Shaofang Wu

MSK1-Mediated β-Catenin Phosphorylation Confers Resistance to PI3K/mTOR Inhibitors in Glioblastoma.

Glioblastoma (GBM) represents a compelling disease for kinase inhibitor therapy because most of these tumors harbor genetic alterations that result in aberrant activation of growth factor–signaling pathways. The PI3K/mammalian target of the rapamycin (mTOR) pathway is dysregulated in over 50% of human GBM but remains a challenging clinical target. Inhibitors against PI3K/mTOR mediators have limited clinical efficacy as single agents. We investigated potential bypass mechanisms to PI3K/mTOR inhibition using gene expression profiling before and after PI3K inhibitor treatment by Affymetrix microarrays. Mitogen- and stress-activated protein kinase 1 (MSK1) was markedly induced after PI3K/mTOR inhibitor treatment and disruption of MSK1 by specific shRNAs attenuated resistance to PI3K/mTOR inhibitors in glioma-initiating cells (GIC). Further investigation showed that MSK1 phosphorylates β-catenin and regulates its nuclear translocation and transcriptional activity. The depletion of β-catenin potentiated PI3K/mTOR inhibitor-induced cytotoxicity and the inhibition of MSK1 synergized with PI3K/mTOR inhibitors to extend survival in an intracranial animal model and decreased phosphorylation of β-catenin at Ser552. These observations suggest that MSK1/β-catenin signaling serves as an escape survival signal upon PI3K/mTOR inhibition and provides a strong rationale for the combined use of PI3K/mTOR and MSK1/β-catenin inhibition to induce lethal growth inhibition in human GBM. Mol Cancer Ther; 15(7); 1656–68. ©2016 AACR.

Activation of WEE1 confers resistance to PI3K inhibition in glioblastoma

Background Oncogenic activation of phosphatidylinositol-3 kinase (PI3K) signaling plays a pivotal role in the development of glioblastoma (GBM). However, pharmacological inhibition of PI3K has so far not been therapeutically successful due to adaptive resistance through a rapid rewiring of cancer cell signaling. Here we identified that WEE1 is activated after transient exposure to PI3K inhibition and confers resistance to PI3K inhibition in GBM. Methods Patient-derived glioma-initiating cells and established GBM cells were treated with PI3K inhibitor or WEE1 inhibitor alone or in combination, and cell proliferation was evaluated by CellTiter-Blue assay. Cell apoptosis was analyzed by TUNEL, annexin V staining, and blotting of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase. Both subcutaneous xenograft and orthotropic xenograft studies were conducted to evaluate the effects of the combination on tumorigenesis; the tumor growth was monitored by bioluminescence imaging, and tumor tissue was analyzed by immunohistochemistry to validate signaling changes. Results PI3K inhibition activates WEE1 kinase, which in turn phosphorylates cell division control protein 2 homolog (Cdc2) at Tyr15 and inhibits Cdc2 activity, leading to G2/M arrest in a p53-independent manner. WEE1 inhibition abrogated the G2/M arrest and propelled cells to prematurely enter into mitosis and consequent cell death through mitotic catastrophe and apoptosis. Additionally, combination treatment significantly suppressed tumor growth in a subcutaneous model but not in an intracranial model due to limited blood-brain barrier penetration. Conclusions Our findings highlight WEE1 as an adaptive resistant gene activated after PI3K inhibition, and inhibition of WEE1 potentiated the effectiveness of PI3K targeted inhibition, suggesting that a combinational inhibition of WEE1 and PI3K might allow successful targeted therapy in GBM.

Preclinical therapeutic efficacy of a novel blood-brain barrier-penetrant dual PI3K/mTOR inhibitor with preferential response in PI3K/PTEN mutant glioma

Glioblastoma (GBM) is an ideal candidate disease for signal transduction targeted therapy because the majority of these tumors harbor genetic alterations that result in aberrant activation of growth factor signaling pathways. Loss of heterozygosity of chromosome 10, mutations in the tumor suppressor gene PTEN, and PI3K mutations are molecular hallmarks of GBM and indicate poor prognostic outcomes in many cancers. Consequently, inhibiting the PI3K pathway may provide therapeutic benefit in these cancers. PI3K inhibitors generally block proliferation rather than induce apoptosis. To restore the sensitivity of GBM to apoptosis induction, targeted agents have been combined with conventional therapy. However, the molecular heterogeneity and infiltrative nature of GBM make it resistant to traditional single agent therapy. Our objectives were to test a dual PI3K/mTOR inhibitor that may cross the blood–brain barrier (BBB) and provide the rationale for using this inhibitor in combination regimens to chemotherapy-induced synergism in GBM. Here we report the preclinical potential of a novel, orally bioavailable PI3K/mTOR dual inhibitor, DS7423 (hereafter DS), in in-vitro and in-vivo studies. DS was tested in mice, and DS plasma and brain concentrations were determined. DS crossed the BBB and led to potent suppression of PI3K pathway biomarkers in the brain. The physiologically relevant concentration of DS was tested in 9 glioma cell lines and 22 glioma-initiating cell (GIC) lines. DS inhibited the growth of glioma tumor cell lines and GICs at mean 50% inhibitory concentration values of less than 250 nmol/L. We found that PI3K mutations and PTEN alterations were associated with cellular response to DS treatment; with preferential inhibition of cell growth in PI3KCA-mutant and PTEN altered cell lines. DS showed efficacy and survival benefit in the U87 and GSC11 orthotopic models of GBM. Furthermore, administration of DS enhanced the antitumor efficacy of temozolomide against GBM in U87 glioma models, which shows that PI3K/mTOR inhibitors may enhance alkylating agent-mediated cytotoxicity, providing a novel regimen for the treatment of GBM. Our present findings establish that DS can specifically be used in patients who have PI3K pathway activation and/or loss of PTEN function. Further studies are warranted to determine the potential of DS for glioma treatment.

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.

APOBEC3G acts as a therapeutic target in mesenchymal gliomas by sensitizing cells to radiation-induced cell death

Genomic, transcriptional, and proteomic analyses of brain tumors reveal that subtypes differ in their pathway activity, progression, and response to therapy. We performed an expression profiling of Glioma Initiating Cells (GICs) and comparative analysis between different groups of GICs indicates major variations in gene expression. Hierarchical clustering analysis revealed groups of GICs reflecting their heterogeneity, and among some of the genes as major regulators of mesenchymal phenotype, we identified ABOBEC3G as one of the most discriminating genes in mesenchymal group. ABOBEC3G revealed a strong correlation with overall survival in TCGA GBM patient cohorts. APOBEC3G regulates cell invasion and silencing of this gene in GICs inhibits cell invasion and also glioma sphere initiation. APOBEC3G controls invasion through TGFβ/Smad2 pathway by regulating Smad2 target genes Thrombospondin 1, matrix metallopeptidase 2 and TIMP metallopeptidase inhibitor 1. We also show that targeting APOBEC3G can sensitize cancer cells to radiation induced cell death by attenuating activation of the DNA repair pathway. This response is mainly shown by decreased pChk2 expression in knockdown APOBEC3G cells. Taken together, we show that APOBEC3G gene is a mesenchymal enriched gene that controls invasion and knockdown of APOBEC3G sensitizes cells to radiation induced cell death, suggesting that APOBEC3G can be considered for use in stratifying patients with GBM for prognostic considerations.Genomic, transcriptional, and proteomic analyses of brain tumors reveal that subtypes differ in their pathway activity, progression, and response to therapy. We performed an expression profiling of Glioma Initiating Cells (GICs) and comparative analysis between different groups of GICs indicates major variations in gene expression. Hierarchical clustering analysis revealed groups of GICs reflecting their heterogeneity, and among some of the genes as major regulators of mesenchymal phenotype, we identified ABOBEC3G as one of the most discriminating genes in mesenchymal group. ABOBEC3G revealed a strong correlation with overall survival in TCGA GBM patient cohorts. APOBEC3G regulates cell invasion and silencing of this gene in GICs inhibits cell invasion and also glioma sphere initiation. APOBEC3G controls invasion through TGFβ/Smad2 pathway by regulating Smad2 target genes Thrombospondin 1, matrix metallopeptidase 2 and TIMP metallopeptidase inhibitor 1. We also show that targeting APOBEC3G can sensitize cancer cells to radiation induced cell death by attenuating activation of the DNA repair pathway. This response is mainly shown by decreased pChk2 expression in knockdown APOBEC3G cells. Taken together, we show that APOBEC3G gene is a mesenchymal enriched gene that controls invasion and knockdown of APOBEC3G sensitizes cells to radiation induced cell death, suggesting that APOBEC3G can be considered for use in stratifying patients with GBM for prognostic considerations.

Abstract 1781: EGFR amplification-A candidate predictive biomarker of PARP inhibitor Talazoparib sensitivity in gliomas

Poly (ADP-ribose) polymerases (PARP) are enzymes involved in DNA-damage repair. Inhibition of PARP is a promising strategy for targeting cancers with defective DNA-damage repair. Several PARP inhibitors are currently in trials in the adjuvant, neoadjuvant, and metastatic settings for the treatment of ovarian, BRCA-mutated breast, and other cancers. Glioma Stem Cells (GSCs) exhibit higher oxidative base damage, single-strand DNA breaks and genomic instability and is reliant on single-strand break repair/base excision repair to tolerate additional stress. PARP critical for maintaining genomic stability by regulating a variety of DNA damage repair mechanisms is constitutively activated in GSCs. In this study, we show that talazoparib – a unique PARP inhibitor exhibits a strong single agent activity to inhibit proliferation of GSCs in vitro and suppress tumor progression in GSCs xenografts. Talazoparib cytotoxic activity was through inhibiting PARylation and by trapping PARP-DNA complexes resulting in accumulation of DNA damage during replication and ultimately leading to apoptosis. More importantly, we found EFGR amplification, which occurs in about 45% of GBM as a biomarker of response to PARPi. Gene expression and RPPA data showed that GSCs with EGFR amplification were associated with increased ROS, basal expression of DNA repair proteins and more DNA damage following talazoparib treatment, which may partially explain the sensitivity of this group of GSCs to talazoparib. EGFR enzyme activity was important for PARPi sensitivity since kinase dead mutant of EGFR or EGFR knock down cell lines were resistant to PARPi as was also shown by decreased PARP/DNA complex formation. Talazoparib showed strong inhibition of tumor growth in subcutaneous glioma model and 20% increase in median survival in an intracranial model with a 10% blood brain penetration of talazoparib. These data provide mechanistic insights into the anti-cancer activity of PARP inhibitors with mechanistic rationale of PARP inhibition and potential EGFR amplification as biomarker in the development for precision cancer. Citation Format: Shaofang Wu, Feng Gao, Xiaolong Li, Jie Ding, Siyuan Zheng, Emmanuel Martinez-Ledesma, Ningping Feng, Erik Sulman, Roel Verhaak, John de Groot, Dimpy Koul, W.K.Alfred Yung. EGFR amplification-A candidate predictive biomarker of PARP inhibitor Talazoparib sensitivity in gliomas [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 1781. doi:10.1158/1538-7445.AM2017-1781

Abstract 2798: Identification of gamma secretase inhibitor (GSI)-responsive GBM patients: Involvement of NOTCH, p53, and PI3K/MAPK signaling

Glioblastoma (GBM) is the most common and lethal primary intracranial tumor. The recent standard-of-care treatment consisting of surgery, followed by radiotherapy and temozolomide, just prolongs the median survival period of GBM patients to 14.6 months. What impedes the survival is due to the extensive heterogeneity among GBM patients at both the cellular and the molecular levels. In our study, we focused on the precision medicine strategy targeting NOTCH signaling. According to recent Phase I/II clinical trial results, only a few GBM patients showed satisfactory response to GSIs. However, little is known regarding how to distinguish GSI-responsive patients. Here, we illustrated that it required (1) high NOTCH activity, (2) wild-type p53 and elevated P53 activity, (3) increased MAPK activity, and (4) decreased PI3K/AKT activity for GBM to respond to GSIs. In our study, we tested a series of patient-derived primary glioma stem cells (GSCs) to two GSIs (RO4929097 and BMS-708163). GSI-sensitive lines were identified and GSI compounds impaired cell viability and sphere formation ability in these cell lines. GSIs also induced cell death of the sensitive GSCs. To find out the signature of the GSI-sensitive GSCs, we did enrichment and correlation analyses against RNA sequencing, RPPA, and methylation data of the GSI-sensitive and resistant GBM lines. We found that GSI-sensitive GSCs harbored significantly elevated Notch activity (high NOTCH1 and NICD1 expression, and low Notch1 methylation). Interestingly, GSI-responsive cells also possessed increased P53 activity (increased expression of P53 targets-P21, BAX and TIGAR), high wild-type p53 frequencies, increased MAPK activity (elevated p-Erk1/2-Thr202/Tyr204 expression), and decreased PI3K/AKT activity (high PTEN expression and low p-AKT-Thr308, RAPTOR, p-4E-BP1-Ser65 expression) (P Citation Format: Chen Zhang, Martinez-Ledesma Juan, Jie Ding, Feng Gao, Shaofang Wu, Xiaolong Li, Erik Philip Sulman, Dimpy Koul, WK Alfred Yung. Identification of gamma secretase inhibitor (GSI)-responsive GBM patients: Involvement of NOTCH, p53, and PI3K/MAPK signaling [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 2798. doi:10.1158/1538-7445.AM2017-2798

Abstract 2122: Downregulation of RGS10 induces PI3K/mTOR inhibitor resistance by Ras activation in glioblastoma

Glioblastoma multiforme (GBM) is the most common form of malignant brain cancer in adults. The prognosis of affected patients is poor and median survival is only one year. The essential role of PI3K signaling in GBM proliferation, metabolism and differentiation has been highlighted. The major mechanism for the oncogenic activity of PI3K is by preventing apoptosis through the downstream activation of Akt and mTOR. The importance of mTOR in the regulation of cell survival downstream of PI3K signaling provides a strong rationale for the use of dual PI3K/mTOR inhibitor. However, the limited effect of dual PI3K/mTOR inhibitor used as single agent is most likely due to activation of collateral pathways, suggesting that additional refinement of this overall approach is necessary. We investigated potential bypass mechanisms to PI3K/mTOR inhibition as a single agent and generated three resistant cell lines to PI3K/mTOR inhibitor with 3 to 5-fold increase in IC50 value and with an aim to identify the potential bypass of survival to PI3K/mTOR inhibitor. We show that the IC50 of the resistant cell lines and parental lines is correlated with s6 phosphorylation and the three resistant cell lines express increased level of phosphorylated ERK1/2, p70S6K and p90RSK protein levels. Further investigation show that RGS10, a regulator of G protein signaling, is down regulated in the resistant cell lines. Down regulation of RGS10 increases the GTP-bound Ras, implying that Ras activation defines the acquired resistance by activating the ERK/p90RSK pathway. Knockdown RGS10 in the parental cells induces ERK1/2, p70S6K and p90RSK phosphorylation and the resistance to PI3K/mTOR inhibitor. Raf1 or p90RSK knockdown can re-sensitize resistant cell lines to PI3K/mTOR inhibition. Combination of PI3K/mTOR inhibitor with Raf inhibitor Sorafinib efficiently inhibited the growth of the resistant cell lines showing Ras/Raf activation serves as an escape mechanism to PI3K/mTOR inhibition. These observations suggest that Ras/Raf signaling serves as an survival signal upon PI3K/mTOR inhibition and provides a strong rationale for the combined use of PI3K/mTOR and Raf1 inhibition to induce lethal growth inhibition in human GBM. Citation Format: Xiaolong Li, Shaofang Wu, Dimpy Koul, W.K. Alfred Yung. Downregulation of RGS10 induces PI3K/mTOR inhibitor resistance by Ras activation in glioblastoma. [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 2122.

Abstract 31: WEE1 kinase inhibition enhances PI3K inhibitor response in p53 deficient gliomas

Glioblastoma multiforme (GBM) is an aggressive brain tumor that is resistant to chemotherapy and radiation, and is almost always fatal. Oncogenic activation of the PI3K signaling pathway plays a pivotal role in the development of GBM and is a major molecular target for therapeutic development. However, clinical results of single-agent PI3K inhibitors have been modest to date. PI3K inhibitors have led to tumor stabilization and some disease responses; however, dramatic tumor regressions are not typical. Multiple recent reports have described mechanisms of resistance to PI3K inhibitors. The goal of the present study was to identify ways to improve the efficacy of PI3K inhibitor-based therapy by overcoming adaptive resistance. In this study we show that PI3K inhibitor BKM induces G2/M cell cycle arrest and inhibits GBM cell proliferation. We identified WEE1 as a putative treatment target of resistance to PI3K inhibition based on its potential function as a mitotic gatekeeper and its specific overexpression in GBM. WEE1 kinase is a key molecule in maintaining G2-cell-cycle checkpoint arrest for premitotic DNA repair. We show that that BKM treatment activates WEE1, which in turn phosphorylates CDC2 at Tyr15, inactivates CDC2 and inhibits CDC2 activity, leading to G2/M arrest in glioma initiating cells (GICs). We show that treatment with WEE1 inhibitor MK1775 inhibited BKM-induced Cdc2 Tyr15 phosphorylation, and forced GICs to enter into the mitotic phase of the cell cycle. Using a panel of GICs, we show that MK1775 synergizes with BKM to inhibit GBM cell proliferation. Further, MK1775 and BKM as a single agent alone partially blocks proliferation, however, combination of MK1775 and BKM induces apoptosis as evaluated by Annexin V positive cells, cleaved PARP and caspase 3, and TUNEL staining. This effect was observed specifically in p53 mutant cells. Finally we show that depletion of WEE1 by shRNAs synergizes with BKM to inhibit GBM cell proliferation and induce apoptosis. These results indicate that WEE1 inhibition potentiated the effectiveness of PI3K targeted inhibition in GBM, and suggest a combinational inhibition of WEE1 and PI3K might allow successful targeted therapy in p53 mutant GBM. Citation Format: Shaofang Wu, Siyuan Zheng, Shuzhen Wang, W.K.Alfred Yung, Dimpy Koul. WEE1 kinase inhibition enhances PI3K inhibitor response in p53 deficient gliomas. [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 31. doi:10.1158/1538-7445.AM2015-31

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