Repurposing an Antiepileptic Drug for the Treatment of Glioblastoma.

PURPOSE: Glioblastoma multiforme (GBM) is a grade IV, highly proliferative, and malignant form of brain tumor with a 5-year survival rate at ~ 5%. Current treatment strategies for GBM include surgery, radiation, and chemotherapy. Major challenges in GBM management include difficulties in surgical resection due to brain's vital functions and GBM metastasis, development of resistance to temozolomide (TMZ), and protection of tumor by blood brain barrier (BBB). Therefore, we aimed to discover a novel therapeutic for GBM by targeting its metabolic reprogramming. METHOD: We screened metabolic inhibitors by their effects on GBM cell viability by MTT assay. We discovered an FDA-approved drug stiripentol (STP) in our screening of metabolic inhibitors in GBM cells. STP is used for Dravet syndrome (a rare epilepsy). We further tested efficacy of STP using proliferation assay, clonogenic assay, in vitro migration assay, cell cycle assay, apoptosis assay, and in U87 3D spheroids. We also tested the toxicity of STP, and combinations used in the study on normal human dermal fibroblasts. RESULTS: STP was effective in decreasing GBM cell viability, proliferation, clonogenic ability, and migration. Moreover, cell cycle changes were involved but robust apoptosis was absent in STP's anticancer effects. STP was effective in 3D spheroid models, and in TMZ-resistant cells. STP showed additive or synergistic effect with TMZ in different anticancer assays on GBM cells and was considerably less toxic in normal cells. CONCLUSION: Our results indicate that STP can be an effective GBM therapeutic that enhances the effects of TMZ on GBM cells. Importantly, STP reduced viability of TMZ-resistant cells. Our results warrant further studies in the mechanistic basis of STP's effects on GBM cells and the preclinical potential of STP in animal models.

CXCL12 regulates platelet activation via the regulator of G-protein signaling 16.

The regulators of G protein signaling (RGS) protein superfamily negatively controls G protein-coupled receptor signal transduction pathways. One of the members of this family, RGS16, is highly expressed in megakaryocytes and platelets. Studies of its function in platelet and megakaryocyte biology have been limited, in part, due to lack of pharmacological inhibitors. For example, RGS16 overexpression inhibited CXC chemokine receptor 4 (CXCR4)-mediated megakaryocyte migration. More recent studies showed that the chemokine stromal cell-derived factor (SDF1α or CXCL12) regulates platelet function via CXCR4. Based on these considerations, the present study investigated the capacity of RGS16 to regulate CXCL12-dependent platelet function, using the RGS16 knockout mouse model (Rgs16(-/-)). RGS16-deficient platelets had increased protease activated receptor 4 and collagen-induced aggregation, as well as increased CXCL12-dependent agonist-induced aggregation, dense and alpha granule secretion, integrin αIIbß3 activation and phosphatidylserine exposure compared to those from WT littermates. CXCL12 alone did not stimulate aggregation or secretion in either RGS16-deficient or WT platelets. Furthermore, platelets from Rgs16(-/-) mice displayed enhanced phosphorylation of ERK and Akt following CXCL12 stimulation relative to controls. Finally, we also found that PKCδ is involved in regulating CXCL12-dependent activation of ERK and Akt, in the Rgs16-deficient platelets. Collectively, our findings provide the first evidence that RGS16 plays an important role in platelet function by modulating CXCL12-dependent platelet activation.

Characterization of a novel function-blocking antibody targeted against the platelet P2Y1 receptor.

OBJECTIVE: Platelet hyperactivity is associated with vascular disease and contributes to the genesis of thrombotic disorders. ADP plays an important role in platelet activation and activates platelets through 2 G-protein-coupled receptors, the Gq-coupled P2Y1 receptor (P2Y1R), and the Gi-coupled P2Y12 receptor. Although the involvement of the P2Y1R in thrombogenesis is well established, there are no antagonists that are currently available for clinical use. APPROACH AND RESULTS: Our goal is to determine whether a novel antibody targeting the ligand-binding domain, ie, second extracellular loop (EL2) of the P2Y1R (EL2Ab) could inhibit platelet function and protect against thrombogenesis. Our results revealed that the EL2Ab does indeed inhibit ADP-induced platelet aggregation, in a dose-dependent manner. Furthermore, EL2Ab was found to inhibit integrin GPIIb-IIIa activation, dense and α granule secretion, and phosphatidylserine exposure. These inhibitory effects translated into protection against thrombus formation, as evident by a prolonged time for occlusion in a FeCl3-induced thrombosis model, but this was accompanied by a prolonged tail bleeding time. We also observed a dose-dependent displacement of the radiolabeled P2Y1R antagonist [(3)H]MRS2500 from its ligand-binding site by EL2Ab. CONCLUSIONS: Collectively, our findings demonstrate that EL2Ab binds to and exhibits P2Y1R-dependent function-blocking activity in the context of platelets. These results add further evidence for a role of the P2Y1R in thrombosis and validate the concept that targeting it is a relevant alternative or complement to current antiplatelet strategies.

The regulator of G-protein signaling 18 regulates platelet aggregation, hemostasis and thrombosis.

Regulators of G protein signaling (RGS) proteins are known to interact with and negatively regulate/turn-off G protein activation. RGS18 is identified as an R4 subfamily member of this family with specific expression in hematopoietic progenitors, myeloerythroid cells, megakaryocytes and platelets. Studies focused on understanding its function in platelet biology have been limited, in part, due to lack of pharmacological inhibitors. Thus, the present study investigated the function of RGS18 in platelets, using the RGS18 knockout mouse model (RGS18(-/-)). We identified phenotypic differences between RGS18(-/-) and wild-type (WT) mice, and show that RGS18 plays a significant role in hemostasis and thrombosis. Hence, RGS18 deficiency markedly shortened bleeding as well as occlusion times (in vivo). Furthermore, RGS18(-/-) platelets displayed hyper-responsiveness with regards to agonist induced aggregation (in vitro). This gain of function phenotype may serve as the mechanism or explain, at least in part, the enhanced hemostasis and thrombosis phenotype observed in the RGS18 deletion mice. Collectively, our findings provide valuable insight and highlight a critical and direct role for RGS18 in modulating platelet function.

Arachidonic acid-derived signaling lipids and functions in impaired healing.

Very little is known about lipid function during wound healing, and much less during impaired healing. Such understanding will help identify what roles lipid signaling plays in the development of impaired/chronic wounds. We took a lipidomics approach to study the alterations in lipid profile in the LIGHT(-/-) mouse model of impaired healing which has characteristics that resemble those of impaired/chronic wounds in humans, including high levels of oxidative stress, excess inflammation, increased extracellular matrix degradation and blood vessels with fibrin cuffs. The latter suggests excess coagulation and potentially increased platelet aggregation. We show here that in these impaired wounds there is an imbalance in the arachidonic acid (AA) derived eicosonoids that mediate or modulate inflammatory reactions and platelet aggregation. In the LIGHT(-/-) impaired wounds there is a significant increase in enzymatically derived breakdown products of AA. We found that early after injury there was a significant increase in the eicosanoids 11-, 12-, and 15-hydroxyeicosa-tetranoic acid, and the proinflammatory leukotrienes (LTD4 and LTE) and prostaglandins (PGE2 and PGF2α ). Some of these eicosanoids also promote platelet aggregation. This led us to examine the levels of other eicosanoids known to be involved in the latter process. We found that thromboxane (TXA2 /B2 ), and prostacyclins 6kPGF1α are elevated shortly after wounding and in some cases during healing. To determine whether they have an impact in platelet aggregation and hemostasis, we tested LIGHT(-/-) mouse wounds for these two parameters and found that, indeed, platelet aggregation and hemostasis are enhanced in these mice when compared with the control C57BL/6 mice. Understanding lipid signaling in impaired wounds can potentially lead to development of new therapeutics or in using existing nonsteroidal anti-inflammatory agents to help correct the course of healing.

Third-hand Smoke: Impact on Hemostasis and Thrombogenesis.

Cigarette smoking is a major risk factor for acute coronary thrombosis. In fact, both active/first-hand smoke and passive/second-hand smoke exposure are known to increase the risk of coronary thrombosis. Although recently a new risk has been identified and termed third-hand smoke (THS), which is the residual tobacco smoke contaminant that remains after a cigarette is extinguished, it remains to be determined whether it can also enhance the risk of thrombogenesis, much like first-hand smoke and second-hand smoke. Therefore, the present studies investigated the impact of THS exposure in the context of platelet biology and related disease states. It was found that THS-exposed mice exhibited an enhanced platelet aggregation and secretion responses as well as enhanced integrin GPIIb-IIIa activation. Furthermore, it was found that THS exposure shortens the tail bleeding time and the occlusion time in a model of thrombosis. Thus, our data demonstrate for the first time (at least in mice) that THS exposure increases the risk of thrombosis-based disease states, which is attributed, at least in part, to their hyperactive platelets.

Novel gene therapy for drug-resistant melanoma: Synergistic combination of PTEN plasmid and BRD4 PROTAC-loaded lipid nanocarriers.

Patients suffering from BRAF mutant melanoma have tumor recurrence within merely 7 months of treatment with a potent BRAF inhibitor (BRAFi) like vemurafenib. It has been proven that diverse molecular pathways driving BRAFi resistance converge to activation of c-Myc in melanoma. Therefore, we identified a novel combinatorial therapeutic strategy by targeting loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor gene and upregulated BRD4 oncoprotein as Myc-dependent vulnerabilities of drug-resistant melanoma. Being promising therapeutic targets, we decided to concomitantly deliver PTEN plasmid and BRD4 targeted PROteolysis-TArgeting Chimera (ARV) to drug the "undruggable" c-Myc in BRAFi-resistant melanoma. Since PTEN plasmid and ARV are distinct in their physicochemical properties, we fabricated PTEN-plasmid loaded lipid nanoparticles (PL-NANO) and ARV-825-loaded nanoliposomes (AL-NANO) to yield a mean particle size of less than 100 nm and greater than 99% encapsulation efficiency for each therapeutic payload. Combination of PL-NANO and AL-NANO displayed synergistic tumor growth inhibition and substantial apoptosis in in vitro two-dimensional and three-dimensional models. Importantly, simultaneous delivery of PL-NANO and AL-NANO achieved significant upregulation of PTEN expression levels and degradation of BRD4 protein to ultimately downregulate c-Myc levels in BRAFi-resistant melanoma cells. Altogether, lipid nanocarriers delivering this novel lethal cocktail stands as one-of-a-kind gene therapy to target undruggable c-Myc oncogene in BRAFi-resistant melanoma.

The effect of hormones insulin and glucagon on ubiquitin modifications elucidated by proteomics in liver cells.

AIMS: Insulin action is intertwined with changing levels of glucose and counter-regulatory hormone glucagon. While insulin lowers blood sugar level, glucagon raises it by promoting the breakdown of the stored glycogen in liver and releases glucose into the bloodstream. The hormones insulin and glucagon are key in the pathogenesis of type 2 diabetes (T2D). Insulin resistance is a primary predisposing factor for diabetes. Phosphorylation of insulin signaling molecules is altered in the insulin-resistant state. However, ubiquitin (Ub) modifications in insulin-resistant state are relatively understudied. To dissect the underlying mechanisms, we performed a proteomics study on hepatoma cells to study the regulation of ubiquitination by insulin and glucagon. MATERIALS AND METHODS: We performed western blotting, immunoprecipitations, and affinity pull down using tandem Ub binding entities (TUBE) reagents on hepatoma cells treated with insulin or glucagon. Next, we performed MS/MS analysis on Ub-linkage specific affinity pull down samples. Gene ontology analysis and protein-protein interaction network analysis was performed using DAVID GO and STRING db, respectively. KEY FINDINGS: The ubiquitination pattern of total Ub, K48-linked Ub, and K63-linked Ub was altered with the treatment of hormones insulin and glucagon. Ubiquitination in immunoprecipitated samples showed enrichment with total Ub and K48-linked Ub but not with K63-linked Ub. Ubiquitination by treatment with hormones mainly enriched key signaling pathways MAPK, Akt, oxidative stress etc. SIGNIFICANCE: Our study identified key altered proteins and signal transduction pathways which aids in understanding the mechanisms of hormonal action on ubiquitination and identify new therapeutic targets for T2D.

Galactose-decorated liver tumor-specific nanoliposomes incorporating selective BRD4-targeted PROTAC for hepatocellular carcinoma therapy.

This research deals with the development of asialoglycoprotein receptors (ASGPR) directed nanoliposomes incorporating a novel BRD4 (Bromodomain-containing protein 4) protein-targeted PROTAC (Proteolysis Targeting Chimera), ARV-825 (ARV) (GALARV), and to investigate the anticancer efficacy of GALARV for specific delivery in hepatocellular carcinoma. GALARV were prepared using the modified hydration method and characterized for their physicochemical properties as well as anticancer activity using 2D and 3D cell culture models. ARV and GALARV (93.83 ± 10.05 nm) showed significant in vitro cytotoxicity and apoptosis in hepatocellular carcinoma cells. GALARV also demonstrated a substantially higher intracellular concentration of ARV compared to non-targeted nanoliposomes (∼3 fold) and ARV alone (∼4.5 fold), showed good physical stability and negligible hemolysis. Immunoblotting results depicted substantial downregulation of target BRD4 protein, oncogenic c-Myc, apoptotic Bcl-2, and survivin proteins. Notably, GALARV treatment resulted in significant apoptosis and subsequent inhibition of the cell viability of 3D tumor spheroids of hepatocellular carcinoma. These results suggest that GALARV is a novel actively targeted PROTAC-based nanotherapeutic approach for hepatocellular carcinoma.

A novel gene therapy for neurodegenerative Lafora disease via EPM2A-loaded DLinDMA lipoplexes.

Aim: To develop novel cationic liposomes as a nonviral gene delivery vector for the treatment of rare diseases, such as Lafora disease - a neurodegenerative epilepsy. Materials & methods: DLinDMA and DOTAP liposomes were formulated and characterized for the delivery of gene encoding laforin and expression of functional protein in HEK293 and neuroblastoma cells. Results: Liposomes with cationic lipids DLinDMA and DOTAP showed good physicochemical characteristics. Nanosized DLinDMA liposomes demonstrated desired transfection efficiency, negligible hemolysis and minimal cytotoxicity. Western blotting confirmed successful expression and glucan phosphatase assay demonstrated the biological activity of laforin. Conclusion: Our study is a novel preclinical effort in formulating cationic lipoplexes containing plasmid DNA for the therapy of rare genetic diseases such as Lafora disease.

A critical role for the transient receptor potential channel type 6 in human platelet activation.

While calcium signaling is known to play vital roles in platelet function, the mechanisms underlying its receptor-operated calcium entry component (ROCE) remain poorly understood. It has been proposed, but never proven in platelets, that the canonical transient receptor potential channel-6 (TRPC6) mediates ROCE. Nonetheless, we have previously shown that the mouse TRPC6 regulates hemostasis, thrombogenesis by regulating platelet aggregation. In the present studies, we used a pharmacological approach to characterize the role of TRPC6 in human platelet biology. Thus, interestingly, we observed that a TRPC6 inhibitor exerted significant inhibitory effects on human platelet aggregation in a thromboxane receptor (TPR)-selective manner; no additional inhibition was observed in the presence of the calcium chelator BAPTA. This inhibitor also significantly inhibited human platelet secretion (dense and alpha granules), integrin IIb-IIIa, Akt and ERK phosphorylation, again, in a TPR-selective manner; no effects were observed in response to ADP receptor stimulation. Furthermore, there was a causal relationship between these inhibitory effects, and the capacity of the TRPC6 inhibitor to abrogate elevation in intracellular calcium, that was again found to be TPR-specific. This effect was not found to be due to antagonism of TPR, as the TRPC6 inhibitor did not displace the radiolabeled antagonist [3H]SQ29,548 from its binding sites. Finally, our studies also revealed that TRPC6 regulates human clot retraction, as well as physiological hemostasis and thrombus formation, in mice. Taken together, our findings demonstrate, for the first time, that TRPC6 directly regulates TPR-dependent ROCE and platelet function. Moreover, these data highlight TRPC6 as a novel promising therapeutic strategy for managing thrombotic disorders.

MALT1-ubiquitination triggers non-genomic NF-κB/IKK signaling upon platelet activation.

We have recently shown that IKK complex plays an important non-genomic role in platelet function, i.e., regulates SNARE machinery-dependent membrane fusion. In this connection, it is well known that MALT1, whose activity is modulated by proteasome, plays an important role in the regulation of IKK complex. Therefore, the present studies investigated the mechanism by which IKK signaling is regulated in the context of the platelet proteasome. It was found that platelets express a functional proteasome, and form CARMA/MALT1/Bcl10 (CBM) complex when activated. Using a pharmacological inhibitor, the proteasome was found to regulate platelet function (aggregation, integrin activation, secretion, phosphatidylserine exposure and changes in intracellular calcium). It was also found to regulate thrombogenesis and physiologic hemostasis. We also observed, upon platelet activation, that MALT1 is ubiquitinated, and this coincides with the activation of the IKK/NF-κB-signaling pathway. Finally, we observed that the proteasome inhibitor blocks CBM complex formation and the interaction of IKKγ and MALT1; abrogates SNARE formation, and the association of MALT1 with TAK1 and TAB2, which are upstream of the CBM complex. Thus, our data demonstrate that MALT1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing platelet signals to the NF-κB pathway.

The antidepressant 5-HT2A receptor antagonists pizotifen and cyproheptadine inhibit serotonin-enhanced platelet function.

There is considerable interest in defining new agents or targets for antithrombotic purposes. The 5-HT2A receptor is a G-protein coupled receptor (GPCR) expressed on many cell types, and a known therapeutic target for many disease states. This serotonin receptor is also known to regulate platelet function. Thus, in our FDA-approved drug repurposing efforts, we investigated the antiplatelet activity of cyproheptadine and pizotifen, two antidepressant 5-HT2A Receptor antagonists. Our results revealed that cyproheptadine and pizotifen reversed serotonin-enhanced ADP-induced platelet aggregation in vitro and ex vivo. And the inhibitory effects of these two agents were found to be similar to that of EMD 281014, a 5-HT2A Receptor antagonist under development. In separate experiments, our studies revealed that these 5-HT2A receptor antagonists have the capacity to reduce serotonin-enhanced ADP-induced elevation in intracellular calcium levels and tyrosine phosphorylation. Using flow cytometry, we also observed that cyproheptadine, pizotifen, and EMD 281014 inhibited serotonin-enhanced ADP-induced phosphatidylserine (PS) exposure, P-selectin expression, and glycoprotein IIb-IIIa activation. Furthermore, using a carotid artery thrombosis model, these agents prolonged the time for thrombotic occlusion in mice in vivo. Finally, the tail-bleeding time was investigated to assess the effect of cyproheptadine and pizotifen on hemostasis. Our findings indicated prolonged bleeding time in both cyproheptadine- and pizotifen-treated mice. Notably, the increases in occlusion and bleeding times associated with these two agents were comparable to that of EMD 281014, and to clopidogrel, a commonly used antiplatelet drug, again, in a fashion comparable to clopidogrel and EMD 281014. Collectively, our data indicate that the antidepressant 5-HT2A antagonists, cyproheptadine and pizotifen do exert antiplatelet and thromboprotective effects, but similar to clopidogrel and EMD 281014, their use may interfere with normal hemostasis.