrate that PRMT5 activity is stimulated upon mTOR inhibition in GBM. Our data further support a signaling cascade in which PRMT5-mediated methylation of hnRNP A1 promotes IRES RNA binding and activation of IRES-mediated protein synthesis and resultant mTOR inhibitor resistance.

Development of an acute, short-term exposure model for phosgene.

Phosgene is classified as a chemical warfare agent, yet data on its short-duration high concentration toxicity in a nose-only exposure rat model is sparse and inconsistent. Hence, an exposure system for short-term/high concentration exposure was developed and characterized. Herein, we report the median lethal concentration (LC50) for a 10-min nasal exposure of phosgene in a 24-h rat survival model. Male Wistar rats (Envigo) weighing 180-210 g on the day of exposure, were exposed to phosgene gas via nose-only inhalation using a system specifically designed to allow the simultaneous exposure and quantification of phosgene. After 24 h, the surviving rats were euthanized, the lung/body mass ratio determined, and lung tissues analyzed for histopathology. Increased terminal airway edema in the lungs located primarily at the alveoli (resulting in an increased lung/body mass ratio) coincided with the observed mortality. An LC50 value of 129.2 mg/m3 for a 10-min exposure was determined. Furthermore, in agreement with other highly toxic compounds, this study reveals a LC50 concentration value supportive of a nonlinear toxic load model, where the toxic load exponent is >1 (ne = 1.17). Thus, in line with other chemical warfare agents, phosgene toxicity is predicted to be more severe with short-duration, high-concentration exposures than long-duration, low-concentration exposures. This model is anticipated to be refined and developed to screen novel therapeutics against relevant short-term high concentration phosgene exposures expected from a terrorist attack, battlefield deployment, or industrial accident.

Combining intracellular antibodies to restore function of mutated p53 in cancer.

TP53 is a tumor suppressor gene that is mutated in 50% of cancers, and its function is tightly regulated by the E3 ligase, Mdm2. Both p53 and Mdm2 are localized in the cell nucleus, a site that is impervious to therapeutic regulation by most antibodies. We identified a cell-penetrating lupus monoclonal anti-DNA antibody, mAb 3E10, that targets the nucleus, and we engineered mAb 3E10 to function as an intranuclear transport system to deliver therapeutic antibodies into the nucleus as bispecific single chain Fv (scFv) fragments. Bispecific scFvs composed of 3E10 include PAb421 (3E10-PAb421) that binds p53 and restores the function of mutated p53, and 3G5 (3E10-3G5) that binds Mdm2 and prevents destruction of p53 by Mdm2. We documented the therapeutic efficacy of these bispecific scFvs separately in previous studies. In this study, we show that combination therapy with 3E10-PAb421 and 3E10-3G5 augments growth inhibition of cells with p53 mutations compared to the effect of either antibody alone. By contrast, no enhanced response was observed in cells with wild-type p53 or in cells homozygous null for p53.

m6A-modification of cyclin D1 and c-myc IRESs in glioblastoma controls ITAF activity and resistance to mTOR inhibition.

A major mechanism conferring resistance to mTOR inhibitors is activation of a salvage pathway stimulating internal ribosome entry site (IRES)-mediated mRNA translation, driving the synthesis of proteins promoting resistance of glioblastoma (GBM). Previously, we found this pathway is stimulated by the requisite IRES-trans-acting factor (ITAF) hnRNP A1, which itself is subject to phosphorylation and methylation events regulating cyclin D1 and c-myc IRES activity. Here we describe the requirement for m6A-modification of IRES RNAs for efficient translation and resistance to mTOR inhibition. DRACH-motifs within these IRES RNAs upon m6A modification resulted in enhanced IRES activity via increased hnRNP A1-binding following mTOR inhibitor exposure. Inhibitor exposure stimulated the expression of m6A-methylosome components resulting in increased activity in GBM. Silencing of METTL3-14 complexes reduced IRES activity upon inhibitor exposure and sensitized resistant GBM lines. YTHDF3 associates with m6A-modified cyclin D1 or c-myc IRESs, regulating IRES activity, and mTOR inhibitor sensitivity in vitro and in xenograft experiments. YTHDF3 interacted directly with hnRNP A1 and together stimulated hnRNP A1-dependent nucleic acid strand annealing activity. These data demonstrate that m6A-methylation of IRES RNAs regulate GBM responses to this class of inhibitors.

Cellular protection from H2O2 toxicity by Fv-Hsp70: protection via catalase and gamma-glutamyl-cysteine synthase.

Heat shock proteins (HSPs), especially Hsp70 (HSPA1), have been associated with cellular protection from various cellular stresses including heat, hypoxia-ischemia, neurodegeneration, toxins, and trauma. Endogenous HSPs are often synthesized in direct response to these stresses but in many situations are inadequate in protecting cells. The present study addresses the transduction of Hsp70 into cells providing protection from acute oxidative stress by H2O2. The recombinant Fv-Hsp70 protein and two mutant Fv-Hsp70 proteins minus the ATPase domain and minus the ATPase and terminal lid domains were tested at 0.5 and 1.0 µM concentrations after two different concentrations of H2O2 treatment. All three recombinant proteins protected SH-SY5Y cells from acute H2O2 toxicity. This data indicated that the protein binding domain was responsible for cellular protection. In addition, experiments pretreating cells with inhibitors of antioxidant proteins catalase and gamma-glutamylcysteine synthase (GGCS) before H2O2 resulted in cell death despite treatment with Fv-Hsp70, implying that both enzymes were protected from acute oxidative stress after treatment with Fv-Hsp70. This study demonstrates that Fv-Hsp70 is protective in our experiments primarily by the protein-binding domain. The Hsp70 terminal lid domain was also not necessary for protection.

BRAF drives synovial fibroblast transformation in rheumatoid arthritis.

Synovial fibroblasts destroy articular cartilage and bone in rheumatoid arthritis, but the mechanism of fibroblast transformation remains elusive. Because gain-of-function mutations of BRAF can transform fibroblasts, we examined BRAF in rheumatoid synovial fibroblasts. The strong gain-of-function mutation, V600R, of BRAF found in melanomas and other cancers was identified in first passage synovial fibroblasts from two of nine rheumatoid arthritis patients and confirmed by restriction site mapping. BRAF-specific siRNA inhibited proliferation of synovial fibroblasts with V600R mutations. A BRAF aberrant splice variant with an intact kinase domain and partial loss of the N-terminal autoinhibitory domain was identified in fibroblasts from an additional patient, and fibroblast proliferation was inhibited by BRAF-specific siRNA. Our finding is the first to establish mechanisms for fibroblast transformation responsible for destruction of articular cartilage and bone in rheumatoid arthritis and establishes a new target for therapeutic intervention.

mTORC2-mediated direct phosphorylation regulates YAP activity promoting glioblastoma growth and invasive characteristics.

The Hippo and mTOR signaling cascades are major regulators of cell growth and division. Aberrant regulation of these pathways has been demonstrated to contribute to gliomagenesis and result in enhanced glioblastoma proliferation and invasive characteristics. Several crosstalk mechanisms have been described between these two pathways, although a complete picture of these signaling interactions is lacking and is required for effective therapeutic targeting. Here we report the ability of mTORC2 to directly phosphorylate YAP at serine 436 (Ser436) positively regulating YAP activity. We show that mTORC2 activity enhances YAP transcriptional activity and the induction of YAP-dependent target gene expression while its ablation via genetic or pharmacological means has the opposite affects on YAP function. mTORC2 interacts with YAP via Sin1 and mutational analysis of serine 436 demonstrates that this phosphorylation event affects several properties of YAP leading to enhanced transactivation potential. Moreover, YAP serine 436 mutants display altered glioblastoma growth, migratory capacity and invasiveness both in vitro and in xenograft experiments. We further demonstrate that mTORC2 is able to regulate a Hippo pathway resistant allele of YAP suggesting that mTORC2 can regulate YAP independent of Hippo signaling. Correlative associations between the expression of these components in GBM patient samples also supported the presence of this signaling relationship. These results advance a direct mTORC2/YAP signaling axis driving GBM growth, motility and invasiveness.

Recombinant Fv-Hsp70 protein mediates neuroprotection after focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: This study investigated the effects of intravenous recombinant Fv-Hsp70 protein on infarction volume and behavior after experimental ischemic stroke. METHODS: Focal cerebral ischemia was produced by occluding the middle cerebral artery using the intraluminal suture technique. Rats subjected to 2 hours of focal ischemia were allowed to survive 24 hours. At 2(1/4) hours and 3 hours after onset of ischemia, Fv-Hsp70 recombinant protein (0.5 mg/kg) or saline was injected through the tail vein. Sensorimotor function and infarction volume were assessed at 24 hours after ischemia. RESULTS: Administration of Fv-Hsp70 after focal cerebral ischemia significantly decreased infarct volume by 68% and significantly improved sensorimotor function compared with the saline-treated control group. Western blots showed Fv-Hsp70 in ischemic but not in control brain; and Fv-Hsp70 suppressed endogenous Hsp70. CONCLUSIONS: Fv-Hsp70 protected the ischemic brain in this experimental stroke model.

Antibody-mediated p53 protein therapy prevents liver metastasis in vivo.

To evaluate the clinical efficacy of monoclonal antibody (mAb) 3E10 Fv antibody-mediated p53 protein therapy, an Fv-p53 fusion protein produced in Pichia pastoris was tested on CT26.CL25 colon cancer cells in vitro and in vivo in a mouse model of colon cancer metastasis to the liver. In vitro experiments showed killing of CT26.CL25 cells by Fv-p53 but not Fv or p53 alone, and immunohistochemical staining confirmed that Fv was required for transport of p53 into cells. Prevention of liver metastasis in vivo was tested by splenic injection of 100 nmol/L Fv-p53 10 min and 1 week after injection of CT26.CL25 cancer cells into the portal vein of BALB/c mice. Mice were sacrificed 1 week after the second injection of Fv-p53 and assigned a quantitative metastasis score. Control mice had an average metastasis score of 3.3 +/- 1.3, whereas mice treated with Fv-p53 had an average metastasis score of 0.8 +/- 0.4 (P = 0.004). These results indicate that Fv-p53 treatment had a profound effect on liver metastasis and represent the first demonstration of effective full-length p53 protein therapy in vivo. mAb 3E10 Fv has significant clinical potential as a mediator of intracellular and intranuclear delivery of p53 for prevention and treatment of cancer metastasis.

Hsp70 associates with Rictor and is required for mTORC2 formation and activity.

mTORC2 is a multiprotein kinase composed of mTOR, mLST8, PRR5, mSIN1 and Rictor. The complex is insensitive to rapamycin and has demonstrated functions controlling cell growth, motility, invasion and cytoskeletal assembly. mTORC2 is the major hydrophobic domain kinase which renders Akt fully active via phosphorylation on serine 473. We isolated Hsp70 as a putative Rictor interacting protein in a yeast two-hybrid assay and confirmed this interaction via co-immunoprecipitation and colocalization experiments. In cells expressing an antisense RNA targeting Hsp70, mTORC2 formation and activity were impaired. Moreover, in cells lacking Hsp70 expression, mTORC2 activity was inhibited following heat shock while controls demonstrated increased mTORC2 activity. These differential effects on mTORC2 activity were specific, in that mTORC1 did not demonstrate Hsp70-dependent alterations under these conditions. These data suggest that Hsp70 is a component of mTORC2 and is required for proper assembly and activity of the kinase both constitutively and following heat shock.

Antibody-mediated Hsp70 protein therapy.

Intracellular Hsp70 provides cytoprotection against a variety of stressful stimuli, and an effective means of increasing intracellular Hsp70 levels could prove beneficial in the prevention and treatment of a variety of human diseases. A novel protein transduction domain consisting of the single chain Fv fragment of an anti-DNA antibody known to penetrate into living cells and tissues, mAb 3E10, has recently been used to deliver functional proteins to cells. The ability of the single chain Fv fragment to deliver Hsp70 into living cells was tested by generating an Fv-Hsp70 fusion protein. Fv-Hsp70 was produced as a secreted protein in both COS-7 cells and the methylotropic yeast strain Pichia pastoris and was shown capable of penetrating into COS-7 cells and primary rat cortical neurons. Pre-treatment with Fv-Hsp70 protected both COS-7 cells and primary rat cortical neurons against subsequent exposure to hydrogen peroxide. These results provide the first evidence that the Fv fragment of mAb 3E10 is capable of delivering proteins to neurons and indicate its potential in the development of Hsp70 protein therapy.

An intracellular delivery vehicle for protein transduction of micro-dystrophin.

The Fv fragment of an antibody that selectively targets and penetrates skeletal muscle in vivo was produced as a fusion protein with a micro-dystrophin for use as a delivery vehicle to transport micro-dystrophin into muscle cells. Fv-micro-dystrophin was produced as a secreted protein by transient transfection of Fv-micro-dystrophin cDNA in COS-7 cells and as a non-secreted protein by permanent transfection in Pichia pastoris. Isolated Fv-micro-dystrophin was shown to be full-length by Western blot analysis. Fv-micro-dystrophin penetrated multiple cell lines in vitro, and it localized to the plasma membrane of a cell line with membrane beta-dystroglycan. In the absence of membrane beta-dystroglycan, it localized to the cytoplasm. Antibody-mediated transduction of micro-dystrophin into muscle cells is a potential therapy for dystrophin-deficient muscular dystrophies.

Antibody mediated transduction of therapeutic proteins into living cells.

Protein therapy refers to the direct delivery of therapeutic proteins to cells and tissues with the goal of ameliorating or modifying a disease process. Current techniques for delivering proteins across cell membranes include taking advantage of receptor-mediated endocytosis or using protein transduction domains that penetrate directly into cells. The most commonly used protein transduction domains are small cell-penetrating peptides derived from such proteins as the HIV-1 Tat protein. A novel protein transduction domain developed as the single chain fragment (Fv) of a murine anti-DNA autoantibody, mAb 3E10, has recently been developed and used to deliver biologically active proteins to living cells in vitro. This review will provide a brief overview of the development of the Fv fragment and provide a summary of recent studies using Fv to deliver therapeutic peptides and proteins (such as a C-terminal p53 peptide, C-terminal p53 antibody fragment, full-length p53, and micro-dystrophin) to cells.

DNA-dependent targeting of cell nuclei by a lupus autoantibody.

A nuclear-penetrating lupus anti-DNA autoantibody, 3E10, has been found to inhibit DNA repair and selectively kill certain cancer cells that are highly vulnerable to DNA damage. In addition, a 3E10 single chain variable fragment (scFv) has been developed for use as a delivery vehicle to carry therapeutic cargo proteins into cell nuclei. A greater understanding of the mechanism by which 3E10 penetrates cell nuclei is needed to help determine the scope of its potential therapeutic applications. Here we show that the presence of extracellular DNA significantly enhances the nuclear uptake of 3E10 scFv. In addition, we find that 3E10 scFv preferentially localizes into tumor cell nuclei in vivo, likely due to increased DNA in the local environment released from ischemic and necrotic regions of tumor. These data provide insight into the mechanism of nuclear penetration by 3E10 and demonstrate the potential for use of 3E10 in therapeutic approaches to diseases ranging from malignancy to ischemic conditions such as stroke.

Antibody-mediated transduction of p53 selectively kills cancer cells.

Some human cancers are caused by functional defects in p53 that are restored by gene therapy with wild-type p53. To circumvent the use of viral vectors, we reconstituted cancer cell lines with p53 by protein transduction. A fusion protein was produced from cDNA constructed from the Fv fragment of an antibody that penetrates living cells and wild-type p53 (Fv-p53). Fv-p53 penetrated and killed cancer cells that do not express p53. Additionally, Fv-p53 killed cancer cells that were malignant as a result of mutations within p53, nuclear exclusion of p53 and over-expression of MDM2. Non-specific toxicity was excluded by showing that Fv-p53 penetrated but did not kill primary cells and cancer cells unresponsive to p53. Fv fragments alone were not cytotoxic, indicating that killing was due to transduction of p53. Fv-p53 was shown to penetrate cancer cells engrafted in vivo. These results support continued efforts to evaluate the potential efficacy of Fv-p53 for the treatment of certain cancers in vivo.

BRAF splice variants in rheumatoid arthritis synovial fibroblasts activate MAPK through CRAF.

Rheumatoid arthritis (RA) is a destructive polyarthritis in which synovial-like fibroblasts (SFs) invade and erode cartilage by expressing membrane-anchored type 1 matrix metalloproteinase (MT1-MMP). The mitogen activated protein kinase (MAPK) pathway is activated in RA SFs, but the mechanism of activation is unknown. Here we identify aberrant BRAF splice variants with deletions in both the kinase domain and RAS-binding domain (RBD) in SFs from the majority of RA patients and show that these BRAF splice variants constitutively activate MAPK through CRAF, increase expression of MT1-MMP, and enhance fibroblast invasion of collagen.

A cell-penetrating bispecific antibody for therapeutic regulation of intracellular targets.

The therapeutic use of antibodies is restricted by the limited access of antibodies to intracellular compartments. To overcome this limitation, we developed a cell-penetrating monoclonal antibody, mAb 3E10, as an intracellular delivery vehicle for the intracellular and intranuclear delivery of antibodies constructed as bispecific single-chain Fv fragments. Because MDM2 is an important target in cancer therapy, we selected monoclonal antibody (mAb) 3G5 for intracellular transport. mAb 3G5 binds MDM2 and blocks binding of MDM2 to p53. Here, we show that the resulting 3E10-3G5 bispecific antibody retains cell-penetrating and MDM2-binding activity, increases tumor p53 levels, and inhibits growth of MDM2-addicted tumors. The use of cell-penetrating bispecific antibodies in targeted molecular therapy will significantly broaden the spectrum of accessible intracellular targets and may have a profound impact in cancer therapy.