Attenuating hypoxia driven malignant behavior in glioblastoma with a novel hypoxia-inducible factor 2 alpha inhibitor

Abstract

Hypoxia inducible factor (HIFs) signaling contributes to malignant cell behavior in glioblastoma (GBM). We investigated a novel HIF2α inhibitor, PT2385, both in vitro, with low-passage patient-derived cell lines, and in vivo, using orthotopic models of glioblastoma. We focused on analysis of HIF2α expression in situ, cell survival/proliferation, and survival in brain tumor-bearing mice treated with PT2385 alone and in combination with standard of care chemoradiotherapy. HIF2α expression increased with glioma grade, with over half of GBM specimens HIF2α positive. Staining clustered in perivascular and perinecrotic tumor regions. Cellular phenotype including proliferation, viability, migration/invasion, and also gene expression were not altered after PT2385 treatment. In the animal model, PT2385 single-agent treatment did improve median overall survival compared to placebo (p = 0.04, n = 21) without a bioluminescence correlate (t = 0.67, p = 0.52). No difference in animal survival was seen in combination treatment with radiation (RT)/temozolomide (TMZ)/PT2385 (p = 0.44, n = 10) or mean tumor bioluminescence (t 1.13, p = 0.32). We conclude that HIF2α is a reasonable novel therapeutic target as expressed in the majority of glioblastomas in our cohort. PT2385 as a single-agent was efficacious in vivo, however, an increase in animal survival was not seen with PT2385 in combination with RT/TMZ. Further study for targeting HIF2α as a therapeutic approach in GBM is warranted.

Figure 1

(A) Immunohistochemistry on a paraffin embedded glioblastoma sample stained for HIF2α expression demonstrating perivascular staining with arrows indicating representative cells expressing HIF2α. (B) Kaplan Meier curve demonstrating higher abundance of HIF2α was associated with an increased hazard for mortality (HR: 2.8, 95% CI 1.00–7.98, log-rank p = 0.04).

Figure 2

Immunohistochemistry on paraffin embedded glioblastoma samples stained for HIF2α expression demonstrating the observation of both nuclear (black arrows) and cytoplasmic (white arrows) staining with the majority of staining observed to be cytoplasmic.

Figure 3

(A) Cell cycle analysis in glioma cells treated with either 10 μM PT2385 or vehicle in normoxic and hypoxic conditions for 72 h demonstrate no measurable difference between treatment groups. (B) Fluorescent microscopy of cell viability testing in renal cell carcinoma and glioma cells treated with either PT2385 or vehicle in hypoxic conditions with viable cells appearing green and dead cells appearing red demonstrate no visual difference between treatment groups, (C) Real-time PCR gene expression fold changes for hypoxia pathway HIF2α downstream genes were decreased after PT2385 treatment in the BTCOE 4536 cell line and unchanged in the BTCOE 4795 cell line.

Figure 4

Original H&E histology of a glioblastoma sample stained for HIF2α using immunohistochemistry along with GFAP and Ki-67 (upper panels). Once this tumor was implanted into mice formation of tumors were verified with bioluminescence and grossly with corresponding histology using H&E, which demonstrated the model retained necrotic features and glioma markers including HIF2α, GFAP, and Ki-67 (lower panels).

Figure 5

(A) Experimental design for single agent (10 mg/kg PO BID PT2385 in 21 days on/7 days off cycles) studies (using model BTCOE 4795). (B) Kaplan Meier survival curve demonstrating animals receiving PT2385 survived longer than animals receiving placebo (p = 0.04). (C) Representative bioluminescent images of a cage receiving placebo treatment and a cage receiving PT2385 treatment after 14 weeks with more animals alive and smaller tumor burdens amongst PT2385 treated animals. (D) Graph of bioluminescent values during 16 weeks of consecutive imaging show similar signal intensities when treatment with vehicle is compared to PT2385 treated animals (t = 0.67, p = 0.52).

Figure 6

(A) Experimental design for chemoradiation (50 mg/kg PO qD TMZ during radiation followed by 5 days on/23 days off cycles) combined with PT2385 (10 mg/kg PO BID PT2385 in 21 days on/7 days off cycles) studies (using model BTCOE 4795). (B) Kaplan Meier survival curve demonstrating no significant difference in survival in animals treated with RT/TMZ/PT2385 versus RT/TMZ/Placebo (p = 0.44). (C) Representative bioluminescent images of a cage receiving placebo treatment and a cage receiving PT2385 treatment after 14 weeks with more animals alive and smaller tumor burdens amongst PT2385 treated animals. (D) Graph of bioluminescent values during 14 weeks of consecutive imaging demonstrate no significant difference between RT/TMZ/Placebo animals compared to RT/TMZ/PT2385 (t 1.13, p = 0.32).