Christopher A. Barker

Radiation-Induced Changes in Gene Expression Involve Recruitment of Existing Messenger RNAs to and away from Polysomes

Although ionizing radiation has been shown to influence gene transcription, little is known about the effects of radiation on gene translational efficiency. To obtain a genome-wide perspective of the effects of radiation on gene translation, microarray analysis was done on polysome-bound RNA isolated from irradiated human brain tumor cells; to allow for a comparison with the effects of radiation on transcription, microarray analysis was also done using total RNA. The number of genes whose translational activity was modified by radiation was approximately 10-fold greater than those whose transcription was affected. The radiation-induced change in a genes translational activity was shown to involve the recruitment of existing mRNAs to and away from polysomes. Moreover, the change in a genes translational activity after irradiation correlated with changes in the level of its corresponding protein. These data suggest that radiation modifies gene expression primarily at the level of translation. In contrast to transcriptional changes, there was considerable overlap in the genes affected at the translational level among brain tumor cell lines and normal astrocytes. Thus, the radiation-induced translational control of a subset of mRNAs seems to be a fundamental component of cellular radioresponse.

The ipsilateral silent period in boys with attention-deficit/hyperactivity disorder

OBJECTIVE Characterize maturation of transcallosal inhibition (ipsilateral silent period [iSP]) in attention deficit/hyperactivity disorder (ADHD) using transcranial magnetic stimulation (TMS). BACKGROUND Maturation of the iSP is related to acquisition of fine motor skills in typically developing children suggesting that dexterous fine motor skills depend upon mature interhemispheric interactions. Since neuromotor maturation is abnormal in boys with ADHD we hypothesized that iSP maturation in these children would be abnormal. We studied iSP maturation in 12 boys with ADHD and 12 age-matched, typically developing boys, 7-13 years of age. METHODS Surface electromyographic activity was recorded from right first dorsal interosseus (FDI). During background activation, focal TMS was delivered at maximal stimulator output over the ipsilateral motor cortex. RESULTS Maturation of finger speed in boys with ADHD was significantly slower than that in the control group. The iSP latency decreased with age in the control group but not in the ADHD group. CONCLUSIONS These findings suggest the presence of a complex relationship between abnormalities of certain interhemispheric interactions (as represented by iSP latency) and delayed maturation of neuromotor skills in boys with ADHD. SIGNIFICANCE These data provide preliminary physiologic evidence supporting delayed or abnormal development of interhemispheric interactions in boys with ADHD.

In vitro and In vivo Radiosensitization Induced by the Ribonucleotide Reductase Inhibitor Triapine (3-Aminopyridine-2-Carboxaldehyde-Thiosemicarbazone)

Purpose: Because ribonucleotide reductase (RR) plays a role in DNA repair, it may serve as a molecular target for radiosensitization. Unlike previously investigated RR inhibitors, Triapine potently inhibits both RR holoenzymes. Therefore, the effects of Triapine on tumor cell radiosensitivity were investigated. Experimental Design: The effects of Triapine on the in vitro radiosensitivity of three human tumor cell lines and one normal cell line were evaluated using a clonogenic assay. Growth delay was used to evaluate the effects of Triapine on in vivo tumor radiosensitivity. The levels of the RR subunits were determined using immunoblot analysis and DNA damage and repair were evaluated using γH2AX foci. Results: Exposure of the tumor cell lines to Triapine before or immediately after irradiation resulted in an increase in radiosensitivity. In contrast, Triapine enhanced the radiosensitivity of the normal fibroblast cell line only when the exposure was before irradiation. There were no consistent differences between cell lines with respect to the expression of the RR subunits. Whereas Triapine had no effect on radiation-induced γH2AX foci at 1 hour, the number of γH2AX foci per cell was significantly greater in the Triapine-treated cells at 24 hours after irradiation, suggesting the presence of unrepaired DNA damage. Triapine administration to mice bearing tumor xenografts immediately after irradiation resulted in a greater than additive increase in radiation-induced tumor growth delay. Conclusions: These results indicate that Triapine can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair.

Abstract 574: Phosphatidylserine targeting antibody in combination with tumor radiation and immune checkpoint blockade promotes anti-tumor activity in mouse B16 melanoma

Phosphatidylserine (PS) is a phospholipid that is exposed on surface of apoptotic cells, tumor cells and tumor endothelium. PS has been shown to promote immunosuppressive signals in the tumor microenvironment. Antibodies that target PS have been shown to reactivate anti-tumor immunity by polarizing tumor associated macrophages into a pro-inflammatory M1 phenotype, reducing the number of MDSCs in tumors and promoting the maturation of dendritic cells into functional APCs. In a mouse B16 melanoma model, targeting PS in combination with immune checkpoint blockade promoted greater anti-tumor activity than either agent alone. This combination was shown to enhance CD4+ and CD8+ T cell infiltration and activation in the tumors of treated animals. Radiation therapy (RT) is an effective focal treatment of primary solid tumors, but is less effective in treating metastatic solid tumors as a monotherapy. There is evidence that RT induces immunogenic tumor cell death and enhances tumor-specific T cell infiltration in treated tumors. The abscopal effect, a phenomenon in which tumor regression occurs outside the site of RT, has been observed in both preclinical and clinical trials when RT is combined with immunotherapy. In this study, we show that irradiation treatment of B16 melanoma causes an increase in PS expression on the surface of viable tumor and immune infiltrates. We subsequently examined the effects of combining RT with an antibody that targets PS (mch1N11) and immune checkpoint blockade (anti-PD-1) in B16 melanoma. We found that treatment with mch1N11 synergizes with RT to improve anti-tumor activity and overall survival in tumor bearing mice. In addition, the triple combination of mch1N11, RT and anti-PD-1 treatment displayed even greater anti-tumor and survival benefit. Analysis of local immune responses in the tumors of treated animals revealed an increase in tumor-associated macrophages with a shift towards a pro-inflammatory M1 phenotype after treatment with RT and mch1N11. In addition, analysis of the systemic immune responses in the spleen and tumor draining lymph nodes revealed an increase in CD8 T cell activation, effector cytokine production and differentiation into effector memory cells in the triple combination. This finding highlights the potential of combining these three agents to improve outcome in patients with advanced-stage melanoma and other cancers and may inform the design of clinical studies combining PS-targeting antibodies with RT and/or checkpoint blockade. Citation Format: Sadna Budhu, Olivier De Henau, Roberta Zappasodi, Rachel Giese, Luis F. Campesato, Christopher Barker, Bruce Freimark, Jeff Hutchins, Jedd D. Wolchok, Taha Merghoub. Phosphatidylserine targeting antibody in combination with tumor radiation and immune checkpoint blockade promotes anti-tumor activity in mouse B16 melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 574. doi:10.1158/1538-7445.AM2017-574

Abstract 4705: CTLA4 blockade with HER2-directed therapy (H) yields clinical benefit in women undergoing radiation therapy (RT) for HER2-positive (HER2+) breast cancer brain metastases (BCBM)

Introduction: BCBM is a devastating complication of breast cancer and women with specific subtypes (i.e. HER2+ disease) are at increased risk. Conventional systemic and local therapies have demonstrated limited effectiveness in preventing and treating BCBM. However, preclinical and clinical data indicate that the local effects of RT may be augmented by immune therapy and that abscopal effects (distant benefits outside of the RT field) may be achieved when combined with CTLA4-blockade. Thus, a pilot study of standard-of-care brain RT with tremelimumab, a CTLA4-directed monoclonal antibody, was undertaken to evaluate the impact on distant (non-CNS) disease control in women with HER2-normal (HER2-) disease and to confirm safety when administered with H in women with HER2+ disease. Methods: Eligible women were age ≥18y, ECOG 0-2, with radiologically confirmed BCBM of any histology requiring whole brain RT (WBRT) or stereotactic radiosurgery (SRS), and non-CNS progression of disease (PD). Twenty women with HER2- disease were enrolled in an efficacy cohort with a primary endpoint of 12-week non-CNS disease control by RECIST v1.1. Six women with HER2+ disease were enrolled in a safety cohort and received concurrent H every 3 weeks. Tremelimumab (10mg/kg) was administered within 5 days prior to or 3 days after the first fraction of RT, then monthly for 6 months, and then every 3 months. Radiographic responses by irRC criteria were explored. Results: Of the 6 women with HER2+ disease: 6 (100%) received WBRT, 1 had a non-CNS partial response (PR) at 12 weeks (-56.9% by RECIST, -86.2% by irRC) after 5 lines of palliative H-chemotherapy that was durable at 6 months; 1 had non-CNS stable disease at 12 weeks that was durable at 6 months; 2 had non-CNS PD at 12 weeks; 1 came off study for non-CNS PD prior to 12 weeks; and 1 died at 5 weeks. Among the 20 women with HER2- disease the best response was non-CNS SD (2 had SD, 9 had PD, 9 came off study due to PD and/or died prior to 12 weeks). The regimen was well tolerated with 15 grade 3 and no grade 4 attributable toxicity events reported. The most common treatment related AEs were diarrhea (12.9%), fatigue (9.7%), and colitis (6.5%). Conclusions: BCBM RT plus tremelimumab with or without H was generally well tolerated. This is a poor prognosis population with 11/26 (42%) women coming off study due to death or rapid PD within 12 weeks of initiating brain RT. Although only modest activity was observed at 12 weeks in the HER2- cohort, durable clinical benefit was observed with concurrent H in 2 women with HER2+ disease. This is the first report of clinical responses to checkpoint blockade with H in breast cancer. The contribution of an abscopal effect due to RT is not known and thus, studies of immune therapy with H +/- RT are underway. Citation Format: Heather McArthur, Kathryn Beal, Darragh Halpenny, Micaela Henrich, Shanu Modi, Sujata Patil, Robert Young, Thomas Kaley, Taha Merghoub, Christopher Barker, Phillip Wong, Nicola Hamilton, Clifford Hudis, Jedd Wolchok, Larry Norton. CTLA4 blockade with HER2-directed therapy (H) yields clinical benefit in women undergoing radiation therapy (RT) for HER2-positive (HER2+) breast cancer brain metastases (BCBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4705. doi:10.1158/1538-7445.AM2017-4705