RESUMEN
BACKGROUND/AIMS: After 9/11, multiple government agencies instituted programs aimed at developing medical radiation countermeasures (MRCs) for two syndromes lethal within weeks of a limited nuclear attack; the hematopoietic-acute radiation syndrome (H-ARS) and the higher-dose gastrointestinal-acute radiation syndrome (GI-ARS). While re-purposing drugs that enhance marrow repopulation treats H-ARS, no mitigator protects GI tract. METHODS: We recently reported anti-ceramide 6B5 single-chain variable fragment (scFv) pre-treatment abrogates ongoing small intestinal endothelial apoptosis to rescue Lgr5+ stem cells, preventing GI-ARS lethality in C57B/L6J mice. Here, with US Department of Defense support, we provide evidence that humanized anti-ceramide scFv (CX-01) is a promising prophylactic MRC for first responders, who risk exposure upon entering a radiation-contaminated site. RESULTS: CX-01, when delivered up to 90 min before irradiation, is highly-effective in preventing small intestinal endothelial apoptosis in mice and lethality in both sexes. Unexpectedly, females require an ~2-fold higher CX-01 dose than males for full protection. CX-01 is effective subcutaneously and intramuscularly, a property critical for battlefield use. Increasing the maximally-effective dose 5-fold does not extend duration of bioeffectiveness. CONCLUSION: While CX-01 prevents GI-ARS lethality, structural modification to extend half-life may be necessary to optimize first responder prophylaxis.
Asunto(s)
Apoptosis , Ceramidas , Ratones Endogámicos C57BL , Anticuerpos de Cadena Única , Animales , Anticuerpos de Cadena Única/inmunología , Femenino , Ratones , Masculino , Ceramidas/metabolismo , Apoptosis/efectos de los fármacos , Síndrome de Radiación Aguda/patología , Síndrome de Radiación Aguda/tratamiento farmacológico , Síndrome de Radiación Aguda/prevención & control , Humanos , Armas Nucleares , Protectores contra Radiación/farmacología , Protectores contra Radiación/uso terapéutico , Intestino Delgado/patología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/efectos de la radiaciónRESUMEN
BACKGROUND/AIMS: It is unknown whether cancer stem cells respond differentially to treatment compared with progeny, potentially providing therapeutic vulnerabilities. Our program pioneered use of ultra-high single dose radiotherapy, which cures diverse metastatic diseases at a higher rate (90-95%) than conventional fractionation (~65%). Single dose radiotherapy engages a distinct biology involving microvascular acid sphingomyelinase/ceramide signaling, which, via NADPH oxidase-2-dependent perfusion defects, initiates an adaptive tumor SUMO Stress Response that globally-inactivates homologous recombination repair of double stand breaks, conferring cure. Accumulating data show diverse stem cells display heightened-dependence on homologous recombination repair to repair resolve double stand breaks. METHODS: Here we use colorectal cancer patient-derived xenografts containing logarithmically-increased Lgr5+ stem cells to explore whether optimizing engagement of this acid sphingomyelinase dependent biology enhances stem cell dependent tumor cure. RESULTS: We show radioresistant colorectal cancer patient-derived xenograft CLR27-2 contains radioresistant microvasculature and stem cells, whereas radiosensitive colorectal cancer patient-derived xenograft CLR1-1 contains radiosensitive microvasculature and stem cells. Pharmacologic or gene therapy enhancement of single dose radiotherapy-induced acid sphingomyelinase/ceramide-mediated microvascular dysfunction dramatically sensitizes CLR27-2 homologous recombination repair inactivation, converting Lgr5+ cells from the most resistant to most sensitive patient-derived xenograft population, yielding tumor cure. CONCLUSION: We posit homologous recombination repair represents a vulnerability determining colorectal cancer stem cell fate, approachable therapeutically using single dose radiotherapy.
Asunto(s)
Neoplasias Colorrectales , Lesiones del Sistema Vascular , Animales , Ceramidas , Neoplasias Colorrectales/genética , Modelos Animales de Enfermedad , Humanos , Células Madre Neoplásicas , Esfingomielina Fosfodiesterasa/genéticaRESUMEN
Fanconi anemia is an inherited genome instability syndrome characterized by interstrand cross-link hypersensitivity, congenital defects, bone marrow failure, and cancer predisposition. Although DNA repair mediated by Fanconi anemia genes has been extensively studied, how inactivation of these genes leads to specific cellular phenotypic consequences associated with Fanconi anemia is not well understood. Here we report that Fanconi anemia stem cells in the C. elegans germline and in murine embryos display marked nonhomologous end joining (NHEJ)-dependent radiation resistance, leading to survival of progeny cells carrying genetic lesions. In contrast, DNA cross-linking does not induce generational genomic instability in Fanconi anemia stem cells, as widely accepted, but rather drives NHEJ-dependent apoptosis in both species. These findings suggest that Fanconi anemia is a stem cell disease reflecting inappropriate NHEJ, which is mutagenic and carcinogenic as a result of DNA misrepair, while marrow failure represents hematopoietic stem cell apoptosis. SIGNIFICANCE: This study finds that Fanconi anemia stem cells preferentially activate error-prone NHEJ-dependent DNA repair to survive irradiation, thereby conferring generational genomic instability that is instrumental in carcinogenesis.
Asunto(s)
Radioisótopos de Cesio/efectos adversos , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Células Madre Embrionarias/patología , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Anemia de Fanconi/patología , Inestabilidad Genómica , Animales , Apoptosis , Caenorhabditis elegans , Reparación del ADN , Células Madre Embrionarias/efectos de la radiación , Anemia de Fanconi/genética , Anemia de Fanconi/radioterapia , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , RatonesRESUMEN
BACKGROUND/AIMS: Despite enormous effort, anti-angiogenic drugs have not lived up to the promise of globally-enhancing anti-cancer therapies. Clinically, anti-angiogenic drugs have been used to persistently suppress vascular endothelial growth factor (VEGF) in order to "normalize" dysfunctional neo-angiogenic microvasculature and prevent recruitment of endothelial progenitors. Recently, we showed that a 1h pre-treatment with anti-angiogenic drugs prior to ultra-high single dose radiotherapy and specific chemotherapies transiently de-represses acid sphingomyelinase (ASMase), leading to enhanced cancer therapy-induced, ceramide-mediated vascular injury and tumor response. Here we formally decipher parameters of chemotherapy induction of endothelial sphingolipid signaling events and define principles for optimizing anti-angiogenic chemosensitization. METHODS: These studies examine the antimetabolite chemotherapeutic gemcitabine in soft tissue sarcoma (STS), a clinically-relevant combination. RESULTS: Initial studies address the theoretic problem that anti-angiogenic drugs such as bevacizumab, an IgG with a 3-week half-life, have the potential for accumulating during the 3-week chemotherapeutic cycles currently standard-of-care for STS treatment. We show that anti-angiogenic ASMase-dependent enhancement of the response of MCA/129 fibrosarcomas in sv129/BL6 mice to gemcitabine progressively diminishes as the level of the VEGFR2 inhibitor DC101, an IgG, accumulates, suggesting a short-acting anti-angiogenic drug might be preferable in multi-cycle chemotherapeutic regimens. Further, we show lenvatinib, a VEGFR2 tyrosine kinase inhibitor with a short half-life, to be superior to DC101, enhancing gemcitabine-induced endothelial cell apoptosis and tumor response in a multi-cycle treatment schedule. CONCLUSION: We posit that a single delivery of a short-acting anti-angiogenic agent at 1h preceding each dose of gemcitabine and other chemotherapies may be more efficacious for repeated sensitization of the ASMase pathway in multi-cycle chemotherapy regimens than current treatment strategies.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/administración & dosificación , Desoxicitidina/análogos & derivados , Compuestos de Fenilurea/administración & dosificación , Quinolinas/administración & dosificación , Sarcoma/tratamiento farmacológico , Neoplasias de los Tejidos Blandos/tratamiento farmacológico , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Desoxicitidina/administración & dosificación , Células Endoteliales/efectos de los fármacos , Masculino , Ratones , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Sarcoma/metabolismo , Neoplasias de los Tejidos Blandos/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , GemcitabinaRESUMEN
Despite great promise, combining anti-angiogenic and conventional anti-cancer drugs has produced limited therapeutic benefit in clinical trials, presumably because mechanisms of anti-angiogenic tissue response remain only partially understood. Here we define a new paradigm, in which anti-angiogenic drugs can be used to chemosensitize tumors by targeting the endothelial acid sphingomyelinase (ASMase) signal transduction pathway. We demonstrate that paclitaxel and etoposide, but not cisplatin, confer ASMase-mediated endothelial injury within minutes. This rapid reaction is required for human HCT-116 colon cancer xenograft complete response and growth delay. Whereas VEGF inhibits ASMase, anti-VEGFR2 antibodies de-repress ASMase, enhancing endothelial apoptosis and drug-induced tumor response in asmase+/+, but not in asmase-/-, hosts. Such chemosensitization occurs only if the anti-angiogenic drug is delivered 1-2h before chemotherapy, but at no other time prior to or post chemotherapy. Our studies suggest that precisely-timed administration of anti-angiogenic drugs in combination with ASMase-targeting anti-cancer drugs is likely to optimize anti-tumor effects of systemic chemotherapy. This strategy warrants evaluation in future clinical trials.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Terapia Molecular Dirigida , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Bovinos , Ceramidas/metabolismo , Sistemas de Liberación de Medicamentos , Endotelio/metabolismo , Activación Enzimática/efectos de los fármacos , Células HCT116 , Humanos , Masculino , Ratones Endogámicos C57BL , Paclitaxel/farmacología , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ glp-1(ar202), a temperature-sensitive gain-of-function C. elegans NOTCH mutant, to delineate NOTCH-driven tumor responses to radiotherapy. At ≤20°C, glp-1(ar202) is wild-type, whereas at 25°C it forms a germline stem cell/progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2/M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Recombinación Homóloga/genética , Neoplasias de Células Germinales y Embrionarias/genética , Receptores Notch/metabolismo , Células Madre/metabolismo , Animales , Apoptosis/efectos de la radiación , Proteínas de Caenorhabditis elegans/genética , Puntos de Control del Ciclo Celular/efectos de la radiación , Reparación del ADN/efectos de la radiación , Femenino , Fase G2/efectos de la radiación , Técnicas de Silenciamiento del Gen , Humanos , Ratones Endogámicos NOD , Ratones SCID , Mutación/genética , Interferencia de ARN/efectos de la radiación , Tolerancia a Radiación/efectos de la radiación , Radiación Ionizante , Receptores Notch/genéticaRESUMEN
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.
Asunto(s)
Adenoviridae/genética , Endotelio Vascular/patología , Fibrosarcoma/prevención & control , Melanoma Experimental/prevención & control , Neovascularización Patológica , Tolerancia a Radiación , Fármacos Sensibilizantes a Radiaciones/farmacología , Esfingomielina Fosfodiesterasa/metabolismo , Animales , Apoptosis , Endotelio Vascular/metabolismo , Endotelio Vascular/efectos de la radiación , Fibrosarcoma/enzimología , Fibrosarcoma/radioterapia , Células Endoteliales de la Vena Umbilical Humana , Humanos , Técnicas para Inmunoenzimas , Masculino , Melanoma Experimental/enzimología , Melanoma Experimental/radioterapia , Ratones , Esfingomielina Fosfodiesterasa/genéticaRESUMEN
BACKGROUND & AIMS: Adult stem cells have been proposed to be quiescent and radiation resistant, repairing DNA double-strand breaks by nonhomologous end joining. However, the population of putative small intestinal stem cells (ISCs) at position +4 from the crypt base contradicts this model, in that they are highly radiosensitive. Cycling crypt base columnar cells (CBCs) at crypt positions +1-3 recently were defined as an alternative population of ISCs. Little is known about the sensitivity of this stem cell population to radiation. METHODS: Radiation-induced lethality of CBCs was quantified kinetically in Lgr5-lacZ transgenic mice. γ-H2AX, BRCA1, RAD51, and DNA-PKcs foci were used as DNA repair surrogates to investigate the inherent ability of CBCs to recognize and repair double-strand breaks. 5-ethynyl-2'-deoxyuridine and 5-bromo-2'-deoxyuridine incorporation assays were used to study patterns of CBC growth arrest and re-initiation of cell cycling. Apoptosis was evaluated by caspase-3 staining. RESULTS: CBCs are relatively radioresistant, repairing DNA by homologous recombination significantly more efficiently than transit amplifying progenitors or villus cells. CBCs undergo apoptosis less than 24 hours after irradiation (32% ± 2% of total lethality) or mitotic death at 24-48 hours. Survival of CBCs at 2 days predicts crypt regeneration at 3.5 days and lethality from gastrointestinal syndrome. Crypt repopulation originates from CBCs that survive irradiation. CONCLUSIONS: Adult ISCs in mice can cycle rapidly yet still be radioresistant. Importantly, homologous recombination can protect adult stem cell populations from genotoxic stress. These findings broaden and refine concepts of the phenotype of adult stem cells.
Asunto(s)
Células Madre Adultas/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN , Yeyuno/efectos de la radiación , Tolerancia a Radiación , Animales , Apoptosis/efectos de la radiación , Médula Ósea/efectos de la radiación , Puntos de Control del Ciclo Celular/efectos de la radiación , Yeyuno/patología , Ratones , Ratones TransgénicosRESUMEN
PURPOSE: To address in vivo the issue of whether Bax and Bak are functionally redundant in signaling apoptosis, capable of substituting for each other. METHODS AND MATERIALS: Mice were exposed to whole-body radiation, and endothelial cell apoptosis was quantified using double immunostaining with TUNEL and anti-CD31 antibody. Crypt survival was determined at 3.5 days after whole-body radiation by the microcolony survival assay. Actuarial animal survival was calculated by the product-limit Kaplan-Meier method, and autopsies were performed to establish cause of death. RESULTS: Radiation exposure of Bax- and Bak-deficient mice, both expressing a wild-type acid sphingomyelinase (ASMase) phenotype, indicated that Bax and Bak are both mandatory, though mutually independent, for the intestinal endothelial apoptotic response. However, neither affected epithelial apoptosis at crypt positions 4-5, indicating specificity toward endothelium. Furthermore, Bax deficiency and Bak deficiency each individually mimicked ASMase deficiency in inhibiting crypt lethality in the microcolony assay and in rescuing mice from the lethal gastrointestinal syndrome. CONCLUSIONS: The data indicate that Bax and Bak have nonredundant functional roles in the apoptotic response of the irradiated intestinal endothelium. The observation that Bax deficiency and Bak deficiency also protect crypts in the microcolony assay provides strong evidence that the microvascular apoptotic component is germane to the mechanism of radiation-induced damage to mouse intestines, regulating reproductive cell death of crypt stem cell clonogens.