Glioblastoma Cancer Stem Cells Research Articles

Abstract CT272: Phase 2 study of dianhydrogalactitol (VAL-083) in patients with MGMT-unmethylated, bevacizumab-naïve glioblastoma in the recurrent or adjuvant setting

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain cancer. Current standard-of-care for glioblastoma (GBM) includes surgery followed by concurrent therapy with radiation and temozolomide (TMZ) followed by adjuvant TMZ (days 1-5 every 28 days). Almost all GBM patients experience recurrent/progressive disease, with a median survival of 3-9 months after recurrence. Second-line treatment for recurrent GBM with bevacizumab (BEV) has not improved survival, and effective therapies for GBM are lacking. Unmethylated promoter status for O6-methylguanine DNA methyltransferase (MGMT) is a validated biomarker for TMZ-resistance and is correlated with poor prognosis. VAL-083 is a bi-functional DNA-targeting agent rapidly inducing interstrand cross-links at N7-guanine, leading to DNA double-strand breaks and cell death. VAL-083's cytotoxicity is independent of MGMT status, and VAL-083 overcomes TMZ-resistance in GBM cell lines, GBM cancer stem cells, and in vivo GBM models. The trial described here is an open-label two-arm biomarker-driven phase 2 clinical trial in MGMT-unmethylated GBM patients with either recurrent disease (Group 1) or newly diagnosed GBM patients requiring adjuvant therapy after chemoirradiation with temozolomide (Group 2). Patients receive VAL-083 IV at 30 or 40 mg/m2/day on days 1, 2, and 3 of a 21-day cycle. The primary objective of this study is to determine the effect of VAL-083 on median overall survival (mOS) in MGMT-unmethylated recurrent GBM patients (Group 1) compared to historical control, and progression-free survival (PFS) in newly diagnosed GBM patients requiring adjuvant therapy after chemoirradiation with temozolomide (Group 2), compared to historical control. Secondary efficacy endpoints include progression-free survival (PFS) (Group 1), overall response rate (ORR), duration of response (DOR), and quality-of-life (QoL). Tumor response will be assessed by MRI approximately every 42 days, as per RANO criteria. The initial starting dose in this study was 40 mg/m2/day, which was subsequently reduced to 30 mg/m2/day to improve tolerance due to myelosuppression. As of 21st January 2020, thirty-five (35) subjects had enrolled at a starting dose of 40 mg/m2/day, and 31 subjects had enrolled at a starting dose of 30 mg/m2/day in Group 1, and 9 subjects enrolled at a starting dose of 30 mg/m2/day in Group 2. As anticipated from prior studies with VAL-083, myelosuppression (thrombocytopenia and neutropenia) has been the most common adverse event observed. Enrollment and safety data update will be provided at the meeting. Clinicaltrials.gov identifier: NCT02717962 Citation Format: Barbara J. O'Brien, Carlos Kamiya-Matsuoka, Shiao-Pei Weathers, Alfred Yung, Monica Loghin, Rebecca Harrison, Nazanin Majd, Jeffrey A. Bacha, Dennis M. Brown, Anne Steino, Gregory Johnson, Sarath Kanekal, John Langlands, Lorena M. Lopez, Richard M. Schwartz, Marta Penas-Prado, John F. de Groot. Phase 2 study of dianhydrogalactitol (VAL-083) in patients with MGMT-unmethylated, bevacizumab-naïve glioblastoma in the recurrent or adjuvant setting [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT272.

Abstract 5725: Epigenetic and transcriptional profiling reveal endosomal protein sorting as a glioblastoma cancer stem cell dependency

Abstract Glioblastoma is a highly lethal primary intrinsic brain tumor for which current standard-of-care therapies are merely palliative. Functionally defined glioblastoma stem cells (GSCs) contribute to this poor prognosis by driving therapeutic resistance, maintenance of cellular heterogeneity, and disease recurrence. A deeper appreciation for the specific features that distinguish normal from neoplastic stem cells may inform selective therapeutic targeting. To further understand the molecular processes essential for maintaining cancer stem cell populations, we interrogated the active enhancer landscapes and transcriptional output of GSCs and normal neural stem cells (NSCs) through H3K27ac ChIP-seq and RNA-seq respectively. GSC-specific targets that held prognostic significance in patient datasets and which were essential in whole genome CRISPR screening data implicated endosomal protein sorting as a key feature in GSC biology. Targeting endosomal sorting impaired GSC viability and proliferation and contributed to apoptosis, while NSCs displayed a reduced cell proliferation deficit. Impaired endosomal protein sorting led to a reduction in the mRNA and protein levels of the stem and neuro-developmental transcription factors SOX2 and OLIG2 and reduced self-renewal capacity of GSCs, suggesting a role in the maintenance of stemness. Mechanistically, regulation of endosomal sorting may impact localization and stability of receptor tyrosine kinases and WNT signaling elements to control GSC maintenance. High expression of this endosomal sorting factor is associated with poor glioblastoma patient prognosis, suggesting its clinical importance. Thus, targeting the endosomal sorting pathway may represent a novel therapeutic approach for glioblastoma treatment. Citation Format: Ryan C. Gimple, Guoxin Zhang, Jeremy N. Rich. Epigenetic and transcriptional profiling reveal endosomal protein sorting as a glioblastoma cancer stem cell dependency [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5725.

Abstract 1505: Platelet derived growth factor receptor alpha oncogene dependency in glioblastoma

Abstract Platelet-derived growth factor receptor alpha (PDGFRA) is a ligand-activated receptor tyrosine kinase (RTK) involved in neurodevelopment. In pediatric and adult glioblastoma (GBM), PDGFRA downstream signaling promotes tumor cell proliferation, stemness and survival. Deregulated oncogenic PDGFRA signaling in these tumors occurs by gene amplification (15% of cases), often times as extrachromosomal elements (ecDNA), or by mutations and transcriptional upregulation. Lack of biomarker-based patient selection has contributed to failure of PDGFRA inhibitors in clinical trials. To provide experimental evidence for selection of patients that can benefit from PDGFRA targeted therapy, we have investigated the mode of PDGFRA deregulation in patient-derived GBM models, which we hypothesize determines the degree of dependency on PDGFRA signaling. PDGFRA copy number and mRNA levels in a panel of 13 GBM tumors, derived cancer stem cells (CSC) and xenografts (PDX) were determined by DNA and RNA seq, respectively. EcDNA location of PDGFRA-amplified CSCs was determined by FISH. Levels of total PDGFRA and activated (phospho-PDGFRA Y754) protein were determined by IHC and reverse phase protein array (RPPA). PDGFRA mRNA overexpression was observed for 3 GBM tumors and their derived models: one GBM carrying PDGFRA ecDNA amplification (HF3253), and two non-amplified GBMs (Tukey p=0.60, 0.37). Messenger RNA correlated to total protein levels for all cases, but corresponding PDGFRA activation was observed only for HF3253. High levels of PDGFRA activation in HF3253 PDX was retained in recurrent tumors after temozolomide treatment. Exploiting intra-tumoral heterogeneity, we have isolated 2 single cell clones from HF3253 CSCs which do not carry PDGFRA ecDNA(-) and tested their self-renewal and tumorigenic potential against the parental ecDNA(+) cells. Reduced self-renewal in both ecDNA(-) clones was observed (p=1e-03, p=0.01). Tumor growth in mouse orthotopic xenografts, measured by symptom-free survival, was extraordinarily reduced in the two ecDNA(-) clones (log-rank, p=1e-03, 2e-04). In contrast to parental HF3253, ecDNA(-) tumors demonstrated diffuse tumor morphology and weak PDGFRA activation. Using RPPA data from four in vitro media conditions (normal growth, minus EGF/FGF, 2% and 10% FBS) in eight GBM CSC models, the effect of growth conditions on PDGFRA activation was significant and strongest under growth factor-depleted conditions (p=5e-04, 2-way ANOVA). In conclusion, we demonstrated the dependency on PDGFRA signaling in a patient-derived GBM model carrying ecDNA PDGFRA amplification. PDGFRA over-expression was observed for 2 GBMs in absence of gene amplification, but it did not result in protein activation, which our data suggest is a determinant of oncogene dependency. PDGFRA activity is preserved by endogenous stressors (removal of growth factors) and exogenous stressors (TMZ). Our data validates PDGFRA as a therapeutic target in a subset of GBM patients and demonstrates that detection of ecDNA-amplified PDGFRA has the potential to be a predictive biomarker of future PDGFRA targeted therapies. Citation Format: Artem D. Berezovsky, Susan M. Irtenkauf, Andrea D. Transou, Laura A. Hasselbach, Tom Mikkelsen, Ana C. deCarvalho. Platelet derived growth factor receptor alpha oncogene dependency in glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1505.

Advances in histone deacetylase inhibitors in targeting glioblastoma stem cells.

Glioblastoma multiforme (GBM) is a lethal grade IV glioma (WHO classification) and widely prevalent primary brain tumor in adults. GBM tumors harbor cellular heterogeneity with the presence of a small subpopulation of tumor cells, described as GBM cancer stem cells (CSCs) that pose resistance to standard anticancer regimens and eventually mediate aggressive relapse or intractable progressive GBM. Existing conventional anticancer therapies for GBM do not target GBM stem cells and are mostly palliative; therefore, exploration of new strategies to target stem cells of GBM has to be prioritized for the development of effective GBM therapy. Recent developments in the understanding of GBM pathophysiology demonstrated dysregulation of epigenetic mechanisms along with the genetic changes in GBM CSCs. Altered expression/activity of key epigenetic regulators, especially histone deacetylases (HDACs) in GBM stem cells has been associated with poor prognosis; inhibiting the activity of HDACs using histone deacetylase inhibitors (HDACi) has been promising as mono-therapeutic in targeting GBM and in sensitizing GBM stem cells to an existing anticancer regimen. Here, we review the development of pan/selective HDACi as potential anticancer agents in targeting the stem cells of glioblastoma as a mono or combination therapy.

A new tyrosine kinase inhibitor K905-0266 inhibits proliferation and sphere formation of glioblastoma cancer cells

Glioblastoma (GBM) is the most prevalent malignant tumour of the central nervous system and carries a poor prognosis; average survival time after diagnosis is 14 months. Because of its unfavourable prognosis, novel therapies are needed. The aim of this study was to assess whether inhibition of GBM and GBM-derived cancer stem cells (CSCs) by a new tyrosine kinase inhibitor (TKI), K905-0266, is possible. To do this, we generated GBM (D54 and U87MG) cells expressing luciferase and characterised the inhibitory effects of the TKI with bioluminescent imaging (BLI) and western blot (WB). The effect of the TKI was then evaluated in CSCs. BLI showed significant inhibition of D54 and U87MG cells by TKI treatment. WB showed that the TKI decreased pERK and Bcl-2 level and increased cleaved caspase-3 level. Sphere formation was significantly reduced by the TKI in CSCs. Our results showed that a new TKI, K905-0266, effectively inhibited GBM and CSCs, making this a candidate for GBM therapy.

JAM-A functions as a female microglial tumor suppressor in glioblastoma.

Glioblastoma (GBM) is the most aggressive primary brain tumor and has a dismal prognosis. Previously, we identified that junctional adhesion molecule A (JAM-A), a cell adhesion molecule, is highly elevated in human GBM cancer stem cells and predicts poor patient prognosis. While JAM-A is also highly expressed in other cells in the tumor microenvironment, specifically microglia and macrophages, how JAM-A expression in these cells affects tumor growth has yet to be determined. The goal of this study was to understand the role of microenvironmental JAM-A in mediating GBM growth. Male and female wild-type (WT) and JAM-A-deficient mice were transplanted intracranially with the syngeneic glioma cell lines GL261 and SB28 and were assessed for differences in survival and microglial activation in tumors and in vitro. RNA-sequencing was performed to identify differentially regulated genes among all genotypes, and differences were validated in vitro and in vivo. We found that JAM-A-deficient female mice succumbed to GBM more quickly compared with WT females and JAM-A-deficient and male WT mice. Analysis of microglia in the tumors revealed that female JAM-A-deficient microglia were more activated, and RNA-sequencing identified elevated expression of Fizz1 and Ifi202b specifically in JAM-A-deficient female microglia. Our findings suggest that JAM-A functions to suppress pathogenic microglial activation in the female tumor microenvironment, highlighting an emerging role for sex differences in the GBM microenvironment and suggesting that sex differences extend beyond previously reported tumor cell-intrinsic differences.

Cross talk between mesenchymal and glioblastoma stem cells: Communication beyond controversies.

Mesenchymal stem cells (MSCs) can be isolated from bone marrow or other adult tissues (adipose tissue, dental pulp, amniotic fluid, and umbilical cord). In vitro, MSCs grow as adherent cells, display fibroblast‐like morphology, and self‐renew, undergoing specific mesodermal differentiation. High heterogeneity of MSCs from different origin, and differences in preparation techniques, make difficult to uniform their functional properties for therapeutic purposes. Immunomodulatory, migratory, and differentiation ability, fueled clinical MSC application in regenerative medicine, whereas beneficial effects are currently mainly ascribed to their secretome and extracellular vesicles. MSC translational potential in cancer therapy exploits putative anti‐tumor activity and inherent tropism toward tumor sites to deliver cytotoxic drugs. However, controversial results emerged evaluating either the therapeutic potential or homing efficiency of MSCs, as both antitumor and protumor effects were reported. Glioblastoma (GBM) is the most malignant brain tumor and its development and aggressive nature is sustained by cancer stem cells (CSCs) and the identification of effective therapeutic is required. MSC dualistic action, tumor‐promoting or tumor‐targeting, is dependent on secreted factors and extracellular vesicles driving a complex cross talk between MSCs and GBM CSCs. Tumor‐tropic ability of MSCs, besides providing an alternative therapeutic approach, could represent a tool to understand the biology of GBM CSCs and related paracrine mechanisms, underpinning MSC‐GBM interactions. In this review, recent findings on the complex nature of MSCs will be highlighted, focusing on their elusive impact on GBM progression and aggressiveness by direct cell‐cell interaction and via secretome, also facing the perspectives and challenges in treatment strategies.

Invitro demonstration of salinomycin as a novel chemotherapeutic agent for the treatment of SOX2‑positive glioblastoma cancer stem cells.

Glioblastoma multiforme (GBM) is the deadliest and most common form of primary brain tumor. Conventional treatments are ineffective at treating GBM due to the heterogeneous cellular makeup of the tumors as well as the existence of drug‑resistant cells known as cancer stem cells(CSCs). CSCs have the ability to initiate tumorigenesis and self‑renew, which can lead to recurrence. Salinomycin, an antibiotic commonly used in agricultural feed, has been revealed to target CSCs in other cancer types. A few studies have suggested salinomycin can be effective at treating glioblastoma stem cells (GSCs); however, no study has examined the effect of salinomycin treatment on GSC markers. In the present study, flow cytometry, RT‑qPCR, and limiting dilution assays were used to further analyze the effects of salinomycin on GSCs. It was revealed that salinomycin decreased the expression of the GSC marker SOX2 at both the transcriptional and translational level. However, the effect of salinomycin on the GSC markers Nestin and CD133 was inconsistent between GBM subtypes. Additionally, the present findings provide initial evidence of caspase‑3‑dependent and independent apoptosis as the method by which salinomycin induces cell death in GBM. The present results indicated that salinomycin is an effective candidate as a chemotherapeutic agent that can treat GBM by targeting both bulk tumor cells as well as CSCs.

Newcastle Disease Virus (NDV) Oncolytic Activity in Human Glioma Tumors Is Dependent on CDKN2A-Type I IFN Gene Cluster Codeletion.

Glioblastoma (GBM) is the most aggressive and frequent primary brain tumor in adults with a median overall survival of 15 months. Tumor recurrence and poor prognosis are related to cancer stem cells (CSCs), which drive resistance to therapies. A common characteristic in GBM is CDKN2A gene loss, located close to the cluster of type I IFN genes at Ch9p21. Newcastle disease virus (NDV) is an avian paramyxovirus with oncolytic and immunostimulatory properties that has been proposed for the treatment of GBM. We have analyzed the CDKN2A-IFN I gene cluster in 1018 glioma tumors and evaluated the NDV oncolytic effect in six GBM CSCs ex vivo and in a mouse model. Our results indicate that more than 50% of GBM patients have some IFN deletion. Moreover, GBM susceptibility to NDV is dependent on the loss of the type I IFN. Infection of GBM with an NDV-expressing influenza virus NS1 protein can overcome the resistance to oncolysis by NDV of type I-competent cells. These results highlight the potential of using NDV vectors in antitumor therapies.

The Rational Development of CD133-Targeting Immunotherapies for Glioblastoma.

CD133 marks self-renewing cancer stem cells (CSCs) in a variety of solid tumors, and CD133+ tumor-initiating cells are known markers of chemo- and radio-resistance in multiple aggressive cancers, including glioblastoma (GBM), that may drive intra-tumoral heterogeneity. Here, we report three immunotherapeutic modalities based on a human anti-CD133 antibody fragment that targets a unique epitope present in glycosylated and non-glycosylated CD133 and studied their effects on targeting CD133+ cells in patient-derived models of GBM. We generated an immunoglobulin G (IgG) (RW03-IgG), a dual-antigen Tcell engager (DATE), and a CD133-specific chimeric antigen receptor Tcell (CAR-T): CART133. All three showed activity against patient-derived CD133+ GBM cells, and CART133 cells demonstrated superior efficacy in patient-derived GBM xenograft models without causing adverse effects on normal CD133+ hematopoietic stem cells in humanized CD34+ mice. Thus, CART133 cells may be a therapeutically tractable strategy to target CD133+ CSCs in human GBM or other treatment-resistant primary cancers.

Hypoxia, Inflammation and Necrosis as Determinants of Glioblastoma Cancer Stem Cells Progression.

Tumor hypoxic microenvironment causes hypoxia inducible factor 1 alpha (HIF-1α) activation and necrosis with alarmins release. Importantly, HIF-1α also controls the expression of alarmin receptors in tumor cells that can bind to and be activated by alarmins. Human tumor tissues possess 1–2% of cancer stem cells (CSCs) residing in hypoxic niches and responsible for the metastatic potential of tumors. Our hypothesis is that hypoxic CSCs express alarmin receptors that can bind alarmins released during necrosis, an event favoring CSCs migration. To investigate this aspect, glioblastoma stem-like cell (GSC) lines were kept under hypoxia to determine the expression of hypoxic markers as well as receptor for advanced glycation end products (RAGE). The presence of necrotic extracts increased migration, invasion and cellular adhesion. Importantly, HIF-1α inhibition by digoxin or acriflavine prevented the response of GSCs to hypoxia alone or plus necrotic extracts. In vivo, GSCs injected in one brain hemisphere of NOD/SCID mice were induced to migrate to the other one in which a necrotic extract was previously injected. In conclusion, our results show that hypoxia is important not only for GSCs maintenance but also for guiding their response to external necrosis. Inhibition of hypoxic pathway may therefore represent a target for preventing brain invasion by glioblastoma stem cells (GSCs).

Regulation of glioblastoma tumor growth and stem cell properties through Gα12: an upstream player in YAP signaling

Glioblastoma multiforme (GBM), the most common and aggressive form of brain tumor, has been associated with the dysregulation of G‐protein coupled receptor signaling. Data from the Cancer Genome Atlas demonstrate remarkable upregulation of mRNA for the Gα12 protein, which is elevated in 26% of GBM patient tumors (the highest rate for all tumor samples surveyed). The Gα12 protein couples GPCRs to guanine nucleotide exchange factors (GEFs) for RhoA and was originally isolated as an oncogene. Furthermore, the subset of GPCRs that couples to Gα12 to activate RhoA includes S1P, LPA, and thrombin, inflammatory mediators that would be generated in the tumor cell environment. GPCR signaling to RhoA leads to activation of the transcriptional co‐activator YAP (Yes‐associated protein 1 gene). The role of YAP in growth of glioblastoma cells and in vivo GBM tumor development was explored in our previous studies (Yu et al. Oncogene 7(41): 5492–5507, 2018) using human glioblastoma cell lines and glioma stem cells (GSCs) derived from patient derived xenografts (PDX). We showed that shRNA mediated knockdown of YAP in GSC‐23 PDX cells significantly attenuated in vitro self‐renewal capability assessed by limiting dilution, oncogene expression, neurosphere formation, and stem cell gene expression. Orthotopic xenografts of the YAP knockdown PDX cells formed significantly smaller tumors and were of lower morbidity than wild‐type cells. Our current work examines the effects of Gα12, which would transduce effects of known and orphan GPCRs upstream that might be activated in the tumor environment to RhoA. We knocked down Gα12 using shRNA in two GBM cancer stem cell lines, GSC‐23 and HK‐281, decreasing Gα12 mRNA levels by 80% without compensatory upregulation in the expression of the homologous Gα13 protein. Loss of Gα12 attenuated GSC‐23 stemness as determined by limiting dilution assay and reduced mRNA levels for canonical stem cells genes (CCND1, NANONG, OCT4, SOX2 and NESTIN) by 35% to 60%. We are currently examining growth of GSC‐23 control and Gα12 knockdown cells following orthotopic injection into mouse brain. Histopathological findings revealed that the deletion of Gα12 reduced tumor‐derived cell migration and invasiveness. Gα12 knockdown tumors also presented with increased areas of necrosis. In vitro transwell assays confirmed that Gα12 knockdown reduced agonist‐stimulated GSC‐23 cell migration. We are exploring the role of YAP‐dependent genes (e.g. Thbs‐1, Dusp‐1, and CCN1) which were selectively decreased in tumors from PDX cells lacking Gα12 to determine their role in tumor cell migration and survival.Support or Funding InformationNIH grant CA218859

Cross Interaction between M2 Muscarinic Receptor and Notch1/EGFR Pathway in Human Glioblastoma Cancer Stem Cells: Effects on Cell Cycle Progression and Survival.

Glioblastomas (GBM) are the most aggressive form of primary brain tumors in humans. A key feature of malignant gliomas is their cellular heterogeneity. In particular, the presence of an undifferentiated cell population of defined Glioblastoma Stem cells (GSCs) was reported. Increased expression of anti-apoptotic and chemo-resistance genes in GCSs subpopulation favors their high resistance to a broad spectrum of drugs. Our previous studies showed the ability of M2 muscarinic receptors to negatively modulate the cell growth in GBM cell lines and in the GSCs. The aim of this study was to better characterize the inhibitory effects of M2 receptors on cell proliferation and survival in GSCs and investigate the molecular mechanisms underlying the M2-mediated cell proliferation arrest and decreased survival. Moreover, we also evaluated the ability of M2 receptors to interfere with Notch1 and EGFR pathways, whose activation promotes GSCs proliferation. Our data demonstrate that M2 receptors activation impairs cell cycle progression and survival in the primary GSC lines analyzed (GB7 and GB8). Moreover, we also demonstrated the ability of M2 receptor to inhibit Notch1 and EGFR expression, highlighting a molecular interaction between M2 receptor and the Notch-1/EGFR pathways also in GSCs.

M2 Receptor Activation Counteracts the Glioblastoma Cancer Stem Cell Response to Hypoxia Condition.

Glioblastoma multiforme (GBM) is the most malignant brain tumor. Hypoxic condition is a predominant feature of the GBM contributing to tumor growth and resistance to conventional therapies. Hence, the identification of drugs able to impair GBM malignancy and aggressiveness is considered of great clinical relevance. Previously, we demonstrated that the activation of M2 muscarinic receptors, through the agonist arecaidine propargyl ester (Ape), arrests cell proliferation in GBM cancer stem cells (GSCs). In the present work, we have characterized the response of GSCs to hypoxic condition showing an upregulation of hypoxia-inducible factors and factors involved in the regulation of GSCs survival and proliferation. Ape treatment in hypoxic conditions is however able to inhibit cell cycle progression, causing a significant increase of aberrant mitosis with consequent decreased cell survival. Additionally, qRT-PCR analysis suggest that Ape downregulates the expression of stemness markers and miR-210 levels, one of the main regulators of the responses to hypoxic condition in different tumor types. Our data demonstrate that Ape impairs the GSCs proliferation and survival also in hypoxic condition, negatively modulating the adaptive response of GSCs to hypoxia.

Abstract B70: Universal adapter CAR-engineered NK-92 cells target patient-derived glioblastoma cancer stem cells

Abstract Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant brain tumor in adults. The current standard-of-care treatment, which includes surgical resection, radiation, and chemotherapy, extends the survival of patients to merely 14.6 months. GBM is virtually incurable due to its heterogeneity, which is represented by tumor cells with different patterns of gene expression (proneural, neural, classical, and mesenchymal GBM), the presence of GBM cancer stem (like) cells (CSC), and immune evasion. Thus, new therapeutic strategies directed at eliminating GBM CSC in particular are urgently needed. Utilizing fresh patient material from surgical resections, GBM CSC were successfully enriched and cultivated in serum-free medium and subsequently differentiated by switching to medium containing FBS. The gene expression pattern of both, GBM CSC and differentiated cells, was characterized. Furthermore, a screening platform was tailored for flow-cytometric characterization of patient-specific antigen expression of all newly established glioblastoma cell lines. In this work we present a promising immunotherapeutic approach towards eradication of GBM CSC by combining the universal targeting and controllability of our recently developed adapter chimeric antigen receptor (AdCAR) system with the “off-the-shelf” properties of the continuously expandable and well-characterized NK cell line NK-92. The AdCAR system is based on the unique properties of a novel scFv targeting a “neo”-epitope-like structure derived from the endogenous vitamin biotin, which can be linked to monoclonal antibodies (mAb). Utilizing these biotinylated mAb as adapter molecules, the system allows precise quantitative (on-/off switch) as well as qualitative (change and combination of target antigen) regulation of immune cell function. AdCAR-transduced NK-92 cells demonstrated significant target cell lysis, which, importantly, is largely independent from activation of endogenously expressed NK receptors and the presence of their ligands on target cells but mainly mediated by AdCAR activation through binding to the respective tumor antigen. In the presence of the respective antigen, first and foremost biotinylated antibodies directed at CD9, CD146, CD276, and EGFR were capable of inducing significant AdCAR NK-92-mediated lysis of GBM cells after 2 hours. Interestingly, due to overexpression of various target antigens on GBM cancer stem cells, CSC lysis was noticeably higher as compared to AdCAR-mediated lysis of differentiated glioblastoma cells. In conclusion, we have successfully generated a CAR-modified NK cell line, AdCAR NK-92, whose effector function can be tightly regulated and redirected against one or multiple antigens, allowing universal and tunable targeting of glioblastoma cells. Most importantly, AdCAR NK-92 cells provide a promising strategy to eradicate GBM cancer stem (like) cells. Citation Format: Stefan Grote, Chun-Ho Chan, Caroline Baden, Stephan M. Huber, Franziska Eckert, Joerg Mittelstaet, Andrew Kaiser, Christian Seitz, Patrick Schlegel, Rupert Handgretinger, Sabine Schleicher. Universal adapter CAR-engineered NK-92 cells target patient-derived glioblastoma cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B70.

Identifying conserved molecular targets required for cell migration of glioblastoma cancer stem cells

Glioblastoma (GBM) is the most prevalent primary malignant brain tumor and is associated with extensive tumor cell infiltration into the adjacent brain parenchyma. However, there are limited targeted therapies that address this disease hallmark. While the invasive capacity of self-renewing cancer stem cells (CSCs) and their non-CSC progeny has been investigated, the mode(s) of migration used by CSCs during invasion is currently unknown. Here we used time-lapse microscopy to evaluate the migratory behavior of CSCs, with a focus on identifying key regulators of migration. A head-to-head migration assay demonstrated that CSCs are more invasive than non-CSCs. Time-lapse live cell imaging further revealed that GBM patient-derived CSC models either migrate in a collective manner or in a single cell fashion. To uncover conserved molecular regulators responsible for collective cell invasion, we utilized the genetically tractable Drosophila border cell collective migration model. Candidates for functional studies were generated using results from a targeted Drosophila genetic screen followed by gene expression analysis of the human homologs in GBM tumors and associated GBM patient prognosis. This strategy identified the highly conserved small GTPase, Rap1a, as a potential regulator of cell invasion. Alteration of Rap1a activity impaired the forward progress of Drosophila border cells during development. Rap1a expression was elevated in GBM and associated with higher tumor grade. Functionally, the levels of activated Rap1a impacted CSC migration speed out of spheres onto extracellular matrix. The data presented here demonstrate that CSCs are more invasive than non-CSCs, are capable of both collective and single cell migration, and express conserved genes that are required for migration and invasion. Using this integrated approach, we identified a new role for Rap1a in the migration of GBM CSCs.

Off-target effect of the BMI1 inhibitor PTC596 drives epithelial-mesenchymal transition in glioblastoma multiforme

Glioblastoma multiforme (GBM) is an incurable primary brain tumor containing a sub-population of cancer stem cells (CSCs). Polycomb Repressive Complex (PRC) proteins BMI1 and EZH2 are enriched in CSCs, promoting clonogenic growth and resistance to genotoxic therapies. We report here that when used at appropriate concentrations, pharmaceutical inhibitors of BMI1 could efficiently prevent GBM colony growth and CSC self-renewal in vitro and significantly extend lifespan in terminally ill tumor-bearing mice. Notably, molecular analyses revealed that the commonly used PTC596 molecule targeted both BMI1 and EZH2, possibly providing beneficial therapeutic effects in some contexts. On the other hand, treatment with PTC596 resulted in instant reactivation of EZH2 target genes and induction of a molecular program of epithelial–mesenchymal transition (EMT), possibly explaining the modified phenotype of some PTC596-treated tumors. Treatment with a related but more specific BMI1 inhibitor resulted in tumor regression and maintenance of cell identity. We conclude that inhibition of BMI1 alone is efficient at inducing GBM regression, and that dual inhibition of BMI1 and EZH2 using PTC596 may be also beneficial but only in specific contexts.

Transforming growth factor-β mimics the key proteome properties of CD133- differentiated and CD133+ cancer stem cells in glioblastoma.

Glioblastoma multiforme is the most aggressive type of primary brain tumor in humans. Its invasive growth is associated with cluster of differentiation (CD)133 cancer stem cells (CSCs) and CD133- differentiated glioblastoma cells (DGCs) with aggressive phenotype, which are developed under the influence of transforming growth factor (TGF)-β. The present study aimed to compare the proteomes of CD133 CSCs and CD133- DGCs stimulated by TGF-β, as well as the expression levels of the main proteins responsible for activating the signaling pathway of receptor interactions with the extracellular matrix (ECM). The U87MG GBM cell line was used in this study. CSCs were extracted from gliomaspheres through magnetic-activated cell sorting based on the expression of CD133 (CD133); CD133- DCGs served as a control. CD133- DGCs of the U87-MG cell line were treated with 10ng/mL TGF-β1, and cell proliferation and migration were analyzed via real-time quantitative microscopy. High-performance liquid chromatography mass spectrometry was used for proteome analysis. The results revealed 589 proteins with significantly changes in expression among CD133 CSCs compared with those in CD133- DGCs (P<0.05). Bioinformatics analysis allowed to attribute 134 differentially expressed proteins to 15 signaling pathways; among these proteins, 14 were involved in signaling cascades associated with the interaction between CSCs and the ECM, and were upregulated >twofold, while four proteins activated this signaling cascade. TGF-β-stimulation increased the mobility, suppressed the proliferation and transformed the proteome profile of CD133- DGCs. Were identified 13 key proteins that activate the signaling pathway of receptor interaction with the ECM and three proteins activating this signaling pathway in CD133- DGCs which had the same values as those of CD133 CSCs. In conclusion, TGF-β increased the expression of proteins that activate the signaling pathway of receptor interaction with the ECM in CD133- DGCs to the level of those in CD133 CSCs.

Discovering Rare Genes Contributing to Cancer Stemness and Invasive Potential by GBM Single-Cell Transcriptional Analysis.

Single-cell RNA sequencing presents the sophisticated delineation of cell transcriptomes in many cancer types and highlights the tumor heterogeneity at higher resolution, which provides a new chance to explore the molecular mechanism in a minority of cells. In this study, we utilized publicly available single-cell RNA-seq data to discover and comprehensively dissect rare genes existing in few glioblastoma (GBM) cells. Moreover, we designed a framework to systematically identify 51 rare protein-coding genes (PCGs) and 47 rare long non-coding RNAs (lncRNAs) in GBM. Patients with high expression levels of rare genes like CYB5R2 and TPPP3 had worse overall survival and disease-free survival, implying their potential implication in GBM progression and prognosis. We found that these rare genes tended to be specifically expressed in GBM cancer stem cells, which emphasized their ability to characterize stem-like cancer cells and implied their contribution to GBM growth. Furthermore, rare genes were enriched in a 17-cell subset, which was located in an individual branch of the pseudotime trajectory of cancer progression and exhibited high cell cycle activity and invasive potential. Our study captures the rare genes highly expressed in few cells, deepens our understanding of special states during GBM tumorigenesis and progression such as cancer stemness and invasion, and proposes potential targets for cancer therapy.

Abstract A058: Dielectric characterization of glioblastoma cancer stem cells

Abstract Glioblastoma (GBM) is the most aggressive type of primary brain tumor, characterized by the intrinsic resistance to chemotherapy due to the presence of a highly aggressive Cancer Stem Cell (CSC) sub-population. The coexistence in the same mass of bulk and CSCs stuck at different stages of differentiation and genetic backgrounds prevents counteracting all cells with single treatments. Through the application of a Dielectrophoretic force (DEP) we provide a successful CSC isolation from a mixed cell population, exploiting their physical properties. Using FACS, the expression of specific stem cell and differentiation markers (CD133, Nestin, Sox1, Sox2, GFAP, Doublecortin (DCX) and Ki67) were analyzed in 17 primary cell lines of GBM. CD133 expression was also exploited as a reliable marker for the identification of the CSC subpopulation and thus used for sorting CD133+ and CD133- GBM primary cells by flow cytometry techniques. CD133+ and CD133- cells were characterized for their ability to self-renew and form neurospheres. In all experiments, CD133+ cells have shown a significant increased efficiency of neurosphere generation that was accompanied by a higher self-renewal potential as measured by the limiting dilution assay (LDA) in vitro. The characterization and isolation of patient-derived GBM cells by FACS allowed constituting a cell library with three distinct cell subpopulation types: CD133+/CD133-/Total population, that have been characterized for their specific Electromagnetic (EM) signature. Determination of EM signature was performed using a sensor that applies electromagnetic fields in the MHz regime to selectively electromanipulate cells with dielectrophoresis (DEP) and allows the monitoring of the induced cell motion generated by the field. By scanning the frequency range of interest, the cell DEP signature and the cross over frequency (frequency for which the DEP force becomes null) were identified. We found that CD133+ and CD133- cell display a significantly different crossover frequency. In particular, CD133+ cells are characterized by a lower cross over frequency than both CD133- and total population. This data suggest a non obvious correlation between crossover frequency and CSCs phenotype which would be potentially exploited to set up a label free system able to efficiently identify and physically separate subpopulation of cells enriched for CSC characteristics. Citation Format: Roberta Bortolozzi, Elena Mariotto, Elena Rampazzo, Elena Porcù, Sofiane Saada, Thomas Provent, Fabrice Lalloué, Arnaud Pothier, Giampietro Viola, Luca Persano. Dielectric characterization of glioblastoma cancer stem cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A058. doi:10.1158/1535-7163.TARG-19-A058