Michael Tobias

Distinct signaling mechanisms of mTORC1 and mTORC2 in glioblastoma multiforme: a tale of two complexes.

Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that functions via two multiprotein complexes, namely mTORC1 and mTORC2, each characterized by different binding partners that confer separate functions. mTORC1 function is tightly regulated by PI3-K/Akt and is sensitive to rapamycin. mTORC2 is sensitive to growth factors, not nutrients, and is associated with rapamycin-insensitivity. mTORC1 regulates protein synthesis and cell growth through downstream molecules: 4E-BP1 (also called EIF4E-BP1) and S6K. Also, mTORC2 is thought to modulate growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases such as Akt and SGK. Recent evidence has suggested that mTORC2 may play an important role in maintenance of normal as well as cancer cells by virtue of its association with ribosomes, which may be involved in metabolic regulation of the cell. Rapamycin (sirolimus) and its analogs known as rapalogues, such as RAD001 (everolimus) and CCI-779 (temsirolimus), suppress mTOR activity through an allosteric mechanism that acts at a distance from the ATP-catalytic binding site, and are considered incomplete inhibitors. Moreover, these compounds suppress mTORC1-mediated S6K activation, thereby blocking a negative feedback loop, leading to activation of mitogenic pathways promoting cell survival and growth. Consequently, mTOR is a suitable target of therapy in cancer treatments. However, neither of these complexes is fully inhibited by the allosteric inhibitor rapamycin or its analogs. In recent years, new pharmacologic agents have been developed which can inhibit these complexes via ATP-binding mechanism, or dual inhibition of the canonical PI3-K/Akt/mTOR signaling pathway. These compounds include WYE-354, KU-003679, PI-103, Torin1, and Torin2, which can target both complexes or serve as a dual inhibitor for PI3-K/mTOR. This investigation describes the mechanism of action of pharmacological agents that effectively target mTORC1 and mTORC2 resulting in suppression of growth, proliferation, and migration of tumor and cancer stem cells.

Targeting cancer stem cells in glioblastoma multiforme using mTOR inhibitors and the differentiating agent all-trans retinoic acid

Glioblastoma multiforme (GBM), the most aggressive primary brain tumor, portends a poor prognosis despite current treatment modalities. Recurrence of tumor growth is attributed to the presence of treatment-resistant cancer stem cells (CSCs). The targeting of these CSCs is therefore essential in the treatment of this disease. Mechanistic target of rapamycin (mTOR) forms two multiprotein complexes, mTORC1 and mTORC2, which regulate proliferation and migration, respectively. Aberrant function of mTOR has been shown to be present in GBM CSCs. All-trans retinoic acid (ATRA), a derivative of retinol, causes differentiation of CSCs as well as normal neural progenitor cells. The purpose of this investigation was to delineate the role of mTOR in CSC maintenance, and to establish the mechanism of targeting GBM CSCs using differentiating agents along with inhibitors of the mTOR pathways. The results demonstrated that ATRA caused differentiation of CSCs, as demonstrated by the loss of the stem cell marker Nestin. These observations were confirmed by western blotting, which demonstrated a time-dependent decrease in Nestin expression following ATRA treatment. This effect occurred despite combination with mTOR (rapamycin), PI3K (LY294002) and MEK1/2 (U0126) inhibitors. Expression of activated extracellular signal-regulated kinase 1/2 (pERK1/2) was enhanced following treatment with ATRA, independent of mTOR pathway inhibitors. Proliferation of CSCs, determined by neurosphere diameter, was decreased following treatment with ATRA alone and in combination with rapamycin. The motility of GBM cells was mitigated by treatment with ATRA, rapamycin and LY29002 alone. However, combination treatment augmented the inhibitory effect on migration suggesting synergism. These findings indicate that ATRA-induced differentiation is mediated via the ERK1/2 pathway, and underscores the significance of including differentiating agents along with inhibitors of mTOR pathways in the treatment of GBM.

Hemorrhagic Ganglioglioma of the Posterior Fossa: Case Report

Gangliogliomas are rare tumors of the central nervous system that are usually found in the supratentorial compartment, although cases throughout the nervous system have been described. They are generally low-grade malignancies that are amenable to cure by surgical resection. Most manifest as seizures, though, based on location, they can present with focal neurological deficits. We present here a rare case of an infratentorial ganglioglioma presenting with hemorrhage. To our knowledge this is the only reported case of a hemorrhagic ganglioglioma and, as such, we examine its possible prognosis.

A Novel Approach Using Electromagnetic Neuronavigation and a Flexible Neuroendoscope for Placement of Ommaya Reservoirs.

OBJECTIVE Placement of intraventricular catheters in oncology patients can be associated with morbidity given their small to slit-like ventricles and underlying hematologic disorders. We studied the accuracy of placing Ommaya reservoirs using neuronavigation and a flexible neuroendoscope to verify catheter positioning. METHODS Ommaya reservoirs placed in 25 oncology patients between 2013 and 2015 were retrospectively reviewed. Twenty-five ventricular catheters were placed using the AxiEM stealth frameless neuronavigation system and a flexible neuroendoscope. Postoperative catheter accuracy, operative complications, and postoperative complications were assessed. We discuss surgical protocol and technical nuances. RESULTS All ventricular catheters were successfully placed into the ipsilateral (84%) or contralateral (16%) foramen of Monro. A single ventricular catheter pass was needed to cannulate the ventricle in 96% of patients. The mean accuracy was 4.09 ± 3.47 mm from the target, the ipsilateral foramen of Monro. One patient had a catheter tract hemorrhage seen on postoperative imaging related to thrombocytopenia. No postoperative neurologic deficits were seen. CONCLUSIONS A combined neuronavigation and neuroendoscopic approach improved catheter tip accuracy compared with accuracy rates described in the literature using other techniques. This approach can be adapted toward routine clinical practice of placing ventricular shunt catheters and Ommaya reservoirs.

International teleconsultation on conjoined twins leading to a successful separation: a case report:

Conjoined twins are identical twins that have incompletely separated in utero. The prognosis for conjoined twins is poor and management in a skilled tertiary care centre is paramount for definitive care. We describe our experience with a telemedical consultation on conjoined twins in The Dominican Republic from our eHealth centre in Valhalla, NY. The patients were two month old, female, pygopagus conjoined twins. A multidisciplinary teleconference was initiated with the patients, their family, the referring paediatrician and our team. Based on this teleconsultation, the team felt as though the twins may be amenable to a surgical separation. They presented to our centre in Valhalla, NY, for a detailed physical examination and series of imaging studies. Soon after, the patients underwent a successful 21 h separation procedure and were discharged 12 weeks later. To our knowledge, this is one of the first reports of an international teleconsultation leading to a successful conjoined twin separation procedure.

Abstract 3125: Therapeutic implications of mTORC1 and mTORC2 inhibitors in genetically heterogeneous glioblastoma

Glioblastoma (GB) is the most common malignant primary brain tumor, which is characterized by marked intra-tumor genetic heterogeneity in receptor tyrosine kinases (RTK) genes. Aberrations in the RTK/PI3K/Akt pathways in GB leads to an abnormal signaling pathway of mechanistic target of Rapamycin (mTOR). mTOR exists in two distinct complexes, namely, mTORC1 and mTORC2, which are involved in regulation of cell survival, growth, and motility. In this investigation, we test the hypothesis that the genetic heterogeneity of GBM cells and its stem cells with respect to the expression of EGFR and/or PDGFRα may define the treatment efficacy of RTK inhibitors with or without PI3K/ mTOR inhibitors. We demonstrated that GB tumors over-expressed PDGFRα and EGFR at range of 80% and 30%, respectively, with varying degree of overlap. Combined treatment with the inhibitors of PDGFRα (AC 710) or EGFR (gefitinib), with or without inhibitors of mTORC1 (rapamycin) or mTORC1/2 (Torin1 and Torin 2), led to suppression of cell proliferation, cell cycle, cell migration and drug resistance in GB cells to varying degrees that correlated with EGFR or PDGFRα expression. Downstream signaling pathway substrates of mTORC 1 and 2, namely pS6k and pAkt, respectively, were suppressed by combined treatments of RTK and mTORC1/2 inhibitors. Furthermore, GB stem cell self-renewal and growth was also suppressed with combined treatments. The combination of AC710 plus mTORC1/2 inhibitor, Torin2, was the most effective. These findings underscore the usefulness of determining PDGFRα and EGFR status when considering viable PI3K/mTOR inhibitors for targeted treatment of GB. Citation Format: Anubhav G. Amin, Arthur Wang, Alex Braun, Michael Tobias, Raj Murali, Meena Jhanwar-Uniyal. Therapeutic implications of mTORC1 and mTORC2 inhibitors in genetically heterogeneous glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3125. doi:10.1158/1538-7445.AM2017-3125

Abstract 1927: Tumor suppressor PTEN regulates cancer stem cells of glioblastoma multiforme: identification of signaling pathways as targets of therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Glioblastoma Multiforme (GBM), a WHO defined grade IV astrocytoma, is a uniformly fatal CNS malignancy. The high rate of recurrence is attributed to the presence of its untreatable cancer stem cells (CSC). Phosphatase and tensin homologue on chromosome 10 (PTEN), a tumor suppressor is a central negative regulator of PI3K/AKT signaling axis that controls multiple cellular functions, including cell growth, survival and proliferation. Recent evidence suggests that PTEN loss is associated with increased CSC in GBM. Furthermore, downstream from PTEN/PI3K/AKT is mTOR, an atypical serine threonine kinase that exists in two multiprotein complexes (mTORC1 and mTORC2), which regulates growth and dissemination of GBM. The molecular mechanism involved in regulation of CSC by PTEN and its interacting pathways remains elusive. The aim of this study is to elucidate the role of PTEN in regulation and maintenance of CSC in GBM, and define the potential downstream targets of therapy. In order to achieve our goals, we evaluated the presence of activated AKT/mTOR, and stem cell marker nestin in surgically resected GBM tumors. GBM cell line U87 (PTEN mutated) was transfected with PTEN plasmids (wild type and 3 phosphorylated mutants) to evaluate its role in CSC self-renewal. Pharmacological treatments included an inhibitor of EGFR, gefitinib (Iressa), with or without inhibitors of PI3K (LY294002; LY) or inhibitors of mTORC1 (rapamycin) or mTORC1/2 (PP242). Expression of mTOR and stem cell markers were assessed using IHC or IF analysis, and protein expression was determined via Western Blotting. Functional analysis included self-renewal, drug withdrawal, differentiation, proliferation and migration. Results demonstrated that a significant number of GBM tumors expressed stem cell markers nestin as well as pAKT and pmTOR. Wild type PTEN expressing CSC had decreased self-renewal compared to PTEN expressing mutant CSCs. PI3K inhibitor, LY, with gefitinib was most effective in suppressing neurosphere formation. Rebound following withdrawal of inhibitors showed a significant upregulation in self-renewal of CSC in cells treated with gefitinib and either LY or PP242. These combinations also significantly suppressed CSC proliferation. mTOR suppression altered the expression of stem cell markers nestin and SOX2. In conclusion, the findings of this study demonstrate that PTEN/PI3K/mTOR signaling forms a complex network that regulates and maintains GBM CSC; this also underscores the combined suppression of interacting signaling pathways, such as EGFR, in targeting GBM CSC. Note: This abstract was not presented at the meeting. Citation Format: Michael LaBagnara, Keith Lambert, Sudeepta Sridhara, Michael Tobias, Raj Murali, Meena Jhanwar-Uniyal. Tumor suppressor PTEN regulates cancer stem cells of glioblastoma multiforme: identification of signaling pathways as targets of therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1927. doi:10.1158/1538-7445.AM2014-1927

Abstract 902: Effective and selective targeting of glioblastoma multiforme using an active-site inhibitor of mTORC1/mTORC2 kinase

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The PI3K-AKT-mTOR signaling axis is central to the transformed phenotype of Glioblastoma multiforme (GBM) cells, due to frequent loss of tumor suppressor PTEN. The mammalian target of rapamycin (mTOR) kinase is present in two cellular multiprotein complexes, mTORC1 and mTORC2, which have distinct subunit composition, substrates and mechanisms of activation. Targeting the mTOR protein is a promising strategy for cancer therapy. However, neither of these complexes are fully inhibited by the allosteric inhibitor rapamycin or its analogs. The aim of this study is to provide evidence that a combined inhibition of mTORC1/2 using an ATP-competitive binding inhibitor, PP242, would effectively suppress GBM growth and dissemination as compared to an allosteric binding inhibitor of mTOR. In addition, we utilized another inhibitor, PI-103, a less selective TORC1/2 inhibitor that also targets PI3K, which is more immunosuppressive than PP242, to deter mTOR pathway. We observed a significant number of GBM tumors showed increased expression of pAKTSer473 and pmTORSer2448, as assessed by immunohistochemistry. GBM cells treated with PP242 significantly reduced the activation of mTORC1 and mTORC2, as shown by reduction in phosphorylation of their substrate levels, p-S6KSer235/236 /4E binding protein-1 (4E-BP1) and Akt, respectively, in a dose dependent manner. Furthermore, insulin induced activation of these kinases was abrogated by pre-treatment with PP242 as compared with rapamycin. The cell cycle analysis using the incorporation of 5-ethynyl-2-deoxyuridine (EdU) into the DNA demonstrated that PP242, but not rapamycin, completely blocked the EdU inclusion. To assess TORC2 function, we studied the phosphorylation forkhead box O (FOXO) transcription factors, which are substrates of both Akt and serum downstream of TORC2. PP242, but not rapamycin, reduced FOXO phosphorylation on Akt consensus sites of FOXO1 correlating with greater inhibition of cell cycling. A combined inhibitor of PI3K and mTOR, p103, an ATP binding inhibitor, caused no change in another substrate of mTORC1, suppressed p-S6KSer235/236 levels. In addition, GBM cell proliferation was significantly suppressed by PP242 which was more pronounced compared to rapamycin. Lastly, migration of GBM cells after treatment with PP242 was significantly suppressed, while analysis of cytoskeleton showed that this migration was associated with cellular behavior rather than cytoskeleton loss. In conclusion, these results suggest a novel combined active-site inhibitor of mTORC1/2 kinase; PP242 suppresses both GBM growth and dissemination, which underscores its potential therapeutic use in treatment of GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 902. doi:1538-7445.AM2012-902

Abstract 3392: Regulation and maintenance of cancer stem cells of glioblastoma multiforme: The role of mTOR/MAPK signaling pathways

Glioblastoma multiforme (GBM), the most aggressive primary brain tumor, remains uniformly incurable despite current treatment modalities. Recurrence of GBM has been associated with therapy resistant cancer stem cells (CSC), which possess the ability to generate tumors of the same genotype and phenotype as parent tumors within in vivo models. The coexistence within the same tumors of cells with different phenotypes and isolation of CSC from gliomas, suggests that the neural stem and/or progenitor cells as the cell of origin for GBM. The use of targeted therapies for GBM has failed due to the presence of chemo-resistant CSCs. Mammalian target of Rapamycin (mTOR) is deregulated in GBM, implying that the mTOR inhibition may have therapeutic potential in suppression of CSC. In addition, studies have shown that MAPK plays an important role in differentiation. We aim to test the hypothesis that: 1) CSC growth and self-renewal in GBM is regulated by mTOR and MAPK, 2) differentiating agents, along with other inhibitors can target CSCs. For this study, we utilized primary tumor and GBM cell line (LN18, U373 U87), possessing CSC properties, as evidenced by neurosphere formation. We observed the presence of neural stem cell marker, nestin in a large number of GBM tumors. Neurospheres expressed the markers of embryonic stem cells such as, SOX2 and nanog, in addition to neural stem cell marker, nestin. Treatment of U87 cells with all-trans retinoic acid (ATRA) decreased the expression of the stem cell markers nanog and nestin, as treatment with ATRA enhanced the differentiation of the CSCs. Further analysis suggested that MAPKinase inhibitor, U0126, increased the expression of nanog and nestin. In addition, inhibition of mTOR altered the expression of nanog and nestin, implying mTOR had effect on maintenance of CSC of GBM. Formation of neurosphere was suppressed by treatment with ATRA. Furthermore, neurophere formation was suppressed by allosteric as well as active-site inhibitor of mTOR, rapamycin (RAPA) and pp242, respectively. The proliferation of neurosphere, as measured by neurosphere diameter was also suppressed by ATRA, RAPA and U0126 treatments. While ATRA decreased neurophere diameter by inducing differentiation, the inhibitors of mTOR and MAPK did so by suppressing the activation of down-stream targets, S6Kinase. The effect of these treatments on the CSCs was further elucidated by scratch migration assay. The migration of LN18 and U373 stem cells was significantly inhibited by ATRA, RAPA, and U0126. These results provide further evidence that a significant number of GBMs express stem cell markers. Moreover, the CSC can be targeted by specific mTOR or MAPK inhibition. Furthermore, suppression of the MAPK pathway by U0126, as made evident by the increased expression of nanog and nestin, suggests the role of this pathway in maintenance of pleuripotency of stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3392. doi:10.1158/1538-7445.AM2011-3392