Radiation-inducible immunotherapy for cancer: senescent tumor cells as a cancer vaccine.

Radiotherapy offers an effective treatment for advanced cancer but local and distant failures remain a significant challenge. Here, we treated melanoma and pancreatic carcinoma in syngeneic mice with ionizing radiation (IR) combined with the poly(ADP-ribose) polymerase inhibitor (PARPi) veliparib to inhibit DNA repair and promote accelerated senescence. Based on prior work implicating cytotoxic T lymphocytes (CTLs) as key mediators of radiation effects, we discovered that senescent tumor cells induced by radiation and veliparib express immunostimulatory cytokines to activate CTLs that mediate an effective antitumor response. When these senescent tumor cells were injected into tumor-bearing mice, an antitumor CTL response was induced which potentiated the effects of radiation, resulting in elimination of established tumors. Applied to human cancers, radiation-inducible immunotherapy may enhance radiotherapy responses to prevent local recurrence and distant metastasis.

Ad.Egr-TNF and local ionizing radiation suppress metastases by interferon-beta-dependent activation of antigen-specific CD8+ T cells.

Ad.Egr-TNF is a radioinducible adenovector currently in phase 3 trials for inoperable pancreatic cancer. The combination of Ad.Egr-TNF and ionizing radiation (IR) contributes to local tumor control through the production of tumor necrosis factor-alpha (TNFalpha) in the tumor microenvironment. Moreover, clinical and preclinical studies with Ad.Egr-TNF/IR have suggested that this local approach suppresses the growth of distant metastatic disease; however, the mechanisms responsible for this effect remain unclear. These studies have been performed in wild-type (WT) and TNFR1,2(-/-) mice to assess the role of TNFalpha-induced signaling in the suppression of draining lymph node (DLN) metastases. The results demonstrate that production of TNFalpha in the tumor microenvironment induces expression of interferon (IFNbeta). In turn, IFNbeta stimulates the production of chemokines that recruit CD8(+) T cells to the tumor. The results further demonstrate that activation of tumor antigen-specific CD8(+) CTLs contributes to local antitumor activity and suppression of DLN metastases. These findings support a model in which treatment of tumors with Ad.Egr-TNF and IR is mediated by local and distant immune-mediated antitumor effects that suppress the development of metastases.

Characterization of response to radiation mediated gene therapy by means of multimodality imaging.

Imaging techniques are under development to facilitate early analysis of spatial patterns of tumor response to combined radiation and antivascular gene therapy. A genetically modified, replication defective adenoviral vector (Ad.EGR-TNFalpha), injected intratumorally, mediates infected cells to express tumor necrosis factor alpha (TNFalpha), which is increased after exposure to radiation. The goal of this study was to characterize an image based "signature" for response to this combined radiation and gene therapy in mice with human prostate xenografts. This study is part of an imaged guided therapy project where such a signature would be useful in guiding subsequent treatments. Changes in the tumor micro-environment were assessed using MRI registered with electron paramagnetic resonance imaging which provides images of tissue oxygenation. Dynamic contrast-enhanced MRI was used to assess tissue perfusion. When compared with null vector (control) treatment, the ratio of contrast agent (Gd-DTPA-BMA) washout rate to uptake rate was lower (P = 0.001) after treatment, suggesting a more balanced perfusion. Concomitantly, oxygenation significantly increased in the treated animals and decreased or did not change in the control animals (P < 0.025). This is the first report of minimally invasive, quantitative, absolute oxygen measurements correlated with tissue perfusion in vivo.

Signal transducer and activator of transcription 1 regulates both cytotoxic and prosurvival functions in tumor cells.

Elsewhere, we reported that multiple serial in vivo passage of a squamous cell carcinoma cells (SCC61) concurrent with ionizing radiation (IR) treatment resulted in the selection of radioresistant tumor (nu61) that overexpresses the signal transducer and activator of transcription 1 (Stat1)/IFN-dependent pathway. Here, we report that (a) the Stat1 pathway is induced by IR, (b) constitutive overexpression of Stat1 is linked with failure to transmit a cytotoxic signal by radiation or IFNs, (c) selection of parental cell line SCC61 against IFN-alpha and IFN-gamma leads to the same IR- and IFN-resistant phenotype as was obtained by IR selection, and (d) suppression of Stat1 by short hairpin RNA renders the IR-resistant nu61 cells radiosensitive to IR. We propose a model that transient induction of Stat1 by IFN, IR, or other stress signals activates cytotoxic genes and cytotoxic response. Constitutive overexpression of Stat1 on the other hand leads to the suppression of the cytotoxic response and induces prosurvival genes that, at high levels of Stat1, render the cells resistant to IR or other inducers of cell death.

HMG-CoA Reductase Inhibition Delays DNA Repair and Promotes Senescence After Tumor Irradiation.

Despite significant advances in combinations of radiotherapy and chemotherapy, altered fractionation schedules and image-guided radiotherapy, many cancer patients fail to benefit from radiation. A prevailing hypothesis is that targeting repair of DNA double strand breaks (DSB) can enhance radiation effects in the tumor and overcome therapeutic resistance without incurring off-target toxicities. Unrepaired DSBs can block cancer cell proliferation, promote cancer cell death, and induce cellular senescence. Given the slow progress to date translating novel DSB repair inhibitors as radiosensitizers, we have explored drug repurposing, a proven route to improving speed, costs, and success rates of drug development. In a prior screen where we tracked resolution of ionizing radiation-induced foci (IRIF) as a proxy for DSB repair, we had identified pitavastatin (Livalo), an HMG-CoA reductase inhibitor commonly used for lipid lowering, as a candidate radiosensitizer. Here, we report that pitavastatin and other lipophilic statins are potent inhibitors of DSB repair in breast and melanoma models both in vitro and in vivo When combined with ionizing radiation, pitavastatin increased persistent DSBs, induced senescence, and enhanced acute effects of radiation on radioresistant melanoma tumors. shRNA knockdown implicated HMG-CoA reductase, farnesyl diphosphate synthase, and protein farnesyl transferase in IRIF resolution, DSB repair, and senescence. These data confirm on-target activity of statins, although via inhibition of protein prenylation rather than cholesterol biosynthesis. In light of prior studies demonstrating enhanced efficacy of radiotherapy in patients taking statins, this work argues for clinical evaluation of lipophilic statins as nontoxic radiosensitizers to enhance the benefits of image-guided radiotherapy. Mol Cancer Ther; 17(2); 407-18. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

Transcriptional control of viral gene therapy by cisplatin.

Ionizing radiation (IR) and radical oxygen intermediates (ROIs) activate the early growth response-1 (Egr1) promoter through specific cis-acting sequences termed CArG elements. Ad.Egr.TNF.11D, a replication-deficient adenoviral vector containing CArG elements cloned upstream of the cDNA for human recombinant TNF-alpha was used to treat human esophageal adenocarcinoma and rat colon adenocarcinoma cells in culture and as xenografts in athymic nude mice. Cisplatin, a commonly used chemotherapeutic agent, causes tumor cell death by producing DNA damage and generating ROIs. The present studies demonstrate induction of TNF-alpha production in tumor cells and xenografts treated with the combination of Ad.Egr.TNF.11D and cisplatin. The results show that the Egr1 promoter is induced by cisplatin and that this induction is mediated in part through the CArG elements. These studies also demonstrate an enhanced antitumor response without an increase in toxicity following treatment with Ad.Egr.TNF.11D and cisplatin, compared with either agent alone. Chemo-inducible cancer gene therapy thus provides a means to control transgene expression while enhancing the effectiveness of commonly used chemotherapeutic agents.

Local gene delivery of tumor necrosis factor alpha can impact primary tumor growth and metastases through a host-mediated response.

TNFerade is a replication incompetent adenovector designed to express human TNFalpha under control of the Egr-1 radiation and chemotherapy enhanced promoter, and is currently in Phase II/III clinical testing. Data from Phase I clinical testing of TNFerade in a limited set of melanoma patients suggested the potential to impact distal metastases following intratumoral injections of TNFerade. These clinical observations and the multiple potential mechanisms of TNFerade led us to hypothesize local treatment with TNFerade + radiation may impact metastatic disease. We explored this hypothesis in preclinical models using the spontaneously metastatic, syngeneic B16F10 murine melanoma model. Established subcutaneous B16F10 tumors were treated with intratumoral injections of TNFerade and localized 2 Gy fractionated radiation therapy, modeling the clinical treatment regimen. Following 10-14 days of treatment, mice were evaluated for metastases development in the iliac and axillary lymph nodes. Comparisons of metastatic burden to control groups indicated TNFerade +/- radiation suppressed the formation of metastases in the lymph nodes. Additional experiments in TNF receptor knockout mice, where the only possible effects are on tumor cells containing the TNFalpha receptor, indicate TNFerade's local and distal activities are critically dependent on a host-mediated response. These data provide direct preclinical evidence local therapy of a solid tumor with TNFerade can also reduce metastatic disease, in addition to effects on the treated lesion. Furthermore, our finding of a host dependant response(s) for TNFerade at both the treated tumor and on lymph node metastases suggest the potential for broad activity independent of tumor histology.

Prolonged treatment with angiostatin reduces metastatic burden during radiation therapy.

Ionizing radiation (IR) and concomitant angiostatin (AS) produce greater than additive local antitumor effects. We examined whether prolonged AS treatment added to IR reduces proliferation of lung metastases from LLC primary tumors. Flank tumors were treated with 40 Gy with or without AS (25 mg/kg/day). IR plus a 14-day course of AS improved local tumor control and blocked the increase in lung weights observed in the group receiving IR plus a 2-day course of AS group. Animals treated with prolonged AS exhibited no increase in lung weight and no macrometastases. These findings suggest that long-term treatment with antiangiogenic compounds may be effective in preventing metastases from IR-treated tumors as well as increasing the local antitumor effects of radiotherapy.

Repurposing cephalosporin antibiotics as pro-senescent radiosensitizers.

Radiation therapy remains a significant therapeutic modality in the treatment of cancer. An attractive strategy would be to enhance the benefits of ionizing radiation (IR)with radiosensitizers. A high-content drug repurposing screen of approved and investigational agents, natural products and other small molecules has identified multiple candidates that blocked repair of IR damage in vitro. Here, we validated a subset of these hits in vitro and then examined effects on tumor growth after IR in a murine tumor model. Based on robust radiosensitization in vivo and other favorable properties of cephalexin, we conducted additional studies with other beta-lactam antibiotics. When combined with IR, each cephalosporin tested increased DNA damage and slowed tumor growth without affecting normal tissue toxicity. Our data implicate reactive oxygen species in the mechanism by which cephalosporins augment the effects of IR. This work provides a rationale for using commonly prescribed beta-lactam antibiotics as non-toxic radiosensitizers to enhance the therapeutic ratio of radiotherapy.

Forphenicinol enhances the antitumor effects of cyclophosphamide in a model of squamous cell carcinoma.

We examined the interaction between forphenicinol (FPL) and cyclophosphamide (CPA) or ionizing radiation (IR) on the growth of murine squamous cell carcinoma tumors SCCVII. Primary tumors were established in C3H mice by injecting SCCVII tumor cells subcutaneously into the right hind limb. FPL (100 mg/kg for 8 days) and/or CPA (25 mg/kg twice) were administered by intraperitoneal injection. Tumors were irradiated to a total dose of 40 Gy (eight 5-Gy fractions). SCCVII tumor growth was inhibited by FPL (P=0.054), IR (P=0.003) and CPA (P<0.001) compared with control. The combination of FPL and CPA inhibited tumor growth additively compared with either treatment alone in both small- and large-volume tumors. FPL did not significantly enhance the antitumor effects of IR, however, when CPA+FPL were combined with IR, significant tumor growth inhibition was observed compared with FPL alone (P<0.001), CPA alone (P=0.002) and IR alone (P=0.002). Due to its low toxicity profile, FPL may be combined with CPA, IR and other cytotoxic therapies to potentially enhance the therapeutic ratio.

Chemoinducible gene therapy: a strategy to enhance doxorubicin antitumor activity.

A replication-defective adenoviral vector, Ad.Egr-TNF.11D, was engineered by ligating the CArG (CC(A/T)6GG) elements of the Egr-1 gene promoter upstream to a cDNA encoding human tumor necrosis factor-alpha. We report here that Ad.Egr-TNF.11D is activated by the clinically important anticancer agents cisplatin, cyclophosphamide, doxorubicin, 5-fluorouracil, gemcitabine, and paclitaxel. N-acetylcysteine, a free radical scavenger, blocked induction of tumor necrosis factor-alpha by anticancer agents, supporting a role for reactive oxygen intermediates in activation of the CArG sequences. Importantly, resistance of PC-3 human prostate carcinoma and PROb rat colon carcinoma tumors to doxorubicin in vivo was reversed by combining doxorubicin with Ad.Egr-TNF and resulted in significant antitumor effects. Treatment with Ad.Egr-TNF.11D has been associated with inhibition of tumor angiogenesis. In this context, a significant decrease in tumor microvessel density was observed following combined treatment with doxorubicin and Ad.Egr-TNF.11D as compared with either agent alone. These data show that Ad.Egr-TNF.11D is activated by diverse anticancer drugs.

Glioblastoma cells block radiation-induced programmed cell death of endothelial cells.

We demonstrate that human umbilical vein endothelial cells (HUVEC) grown in co-culture (CC) with U87 glioblastoma cells transfected with green fluorescent protein (GFP-U87) exhibit resistance to radiation-mediated apoptosis. cDNA macroarray analysis reveals increases in the accumulation of RNAs for HUVEC genes encoding cell adhesion molecules, growth factor-related proteins, and cell cycle regulatory/DNA repair proteins. An increase in protein expression of integrin alphav, integrin beta1, MAPK(p42), Rad51, DNA-PK(CS), and ataxia telangiectasia gene (ATM) was detected in HUVEC grown in CC with GFP-U87 cells compared with HUVEC grown in mono-culture. Treatment with anti-VEGF antibody decreases the expression of integrin alphav, integrin beta1, DNA-PK(CS) and ATM with a corresponding increase in ionizing radiation (IR)-induced apoptosis. These data support the concept that endothelial cells growing in the tumor microenvironment may develop resistance to cytotoxic therapies due to the up-regulation by tumor cells of endothelial cells genes associated with survival.

Control of gene therapy by MDR1 and EGR1 promoter sequences in transcriptional targeting by chemotherapy (Review).

The promising benefits of cancer gene therapy have been limited by the inability to deliver therapeutic genes homogeneously throughout the tumor mass and to control gene expression within the tumor cells. Transcriptional targeting, the use of DNA regulatory promoter sequences to localize transgene expression, has been employed as a solution to circumvent these limitations. TNF-alpha is a cytokine that exhibits potent anticancer properties, but its utility following systemic administration is limited by toxicity. We review a strategy whereby ligating TNF-alpha to segments of the chemo-inducible EGR1 or MDR1 promoters activates expression of TNF-alpha cDNA and enhances effectiveness of gene therapy and chemotherapy.

Angiostatin potentiates cyclophosphamide treatment of metastatic disease.

PURPOSE: We examined the interaction between cyclophosphamide (CPA) and angiostatin (AS) on the growth of primary Lewis lung carcinoma (LLC) tumors and on the development of LLC pulmonary metastases. We studied the effects of AS and CPA on the stages of angiogenesis employing in vitro assays. METHODS: Primary tumor growth and pulmonary metastases were measured to evaluate the effects of treatment with AS alone, CPA alone or the combination of CPA and AS. We examined the effects of CPA plus AS on endothelial cell (HUVEC) survival, migration and tube formation. RESULTS: Combined treatment with CPA and AS did not significantly affect primary tumor growth when compared with CPA treatment alone. However, a significant decrease in the number of pulmonary metastases was observed following CPA plus AS treatment when compared with CPA treatment alone ( P<0.001). AS did not enhance CPA-mediated HUVEC cytotoxicity, and CPA failed to enhance AS-mediated inhibition of migration. However, tube formation was inhibited following combined treatment with CPA and AS when compared with either treatment alone. CONCLUSIONS: AS enhanced the antimetastatic effects of CPA without significantly influencing the effects of CPA on primary tumor growth. CPA plus AS inhibited tube formation, suggesting that interrupting specific steps in the angiogenesis process might be an effective approach to the treatment of subclinical distant metastases.

Dual induction of the Epo-Egr-TNF-alpha- plasmid in hypoxic human colon adenocarcinoma produces tumor growth delay.

Gene therapy is a modality for the treatment of solid tumors that involves the introduction of a suicide gene into the tumor cells. Genetic radiotherapy involves the placement of a radiation-sensitive promoter upstream from a suicide gene. Because of their irregular vasculature some solid tumors are chronically hypoxic and hence are resistant to conventional treatment with chemotherapy and ionizing radiation (IR). The purpose of this study was to demonstrate that regional tumor hypoxia could be exploited to improve local tumor control. The cDNA coding the erythropoietin hypoxia-responsive element (EPO) was placed upstream from the Egr-TNF-alpha construct. WIDR human colon adenocarcinoma cells were injected into the right hind limb of nude mice and treated with Epo-Egr-TNF-alpha plasmid with or without IR. Tumor volumes were measured by calipers and tumor necrosis factor (TNF)-alpha content of the tumor was determined by enzyme-linked immunosorbent assay. Treatment with the combined regimen of Epo-Egr-TNF-alpha plasmid + IR resulted in significant tumor growth delay. Tumor TNF-alpha content was increased by 30 per cent in the combined treatment group compared with each treatment alone. Regional tumor hypoxia can be exploited successfully to induce tumor growth delay, enhance local control, and enhance the therapeutic ratio.

A manganese superoxide dismutase (SOD2)-mediated adaptive response.

Very low doses of ionizing radiation, 5 to 100 mGy, can induce adaptive responses characterized by elevation in cell survival and reduction in micronuclei formation. Utilizing these end points, RKO human colon carcinoma and transformed mouse embryo fibroblasts (MEF), wild-type or knockout cells missing TNF receptors 1 and 2 (TNFR1(-)R2(-)), and C57BL/6 and TNFR1(-)R2(-) knockout mice, we demonstrate that intact TNF signaling is required for induction of elevated manganese superoxide dismutase (SOD2) activity (P < 0.001) and the subsequent expression of these SOD2-mediated adaptive responses when cells are challenged at a later time with 2 Gy. In contrast, amifostine's free thiol form WR1065 can directly activate NF-κB giving rise to elevated SOD2 activity 24 h later and induce an adaptive response in both MEF wild-type and TNF signaling defective TNFR1(-)R2(-) cells. Transfection of cells with SOD2 siRNA completely abolishes both the elevation in SOD2 activity and expression of the adaptive responses. These results were confirmed in vivo using a micronucleus assay in splenocytes derived from C57BL/6 and TNFR1(-)R2(-) knockout mice that were exposed to 100 mGy or 400 mg/kg amifostine 24 h prior to exposure to a 2 Gy whole-body dose. A dose of 100 mGy also conferred enhanced protection to C57BL/6 mice exposed 24 h later to 100 mg/kg of N-Ethyl-N-nitrosourea (ENU). While very low radiation doses require an intact TNF signaling process to induce a SOD2-mediated adaptive response, amifostine can induce a similar adaptive response in both TNF receptor competent and knockout cells, respectively.

A survivin-associated adaptive response in radiation therapy.

Adaptive responses can be induced in cells by very low doses of ionizing radiation resulting in an enhanced resistance to much larger exposures. The inhibitor of apoptosis protein, survivin, has been implicated in many adaptive responses to cellular stress. Computerized axial tomography used in image-guided radiotherapy to position and monitor tumor response uses very low radiation doses ranging from 0.5 to 100 mGy. We investigated the ability of these very low radiation doses administered along with two 2 Gy doses separated by 24 hours, a standard conventional radiotherapy dosing schedule, to initiate adaptive responses resulting in the elevation of radiation resistance in exposed cells. Human colon carcinoma (RKO36), mouse sarcoma (SA-NH), along with transformed mouse embryo fibroblasts, wild type or cells lacking functional tumor necrosis factor receptors 1 and 2 were used to assess their relative ability to express an adaptive response when grown either to confluence in vitro or as tumors in the flank of C57BL/6 mice. The survival of each of these cells was elevated from 5% to 20% (P ≤ 0.05) as compared to cells not receiving a 100 mGy or lesser dose. In addition, the cells exposed to 100 mGy exhibited elevations in survivin levels, reductions in apoptosis frequencies, and loss of an adaptive response if transfected with survivin siRNA. This survivin-mediated adaptive response has the potential for affecting outcomes if regularly induced throughout a course of image guided radiation therapy.

Everolimus exhibits efficacy as a radiosensitizer in a model of non-small cell lung cancer.

Signaling pathways that activate mTOR (mammalian target of rapamycin) are altered in many human cancers and these alterations are associated with prognosis and treatment response. mTOR inhibition can restore sensitivity to DNA damaging agents such as cisplatin. The rapamycin derivative everolimus exhibits antitumor activity and is approved for patients with renal cell cancer. Clinically, everolimus has also been evaluated in patients with advanced non-small cell lung cancer (NSCLC) that were refractory to chemotherapy and epidermal growth factor receptor tyrosine kinase inhibitors. We tested the effects of combined treatment with everolimus (RAD001) and fractionated radiation using a xenograft model of human NSCLC (A549 cells). In growth studies, mean tumor volume was reduced in the everolimus plus 30 Gy cohort with significant tumor growth suppression compared to 30 Gy alone (p=0015), or everolimus alone (p<0.001, ANOVA). everolimus (20 nM) significantly reduced protein levels of the mTOR downstream effector p70-S6K compared with radiation and vehicle (p=0.05, ANOVA) and significantly suppressed phospho-p70-S6K levels compared with all other treatments (p<0.001, ANOVA). We also evaluated everolimus and radiation effects on gene expression in A549 cells. Everolimus ± 5 Gy suppressed endothelin 1 and lactate dehydrogenase expression and increased VEGFA, p21, hypoxia-inducible factor-1α and SLC2A1 (facilitated glucose transporter 1). mTOR mRNA levels were unaffected while TNF-α levels were increased with everolimus + 5 Gy compared to either treatment alone. These findings suggest that everolimus increases the antitumor activity of radiation. Clinical trials combining everolimus with fractionated radiation in patients with NSCLC are warranted.

Tumor endothelial inflammation predicts clinical outcome in diverse human cancers.

BACKGROUND: Vascular endothelial cells contribute to the pathogenesis of numerous human diseases by actively regulating the stromal inflammatory response; however, little is known regarding the role of endothelial inflammation in the growth of human tumors and its influence on the prognosis of human cancers. METHODS: Using an experimental model of tumor necrosis factor-alpha (TNF-α)-mediated inflammation, we characterized inflammatory gene expression in immunopurified tumor-associated endothelial cells. These genes formed the basis of a multivariate molecular predictor of overall survival that was trained and validated in four types of human cancer. RESULTS: We report that expression of experimentally derived tumor endothelial genes distinguished pathologic tissue specimens from normal controls in several human diseases associated with chronic inflammation. We trained these genes in human cancer datasets and defined a six-gene inflammatory signature that predicted significantly reduced overall survival in breast cancer, colon cancer, lung cancer, and glioma. This endothelial-derived signature predicted outcome independently of, but cooperatively with, standard clinical and pathological prognostic factors. Consistent with these findings, conditioned culture media from human endothelial cells stimulated by pro-inflammatory cytokines accelerated the growth of human colon and breast tumors in immunodeficient mice as compared with conditioned media from untreated endothelial cells. CONCLUSIONS: This study provides the first prognostic cancer gene signature derived from an experimental model of tumor-associated endothelial inflammation. These findings support the notion that activation of inflammatory pathways in non-malignant tumor-infiltrating endothelial cells contributes to tumor growth and progression in multiple human cancers. Importantly, these results identify endothelial-derived factors that could serve as potential targets for therapy in diverse human cancers.

Response of human prostate cancer cells and tumors to combining PARP inhibition with ionizing radiation.

Radiation therapy remains a promising modality for curative treatment of localized prostate cancer, but dose-limiting toxicities significantly limit its effectiveness. Agents that enhance efficacy at lower radiation doses might have considerable value in increasing tumor control without compromising organ function. Here, we tested the hypothesis that the PARP inhibitor ABT-888 (veliparib) can enhance the response of prostate cancer cells and tumors to ionizing radiation (IR). Following exposure of DU-145 and PC-3 prostate cancer cell lines to the combination of 10 µmol/L ABT-888 and 6 Gy, we observed similar persistence between both cell lines of DNA damage foci and in vitro radiosensitization. We have previously observed that persistent DNA damage foci formed after ABT-888 plus IR efficiently promote accelerated cell senescence, but only PC-3 cells displayed the expected senescent response of G(2)-M arrest, induction of p21 and ß-galactosidase expression, and accumulation as large flat cells. In turn, combining ABT-888 with 6 Gy resulted in delayed tumor regrowth compared with either agent alone only in PC-3 xenograft tumors, whereas DU-145 tumors continued to grow. By 7 days after treatment with ABT-888 plus IR, PC-3 tumors contained abundant senescent cells displaying persistent DNA damage foci, but no evidence of senescence was noted in the DU-145 tumors. That equivalent radiosensitization by ABT-888 plus IR in vitro failed to predict comparable results with tumors in vivo suggests that the efficacy of PARP inhibitors may partially depend on a competent senescence response to accumulated DNA damage.