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These studies define a new mechanism-based approach to radiosensitize tumor cure by single dose radiotherapy (SDRT). Published evidence indicates that SDRT induces acute microvascular endothelial apoptosis initiated via acid sphingomyelinase (ASMase) translocation to the external plasma membrane. Ensuing microvascular damage regulates radiation lethality of tumor stem cell clonogens to effect tumor cure. Based on this biology, we engineered an ASMase-producing vector consisting of a modified pre-proendothelin-1 promoter, PPE1(3x), and a hypoxia-inducible dual-binding HIF-2α-Ets-1 enhancer element upstream of the asmase gene, inserted into a replication-deficient adenovirus yielding the vector Ad5H2E-PPE1(3x)-ASMase. This vector confers ASMase over-expression in cycling angiogenic endothelium in vitro and within tumors in vivo, with no detectable enhancement in endothelium of normal tissues that exhibit a minute fraction of cycling cells or in non-endothelial tumor or normal tissue cells. Intravenous pretreatment with Ad5H2E-PPE1(3x)-ASMase markedly increases SDRT cure of inherently radiosensitive MCA/129 fibrosarcomas, and converts radiation-incurable B16 melanomas into biopsy-proven tumor cures. In contrast, Ad5H2E-PPE1(3x)-ASMase treatment did not impact radiation damage to small intestinal crypts as non-dividing small intestinal microvessels did not overexpress ASMase and were not radiosensitized. We posit that combination of genetic up-regulation of tumor microvascular ASMase and SDRT provides therapeutic options for currently radiation-incurable human tumors.

While there is significant interest in combining anti-angiogenesis therapy with conventional anti-cancer treatment, clinical trials have as of yet yielded limited therapeutic gain, mainly because mechanisms of anti-angiogenic therapy remain to a large extent unknown. Currently, anti-angiogenic tumor therapy is conceptualized to either “normalize” dysfunctional tumor vasculature, or to prevent recruitment of circulating endothelial precursors into the tumor. An alternative biology, restricted to delivery of anti-angiogenics immediately prior to single dose radiotherapy (radiosurgery), is provided in the present study.Genetic data indicate an acute wave of ceramide-mediated endothelial apoptosis, initiated by acid sphingomyelinase (ASMase), regulates tumor stem cell response to single dose radiotherapy, obligatory for tumor cure. Here we show VEGF prevented radiation-induced ASMase activation in cultured endothelium, occurring within minutes after radiation exposure, consequently repressing apoptosis, an event reversible with exogenous C16-ceramide. Anti-VEGFR2 acts conversely, enhancing ceramide generation and apoptosis. In vivo, MCA/129 fibrosarcoma tumors were implanted in asmase+/+ mice or asmase−/− littermates and irradiated in the presence or absence of anti-VEGFR2 DC101 or anti-VEGF G6-31 antibodies. These anti-angiogenic agents, only if delivered immediately prior to single dose radiotherapy, de-repressed radiation-induced ASMase activation, synergistically increasing the endothelial apoptotic component of tumor response and tumor cure. Anti-angiogenic radiosensitization was abrogated in tumors implanted in asmase−/− mice that provide apoptosis-resistant vasculature, or in wild-type littermates pre-treated with anti-ceramide antibody, indicating that ceramide is necessary for this effect.These studies show that angiogenic factors fail to suppress apoptosis if ceramide remains elevated while anti-angiogenic therapies fail without ceramide elevation, defining a ceramide rheostat that determines outcome of single dose radiotherapy. Understanding the temporal sequencing of anti-angiogenic drugs and radiation enables optimized radiosensitization and design of innovative radiosurgery clinical trials.

While there is significant interest in combining anti-angiogenesis therapy with conventional anti-cancer treatment, clinical trials have as of yet yielded limited therapeutic gain, mainly because mechanisms of anti-angiogenic therapy remain to a large extent unknown. Currently, anti-angiogenic tumor therapy is conceptualized to either "normalize" dysfunctional tumor vasculature, or to prevent recruitment of circulating endothelial precursors into the tumor. An alternative biology, restricted to delivery of anti-angiogenics immediately prior to single dose radiotherapy (radiosurgery), is provided in the present study.Genetic data indicate an acute wave of ceramide-mediated endothelial apoptosis, initiated by acid sphingomyelinase (ASMase), regulates tumor stem cell response to single dose radiotherapy, obligatory for tumor cure. Here we show VEGF prevented radiation-induced ASMase activation in cultured endothelium, occurring within minutes after radiation exposure, consequently repressing apoptosis, an event reversible with exogenous C(16)-ceramide. Anti-VEGFR2 acts conversely, enhancing ceramide generation and apoptosis. In vivo, MCA/129 fibrosarcoma tumors were implanted in asmase(+/+) mice or asmase(-/-) littermates and irradiated in the presence or absence of anti-VEGFR2 DC101 or anti-VEGF G6-31 antibodies. These anti-angiogenic agents, only if delivered immediately prior to single dose radiotherapy, de-repressed radiation-induced ASMase activation, synergistically increasing the endothelial apoptotic component of tumor response and tumor cure. Anti-angiogenic radiosensitization was abrogated in tumors implanted in asmase(-/-) mice that provide apoptosis-resistant vasculature, or in wild-type littermates pre-treated with anti-ceramide antibody, indicating that ceramide is necessary for this effect.These studies show that angiogenic factors fail to suppress apoptosis if ceramide remains elevated while anti-angiogenic therapies fail without ceramide elevation, defining a ceramide rheostat that determines outcome of single dose radiotherapy. Understanding the temporal sequencing of anti-angiogenic drugs and radiation enables optimized radiosensitization and design of innovative radiosurgery clinical trials.