Shwetal Mehta

PNIPAAm-co-Jeffamine® (PNJ) scaffolds as in vitro models for niche enrichment of glioblastoma stem-like cells

Glioblastoma (GBM) is the most common adult primary brain tumor, and the 5-year survival rate is less than 5%. GBM malignancy is driven in part by a population of GBM stem-like cells (GSCs) that exhibit indefinite self-renewal capacity, multipotent differentiation, expression of neural stem cell markers, and resistance to conventional treatments. GSCs are enriched in specialized niche microenvironments that regulate stem phenotypes and support GSC radioresistance. Therefore, identifying GSC-niche interactions that regulate stem phenotypes may present a unique target for disrupting the maintenance and persistence of this treatment resistant population. In this work, we engineered 3D scaffolds from temperature responsive poly(N-isopropylacrylamide-co-Jeffamine M-1000® acrylamide), or PNJ copolymers, as a platform for enriching stem-specific phenotypes in two molecularly distinct human patient-derived GSC cell lines. Notably, we observed that, compared to conventional neurosphere cultures, PNJ cultured GSCs maintained multipotency and exhibited enhanced self-renewal capacity. Concurrent increases in expression of proteins known to regulate self-renewal, invasion, and stem maintenance in GSCs (NESTIN, EGFR, CD44) suggest that PNJ scaffolds effectively enrich the GSC population. We further observed that PNJ cultured GSCs exhibited increased resistance to radiation treatment compared to GSCs cultured in standard neurosphere conditions. GSC radioresistance is supported inxa0vivo by niche microenvironments, and this remains a significant barrier to effectively treating these highly tumorigenic cells. Taken in sum, these data indicate that the microenvironment created by synthetic PNJ scaffolds models niche enrichment of GSCs in patient-derived GBM cell lines, and presents tissue engineering opportunities for studying clinically important behaviors such as radioresistance inxa0vitro.

Developmentally regulated signaling pathways in glioma invasion

Malignant gliomas are the most common, infiltrative, and lethal primary brain tumors affecting the adult population. The grim prognosis for this disease is due to a combination of the presence of highly invasive tumor cells that escape surgical resection and the presence of a population of therapy-resistant cancer stem cells found within these tumors. Several studies suggest that glioma cells have cleverly hijacked the normal developmental program of neural progenitor cells, including their transcriptional programs, to enhance gliomagenesis. In this review, we summarize the role of developmentally regulated signaling pathways that have been found to facilitate glioma growth and invasion. Furthermore, we discuss how the microenvironment and treatment-induced perturbations of these highly interconnected signaling networks can trigger a shift in cellular phenotype and tumor subtype.

Immediate Label-Free Ex Vivo Evaluation of Human Brain Tumor Biopsies With Confocal Reflectance Microscopy

Confocal microscopy utilizing fluorescent dyes is widely gaining use in the clinical setting as a diagnostic tool. Reflectance confocal microscopy is a method of visualizing tissue specimens without fluorescent dyes while relying on the natural refractile properties of cellular and subcellular structures. We prospectively evaluated 76 CNS lesions with confocal reflectance microscopy (CRM) to determine cellularity, architecture, and morphological characteristics. A neuropathologist found that all cases showed similar histopathological features when compared to matched hematoxylin and eosin-stained sections. RNA isolated from 7 tissues following CRM imaging retained high RNA integrity, suggesting that CRM does not alter tissue properties for molecular studies. A neuropathologist and surgical pathologist masked to the imaging results independently evaluated a subset of CRM images. In these evaluations, 100% of images reviewed by the neuropathologist and 95.7% of images reviewed by the surgical pathologist were correctly diagnosed as lesional or nonlesional. Furthermore, 97.9% and 91.5% of cases were correctly diagnosed as tumor or not tumor by the neuropathologist and surgical pathologist, respectively, while 95.8% and 85.1% were identified with the correct diagnosis. Our data indicate that CRM is a useful tool for rapidly screening patient biopsies for diagnostic adequacy, molecular studies, and biobanking.

Atypical Teratoid Rhabdoid Tumor: Two Case Reports and an Analysis of Adult Cases with Implications for Pathophysiology and Treatment

We present the first quantitative analysis of atypical teratoid rhabdoid tumors (ATRT) in adults, including two patients from our own institutions. These are of interest as one occurred during pregnancy and one is a long-term survivor. Our review of pathological findings of 50 reported cases of adult ATRT leads us to propose a solely ectodermal origin for the tumor and that epithelial–mesenchymal transition (EMT) is a defining feature. Thus, the term ATRT may be misleading. Our review of clinical findings shows that ATRT tends to originate in mid-line structures adjacent to the CSF, leading to a high rate of leptomeningeal dissemination. Thus, we hypothesize that residual undifferentiated ectoderm in the circumventricular organs, particularly the pituitary and pineal glands, is the most common origin for these tumors. We note that if growth is not arrested soon after diagnosis, or after the first relapse/progression, death is almost universal. While typically rapidly fatal (as in our first case), long-term remission is possible (as in our second). Significant predictors of prognosis were the extent of resection and the use of chemotherapy. Glial differentiation (GFAP staining) was strongly associated with leptomeningeal metastases (chi-squared pu2009=u20090.02) and both predicted markedly worse outcomes. Clinical trials including adults are rare. ATRT is primarily a disease of infancy and radiotherapy is generally avoided in those aged less than 3u2009years old. Treatment options in adults differ from infants in that cranio-spinal irradiation is a viable adjunct to systemic chemotherapy in the adult population. Given the grave prognosis, this combined approach appears reasonable. As effective chemotherapy is likely to cause myelosuppression, we recommend that stem-cell rescue be available locally.

Targetable gene fusions associate with the IDH wild-type astrocytic lineage in adult gliomas

Abstract Gene fusions involving oncogenes have been reported in gliomas and may serve as novel therapeutic targets. Using RNA-sequencing, we interrogated a large cohort of gliomas to assess for the incidence of targetable genetic fusions. Gliomas (nu2009=u2009390) were profiled using the ArcherDx FusionPlex Assay. Fifty-two gene targets were analyzed and fusions with preserved kinase domains were investigated. Overall, 36 gliomas (9%) harbored a total of 37 potentially targetable fusions, the majority of which were found in astrocytomas (nu2009=u200934). Within this lineage 11% (25/235) of glioblastomas, 12% (5/42) of anaplastic astrocytomas, 8% (2/25) of grade II astrocytomas, and 33% (2/6) of pilocytic astrocytoma harbored targetable fusions. Fusions were significantly more frequent in IDH wild-type tumors (12%, nu2009=u200931/261) relative to IDH mutants (4%; nu2009=u20094/109) (pu2009=u20090.011). No fusions were seen in oligodendrogliomas. The most frequently observed therapeutically targetable fusions were in FGFR (nu2009=u200912), MET (nu2009=u200911), and NTRK (nu2009=u20098). Several additional novel fusions that have not been previously described in gliomas were identified including EGFR:VWC2 and FGFR3:NBR1. In summary, targetable gene fusions are enriched in IDH wild-type high-grade astrocytic tumors, which will influence enrollment in and interpretation of clinical trials of glioma patients.

Phase 0 Trial of AZD1775 in First-Recurrence Glioblastoma Patients

Purpose: AZD1775 is a first-in-class Wee1 inhibitor with dual function as a DNA damage sensitizer and cytotoxic agent. A phase I study of AZD1775 for solid tumors suggested activity against brain tumors, but a preclinical study indicated minimal blood–brain barrier penetration in mice. To resolve this controversy, we examined the pharmacokinetics and pharmacodynamics of AZD1775 in patients with first-recurrence, glioblastoma. Patients and Methods: Twenty adult patients received a single dose of AZD1775 prior to tumor resection and enrolled in either a dose-escalation arm or a time-escalation arm. Sparse pharmacokinetic blood samples were collected, and contrast-enhancing tumor samples were collected intraoperatively. AZD1775 total and unbound concentrations were determined by a validated LC/MS-MS method. Population pharmacokinetic analysis was performed to characterize AZD1775 plasma pharmacokinetic profiles. Pharmacodynamic endpoints were compared to matched archival tissue. Results: The AZD1775 plasma concentration–time profile following a single oral dose in patients with glioblastoma was well-described by a one-compartment model. Glomerular filtration rate was identified as a significant covariate on AZD1775 apparent clearance. AZD1775 showed good brain tumor penetration, with a median unbound tumor-to-plasma concentration ratio of 3.2, and achieved potential pharmacologically active tumor concentrations. Wee1 pathway suppression was inferred by abrogation of G2 arrest, intensified double-strand DNA breakage, and programmed cell death. No drug-related adverse events were associated with this study. Conclusions: In contrast to recent preclinical data, our phase 0 study of AZD 1775 in recurrent glioblastoma indicates good human brain tumor penetration, provides the first evidence of clinical biological activity in human glioblastoma, and confirms the utility of phase 0 trials as part of an accelerated paradigm for drug development in patients with glioma. Clin Cancer Res; 24(16); 3820–8. ©2018 AACR. See related commentary by Vogelbaum, p. 3790

pH driven precipitation of quisinostat onto PLA-PEG nanoparticles enables treatment of intracranial glioblastoma

Histone deacetylases (HDACs) are known to be key enzymes in cancer development and progression through their modulation of chromatin structure and the expression and post-translational modification of numerous proteins. Aggressive dedifferentiated tumors, like glioblastoma, frequently overexpress HDACs, while HDAC inhibition can lead to cell cycle arrest, promote cellular differentiation and induce apoptosis. Although multiple HDAC inhibitors, such as quisinostat, are of interest in oncology due to their potent in vitro efficacy, their failure in the clinic as monotherapies against solid tumors has been attributed to poor delivery. Thus, we were motivated to develop quisinostat loaded poly(D,L-lactide)-b-methoxy poly(ethylene glycol) nanoparticles (NPs) to test their ability to treat orthotopic glioblastoma. In developing our NP formulation, we identified a novel, pH-driven approach for achieving over 9% (w/w) quisinostat loading. We show quisinostat-loaded NPs maintain drug potency in vitro and effectively slow tumor growth in vivo, leading to a prolonged survival compared to control mice.

Abstract B21: Use of polymeric nanoparticles for the delivery of YM155 to glioma cells in vitro and in vivo

YM155 (Sepantronium Bromide) is a potent survivin inhibitor that is the subject of clinical trials for multiple types of cancer including melanoma, lypmphoma, lung cancer, prostate and breast cancer. YM155 is a therapeutic candidate for the treatment of glioblastoma (GBM), however, its rapid plasma clearance and minimal blood-brain barrier penetration are obstacles to clinical translation (1). The encapsulation of drugs in nanoparticles can help increase drug circulation time by reducing clearance, and nanoparticles have the ability to increase drug concentration in the tumor through the enhanced permeability and retention effect (2). Here, we encapsulated YM155 in poly(lactic acid)-b-poly(ethylene glycol) nanoparticles (NPs) and evaluated the potency of YM155 in 4 glioma cell lines in vitro. Additionally, we investigated the ability of systemically administered NPs to inhibit tumor growth in vivo in mice bearing orthotopic gliomas. In vitro potency of YM155 was measured by CellTiter Glo in 3 immortalized GBM lines (GL261, U118, U87), as well as in patient derived tumor cells (GB7). Tumor growth inhibition was evaluated in C57 albino mice bearing orthotopic GL261-luc2 tumors. Mice were treated for 3 consecutive days with saline, free YM155 or NPs by tail vein injection, and tumor growth was measured by bioluminescence using an IVIS imaging system. All cells tested were sensitive to both free and encapsulated YM155. GL261 was the least sensitive of the cells tested, with an IC50 of 196 nM, while U118 and U87 had IC50s of 61 and 32 nM, respectively. The patient derived GB7 cells were the most sensitive to YM155, with an IC50 of 22 nM. Tumors grew exponentially for mice treated with free YM155 or saline, whereas tumor growth was halted in 2/5 subjects after one cycle of treatment with NPs. This work shows the potency of YM155 against multiple glioma cell lines in vitro. In vivo, free YM155 does not affect the growth of an orthotopic glioma; however, our initial treatment study provides evidence systemically administered YM155-loaded NPs allow for the delivery of a therapeutically relevant dose to orthotopic GBM. Success of this work shows the ability to encapsulate a water soluble drug in polymeric nanoparticles, which can be used to generate new options in the treatment of GBM. Further work will evaluate drug efficacy in additional patient derived cells and investigate the ability of NP encapsulated YM155 alone or in conjunction with radiation to treat mice bearing patient derived xenograft tumors. References: 1. Minematsu T, Sonoda T, Hashimoto T, Iwai M, Oppeneer T, Felder L, et al. Pharmacokinetics, distribution and excretion of YM155 monobromide, a novel small-molecule survivin suppressant, in male and pregnant or lactating female rats. Biopharm Drug Dispos. 2012;33:160–9. 2. Householder KT, DiPerna DM, Chung EP, Wohlleb GM, Dhruv HD, Berens ME, et al. Intravenous delivery of camptothecin-loaded PLGA nanoparticles for the treatment of intracranial glioma. Int J Pharm. 2015;479:374–80. Citation Format: Kyle T. Householder, Danielle M. DiPerna, Jonathan T. Yamaguchi, Nader Sanai, Shwetal Mehta, Rachael W. Sirianni. Use of polymeric nanoparticles for the delivery of YM155 to glioma cells in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B21.

Abstract B20: Targeting Olig2 co-regulators for malignant glioma therapy

The transcription factor OLIG2 is expressed within the highly tumorigenic sub-population of stem-like cells in malignant glioma and required for tumor formation in orthotopic grafts of human glioma as well as genetically defined murine glioma models. Our work (recently confirmed and extended by Suva et al., 2014) suggests that small molecule OLIG2 antagonist could serve as targeted therapy for high grade gliomas – either as a mono therapeutic or as an adjuvant to conventional radiotherapy or cytotoxic drugs. However, transcription factors in general are considered to be unattractive targets for drug development because their interactions with DNA and co-regulatory proteins involve large and complex surface area contacts. Our recent data suggests that OLIG2 interacts with distinct partner proteins based on its phosphorylation state, which in turn regulates its pro-mitogenic functions. Here we employed two independent proteomics screens (mass spectrometry and Nucleic Acid Programmable Protein Arrays, NAPPA) to identify OLIG2 co-regulators as possible surrogate targets to suppress OLIG2 function. These screens led to identification of two novel OLIG2 partner proteins: HDAC1 and proto-oncogene FYN kinase. OLIG2 is known to be a negative regulator of gene expression and histone deacetylation is required for suppression of gene expression. We demonstrate that OLIG2 functions as an HDAC1 carrier, delivering this enzyme to specific target genes (e.g., p21) and non-histone proteins like p53. HDACs are ubiquitously expressed and are known to be redundant, but we show that ablation of HDAC1 alone (using shRNA) significantly decreases the proliferation of glioma initiating cells with minimal impact on normal neural stem cells. In addition, we show that FYN kinase interacts with OLIG2 and phosphorylates it at tyrosine residues. Ablation of FYN decreases phosphorylation of OLIG2 at tyrosine residues and affects OLIG2-mediated expression of oncogenic target genes like EGFR and WNT5a. This work provides two potential co-regulators of OLIG2 which could serve as druggable surrogate targets to block OLIG2 function in malignant glioma. Further work will evaluate the efficacy of HDAC1 and FYN-specific inhibitor as single agents or as adjuvant therapy to radiation and temozolomide in orthotopic patient-derived xenograft models. Targeting factors regulating OLIG2-mediated tumor growth will have practical overtones for targeted therapies for malignant gliomas. Citation Format: Robert Kupp, Costanza Lo Cascio, Femina Rauf, Dimphna Meijer, Charles D. Stiles, Nader Sanai, Joshua LaBaer, Shwetal V. Mehta. Targeting Olig2 co-regulators for malignant glioma therapy. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B20.

Abstract 510: A novel role for Olig2 in the regulation of glioma invasion

Glioblastoma multiforme (GBM) are highly infiltrative, aggressive and lethal primary brain tumors in adults that are resistant to conventional treatments. A major clinical obstacle in the treatment of GBMs is the ability of glioma cells to invade the surrounding brain parenchyma, preventing their complete surgical resection. Several studies have shown that invasive glioma stem cells (GSCs) extend beyond the surgically resected regions. These invasive cells are the cause for tumor recurrence and poor prognosis in GBM patients. The molecular and cellular mechanism involved in the regulation of the switch from proliferation to invasion/migration in GBM is poorly understood. We investigated the role of Olig2, a Central Nervous System (CNS) specific transcription factor, in promoting the switch from proliferating to invading glioma cell. Genetically relevant murine glioma model as well as patient-derived glioma stem cells (GSCs) were utilized to identify the effect of Olig2 on glioma invasion. Functionally, Olig2-mediated invasion was assessed by 3D-transwell invasion assays, organotypic slice cultures as well as PDX tumors in immunocompromised mice. In addition, murine and patient-derived GSCs were utilized to identify direct genetic targets of Olig2 involved in glioma invasion by ChIP-seq and RNA-seq. Our data demonstrate that Olig2-expressing murine glioma stem/progenitor-like cells are highly invasive as compared to Olig2 knockout cells. We further corroborated the requirement of Olig2 for glioma invasion in patient-derived GSCs by utilizing shRNA-mediated knockdown of Olig2. Olig2 depletion resulted in reduced glioma cell invasion in at least four independent human GSCs. Recent studies have shown that activation of transcription factors such as Zeb1 and Twist1 occurs in invasive high grade gliomas. We show that knockdown of Olig2 in murine as well as human GSCs leads to a significant decrease in the expression of invasion genes such as Zeb1, Vimentin, Twist1, and Stat3 as compared to control cells. In conclusion, our studies highlight a novel role for the Olig2 signalling network in promoting the switch from proliferation to invasion in GBM, and provide a plausible target for inhibiting glioma invasion. Citation Format: Shiv K. Singh, Nicole Giannonatti, Robert Kupp, Costanza Lo Cascio, Shwetal Mehta. A novel role for Olig2 in the regulation of glioma invasion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 510. doi:10.1158/1538-7445.AM2015-510