Suppression of RAF/MEK or PI3K synergizes cytotoxicity of receptor tyrosine kinase inhibitors in glioma tumor-initiating cells

Additional file 5. Data of RPPA analysis

Depletion of CLK2 sensitizes glioma stem-like cells to PI3K/mTOR and FGFR inhibitors.

The Cdc2-like kinases (CLKs) regulate RNA splicing and have been shown to suppress cell growth. Knockdown of CLK2 was found to block glioma stem-like cell (GSC) growth in vivo through the AKT/FOXO3a/p27 pathway without activating mTOR and MAPK signaling, suggesting that these pathways mediate resistance to CLK2 inhibition. We identified CLK2 binding partners using immunoprecipitation assays and confirmed their interactions in vitro in GSCs. We then tested the cellular viability of several signaling inhibitors in parental and CLK2 knockdown GSCs. Our results demonstrate that CLK2 binds to 14-3-3τ isoform and prevents its ubiquitination in GSCs. Stable CLK2 knockdown increased PP2A activity and activated PI3K signaling. Treatment with a PI3K/mTOR inhibitor in CLK2 knockdown cells led to a modest reduction in cell viability compared to drug treatment alone at a lower dose. However, FGFR inhibitor in CLK2 knockdown cells led to a decrease in cell viability and increased apoptosis. Reduced expression of CLK2 in glioblastoma, in combination with FGFR inhibitors, led to synergistic apoptosis induction and cell cycle arrest compared to blockade or either kinase alone

The polo-like kinase 1 inhibitor volasertib synergistically increases radiation efficacy in glioma stem cells.

Background: Despite the availability of hundreds of cancer drugs, there is insufficient data on the efficacy of these drugs on the extremely heterogeneous tumor cell populations of glioblastoma (GBM). Results: The PKIS of 357 compounds was initially evaluated in 15 different GSC lines which then led to a more focused screening of the 21 most highly active compounds in 11 unique GSC lines using HTS screening for cell viability. We further validated the HTS result with the second-generation PLK1 inhibitor volasertib as a single agent and in combination with ionizing radiation (IR). Conclusions: Our results reinforce the potential therapeutic efficacy of volasertib in combination with radiation for the treatment of GBM. Methods: We used high-throughput screening (HTS) to identify drugs, out of 357 compounds in the published Protein Kinase Inhibitor Set, with the greatest efficacy against a panel of glioma stem cells (GSCs), which are representative of the classic cancer genome atlas (TCGA) molecular subtypes. Oncotarget 2018; 9(8):10497-10509

An inhibitor of oxidative phosphorylation exploits cancer vulnerability.

Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors

Abstract 1612: Targeting intercellular adhesion molecule-1 (ICAM-1) prolongs glioblastoma survival in combination with bevacizumab .

Abstract Glioblastoma is the most common malignant brain tumor and is characterized by cellular heterogeneity, vascular proliferation and extensive tissue infiltration. Analysis of gene expression data from orthotopic glioma stem cells with acquired resistance to bevacizumab therapy identified over expression of intercellular adhesion molecule 1 (ICAM1, CD54) in resistant tumors. ICAM1 is a cell adhesion glycoprotein of the immunoglobulin supergene family which interacts with β2 integrins, mediates leukocyte transendothelial migration and T cell activation during inflammatory processes. ICAM1 over expression in tumors resistant to antiangiogenic therapy was validated by real time PCR and Western blot. In a panel of glioma stem cell lines, ICAM1 expression was higher in mesenchymal compared to proneural cell lines. GFP-tagged ICAM1 shRNA lentivirus was used to knockdown ICAM1 in multiple cell lines. We injected shICAM1 NSC11 and scramble glioma stem cells into the brain of nude mice. Mice bearing tumors formed from shICAM1 NSC11 cells survived significantly longer than mice injected with scramble control cells. Tumor size was significantly decreased in mice bearing tumors formed from shICAM1 cells than that in mice bearing tumors from GFP-tagged NSC11 control cells. Knocking-down ICAM1 suppresses tumor invasion both in vitro and in vivo. We next examined the mechanism of ICAM1 over expression after treated with anti-VEGF therapy. We observed that ICAM1 over expression correlated with hypoxia-inducible factor by immnofluorescence analysis in tumor tissue. Furthermore, we found that ICAM1 protein expression was markedly increased after NSC11 and NSC17 cells under hypoxic condition in a time-dependent manner. Furthermore, hypoxia-induced p-STAT3 increased transcription of ICAM1, which was inhibited by an inhibitor of the JAK/STAT3 pathway. Our studies identify ICAM1 as a potentially important mediator of tumor migration/invasion in glioblastoma. Targeting ICAM1 may provide a new strategy to prolong the efficacy of antiangiogenic therapy and prevent the invasive phenotype. Citation Format: Yuji Piao, Ji Liang, Verlene Henry, Lindsay Holmes, John F. de Groot. Targeting intercellular adhesion molecule-1 (ICAM-1) prolongs glioblastoma survival in combination with bevacizumab . [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 1612. doi:10.1158/1538-7445.AM2013-1612</jats:p

Abstract 1388: Acquired resistance to anti-VEGF therapy in glioblastoma is associated with an inflammatory and mesenchymal phenotype

Abstract Glioblastoma is the most common malignant brain tumor and is characterized by rapid angiogenesis-dependent (re)growth, cell heterogeneity, and extensive local tissue infiltration. Antiangiogenic therapy, among the most promising treatment developments, rapidly reduces vascular permeability and delays glioblastoma progression but the therapy itself ultimately promotes an aggressive treatment-resistant phenotype. The aim of the present study was to identify mechanisms responsible for glioblastoma resistance to antiangiogenic therapy. When mice bearing U87 and NSC11 xenografts treated with bevacizumab became moribund, tumor tissue was extracted to obtain tumor cells with acquired resistance to bevacizumab. U87 and NSC11 cells were dissociated and cultured in serum-containing and stem cell media, respectively. To verify resistance, the resistant cells were expanded in vitro and then re-implanted orthotopically in nude mice. Mice were randomized to receive bevacizumab or vehicle and followed for survival. As anticipated, tumors formed by parental U87 and NSC11 cells were sensitive, whereas tumors obtained at the time of acquired resistance to anti-VEGF therapy were resistant to bevacizumab and did not have prolongation of survival compared to untreated controls. To elucidate the molecular mechanisms underlying resistance to antiangiogenic treatment, we performed gene expression profiling using Affymetrix U133Plus_2.0 arrays comparing untreated and bevacizumab-treated tumors. Comparison of anti-VEGF therapy-resistant cell lines to untreated controls demonstrated an increase in genes associated with a mesenchymal phenotype. Antiangiogenic therapy increased the expression of genes of mesenchymal origin, cellular migration/invasion, and inflammation including chemokine secretion, myeloid cell chemotaxis and multiple markers reflecting a pro-inflammatory environment. Consistent with these data, mice bearing U87 resistant tumors showed significantly greater infiltration of F4/80+ myeloid cells compared to wild-type tumors. Comparing the mesenchymal gene signature from Phillips et al with the gene changes in our experiments using Gene Set Enrichment Analysis (GSEA v2.07, Broad Institute, MIT), we show that NSC11 tumors treated with bevacizumab showed a highly significant correlation to the published mesenchymal gene signature. In addition to the pro-inflammatory changes, invasion-related genes were also upregulated. We found the U87 and NSC11 resistant cells have significantly higher invasion rates in vitro compared with U87 and NSC11 parental cell lines. Our studies identify prominent mechanisms of glioblastoma resistance to antiangiogenic therapy. A better understanding of resistance may provide new strategies to prolong the efficacy of this therapy and accelerate the integration of combination therapies into clinical trials. 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 1388. doi:1538-7445.AM2012-1388</jats:p

Abstract LB-314: CSF-1R inhibitor JNJ-28312141 reduces antiangiogenic therapy-induced myeloid cell infiltration and mesenchymal shift

Abstract Glioblastoma is the most common and malignant primary brain tumor in adults. Despite maximal tumor resection, radiation and chemotherapy, the tumor's high degree of infiltration leads to 100% recurrence and an average life expectancy after diagnosis of about 12 to 14 months. Vascular endothelial growth factor (VEGF) and other pro-angiogenic factors have been identified as critical mediators of angiogenesis in glioblastoma. Inhibition of angiogenesis results in a high radiographic response rate but ultimately all patients’ progress on this therapy. An increase in monocyte/macrophage recruitment has been associated with resistance to antiangiogenic therapy in preclinical mouse xenograft models and in patients. We hypothesized that combining antiangiogenic therapy with therapies inhibiting macrophage infiltration may delay resistance and prolong animal survival. In our glioma stem cell model, we evaluated the impact of bevacizumab alone and in combination with JNJ-28312141 (a novel colony-stimulating factor-1 receptor/FMS-related receptor tyrosine kinase-3 inhibitor) on the recruitment of myeloid cells, microvascular density, and animal survival. Four days after orthotopic implantation of 1× 105 NSC11 glioma stem cells, mice were randomized to treatment with bevacizumab, JNJ-28312141, or the combination of bevacizumab plus JNJ-28312141 and followed for survival (n=8-10 per group). JNJ-28312141 did not prolong survival compared to control. However, JNJ-28312141 combined with bevacizumab significantly reduced tumor vascularity (microvascular density measured using Factor VIII) and prolonged survival compared with bevacizumab alone (p=0.04). We compared the F4/80+ myeloid cell population in xenograft tumors using immunohistochemistry and immunofluorescence. The number of F4/80+ myeloid cells was significantly less in mice treated with JNJ-28312141 and bevacizumab compared with mice treated with bevacizumab alone (p&amp;lt;0.01). To evaluate the impact of treatment on the expression of mesenchymal markers, we quantified YKL-40 protein expression using immunofluorescence. Bevacizumab increased the expression of YKL-40 compared to controls and this increase in expression was significant blocked in the mice treated with the combination of bevacizumab and JNJ -28312141. These results suggest that resistance to antiangiogenic therapy can be partly overcome in glioblastoma by combining anti-VEGF therapy with an inhibitor of macrophages. In our orthotopic glioma model, CSF-1R inhibition is associated with a decrease in the recruitment and infiltration of F4/80+ cells into tumor and a decrease in mesenchymal marker expression. Our studies provide a new strategy to prolong the efficacy of antiangiogenic therapy and accelerate the integration of combination therapies into clinical trials. 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 LB-314. doi:1538-7445.AM2012-LB-314</jats:p

Abstract 1385: TGF beta regulates tumor resistance to antiangiogenic therapy through POSTN in glioma stem cell models

Abstract Periostin (osteoblast-specific factor 2, POSTN) is a 90-kDa ECM (extracellular matrix) protein containing an amino-terminal EMI, tandem repeat of four fascilin-domains and carboxyl-terminal domain including a heparin-binding site. Stromal POSTN plays a key role in regulating cancer stem cell (CSC) maintenance and expansion during metastatic colonization. Recently, it has been reported that POSTN functions as a progression associated and prognostic biomarker in glioma via inducing an invasive and proliferative phenotype. POSTN mRNA expression was significantly higher in grade IV gliomas than in grade II and grade III tumors. POSTN interacts with several integrin receptors such as αvβ1 and αvβ3 to regulate cellular response including cell proliferation, EMT (epithelial-mesenchymal transition) and cell migration. We demonstrated in previous studies that bevacizumab (Bevacizumab, Roche/Genentech) increased glioblastoma invasion in vivo. In the present study, we investigated the role of POSTN and its receptor in tumor invasion and resistance to antiangiogenic therapy. POSTN expression after treatment with avastin in vivo was increased as measured by Western blot and immunofluorescence. Stable knockdown of POSTN expression using specific shRNA abrogated expression of EMT (CAMK2N1, COL1A2, KRT14, COL3A1 and MMP-9) and angiogenesis-related (ANGPTL4, VEGFA, CXCL5, HPSE and EFNA3) genes compared to controls. VEGF expression was decreased in POSTN shRNA, and POSTN shRNA infected cells decreased invasion in both GSC11 and GSC272 glioma stem cell lines. TGF beta1 increased secretion of POSTN and phosphorylation of smad3 through a decrease in POSTN binding to smad3. Moreover, recombinant POSTN increased glioma cell invasion in an intergrin β1 receptor-dependent fashion. In animal experiments, median survival of animals implanted with GSC272-control cells was 63 days, while GSC272-bevacizumab was 84.5 days and knock down of POSTN was 84 days (p&amp;lt;0.05). Treatment with bevacizumab increased survival of POSTN shPOSTN tumors to 114.5 days compare to control (p&amp;lt;0.01). Tumor volume was decreased in the bevacizumab treatment (1.35 mm3) and POSTN shRNA (0.05 mm3) groups compared to controls (7.04 mm3). However, no tumor was observed in mice treated with both POSTN shRNA and bevacizumab. Bevacizumab increased TGF beta expression in GSC272 tumor tissue whereas tumors from POSTN shRNA mice treated with bevacizumab did not show evidence of TGF beta expression. Collectively, our data suggests that TGF beta increases POSTN secretion which appears to play an important role in glioma invasion and resistance to antiangiogenic therapy. Citation Format: Soon Young Park, Yuji Piao, Ningyi Tiao, Verlene Henry, Jianwen Dong, John Frederick de Groot. TGF beta regulates tumor resistance to antiangiogenic therapy through POSTN in glioma stem cell models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1385. doi:10.1158/1538-7445.AM2015-1385</jats:p

Acquired Resistance to Anti-VEGF Therapy in Glioblastoma Is Associated with a Mesenchymal Transition

Abstract Purpose: Antiangiogenic therapy reduces vascular permeability and delays progression but may ultimately promote an aggressive treatment-resistant phenotype. The aim of the present study was to identify mechanisms responsible for glioblastoma resistance to antiangiogenic therapy. Experimental Design: Glioma stem cell (GSC) NSC11 and U87 cell lines with acquired resistance to bevacizumab were developed from orthotopic xenografts in nude mice treated with bevacizumab. Genome-wide analyses were used to identify changes in tumor subtype and specific factors associated with resistance. Results: Mice with established parental NSC11 and U87 cells responded to bevacizumab, whereas glioma cell lines derived at the time of acquired resistance to anti-VEGF therapy were resistant to bevacizumab and did not have prolongation of survival compared with untreated controls. Gene expression profiling comparing anti-VEGF therapy-resistant cell lines to untreated controls showed an increase in genes associated with a mesenchymal origin, cellular migration/invasion, and inflammation. Gene-set enrichment analysis showed that bevacizumab-treated tumors showed a highly significant correlation to published mesenchymal gene signatures. Mice bearing resistant tumors showed significantly greater infiltration of myeloid cells in NSC11- and U87-resistant tumors. Invasion-related genes were also upregulated in both NSC11 and U87 resistant cells which had higher invasion rates in vitro compared with their respective parental cell lines. Conclusions: Our studies identify multiple proinflammatory factors associated with resistance and identify a proneural to mesenchymal transition in tumors resistant to antiangiogenic therapy. Clin Cancer Res; 19(16); 4392–403. ©2013 AACR.</jats:p

Supplementary Figure 6 from Acquired Resistance to Anti-VEGF Therapy in Glioblastoma Is Associated with a Mesenchymal Transition

PDF file, 809K, TGF-beta increases following the acquisition of bevacizumab resistance.</p