miR-18a Inhibits BMP4 and HIF-1α Normalizing Brain Arteriovenous Malformations.

RATIONALE: Brain arteriovenous malformations (AVMs) are abnormal tangles of vessels where arteries and veins directly connect without intervening capillary nets, increasing the risk of intracerebral hemorrhage and stroke. Current treatments are highly invasive and often not feasible. Thus, effective noninvasive treatments are needed. We previously showed that AVM-brain endothelial cells (BECs) secreted higher VEGF (vascular endothelial growth factor) and lower TSP-1 (thrombospondin-1) levels than control BEC; and that microRNA-18a (miR-18a) normalized AVM-BEC function and phenotype, although its mechanism remained unclear. OBJECTIVE: To elucidate the mechanism of action and potential clinical application of miR-18a as an effective noninvasive treatment to selectively restore the phenotype and functionality of AVM vasculature. METHODS AND RESULTS: The molecular pathways affected by miR-18a in patient-derived BECs and AVM-BECs were determined by Western blot, RT-qPCR (quantitative reverse transcription polymerase chain reaction), ELISA, co-IP, immunostaining, knockdown and overexpression studies, flow cytometry, and luciferase reporter assays. miR-18a was shown to increase TSP-1 and decrease VEGF by reducing PAI-1 (plasminogen activator inhibitor-1/SERPINE1) levels. Furthermore, miR-18a decreased the expression of BMP4 (bone morphogenetic protein 4) and HIF-1α (hypoxia-inducible factor 1α), blocking the BMP4/ALK (activin-like kinase) 2/ALK1/ALK5 and Notch signaling pathways. As determined by Boyden chamber assays, miR-18a also reduced the abnormal AVM-BEC invasiveness, which correlated with a decrease in MMP2 (matrix metalloproteinase 2), MMP9, and ADAM10 (ADAM metallopeptidase domain 10) levels. In vivo pharmacokinetic studies showed that miR-18a reaches the brain following intravenous and intranasal administration. Intranasal co-delivery of miR-18a and NEO100, a good manufacturing practices-quality form of perillyl alcohol, improved the pharmacokinetic profile of miR-18a in the brain without affecting its pharmacological properties. Ultra-high-resolution computed tomography angiography and immunostaining studies in an Mgp-/- AVM mouse model showed that miR-18a decreased abnormal cerebral vasculature and restored the functionality of the bone marrow, lungs, spleen, and liver. CONCLUSIONS: miR-18a may have significant clinical value in preventing, reducing, and potentially reversing AVM.

Blocking Ras inhibition as an antitumor strategy.

Ras proteins are among the most frequently mutated drivers in human cancer and remain an elusive pharmaceutical targeting. Previous studies have improved the understanding of Ras structure, processing, and signaling pathways in cancer cells and have opened new possibilities for inhibiting Ras function. In this review we discuss the most recent advances towards inhibiting Ras activity with small molecules, highlighting the two approaches: (i) compounds that bind directly to Ras protein and (ii) inhibitors of the enzymes involved in the post-translational modifications of Ras. In the former, we analyze the most recent contributions in each of the main classes of Ras direct binders, including the different types of nucleotide exchange inhibitors, allosteric compounds, and molecules that interfere with the interaction between Ras and its effectors. In the latter, we examine the compounds that inhibit Ras activation by blocking any of its post-translational modifications. Also, a special focus is made on those molecules that have progressed the farthest from medicinal chemistry and drug development points of view. Finally, the current scene regarding the clinical trials of Ras inhibitors, together with the future promising avenues for further development of the challenging Ras field are reviewed.

Development of a Nucleotide Exchange Inhibitor That Impairs Ras Oncogenic Signaling.

Despite more than three decades of intense effort, no anti-Ras therapies have reached clinical application. Contributing to this failure has been an underestimation of Ras complexity and a dearth of structural information. In this regard, recent studies have revealed the highly dynamic character of the Ras surface and the existence of transient pockets suitable for small-molecule binding, opening up new possibilities for the development of Ras modulators. Herein, a novel Ras inhibitor (compound 12) is described that selectively impairs mutated Ras activity in a reversible manner without significantly affecting wild-type Ras, reduces the Ras-guanosine triphosphate (GTP) levels, inhibits the activation of the mitogen-activated protein kinase (MAPK) pathway, and exhibits remarkable cytotoxic activity in Ras-driven cellular models. The use of molecular dynamics simulations and NMR spectroscopy experiments has enabled the molecular bases responsible for the interactions between compound 12 and Ras protein to be explored. The new Ras inhibitor binds partially to the GTP-binding region and extends into the adjacent hydrophobic pocket delimited by switch II. Hence, Ras inhibitor 12 could represent a new compound for the development of more efficacious drugs to target Ras-driven cancers; a currently unmet clinical need.

NEO100 enables brain delivery of blood‒brain barrier impermeable therapeutics.

BACKGROUND: Intracarotid injection of mannitol has been applied for decades to support brain entry of therapeutics that otherwise do not effectively cross the blood-brain barrier (BBB). However, the elaborate and high-risk nature of this procedure has kept its use restricted to well-equipped medical centers. We are developing a more straightforward approach to safely open the BBB, based on the intra-arterial (IA) injection of NEO100, a highly purified version of the natural monoterpene perillyl alcohol. METHODS: In vitro barrier permeability with NEO100 was evaluated by transepithelial/transendothelial electrical resistance and antibody diffusion assays. Its mechanism of action was studied by western blot, microarray analysis, and electron microscopy. In mouse models, we performed ultrasound-guided intracardiac administration of NEO100, followed by intravenous application of Evan's blue, methotrexate, checkpoint-inhibitory antibodies, or chimeric antigen receptor (CAR) T cells. RESULTS: NEO100 opened the BBB in a reversible and nontoxic fashion in vitro and in vivo. It enabled greatly increased brain entry of all tested therapeutics and was well tolerated by animals. Mechanistic studies revealed effects of NEO100 on different BBB transport pathways, along with translocation of tight junction proteins from the membrane to the cytoplasm in brain endothelial cells. CONCLUSION: We envision that this procedure can be translated to patients in the form of transfemoral arterial catheterization and cannulation to the cerebral arteries, which represents a low-risk procedure commonly used in a variety of clinical settings. Combined with NEO100, it is expected to provide a safe, widely available approach to enhance brain entry of any therapeutic.

Enhanced brain delivery and therapeutic activity of trastuzumab after blood-brain barrier opening by NEO100 in mouse models of brain-metastatic breast cancer.

BACKGROUND: The antitumor efficacy of human epidermal growth factor receptor 2 (HER2)-targeted therapies, such as humanized monoclonal antibody trastuzumab (Herceptin®, Roche), in patients with breast-to-brain cancer metastasis is hindered by the low permeability of the blood-brain barrier (BBB). NEO100 is a high-purity version of the natural monoterpene perillyl alcohol, produced under current good manufacturing practice (cGMP) regulations, that was shown previously to reversibly open the BBB in rodent models. Here we investigated whether NEO100 could enable brain entry of trastuzumab to achieve greater therapeutic activity. METHODS: An in vitro BBB, consisting of human astrocytes and brain endothelial cells, was used to determine trastuzumab penetration in the presence or absence of NEO100. For in vivo studies, we administered intravenous (IV) trastuzumab or the trastuzumab-drug conjugate ado-trastuzumab emtansine (T-DM1; Kadcyla®, Roche), to mouse models harboring intracranial HER2+ breast cancer, with or without BBB opening via IA NEO100. Brain and tumor tissues were examined for the presence of trastuzumab and infiltration of immune cells. Therapeutic impact was evaluated based on overall survival. RESULTS: NEO100 greatly increased trastuzumab penetration across an in vitro BBB. In vivo, IA NEO100-mediated BBB opening resulted in brain tumor-selective accumulation of trastuzumab, without detectable presence in normal brain tissue, along with increased presence of immune cell populations. IV delivery of trastuzumab or T-DM1 achieved significantly greater overall survival of tumor-bearing mice when combined with IA NEO100. CONCLUSION: IA NEO100 facilitates brain tumor entry of trastuzumab and T-DM1 and significantly enhances their therapeutic efficacy, along with increased antibody-dependent immune cell recruitment.

Isoprenylcysteine Carboxylmethyltransferase-Based Therapy for Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford progeria syndrome (HGPS, progeria) is a rare genetic disease characterized by premature aging and death in childhood for which there were no approved drugs for its treatment until last November, when lonafarnib obtained long-sought FDA approval. However, the benefits of lonafarnib in patients are limited, highlighting the need for new therapeutic strategies. Here, we validate the enzyme isoprenylcysteine carboxylmethyltransferase (ICMT) as a new therapeutic target for progeria with the development of a new series of potent inhibitors of this enzyme that exhibit an excellent antiprogeroid profile. Among them, compound UCM-13207 significantly improved the main hallmarks of progeria. Specifically, treatment of fibroblasts from progeroid mice with UCM-13207 delocalized progerin from the nuclear membrane, diminished its total protein levels, resulting in decreased DNA damage, and increased cellular viability. Importantly, these effects were also observed in patient-derived cells. Using the Lmna G609G/G609G progeroid mouse model, UCM-13207 showed an excellent in vivo efficacy by increasing body weight, enhancing grip strength, extending lifespan by 20%, and decreasing tissue senescence in multiple organs. Furthermore, UCM-13207 treatment led to an improvement of key cardiovascular hallmarks such as reduced progerin levels in aortic and endocardial tissue and increased number of vascular smooth muscle cells (VSMCs). The beneficial effects go well beyond the effects induced by other therapeutic strategies previously reported in the field, thus supporting the use of UCM-13207 as a new treatment for progeria.

Pharmacokinetic properties of the temozolomide perillyl alcohol conjugate (NEO212) in mice.

BACKGROUND: NEO212 is a novel small-molecule anticancer agent that was generated by covalent conjugation of the natural monoterpene perillyl alcohol (POH) to the alkylating agent temozolomide (TMZ). It is undergoing preclinical development as a therapeutic for brain-localized malignancies. The aim of this study was to characterize metabolism and pharmacokinetic (PK) properties of NEO212 in preclinical models. METHODS: We used mass spectrometry (MS) and modified high-performance liquid chromatography to identify and quantitate NEO212 and its metabolites in cultured glioblastoma cells, in mouse plasma, brain, and excreta after oral gavage. RESULTS: Our methods allowed identification and quantitation of NEO212, POH, TMZ, as well as primary metabolites 5-aminoimidazole-4-carboxamide (AIC) and perillic acid (PA). Intracellular concentrations of TMZ were greater after treatment of U251TR cells with NEO212 than after treatment with TMZ. The half-life of NEO212 in mouse plasma was 94 min. In mice harboring syngeneic GL261 brain tumors, the amount of NEO212 was greater in the tumor-bearing hemisphere than in the contralateral normal hemisphere. The brain:plasma ratio of NEO212 was greater than that of TMZ. Excretion of unaltered NEO212 was through feces, whereas its AIC metabolite was excreted via urine. CONCLUSIONS: NEO212 preferentially concentrates in brain tumor tissue over normal brain tissue, and compared to TMZ has a higher brain:plasma ratio, altogether revealing favorable features to encourage its further development as a brain-targeted therapeutic. Its breakdown into well-characterized, long-lived metabolites, in particular AIC and PA, will provide useful equivalents for PK studies during further drug development and clinical trials with NEO212.

NEO212, a conjugate of temozolomide and perillyl alcohol, blocks the endothelial-to-mesenchymal transition in tumor-associated brain endothelial cells in glioblastoma.

As the endothelial-to-mesenchymal transition (EndMT) supports the pro-angiogenic and invasive characteristics of glioblastoma multiforme (GBM), blocking this process would be a promising approach to inhibit tumor progression and recurrence. Here, we demonstrate that glioma stem cells (GSC) induce EndMT in brain endothelial cells (BEC). TGF-ß signaling is necessary, but not sufficient to induce this EndMT process. Cell-to-cell contact and the contribution of Notch signaling are also required. NEO212, a conjugate of temozolomide and perillyl alcohol, blocks EndMT induction and reverts the mesenchymal phenotype of tumor-associated BEC (TuBEC) by blocking TGF-ß and Notch pathways. Consequently, NEO212 reduces the invasiveness and pro-angiogenic properties associated with TuBEC, without affecting control BEC. Intracranial co-implantation of BEC and GSC in athymic mice showed that EndMT occurs in vivo, and can be blocked by NEO212, supporting the potential clinical value of NEO212 for the treatment of GBM.

Safety and Tolerability of c-MET Inhibitors in Cancer.

The role of aberrant hepatocyte growth factor receptor (c-MET, also known as tyrosine-protein kinase MET)/hepatocyte growth factor (HGF) signaling in cancer progression and invasion has been extensively studied. c-MET inhibitors have shown promising pre-clinical and early phase clinical trial anti-tumor activity in several tumor types, although results of most phase III trials with these agents have been negative. To date, two small molecule c-MET inhibitors, cabozantinib and crizotinib, have been approved by regulatory authorities for the treatment of selected cancer types, but several novel c-MET inhibitors (either monoclonal antibodies or small molecule c-MET tyrosine kinase inhibitors) and treatment combinations are currently under study in different settings. Here we provide an overview of the mechanism of action and rationale of c-MET inhibition in cancer, the efficacy of approved agents, and novel promising c-MET-inhibitors and novel targeted combination strategies under development in different cancer types, with a focus on the safety profile and tolerability of these compounds.

Inhibition of motility by NEO100 through the calpain-1/RhoA pathway.

OBJECTIVE: Glioblastoma (GBM) is the most aggressive type of brain tumor with a high rate of tumor recurrence, and it often develops resistance over time to current standard of care chemotherapy. Its highly invasive nature plays an essential role in tumor progression and recurrence. Glioma stem cells (GSCs) are a subpopulation of glioma cells highly resistant to treatments and are considered responsible for tumor recurrence. METHODS: Patient-derived populations of GSCs were analyzed by western blot, MTT, and cytoplasmic calcium labeling to determine the cytotoxicity of NEO100. High-performance liquid chromatography was used to evaluate the levels of NEO100 in the cell culture supernatants. The effects of the compound on GSC motility were studied using Boyden chamber migration, 3D spheroid migration and invasion assays, and an mRNA expression PCR array. A RhoA activation assay, western blot, and immunofluorescence techniques were employed to confirm the signaling pathways involved. Intracranial implantation of GSCs in athymic mice was used to evaluate the effects of NEO100 in vivo on tumor progression and overall survival. RESULTS: Here, the authors show how NEO100, a highly purified good manufacturing practices-quality form of perillyl alcohol, is cytotoxic for different subtypes of GSCs, regardless of the mechanisms of DNA repair present. At doses similar to the IC50 (half maximal inhibitory concentration) values, NEO100 induces ER stress and activates apoptotic pathways in all GSC populations tested. At subcytotoxic doses in the micromolar range, NEO100 blocks migration and invasion of GSCs. These results correlate with a decrease in calpain-1 expression and an increase in RhoA activation, leading to enhanced contractility of the GSCs. In addition, NEO100 blocks the activation of the kinases Src, p42/44 MAPK, Akt, and Stat3, all related to cell proliferation and migration. Intranasal administration of NEO100 in mice with GSC-derived intracranial tumors led to a decrease in tumor progression and a 32% increase in overall survival. Immunostaining studies showed that NEO100 induces apoptosis and reduces GSC invasion in vivo. CONCLUSIONS: NEO100 could have significant value targeting GSCs and could be used for GBM therapy as either monotherapy or a coadjuvant therapy during temozolomide rest cycles.

The Rolipram-Perillyl Alcohol Conjugate (NEO214) Is A Mediator of Cell Death through the Death Receptor Pathway.

Glioblastoma (GBM) is a highly aggressive primary brain tumor with a poor prognosis. Treatment with temozolomide, standard of care for gliomas, usually results in drug resistance and tumor recurrence. Therefore, there is a great need for drugs that target GBM. NEO214 was generated by covalently linking rolipram to perillyl alcohol (POH) via a carbamate bond to form the rolipram-perillyl alcohol conjugate. We show here that NEO214 is effective against both temozolomide-sensitive and temozolomide-resistant glioma cells. Furthermore, NEO214 is effective for different mechanisms of temozolomide resistance: overexpression of MGMT (O6-methylguanine methyl-transferase); deficiency in specific mismatch repair proteins; and overexpression of base excision repair (BER) proteins. NEO214-induced cytotoxicity involves apoptosis triggered by endoplasmic reticulum (ER) stress, as well as activating the Death Receptor 5 (DR5)/TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) pathway. In vitro studies show that glioma cells treated with NEO214 express DR5 and exhibit cell death in the presence of recombinant TRAIL, a growth factor constitutively produced by astrocytes. Our in vitro 3D coculture data show that induction of DR5 in glioma cells with NEO214 and TRAIL cause tumor cell death very effectively and specifically for glioma cells. In vivo studies show that NEO214 has antitumor efficacy in orthotropic syngeneic rodent tumor models. Furthermore, NEO214 has therapeutic potential especially for brain tumors because this drug can cross the blood-brain barrier (BBB), and is effective in the TRAIL-rich astrocyte microenvironment. NEO214 is a strong candidate for use in the treatment of GBMs.

A Potent Isoprenylcysteine Carboxylmethyltransferase (ICMT) Inhibitor Improves Survival in Ras-Driven Acute Myeloid Leukemia.

Blockade of Ras activity by inhibiting its post-translational methylation catalyzed by isoprenylcysteine carboxylmethyltransferase (ICMT) has been suggested as a promising antitumor strategy. However, the paucity of inhibitors has precluded the clinical validation of this approach. In this work we report a potent ICMT inhibitor, compound 3 [UCM-1336, IC50 = 2 µM], which is selective against the other enzymes involved in the post-translational modifications of Ras. Compound 3 significantly impairs the membrane association of the four Ras isoforms, leading to a decrease of Ras activity and to inhibition of Ras downstream signaling pathways. In addition, it induces cell death in a variety of Ras-mutated tumor cell lines and increases survival in an in vivo model of acute myeloid leukemia. Because ICMT inhibition impairs the activity of the four Ras isoforms regardless of its activating mutation, compound 3 surmounts many of the common limitations of available Ras inhibitors described so far. In addition, these results validate ICMT as a valuable target for the treatment of Ras-driven tumors.

NEO212 Inhibits Migration and Invasion of Glioma Stem Cells.

Glioblastoma multiforme is a malignant brain tumor noted for its extensive vascularity, aggressiveness, and highly invasive nature, suggesting that cell migration plays an important role in tumor progression. The poor prognosis in GBM is associated with a high rate of tumor recurrence, and resistance to the standard of care chemotherapy, temozolomide (TMZ). The novel compound NEO212, a conjugate of TMZ and perillyl alcohol (POH), has proven to be 10-fold more cytotoxic to glioma stem cells (GSC) than TMZ, and is active against TMZ-resistant tumor cells. In this study, we show that NEO212 decreases migration and invasion of primary cultures of patient-derived GSCs, in both mesenchymal USC02 and proneural USC04 populations. The mechanism by which NEO212 reduces migration and invasion appears to be independent of its DNA alkylating effects, which cause cytotoxicity during the first hours of treatment, and is associated with a decrease in the FAK/Src signaling pathway, an effect not exhibited by TMZ. NEO212 also decreases the production of matrix metalloproteinases MMP2 and MMP9, crucial for GSC invasion. Gene expression analysis of epithelial and mesenchymal markers suggests that NEO212 increases the expression of epithelial-like characteristics, suggesting a reversion of the epithelial-to-mesenchymal transition process. Furthermore, in an in vivo orthotopic glioma model, NEO212 decreases tumor progression by reducing invasion of GSCs, thereby increasing survival time of mice. These studies indicate that NEO212, in addition to cytotoxicity, can effectively reduce migration and invasion in GSCs, thus exhibiting significant clinical value in the reduction of invasion and malignant glioma progression. Mol Cancer Ther; 17(3); 625-37. ©2018 AACR.

Intratumoral delivery of bortezomib: impact on survival in an intracranial glioma tumor model.

OBJECTIVE Glioblastoma (GBM) is the most prevalent and the most aggressive of primary brain tumors. There is currently no effective treatment for this tumor. The proteasome inhibitor bortezomib is effective for a variety of tumors, but not for GBM. The authors' goal was to demonstrate that bortezomib can be effective in the orthotopic GBM murine model if the appropriate method of drug delivery is used. In this study the Alzet mini-osmotic pump was used to bring the drug directly to the tumor in the brain, circumventing the blood-brain barrier; thus making bortezomib an effective treatment for GBM. METHODS The 2 human glioma cell lines, U87 and U251, were labeled with luciferase and used in the subcutaneous and intracranial in vivo tumor models. Glioma cells were implanted subcutaneously into the right flank, or intracranially into the frontal cortex of athymic nude mice. Mice bearing intracranial glioma tumors were implanted with an Alzet mini-osmotic pump containing different doses of bortezomib. The Alzet pumps were introduced directly into the tumor bed in the brain. Survival was documented for mice with intracranial tumors. RESULTS Glioma cells were sensitive to bortezomib at nanomolar quantities in vitro. In the subcutaneous in vivo xenograft tumor model, bortezomib given intravenously was effective in reducing tumor progression. However, in the intracranial glioma model, bortezomib given systemically did not affect survival. By sharp contrast, animals treated with bortezomib intracranially at the tumor site exhibited significantly increased survival. CONCLUSIONS Bypassing the blood-brain barrier by using the osmotic pump resulted in an increase in the efficacy of bortezomib for the treatment of intracranial tumors. Thus, the intratumoral administration of bortezomib into the cranial cavity is an effective approach for glioma therapy.

New inhibitors of angiogenesis with antitumor activity in vivo.

Angiogenesis is a requirement for the sustained growth and proliferation of solid tumors, and the development of new compounds that induce a sustained inhibition of the proangiogenic signaling generated by tumor hypoxia still remains as an important unmet need. In this work, we describe a new antiangiogenic compound (22) that inhibits proangiogenic signaling under hypoxic conditions in breast cancer cells. Compound 22 blocks the MAPK pathway, impairs cellular migration under hypoxic conditions, and regulates a set of genes related to angiogenesis. These responses are mediated by HIF-1α, since the effects of compound 22 mostly disappear when its expression is knocked-down. Furthermore, administration of compound 22 in a xenograft model of breast cancer produced tumor growth reductions ranging from 46 to 55% in 38% of the treated animals without causing any toxic side effects. Importantly, in the responding tumors, a significant reduction in the number of blood vessels was observed, further supporting the mechanism of action of the compound. These findings provide a rationale for the development of new antiangiogenic compounds that could eventually lead to new drugs suitable for the treatment of some types of tumors either alone or in combination with other agents.