Jun Fu

Antitumor Activity of NVP-BKM120—A Selective Pan Class I PI3 Kinase Inhibitor Showed Differential Forms of Cell Death Based on p53 Status of Glioma Cells

Purpose: The aim of this study was to show preclinical efficacy and clinical development potential of NVP-BKM120, a selective pan class I phosphatidylinositol-3 kinase (PI3K) inhibitor in human glioblastoma (GBM) cells in vitro and in vivo. Experimental Design: The effect of NVP-BKM120 on cellular growth was assessed by CellTiter-Blue assay. Flow cytometric analyses were carried out to measure the cell-cycle, apoptosis, and mitotic index. Mitotic catastrophe was detected by immunofluorescence. The efficacy of NVP-BKM120 was tested using intracranial U87 glioma model. Results: We tested the biologic effects of a selective PI3K inhibitor NVP-BKM120 in a set of glioma cell lines. NVP-BKM120 treatment for 72 hours resulted in a dose-dependent growth inhibition and effectively blocked the PI3K/Akt signaling cascade. Although we found no obvious relationship between the cell lines sensitivity to NVP-BKM120 and the phosphatase and tensin homolog (PTEN) and epidermal growth factor receptor (EGFR) statuses, we did observe a differential sensitivity pattern with respect to p53 status, with glioma cells containing wild-type p53 more sensitive than cells with mutated or deleted p53. NVP-BKM120 showed differential forms of cell death on the basis of p53 status of the cells with p53 wild-type cells undergoing apoptotic cell death and p53 mutant/deleted cells having a mitotic catastrophe cell death. NVP-BKM120 mediates mitotic catastrophe mainly through Aurora B kinase. Knockdown of p53 in p53 wild-type U87 glioma cells displayed microtubule misalignment, multiple centrosomes, and mitotic catastrophe cell death. Parallel to the assessment of the compound in in vitro settings, in vivo efficacy studies using an intracranial U87 tumor model showed an increased median survival from 26 days (control cohort) to 38 and 48 days (treated cohorts). Conclusion: Our present findings establish that NVP-BKM120 inhibits the PI3K signaling pathways, leading to different forms of cell death on the basis of p53 statuses. Further studies are warranted to determine if NVP-BKM120 has potential as a glioma treatment. Clin Cancer Res; 18(1); 184–95. ©2011 AACR.

A High Notch Pathway Activation Predicts Response to γ Secretase Inhibitors in Proneural Subtype of Glioma Tumor‐Initiating Cells

Genomic, transcriptional, and proteomic analyses of brain tumors reveal subtypes that differ in pathway activity, progression, and response to therapy. However, a number of small molecule inhibitors under development vary in strength of subset and pathway‐specificity, with molecularly targeted experimental agents tending toward stronger specificity. The Notch signaling pathway is an evolutionarily conserved pathway that plays an important role in multiple cellular and developmental processes. We investigated the effects of Notch pathway inhibition in glioma tumor‐initiating cell (GIC, hereafter GIC) populations using γ secretase inhibitors. Drug cytotoxicity testing of 16 GICs showed differential growth responses to the inhibitors, stratifying GICs into responders and nonresponders. Responder GICs had an enriched proneural gene signature in comparison to nonresponders. Also gene set enrichment analysis revealed 17 genes set representing active Notch signaling components NOTCH1, NOTCH3, HES1, MAML1, DLL‐3, JAG2, and so on, enriched in responder group. Analysis of The Cancer Genome Atlas expression dataset identified a group (43.9%) of tumors with proneural signature showing high Notch pathway activation suggesting γ secretase inhibitors might be of potential value to treat that particular group of proneural glioblastoma (GBM). Inhibition of Notch pathway by γ secretase inhibitor treatment attenuated proliferation and self‐renewal of responder GICs and induces both neuronal and astrocytic differentiation. In vivo evaluation demonstrated prolongation of median survival in an intracranial mouse model. Our results suggest that proneural GBM characterized by high Notch pathway activation may exhibit greater sensitivity to γ secretase inhibitor treatment, holding a promise to improve the efficiency of current glioma therapy. Stem Cells 2014;32:301–312

A survey of intragenic breakpoints in glioblastoma identifies a distinct subset associated with poor survival

With the advent of high-throughput sequencing technologies, much progress has been made in the identification of somatic structural rearrangements in cancer genomes. However, characterization of the complex alterations and their associated mechanisms remains inadequate. Here, we report a comprehensive analysis of whole-genome sequencing and DNA copy number data sets from The Cancer Genome Atlas to relate chromosomal alterations to imbalances in DNA dosage and describe the landscape of intragenic breakpoints in glioblastoma multiforme (GBM). Gene length, guanine-cytosine (GC) content, and local presence of a copy number alteration were closely associated with breakpoint susceptibility. A dense pattern of repeated focal amplifications involving the murine double minute 2 (MDM2)/cyclin-dependent kinase 4 (CDK4) oncogenes and associated with poor survival was identified in 5% of GBMs. Gene fusions and rearrangements were detected concomitant within the breakpoint-enriched region. At the gene level, we noted recurrent breakpoints in genes such as apoptosis regulator FAF1. Structural alterations of the FAF1 gene disrupted expression and led to protein depletion. Restoration of the FAF1 protein in glioma cell lines significantly increased the FAS-mediated apoptosis response. Our study uncovered a previously underappreciated genomic mechanism of gene deregulation that can confer growth advantages on tumor cells and may generate cancer-specific vulnerabilities in subsets of GBM.

Novel HSP90 Inhibitor NVP-HSP990 Targets Cell-Cycle Regulators to Ablate Olig2-Positive Glioma Tumor–Initiating Cells

Genetic heterogeneity and signaling alterations diminish the effectiveness of single-agent therapies in glioblastoma multiforme (GBM). HSP90 is a molecular chaperone for several signaling proteins that are deregulated in glioma cells. Thus, HSP90 inhibition may offer an approach to coordinately correct multiple signaling pathways as a strategy for GBM therapy. In this study, we evaluated the effects of a novel HSP90 inhibitor, NVP-HSP990, in glioma tumor-initiating cell (GIC) populations, which are strongly implicated in the root pathobiology of GBM. In GIC cultures, NVP-HSP990 elicited a dose-dependent growth inhibition with IC50 values in the low nanomolar range. Two GIC subgroups with different responses were observed with an Olig2-expressing subset relatively more sensitive to treatment. We also showed that Olig2 is a functional marker associated with cell proliferation and response to NVP-HSP990, as NVP-HSP990 attenuated cell proliferation in Olig2-high GIC lines. In addition, NVP-HSP990 disrupted cell-cycle control mechanism by decreasing CDK2 and CDK4 and elevating apoptosis-related molecules. Mechanistic investigations revealed molecular interactions between CDK2/CDK4 and Olig2. Inhibition of CDK2/CDK4 activity disrupted Olig2-CDK2/CDK4 interactions and attenuated Olig2 protein stability. In vivo evaluation showed a relative prolongation of median survival in an intracranial model of GIC growth. Our results suggest that GBM characterized by high-expressing Olig2 GIC may exhibit greater sensitivity to NVP-HSP990 treatment, establishing a foundation for further investigation of the role of HSP90 signaling in GBM.

Abstract 235: Oncogene addiction switch from NOTCH to PI3K/AKT requires simultaneous targeting of NOTCH and PI3K pathway inhibition in glioblastoma.

Notch signaling pathway regulates normal stem cells in the brain and glioma stem cells (GSCs) with high Notch activity. However, blocking the proteolytic activation of NOTCH with γ-secretase inhibitors (GSIs) fails to alter the growth of some GSCs as GSIs seem to be active in only a fraction of GSCs lines with constitutive NOTCH activity and limiting the efficacy of this strategy. Linking the molecular aberrations of cancer to drug responses could guide treatment choice and identification of new therapeutic applications. Here we report loss of PTEN as a critical event leading to resistance to NOTCH inhibition, which causes the transfer of “oncogene addiction” from the NOTCH to the phosphoinositol-3 kinase (PI3K)/AKT pathway. This novel observation suggests the need to simultaneous inhibition of both pathways as a means to improve therapeutic efficacy in human GBMs. We investigated the effects of Notch pathway inhibition in GSC using GSI. Drug cytotoxicity test on 16 GSCs show differential growth response to GSI stratifying GSCs into two groups: responders (6 cell lines) vs non-responders (10 cell lines). Active Notch signaling seems to be important features for the GSC as Notch inhibition only affected GSCs defined as having increased endogenous Notch activity. However in the responder group GSCs with the PTEN mutation seems to be less sensitive to GSI treatment. Here we show that NOTCH regulates the expression of PTEN and the activity of the PI3K/AKT signaling pathway in GSCs since treatment with GSI attenuated Notch intracellular domain (NICD), Hes-1, Hes-3, and Hes-5 and increases PTEN expression. NOTCH regulates PTEN expression via Hes-1 as knockdown of either Notch or Hes1 led to increase expression of PTEN. In contrast, PTEN knockdown decreases the response of GSCs to GSI in PTEN wild type GSCs. Our data shows that PTEN is an important mediator of GSI induced attenuation of cell growth suggesting that there might be a regulatory circuit-linking Notch signaling with PTEN/PI3K/Akt pathway, providing the basis for the design of new therapeutic strategies and suggests the need to simultaneously inhibit both pathways as a means to improve therapeutic efficacy in human GBMs. Our preliminary data reveal the synergistic attenuation of cell growth using combination of GSI and PI3K inhibitors in PTEN mutant GSCs. Our results warrant further studies to test the efficacy of combined PI3K/AKT- and NOTCH-directed therapies in human cancer. Citation Format: Norihiko Saito, Jun Fu, Shuzhen Wang, Erik P. Sulman, Frederick F. Lang, Wk Alfred Yung, Dimpy Koul. Oncogene addiction switch from NOTCH to PI3K/AKT requires simultaneous targeting of NOTCH and PI3K pathway inhibition in glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 235. doi:10.1158/1538-7445.AM2013-235

Abstract 1720: MSK1 mediates PI3K inhibition resistance by induction of β-catenin phosphorylation in glioblastoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Glioblastoma (GBM), the most common malignant brain tumor in adults, represents a compelling disease for kinase inhibitor therapy because majority of these tumors harbor genetic alterations that result in aberrant activation of growth factor signaling pathways. PI3K/mTOR-pathway is dysregulated in over 50% of human glioblastomas (GBM) and represents an attractive target for therapy. One major challenge for single pathway PI3K inhibition targeting therapy is intrinsic resistance. To study the mechanism of intrinsic resistance to PI3K inhibition, we analyzed the pre- and post-treatment reverse phase protein arrays (RPPA) data of both giloma cells and giloma initiating cells (GIC). Mitogen- and stress-activated protein kinase 1 (MSK1) was markedly induced after PI3K inhibitor treatment and it was linked with increase expression of β-catenin. We further found that MSK1 directly interacts with β-catenin and stabilizes β-catenin protein level. MSK1 also regulates β-catenin nuclear translocation and β-catenin transcriptional activity. More interestingly, using in vitro kinase assay, we identified MSK1 is a kinase that phosphorylates β-catenin. Finally, we showed that depletion of β-catenin potentiates PI3K inhibitors induced cytotoxity in a series of GICs. Taken together, our results suggest that MSK1/β-catenin signaling may serve as an escape survival signaling upon PI3K inhibition, and combination of PI3K inhibition and MSK1/β-catenin inhibition is required to induce lethal growth inhibition in human glioblastoma cells. Citation Format: Shaofang Wu, Jun Fu, Siyuan Zheng, Roel G. Verhaak, W. K Yung, Dimpy Koul. MSK1 mediates PI3K inhibition resistance by induction of β-catenin phosphorylation in glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1720. doi:10.1158/1538-7445.AM2014-1720

Abstract 4170: Oncogenic activation of LMO2 by EGFR signaling regulates STAT3 phosphorylation and transcriptional activity in human glioma

Glioblastoma multiforme (GBM) is a highly heterogeneous disease characterized by multiple genetic abnormalities. Amplification and mutation of the epidermal growth factor receptor (EGFR) gene represent signature genetic abnormalities occurred in over 60% of GBM. The successful application of EGFR-targeted therapy for the treatment of GBM is thus dependent on a thorough understanding of the intricate signal pathways cross-linkage that underlies the pathobiology of this disease. The LIM-domain only factor 2 (LMO2) gene is a crucial oncogene involved in hematopoietic development and leukemogenesis. Herein, we showed, from the TCGA GBM data, that LMO2 is highly expressed in EGFR-aberrant GBM samples and might serve as a downstream effector of EGFR signaling in GBM. Depletion of LMO2 expression impairs the maintenance of glioma cell proliferation in vitro and in vivo. In addition, EGFR signaling induces LMO2 nuclear translocation and enables LMO2 to form a triplex with EGFR and Signal transducer and activator of transcription 3 (STAT3). More importantly, LMO2 serves as a bridging molecule to promote the formation of EGFR/STAT3 complex and to enhance EGFR-mediated STAT3 Y705 phosphorylation upon EGF stimulation, which in turn up-regulates the expression of STAT3-responsive genes involved in the regulation of cell proliferation and tumor development. In conclusion, these findings reveal that LMO2 might serve as a critical effector of EGFR signaling in human glioma and highlight the essential function of LMO2 in EGF-stimulated STAT3 activation and tumorigenesis. Citation Format: Jun Fu, Dimpy Koul, Shaofang Wu, Siyuan Zheng, Roel G. Verhaak, W.K Yung. Oncogenic activation of LMO2 by EGFR signaling regulates STAT3 phosphorylation and transcriptional activity in human glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4170. doi:10.1158/1538-7445.AM2014-4170

Abstract 4909: Olig2 phosphorylation by CDK2 implicated in glioma proliferation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Oligodendrocyte transcription factor 2 (Olig2) is an important proneural transcription factor that regulates early development of the central nervous system and formation of glioma. Although recent studies established the role of developmentally regulated Olig2 phosphorylation in promoting the proliferation of both normal and malignant neural stem/progenitor cells, the molecular mechanisms by which Olig2 phosphorylation is regulated and functions as a pro-proliferative factor are not completely understood. Here we identified Olig2 as a critical phosphorylation target for cyclin-dependent kinase 2 (CDK2). CDK2 stabilizes and activates Olig2, thereby regulating G1-to-S phase progression by repressing the expression of Cyclin-dependent kinase inhibitor p27kip1. After phosphorylation by CDK2, Olig2 exerts pro-proliferative effects that are reflected in normal neural/glioma stem cells and in murine xenograft models of glioma. Olig2 phosphorylation regulates its binding to E-box sequence in p27 gene promoter and represses p27 expression. We show that constitutive p27 expression antagonizes CDK2-mediated Olig2 phosphorylation and attenuates cell proliferation both in in-vitro and in-vivo glioma models. Expression analysis of TCGA GBM samples also showed that high Olig2 expression is always accompanied with high CDK2 expression in the proneural, classical, and neural subclasses, which could provide a convincing evidence to use a CDK2 inhibitor to treat Olig2-high gliomas. Treatment with CDK2 inhibitor preferentially suppresses proliferation of Olig2-high glioma cells. Thus, we conclude that a CDK2 inhibitor could be of great therapeutic value in patients with Olig2-high glioma and Olig2 might be used as a biological marker for CDK2 inhibitors in the future studies. Citation Format: Jun Fu, Dimpy Koul, Siyuan Zheng, Vivek Singh, Jun Yao, Yanhua Zheng, Zhimin Lu, Erik P. Sulman, Frederick F. Lang, Alfred W.K Yung. Olig2 phosphorylation by CDK2 implicated in glioma proliferation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4909. doi:10.1158/1538-7445.AM2013-4909

Abstract 3347: A proneural signature profile predicts response to Notch inhibition in glioma stem cells

The Cancer Genome Atlas Network described genomic abnormalities and gene expression-based molecular subtypes of GBM that showed a strong relationship between subtypes, genomic alterations and different neural lineages. Future studies are required to elucidate the intricate relationship between tumor subtypes and treatment response. We have thus embarked on a comprehensive effort to detecting expression signatures that are associated with response to the therapy and these signatures may allow prospective selection of patients with high likelihood of responding to therapy. Notch signaling pathway is an evolutionarily conserved pathway that plays an important role in multiple cellular and developmental processes including cell fate decision, differentiation, proliferation, survival, angiogenesis and migration. Recent studies indicate that the Notch signaling pathway regulates normal stem cells in the brain, and glioma stem Cells (GSCs) with high Notch activity. We investigated the effects of Notch pathway inhibition on GSCs growth using commercially available inhibitors. Drug cytotoxicity test on 16 GSCs show differential growth response to Notch inhibitors stratifying GSCs into two groups: responders (6 cell lines) vs non-responders (10 cell lines). Gene expression clustering identified a responder gene signature enriched in 1: proneural genes such as Olig2, Sox-9, NKX2-2, and 2: Notch signaling component such as Notch-1, Notch-3, Hes-3 and Hes-5. These signature were validated by western blot analysis. Treatment with Notch inhibitors reduced neurosphere formation in vitro accompanied with attenuated Notch intracellular domain (NICD), Hes-1, Hes-3, and Hes-5. In addition, the expression of nestin-the stem cell marker was reduced and an increased expression of lineage differentiation markers TuJ1 and GFAP was observed following Notch inhibition. In conclusion, we have identified a responder signature for a group of glioma stem cells that can be targeted by Notch inhibitors and this may allow the selection of patients with high likelihood of responding to this therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3347. doi:1538-7445.AM2012-3347

Abstract LB-114: Molecular and lineage analysis of glioblastoma stem cells identifies clinically relevant models of glioblatoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The Cancer Genome Atlas project described a robust gene expression-based molecular classification of GBM. However, the functional and biological significance of the molecular subclasses are being determined. In this study, we hypothesize that Glioma Stem Cells (GSCs) isolated from individual patient tumor samples will recapitulate the molecular characteristics of tumor samples and provide a relevant model for functional analysis of the molecular subclasses. Thus we conducted a comprehensive analysis of 26 GSC lines with expression array, RPPA, and a series of neural lineage markers. Analysis of the expression data classified the 26 GSC lines into four subtypes ( Classical, Proneural 1, Proneural 2 and Mesenchymal ,) closely similar to the TCGA subclasses with a distinct profile for each subtype (c-Myc, Cyclin D2 for Classical ; Olig2, NKX2–2, Notch-1, Notch-3 for Proneural 2 ; BMP4, DCX, p16INK4a, ID2 for Proneural 1 ; CD44, CAV1, TGFBR2 for Mesenchymal ). Further analysis showed that GSC subtypes exhibit divergent patterns of signaling pathway activation. The major pathways activated in 4 subtypes were Notch pathway in Proneural 2 , Wnt/β-Catenin in Proneural 2 and Classical , while FGF/VEGF and TGF-β in Mesenchymal. In vitro treatment with SB 431542, a TGF-βR inhibitor, showed that Mesenchymal subtype is more sensitive than other subtypes. More importantly, lineage analysis of GSCs subtypes show that Proneural and Classical GSCs differentially express lineage markers for neural stem/progenitor cells and were responsive to differentiating agent retinoic acid. In addition, GSC subtypes exhibit distinct biological behaviors in self-renewal capacity, proliferation, invasiveness, angiogenic potential, response to growth factor stimulus, and differentiation in vitro and in vivo with Classical subtype being more proliferative and Mesenchymal subtype being more angiogenic and invasive. In conclusion, our comprehensive analysis showed that GSCs reflect patient tumor subclass and these subtypes showed distinct regulatory pathway activation, lineage profile and biological behaviors. Thus GSC is biologically and molecularly a more relevant model system for preclinical studies of therapeutic intervention and to expand our molecular understanding of human GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-114. doi:10.1158/1538-7445.AM2011-LB-114