Patient-derived Glioblastoma Stem-like Cells Research Articles

MicroRNA-Mediated Dynamic Bidirectional Shift between the Subclasses of Glioblastoma Stem-like Cells

Large-scale transcriptomic profiling of glioblastoma(GBM) into subtypes has provided remarkable insight into the pathobiology and heterogeneous nature of this disease. The mechanisms of speciation and inter-subtype transitions of these molecular subtypes require better characterization to facilitate the development of subtype-specific targeting strategies. The deregulation of microRNA expression among GBM subtypes and their subtype-specific targeting mechanisms are poorly understood. To reveal the underlying basis of microRNA-driven complex subpopulation dynamics within the heterogeneous intra-tumoral ecosystem, we characterized the expression of the subtype-enriched microRNA-128 (miR-128) in transcriptionally and phenotypicallydiverse subpopulations of patient-derived glioblastoma stem-like cells. Because microRNAs are capable of re-arranging the molecular landscape in a cell-type-specific manner, we argue that alterations in miR-128 levels are a potent mechanism of bidirectional transitions between GBM subpopulations, resulting in intermediate hybrid stages and emphasizing highly intricate intra-tumoral networking.

MicroRNA Signatures and Molecular Subtypes of Glioblastoma: The Role of Extracellular Transfer.

SummaryDespite the importance of molecular subtype classification of glioblastoma (GBM), the extent of extracellular vesicle (EV)-driven molecular and phenotypic reprogramming remains poorly understood. To reveal complex subpopulation dynamics within the heterogeneous intratumoral ecosystem, we characterized microRNA expression and secretion in phenotypically diverse subpopulations of patient-derived GBM stem-like cells (GSCs). As EVs and microRNAs convey information that rearranges the molecular landscape in a cell type-specific manner, we argue that intratumoral exchange of microRNA augments the heterogeneity of GSC that is reflected in highly heterogeneous profile of microRNA expression in GBM subtypes.

PNIPAAm-co-Jeffamine® (PNJ) scaffolds as invitro 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 invivo 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 invitro.

M6A Demethylase ALKBH5 Maintains Tumorigenicity of Glioblastoma Stem-like Cells by Sustaining FOXM1 Expression and Cell Proliferation Program

The dynamic and reversible N6-methyladenosine (m6A) RNA modification installed and erased by N6-methyltransferases and demethylases regulates gene expression and cell fate. We show that the m6A demethylase ALKBH5 is highly expressed in glioblastoma stem-like cells (GSCs). Silencing ALKBH5 suppresses the proliferation of patient-derived GSCs. Integrated transcriptome and m6A-seq analyses revealed altered expression of certain ALKBH5 target genes, including the transcription factor FOXM1. ALKBH5 demethylates FOXM1 nascent transcripts, leading to enhanced FOXM1 expression. Furthermore, a long non-coding RNA antisense to FOXM1 (FOXM1-AS) promotes the interaction of ALKBH5 with FOXM1 nascent transcripts. Depleting ALKBH5 and FOXM1-AS disrupted GSC tumorigenesis through the FOXM1 axis. Our work uncovers a critical function for ALKBH5 and provides insight into critical roles of m6A methylation in glioblastoma.

Dissecting inherent intratumor heterogeneity in patient-derived glioblastoma culture models

Molecular profile of glioblastoma multiforme (GBM) revealed 4 subtypes, 2 of which, proneural and mesenchymal, have been predominantly observed, with the latter displaying a more aggressive phenotype and increased therapeutic resistance. Single-cell RNA sequencing revealed that multiple subtypes actually reside within the same tumor, suggesting cellular heterogeneity in GBM. Further, plasticity between these 2 subtypes is observed during tumor recurrence and in response to radiation therapy. Patient-derived GBM stemlike cells were cultured as neurospheres. These cells were differentiated in serum by attaching to the culture dishes. The "floating" cells that were not attached/differentiated were harvested from the conditioned medium. The characteristics of these cells were studied with limiting dilution assays and immunofluorescence staining. Cell growth and nuclear factor-kappaB (NFkB) activation were monitored using bioluminescent assays as well as quantitative polymerase chain reaction and western blotting. In vivo tumorigenesis was evaluated in orthotopic xenograft models using bioluminescence imaging. Patient-derived GBM stemlike cells undergo differentiation by attaching to the culture dish in serum-containing medium. We observed that a small subset of these cells escape this adhesion/differentiation and grow as floating cells. These cells displayed enhanced cancer stem cell properties with a molecular and phenotypic mesenchymal signature, including resistance to radiation and targeted therapies, a more aggressive tumor formation, and NFkB activation. Our results endorse inherent intratumor molecular subtype heterogeneity in glioblastoma and provide a valuable approach to study phenotypic plasticity, which could be applied to find novel therapeutic strategies to eradicate this aggressive tumor and can be extended to other cancer types.

An NAD+-dependent transcriptional program governs self-renewal and radiation resistance in glioblastoma

Accumulating evidence suggests cancer cells exhibit a dependency on metabolic pathways regulated by nicotinamide adenine dinucleotide (NAD+). Nevertheless, how the regulation of this metabolic cofactor interfaces with signal transduction networks remains poorly understood in glioblastoma. Here, we report nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting step in NAD+ synthesis, is highly expressed in glioblastoma tumors and patient-derived glioblastoma stem-like cells (GSCs). High NAMPT expression in tumors correlates with decreased patient survival. Pharmacological and genetic inhibition of NAMPT decreased NAD+ levels and GSC self-renewal capacity, and NAMPT knockdown inhibited the in vivo tumorigenicity of GSCs. Regulatory network analysis of RNA sequencing data using GSCs treated with NAMPT inhibitor identified transcription factor E2F2 as the center of a transcriptional hub in the NAD+-dependent network. Accordingly, we demonstrate E2F2 is required for GSC self-renewal. Downstream, E2F2 drives the transcription of members of the inhibitor of differentiation (ID) helix-loop-helix gene family. Finally, we find NAMPT mediates GSC radiation resistance. The identification of a NAMPT-E2F2-ID axis establishes a link between NAD+ metabolism and a self-renewal transcriptional program in glioblastoma, with therapeutic implications for this formidable cancer.

The effects of tumor treating fields and temozolomide in MGMT expressing and non-expressing patient-derived glioblastoma cells

A recent Phase 3 study of newly diagnosed glioblastoma (GBM) demonstrated the addition of tumor treating fields (TTFields) to temozolomide (TMZ) after combined radiation/TMZ significantly increased survival and progression free survival. Preliminary data suggested benefit with both methylated and unmethylated O-6-methylguanine-DNA methyl-transferase (MGMT) promoter status. To date, however, there have been no studies to address the potential interactions of TTFields and TMZ. Thus, the effects of TTFields and TMZ were studied in vitro using patient-derived GBM stem-like cells (GSCs) including MGMT expressing (TMZ resistant: 12.1 and 22GSC) and non-MGMT expressing (TMZ sensitive: 33 and 114GSC) lines. Dose-response curves were constructed using cell proliferation and sphere-forming assays. Results demonstrated a ⩾10-fold increase in TMZ resistance of MGMT-expressing (12.1GSCs: IC50=160μM; 22GSCs: IC50=44μM) compared to MGMT non-expressing (33GSCs: IC50=1.5μM; 114GSCs: IC50=5.2μM) lines. TTFields inhibited 12.1 GSC proliferation at all tested doses (50–500kHz) with an optimal frequency of 200kHz. At 200kHz, TTFields inhibited proliferation and tumor sphere formation of both MGMT GSC subtypes at comparable levels (12.1GSC: 74±2.9% and 38±3.2%, respectively; 22GSC: 61±11% and 38±2.6%, respectively; 33GSC: 56±9.5% and 60±7.1%, respectively; 114 GSC: 79±3.5% and 41±4.3%, respectively). In combination, TTFields (200kHz) and TMZ showed an additive anti-neoplastic effect with equal efficacy for TTFields in both cell types (i.e., ± MGMT expression) with no effect on TMZ resistance. This is the first demonstration of the effects of TTFields on cancer stem cells. The expansion of such studies may have clinical implications.

CSIG-20. IDENTIFICATION OF A NAMPT-E2F2-ID1 SIGNALING AXIS IN GLIOBLASTOMA

Emerging evidence shows that nicotinamide adenine dinucleotide (NAD+)-dependent signaling plays a critical role in a variety of biological contexts including cancer. The rate-limiting enzyme of the mammalian NAD+ synthesis pathway, nicotinamide phosphoribosyltransferase (NAMPT), governs NAD+-dependent processes by catalyzing production of nicotinamide mononucleotide (NMN), a key NAD+ precursor. However, the role and molecular mechanisms of NAMPT in the biology of human glioblastoma have remained poorly understood. Here, we demonstrate that NAMPT is highly expressed in glioblastoma tumor specimens and patient-derived glioblastoma stem-like cells (GSCs). High NAMPT expression in glioblastoma tumors correlates with poor prognosis in patients. NAMPT inhibition using FK866 as well as RNA interference (RNAi) depleted NAD+ levels in GSCs and substantially decreased GSC self-renewal. Importantly, NAMPT knockdown diminished the in vivo tumorigenicity of GSCs and prolonged the survival of animals in an orthotopic xenograft model. Bioinformatic analysis of RNA-sequencing data derived from FK866-treated GSCs revealed E2F family transcription factor E2F2 as a potential hub of the NAD+-dependent transcriptome. Accordingly, E2F2 knockdown decreased the self-renewal capacity of GSCs. Downstream, we find that E2F2 directly controls the transcription of helix-loop-helix protein inhibitor of differentiation 1 (ID1), a known regulator of self-renewal. Finally, we show that NAMPT promotes radiation resistance of GSCs in culture, highlighting the potential therapeutic relevance of this pathway. The identification of a NAMPT-E2F2-ID1 signaling pathway, which regulates GSC maintenance, has ramifications for the development of novel therapeutic strategies for glioblastoma patients.

TP53 mutated glioblastoma stem-like cell cultures are sensitive to dual mTORC1/2 inhibition while resistance in TP53 wild type cultures can be overcome by combined inhibition of mTORC1/2 and Bcl-2.

BackgroundGlioblastoma is the most malignant tumor of the central nervous system and still lacks effective treatment. This study explores mutational biomarkers of 11 drugs targeting either the RTK/Ras/PI3K, the p53 or the Rb pathway using 25 patient-derived glioblastoma stem-like cell cultures (GSCs).ResultsWe found that TP53 mutated GSCs were approximately 3.5 fold more sensitive to dual inhibition of mammalian target of rapamycin complex 1 and 2 (mTORC1/2) compared to wild type GSCs. We identified that Bcl-2(Thr56/Ser70) phosphorylation contributed to the resistance of TP53 wild type GSCs against dual mTORC1/2 inhibition. The Bcl-2 inhibitor ABT-263 (navitoclax) increased sensitivity to the mTORC1/2 inhibitor AZD8055 in TP53 wild type GSCs, while sensitivity to AZD8055 in TP53 mutated GSCs remained unchanged.ConclusionOur data suggest that Bcl-2 confers resistance to mTORC1/2 inhibitors in TP53 wild type GSCs and that combined inhibition of both mTORC1/2 and Bcl-2 is worthwhile to explore further in TP53 wild type glioblastomas, whereas in TP53 mutated glioblastomas dual mTORC1/2 inhibitors should be explored.

Abstract 4370: Genome-wide CRISPR-Cas9 screens reveal loss of redundancy between PKMYT1 and WEE1 in patient-derived glioblastoma stem-like cells

Abstract Glioblastoma multiforme (GBM) is the most aggressive and common form of brain cancer in adults. There are currently no effective therapies for GBM. Even with standard of care treatments, such as surgery, radiation, and chemotherapy, ∼90% of adult patients die within 2 years of diagnosis. To identify therapeutic targets for Glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 “knockout” (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit Cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, likely as a result of oncogenic signaling, causing GBM-specific lethality. From a biological standpoint, our results help re-discover PKMYT1 function in human cells. From a cancer standpoint, these results suggest that targeting PKMYT1 is a glioma-lethal gene and that tandem or sequential use of PKMYT1 and WEE1 inhibitors may illicit therapeutic responses in GBM. In addition to these results we will also present retest data for other candidate glioma-lethal genes and networks in multiple patient samples. Citation Format: Chad Toledo, Ding Yu, Pia Hoellerbauer, Ryan Davis, Ryan Basom, Emily Girard, Philip Corrin, Hamid Bolouri, Jerry Davison, Qing Zhang, Do-Hyun Nam, Jeongwu Lee, Steven Pollard, Jeffery Delrow, Bruce Clurman, James Olson, Patrick Paddison. Genome-wide CRISPR-Cas9 screens reveal loss of redundancy between PKMYT1 and WEE1 in patient-derived glioblastoma stem-like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4370.

Abstract 2523: Transcriptional profiling of glioblastoma stem-like cells reveals enrichment of ion channels with functional implications for malignancy

Abstract Glioblastoma Multiforme (GBM) is the most prevalent and aggressive form of cancer in the adult central nervous system. Ion channels are increasingly being linked to cancer progression through the regulation of cell proliferation and migration. However, the role of ion channels in GBM tumor formation and progression is not well understood. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence, suggesting that ion channel expression may be perturbed in this population. Here, we used RNA-sequencing to assess the expression patterns of ion channels and transporters and identify uniquely enriched ion channels in GSCs. Twenty-two patient-derived GSC samples were expression profiled along with human neural stem cell (NSC) and astrocyte control cell populations. Differential expression analysis revealed a set of ion channels highly enriched in GSCs compared with controls. Real-time PCR analysis confirmed the increased expression of ion channels of interest across selected GSC lines compared to NSCs. The expression pattern of ion channel candidates was also more likely to be associated with distinct GBM molecular subtypes. Next, an integrative approach was taken to further identify ion channels unique to GSCs; the abovementioned findings were compared to results from transcriptome analyses of GBM bulk tumor cells (The Cancer Genome Atlas) and region-specific GBM cells (Ivy Glioblastoma Atlas Project). Ion channels that were identified in these analyses were associated with altered clinical outcomes. The functional implications of these expression changes were further assessed with targeted drug screening of GSCs and NSCs. Pharmacological antagonists of GSC-enriched ion channels suppressed stem cell viability. These antagonists similarly hindered BrdU incorporation of dividing GSCs, suggesting that these channels play a role in stem cell proliferative capacity. Finally, calcium imaging was used to test the real-time functional responses of GSCs to channel blockers, and these outcomes will be discussed. Collectively, these findings suggest the presence of ion channels that uniquely identify GSCs from other neural cell types and influence GSC proliferation, a hallmark of GBM tumor recurrence. Citation Format: Julia Pollak, Karan G. Rai, Patrick J. Paddison, Robert C. Rostomily, Jan-Marino Ramirez. Transcriptional profiling of glioblastoma stem-like cells reveals enrichment of ion channels with functional implications for malignancy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2523.

EphrinB2 drives perivascular invasion and proliferation of glioblastoma stem-like cells.

Glioblastomas (GBM) are aggressive and therapy-resistant brain tumours, which contain a subpopulation of tumour-propagating glioblastoma stem-like cells (GSC) thought to drive progression and recurrence. Diffuse invasion of the brain parenchyma, including along preexisting blood vessels, is a leading cause of therapeutic resistance, but the mechanisms remain unclear. Here, we show that ephrin-B2 mediates GSC perivascular invasion. Intravital imaging, coupled with mechanistic studies in murine GBM models and patient-derived GSC, revealed that endothelial ephrin-B2 compartmentalises non-tumourigenic cells. In contrast, upregulation of the same ephrin-B2 ligand in GSC enabled perivascular migration through homotypic forward signalling. Surprisingly, ephrin-B2 reverse signalling also promoted tumourigenesis cell-autonomously, by mediating anchorage-independent cytokinesis via RhoA. In human GSC-derived orthotopic xenografts, EFNB2 knock-down blocked tumour initiation and treatment of established tumours with ephrin-B2-blocking antibodies suppressed progression. Thus, our results indicate that targeting ephrin-B2 may be an effective strategy for the simultaneous inhibition of invasion and proliferation in GBM.

Modulation of Natural Killer Cell Activity in the Setting of Oncolytic Virotherapy and with a Chimeric Antigen Receptor

Natural killer (NK) cells are innate lymphocytes that can rapidly eradicate tumor cells, especially those lacking MHC Class I molecules. NK cells can also rapidly eradicate herpes virus-infected cells. We designed an oncolytic herpes virus (oHSV) to selectively infect, replicate within, and lyse glioblastoma (GBM), a devastating brain tumor with a median survival of only 15 months following diagnosis. We have shown that the rapid influx of NK cells limits oHSV efficacy in GBM as they impede oHSV replication and spread [Alvarez-Breckenridge et al., Nat Med, 2012, 18(12):1827-34]. In the current study, we developed NK cell-based novel GBM therapies by decreasing the brain influx of NK cells to enhance the efficacy of oHSV, while arming NK cells in the brain with a chimeric antigen receptor (CAR) that targets both the wild-type EGFR and its mutant form EGFRvIII, two GBM tumor-associated antigens. We then investigated the synergistic effects between EGFR-CAR NK cells and oHSV.Transforming growth factor (TGF)-β is a potent immunosuppressive cytokine of NK cells [Yu et al, Immunity, 2006, 24(5):575-90]. We first determined if oHSV efficacy for treatment of GBM would be augmented by inhibiting anti-oHSV activity of NK cells with TGF-β pre-treatment. In vitro, NK cells pre-treated with TGF-β displayed less cytolytic capacity against oHSV-infected GBM cell lines and patient-derived GBM stem-like cells. In viral replication assays, co-culturing oHSV-infected GBM cells with NK cells pre-treated with TGF-β significantly increased virus titers. In an immunocompetent syngeneic GBM mouse model,administration of TGF-β to GBM-bearing mice prior to oHSV injection significantly inhibited intracranial infiltration and activation of NK cells (P < 0.05). In orthotopic human GBM xenograft mouse models and in syngeneic GBM mouse models, TGF-β treatment in vivo prior to oHSV therapy resulted in inhibition of NK cell infiltration, suppression of tumor growth and significantly prolonged survival of GBM-bearing mice (P < 0.05). Furthermore, depletion of NK cells incompletely blocked the positive effects of in vivo treatment of GBM with TGF-β on survival, suggesting that TGF-β may also directly act on other innate immune cells such as macrophages/microglia. These data demonstrate a single dose of TGF-β prior to oHSV administration enhances anti-tumor efficacy for GBM at least in part through the transient inhibition of the innate immune responses to oHSV infection.We next investigated whether NK cell activity could be enhanced to more directly target brain tumors while sparing eradication of oHSV. We therefore infected both human NK-92 cells and primary human NK cells to express the second generation CAR targeting both EGFR and EGFRvIII that we designed. Further, we asked if the treatment with EGFR-CAR NK cells plus oHSV could create a therapeutic synergy for the treatment to brain tumors. In vitro, compared with mock-transduced CAR-NK-cells, EGFR-CAR NK cells exhibited significantly higher cytotoxicity and IFN-γ production when co-cultured with tumor cells, for both NK-92 and primary NK cells (P < 0.01). Further, significantly higher cytolytic activity against tumor cells was obtained when CAR NK cells were combined with oHSV-1 infection of tumor cells, compared to either of the monotherapies alone (P < 0.05). In mice, to avoid oHSV clearance by the EGFR-CAR NK cells following the inoculation of the mouse with tumor cells, we administered these two agents sequentially; administering EGFR-CAR NK cells directly into the tumor first as a single injection of 2 × 106 cells, followed by intracranial infection with 2 × 105 plaque-forming units oHSV five days later, presumably after EGFR-CAR NK survival has diminished. Compared to vehicle controls, intracranial administration of either EGFR-CAR NK cells or oHSV blunted tumor growth. However, the combination of EGFR-CAR NK cells followed by oHSV infection resulted in significantly more efficient killing of tumor cells (P < 0.05) and significantly longer survival for tumor-bearing mice when compared to either monotherapy alone.Collectively, our studies demonstrate that in animal tumor models, we can combine novel NK cell and oHSV therapies to significantly improve survival. DisclosuresNo relevant conflicts of interest to declare.

Abstract C159: Genome-wide CRISPR-Cas9 screens uncover therapeutic targets and tumor suppressor genes in glioblastoma multiforme

Abstract Precision oncology is currently based on the notion that genomic analysis of descriptive molecular signatures from patient tumors will lead to actionable therapeutic targets following the patient's arrival into the clinic. However, this approach has failed to deliver new effective treatments for glioblastoma multiforme (GBM), which is the most common and aggressive form of brain cancer; and thus, ∼90% of adult GBM patients receiving standard of care therapies continue to die within 2 years of diagnosis. In addition, this approach has neglected the importance for using functional genetics in precision oncology. To identify new therapeutic targets for GBM, we applied functional genetics to perform lethal genome-wide CRISPR-Cas9 knockout screens in patient-derived GBM stem-like cells (GSCs) and also non-transformed human neural stem cells (NSCs), non-neoplastic tissue of origin controls. Here, we present our latest findings from these screens, which include multiple novel GBM-specific lethal genes that were validated by both in vitro and in vivo preclinical studies. Knockout of GBM-specific lethal genes, including the WEE1-like kinase, PKMYT1/Myt1, lead to lethality in GSCs, but not NSCs. Focused mechanistic studies revealed that PKMYT1 acts redundantly with WEE1 to phosphorylate CDK1-Y15 and to promote timely completion of mitosis in NSCs, but that this redundancy is lost in most GBM isolates and in NSCs harboring activated alleles of EGFR and AKT1, which are commonly altered signaling pathways in GBM. Moreover, PKMYT1 depletion in GSCs and genetically altered NSCs requiring PKMYT1 lead to cytokinesis failure and cell death during mitosis. In addition to lethal genes, genes promoting in vitro expansion of NSCs upon knockout were examined. For this category, we validated multiple genes that are candidate tumor suppressors and involved in: the negative regulation of Hippo signaling, TP53 signaling, epigenetic regulation, promoting neural development, and other cellular functions. Knockout of these genes caused shortened cell cycle transit times and drastic growth advantages in NSCs, and in the case of CREBBP knockout, caused precious entry into S-phase and deregulation of cell cycle gene expression. The identification of these potential tumor suppressors here reveal new genetic drivers in glioma/GBM, which contributes to a growing body of work that will help redefine GBM gene signatures. Furthermore, we found that the molecular signatures of pathways and genes commonly altered in GBM are not ideal GBM therapeutic targets since most of these targets failed to score as GBM-specific lethal hits in our knockout screens and likely are non-essential or essential in both the GSCs and NSCs. Nonetheless, these common GBM alterations give rise to cancer-specific vulnerabilities, which then lead to genes, such as PKMYT1, that are required to overcome functional impairments in cancer cells. Taken together, we demonstrated here that genomics and functional genetics are equally important for precision oncology, as the combination of both tools can identify genetically altered genes, cancer-specific therapeutic targets, and tumor suppressors. These results are part of a rapidly growing body of work by the science community that will one day allow oncologists to tailor therapies for each patient based upon his or her tumor genetic profile and characteristics. Citation Format: Chad M. Toledo, Yu Ding, Pia Hoellerbauer, Ryan J. Davis, Ryan Basom, Emily J. Girard, Eunjee Lee, Philip Corrin, Hamid Bolouri, Jerry Davison, Qing Zhang, Do-Hyun Nam, Jeongwu Lee, Steven M. Pollard, Jun Zhu, Jeffery J. Delrow, Bruce E. Clurman, James M. Olson, Patrick J. Paddison. Genome-wide CRISPR-Cas9 screens uncover therapeutic targets and tumor suppressor genes in glioblastoma multiforme. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C159.

Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells.

To identify therapeutic targets for glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 knockout (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, most likely as a result of oncogenic signaling, causing GBM-specific lethality.

ATPS-60COMPREHENSIVE IDENTIFICATION OF GLIOBLASTOMA MULTIFORME THERAPEUTIC TARGETS AND GROWTH SUPPRESSOR GENES USING GENE EDITING

Glioblastoma multiforme (GBM) is the most aggressive and common form of brain cancer in adults and is currently incurable. To identify new therapeutic targets for GBM, we performed genome-wide CRISPR-Cas9 knockout (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem cells (NSCs), non-neoplastic tissue of origin controls. These screens revealed thousands of gene candidates acting to promote or limit expansion of GSCs and NSCs. In vitro and in vivo validation studies revealed several novel therapeutic targets, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies revealed that PKMYT1 acts redundantly with WEE1 to facilitate mitosis in NSCs, but that this redundancy is lost in most GBM isolates and in NSCs harboring activated alleles of EGFR and AKT1. In addition to lethal genes, KOs promoting in vitro expansion of NSCs were also examined. For this category, we validated multiple genes, including: ARID1A, ARID1B, CREBBP, EP300, NF2, PDCD10, PTPN14, TAOK1, TGFBR2, and TP53, whose inhibition shortens NSC cell cycle transit times, causing dramatic growth advantages, and, in the case of CREBBP KO, dramatic deregulation of cell cycle gene expression and precocious S-phase entry. Taken together, these results demonstrate the use of CRISPR-Cas9 gene editing to identify novel cancer therapeutic targets and growth suppressor genes in human GBM isolates and NSCs. The results also inform precision medicine approaches for GBM, since most, if not all, candidate therapeutic targets could not be predicted from mutation, CNV, or gene expression analysis of the patient isolates alone.

STEM-11MECHANISMS OF CDC20-ANAPHASE-PROMOTING COMPLEX ACTION IN HUMAN GLIOBLASTOMA STEM-LIKE CELLS

Recent studies have shown that the major mitotic regulator CDC20-Anaphase Promoting Complex (APC) is required for the maintenance of human glioblastoma stem-like cells (GSCs), a clinically important subpopulation of glioblastoma cells that are believed to underlie tumor recurrence. We have previously shown that CDC20-APC operates through pluripotency transcription factor SOX2 to promote GSC self-renewal and invasion in vitro (Mao, Gujar et al. Cell Reports. 2015). Using patient-derived GSCs, we herein show that APC co-activator CDC20 is required for GSC tumorigenicity in an orthotopic xenograft model. We further demonstrate through in vivo epistasis experiments that CDC20 acts through SOX2 to control GSC tumorigenicity. To test if CDC20-APC activity might dictate GSC responsiveness to standard-of-care chemotherapy agent temozolomide (TMZ), we utilized pharmacological and genetic approaches to inhibit CDC20-APC activity. Intriguingly, both APC inhibitor ProTAME and lentiviral CDC20 RNA interference augmented TMZ cytotoxicity in human GSCs. Collectively, these data suggest that CDC20-APC controls the tumor-initiating potential of GSCs in vivo through SOX2 and that CDC20-APC inhibitory strategies may not only disrupt the GSC state but also enhance the efficacy of chemotherapy.

MiR-135b suppresses tumorigenesis in glioblastoma stem-like cells impairing proliferation, migration and self-renewal.

Glioblastoma multiforme (GBM) is the most common and fatal malignant adult primary brain tumor. Currently, the overall prognosis for GBM patients remains poor despite advances in neurosurgery and adjuvant treatments. MicroRNAs (miRNAs) contribute to the pathogenesis of various types of tumor, including GBM. In this study we analyzed the expression of a panel of miRNAs, which are known to be differentially expressed by the brain and GBM tumor, in a collection of patient-derived GBM stem-like cells (GSCs). Notably, the average expression level of miR-135b, was the most downregulated compared to its normal counterpart, suggesting a potential role as anti-oncogene.Restoration of miR-135b in GSCs significantly decreased proliferation, migration and clonogenic abilities. More importantly, miR-135b restoration was able to significantly reduce brain infiltration in mouse models of GBM obtained by intracerebral injection of GSC lines. We identified ADAM12 and confirmed SMAD5 and GSK3β as miR-135b targets and potential mediators of its effects. The whole transcriptome analysis ascertained that the expression of miR-135b downmodulated additional genes driving key pathways in GBM survival and infiltration capabilities.Our results identify a critical role of miR-135b in the regulation of GBM development, suggesting that miR-135b might act as a tumor-suppressor factor and thus providing a potential candidate for the treatment of GBM patients.

Abstract B39: Targeting cancer stem cells via inhibition of PI3K/AKT pathway alone and in combination with autophagy blockade

Abstract Background: Glioblastoma Multiforme (GBM) is the most aggressive form of malignant primary brain tumor. Despite an aggressive treatment regimen consisting of surgery, radiation and chemotherapy, individuals with GBM survive on average 15 months. A subset of glioblastoma cells, known as glioblastoma stem-like cells (GSCs), are chemotherapy and radiotherapy-resistent and are hypothesized to be responsible for tumor formation, maintenance, and recurrence. The PI3K/AKT pathway is overactive in GBM and has been shown to play an important role in cancer stem cell maintenance. Therefore we sought to determine the efficacy of drugs targeting the AKT pathway in multiple patient-derived GSC lines. In addition, we evaluated the cytotxicity of the most promising therapy on medulloblastoma and neuroblastoma stem cells. Methods: GSC lines were generated from patient's tumors, propagated in neurosphere media and analyzed for stem cell markers by immunocytochemistry. Effect of growth factor receptor inhibitors (erlotinib, gefetinib, sunitinib), the intracellular PI3K/AKT pathway inhibitors (GDC-0941, OSU-03012, perifosine, NVP-BEZ235, KU-0063794 and everolimus) and autophagy inhibitor (chloroquine) on cell viability was determined with MTS assay. Caspase 3/7 activity was determined using CellEvent caspase reagent. The effect of the inhibitors on cell signaling pathways was evaluated by western blot analysis. Medulloblastoma and neuroblastoma stem cells were derived from Daoy and NB1691 cells respectively via propagation in neurosphere media. Results: GSC's expressed nestin, CD133, GFAP, musashi, BMi1, and SOX2. In addition, transplantation of GSC's into nude mice generated tumors. Of 11 GSC lines examined, all 11 demonstrated activation of the AKT pathway as indicated by robust phosphorylation of AKT (S473) and BAD (S136) and 10/11 demonstrated phosphorylation of mTOR (S2481). Growth factor receptor inhibition failed to induce significant reduction in cell viability. Intracellular inhibitors produced variable results except for dual PI3K/mTOR inhibitor NVB-BEZ235 (100nM) which consistantly induced approximately 50% reduction in cell viability. While the inhibition of autophagy with chloroquine significantly increased inhibitor induced cell death in some cell lines, combined PDK1 and autophagy inhibition induced robust caspase activity and significantly reduced viability in all six GSCs examined to an average of 6.1±2.7% viability compared to non-treated controls (100±1.9%). Similarly the combination significantly reduced both DAOY and NB1691 stem cell viability. In contrast the combination had no effect on primary neuronal cultures. Conclusion: The AKT/PI3K pathway is overactive in GSCs and may contribute to stem cell maintenance as well as inhibition of apoptosis. Our data indicates that inhibiting autophagy concommitantly with PI3K/AKT pathway inhibition can potentiate their cytotoxic effect. Furthermore combined chloroquine and OSU-03012 resulted in a substantial decrease cell viability accross multiple GSC lines and in medulloblastoma and neuroblastoma stem cells suggesting that this combination may represent a potential adjuvant therapy these cancers. Citation Format: Regina M. Graham, Andrew Middleton, Daniel A. Benito, Raisa Uddin, Baoyu Zhang, Winston Walters, Amade Bregy, Steven Vanni, Ricardo J. Komotar. Targeting cancer stem cells via inhibition of PI3K/AKT pathway alone and in combination with autophagy blockade. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B39.

The HDAC Inhibitors Scriptaid and LBH589 Combined with the Oncolytic Virus Delta24-RGD Exert Enhanced Anti-Tumor Efficacy in Patient-Derived Glioblastoma Cells.

BackgroundA phase I/II trial for glioblastoma with the oncolytic adenovirus Delta24-RGD was recently completed. Delta24-RGD conditionally replicates in cells with a disrupted retinoblastoma-pathway and enters cells via αvβ3/5 integrins. Glioblastomas are differentially sensitive to Delta24-RGD. HDAC inhibitors (HDACi) affect integrins and share common cell death pathways with Delta24-RGD. We studied the combination treatment effects of HDACi and Delta24-RGD in patient-derived glioblastoma stem-like cells (GSC), and we determined the most effective HDACi.MethodsSAHA, Valproic Acid, Scriptaid, MS275 and LBH589 were combined with Delta24-RGD in fourteen distinct GSCs. Synergy was determined by Chou Talalay method. Viral infection and replication were assessed using luciferase and GFP encoding vectors and hexon-titration assays. Coxsackie adenovirus receptor and αvβ3 integrin levels were determined by flow cytometry. Oncolysis and mechanisms of cell death were studied by viability, caspase-3/7, LDH and LC3B/p62, phospho-p70S6K. Toxicity was studied on normal human astrocytes. MGMT promotor methylation status, TCGA classification, Rb-pathway and integrin gene expression levels were assessed as markers of responsiveness.ResultsScriptaid and LBH589 acted synergistically with Delta24-RGD in approximately 50% of the GSCs. Both drugs moderately increased αvβ3 integrin levels and viral infection in responding but not in non-responding GSCs. LBH589 moderately increased late viral gene expression, however, virus titration revealed diminished viral progeny production by both HDACi, Scriptaid augmented caspase-3/7 activity, LC3B conversion, p62 and phospho-p70S6K consumption, as well as LDH levels. LBH589 increased LDH and phospho-p70S6K consumption. Responsiveness correlated with expression of various Rb-pathway genes and integrins. Combination treatments induced limited toxicity to human astrocytes.ConclusionLBH589 and Scriptaid combined with Delta24-RGD revealed synergistic anti-tumor activity in a subset of GSCs. Both HDACi moderately augmented viral infection and late gene expression, but slightly reduced progeny production. The drugs differentially activated multiple cell death pathways. The limited toxicity on astrocytes supports further evaluation of the proposed combination therapies.