Combinatorial targeting of angiogenesis with inducers of autophagy in the treatment of glioblastoma multiforme

Malignant gliomas represent 80% of tumors developing in the central nervous system, with 50% being glioblastomas (GBM), the most aggressive form of the disease. The benefit from current therapies is very limited, the overall 5-year survival rate of patients with GBM being less than 5%. Thus, there is an urgent need for better therapies. It has been reported that the use of tricyclic antidepressants (TCAs) is linked with reduced incidence of gliomas. In line with these observations, our lab previously demonstrated that combining imipramine (IM), a TCA that activates adenylate cyclase and elevates production of cAMP, with ticlopidine (TIC), an inhibitor of the purinergic receptor P2Y12 that otherwise suppresses cAMP, results in a concerted increase in the levels of intracellular cAMP, which consequently stimulates increased autophagy in glioma cells, leading to autophagy-associated cell death (AACD) in culture and during orthotopic tumor growth. In several mouse models of glioma, the combination improved survival and reduced malignant grade. Seeking to assess the therapeutic translational potential of these repurposed drugs, particularly IM, we performed pre-clinical trials combining IM+/-TIC with anti-VEGF therapy, which is commonly used in patients with GBM; such anti-angiogenic therapy is associated with a transitory improvement in progression-free survival and quality of life, but does not translate into an overall survival benefit. Using genetic mouse models with enabling bioluminescent monitoring of the cancer cells, we treated established GBMs with imipramine, ticlopidine, and B20S, an anti-mouse VEGF monoclonal antibody. We observed transient stabilization of tumor growth, and the overall survival was significantly extended in the regimens combining B20S with IM +/- TIC compared to control and single treatments. Similar to clinical observations in GBM patients treated with anti-angiogenic therapy, mice treated with B20S alone had no survival benefit, implicating a synergetic effect for the combination of anti-VEGF treatment with imipramine and ticlopidine. We found that the combinatorial therapy elicited further increases in autophagy and displayed some indication of blood vessel normalization, concomitant with an elevation of CD8+T lymphocyte infiltration into the orthotopic tumors. Notably, we could partially reverse the beneficial effect of the combinations using a CD8+T cell-depleting antibody, revealing the involvement of the recruited CD8+T cells in the efficacy of the combinatorial treatment, in addition to the cell-intrinsic autophagy-associated death that is demonstrable in culture and in tumors. Together, these results suggest a strategy to improve anti-angiogenic therapy in patients with glioblastoma, by using repurposed FDA-approved drugs that markedly elevate autophagy in concert with anti-VEGF therapy. The recruitment of CD8+ T cells into the treated tumors and their evident contribution to therapeutic efficacy motivates ongoi