Citations

Abstract In this study, we investigated the precursor and active forms of a p53 small-molecule inhibitor for their effects on temozolomide (TMZ) antitumor activity against glioblastoma (GBM), using both in vitro and in vivo experimental approaches. Results from in vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased when p53 wild-type (p53wt) GBMs were cotreated with the active form of p53 inhibitor, and this heightened cytotoxic response was accompanied by increased poly(ADP-ribose) polymerase cleavage as well as elevated cellular phospho-H2AX. Analysis of the same series of GBMs, as intracranial xenografts in athymic mice, and administering corresponding p53 inhibitor precursor, which is converted to the active compound in vivo, yielded results consistent with the in vitro analyses: TMZ + p53 inhibitor precursor cotreatment of three distinct p53wt GBM xenografts resulted in significant enhancement of TMZ antitumor effect relative to treatment with TMZ alone, as indicated by serial bioluminescence monitoring as well as survival analysis (P < 0.001 for cotreatment survival benefit in each case). Mice receiving intracranial injection with p53null GBM showed similar survival benefit from TMZ treatment regardless of the presence or absence of p53 inhibitor precursor. In total, our results indicate that the p53 active and precursor inhibitor pair enhances TMZ cytotoxicity in vitro and in vivo, respectively, and do so in a p53-dependent manner. [Cancer Res 2008;68(24):10034–8]

Purpose Epidermal growth factor receptor (EGFR) amplification in glioblastoma multiforme (GBM) is a common occurrence and is associated with treatment resistance. Erlotinib, a selective EGFR inhibitor, was combined with temozolomide (TMZ) and radiotherapy (RT) in a phase I/II trial. Patients and Methods Adults not taking enzyme-inducing anticonvulsants after resection or biopsy of GBM were treated with erlotinib (150 mg daily) until progression. Erlotinib was delivered alone for 1 week, then concurrently with TMZ (75 mg mg/m2 daily) and RT (60 Gy), and finally, concurrently with up to six cycles of adjuvant TMZ (200 mg/m2 daily for 5 days every 28 days). The primary end point was survival at 1 year. Results Ninety-seven eligible patients were accrued with a median follow-up time of 22.2 months. By definition, the primary end point was successfully met with a median survival time of 15.3 months. However, there was no sign of benefit in overall survival when comparing N0177 with the RT/TMZ arm of the European Organisation for Research and Treatment of Cancer/National Cancer Institute of Canada trial 26981/22981 (recursive partitioning analysis [RPA] class III, 19 v 21 months; RPA class IV, 16 v 16 months; RPA class V, 8 v 10 months, respectively). Presence of diarrhea, rash, and EGFRvIII, p53, phosphatase and tensin homolog (PTEN), combination EGFR and PTEN, and EGFR amplification status were not predictive (P > .05) of survival. Conclusion Although the primary end point was successfully met using nitrosourea-based (pre-TMZ) chemotherapy era historic controls, there was no sign of benefit compared with TMZ era controls. Analyses of molecular subsets did not reveal cohorts of patients sensitive to erlotinib. TMZ chemotherapy combined with RT resulted in improved outcomes compared with historical controls who received nitrosourea-based chemotherapies.

Abstract Purpose: Hyperactivation of the phosphatidylinositol 3-kinase/Akt signaling through disruption of PTEN function is common in glioblastoma multiforme, and these genetic changes are predicted to enhance sensitivity to mammalian target of rapamycin (mTOR) inhibitors such as RAD001 (everolimus). Experimental Design: To test whether PTEN loss could be used as a predictive marker for mTOR inhibitor sensitivity, the response of 17 serially transplantable glioblastoma multiforme xenografts was evaluated in an orthotopic therapy evaluation model. Of these 17 xenograft lines, 7 have either genomic deletion or mutation of PTEN. Results: Consistent with activation of Akt signaling, there was a good correlation between loss of PTEN function and elevated levels of Akt phosphorylation. However, of the 7 lines with disrupted PTEN function, only 1 tumor line (GBM10) was significantly sensitive to RAD001 therapy (25% prolongation in median survival), whereas 1 of 10 xenograft lines with wild-type PTEN was significantly sensitive to RAD001 (GS22; 34% prolongation in survival). Relative to placebo, 5 days of RAD001 treatment was associated with a marked 66% reduction in the MIB1 proliferation index in the sensitive GBM10 line (deleted PTEN) compared with a 25% and 7% reduction in MIB1 labeling index in the insensitive GBM14 (mutant PTEN) and GBM15 (wild-type PTEN) lines, respectively. Consistent with a cytostatic antitumor effect, bioluminescent imaging of luciferase-transduced intracranial GBM10 xenografts showed slowed tumor growth without significant tumor regression during RAD001 therapy. Conclusion: These data suggest that loss of PTEN function is insufficient to adequately predict responsiveness to mTOR inhibitors in glioblastoma multiforme.

Abstract Intrinsic or acquired chemoresistance to alkylating agents is a major cause of treatment failure in patients with malignant brain tumors. Alkylating agents, the mainstay of treatment for brain tumors, damage the DNA and induce apoptosis, but the cytotoxic activity of these agents is dependent on DNA repair pathways. For example, O6-methylguanine DNA adducts can cause double-strand breaks, but this is dependent on a functional mismatch repair pathway. Thus, tumor cell lines deficient in mismatch repair are resistant to alkylating agents. Perhaps the most important mechanism of resistance to alkylating agents is the DNA repair enzyme O6-methylguanine methyltransferase, which can eliminate the cytotoxic O6-methylguanine DNA adduct before it causes harm. Another mechanism of resistance to alkylating agents is the base excision repair (BER) pathway. Consequently, efforts are ongoing to develop effective inhibitors of BER. Poly(ADP-ribose)polymerase plays a pivotal role in BER and is an important therapeutic target. Developing effective strategies to overcome chemoresistance requires the identification of reliable preclinical models that recapitulate human disease and which can be used to facilitate drug development. This article describes the diverse mechanisms of chemoresistance operating in malignant glioma and efforts to develop reliable preclinical models and novel pharmacologic approaches to overcome resistance to alkylating agents.

Object Bioluminescence imaging (BLI) offers a rapid and accurate means for longitudinal study of tumor cell growth and response to therapy in rodent models. Because this technology has only recently come into use in the field of small animal imaging, applications in this area have been limited. In the current study we have applied BLI to the analysis of clinically relevant issues involving use of the DNA methylating agent temozolomide (TMZ) in a mouse model. Methods An invasive glioblastoma multiforme xenograft was modified for BLI via transduction with a luciferase-encoding lentivirus. Supratentorial tumors were established in athymic nude mice that were subsequently assigned randomly to control and TMZ treatment groups, and the extent of intracranial tumor was monitored using BLI. Results In an experiment designed to compare the extent of antitumor effect between a single high-dose TMZ treatment and a protracted low-dose TMZ regimen, BLI revealed the protracted regimen as having superior antitumor effect, and this interpretation was consistent with results from a survival comparison between the two TMZ treatment groups. In a second experiment designed to assess the utility of BLI for testing therapies against recurrent glioblastoma multiforme, mice with intracranial tumors were retreated with TMZ at a time when BLI monitoring revealed tumor regrowth following initial TMZ treatment, and retreatment was successful in providing additional survival benefit. Conclusion The results of these experiments indicate that BLI monitoring can be used as a surrogate for predicting survival benefit from TMZ treatment, permits early determination of relative survival benefit associated with distinct TMZ therapeutic regimens, and offers a means of investigating secondary/salvage therapy efficacy following tumor regrowth from initial therapy.

Abstract Purpose: Glioblastoma multiforme is the most frequent primary brain tumor in adults and represents one of the most lethal malignancies with a median survival of 12-16 months. We have previously shown that an oncolytic measles virus derivative expressing soluble human carcinoembryonic antigen (MV-CEA) has significant antitumor activity against glioblastoma multiforme cell lines and xenografts. Radiation therapy (RT) represents one of the mainstays of glioma treatment. Here we tested the hypothesis that the combination of RT with MV-CEA would have synergistic activity against gliomas. Experimental Design: 3-(4,5-Dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and clonogenic assays were used to test cytoxicity of the combination treatment in vivo. To examine the mechanism of synergy, one-step viral growth curves, terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assays, and Western blot analyses were performed. In vivo assessment of synergistic antitumor activity was conducted in a U87 glioma model. Results: MTS and clonogenic assays showed a strong synergistic interaction between MV-CEA and RT in glioblastoma multiforme cells including both primary and established glioma lines. Furthermore, significant antitumor efficacy was observed in vivo in a subcuteneous U87 xenograph model. There was significant prolongation of survival (P = 0.001) in the combination treatment group as compared with single modality– or control-treated animals. One-step viral growth curves showed increased viral burst size by up to 2 log in MV/RT combination–treated cells, as compared with single agent MV-CEA–treated glioma cells. Changes in CEA levels and expression of viral N and H protein were also consistent with increased viral production. Furthermore, TUNEL assays and Western blot analysis showed increase in apoptosis in MV/RT combination–treated cells. The pan-caspase inhibitor Z-VAD-FMK and the caspase-8 inhibitor Z-IETD-FMK, but not the caspase-9 inhibitor Z-IEHD-FMK, protected glioma cells from MV-CEA/RT–induced cleavage of poly(ADP-ribose) polymerase (PARP), indicating that the apoptotic death in combination-treated cells is mostly mediated via the extrinsic caspase pathway. The Fas/Fas ligand interaction blocking antibody NOK-1 blocked MV/RT–induced PARP cleavage whereas the Fas agonistic antibody CH11 increased PARP cleavage in MV/RT combination–treated cells. Reverse transcription-PCR, fluorescence-activated cell sorting analysis and immunohistochemistry showed up-regulation of Fas in combination-treated tumor in vitro and in vivo cells. Conclusions: There is synergy between MV-CEA and RT in vitro and in vivo. The synergistic effect of the combination seems to be due to increase in viral burst size and increase in apoptotic cell death. This latter effect is mostly mediated via the extrinsic caspase-8 pathway, activated via increased signaling through the Fas death receptor pathway. These results could have translational implications in glioma therapy.

Abstract In the current study, we examined a panel of serially passaged glioblastoma xenografts, in the context of an intracranial tumor therapy response model, to identify associations between glioblastoma molecular characteristics and tumor sensitivity to the epidermal growth factor receptor (EGFR) kinase inhibitor erlotinib. From an initial evaluation of 11 distinct glioblastoma xenografts, two erlotinib-sensitive tumors were identified, each having amplified EGFR and expressing wild-type PTEN. One of these tumors expressed truncated EGFRvIII, whereas the other expressed full-length EGFR. Subsequent cDNA sequence analysis revealed the latter tumor as expressing an EGFR sequence variant with arginine, rather than leucine, at amino acid position 62; this was the only EGFR sequence variant identified among the 11 xenografts, other than the aforementioned vIII sequence variant. EGFR cDNAs were then examined from 12 more xenografts to determine whether additional missense sequence alterations were evident, and this analysis revealed one such case, expressing threonine, rather than alanine, at amino acid position 289 of the extracellular domain. This glioblastoma was also amplified for EGFR, but did not display significant erlotinib sensitivity, presumably due to its lacking PTEN expression. In total, our study identified two erlotinib-sensitive glioblastoma xenografts, with the common molecular characteristics shared by each being the expression of wild-type PTEN in combination with the expression of amplified and aberrant EGFR. [Mol Cancer Ther 2007;6(3):1167–74]

Abstract This study was undertaken to characterize preclinical cytotoxic interactions for human malignancies between the multikinase inhibitor sorafenib (BAY 43-9006) and proteasome inhibitors bortezomib or MG132. Multiple tumor cell lines of varying histiotypes, including A549 (lung adenocarcinoma), 786-O (renal cell carcinoma), HeLa (cervical carcinoma), MDA-MB-231 (breast), K562 (chronic myelogenous leukemia), Jurkat (acute T-cell leukemia), MEC-2 (B-chronic lymphocytic leukemia), and U251 and D37 (glioma), as well as cells derived from primary human glioma tumors that are likely a more clinically relevant model were treated with sorafenib or bortezomib alone or in combination. Sorafenib and bortezomib synergistically induced a marked increase in mitochondrial injury and apoptosis, reflected by cytochrome c release, caspase-3 cleavage, and poly(ADP-ribose) polymerase degradation in a broad range of solid tumor and leukemia cell lines. These findings were accompanied by several biochemical changes, including decreased phosphorylation of vascular endothelial growth factor receptor-2, platelet-derived growth factor receptor-β, and Akt and increased phosphorylation of stress-related c-Jun NH2-terminal kinase (JNK). Inhibition of Akt was required for synergism, as a constitutively active Akt protected cells against apoptosis induced by the combination. Alternatively, the JNK inhibitor SP600125 could also protect cells from apoptosis induced by the combination, indicating that both inhibition of Akt and activation of JNK were required for the synergism. These findings show that sorafenib interacts synergistically with bortezomib to induce apoptosis in a broad spectrum of neoplastic cell lines and show an important role for the Akt and JNK pathways in mediating synergism. Further clinical development of this combination seems warranted. [Mol Cancer Ther 2006;5(9):2378–87]

Abstract Purpose: The influence of epidermal growth factor receptor (EGFR) amplification on glioblastoma patient prognosis following definitive radiotherapy has been extensively investigated in clinical studies, and yet the relationship between EGFR status and radiation response remains unclear. The intent of the current study was to address this relationship using several EGFR-amplified glioblastoma xenografts in an orthotopic athymic mouse model. Experimental Design: We examined the effect of radiation on the survival of nude mice with intracranial xenografts derived from 13 distinct patient tumors, 7 of which have amplified EGFR. Mice with established intracranial tumors were randomized to sham treatment or 12-Gy radiation in six fractions delivered over 12 days. Results: For six of the xenografts, radiation of mice with intracranial tumor significantly extended survival, and four of these xenografts had EGFR amplification. For seven other xenografts, radiation treatment did not significantly extend survival, and three of these, including GBM12, had EGFR amplification. Similar to EGFR, the tumor genetic status of p53 or PTEN did not show preferential association with radiation-sensitive or radiation-resistant xenografts whereas hyperphosphorylation of Akt on Ser473 was associated with increased radioresistance. To specifically investigate whether inhibition of EGFR kinase activity influences radiation response, we examined combined radiation and EGFR inhibitor treatment in mice with intracranial GBM12. The combination of oral erlotinib administered concurrently with radiation resulted only in additive survival benefit relative to either agent alone. Conclusions: Our results indicate that EGFR amplification, as a biomarker, is not singularly predictive of glioblastoma response to radiation therapy, nor does the inhibition of EGFR enhance the intrinsic radiation responsiveness of glioblastoma tumors. However, efficacious EGFR inhibitor and radiation monotherapy regimens can be used in combination to achieve additive antitumor effect against a subset of glioblastoma.