Citations

BACKGROUNDMitogen‐activated protein kinase (MAPK) activation and mammalian target of rapamycin (mTOR)‐dependent signaling are hallmarks of glioblastoma. In the current study, the authors conducted a phase 1/2 study of sorafenib (an inhibitor of Raf kinase and vascular endothelial growth factor receptor 2 [VEGFR‐2]) and the mTOR inhibitor temsirolimus in patients with recurrent glioblastoma.METHODSPatients with recurrent glioblastoma who developed disease progression after surgery or radiotherapy plus temozolomide and with ≤2 prior chemotherapy regimens were eligible. The phase 1 endpoint was the maximum tolerated dose (MTD), using a cohorts‐of‐3 design. The 2‐stage phase 2 study included separate arms for VEGF inhibitor (VEGFi)–naive patients and patients who progressed after prior VEGFi.RESULTSThe MTD was sorafenib at a dose of 200 mg twice daily and temsirolimus at a dose of 20 mg weekly. In the first 41 evaluable patients who were treated at the phase 2 dose, there were 7 who were free of disease progression at 6 months (progression‐free survival at 6 months [PFS6]) in the VEGFi‐naive group (17.1%); this finding met the prestudy threshold of success. In the prior VEGFi group, only 4 of the first 41 evaluable patients treated at the phase 2 dose achieved PFS6 (9.8%), and this did not meet the prestudy threshold for success. The median PFS for the 2 groups was 2.6 months and 1.9 months, respectively. The median overall survival for the 2 groups was 6.3 months and 3.9 months, respectively. At least 1 adverse event of grade ≥3 was observed in 75.5% of the VEGFi‐naive patients and in 73.9% of the prior VEGFi patients.CONCLUSIONSThe limited activity of sorafenib and temsirolimus at the dose and schedule used in the current study was observed with considerable toxicity of grade ≥3. Significant dose reductions that were required in this treatment combination compared with tolerated single‐agent doses may have contributed to the lack of efficacy. Cancer 2018;124:1455‐63. © 2018 American Cancer Society.

Abstract Poly ADP-ribose polymerase (PARP) inhibitors, including talazoparib, potentiate temozolomide efficacy in multiple tumor types; however, talazoparib-mediated sensitization has not been evaluated in orthotopic glioblastoma (GBM) models. This study evaluates talazoparib ± temozolomide in clinically relevant GBM models. Talazoparib at 1–3 nmol/L sensitized T98G, U251, and GBM12 cells to temozolomide, and enhanced DNA damage signaling and G2–M arrest in vitro. In vivo cyclical therapy with talazoparib (0.15 mg/kg twice daily) combined with low-dose temozolomide (5 mg/kg daily) was well tolerated. This talazoparib/temozolomide regimen prolonged tumor stasis more than temozolomide alone in heterotopic GBM12 xenografts [median time to endpoint: 76 days versus 50 days temozolomide (P = 0.005), 11 days placebo (P < 0.001)]. However, talazoparib/temozolomide did not accentuate survival beyond that of temozolomide alone in corresponding orthotopic xenografts [median survival 37 vs. 30 days with temozolomide (P = 0.93), 14 days with placebo, P < 0.001]. Average brain and plasma talazoparib concentrations at 2 hours after a single dose (0.15 mg/kg) were 0.49 ± 0.07 ng/g and 25.5±4.1 ng/mL, respectively. The brain/plasma distribution of talazoparib in Bcrp−/− versus wild-type (WT) mice did not differ, whereas the brain/plasma ratio in Mdr1a/b−/− mice was higher than WT mice (0.23 vs. 0.02, P < 0.001). Consistent with the in vivo brain distribution, overexpression of MDR1 decreased talazoparib accumulation in MDCKII cells. These results indicate that talazoparib has significant MDR1 efflux liability that may restrict delivery across the blood–brain barrier, and this may explain the loss of talazoparib-mediated temozolomide sensitization in orthotopic versus heterotopic GBM xenografts. Mol Cancer Ther; 16(12); 2735–46. ©2017 AACR.

Abstract Purpose: CD90 (Thy-1) is a glycophosphatidylinositol-anchored glycoprotein considered as a surrogate marker for a variety of stem cells, including glioblastoma (GBM) stem cells (GSC). However, the molecular and cellular functions of CD90 remain unclear. Experimental Design: The function of CD90 in GBM was addressed using cellular models from immortalized and primary GBM lines, in vivo orthotopic mouse models, and GBM specimens' transcriptome associated with MRI features from GBM patients. CD90 expression was silenced in U251 and GBM primary cells and complemented in CD90-negative U87 cells. Results: We showed that CD90 is not only expressed on GSCs but also on more differentiated GBM cancer cells. In GBM patients, CD90 expression was associated with an adhesion/migration gene signature and with invasive tumor features. Modulation of CD90 expression in GBM cells dramatically affected their adhesion and migration properties. Moreover, orthotopic xenografts revealed that CD90 expression induced invasive phenotypes in vivo. Indeed, CD90 expression led to enhanced SRC and FAK signaling in our GBM cellular models and GBM patients' specimens. Pharmacologic inhibition of these signaling nodes blunted adhesion and migration in CD90-positive cells. Remarkably, dasatinib blunted CD90-dependent GBM cell invasion in vivo and killed CD90high primary GSC lines. Conclusions: Our data demonstrate that CD90 is an actor of GBM invasiveness through SRC-dependent mechanisms and could be used as a predictive factor for dasatinib response in CD90high GBM patients. Clin Cancer Res; 23(23); 7360–74. ©2017 AACR.

Abstract Purpose: Bevacizumab, a humanized monoclonal antibody to VEGF, is used routinely in the treatment of patients with recurrent glioblastoma (GBM). However, very little is known regarding the effects of bevacizumab on the cells in the perivascular space in tumors. Experimental Design: Established orthotopic xenograft and syngeneic models of GBM were used to determine entry of monoclonal anti-VEGF-A into, and uptake by cells in, the perivascular space. Based on the results, we examined CD133+ cells derived from GBM tumors in vitro. Bevacizumab internalization, trafficking, and effects on cell survival were analyzed using multilabel confocal microscopy, immunoblotting, and cytotoxicity assays in the presence/absence of inhibitors. Results: In the GBM mouse models, administered anti-mouse-VEGF-A entered the perivascular tumor niche and was internalized by Sox2+/CD44+ tumor cells. In the perivascular tumor cells, bevacizumab was detected in the recycling compartment or the lysosomes, and increased autophagy was found. Bevacizumab was internalized rapidly by CD133+/Sox2+-GBM cells in vitro through macropinocytosis with a fraction being trafficked to a recycling compartment, independent of FcRn, and a fraction to lysosomes. Bevacizumab treatment of CD133+ GBM cells depleted VEGF-A and induced autophagy thereby improving cell survival. An inhibitor of lysosomal acidification decreased bevacizumab-induced autophagy and increased cell death. Inhibition of macropinocytosis increased cell death, suggesting macropinocytosis of bevacizumab promotes CD133+ cell survival. Conclusions: We demonstrate that bevacizumab is internalized by Sox2+/CD44+-GBM tumor cells residing in the perivascular tumor niche. Macropinocytosis of bevacizumab and trafficking to the lysosomes promotes CD133+ cell survival, as does the autophagy induced by bevacizumab depletion of VEGF-A. Clin Cancer Res; 23(22); 7059–71. ©2017 AACR.

Abstract 3D biomaterial models have potential to explore the influence of the tumor microenvironment on aberrant signaling pathways and compensatory signals using patient‐derived cells. Glioblastoma (GBM) tumors are highly heterogeneous, with both cell composition and extracellular matrix biophysical factors seen as key regulators of malignant phenotype and treatment outcomes. Amplification, overexpression, and mutation of the epidermal growth factor receptor (EGFR) tyrosine kinase have been identified in 50% of GBM patients. Here, hyaluronic acid (HA) decorated methacrylamide‐functionalized gelatin (GelMA) hydrogels are used to examine the synergies between microenvironmental factors and a model EGFR tyrosine kinase inhibitor (TKI) using patient‐derived xenograft cells. The in vitro behavior of 3 patient‐derived xenografts that reflect a clinically relevant range of EGFR variants is characterized: GBM10 (EGFR, wild type), GBM12 (EGFR+), and GBM6 (EGFRvIII). GelMA hydrogels support xenograft culture; cells remain viable, active, respond to matrix‐immobilized HA, and upregulate genes associated with matrix remodeling and tumor growth. Interestingly, matrix‐immobilized HA alters the response of GBM cells to a model tyrosine kinase inhibitor, erlotinib. While constitutively activated EGFRvIII cells are sensitive to TKI in gelatin hydrogels, hyaluronic acid mediated adhesive signaling interacts with EGFRvIII signaling to increase cell metabolic activity, increase soluble hyaluronic acid synthesis, and modify response to erlotinib exposure.

Abstract Introduction Glioblastoma (GBM) is the most common and lethal form of brain cancer. Poor survival is linked to diffuse infiltration throughout the brain, suggesting the need to examine the effect of the tissue environment at the margins of the GBM tumor on processes of GBM invasion. Further, the presence of GBM cancer stem cells (GSCs) in primary tumor specimens is believe to play a significant role in GBM spreading, recurrence, and therapeutic resistance. While biophysical properties of the tumor microenvironment, such as spatial gradients in matrix stiffness and hyaluronic acid (HA) content as well as regional hypoxia, may affect GBM invasive phenotypes, limited tools are available to rigorously examine the relationship between tissue microenvironment and tumor growth, invasion, and therapeutic outcomes. This work focuses on the integration of HA-functionalized gelatin hydrogels with 3D cell invasion assays to investigate the impact of tissue microenvironment on GBM invasion and GSC population. Methods Hydrogels are generated from methacrylamide functionalized gelatin (GelMA) and methacrylated HA. Prepolymer mixture was crosslinked under UV light in the presence of a photoinitiator. GBM cells, either U87 cell lines or patient-derived xenografts (PDX), were mixed with prepolymer solution prior to polymerization and collected for up to 7 days in culture. To assess invasion, cells were seeded onto dextran beads overnight, with populational invasion metrics calculated after 3 and 7 days, while live cell tracing via confocal microscopy was employed for single cell invasion metrics. Samples were cultured with 5% CO2 and ~20% O2 (normoxia) or 1% O2(hypoxia). Results The invasion of U87 and U87viii (constitutively active EGFR) cells increased significantly in GelMA hydrogels lacking HA, consistent with our previous findings that compensatory secretion of soluble HA in the absence of matrix-bound HA was associated with upregulated invasion. Heightened U87 invasion was observed in the presence of hypoxia for all hydrogel compositions, and was associated with upregualted markers of invasion (gene expression: MMP-2, VEGF; protein expression: ERK1/2) while PI3K was downregulated, suggesting cells respond to hypoxia by alternating invasion and proliferation behaviors. We subsequently examined the effect of matrix biophysical properties and hypoxia on the expansion of GSC populations within PDX cells (a gift of Jann Sarkaria, Mayo Clinic). Here, the expansion of the GSC population in EGFRviii mutant GBM6 was a function of hypoxia as well as matrix-bound HA. Conclusions This work demonstrates that GBM cells respond dynamically to their matrix biophysical (Stiffness, HA content, hypoxia) properties. Using PDX cells, we provide evidence that the expansion of GSC is related with both metabolic environment (O2%) and matrix composition (+/- HA). Ongoing work seeks to understand how GSCs contribute to the invasive phenotype of GBM. Citation Format: Jee-wei E. Chen, Jan C. Lumibao, H Rex Gaskins, Jann N. Sarkaria, Brendan A. Harley. Extracellular matrix stimuli regulate cancer stem cell population and migratory potential in glioblastoma [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 931. doi:10.1158/1538-7445.AM2017-931

Abstract The extracellular matrix (ECM) is increasingly recognized as having a key role in cancer development. We have developed a biomaterial tumor mimic that recapitulates systematically the main characteristics and heterogeneities of glioblastoma ECM. With the use of these 3D in vitro platforms we seek to understand the key features that make this cancer so challenging to treat. We are using this device in three different ways: (1) to further understand how particularities of brain tumor ECM affect cancer cells and assist in the diagnostic of different glioblastoma (GBM) tumor types, (2) provide new models for drug screening to afford more personalized therapies and (3) study the mechanisms of resistance to current therapies in order to identify more effective combinatorial treatments and new therapeutic targets. Hyaluronic acid (HA), is the main component of the brain ECM and GBM is associated with aberrant HA secretion and overexpression of receptors associated with HA, such as CD44 and EGFR. We cultured GBM patient-derived xenograft (PDX) cells within gelatin based hydrogels that contained variable concentrations of HA. Cell proliferation and gene expression analyses demonstrate that biomimetic hydrogels support xenograft culture and cells upregulate matrix remodeling genes and others related to tumor growth in response to matrix biophysical properties. We also used these platforms to evaluate the mechanisms of cell resistance to tyrosine kinase inhibitors. For this purpose, we first focused on the epidermal growth factor receptor (EGFR), which has been identified as a molecular target and associated with worse clinical outcomes. We characterized the 3D in vitro behavior of 3 PDX that represent these EGFR variants: GBM10 (EGFR, wild type), GBM12 (EGFR+) and GBM6 (EGFRvIII). We studied the relationship between the HA contained in the surrounding matrix and response of GBM cells to a tyrosine kinase inhibitor (TKI), erlotinib. Results indicate that while EGFR+ cells are sensitive to TKI in HA hydrogels, HA seems to collaborate with EGFRvIII signaling to stir cell activity. Immunoblots demonstrate that this enhanced cell activity is related to a significant increase in PDGFR concentration. Blockade of the CD44 receptor, in combination with erlotinib treatment, does not affect phosphorylation rates of PI3K or ERK, however, it leads to a significant decrease of STAT3 phosphorylation. This is translated into a lower tumor cell metabolic activity in HA matrices. Finally, we show that, patient-derived xenograft cells resistant to erlotinib in vivo (GBM10), become sensitive when CD44 is blocked in HA-containing matrices, as demonstrated by a decrease of phosphorylated PI3K. In summary, we highlight the importance of extracellular HA in EGFR inhibition efficiency. We demonstrate that this biomaterial tumor model can be used as a valuable tool in the mechanistic studies of tumor development and prediction of tyrosine kinase inhibitors efficacy. Citation Format: Sara Pedron, Gabrielle L. Wolter, Emily Chen, Jann N. Sarkaria, Brendan A. Harley. Glioblastoma tumor model to analyze the mechanisms of resistance to tyrosine kinase inhibitors [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 5776. doi:10.1158/1538-7445.AM2017-5776

Abstract Glioblastoma (GBM) is the most common and most lethal primary malignant brain tumor. Two common alterations in GBM are loss of the tumor suppressor PTEN and upregulation of the oncogenic receptor tyrosine kinase MET. We uncovered a new connection between PTEN and MET expressions and functions in GBM. We found that PTEN expression in GBM cell lines (U87, U373, A172, LNZ-308) and primary cells (GBM8) inhibits MET expression. We uncovered the molecular basis and functional significance of this connection. Based on our previous work and published literature, PTEN stabilizes p53, p53 induces miR-34a expression, and miR-34a inhibits MET expression. We therefore hypothesized that PTEN inhibits MET by regulating p53 and miR-34a and that the PTEN tumor suppressive effects are partly mediated by MET downregulation. To verify the hypothesis, we first showed that PTEN restoration leads to miR-34a upregulation in GBM cells. We then showed that this latter effect is p53-dependent as p53 knockdown with a specific shRNA partly reversed PTEN induction of miR-34a. We also showed that PTEN-induced downregulation of MET expression can be rescued by inhibition of miR-34a with an anti-miR3a. We therefore established a new molecular cascade that links PTEN, p53, miR-34a and MET in GBM. To determine the functional relevance of this cascade, we assessed the effects of PTEN on GBM cell death and invasion also in the settings of shRNA-mediated knockdown of p53 and anti-miR-mediated inhibition of miR-34a. PTEN strongly induced GBM cell death (measured by Annexin V/7AAD flow cytometry) and inhibited transwell invasion though a collagen IV matrix. The PTEN effects on cell death and invasion were partly reduced by p53 knock-down and miR-34a inhibition. This demonstrates that the tumor suppressive effects of PTEN in GBM are partly mediated by the PTEN/p53/miR-34a/MET cascade. In ongoing experiments, we are investigating the expressions of PTEN, MET, p53 and miR-34a in human GBM tissues to determine if there are potential correlations that are consistent with our above findings. Altogether, this study establishes a new molecular and functional link between PTEN and MET that involves p53 and miR-34a in GBM. Citation Format: Fadilla Guessous, Nichola A. Cruickshanks, Ying Zhang, David Schiff, Jann Sarkaria, Roger Abounader. PTEN tumor suppressive effects in glioblastoma are partly mediated by MET downregulation via a p53-miR-34a axis [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 345. doi:10.1158/1538-7445.AM2017-345