Science Spotlight

Anticipation of VEGF-mediated resistance to oncolytic viruses in glioblastoma

From the Holland lab, Human Biology Division

Angiogenesis plays various, sometimes seemingly opposing roles in tumor progression. The neogenesis of blood vessels in tumors improves the delivery of oxygen and nutrients for tumor cell growth and constitutes a potential metastatic route. Vascular Endothelial Growth Factor (VEGF) is one of the main drivers of angiogenesis, and several therapeutic agents blocking its activity have been developed, such as the blocking antibody bevacizumab. Glioblastoma figures among the diseases benefiting from VEGF blockade, mostly because it prevents cerebral edema. Glioblastoma (GM) is one of the most aggressive type of brain tumor.  The GM isocitrate dehydrogenase (IDH) wild-type subgroup belongs to the so-called T-cell deserted tumors, which lack immune infiltrates and for which anti-tumor immunity must be triggered in order for checkpoint inhibitor therapies to be effective.  This was demonstrated by the recent failures of anti-PD-1 and anti-CTLA-4 in IDH wild-type GM clinical trials. Recently, Science Spotlight covered a study by the Holland lab where the team succeeded in triggering abscopal immunity using oncolytic herpes simplex viruses encoding ULBP3 (oHSVULBP3)1, a protein that reeducates the abundant tumor macrophages to stimulate T cell infiltration of the tumor. Not only did this strategy improve survival of an IDH-wild type GM mouse model closely recapitulating the human disease, but the combination with PD-1 blocking antibodies enhanced this anti-tumor effect. As VEGF is also a potent immunosuppressive signal, the Holland lab investigated whether the angiogenic factor could be an additional barrier to o-HSVULBP3-induced anti-tumor immunity.  The results of their investigation were published in a recent issue of the journal Neuro-Oncology2.

Dr. Hans-Georg Wirsching, first author of the study, and his colleagues first assessed the expression of VEGF in tumors treated with PBS or oHSVULBP3 and found that VEGF was overexpressed when oncolytic viruses were injected. As secreted VEGF is trapped in the extracellular matrix and matrix metallo-proteinases (MMP) 3 and 9 are required to release the active form of VEGF, MMP3 and MMP9 expression was similarly assessed. The researchers found that MMP9 was also upregulated at the protein level in the oncolytic virus condition, although the expression level remained very low.

To understand if release of VEGF by MMP9 is a barrier to the therapeutic benefit of oHSVULBP3, a survival study was conducted with glioblastoma-bearing animals injected intratumorally with either PBS, oHSVULBP3, or oHSVULBP3 additionally carrying an MMP9 expression cassette (oHSVULBP3-MMP9). Animals treated with oHSVULBP3-MMP9 lived as long as animals injected with PBS, whereas oHSVULBP3 prolonged survival. To test the hypothesis that the release of VEGF is responsible of the lost survival benefit, the authors treated the animals with either B20, an antibody blocking VEGF, or oHSVULBP3-MMP9 + B20. The latter combination prolonged the survival of animals similarly to oHSVULBP3, confirming that VEGF release by MMP9 ablates the anti-tumor effect of oHSVULBP3.  Gene expression analysis of oHSVULBP3-MMP9 vs oHSVULBP3 treated tumors revealed that Toll-like receptor (TLR) signaling and T cell activation signatures were downregulated in oHSVULBP3-MMP9 tumors, synonym of reduced innate and adaptive immunity respectively.

Survival plots showing that MMP9 expression in oncolytic viruses engineered to already expressing ULBP3 (oHSVULBP3) inhibits the therapeutic benefit of these viruses. However, blocking VEGF with the B20 antibody restores the therapeutic benefit, suggesting that VEGF release by MMP9 is responsible for the therapeutic resistance to oHSVULBP3.
Survival plots showing that MMP9 expression in oncolytic viruses engineered to already expressing ULBP3 (oHSVULBP3) inhibits the therapeutic benefit of these viruses. However, blocking VEGF with the B20 antibody restores the therapeutic benefit, suggesting that VEGF release by MMP9 is responsible for the therapeutic resistance to oHSVULBP3. Illustration from publication.

These results have two main implications for the future design of preclinical and clinical trials involving oncolytic viruses in glioblastoma. First, MMP9 upregulation may be an escape route for tumors to resist oncolytic viral therapies. In these circumstances, layering VEGF blockade strategy on the top of oncolytic virus seems a promising approach. Second, generation of oncolytic viruses producing MMP-9 to increase viral spread is no longer recommended, since the consequential release of VEGF sets up a highly immunosuppressive environment. Rigorous analysis of samples from clinical trials will help resolve whether these findings also apply to the human disease.

This work was supported by the Swiss National Science Foundation, Oncorus and the National Institutes of Health.

Fred Hutch/UW Cancer Consortium members Drs. Houghton and Holland contributed to this research.

1. Wirsching H-G, Zhang H., Szulzewsky F, Arora S, Grandi P, Cimino PJ, Amankulor N, Campbell JS, McFerrin L, Pattwell SS,  Ene C, Hicks A, Ball M, Yan J, Zhang J, Kumasaka D, Pierce RH, Weller M, Finer M, Quéva C, Glorioso JC, Houghton AM, Holland E. Arming oHSV with ULBP3 drives abscopal immunity in lymphocyte-depleted glioblastoma. JCI Insight. 2019;4(13):e128217. https://doi.org/10.1172/jci.insight.128217.

2. Wirsching HG, Arora S, Zhang H, Szulzewsky F, Cimino PJ, Quéva C, Houghton AM, Glorioso JC, Weller M, Holland EC. 2019. Cooperation of oncolytic virotherapy with VEGF-neutralizing antibody treatment in IDH wildtype glioblastoma dependends on MMP9. Neuro Oncol. 2019 Aug 15. pii: noz145. doi: 10.1093/neuonc/noz145.

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