Ruby Kuang

Macrophage migration inhibitory factor downregulation: a novel mechanism of resistance to anti-angiogenic therapy

Anti-angiogenic therapies for cancer such as VEGF neutralizing antibody bevacizumab have limited durability. While mechanisms of resistance remain undefined, it is likely that acquired resistance to anti-angiogenic therapy will involve alterations of the tumor microenvironment. We confirmed increased tumor-associated macrophages in bevacizumab-resistant glioblastoma patient specimens and two novel glioblastoma xenograft models of bevacizumab resistance. Microarray analysis suggested downregulated macrophage migration inhibitory factor (MIF) to be the most pertinent mediator of increased macrophages. Bevacizumab-resistant patient glioblastomas and both novel xenograft models of resistance had less MIF than bevacizumab-naive tumors, and harbored more M2/protumoral macrophages that specifically localized to the tumor edge. Xenografts expressing MIF-shRNA grew more rapidly with greater angiogenesis and had macrophages localizing to the tumor edge which were more prevalent and proliferative, and displayed M2 polarization, whereas bevacizumab-resistant xenografts transduced to upregulate MIF exhibited the opposite changes. Bone marrow-derived macrophage were polarized to an M2 phenotype in the presence of condition-media derived from bevacizumab-resistant xenograft-derived cells, while recombinant MIF drove M1 polarization. Media from macrophages exposed to bevacizumab-resistant tumor cell conditioned media increased glioma cell proliferation compared with media from macrophages exposed to bevacizumab-responsive tumor cell media, suggesting that macrophage polarization in bevacizumab-resistant xenografts is the source of their aggressive biology and results from a secreted factor. Two mechanisms of bevacizumab-induced MIF reduction were identified: (1) bevacizumab bound MIF and blocked MIF-induced M1 polarization of macrophages; and (2) VEGF increased glioma MIF production in a VEGFR2-dependent manner, suggesting that bevacizumab-induced VEGF depletion would downregulate MIF. Site-directed biopsies revealed enriched MIF and VEGF at the enhancing edge in bevacizumab-naive patients. This MIF enrichment was lost in bevacizumab-resistant glioblastomas, driving a tumor edge M1-to-M2 transition. Thus, bevacizumab resistance is driven by reduced MIF at the tumor edge causing proliferative expansion of M2 macrophages, which in turn promotes tumor growth.

GLUT3 upregulation promotes metabolic reprogramming associated with antiangiogenic therapy resistance

Clinical trials revealed limited response duration of glioblastomas to VEGF-neutralizing antibody bevacizumab. Thriving in the devascularized microenvironment occurring after antiangiogenic therapy requires tumor cell adaptation to decreased glucose, with 50% less glucose identified in bevacizumab-treated xenografts. Compared with bevacizumab-responsive xenograft cells, resistant cells exhibited increased glucose uptake, glycolysis, 13C NMR pyruvate to lactate conversion, and survival in low glucose. Glucose transporter 3 (GLUT3) was upregulated in bevacizumab-resistant versus sensitive xenografts and patient specimens in a HIF-1α-dependent manner. Resistant versus sensitive cell mitochondria in oxidative phosphorylation-selective conditions produced less ATP. Despite unchanged mitochondrial numbers, normoxic resistant cells had lower mitochondrial membrane potential than sensitive cells, confirming poorer mitochondrial health, but avoided the mitochondrial dysfunction of hypoxic sensitive cells. Thin-layer chromatography revealed increased triglycerides in bevacizumab-resistant versus sensitive xenografts, a change driven by mitochondrial stress. A glycogen synthase kinase-3β inhibitor suppressing GLUT3 transcription caused greater cell death in bevacizumab-resistant than -responsive cells. Overexpressing GLUT3 in tumor cells recapitulated bevacizumab-resistant cell features: survival and proliferation in low glucose, increased glycolysis, impaired oxidative phosphorylation, and rapid in vivo proliferation only slowed by bevacizumab to that of untreated bevacizumab-responsive tumors. Targeting GLUT3 or the increased glycolysis reliance in resistant tumors could unlock the potential of antiangiogenic treatments.

Improved Survival with Decreased Wait Time to Surgery in Glioblastoma Patients Presenting with Seizure

BACKGROUND: Preoperative seizure is reported to confer favorable prognosis in glioblastoma patients, but studies to date have not investigated how broadly applicable seizure is as a prognostic factor. OBJECTIVE: To investigate if prompter surgical intervention affects the relationship between preoperative seizure and prognosis in glioblastoma patients, focusing on the development of tumor growth and/or additional preoperative symptoms after seizure. METHODS: Retrospective analysis of 443 patients (mean age = 60.2; 60% male) undergoing first glioblastoma resection at our institution (2005‐2011). RESULTS: Preoperative seizure(s) occurred in 28% of patients (n = 124), of which 63 (51%) had only seizure at presentation. Patients experiencing seizure as their only preoperative symptom (“seizure‐only”; n = 45) survived over twice as long as patients who presented with seizure and then later developed additional preoperative symptoms (n = 18; “other symptoms postseizure”; 26.8 vs 10.2 months, P < .001) and patients without preoperative seizure (“no seizure”; 26.8 vs 13.1 months, P < .001). Multivariate stepwise analysis revealed preoperative seizures only (hazard ratio 0.54 [0.37‐0.75]; P < .001) to be independently associated with increased survival. Longer wait time from presentation (ie, diagnostic magnetic resonance imaging) to surgery was a risk factor for developing additional symptoms. Eleven “other symptoms postseizure” patients (69%) vs 6 of the “seizure‐only” patients (15%) had wait times >45 days (P < .001). CONCLUSION: Seizure as the only preoperative symptom independently improved survival, however, when patients developed additional preoperative symptoms, typically due to surgical delay, no prognostic benefit was observed. Prompt diagnosis and neurosurgical intervention is warranted in patients with seizures without other preoperative symptoms to preserve their favorable prognosis.

Abstract 3271: Gene expression changes underlying glioblastoma resistance to anti-angiogenic therapy

Introduction: Despite positive pre-clinical and clinical trials, treatment of glioblastoma with bevacizumab has been limited by acquired resistance and transient response. To study gene expression changes underlying tumor resistance and progression during bevacizumab therapy, we performed microarray gene expression analysis on a novel multigenerational xenograft model of acquired bevacizumab resistance. Methods: Using a two-component normal mixture model, we identified a set of genes exhibiting significant inter-generational variance. Protein-protein interaction scores among these genes were extracted from the String 10 database and used as undirected edge weights in a network representation of the significant gene set. Gene set over-representation (GSO) analysis via ConsensusPathDB of gene clusters identified biologically meaningful clusters and subnetworks mediating distinct functions and molecular pathways. Results: Gene set enrichment analysis revealed significant overexpression across generations of previously identified gene signatures of the mesenchymal subtype, as well as a tumor mesenchymal metabolic signature. Key mesenchymal markers, including putative tumor-stemness marker CD44, NT5E, SNAI2, and ZEB2, were found to be upregulated across generations. These results suggest tumor progression under bevacizumab challenge to be accompanied by a shift in gene expression towards the mesenchymal subtype, a subtype of GBM associated with enhanced invasiveness, resistance, and worse outcome. Our analysis also revealed expression changes in pathways related to angiogenesis. Pro-angiogenic genes FGF2, MMP1, HIF1A, UGCG, LPAR1, and ITGB3 were found to be upregulated. Furthermore, GSO analysis identified angiogenesis as a significantly enriched ontology within the inflammatory response and ECM remodeling subnetworks identified by spectral clustering. Angiogenesis related genes identified via GSO included highly upregulated inflammatory mediators such as COX2, IL6, IL1A, upregulated pro-angiogenic factors TGFA, WNT5A, FGF2, and a downregulated anti-angiogenic gene, SPARC. Conclusions: These results suggest a mesenchymal and pro-angiogenic response to bevacizumab treatment supported by multiple converging pathways involving inflammation, hypoxia, and ECM remodeling. Strategies preventing the evolution of these responses should be developed and tested in the context of our novel xenograft model in order to improve the durability of response to these therapies and allow them to fulfill their therapeutic promise. Citation Format: William C. Chen, Arman Jahangiri, Garima Yagnik, Maxim Sidorov, Jonathan Rick, Ruby Kuang, Michael DeLay, Manish K. Aghi. Gene expression changes underlying glioblastoma resistance to anti-angiogenic therapy. [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 3271.