Jianwen Dong

Periostin (POSTN) regulates tumor resistance to antiangiogenic therapy in glioma models

Periostin (POSTN) interacts with multiple integrins to coordinate a variety of cellular processes, including epithelial-to-mesenchymal transition (EMT) and cell migration. In our previous study, anti-VEGF-A therapy was associated with resistance and EMT. This study sought to determine the role of POSTN in the resistance of glioma stem cells (GSC) to antiangiogenic therapy. In mouse xenograft models of human glioma, POSTN expression was associated with acquired resistance to anti-VEGF-A therapy and had a synergistic effect with bevacizumab in prolonging survival and decreasing tumor volume. Resistance to anti-VEGF-A therapy regulated by POSTN was associated with increased expression of TGFβ1 and hypoxia-inducible factor-1α (HIF1α) in GSCs. At the molecular level, POSTN regulated invasion and expression of EMT (caveolin-1) and angiogenesis-related genes (HIF1α and VEGF-A) through activation of STAT3. Moreover, recombinant POSTN increased GSC invasion. Collectively, our findings suggest that POSTN plays an important role in glioma invasion and resistance to antiangiogenic therapy. Mol Cancer Ther; 15(9); 2187–97. ©2016 AACR.

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). In vitro studies showed that volasertib inhibited cell viability, and high levels of the anti-apoptotic protein Bcl-xL expression were highly correlated with volasertib resistance. Volasertib sensitized GSCs to radiation therapy by enhancing G2/M arrest and by inducing apoptosis. Colony-formation assay demonstrated that volasertib plus IR synergistically inhibited colony formation. In intracranial xenograft mouse models, the combination of volasertib and radiation significantly inhibited GSC tumor growth and prolonged median survival compared with radiation treatment alone due to inhibition of cell proliferation, enhancement of DNA damage, and induction of apoptosis. 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.

Abstract 2945: Arsenic trioxide sensitizes glioma stem cells to brain penetrant PI3K and mTOR inhibitor GDC-0084

Glioblastoma is the most aggressive primary malignant brain tumor with few effective therapies. The current study evaluated arsenic trioxide (As2O3, ATO), a small-molecular agent that inhibits tumor growth via promoting promyelocytic leukemia protein (PML) degradation, in combination with multiple PI3K/mTOR inhibitors using high-throughput screening (HTS) to validate if ATO reverses glioblastoma resistance to PI3K/mTOR-targeted therapy. Quantitative single-agent and 2-drug combinations (5 drug doses, maximal concentration of 1 uM of each agent) were evaluated in 20 patient-derived glioma stem-like cells (GSCs). ATO was applied as an “anchor” drug and several mTOR and EGFR inhibitors as “probe” drugs to explore potential combination efficacy. Data from single-agent screening demonstrated that brain penetrant PI3K/mTOR inhibitor GDC-0084 potently inhibited cell viability with an IC50 ranging from 0.12μM to 5.78μM under normoxic conditions. Under hypoxic conditions, 10 of the 16 GSC cell lines remained sensitive, indicating less efficacy of GDC-0084 in the setting of a hypoxic microenvironment. Evaluation of drug combinations identified ATO and GDC-0084 as the most effective combination in vitro. ATO was synergistic with GDC-0084 in several GSCs resistant to GDC-0084 monotherapy. GSC sensitivity to GDC-0084 as single agent and in combination correlated with apoptosis, angiogenesis and PI3K/Akt pathways using gene set enrichment analyses (GSEA). In vitro combination treatment significantly inhibited PML, p-S6, p-AKT and p-mTOR expressions compared to single agent. In an orthotopic mouse model of glioma, targeting PI3K/mTOR with GDC-0084 prolonged the median survival to 81 days compared to 69 days in the control group. The efficacy of combining ATO and GDC-0084 in an orthotopic GSC mouse model is ongoing. Our studies confirm prior work demonstrating the efficacy of combining GDC-0084 with ATO, which now requires clinical validation. Citation Format: Jianwen Dong, Emmanuel Martinez-Ledesma, Nghi Nguyen, Caroline Carrillo, Yuji Piao, Verlene Henry, Soon Young Park, Ningyi Tiao, Clifford Stephan, Roel Verhaak, Erik Sulman, Veerakumar Balasubramaniyan, John F. de Groot. Arsenic trioxide sensitizes glioma stem cells to brain penetrant PI3K and mTOR inhibitor GDC-0084 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2945.

Abstract LB-063: Anti-tumor activity of the novel dual p70S6KAKT inhibitor M2698 in glioma stem cell mouse models

The efficacy of the dual p70S6K/AKT inhibitor M2698 was evaluated in a panel of patient-derived glioma stem cell (GSC) lines. The IC50 was determined from a 7-dose cell viability assay using Celltiter Glo. GSCs were classified as sensitive (IC50 ≤ 1µM) or resistant (IC50 >1µM). Two sensitive cell lines (GSC17 and GSC231) and two resistant cell lines (GSC272 and GSC20) were selected for in vivo experiments. To determine the efficacy of M2698 in orthotopic models of glioblastoma, GSCs were implanted into the basal ganglia of nude mice. After four days, mice were treated with vehicle (control) or M2698 at 20 mg/kg daily M-F. In the cell line GSC231 (sensitive in vitro), the median animal survival time was 47 days in the control-treated mice and 62 days in the M2698-treated mice (p=0.0288). The tumor volumes at 6, 7.5, 10 weeks were 4.98 ± 6.17 mm3, 48.48 ± 10.57 mm3, and 61.38 ± 28.00 mm3, respectively, in the control-treated mice and 0.33 ± 0.45 mm3, 9.96 ± 6.89 mm3, and 18.00 ± 5.59 mm3, respectively, in the M2698 -treated mice (p=0.322, p In the GSC272 (resistant line) xenograft mouse model, the median survival time was 72 days in the control-treated mice and 76 days in the M2698-treated mice. Although this difference was statistically significant (p=0.0022), the 4 day improvement in survival was minimal compared to the sensitive line GSC231. The tumor volumes at 6, 7.5, 10 weeks were 7.48 ± 4.10 mm3, 126.37 ± 41.98 mm3, and 158.79 ± 39.53 mm3, respectively in the control mice and 0.54 ± 0.47 mm3, 8.62 ± 3.88 mm3, and 64.3 ± 5.55 mm3, respectively in the M2698-treated mice (p=0.014, p treated mice and 1.40 ± 1.06% per field, 4.30 ± 2.26% per field, and 5.13 ± 1.02% per field, respectively, in the M2698-treated mice (p=0.302, p=0.005, and p=0.036, respectively). GSC17 and GSC20 lines that were resistant to M2698 were further evaluated to elucidate potential mechanisms of resistance to p70S6K/AKT inhibitor treatment. Reverse Phase Protein Array (RPPA) assay was performed 24 hours after treatment with M2698 in GSC17 and GSC20 in vitro. M2698 successfully prevented S6 phosphorylation, but also resulted in an increase in P-ERK1/2 and P-MAPK expression suggesting compensatory activation of the Ras/MEK/ERK signaling pathway. To validate these results in vivo, we performed immunohistochemistry staining of P-S6 and P-ERK1/2 in tumor tissue from the GSC17 xenograft model at the latest time points. The data revealed that tumors from M2698-treated mice had lower levels of P-S6 expression and high level of P-ERK1/2 expression compared with the untreated control group. These results suggest that tumors not responsive to p70S6K/AKT inhibitor monotherapy might benefit from combination treatment with a MEK inhibitor. Citation Format: yuji piao, Craig Thomas, Verlene Henry, Ningyi Tiao, Soon Young park, Martinez-ledesma Juan, Jianwen Dong, John frank de groot. Anti-tumor activity of the novel dual p70S6KAKT inhibitor M2698 in glioma stem cell mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-063. doi:10.1158/1538-7445.AM2017-LB-063

Abstract 2815: Antitumor activity of CDK4/6 inhibition in combination with radiation therapy on glioblastoma stem cells

Almost 80% of glioblastoma tumors have alterations in the CNKN2A-CDK-Rb pathway. Cyclin dependent kinases 4 and 6 (CDK4/6) act as cell cycle regulators and have been recognized as therapeutic targets for glioblastoma (GBM). In the current study, we determined the antitumor activity of CDK4/6 inhibition (palbociclib and abemaciclib) alone and in combination with radiation on a panel of glioblastoma stem cells (GSCs). The GSCs were molecularly characterized using multiple ‘omimcs’ techniques and Western blots identified specific phospho- and total protein levels. Approximately 50% of cell lines have homozygous deletion of CDKN2A although CDK4 and CDK6 protein expression was significantly elevated in most of the GSCs. RB protein was constitutively phosphorylated in only a subset (45%): GSC11, GSC262, GSC231, GSC304 and GSC7-2. In our panel of GSCs, there were total of three cell lines with CDKN2A loss, RB phosphorylation and CDK4/6 overexpression (GSC262, GSC11 and GSC231). Our data showed that GSC262, GSC11, GSC231 (CDKN2A deletion, RB intact), GSC7-2 and GSC300 (CDKN2A WT, RB intact) were sensitive to CDK4/6 treatment (IC50 ≤ 1μM). CDK4/6 inhibitor treated GSCs showed G1 cell cycle arrest and decreased phospho-RB levels in GSC262 and GSC7-2 cells. We next determined whether CDK4/6 inhibition combined with radiation has additive or synergistic antitumor effect. A clonogenic assay demonstrated that neurosphere formation was significantly decreased by CDK4/6 inhibition in combination with radiation compared to either treatment in GSC262 (resistant to radiation treatment) or GSC7-2 (sensitive to radiation treatment) cell lines. The radiation resistant GSC (GSC262) treated with radiation and CDK4/6 inhibitor became sensitive to radiation therapy whereas radiation sensitive GSC (GSC7-2) had a significant decrease in colony formation compared to radiation alone. CDK4/6 combined with radiation increased γ-H2AX protein expression levels and decreased cyclin D1 protein expression in a time dependent manner compared to single drug treatment. These data indicate synergistic antitumor effects of CDK4/6 inhibition combined with radiation therapy in GSCs in vitro. Further investigation is ongoing to evaluate this combination therapy. Citation Format: Christopher Nguyen, Yuji Piao, Juan Emmanuel Martinez-Ledesma, Jianwen Dong, Soon Yeung Park, Roel Verhaak, Erik Sulman, John F. De Groot. Antitumor activity of CDK4/6 inhibition in combination with radiation therapy on glioblastoma stem 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 2815.

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

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 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

Abstract 695: Synergistic antitumor effects of polo like kinase inhibitor volasertib in combination with ionizing radiation in glioblastoma

Despite the availability of hundreds of drugs, there is little data on the efficacy of these agents in the extremely heterogeneous populations of tumor cells observed in glioblastoma. In this study, a high-throughput compound-screening (HTS) assay was used to identify drug sensitivities of a panel of 15 glioblastoma stem cell (GSC) lines, which are representative of the classic the cancer genome atlas (TCGA) molecular subtypes, to 21 compounds in Published Protein Kinase Inhibitor Set (PKSI). This HTS screen identified sensitivity of GSCs to inhibition of polo like kinase-1 (PLK-1), a key regulator of mitosis. Given that PLK-1 is often overexpressed in a broad spectrum of cancers, and with highest expression levels being correlated with poor prognosis in several cancer types as well, we further verified the HTS result with the second-generation PLK1 inhibitor volasertib as a single agent or in combination with ionizing radiation in GSCs. Efficacy of volasertib was analyzed by Cell-Titer Glo 5 days after treatment. Morphological and molecular changes were approached by immunoblotting and flow cytometry after volasertib treatment, and in combination with ionizing radiation at 2 Gy. In vitro studies showed that volasertib inhibited cell viability with an IC50 ranging from 44.3nM to 4.36μM. Volasertib induced G2/M arrest accompanied by high levels of PLK-1, Aurora B and phosphor-histone 3 and prominent cleaved poly ADP ribose polymerase (PARP) in a dose- and time-dependent manner. Colony formation assay demonstrated that volasertib and ionizing radiation had synergistic effects on colony formation inhibition, suggesting that GSCs arrested in M phase by volasertib are more sensitive to ironizing radiation. The promising in vitro results of volasertib in GSCs led to further investigate the drug efficacy in intracranial xenograft models by volasertib alone, and in combination with radiation. Taken together, our results reinforce the potential therapeutic candidate of volasertib as a single agent and in combination with ionizing radiation in glioblastoma. Citation Format: Jianwen Dong, Nghi Nguyen, Ravesanker Ezhilarasan, Shaofang Wu, Yuji Piao, Soon Young Park, Ningyi Tiao, Clifford Stephan, Erik P. Sulman, John F. de Groot. Synergistic antitumor effects of polo like kinase inhibitor volasertib in combination with ionizing radiation in glioblastoma. [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 695. doi:10.1158/1538-7445.AM2015-695