Theo Sottero

Inhibition of PI3K/mTOR pathways in glioblastoma and implications for combination therapy with temozolomide.

Due to its molecular heterogeneity and infiltrative nature, glioblastoma multiforme (GBM) is notoriously resistant to traditional and experimental therapeutics. To overcome these hurdles, targeted agents have been combined with conventional therapy. We evaluated the preclinical potential of a novel, orally bioavailable PI3K/mTOR dual inhibitor (XL765) in in vitro and in vivo studies. In vivo serially passaged human GBM xenografts that are more genetically stable than GBM cell lines in culture were used for all experiments. Biochemical downstream changes were evaluated by immunoblot and cytotoxicity by colorimetric ATP-based assay. For in vivo experiments, human xenograft GBM 39 grown intracranially in nude mice was altered to express luciferase to monitor tumor burden by optical imaging. XL765 resulted in concentration-dependent decreases in cell viability in vitro. Cytotoxic doses resulted in specific inhibition of PI3K signaling. Combining XL765 with temozolomide (TMZ) resulted in additive toxicity in 4 of 5 xenografts. In vivo, XL765 administered by oral gavage resulted in greater than 12-fold reduction in median tumor bioluminescence compared with control (Mann-Whitney test p = 0.001) and improvement in median survival (logrank p = 0.05). TMZ alone showed a 30-fold decrease in median bioluminescence, but the combination XL765 + TMZ yielded a 140-fold reduction in median bioluminescence (Mann-Whitney test p = 0.05) with a trend toward improvement in median survival (logrank p = 0.09) compared with TMZ alone. XL765 shows activity as monotherapy and in combination with conventional therapeutics in a range of genetically diverse GBM xenografts.

Cooperation of the HDAC inhibitor vorinostat and radiation in metastatic neuroblastoma: Efficacy and underlying mechanisms

Histone deacetylase (HDAC) inhibitors can radiosensitize cancer cells. Radiation is critical in high-risk neuroblastoma treatment, and combinations of HDAC inhibitor vorinostat and radiation are proposed for neuroblastoma trials. Therefore, we investigated radiosensitizing effects of vorinostat in neuroblastoma. Treatment of neuroblastoma cell lines decreased cell viability and resulted in additive effects with radiation. In a murine metastatic neuroblastoma in vivo model vorinostat and radiation combinations decreased tumor volumes compared to single modality. DNA repair enzyme Ku-86 was reduced in several neuroblastoma cells treated with vorinostat. Thus, vorinostat potentiates anti-neoplastic effects of radiation in neuroblastoma possibly due to down-regulation of DNA repair enzyme Ku-86.

Abstract SY28-04: Rational incorporation of novel agents into multimodality treatment of glioma and neuroblastoma

Background and Objectives: We sought to incorporate novel agents into multimodality therapy for poor-prognosis human malignancies, specifically neuroblastoma and glioma. Neuroblastoma is the most common extracranial tumor of childhood and high-risk patients with evidence of metastases have an overall survival rate of less than 40% despite intensive multi-modality treatment and therefore new treatment strategies are urgently needed. For neuroblastoma, targeted radiotherapy with 131I-metaiodobenzylguanidine (MIBG) is a promising new treatment approach with 30% response rates in refractory disease. MIBG, which is a norepinephrine analogue, is taken up by the norepinephrine transporter (NET) that is abundantly expressed on neuroblastoma cells. We have focused on combining 131I-MIBG with novel drugs, likely to function as radiosensitizing agents. Specifically, we investigated the addition investigated the radiosensitizing effect of vorinostat (suberoylanilide hydroxamic acid) and radiation in the treatment of metastatic neuroblastoma in vitro and in vivo and evaluated potential mechanisms underlying these interactions. Due to their molecular heterogeneity and infiltrative nature, glioblastomas (GBM) have been notoriously difficult to treat with traditional and experimental therapeutics. Similarly, neuroblastoma the most common extracranial tumor of childhood, often presents with high-risk disease, and such patients have an overall survival rate of less than 40% despite intensive multi-modality treatment. Thus, for both glioblastoma and neuroblastoma new treatment strategies are urgently needed. To overcome the hurdles of resistance to treatment, many have suggested the use of targeted agents in combination with conventional therapy and we have sought a rational approach to the choice of targeted agent for each of these malignancies. Since the PI3K/mTOR pathway is known to play a critical role in glioma pathogenesis, we chose to target this pathway using an orally bioavailable PI3K/mTOR inhibitor (XL765). We evaluated the preclinical potential of XL765 in the treatment of gliomas, in in vitro and in vivo models, as a single agent and in combination with radiation, temozolomide, and erlotinib. Methods: For the study of neuroblastoma, various neuroblastoma cell lines were exposed to radiation, vorinostat, and combinations thereof. Response to therapy in vitro was assessed using an ATP-based proliferation assay and clonogenic survival curves. To establish the effects of vorinostat and radiation in vivo, a metastatic neuroblastoma model was established by tail vein injection of NB1691luc cells into athymic mice (a generous gift from Dr. Dickson, St. Jude Children9s Research Hospital). Mice developed widely metastatic tumor that was visible by bioluminesence 21 days post-injection in 100% of animals. Mice were treated with vorinostat 150 mg/kg intraperitoneal (IP), radiation, or combinations thereof. Each treatment group contained 10 mice. Since in all tumor-bearing mice metastatic neuroblastoma was detected in all parts of the body except the head, 1 Gy of radiation was administered to the entire animal9s body while shielding the head. Radiation was performed 1 hour after IP administration of either vorinostat or DMSO as a control. Phosphorylated H2AX after treatment with vorinostat and radiation was determined by flow cytometry. Western blot analysis was performed to detect variations in expression levels of DNA repair enzymes Rad51 and Ku-86. For the study of glioblastoma, we have obtained a series of in vivo serially passaged human GBM xenografts termed GBM6 (EGFR VIII amplified, PTEN wt), GBM8 (EGFR wt amplified, PTEN null), GBM12 (EGFR wt amplified, PTEN wt), GBM 39 (EGFR vIII amplified, PTEN wt), and GBM GS-2 (EGFR wt, PTEN null) that are more genetically stable than GBM cell lines in culture. Using XL765 in vitro we have evaluated downstream molecular changes by Western blot and cytotoxicity by colorimetric ATP-based assay. Based on these results we have combined XL765 with erlotinib (ERL), temozolomide (TMZ) and radiation (XRT) using the same assays to test for combinatorial effects. Finally, we injected xenograft GBM39 (EGFR VIII amplified, PTEN wt) intracranially into nude mice and tested various combinations of the above agents. GBM39, altered to express luciferase, was used so that intracranial tumor burden could be monitored using optical imaging. Results: For studies focusing on neuroblastoma, vorinostat resulted in dose-dependent decreased viability in all three independent neuroblastoma cell lines tested. Vorinostat had radiosensitizing effects in vitro in all three cell lines investigated. Animals treated with a combination of vorinostat and radiation showed decreased tumor volumes compared to single modality treatments (p(2) = 0.04). Phosphorylated H2AX was increased after treatment with vorinostat and radiation compared to radiation or drug alone. Expression levels of Ku-86 were reduced in neuroblastoma cells treated with vorinostat, whereas Rad51 expression did not change. In the study of glioblastoma and the PI3K/mTOR inhibitor XL765, for all xenografts tested in vitro, XL765 inhibited the PI3K pathway in a concentration- and time-dependent manner and these molecular changes correlated with increasing cytotoxicity. Treatment of GBM6 with XL765 + TMZ + XRT resulted in combined cytotoxicity that was significantly greater than any monotherapy or dual therapy combinations (p = 0.002). Similarly, in GBM8 combination of XL765 + TMZ demonstrated increased cytotoxicity compared to either agent alone (p 60-fold reduction in bio-luminescence (p(2) Conclusions: In the multimodality treatment of high-risk neuroblastoma, vorinostat has a radiosensitizing effect on neuroblastoma cells in vitro and in vivo. A possible mechanism of radiosensitizing is down-regulation of the DNA double strand repair enzyme Ku-86. These preclinical results have been translated into a phase 1 clinical trial of the combination of vorinostat and 131I-MIBG. In the multimodality treatment of gliomas, the PI3K/mTOR inhibitor XL765 showed excellent single agent cytotoxicity and resulted in supra-additive anti-tumor activity when combined with temozolomide in the treatment of a xenograft with MGMT hypermethylation. A glioma with MGMT hypermethylation would be expected to be relatively sensitive to temozolomide and we are currently asking whether such cooperative anti-tumor activity will also be evident in gliomas without MGMT methylation. Conversely, XL765 did not add to the cytotoxicity of erlotinib, again, as predicted by genotype. Against the permissive genotype of both EGFR vIII amplification and wild type PTEN, erlotinib alone was exceedingly effective as a single agent and did not require inhibition of PI3K to enhance its anti-tumor activity. We predict that XL765 will cooperative with erlotinib in decreasing tumor growth in gliomas with NON-permissive genotypes such as those with PTEN mutations and we are currently testing this hypothesis in vivo. Citation Format: Daphne A. Haas-Kogan, Gautam Prasad, Sabine Mueller, Xiaodong Yang, Steven DuBois, Theo L. Sottero, Mei-Yin Polley, David James, Mitchel S. Berger, Michael D. Prados, Dana T. Aftab, Katherine K. Matthay. Rational incorporation of novel agents into multimodality treatment of glioma and neuroblastoma [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY28-04