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Programmed cell death protein 1 (PD-1) immune checkpoint blockade therapy has revolutionized cancer treatment. Although PD-1 blockade is effective in a subset of patients with cancer, many fail to respond because of either primary or acquired resistance. Thus, next-generation strategies are needed to expand the depth and breadth of clinical responses. Toward this end, we designed a human primary T cell phenotypic high-throughput screening strategy to identify small molecules with distinct and complementary mechanisms of action to PD-1 checkpoint blockade. Through these efforts, we selected and optimized a chemical series that showed robust potentiation of T cell activation and combinatorial activity with αPD-1 blockade. Target identification was facilitated by chemical proteomic profiling with a lipid-based photoaffinity probe, which displayed enhanced binding to diacylglycerol kinase α (DGKα) in the presence of the active compound, a phenomenon that correlated with the translocation of DGKα to the plasma membrane. We further found that optimized leads within this chemical series were potent and selective inhibitors of both DGKα and DGKζ, lipid kinases that constitute an intracellular T cell checkpoint that blunts T cell signaling through diacylglycerol metabolism. We show that dual DGKα/ζ inhibition amplified suboptimal T cell receptor signaling mediated by low-affinity antigen presentation and low major histocompatibility complex class I expression on tumor cells, both hallmarks of resistance to PD-1 blockade. In addition, DGKα/ζ inhibitors combined with αPD-1 therapy to elicit robust tumor regression in syngeneic mouse tumor models. Together, these findings support targeting DGKα/ζ as a next-generation T cell immune checkpoint strategy.

Background: Transesophageal echocardiography (TEE) and cardiac computed tomography angiography (CCTA) are currently utilized for left atrial appendage closure (LAAC) planning. During the recent global iodine contrast media shortage in 2022, cardiac magnetic resonance imaging (CMR) was utilized for the first time for LAAC planning. This study sought to assess the utility of CMR versus TEE for LAAC planning., Methods: This single center retrospective study consisted of all patients who underwent preoperative CMR for LAAC with Watchman FLX or Amplatzer Amulet. Key measures were accuracy of LAA thrombus exclusion, ostial diameter, depth, lobe count, morphology, accuracy of predicted device size, and devices deployed per case. Bland-Altman Analysis was used to compare CMR versus TEE measurements of LAA ostial diameter and depth., Results: 25 patients underwent preoperative CMR for LAAC planning. A total of 24 (96%) cases were successfully completed with 1.2 ± 0.5 devices deployed per case. Among the 18 patients who underwent intraoperative TEE, there was no significant difference between CMR versus TEE in LAA thrombus exclusion (CMR 83% vs. TEE 100% cases, p = .229), lobe count (CMR 1.7 ± 0.8 vs. TEE 1.4 ± 0.6, p = .177), morphology (p = .422), and accuracy of predicted device size (CMR 67% vs. TEE 72% cases, p = 1.000). When comparing the difference between CMR and TEE measurements, Bland-Altman analysis demonstrated no significant difference in LAA ostial diameter (CMR-TEE bias 0.7 mm, 95% CI [-1.1, 2.4], p = .420), but LAA depth was significantly larger with CMR versus TEE (CMR-TEE bias 7.4 mm, 95% CI [1.6, 13.2], p = .015)., Conclusions: CMR is a promising alternative for LAAC planning in cases where TEE or CCTA are contraindicated or unavailable., (© 2023 Wiley Periodicals LLC.)

We describe a phenotypic screening and optimization strategy to discover compounds that block intracellular checkpoint signaling in T-cells. We identified dual DGKα and ζ inhibitors notwithstanding the modest similarity between α and ζ relative to other DGK isoforms. Optimized compounds produced cytokine release and T-cell proliferation consistent with DGK inhibition and potentiated an immune response in human and mouse T-cells. Additionally, lead inhibitor BMS-502 demonstrated dose-dependent immune stimulation in the mouse OT-1 model, setting the stage for a drug discovery program., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

Two isoforms of diacylglycerol kinases (DGKs), DGKα and DGKζ, are primarily responsible for terminating DAG-mediated activation of Ras and PKCθ pathways in T cells. A direct comparison of tumor growth between mice lacking each isoform has not been undertaken. We evaluated the growth of three syngeneic tumor cell lines in mice lacking either DGKα or DGKζ in the presence or absence of treatment with anti-PD1 and determined that (i) mice deficient in DGKζ conferred enhanced control of tumor relative to mice deficient in DGKα and (ii) deficiency of DGKζ acted additively with anti-PD1 in tumor control. Consistent with this finding, functional and RNA-sequencing analyses revealed greater changes in stimulated DGKζ-deficient T cells compared with DGKα-deficient T cells, which were enhanced relative to wildtype T cells. DGKζ also imparted greater regulation than DGKα in human T cells. Together, these data support targeting the ζ isoform of DGKs to therapeutically enhance T cell anti-tumor activity., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

C-terminal Src kinase (CSK) functions as a negative regulator of T cell activation through inhibitory phosphorylation of LCK, so inhibitors of CSK are of interest as potential immuno-oncology agents. Screening of an internal kinase inhibitor collection identified pyridazinone lead 1 , and a series of modifications led to optimized compound 13 . Compound 13 showed potent activity in biochemical and cellular assays in vitro and demonstrated the ability to increase T cell proliferation induced by T cell receptor signaling. Compound 13 gave extended exposure in mice upon oral dosing and produced a functional response (decrease in LCK phosphorylation) in mouse spleens at 6 h post dose., Competing Interests: The authors declare the following competing financial interest(s): The authors of this manuscript are employees of Bristol-Myers Squibb., (Copyright © 2019 American Chemical Society.)

Engineered domains of human fibronectin (Adnectins™) were used to generate a bispecific Adnectin targeting epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR), two transmembrane receptors that mediate proliferative and survival cell signaling in cancer. Single-domain Adnectins that specifically bind EGFR or IGF-IR were generated using mRNA display with a library containing as many as 10 ( 13) Adnectin variants. mRNA display was also used to optimize lead Adnectin affinities, resulting in clones that inhibited EGFR phosphorylation at 7 to 38 nM compared to 2.6 μM for the parental clone. Individual, optimized, Adnectins specific for blocking either EGFR or IGF-IR signaling were engineered into a single protein (EI-Tandem Adnectin). The EI-Tandems inhibited phosphorylation of EGFR and IGF-IR, induced receptor degradation, and inhibited down-stream cell signaling and proliferation of human cancer cell lines (A431, H292, BxPC3 and RH41) with IC 50 values ranging from 0.1 to 113 nM. Although Adnectins bound to EGFR at a site distinct from those of anti-EGFR antibodies cetuximab, panitumumab and nimotuzumab, like the antibodies, the anti-EGFR Adnectins blocked the binding of EGF to EGFR. PEGylated EI-Tandem inhibited the growth of both EGFR and IGF-IR driven human tumor xenografts, induced degradation of EGFR, and reduced EGFR phosphorylation in tumors. These results demonstrate efficient engineering of bispecific Adnectins with high potency and desired specificity. The bispecificity may improve biological activity compared to monospecific biologics as tumor growth is driven by multiple growth factors. Our results illustrate a technological advancement for constructing multi-specific biologics in cancer therapy.

The biology of IGF-IR/IR signaling was studied in normal mouse embryonic fibroblasts (MEFs) that were either wild type (wt), heterozygous (het), or null for the IGF-IR. The ability of IGF-I, IGF-II, or insulin to stimulate serum-starved MEFs was characterized by gene expression profiling and biochemical analyses for activation of downstream signals. Each genotypic group of MEFs exhibited distinct patterns of expression both while resting and in response to stimulation. The insulin receptor (IR) pathway in IGF-IR null MEFs was hypersensitive to insulin ligand stimulation resulting in greater AKT phosphorylation than in wt or het MEFs stimulated with the same ligand. Interestingly, the IR pathway hypersensitivity in IGF-IR null MEFs occurred with no observed changes in the levels of IR isoforms A or B. A new small molecule IGF-IR inhibitor (BMS-754807), having equipotent activity against both IGF-IR and IR, proved effective in suppressing both AKT and ERK phosphorylation from both the IGF-IR and IR pathways by all three ligands tested in wt, het, and null MEFs. The use of a dual IGF-IR/IR inhibitor addresses concerns about the use of growth inhibiting therapies directed against the IGF-IR receptor in certain cancers. Lastly, comparison of the antiproliferative effects (IC(50)s) of various compounds in wt vs. null MEFs demonstrates that genetically characterized MEFs provide a simple and inexpensive tool with which to define compounds as having mostly on-target or off-target IGF-IR activities because off-target compounds affect both wt and null MEFs equally.

BMS-754807 is a potent and reversible inhibitor of the insulin-like growth factor 1 receptor/insulin receptor family kinases (Ki, <2 nmol/L). It is currently in phase I development for the treatment of a variety of human cancers. BMS-754807 effectively inhibits the growth of a broad range of human tumor types in vitro, including mesenchymal (Ewing's, rhabdomyosarcoma, neuroblastoma, and liposarcoma), epithelial (breast, lung, pancreatic, colon, gastric), and hematopoietic (multiple myeloma and leukemia) tumor cell lines (IC50, 5-365 nmol/L); the compound caused apoptosis in a human rhabdomyosarcoma cell line, Rh41, as shown by an accumulation of the sub-G1 fraction, as well as by an increase in poly ADP ribose polymerase and Caspase 3 cleavage. BMS-754807 is active in vivo in multiple (epithelial, mesenchymal, and hematopoietic) xenograft tumor models with tumor growth inhibition ranging from 53% to 115% and at a minimum effective dose of as low as 6.25 mg/kg dosed orally daily. Combination studies with BMS-754807 have been done on multiple human tumor cell types and showed in vitro synergies (combination index, <1.0) when combined with cytotoxic, hormonal, and targeted agents. The combination of cetuximab and BMS-754807 in vivo, at multiple dose levels, resulted in improved clinical outcome over single agent treatment. These data show that BMS-754807 is an efficacious, orally active growth factor 1 receptor/insulin receptor family-targeted kinase inhibitor that may act in combination with a wide array of established anticancer agents.

Purpose: Poly(ADP-ribose) polymerase-1 (PARP-1) is the founding member of a family of enzymes that catalyze the addition of ADP-ribose units to proteins that mediate DNA repair pathways. Ionizing radiation induces DNA strand breaks, suggesting that PARP-1 inhibition may sensitize tumor cells to radiation., Experimental Design: We investigated the combination of PARP-1 inhibition with radiation in lung cancer models. ABT-888, a novel potent PARP-1 inhibitor, was used to explore the effects of PARP-1 inhibition on irradiated tumors and tumor vasculature., Results: ABT-888 reduced clonogenic survival in H460 lung cancer cells, and inhibited DNA repair as shown by enhanced expression of DNA strand break marker histone gamma-H2AX. Both apoptosis and autophagy contributed to the mechanism of increased cell death. Additionally, ABT-888 increased tumor growth delay at well-tolerated doses in murine models. For a 5-fold increase in tumor volume, tumor growth delay was 1 day for ABT-888 alone, 7 days for radiation alone, and 13.5 days for combination treatment. Immunohistochemical staining of tumor sections revealed an increase in terminal deoxyribonucleotide transferase-mediated nick-end labeling apoptotic staining, and a decrease in Ki-67 proliferative staining after combination treatment. Matrigel assay showed a decrease in in vitro endothelial tubule formation with ABT-888/radiation combination treatment, and von Willebrand factor staining of tumor sections revealed decreased vessel formation in vivo, suggesting that this strategy may also target tumor angiogenesis., Conclusions: We conclude that PARP-1 inhibition shows promise as an effective means of enhancing tumor sensitivity to radiation, and future clinical studies are needed to determine the potential of ABT-888 as a radiation enhancer.

Purpose: Survivin, a member of the inhibitor of apoptosis gene family, has also been shown to regulate mitosis. It binds Aurora B kinase and the inner centromere protein to form the chromosome passenger complex. Both Aurora B and survivin are overexpressed in many tumors. In this study, we examined whether irradiation affected survivin and Aurora B expression in mesothelioma cells, and how inhibition of these molecules affected radiosensitivity., Methods and Materials: ZM447439 and survivin antisense oligonucleotides were used to inhibit survivin and Aurora B kinase respectively. Western blot was performed to determine the expression of survivin, Aurora B, phosphorylated-histone H3 (Ser 10), and caspase cleavage. Multinucleated cells were counted using flow cytometry, and cell survival after treatment was determined using clonogenic assay., Results: At 3-Gy irradiation an increase was observed in levels of survivin and Aurora B as well as the kinase activity of Aurora B, with an increase in G2/M phase. The radiation-induced upregulation of these molecules was effectively attenuated by antisense oligonucleotides against survivin and a small-molecule inhibitor of Aurora B, ZM447439. Dual inhibition of survivin and Aurora B synergistically radiosensitized mesothelioma cells with a dose enhancement ratio of 2.55. This treatment resulted in increased formation of multinucleated cells after irradiation but did not increase levels of cleaved caspase 3., Conclusion: Inhibition of survivin and Aurora B induces mitotic cell arrest in mesothelioma cells after irradiation. These two proteins may be potential therapeutic targets for the enhancement of radiotherapy in malignant pleural mesothelioma.

The vascular endothelial growth factor receptor (VEGFR) tyrosine kinases are being explored as targets for antiangiogenic cancer therapy. Radiotherapy also inhibits tumor growth and affects vasculature. We investigated the combination of the potent VEGFR tyrosine kinase inhibitor AZD2171 and ionizing radiation in cell culture and mouse models of lung cancer. We show that ionizing radiation induces expression of phosphorylated VEGFR-2 (Flk-1) in endothelial cells and that this phosphorylation is inhibited by AZD2171. Human umbilical vascular endothelial cells become more sensitive to radiation after treatment with AZD2171 as determined by clonogenic assay. Matrigel assay showed a decrease in in vitro endothelial tubule formation with AZD2171/radiation combination treatment. When similar combination was applied to the H460 lung cancer xenograft model in nude mice, loss of radiation-induced phosphorylated Flk-1 was observed in the combination treatment group, which also showed a large decrease in tumor vascular density by staining of the von Willebrand factor. H460 tumor growth delay was enhanced in the combination treatment group compared with the groups treated with AZD2171 or radiation alone. Additionally, after therapy, Ki67 index showed >4-fold reduction of tumor proliferation in the combination therapy group, which also showed increased intratumoral apoptotic index by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. In conclusion, AZD2171 sensitizes lung tumor xenografts to radiation and inhibits angiogenesis both in vitro and in vivo. When used as a radiation enhancer, AZD2171 has the potential to improve tumor growth delay by inhibiting tumor proliferation and promoting apoptosis. Clinical trials are needed to determine the potential of this combination therapy in patients with locally advanced lung cancer.

Autophagy is an alternative cell death pathway that is induced by mammalian target of rapamycin (mTOR) inhibitors and up-regulated when apoptosis is defective. We investigated radiation-induced autophagy in the presence or absence of Bax/Bak with or without an mTOR inhibitor, Rad001. Two isogenic cell lines, wild type (WT) and Bak/Bak(-/-) mouse embryonic fibroblasts and tumor cell lines were used for this study. Irradiated Bak/Bak(-/-) cells had a decrease of Akt/mTOR signaling and a significant increase of pro-autophagic proteins ATG5-ATG12 COMPLEX and Beclin-1. These molecular events resulted in an up-regulation of autophagy. Bax/Bak(-/-) cells were defective in undergoing apoptosis but were more radiosensitive than the WT cells in autophagy. Both autophagy and sensitization of Bak/Bax(-/-) cells were further enhanced in the presence of Rad001. In contrast, inhibitors of autophagy rendered the Bak/Bax(-/-) cells radioresistant, whereas overexpression of ATG5 and Beclin-1 made the WT cells radiosensitive. When this novel concept of radiosensitization was tested in cancer models, small interfering RNAs against Bak/Bax also led to increased autophagy and sensitization of human breast and lung cancer cells to gamma radiation, which was further enhanced by Rad001. This is the first report to demonstrate that inhibition of pro-apoptotic proteins and induction of autophagy sensitizes cancer cells to therapy. Therapeutically targeting this novel pathway may yield significant benefits for cancer patients.

Background: -160C-->A and -347G-->GA polymorphisms in the promoter region decrease E-cadherin gene transcription. Decreased E-cadherin expression predicts poor outcome among patients with cancer. We sought to investigate whether -160C-->A and/or -347G-->GA polymorphisms were associated with the aggressiveness of prostate cancer., Methods: TaqMan single nucleotide polymorphism genotyping assay (Applied Biosystems, Foster City, CA) was used to detect -160C-->A and -347G-->GA polymorphisms in deoxyribonucleic acid from the paraffin-embedded prostate tissues of 98 Caucasian patients., Results: The genotype frequencies were -160C/C: 48% (47 of 98); -160C/A: 44% (43 of 98); -160A/A: 8% (8 of 98); -347G/G: 68% (67 of 98); -347G/GA: 28% (27 of 98); and -347GA/GA: 4% (4 of 98). Using the chi-square test, we found that the polymorphisms -160C-->A and -347G-->GA were not related to other clinical and pathologic parameters (i.e., age, prostate-specific antigen level, Gleason grade, and clinical stage) (P > 0.05). In combination analysis, there was no significant relationship between patients with both -160C/C and -347G/G, and these same parameters (P > 0.05). Using the log-rank test, we found no significant difference in relapse-free survival and overall survival between patients with -160C/C and those with -160A/C or -160A/A (P = 0.0764 and 0.2746, respectively), and also no significant difference between patients with -347G/G and those with -347GA/G or -347GA/GA (P = 0.9416 and 0.7367, respectively). There was also no significant difference in relapse-free survival and overall survival between patients with homozygosities of -160C/-347G and patients with other genotypes (P = 0.1418 and 0.2434, respectively)., Conclusion: We conclude that E-cadherin -160C-->A and/or -347G-->GA polymorphisms are not associated with the aggressiveness of prostate cancer in Caucasian patients.

Signal transducer and activator of transcription 3 (Stat3) and Survivin are constitutively up-regulated in various human tumor cells. We previously found Survivin to be significantly reduced in response to radiation in human umbilical vein endothelial cells (HUVEC) but not in tumor cell lines. In this study, we examined the effect of Stat3 on Survivin expression in irradiated HUVECs and breast cancer cells. We also studied how inhibition of Stat3 and Survivin activity affects cell survival and angiogenesis following irradiation. We determined that Survivin was significantly increased by overexpression of an active Stat3 (Stat3-C). Following irradiation, the level of phospho-Stat3 Tyr(705), but not phospho-Stat3 Ser(727), was reduced in HUVECs, whereas it remained unchanged in irradiated breast cancer cells. Correspondingly, Stat3 DNA-binding activity following irradiation was specifically down-regulated in HUVECs but not in breast cancer cells. Mutation of Tyr(705) abolished radiation-induced down-regulation of Survivin. Clonogenic and endothelial cell morphogenesis assays suggested that DN-Stat3 and DN-Survivin together resulted in the greatest radiosensitization of MDA-MB-231, decreasing angiogenesis and cell survival. In summary, Stat3 modulates Survivin, and both are potential therapeutic targets for radiation sensitization in breast cancer.

The phosphatidylinositol 3-kinase/Akt pathway plays a critical role in oncogenesis, and dysregulation of this pathway through loss of PTEN suppression is a particularly common phenomenon in aggressive prostate cancers. The mammalian target of rapamycin (mTOR) is a downstream signaling kinase in this pathway, exerting prosurvival influence on cells through the activation of factors involved in protein synthesis. The mTOR inhibitor rapamycin and its derivatives are cytotoxic to a number of cell lines. Recently, mTOR inhibition has also been shown to radiosensitize endothelial and breast cancer cells in vitro. Because radiation is an important modality in the treatment of prostate cancer, we tested the ability of the mTOR inhibitor RAD001 (everolimus) to enhance the cytotoxic effects of radiation on two prostate cancer cell lines, PC-3 and DU145. We found that both cell lines became more vulnerable to irradiation after treatment with RAD001, with the PTEN-deficient PC-3 cell line showing the greater sensitivity. This increased susceptibility to radiation is associated with induction of autophagy. Furthermore, we show that blocking apoptosis with caspase inhibition and Bax/Bak small interfering RNA in these cell lines enhances radiation-induced mortality and induces autophagy. Together, these data highlight the emerging importance of mTOR as a molecular target for therapeutic intervention, and lend support to the idea that nonapoptotic modes of cell death may play a crucial role in improving tumor cell kill.

Purpose: Integrins alpha v beta 3 and alpha v beta 5 are important in tumor growth and angiogenesis and have been recently explored as targets for cancer therapy. Radiotherapy also inhibits tumor growth and affects vasculature. We explored the combination of integrin antagonist Cilengitide (EMD 121974) and ionizing radiation., Methods and Materials: Levels of alpha v beta 3 were determined for human umbilical vein endothelial cells (HUVEC), as well as H157 and H460 human non-small-cell lung cancer cells, using FACS analysis and immunofluorescence imaging. Clonogenic assays, Western immunoblots probed for cleaved caspase 3, and Annexin-V probing were used to evaluate cell survival and apoptosis. A cell detachment assay and matrigel assay were used to further examine the effects of treatment., Results: Human umbilical vein endothelial cells had the highest alpha v beta 3 level, followed by H157, and H460. Interestingly, we found that 5 Gy irradiation induced expression of alpha v beta 3 in all cell lines. Clonogenic assays showed a radiosensitizing effect with Cilengitide, and calculation of the dose enhancement ratio showed that the effect was highest in HUVECs (1.38), followed by H157 (1.19), and H460 (1.10), corresponding to the levels of target expression. There was an increase in apoptotic cells after combination treatment with Cilengitide and radiation, and there was an increase in detached cells after treatment with Cilengitide. Additionally, there was decreased endothelial tubule formation after combination treatment., Conclusions: We conclude that radiation induces expression of alpha v beta 3 integrin in endothelial and non-small-cell lung cancer models, and that integrin antagonist Cilengitide is a radiosensitizer in proportion to the levels of target integrin expression.

Objectives: ATM is an important protein that protects the genome from double-stranded DNA breaks. Mutations and polymorphisms in the ATM gene have been associated with an increased risk of certain types of malignancies, especially breast cancer., Methods: We analyzed tissue from 98 white patients with prostate cancer for the presence of IVS62+60G>A polymorphism in the ATM gene., Results: The frequency of the homozygous IVS62+60G/G, heterozygous IVS62+60G/A, and homozygous IVS62+60A/A was 18.37% (18 of 98), 30.61% (30 of 98), and 51.02% (50 of 98), respectively. Using Fisher's exact test, we found the polymorphism IVS62+60G>A was not significantly related to age, tumor grade, prostate-specific antigen level, or clinical stage (P >0.05). No difference was found in relapse-free survival between patients with IVS62+60G/G and those with IVS62+60G/A or IVS62+60A/A (P = 0.4533)., Conclusions: The results of our study indicate that this ATM polymorphism is not associated with the aggressiveness of prostate cancer in white men.

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is known to be activated by radiation. The mammalian target of rapamycin (mTOR) is downstream of Akt, and we investigated the effects of radiation on Akt/mTOR signaling in breast cancer cell models. RAD001 (everolimus), a potent derivative of the mTOR inhibitor rapamycin, was used to study the effects of mTOR inhibition, as the role of mTOR inhibition in enhancing radiation remains unexplored. RAD001 decreased clonogenic cell survival in both breast cancer cell lines MDA-MB-231 and MCF-7, although the effect is greater in MDA-MB-231 cells. Irradiation induced Akt and mTOR signaling, and this signaling is attenuated by RAD001. The radiation-induced signaling activation is mediated by PI3K because inhibition of PI3K with LY294002 inhibited the increase in downstream mTOR signaling. Additionally, caspase-dependent apoptosis is an important mechanism of cell death when RAD001 is combined with 3 Gy radiation, as shown by induction of caspase-3 cleavage. An increase in G(2)-M cell cycle arrest was seen in the combination treatment group when compared with controls, suggesting that cell cycle arrest may have been a contributing factor in the increased radiosensitization seen in this study. We conclude that RAD001 attenuates radiation-induced prosurvival Akt/mTOR signaling and enhances the cytotoxic effects of radiation in breast cancer cell models, showing promise as a method of radiosensitization of breast cancer.

p53 plays a critical role in cell cycle arrest and induction of apoptosis. Certain malignancies carry wild-type p53, which is frequently down-regulated by murine double minute 2 (MDM2) overexpression. Availability of a small-molecule inhibitor against MDM2, nutlin, has made it feasible to evaluate the anti-MDM2-based therapeutic strategies. The rationale for the current study is that functional p53 has been linked with improved responses to radiation treatment. Hence, this study evaluates the use of nutlin, a small-molecule inhibitor that blocks the interaction of p53 and MDM2, in sensitizing cancer cells to radiation. Expression of MDM2, p53, and p21 in both p53 wild-type and p53-defective lung cancer cell lines was examined. Clonogenic and 7-amino-actinomycin D studies were used to determine possible mechanisms of cell death. The combined effect of MDM2 inhibition and radiation on cell cycle was also studied. We found that radiosensitization by nutlin occurs in lung cancer cells with wild-type p53. There were increased apoptosis and cell cycle arrest following administration of nutlin and radiation. Furthermore, the combination of nutlin and radiation decreased the ability of endothelial cells to form vasculature, as shown by Matrigel assays. Our data suggest that nutlin is an effective radiosensitizer of p53 wild-type cells. The radiosensitizing effect seems to be at least partially due to induction of apoptosis and cell cycle arrest. In addition, nutlin may be an effective radiosensitizer of tumor vasculature.

Epidermal growth factor receptor (HER1/EGFR)-mediated signal transduction pathways are important in cellular response to ionizing radiation. High HER1/EGFR expression on cancer cells may contribute to radioresistance. In this pre-clinical study, we evaluated the radiosensitizing effect of erlotinib, a small molecule HER1/EGFR inhibitor in three human cancer cell lines with different HER1/EGFR expression--A431 (very high expression), H157 (moderate expression) and H460 (low expression). Our results demonstrated that A431 was the most radioresistant, while H460 was the most radiosensitive. However, A431 cells were the most sensitive to erlotinib (IC50 = 300 nM) and H460 cells the most resistant (IC50 = 8 microM). H157 had intermediate sensitivity to radiation and erlotinib (IC50 = 3 microM). With 300 nM erlotinib, the radiation dose enhancement ratios (DER) were 1.40, 1.17 and 1.04 in A431, H157 and H460, respectively. Treatment with erlotinib for 24 hr at 300 nM increased G1 arrest by 18.6, 2.0 and 4.8% in A431, H157 and H460, respectively. Erlotinib-induced apoptosis was augmented by radiation in A431 cells only. In conclusion, high HER1/EGFR expression may result in a high degree of radiosensitization with erlotinib combined with radiation. The extent of erlotinib-induced radiosensitization was proportional to HER1/EGFR expression, as well as autophosphorylation of the human epidermal growth factor receptor (HER1/EGFR).

Purpose: Clusterin plays important roles in cell survival and death. Inactivation of clusterin enhances the therapeutic efficacy of chemotherapy in lung cancer models. The purpose of this study was to determine whether inhibition of clusterin by an antisense-based investigative drug enhances radiation sensitization in a lung cancer model., Methods and Materials: Cells were transfected with an antisense oligonucleotide (ASO) against clusterin (OGX-011). Apoptosis was determined by 7-aminoactinomycin D staining. Cell survival was examined by 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) and clonogenic assay. Xenograft model was used to demonstrate tumor growth and tumor blood flow., Results: OGX-011 specifically attenuated the expression of secreted clusterin (prosurvival), with no apparent effect on the expression of nuclear clusterin (proapoptotic). Apoptosis was significantly increased when H460 lung cancer cells were treated with OGX-011 plus radiation. Inhibition of clusterin followed by radiation greatly decreased cell survival. H460 xenografts that were treated with OGX-011 plus radiotherapy demonstrated growth delay beyond 17 days. Doppler studies showed that tumor blood flow was compromised when mice bearing H460 xenografts were treated with OGX-011 and radiation., Conclusion: A combination of radiotherapy and OGX-011 improved control of tumor growth and vascular regression in the H460 lung cancer model.

It is known that radiation activates the phosphoinositol-3 kinase (PI3K)/Akt pathway and that inhibition of PI3K or Akt sensitizes tumor vasculature to radiotherapy. Mammalian target of rapamycin (mTOR) is a downstream target of Akt, and we hypothesized that irradiation activates mTOR signaling in both glioma and endothelial cells (ECs) and that radiosensitization results from inhibiting mTOR signaling. mTOR inhibitors, rapamycin and RAD001 (everolimus) were found to radiosensitize vascular ECs, but failed to sensitize glioma cells as determined by clonogenic assay. Therefore, we investigated the anti-angiogenic effects of mTOR inhibitors. Increased phospho-mTOR protein was detected in irradiated human umbilical vein endothelial cells (HUVEC), but not in GL261 glioma cells. Phospho-S6, a biomarker for mTOR signaling, was also found to be induced following irradiation in HUVEC and this effect was inhibited by PI3K or mTOR inhibitors. Significant increase in cleaved caspase 3 was detected when Rad001 was combined with radiation. Endothelial tube formation was significantly diminished following treatment with rapamycin and 3 Gy of radiation. Histological sections of GL261 tumors from mice showed a greatly reduced vascular density when treated with RAD001 and radiation. Power Weighted Doppler of glioma xenografts in mice showed a significant reduction in vasculature and blood flow compared with mice treated with 3 Gy or RAD001 alone. We conclude that irradiation activates mTOR signaling in vascular endothelium and that rapamycin and RAD001 increased apoptosis of ECs in response to radiation. To the authors' best knowledge this is the first study which demonstrates that mTOR inhibitors may be a way to target the vasculature by radiosensitizing the vascular endothelium resulting in better tumor control as seen in experiments demonstrating increased tumor growth delay in mice treated with rapamycin with radiation compared with mice treat with either treatment alone. We conclude that mTOR inhibitors have increased efficacy as antiangiogenics when combined with radiation.

Murine double minute 2 (MDM2) inhibits p53-mediated functions, which are essential for therapies using DNA-damaging agents. The purpose of this study was to determine whether MDM2 inhibition enhances the radiosensitivity of a lung cancer model. The effects of MDM2 inhibition on tumor vasculature were also studied. Transient transfection of H460 lung cancer cells and human umbilical vascular endothelial cells (HUVEC) with antisense oligonucleotides (ASODN) against MDM2 resulted in a reduced level of MDM2 and increased levels of p21 and p53. Clonogenic assays showed that inhibition of MDM2 greatly decreased cell survival following irradiation. Quantification of apoptotic cells by 7-aminoactinomycin D staining and of senescent cells by X-gal staining showed that both processes were significantly increased in H460 cells treated with MDM2-specific ASODN and radiation. H460 xenografts that were treated with MDM2 ASODN plus radiotherapy also showed significant growth delay (P < 0.001) and increased apoptosis by terminal deoxynucleotidyl transferase-mediated nick end labeling staining. HUVECs transfected with MDM2-specific ASODN showed impaired viability and migration with decreased tube formation. Doppler studies showed that tumor blood flow was compromised when H460 xenografts were treated with MDM2-specific ASODN and radiation. A combination of radiotherapy and inhibition of MDM2 through the antisense approach results in improved tumor control in the H460 lung cancer model. This implies that a similar strategy should be investigated among patients with locally advanced lung cancer, receiving thoracic radiotherapy.

Endostatin is an important inhibitory molecule which mediates the sequential steps involved in angiogenesis. Lower level or impaired function of endostatin is associated with a higher risk of developing malignant solid tumors and with a worse prognosis of the disease. The endostatin N104 polymorphism might be associated with an impaired ability to inhibit angiogenesis. We analyzed the tissues from 98 Caucasian prostate cancer patients for the presence of D104N polymorphism. The frequencies of homozygous 4349G/G(104D/D), and heterozygous 4349G/A(104D/N) were 83.67%(82/98) and 16.33%(16/98), respectively; no individuals were homozygous 4349A/A(104N/N). With the Fisher's exact test we found the genotype of D104N was not significantly related to age, tumor grade, PSA and clinical stage (P > 0.05). There was no difference in relapse free survival(RFS) or overall survival(OS) between patients with 104D/N and those with 104D/D (P = 0.8283, 0.3713 respectively). We concluded that endostatin polymorphism was not associated with the aggressiveness of prostate cancer in Caucasian patients.

Survivin and XIAP are members of inhibitors of apoptosis (IAPs) family. They are upregulated in various malignancies. Inactivation of these molecules has resulted in chemosensitization. The purpose of this study was to determine whether inhibition of survivin, XIAP, or both enhances radiotherapy in a lung cancer model. Transient transfection of H460 cells with antisense oligonucleotides (ASOs) against either molecule has specifically reduced their expression, by Western analysis. Results from 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and clonogenic assays suggest that inhibition of survivin or XIAP greatly decreased cell survival following irradiation. A significantly increased number of apoptotic cells were detected when H460 cells were treated with either antisurvivin, anti-XIAP or both ASOs (P=0.03, 0.0003 and 0.01, respectively) plus irradiation. H460 xenografts that were treated with ASOs plus radiotherapy demonstrated growth delay beyond 15 days. Growth delay in the groups of combined treatment was greater than that in other groups. However, treatment with ASOs alone did not affect tumor growth delay in mice, but decreased the survival of H460 cells in culture. Antisense treatment did not cause any mortality or weight loss during the 32 days of study. These data suggest that inhibition of survivin or XIAP radiosensitizes H460 lung cancer cells by upregulating apoptosis and downregulating cell survival. Combination of radiotherapy and inhibition of survivin and XIAP through the antisense approach results in improved tumor control by radiotherapy in a mouse model of lung cancer.

Expression of survivin is elevated in most malignancies, especially in radiation-resistant cell lines. In this study, we investigated how radiation affects survivin expression in primary endothelial cells as well as in malignant cell lines. We found that 3 Gy significantly reduced survivin protein level in human umbilical vein endothelial cells (HUVECs) but not in tumor cell lines. Flow cytometry studies suggest that the down-regulation of survivin is independent of cell cycle. In addition, survivin mRNA level was also down-regulatable by irradiation. However, it was abrogated by actinomycin D-mediated inhibition of gene transcription. Luciferase reporter gene assays suggest that irradiation suppressed the survivin promoter. p53 overexpression reduced survivin expression, but overexpression of a p53 mutant failed to abolish the radiation-induced down-regulation in HUVECs. Alteration of p53 status in Val138 lung cancer cell line also failed to restore the radiation-inducible down-regulation. Overexpression of survivin in 293 cells prevented apoptosis induced by irradiation and increased cell viability after irradiation. The inhibition of survivin using antisense oligonucleotides caused a significant decrease in cell viability of irradiated H460 lung cancer cells. These data suggest that radiation transcriptionally down-regulates survivin in HUVECs. This regulatory mechanism is defective in malignancies and is not mediated by p53. Survivin overexpression may lead to resistance to radiotherapy by inhibiting apoptosis and enhancing cell viability. The inhibition of survivin results in sensitization of H460 lung cancer cells to radiation. These studies suggest that survivin may be a target for cancer therapy.

Purpose: Recent studies have demonstrated radiosensitization by inhibiting receptor tyrosine kinases (RTKs). Irradiation activates RTKs and their downstream prosurvival molecule, Akt. In this study, we investigated the mechanism by which SU6668, an inhibitor of RTKs involved in angiogenic pathways, enhances effects of irradiation., Methods and Materials: Western blots were used to determine Akt phosphorylation. Clonogenic assays were performed to determine endothelial survival after combination of SU6668 and irradiation. This combination therapy was also tested in mouse models with Lewis lung carcinoma or glioblastoma multiforme (GL261) for inhibition of tumor growth and tumor vasculature by examining tumor volume, tumor vascular window, and blood flow., Results: We found that SU6668 inhibited the Akt activation inducible by irradiation. Clonogenic survival of endothelial cells was decreased after the combined therapy compared with radiotherapy alone. In vivo studies demonstrated reduction of tumor vasculature and blood flow. In addition, 21 Gy in 7 fractions given concurrently with SU6668 resulted in tumor growth delay compared to either treatment alone., Conclusion: These data suggest that the combination therapy was more effective in destroying tumor vasculature than either treatment alone. SU6668 augments tumor-suppressive effects of radiotherapy in Lewis lung carcinoma and GL261 xenographs, possibly through reducing the survival of tumor endothelium.

Increased expression of cyclooxygenase (COX) 2 and the production of PGs appear to provide a survival advantage to transformed cells through the inhibition of apoptosis, increased attachment to extracellular matrix, increased invasiveness and the stimulation of angiogenesis. The purpose of this study was to determine whether an angiogenic antagonist, SU5416, could inhibit endogenous and phorbol 12-myristate 13-acetate (PMA)-mediated induction of COX-2 expression. SU5416 (5 micro M) inhibited endogenous as well as PMA-mediated induction of COX-2 expression when analyzed by immunoblot and Northern blot analysis. However, COX-1 expression remained unchanged under similar conditions. PMA is a potent inducer of reactive oxygen species that can play an important role during the induction of COX-2 expression. Our results demonstrated that PMA-mediated induction of COX-2 expression was found to be dependent on NADPH oxidase activity. An inhibitor of NADPH oxidase (diphenyleneiodonium chloride) blocked the PMA-mediated induction of COX-2 expression. The oxidase complex exhibited a temporal pattern of activation after exposure to PMA in which maximum activation was observed at 30 min after the addition of PMA. Activation of NADPH oxidase was also inhibited by SU5416, whereas an inhibitor of epidermal growth factor receptor signaling was unable to prevent the PMA-mediated induction of NADPH oxidase activity. When we blocked the PMA-mediated production of reactive oxygen species by blocking NADPH oxidase with SU5416, COX-2 expression and PGE(2) synthesis were also inhibited. Our results suggest that inhibition of NADPH oxidase activity, blocking of COX-2 expression, and PGE(2) synthesis may represent novel targets for SU5416.