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Purpose: The phase II CAPTIVATE study investigated first-line treatment with ibrutinib plus venetoclax for chronic lymphocytic leukemia in two cohorts: minimal residual disease (MRD)-guided randomized treatment discontinuation (MRD cohort) and fixed duration (FD cohort). We report tumor debulking and tumor lysis syndrome (TLS) risk category reduction with three cycles of single-agent ibrutinib lead-in before initiation of venetoclax using pooled data from the MRD and FD cohorts. Patients and Methods: In both cohorts, patients initially received three cycles of ibrutinib 420 mg/day then 12 cycles of ibrutinib plus venetoclax (5-week ramp-up to 400 mg/day). Results: In the total population (N = 323), the following decreases from baseline to after ibrutinib lead-in were observed: percentage of patients with a lymph node diameter ≥5 cm decreased from 31% to 4%, with absolute lymphocyte count ≥25 × 109/L from 76% to 65%, with high tumor burden category for TLS risk from 23% to 2%, and with an indication for hospitalization (high TLS risk, or medium TLS risk and creatinine clearance Conclusions: Three cycles of ibrutinib lead-in before venetoclax initiation provides effective tumor debulking, decreases the TLS risk category and reduces the need for hospitalization for intensive monitoring for TLS.

Purpose:The phase II CAPTIVATE study investigated first-line treatment with ibrutinib plus venetoclax for chronic lymphocytic leukemia in two cohorts: minimal residual disease (MRD)-guided randomized treatment discontinuation (MRD cohort) and fixed duration (FD cohort). We report tumor debulking and tumor lysis syndrome (TLS) risk category reduction with three cycles of single-agent ibrutinib lead-in before initiation of venetoclax using pooled data from the MRD and FD cohorts.Patients and Methods:In both cohorts, patients initially received three cycles of ibrutinib 420 mg/day then 12 cycles of ibrutinib plus venetoclax (5-week ramp-up to 400 mg/day).Results:In the total population (N = 323), the following decreases from baseline to after ibrutinib lead-in were observed: percentage of patients with a lymph node diameter ≥5 cm decreased from 31% to 4%, with absolute lymphocyte count ≥25 × 109/L from 76% to 65%, with high tumor burden category for TLS risk from 23% to 2%, and with an indication for hospitalization (high TLS risk, or medium TLS risk and creatinine clearance Conclusions:Three cycles of ibrutinib lead-in before venetoclax initiation provides effective tumor debulking, decreases the TLS risk category and reduces the need for hospitalization for intensive monitoring for TLS.

Supplementary Data from Effective Tumor Debulking with Ibrutinib Before Initiation of Venetoclax: Results from the CAPTIVATE Minimal Residual Disease and Fixed-Duration Cohorts

Purpose: Activating PIK3CA genomic alterations are frequent in head and neck squamous cell carcinoma (HNSCC), and there is an association between phosphoinositide 3-kinase (PI3K) signaling and radioresistance. Hence, we investigated the therapeutic efficacy of inhibiting PI3K with GDC-0032, a PI3K inhibitor with potent activity against p110α, in combination with radiation in HNSCC.Experimental Design: The efficacy of GDC-0032 was assessed in vitro in 26 HNSCC cell lines with crystal violet proliferation assays, and changes in PI3K signaling were measured by Western blot analysis. Cytotoxicity and radiosensitization were assessed with Annexin V staining via flow cytometry and clonogenic survival assays, respectively. DNA damage repair was assessed with immunofluorescence for γH2AX foci, and cell cycle analysis was performed with flow cytometry. In vivo efficacy of GDC-0032 and radiation was assessed in xenografts implanted into nude mice.Results: GDC-0032 inhibited potently PI3K signaling and displayed greater antiproliferative activity in HNSCC cell lines with PIK3CA mutations or amplification, whereas cell lines with PTEN alterations were relatively resistant to its effects. Pretreatment with GDC-0032 radiosensitized PIK3CA-mutant HNSCC cells, enhanced radiation-induced apoptosis, impaired DNA damage repair, and prolonged G2–M arrest following irradiation. Furthermore, combined GDC-0032 and radiation was more effective than either treatment alone in vivo in subcutaneous xenograft models.Conclusions: GDC-0032 has increased potency in HNSCC cell lines harboring PIK3CA-activating aberrations. Further, combined GDC-0032 and radiotherapy was more efficacious than either treatment alone in PIK3CA-altered HNSCC in vitro and in vivo. This strategy warrants further clinical investigation. Clin Cancer Res; 22(8); 2009–19. ©2015 AACR.

Supplementary Figure Legends

Supplementary Figure S1. GDC-0032 downregulated PI3K signaling in a dose dependent fashion. Supplementary Figure S2. BYL719 enhances radiation induced cell death in PIK3CA activated cell lines. Supplementary Figure S3. A66 enhances radiation induced cell death in PIK3CA activated cell lines Supplementary Figure S4. PI3K pathway inhibition enhances cell death in HSC-2 (PIK3CA mutated) following irradiation. Cells were treated with the given drugs for 24 hours, irradiated with 4 Gy, then assessed for Annexin V staining 72 hours after irradiation. Supplementary Figure S5. GDC-0032 radiosensitizes LB-771 (PIK3CA amplified) cells in vitro as assessed via the clonogenic survival assay. Supplementary Figure S6. GDC-0032 enhances DNA damage and apoptosis following irradiation. Supplementary Figure S7. GDC-0032 does not significantly alter the early G2/M checkpoint. Supplementary Figure S8. ATR inhibition partially blocks the early G2/M checkpoint. Supplementary Figure S9. Wee1 inhibition partially blocks the early G2/M checkpoint. Supplementary Figure S10. Wee1 inhibition doesn't reverse the enhanced G2/M checkpoint induced by GDC-0032 following irradiation. 3 Supplementary Figure S11. GDC-0032 and radiation is superior to either treatment alone in an HPV(+) oropharyngeal squamous cell carcinoma patient derived xenograft.

TP53 aberrations [del(17p) or TP53 mutation] predict poor survival with chemoimmunotherapy in patients with chronic lymphocytic leukaemia (CLL). We evaluated long-term efficacy and safety of first-line ibrutinib-based therapy in patients with CLL bearing TP53 aberrations in a pooled analysis across four studies: PCYC-1122e, RESONATE-2 (PCYC-1115/16), iLLUMINATE (PCYC-1130) and ECOG-ACRIN E1912. The pooled analysis included 89 patients with TP53 aberrations receiving first-line treatment with single-agent ibrutinib (n = 45) or ibrutinib in combination with an anti-CD20 antibody (n = 44). All 89 patients had del(17p) (53% of 89 patients) and/or TP53 mutation (91% of 58 patients with TP53 sequencing results available). With a median follow-up of 49·8 months (range, 0·1-95·9), median progression-free survival was not reached. Progression-free survival rate and overall survival rate estimates at four years were 79% and 88%, respectively. Overall response rate was 93%, including complete response in 39% of patients. No new safety signals were identified in this analysis. Forty-six percent of patients remained on ibrutinib treatment at last follow-up. With median follow-up of four years (up to eight years), results from this large, pooled, multi-study data set suggest promising long-term outcomes of first-line ibrutinib-based therapy in patients with TP53 aberrations. Registered at ClinicalTrials.gov (NCT01500733, NCT01722487, NCT02264574 and NCT02048813).

The cardiovascular biology of proton radiotherapy is not well understood. We aimed to compare the genomic dose-response to proton and gamma radiation of the mouse aorta to assess whether their vascular effects may diverge. We performed comparative RNA sequencing of the aorta following (4 hrs) total-body proton and gamma irradiation (0.5-200 cGy whole body dose, 10 dose levels) of conscious mice. A trend analysis identified genes that showed a dose response. While fewer genes were dose-responsive to proton than gamma radiation (29 vs. 194 genes; q-value ≤ 0.1), the magnitude of the effect was greater. Highly responsive genes were enriched for radiation response pathways (DNA damage, apoptosis, cellular stress and inflammation; p-value ≤ 0.01). Gamma, but not proton radiation induced additionally genes in vasculature specific pathways. Genes responsive to both radiation types showed almost perfectly superimposable dose-response relationships. Despite the activation of canonical radiation response pathways by both radiation types, we detected marked differences in the genomic response of the murine aorta. Models of cardiovascular risk based on photon radiation may not accurately predict the risk associated with proton radiation.

Purpose. The cardiovascular biology of proton radiotherapy is not well understood. We aimed to compare the genomic dose-response to proton and gamma radiation of the mouse aorta to assess whether their vascular effects may diverge.Materials and methods.We performed comparative RNA sequencing of the aorta following (4 hrs) total-body proton and gamma irradiation (0.5 - 200 cGy whole body dose, 10 dose levels) of conscious mice. A trend analysis identified genes that showed a dose response.Results.While fewer genes were dose-responsive to proton than gamma radiation (29 vs. 194 genes;q-value ≤ 0.1), the magnitude of the effect was greater. Highly responsive genes were enriched for radiation response pathways (DNA damage, apoptosis, cellular stress and inflammation;p-value ≤ 0.01). Gamma, but not proton radiation induced additionally genes in vasculature specific pathways. Genes responsive to both radiation types showed almost perfectly superimposable dose-response relationships.Conclusions.Despite the activation of canonical radiation response pathways by both radiation types, we detected marked differences in the genomic response of the murine aorta. Models of cardiovascular risk based on photon radiation may not accurately predict the risk associated with proton radiation.

Patients with oncogene driven tumors are treated with targeted therapeutics including EGFR inhibitors. Genomic data from The Cancer Genome Atlas (TCGA) demonstrates molecular alterations to EGFR, MAPK, and PI3K pathways in previously untreated tumors. Therefore, this study uses bioinformatics algorithms to delineate interactions resulting from EGFR inhibitor use in cancer cells with these genetic alterations. We modify the HaCaT keratinocyte cell line model to simulate cancer cells with constitutive activation of EGFR, HRAS, and PI3K in a controlled genetic background. We then measure gene expression after treating modified HaCaT cells with gefitinib, afatinib, and cetuximab. The CoGAPS algorithm distinguishes a gene expression signature associated with the anticipated silencing of the EGFR network. It also infers a feedback signature with EGFR gene expression itself increasing in cells that are responsive to EGFR inhibitors. This feedback signature has increased expression of several growth factor receptors regulated by the AP-2 family of transcription factors. The gene expression signatures for AP-2alpha are further correlated with sensitivity to cetuximab treatment in HNSCC cell lines and changes in EGFR expression in HNSCC tumors with low CDKN2A gene expression. In addition, the AP-2alpha gene expression signatures are also associated with inhibition of MEK, PI3K, and mTOR pathways in the Library of Integrated Network-Based Cellular Signatures (LINCS) data. These results suggest that AP-2 transcription factors are activated as feedback from EGFR network inhibition and may mediate EGFR inhibitor resistance.

Bioinformatics techniques to analyze time course bulk and single cell omics data are advancing. The absence of a known ground truth of the dynamics of molecular changes challenges benchmarking their performance on real data. Realistic simulated time-course datasets are essential to assess the performance of time course bioinformatics algorithms. We develop an R/Bioconductor package, CancerInSilico, to simulate bulk and single cell transcriptional data from a known ground truth obtained from mathematical models of cellular systems. This package contains a general R infrastructure for running cell-based models and simulating gene expression data based on the model states. We show how to use this package to simulate a gene expression data set and consequently benchmark analysis methods on this data set with a known ground truth. The package is freely available via Bioconductor: http://bioconductor.org/packages/CancerInSilico/

Epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and cetuximab, a monoclonal antibody targeting this receptor, is widely used to treat these patients. In the following investigation, we examined the role of SMAD4 down-regulation in mediating epithelial-to-mesenchymal transition (EMT) and cetuximab resistance in HNSCC. We determined that SMAD4 downregulation was significantly associated with increased cell motility, increased expression of vimentin, and cetuximab resistance in HNSCC cell lines. In the HNSCC genomic dataset obtained from The Cancer Genome Atlas, SMAD4 was altered in 20/279 (7%) of HNSCC via homozygous deletion, and nonsense, missense, and silent mutations. When SMAD4 expression was compared with respect to human papillomavirus (HPV) status, HPV-positive tumors had higher expression compared to HPV-negative tumors. Furthermore, higher SMAD4 expression also correlated with higher CDKN2A (p16) expression. Our data suggest that SMAD4 down-regulation plays an important role in the induction of EMT and cetuximab resistance. Patients with higher SMAD4 expression may benefit from cetuximab use in the clinic.

Astronauts could be exposed to solar particle event (SPE) radiation, which is comprised mostly of proton radiation. Proton radiation is also a treatment option for certain cancers. Both astronauts and clinical patients exposed to ionizing radiation are at risk for white blood cell (WBC) loss, which are the body’s main defense against infection. In this report, the effect of Neulasta treatment, a granulocyte colony stimulating factor, after proton radiation exposure is discussed. Mini pigs exposed to total body proton irradiation at a dose of 2 Gy received 4 treatments of either Neulasta or saline injections. Peripheral blood cell counts and thromboelastography parameters were recorded up to 30 days post-irradiation. Neulasta significantly improved white blood cell (WBC), specifically neutrophil, loss in irradiated animals by approximately 60% three days after the first injection, compared to the saline treated irradiated animals. Blood cell counts quickly decreased after the last Neulasta injection, suggesting a transient effect on WBC stimulation. Statistically significant changes in hemostasis parameters were observed after proton radiation exposure in both the saline and Neulasta treated irradiated groups, as well internal organ complications such as pulmonary changes. In conclusion, Neulasta treatment temporarily alleviates proton radiation-induced WBC loss, but has no effect on altered hemostatic responses.

Recent evidence has suggested that disseminated intravascular coagulation (DIC) plays an integral role in death at the LD 50 dose of either gamma or solar particle event (SPE)-like proton radiation in ferrets. In these studies, Yucatan minipigs were evaluated to determine whether they were susceptible to the development of radiation induced DIC. Yucatan minipigs were exposed to a dose of 2.5 Gray (Gy) with X-rays and monitored over the course of 30 days. Evidence of DIC was evaluated by way of thromboelastometry parameters, platelet counts, fibrinogen concentration, and the d-dimer assay. Pigs exposed to X-rays developed signs of DIC within 2 days' post-irradiation. The development of DIC was exacerbated over the course of the studies, and one of the pigs died at day 14 and another had to be euthanized on day 16 post-irradiation. For both of these pigs, DIC was evident at the time of death. The following observations were indicated or were suggestive of DIC: whole blood clotting was impaired (as evidenced by thromboelastometry alterations), there were decreased platelet counts, elevated d-dimer concentrations in the blood, and/or hemorrhaging and the presence of fibrin in tissues observed during post-mortem examination. The extrapolation of data from these studies, in combination with other published data, have led to the hypothesis that there could be a correlation between the propensity to develop DIC, as indicated by hemorrhaging at death at relatively low doses of radiation, and the LD 50 for a particular species. Our data suggest that the development of DIC may contribute to death at the LD 50 dose in large mammals.

Head and neck squamous cell carcinomas have distinct mutation and copy-number profiles depending on human papillomavirus status. Although several challenges remain in biomarker implementation and clinical trial feasibility, incorporating available genomic data will expedite the development of novel therapeutics and predictive biomarker-driven clinical trials. Clin Cancer Res; 21(3); 495–7. ©2014 AACR. See related article by Seiwert et al., p. 632

Purpose : To determine whether proton radiation affects coagulation. Material and methods : Ferrets were exposed to solar particle event-like proton radiation at doses of 0, 25, 100, or 200 centigray (cGy), and dose rates of 50 cGy/minute (high dose rate or HDR) or 50 cGy/hour (low dose rate or LDR). Plasma was isolated from blood collected prior to radiation exposure and at 3 – 7 h postradiation. Prothrombin time (PT) assays and activated partial thromboplastin time (aPTT) assays were performed as were mixing studies to determine the coagulation factors involved. Results : HDR and LDR exposure led to statistically signifi cant increases in PT values. It was determined that the HDR-induced increase in PT was due to Factor VII, while Factors II, V, and VII contributed to the LDR-induced increase in PT values. Only acute LDR exposure caused an increase in aPTT values, which remained elevated for 48 h post-irradiation (which was the latest time assayed in these studies). Mixing studies revealed that Factor IX contributed to the increased aPTT values. A majority of the animals exposed at the LDR had an International Normalized Ratio approaching or surpassing 2.0. Conclusions : PT/aPTT assays resulted in increased clotting times due to diff erent coagulation factors, indicating potential radiation-induced coagulopathy.

Animal models are frequently used to assist in the determination of the long- and short-term effects of space flight. The space environment, including microgravity, can impact many physiological and immunological system parameters. It has been found that ground based models of microgravity produce changes in white blood cell counts, which negatively affects immunologic function. As part of the Center of Acute Radiation Research (CARR), we compared the acute effects on white blood cell parameters induced by the more traditionally used animal model of hindlimb unloading (HU) with a recently developed reduced weightbearing analog known as partial weight suspension (PWS). Female ICR mice were either hindlimb unloaded or placed in the PWS system at 16% quadrupedal weightbearing for 4 h, 1, 2, 7 or 10 days, at which point complete blood counts were obtained. Control animals (jacketed and non-jacketed) were exposed to identical conditions without reduced weightbearing. Results indicate that significant changes in total white blood cell (WBC), neutrophil, lymphocyte, monocyte and eosinophil counts were observed within the first 2 days of exposure to each system. These differences in blood cell counts normalized by day 7 in both systems. The results of these studies indicate that there are some statistically significant changes observed in the blood cell counts for animals exposed to both the PWS and HU simulated microgravity systems.

The present study evaluated the acute effects of radiation dose, dose rate and fractionation as well as the energy of protons in hematopoietic cells of irradiated mice. The mice were irradiated with a single dose of 51.24 MeV protons at a dose of 2 Gy and a dose rate of 0.05–0.07 Gy/min or 1 GeV protons at doses of 0.1, 0.2, 0.5, 1, 1.5 and 2 Gy delivered in a single dose at dose rates of 0.05 or 0.5 Gy/min or in five daily dose fractions at a dose rate of 0.05 Gy/min. Sham-irradiated animals were used as controls. The results demonstrate a dose-dependent loss of white blood cells (WBCs) and lymphocytes by up to 61% and 72%, respectively, in mice irradiated with protons at doses up to 2 Gy. The results also demonstrate that the dose rate, fractionation pattern and energy of the proton radiation did not have significant effects on WBC and lymphocyte counts in the irradiated animals. These results suggest that the acute effects of proton radiation on WBC and lymphocyte counts are determined mainly by the radiation dose, with very little contribution from the dose rate (over the range of dose rates evaluated), fractionation and energy of the protons.

Four new coordination polymer compounds have been prepared using 3-amino-1,2,4-triazole (AmTRZ) as an organic linker, namely, ZnCl(AmTRZ) (1), Zn(HCO2)(AmTRZ) (2), Zn5(OH)2(AmTRZ)6(NO3)2·6H2O (3), and Zn5(OH)(AmTRZH)(AmTRZ)6(HCO2)3·5H2O (4). They were synthesized under mild hydro- or solvothermal conditions using water or dimethylformamide as solvent. Their crystal structures were characterized by single-crystal X-ray diffraction techniques, and thermal properties of all compounds were examined by thermogravimetric analysis. Isostructural compounds 1 and 2 (Pbca, a = 9.389(1) A, b = 10.115(1) A, c = 11.571(2) A, V = 1098.9(3) A3, Z = 8 for 1 and a = 9.198(1) A, b = 10.341(1) A, c = 12.577(2) A, V = 1196.2(3) A, Z = 8 for 2) display a two-dimensional layer structure where Zn centers are linked by AmTRZ molecules to form gridlike layers containing eight-membered rings. Structure 3 (Pa3, a = 15.0448(6) A, V = 3405.3(2) A3, Z = 4) is a dense three-dimensional framework, featuring seven-membered channels wher...

Two new Zn-based coordination polymers based polymers, namely, Zn7(TRZ)8(SIP)2·7H2O (1) and Zn5Na2(DMA)4(TRZ)4(SIP)4·5H2O (2) (TRZ = 1,2,4-triazole, SIP = sulfoisophthalate, and DMA = dimethylamine), were synthesized under mild solvothermal conditions. Their crystal structures were characterized by single-crystal X-ray diffraction, and their thermal properties were examined by thermogravimetric analysis. Structure 1 crystallizes in monoclinic P21/n space group (a = 14.050(3) A, b = 12.517(3) A, c = 15.377(3) A, β = 97.95(3)°, V = 2678.3(9) A3, Z = 2), while structure 2 is described as triclinic P1 (a = 9.701(2) A, b = 12.442(3) A, c = 17.218(3) A, α = 89.83(3)°, β = 75.01(3)°, γ = 73.33(3)°, V = 1917.5(7) A3, Z = 1). Structure 1 contains 8- and 16-membered channels made of Zn polyhedra linked by TRZ, which are occupied by water molecules and SIP ligands. Structure 2 consists of one-dimensional chains of Zn−TRZ moieties that are linked to each other via SIP. It is a negatively charged open framework in wh...

Purpose: Activating PIK3CA genomic alterations are frequent in head and neck squamous cell carcinoma (HNSCC), and there is an association between phosphoinositide 3-kinase (PI3K) signaling and radioresistance. Hence, we investigated the therapeutic efficacy of inhibiting PI3K with GDC-0032, a PI3K inhibitor with potent activity against p110α, in combination with radiation in HNSCC. Experimental Design: The efficacy of GDC-0032 was assessed in vitro in 26 HNSCC cell lines with crystal violet proliferation assays, and changes in PI3K signaling were measured by Western blot analysis. Cytotoxicity and radiosensitization were assessed with Annexin V staining via flow cytometry and clonogenic survival assays, respectively. DNA damage repair was assessed with immunofluorescence for γH2AX foci, and cell cycle analysis was performed with flow cytometry. In vivo efficacy of GDC-0032 and radiation was assessed in xenografts implanted into nude mice. Results: GDC-0032 inhibited potently PI3K signaling and displayed greater antiproliferative activity in HNSCC cell lines with PIK3CA mutations or amplification, whereas cell lines with PTEN alterations were relatively resistant to its effects. Pretreatment with GDC-0032 radiosensitized PIK3CA-mutant HNSCC cells, enhanced radiation-induced apoptosis, impaired DNA damage repair, and prolonged G2–M arrest following irradiation. Furthermore, combined GDC-0032 and radiation was more effective than either treatment alone in vivo in subcutaneous xenograft models. Conclusions: GDC-0032 has increased potency in HNSCC cell lines harboring PIK3CA-activating aberrations. Further, combined GDC-0032 and radiotherapy was more efficacious than either treatment alone in PIK3CA-altered HNSCC in vitro and in vivo. This strategy warrants further clinical investigation. Clin Cancer Res; 22(8); 2009–19. ©2015 AACR.

The mechanism by which radiation exposure leads to death in mammalian organisms remains unknown, although numerous hypotheses have been discussed. At the lowest total body radiation doses leading to mammalian mortality, death occurs from the hematopoietic syndrome (HS). HS is thought to result from the cell killing effects of radiation in the bone marrow that lead to low numbers of circulating blood cells and the resultant HS symptoms, such as infection [from the loss of white blood cells (WBC)] and bleeding (presumably from the loss of platelets). Over approximately the last half century, the dose of ionizing radiation that kills half of an experimental group/exposed population, known as the LD50, has been used as a parameter to compare the radiation sensitivity of various mammalian species. It is well known that the LD50 is highly variable for different mammalian species; however, the bone marrow cells of different species, strains and individuals are known to have remarkably similar sensitivities to the cell killing effects of ionizing radiation (1, 2). These results suggest that the lethal effects of radiation in blood cells may not be the primary mechanism by which the HS causes death. Our results have suggested that radiation induced activation of the coagulation cascade, resulting in a condition known as disseminated intravascular coagulation (DIC), could be the major mechanism by which relatively low doses of radiation could lead to animal, including human, mortality.

The effectiveness of simulated solar particle event (SPE) proton radiation to induce retching and vomiting was evaluated in the ferret experimental animal model. The endpoints measured in the study included: (1) the fraction of animals that retched or vomited, (2) the number of retches or vomits observed, (3) the latency period before the first retch or vomit and (4) the duration between the first and last retching or vomiting events. The results demonstrated that γ ray and proton irradiation delivered at a high dose rate of 0.5 Gy/min induced dose-dependent changes in the endpoints related to retching and vomiting. The minimum radiation doses required to induce statistically significant changes in retching- and vomiting-related endpoints were 0.75 and 1.0 Gy, respectively, and the relative biological effectiveness (RBE) of proton radiation at the high dose rate did not significantly differ from 1. Similar but less consistent and smaller changes in the retching- and vomiting-related endpoints were observed for groups irradiated with γ rays and protons delivered at a low dose rate of 0.5 Gy/h. Since this low dose rate is similar to a radiation dose rate expected during a SPE, these results suggest that the risk of SPE radiation-induced vomiting is low and may reach statistical significance only when the radiation dose reaches 1 Gy or higher.

Acute radiation sickness (ARS) is expected to occur in astronauts during large solar particle events (SPEs). One parameter associated with ARS is the hematopoietic syndrome, which can result from decreased numbers of circulating blood cells in those exposed to radiation. The peripheral blood cells are critical for an adequate immune response, and low blood cell counts can result in an increased susceptibility to infection. In this study, Yucatan minipigs were exposed to proton radiation within a range of skin dose levels expected for an SPE (estimated from previous SPEs). The proton-radiation exposure resulted in significant decreases in total white blood cell count (WBC) within 1 day of exposure, 60% below baseline control value or preirradiation values. At the lowest level of the blood cell counts, lymphocytes, neutrophils, monocytes and eosinophils were decreased up to 89.5%, 60.4%, 73.2% and 75.5%, respectively, from the preirradiation values. Monocytes and lymphocytes were decreased by an average of 70% (compared to preirradiation values) as early as 4 h after radiation exposure. Skin doses greater than 5 Gy resulted in decreased blood cell counts up to 90 days after exposure. The results reported here are similar to studies of ARS using the nonhuman primate model, supporting the use of the Yucatan minipig as an alternative. In addition, the high prevalence of hematologic abnormalities resulting from exposure to acute, whole-body SPE-like proton radiation warrants the development of appropriate countermeasures to prevent or treat ARS occurring in astronauts during space travel.

Immune system adaptation during spaceflight is a concern in space medicine. Decreased circulating leukocytes observed during and after space flight infer suppressed immune responses and susceptibility to infection. The microgravity aspect of the space environment has been simulated on Earth to study adverse biological effects in astronauts. In this report, the hindlimb unloading (HU) model was employed to investigate the combined effects of solar particle event-like proton radiation and simulated microgravity on immune cell parameters including lymphocyte subtype populations and activity. Lymphocytes are a type of white blood cell critical for adaptive immune responses and T lymphocytes are regulators of cell-mediated immunity, controlling the entire immune response. Mice were suspended prior to and after proton radiation exposure (2 Gy dose) and total leukocyte numbers and splenic lymphocyte functionality were evaluated on days 4 or 21 after combined HU and radiation exposure. Total white blood cell (WBC), lymphocyte, neutrophil, and monocyte counts are reduced by approximately 65%, 70%, 55%, and 70%, respectively, compared to the non-treated control group at 4 days after combined exposure. Splenic lymphocyte subpopulations are altered at both time points investigated. At 21 days post-exposure to combined HU and proton radiation, T cell activation and proliferation were assessed in isolated lymphocytes. Cell surface expression of the Early Activation Marker, CD69, is decreased by 30% in the combined treatment group, compared to the non-treated control group and cell proliferation was suppressed by approximately 50%, compared to the non-treated control group. These findings reveal that the combined stressors (HU and proton radiation exposure) result in decreased leukocyte numbers and function, which could contribute to immune system dysfunction in crew members. This investigation is one of the first to report on combined proton radiation and simulated microgravity effects on hematopoietic, specifically immune cells.

Recombinant tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is an antitumor protein that is in clinical trials as a potential anticancer therapy but suffers from drug properties that may limit efficacy such as short serum half-life, stability, cost, and biodistribution, particularly with respect to the brain. To overcome such limitations, we identified TRAIL-inducing compound 10 (TIC10), a potent, orally active, and stable small molecule that transcriptionally induces TRAIL in a p53-independent manner and crosses the blood-brain barrier. TIC10 induces a sustained up-regulation of TRAIL in tumors and normal cells that may contribute to the demonstrable antitumor activity of TIC10. TIC10 inactivates kinases Akt and extracellular signal–regulated kinase (ERK), leading to the translocation of Foxo3a into the nucleus, where it binds to the TRAIL promoter to up-regulate gene transcription. TIC10 is an efficacious antitumor therapeutic agent that acts on tumor cells and their micro-environment to enhance the concentrations of the endogenous tumor suppressor TRAIL.

Next generation sequencing technologies enable precise personalized medicine. Thus, patients with oncogene driven tumors are currently treated with targeted therapeutics such as EGFR inhibitors. However, drug interactions with other activated signaling pathways in treated tumors often alter predicted therapeutic response. Therefore, bioinformatics algorithms are needed to infer unanticipated molecular interactions from anticipated molecular response to targeted therapeutics in diverse genetic backgrounds. To model heterogeneous genetic backgrounds in HNSCC, we use HaCaT cells with forced overexpression of EGFR, HRAS, and PIK3CA. Previously, the CoGAPS matrix factorization algorithm was shown to infer the specific signaling pathways that were activated in these HaCaT knock-in constructs from gene expression data. In this study, we evaluated whether CoGAPS could also delineate unanticipated signaling changes from anticipated cellular signaling response caused by targeted therapeutic in diverse genetic backgrounds. To delineate these signaling responses, we measured gene expression after treating the modified HaCaT cells with three EGFR targeted agents (gefitinib, cetuximab and afatinib) for 24 hours. The CoGAPS matrix factorization algorithm distinguished a gene expression signature associated with the anticipated silencing of the EGFR network and a signature associated with unanticipated transcriptional feedback in HaCaT constructs that were sensitive to EGFR inhibitors. Notably, the feedback signature showed that EGFR gene expression itself increased in cells that were responsive to EGFR inhibitors. The CoGAPS algorithm further associated such feedback with increased expression of several growth factor receptors by the AP-2 family of transcription factors. Once transcribed, these growth factor receptors may ultimately compensate for EGFR inhibition in these sensitive cells. Our data suggest, that CoGAPS gene expression signatures delineate on target and feedback effects of drugs related to therapeutic sensitivity in diverse genetic backgrounds. Citation Format: Elana J. Fertig, Hiroyuki Ozawa, Manjusha Thakar, Jason Howard, Gabriel Krigsfeld, Alexander V. Favorov, Daria A. Gaykalova, Michael F. Ochs, Christine H. Chung. The CoGAPS matrix factorization algorithm infers feedback mechanisms from therapeutic inhibition of EGFR that increases expression of growth factor receptors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 777.

A major solar particle event (SPE) may place astronauts at significant risk for the acute radiation syndrome (ARS), which may be exacerbated when combined with other space flight stressors, such that the mission or crew health may be compromised. The National Space Biomedical Research Institute (NSBRI) Center of Acute Radiation Research (CARR) is focused on the assessment of risks of adverse biological effects related to the ARS in animals exposed to space flight stressors combined with the types of radiation expected during an SPE. The CARR studies are focused on the adverse biological effects resulting from exposure to the types of radiation, at the appropriate energies, doses and dose-rates, present during an SPE (and standard reference radiations: gamma rays or electrons). All animal studies described have been approved by the University of PA IACUC. Some conclusions from recent CARR investigations are as follows: (i) the relative biological effectiveness (RBE) values for SPE-like protons compared with standard reference radiations (gammas or electrons) for white blood cells (WBCs) vary greatly between mice, ferrets and pigs, with the RBE values being greater in ferrets than those in mice, and considerably greater in pigs compared with those in ferrets or mice [1, 2]. This trend for the data suggests that the RBE values for WBCs in humans could be considerably greater than those observed in small mammals, and SPE proton radiation may be far more hazardous to humans than previously estimated from small animal studies. (ii) Very low doses of SPE proton radiation (25 cGy) increase blood clotting times in ferrets, and the low SPE-like dose rate has more severe effects than high dose rate radiation [3]. (iii) Results from pig and ferret studies suggest that disseminated intravascular coagulation is a major cause of death at doses near the LD50 level for SPE-like proton and gamma radiation. (iv) Exposure to SPE-like proton or gamma radiation, in combination with simulated microgravity (hindlimb suspension), leads to a very high level of morbidity/mortality in mice given a bacterial challenge with non-toxic levels of Pseudomonas aeruginosa or Klebsiella pneumoniae; the threshold for this effect was 1.5 Gy. (v) T-cell activation was reduced in mice exposed to SPE-like radiation with or without simulated hypogravity (either partial weight suspension or hindlimb suspension) (e.g. [4]). (vi) Radiation and simulated hypogravity had synergistic effects on immune system biological endpoints (e.g. [5]). (vii) Pigs exposed to simulated SPE radiation exhibited increases in intracranial pressure that remained elevated over the 90-day experimental period. (viii) A major sparing effect of SPE-like low dose rate radiation (compared with the results for high dose rate radiation) was observed for ferret emesis parameters, such that the differences between the results for ferret exposure to low dose rate radiation (50 cGy/h) and controls were not statistically significant (for doses up to 2 Gy). For high dose rate SPE proton radiation, the threshold value for retching was 75 cGy, and for ferret vomiting, it was 1 Gy.

We have identified TNFSF10 (TRAIL) as a p53-transcriptional target gene. There are two p53 DNA-binding sites in the human TNFSF10 promoter region, at 346 and 625 bp upstream of the transcription start site. A human p53-expressing adenovirus (Ad-p53) induced TRAIL mRNA and protein expression in HCT116 p53-/- human colon cancer cells. A human TRAIL-promoter reporter assay showed increased luciferase activity with the promoter vector that contains two p53 DNA-binding motifs,following Ad-p53 infection, compared to the control adenovirus infection. Using HCT116 cells, gene silencing of TNFSF10 by siRNA suppressed caspase 3 and 7 activity, even after treatment with the DNA-damaging chemotherapeutic agent adriamycin. TRAIL protein expression was elevated in adriamycin-treated breast cancer cells. In vivo, TRAIL expression was induced in mouse natural killer cells at 24 hours after systemic treatment with 5-Fluorouracil. p53-dependent TRAIL induction in natural killer cells after chemotherapy exposure provides a link between the tumor suppressor p53 and the host immune response during cancer therapy as well as a paracrine-mediated cell-extrinsic death response. Our findings provide new mechanistic insights into the signaling of p53-dependent cell death and tumor suppression, including the involvement of the host immune system and natural killer cells in vivo in the anti-tumor efficacy of chemotherapy.

Immune system adaptation during spaceflight is a concern in space medicine. Decreased circulating leukocytes observed during and after space flight infer suppressed immune responses and susceptibility to infection. The microgravity aspect of the space environment has been simulated on Earth to study adverse biological effects in astronauts. In this report, the hindlimb unloading (HU) model was employed to investigate the combined effects of solar particle event-like proton radiation and simulated microgravity on immune cell parameters, including lymphocyte subtype populations and activity. Lymphocytes are a type of white blood cell critical for adaptive immune responses and T lymphocytes are regulators of cell-mediated immunity, controlling the entire immune response. Mice were suspended prior to and after proton radiation exposure (0 or 2 Gy doses) and total leukocyte numbers and splenic lymphocyte functionality were evaluated on days 4 or 21 after combined HU and radiation exposure. Total white blood cell (WBC), lymphocyte, neutrophil and monocyte counts are reduced by ∼65, 70, 55 and 70%, respectively, compared with the non-treated control group 4 days after combined exposure. Splenic lymphocyte subpopulations are altered at both time points investigated. At 21 days post-exposure to combined HU and proton radiation, T-cell activation and proliferation were assessed in isolated lymphocytes. Cell surface expression of the Early Activation Marker, CD69, is decreased by 30% in the combined treatment group, compared with the non-treated control group and cell proliferation was suppressed by ∼50%, compared with the non-treated control group. These findings reveal that the combined stressors (HU and proton radiation exposure) induce decreased leukocyte numbers and function, contributing to immune system dysfunction in crew members. This research was supported by the NIH Training Grant 2T32CAO9677 and the National Space Biomedical Research Institute (NSBRI) Center of Acute Radiation Research (CARR) grant. The NSBRI is funded through the National Aeronautics and Space Administration (NASA) Class Code (NCC) 9-58. The authors declare that this work has been published: Sanzari JK, Romero-Weaver AL, James G, Krigsfeld G, Lin L, Diffenderfer ES, Kennedy AR. Leukocyte activity is altered in a ground based murine model of microgravity and proton radiation exposure. PLoS One. 2013;8(8):e71757. doi:10.1371/journal.pone.0071757.

TNF-related apoptosis-inducing ligand (TRAIL; Apo2L) is an endogenous protein that induces tumor-selective apoptosis and is currently being tested in clinical trials as an anti-tumor agent. Despite promising preclinical activity, recombinant human TRAIL suffers from efficacy-limiting properties such as short serum half-life, stability, cost, and limitations in delivery, e.g. to the central nervous system. We hypothesized that induction of the human TRAIL gene by small molecules may overcome these limitations of TRAIL-based protein therapy. A cell-based luciferase reporter screen using the human TRAIL promoter identified TIC10 as a p53-independent small molecule inducer of the TRAIL gene. TIC10 induces TRAIL on the surface of normal and tumor cells, in sera of non-tumor-bearing mice, and throughout the tumor microenvironment. Co-culture experiments suggest that TRAIL upregulation by normal cells can contribute to the anti-tumor response of TIC10 through a TRAIL-mediated bystander effect. TIC10-induced TRAIL causes tumor-specific and TRAIL-mediated cell death as indicated by inhibition of cytotoxic effects following TRAIL gene knockdown in vitro and in vivo. Expression profiling of TIC10-induced transcriptional changes revealed altered expression levels of Foxo3a target genes, including DR5. We found that TIC10 causes the nuclear translocation of Foxo3a, but not Foxo1, to upregulate TRAIL gene transcription as determined by chromatin-immunoprecipitation assays. Western blot analysis indicated that TIC10 potently inhibits the phosphorylation of Foxo3a at Ser253 and Ser294 that normally docks the transcription factor to 14-3-3 proteins in the cytosol. Dual inhibition of Akt and ERK by TIC10, by other individual small molecule kinase inhibitors in combination or targeted siRNA inhibited the phosphorylation of these critical serine residues and synergistically induced Foxo3a-dependent TRAIL and TRAIL-mediated cell death. TIC10 is a promising first-in-class anti-tumor agent with a broad range of anti-tumor effects in preclinical animal models including othrotopic GBM, immunocompetent lymphoma, xenografted colon, lung, or breast cancers, and exhibits synergy with taxanes as well as bevacizumab among other agents. TIC10 appears to have no measurable toxic effects in mice including evaluation of serum chemistries, liver function studies, LDH, tissue histology, weight of the animals or behavioral changes. We are pursuing the clinical translation of these important findings that indicate a favorable and wide therapeutic index for a novel anti-tumor agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1935. doi:1538-7445.AM2012-1935

TRAIL is an attractive anti-neoplastic agent due to its minimal toxicity and its potent ability to induce apoptosis in cancer cells. Clinical trials with intravenous administration of recombinant human TRAIL in cancer patients have been limited by the short serum half-life of TRAIL and molecular resistance mechanisms including loss of cell surface death receptor expression and/or pro-survival expression changes in proteins that regulate apoptosis such as Mcl-1. Although TRAIL death receptor-targeted agonistic antibodies have an increased half-life, they act on one of the two pro-apoptotic TRAIL receptors DR4 (TRAIL-R1) or DR5 (TRAIL-R2). In order to pharmacologically increase tumor and host TRAIL production as a novel anti-neoplastic mechanism, to improve the pharmacokinetic properties of TRAIL by increasing its effective half-life, and perhaps its local concentration within the tumor microenvironment, we screened for small molecules capable of inducing the endogenous TRAIL gene. A cell-based bioluminescence reporter screen was conducted in TRAIL-resistant Bax-null human HCT116 colon cancer cells using the TRAIL gene promoter yielded TRAIL-inducing compound 10 (TIC10). TIC10 induces TRAIL at the message, membrane-bound, and soluble levels in a time- and dose-dependent manner in TRAIL-sensitive and TRAIL-resistant human cancer cell lines as well as normal human fibroblasts without any effect on cell cycle profile. TIC10 induces cell death in a number of cancer cell lines without evidence of cell cycle arrest, a characteristic of HDAC inhibitors that are the only other pharmacological agent currently shown to upregulate TRAIL. Lack of structural homology with HDAC inhibitors along with this observation suggest a novel mechanism of TRAIL-upregulation. Western blot analysis revealed that TIC10 sensitizes to TRAIL-mediated apoptosis by tumor cell-specific upregulation of DR5 and downregulation of Mcl-1. Human xenograft experiments demonstrate that TIC10 is highly effective as a multi- or single-dose agent with similar efficacy to TRAIL in TRAIL-sensitive xenografts. Some TRAIL-resistant xenografts such as RKO demonstrate TIC10 superiority to TRAIL with no incidence of toxicity. TIC10 causes highly increased protein levels of TRAIL and cleaved caspase-8, increased TUNEL staining indicating apoptosis, in addition to striking decrease in tumor cell density by histology in tumors of human xenografts in mice. Mice lacking tumors demonstrate an upregulation of TRAIL protein levels in serum. The effect of TIC10 on normal cell production of TRAIL provides a novel mechanism for killing tumor cells through a bystander effect in the tumor microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4502. doi:10.1158/1538-7445.AM2011-4502

TNF-related apoptosis-inducing ligand (TRAIL) has been hailed as a promising cancer drug target due to its apoptosis-inducing capabilities which are aimed selectively at cancer cells. Methods of inducing endogenous TRAIL as well as introducing exogenous recombinant human TRAIL (rhTRAIL) are being explored in preclinical studies and in the case of the latter, clinical trials. While the gene encoding for DR5, a TRAIL death receptor, has been known to be regulated by p53, we have recently shown that TRAIL is regulated by p53 at base pairs 504-1102 of its promoter (Kuribayashi et al., Cancer Biology and Therapy. 7:2034-2038, 2008). p53 is mutated in over half of all cancers; therefore, a method of upregulating endogenous TRAIL via a mechanism independent of p53 holds great potential therapeutic value for cancer. We searched for small molecule p53-independent upregulators of TRAIL using a luciferase reporter assay which utilized a plasmid with the gene encoding for firefly luciferase under the transcriptional control of the first 504 base pairs of the human TRAIL promoter. Screening with the NCI Diversity Set II library yielded several hits with >2-fold TRAIL induction which were repeated and further analyzed for use as a drug and to gain mechanistic insight. Interestingly, one hit from this screen was breflate, the prodrug of Brefeldin A (BFA). BFA inhibits retrograde protein transport from the Golgi apparatus to the endoplasmic reticulum and is currently being investigated as a cancer therapeutic. Shao et al. (Exp. Cell Res. 227:190-196, 1996) have previously reported that BFA is a potent inducer of apoptosis in some cancer cell lines independently of p53, though the mechanistic relationship was not clear. Breflate clearly increased intracellular levels of TRAIL in HCT116 Bax−/− cells as determined by Western blot analysis, and in addition to the original reporter assay this is being corroborated by reverse transcriptase polymerase chain reaction (RT-PCR). Moreover, sub-G1 analysis found that breflate induced levels of apoptosis >2-fold relative to control levels at 10μM after 24 hours in HCT116 p53−/− cells. Further mechanistic and ongoing in vivo studies are being conducted with breflate alone and in combination with chemotherapy or targeted cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5467.

Background We previously reported the identification of ONC201/TIC10, a novel small molecule inducer of the human TRAIL gene that improves efficacy-limiting properties of recombinant TRAIL and is in clinical trials in advanced cancers based on its promising safety and antitumor efficacy in several preclinical models. Methods We performed a high throughput luciferase reporter screen using the NCI Diversity Set II to identify TRAIL-inducing compounds. Results Small molecule-mediated induction of TRAIL reporter activity was relatively modest and the majority of the hit compounds induced low levels of TRAIL upregulation. Among the candidate TRAIL-inducing compounds, TIC9 and ONC201/TIC10 induced sustained TRAIL upregulation and apoptosis in tumor cells in vitro and in vivo. However, ONC201/TIC10 potentiated tumor cell death while sparing normal cells, unlike TIC9, and lacked genotoxicity in normal fibroblasts. Investigating the effects of TRAIL-inducing compounds on cell signaling pathways revealed that TIC9 and ONC201/TIC10, which are the most potent inducers of cell death, exclusively activate Foxo3a through inactivation of Akt/ERK to upregulate TRAIL and its pro-apoptotic death receptor DR5. Conclusion These studies reveal the selective activity of ONC201/TIC10 that led to its selection as a lead compound for this novel class of antitumor agents and suggest that ONC201/TIC10 is a unique inducer of the TRAIL pathway through its concomitant regulation of the TRAIL ligand and its death receptor DR5.