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Purpose: This study examined the feasibility, efficacy (abscopal effect), and immune effects of TGFβ blockade during radiotherapy in metastatic breast cancer patients.Experimental Design: Prospective randomized trial comparing two doses of TGFβ blocking antibody fresolimumab. Metastatic breast cancer patients with at least three distinct metastatic sites whose tumor had progressed after at least one line of therapy were randomized to receive 1 or 10 mg/kg of fresolimumab, every 3 weeks for five cycles, with focal radiotherapy to a metastatic site at week 1 (three doses of 7.5 Gy), that could be repeated to a second lesion at week 7. Research bloods were drawn at baseline, week 2, 5, and 15 to isolate PBMCs, plasma, and serum.Results: Twenty-three patients were randomized, median age 57 (range 35–77). Seven grade 3/4 adverse events occurred in 5 of 11 patients in the 1 mg/kg arm and in 2 of 12 patients in the 10 mg/kg arm, respectively. Response was limited to three stable disease. At a median follow up of 12 months, 20 of 23 patients are deceased. Patients receiving the 10 mg/kg had a significantly higher median overall survival than those receiving 1 mg/kg fresolimumab dose [hazard ratio: 2.73 with 95% confidence interval (CI), 1.02–7.30; P = 0.039]. The higher dose correlated with improved peripheral blood mononuclear cell counts and a striking boost in the CD8 central memory pool.Conclusions: TGFβ blockade during radiotherapy was feasible and well tolerated. Patients receiving the higher fresolimumab dose had a favorable systemic immune response and experienced longer median overall survival than the lower dose group. Clin Cancer Res; 24(11); 2493–504. ©2018 AACR.

Supplementary Table S1. Antibodies and dextramers used for flow cytometric analysis Supplementary Table S2. Patients with one or more treatment positively-related (maximum grade possible, probable, definite) AEs by treatment arm Supplementary Table S3. Levels of circulating tumor-specific CD8+ T cells with reactivity for JARID1B, Muc1 or Her2 in breast cancer patients with HLA-A*0201 status Supplementary Table S4. Humoral immune responses were not significantly affected following TGFβ blockade and radiation Supplementary Figure S1: Study schema Supplementary Figure S2. Gating strategy in panel 1. Supplementary Figure S3. Gating strategy in panel 2. Supplementary Figure S4. Changes in PBMC, survivin- and JARID1B-reactive CD8 T cells in response to TGFβ blockade and radiation. Supplementary Figure S5. Diametrically opposing dynamics in the myeloid suppressor compartment versus the suppressor T cell pool in response to TGFβ blockade and radiation. Supplementary Figure S6. Changes in plasma levels of tryptophan and kynurenine in response to TGFβ blockade and radiation.

Acute radiation exposure of the thorax can lead to late serious, and even life-threatening, pulmonary and cardiac damage. Sporadic in nature, late complications tend to be difficult to predict, which prompted this investigation into identifying non-invasive, tissue-specific biomarkers for the early detection of late radiation injury. Levels of circulating microRNA (miRNA) were measured in C3H and C57Bl/6 mice after whole thorax irradiation at doses yielding approximately 70% mortality in 120 or 180 days, respectively (LD70/120 or 180). Within the first two weeks after exposure, weight gain slowed compared to sham treated mice along with a temporary drop in white blood cell counts. 52% of C3H (33 of 64) and 72% of C57Bl/6 (46 of 64) irradiated mice died due to late radiation injury. Lung and heart damage, as assessed by computed tomography (CT) and histology at 150 (C3H mice) and 180 (C57Bl/6 mice) days, correlated well with the appearance of a local, miRNA signature in the lung and heart tissue of irradiated animals, consistent with inherent differences in the C3H and C57Bl/6 strains in their propensity for developing radiation-induced pneumonitis or fibrosis, respectively. Radiation-induced changes in the circulating miRNA profile were most prominent within the first 30 days after exposure and included miRNA known to regulate inflammation and fibrosis. Importantly, early changes in plasma miRNA expression predicted survival with reasonable accuracy (88-92%). The miRNA signature that predicted survival in C3H mice, including miR-34a-5p, -100-5p, and -150-5p, were associated with pro-inflammatory NF-κB-mediated signaling pathways, whereas the signature identified in C57Bl/6 mice (miR-34b-3p, -96-5p, and -802-5p) was associated with TGF-β/SMAD signaling. This study supports the hypothesis that plasma miRNA profiles could be used to identify individuals at high risk of organ-specific late radiation damage, with applications for radiation oncology clinical practice or in the context of a radiological incident.

Purpose: This study examined the feasibility, efficacy (abscopal effect), and immune effects of TGFβ blockade during radiotherapy in metastatic breast cancer patients. Experimental Design: Prospective randomized trial comparing two doses of TGFβ blocking antibody fresolimumab. Metastatic breast cancer patients with at least three distinct metastatic sites whose tumor had progressed after at least one line of therapy were randomized to receive 1 or 10 mg/kg of fresolimumab, every 3 weeks for five cycles, with focal radiotherapy to a metastatic site at week 1 (three doses of 7.5 Gy), that could be repeated to a second lesion at week 7. Research bloods were drawn at baseline, week 2, 5, and 15 to isolate PBMCs, plasma, and serum. Results: Twenty-three patients were randomized, median age 57 (range 35–77). Seven grade 3/4 adverse events occurred in 5 of 11 patients in the 1 mg/kg arm and in 2 of 12 patients in the 10 mg/kg arm, respectively. Response was limited to three stable disease. At a median follow up of 12 months, 20 of 23 patients are deceased. Patients receiving the 10 mg/kg had a significantly higher median overall survival than those receiving 1 mg/kg fresolimumab dose [hazard ratio: 2.73 with 95% confidence interval (CI), 1.02–7.30; P = 0.039]. The higher dose correlated with improved peripheral blood mononuclear cell counts and a striking boost in the CD8 central memory pool. Conclusions: TGFβ blockade during radiotherapy was feasible and well tolerated. Patients receiving the higher fresolimumab dose had a favorable systemic immune response and experienced longer median overall survival than the lower dose group. Clin Cancer Res; 24(11); 2493–504. ©2018 AACR.

The ability to recognize and respond to universal molecular patterns on invading microorganisms allows our immune system to stay on high alert, sensing danger to our self-integrity. Our own damaged cells and tissues in pathological situations activate similar warning systems as microbes. In this way, the body is able to mount a response that is appropriate to the danger. Toll-like receptors are at the heart of this pattern recognition system that initiates innate pro-oxidant, pro-inflammatory signaling cascades and ultimately bridges recognition of danger to adaptive immunity. The acute inflammatory lesions that are formed segue into resolution of inflammation, repair and healing or, more dysfunctionally, into chronic inflammation, autoimmunity, excessive tissue damage and carcinogenesis. Redox is at the nexus of this decision making process and is the point at which ionizing radiation initially intercepts to trigger similar responses to self-damage. In this review we discuss our current understanding of how radiation-damaged cells interact with Toll-like receptors and how the immune systems interprets these radiation-induced danger signals in the context of whole-body exposures and during local tumor irradiation.

The transcription factor NF-E2-related factor 2 (Nrf2) binds the antioxidant DNA response element (ARE) to activate important cellular cytoprotective defense systems. Recently several types of cancers have been shown to overexpress Nrf2, but its role in the cellular response to radiation therapy has yet to be fully determined. In this study, we report that single doses of ionizing radiation from 2 to 8 Gy activate ARE-dependent transcription in breast cancer cells in a dose-dependent manner, but only after a delay of five days. Clinically relevant daily dose fractions of radiation also increased ARE-dependent transcription, but again only after five days. Downstream activation of Nrf2-ARE-dependent gene and protein markers, such as heme oxygenase-1, occurred, whereas Nrf2-deficient fibroblasts were incapable of these responses. Compared with wild-type fibroblasts, Nrf2-deficient fibroblasts had relatively high basal levels of reactive oxygen species that increased greatly five days after radiation exposure. Further, in vitro clonogenic survival assays and in vivo sublethal whole body irradiation tests showed that Nrf2 deletion increased radiation sensitivity, whereas Nrf2-inducing drugs did not increase radioresistance. Our results indicate that the Nrf2-ARE pathway is important to maintain resistance to irradiation, but that it operates as a second-tier antioxidant adaptive response system activated by radiation only under specific circumstances, including those that may be highly relevant to tumor response during standard clinical dose-fractionated radiation therapy. Cancer Res; 70(21); 8886–95. ©2010 AACR.

A small library of monovalent and bivalent Smac mimics was synthesized based on 2 types of monomers, with general structure NMeAla-Xaa-Pro-BHA (Xaa=Cys or Lys). Position 2 of the compounds was utilized to dimerize both types of monomers employing various bis-reactive linkers, as well as to modify selected compounds with lipids. The resulting library was screened in vitro against metastatic human breast cancer cell line MDA-MB-231, and the two most active compounds selected for in vivo studies. The most active lipid-conjugated analogue M11, showed in vivo activity while administered both subcutaneously and orally. Collectively, our findings suggest that lipidation may be a viable approach in the development of new Smac-based therapeutic leads.

Our ability to use ionizing radiation as an energy source, as a therapeutic agent, and, unfortunately, as a weapon, has evolved tremendously over the past 120 years, yet our tool box to handle the consequences of accidental and unwanted radiation exposure remains very limited. We have identified a novel group of small molecule compounds with a 4-nitrophenylsulfonamide (NPS) backbone in common that dramatically decrease mortality from the hematopoietic acute radiation syndrome (hARS). The group emerged from an in vitro high throughput screen (HTS) for inhibitors of radiation-induced apoptosis. The lead compound also mitigates against death after local abdominal irradiation and after local thoracic irradiation (LTI) in models of subacute radiation pneumonitis and late radiation fibrosis. Mitigation of hARS is through activation of radiation-induced CD11b+Ly6G+Ly6C+ immature myeloid cells. This is consistent with the notion that myeloerythroid-restricted progenitors protect against WBI-induced lethality and extends the possible involvement of the myeloid lineage in radiation effects. The lead compound was active if given to mice before or after WBI and had some anti-tumor action, suggesting that these compounds may find broader applications to cancer radiation therapy.

The immune system has the power to modulate the expression of radiation-induced normal and tumor tissue damage. On the one hand, it can contribute to cancer cure, and on the other hand, it can influence acute and late radiation side effects, which in many ways resemble acute and chronic inflammatory disease states. The way radiation-induced inflammation feeds into adaptive antigen-specific immune responses adds another dimension to the tumor-host cross talk during radiation therapy and to possible radiation-driven autoimmune responses. Understanding how radiation affects inflammation and immunity is therefore critical if we are to effectively manipulate these forces for benefit in radiation oncology treatments.

Purpose: This is a pilot study combining focal irradiation and systemic TGFβ blockade in metastatic breast cancer. The rationale for using TGFβ blockade was to limit tumor growth and further spread as well as to curb systemic immune suppression. Combining this systemic approach with hypofractionated radiation of selective tumor metastasis aimed at vaccinating each patient in vivo with her own, relevant tumor antigens by local radiation damage and allow T cells to reach their full potential while escaping TGFβ's grip. Experimental Design: Serial blood samples from 22 patients undergoing treatment with 1mg or 10mg Fresolimumab and Radiation at the New York University School of Medicine (n=15) and at the David Geffen School of Medicine, University of California, Los Angeles (n=7) were immunophenotyped based on flow cytometric analysis of 21 surface antigens. Results: There were significant differences between the 1mg and 10mg groups with respect to several immune parameters, especially the rise in circulating central memory CD8s at the higher dose level (relative increase at 2 weeks 10mg vs 1mg, p=0.027). Regulatory networks responded to the 10mg treatment regime with a consistent expansion in CD4 Tregs while mMDSCs declined (ratio Tregs/mMDSC rising in 10mg vs 1mg, p=0.026). An overall survival benefit was seen in the 10mg Fresolimumab arm (median OS 64.1 weeks versus 20 weeks, p=0.015 log rank test) albeit not striking. CART analysis allowed accurate survival classification based on 2-week changes in CD8/mMDSC ratios and plasma Tryptophan (ROC AUC 0.979). Conclusion: Inhibiting TGFβ in the context of focal irradiation seems to create a favorable systemic immune landscape that drives T cell memory differentiation while limiting myeloid suppression. Citation Format: Dörthe Schaue, Michael W. Xie, Josephine A. Ratikan, Ewa D. Micewicz, Lin Hwang, Kym F. Faull, James W. Sayre, Percy P. Lee, John A. Glaspy, Sandra Demaria, Silvia C. Formenti, William H. McBride. Radiation and TGFβ blockade bring back memories in metastatic breast cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B86.

Purpose This study used chloroquine to direct radiation-induced tumor cell death pathways to harness the antitumor activity of the immune system. Methods and Materials Chloroquine given immediately after tumor irradiation increased the cure rate of MCaK breast cancer in C3H mice. Chloroquine blocked radiation-induced autophagy and drove MCaK cells into a more rapid apoptotic and more immunogenic form of cell death. Results Chloroquine treatment made irradiated tumor vaccines superior at inducing strong interferon gamma-associated immune responses in vivo and protecting mice from further tumor challenge. In vitro, chloroquine slowed antigen uptake and degradation by dendritic cells, although T-cell stimulation was unaffected. Conclusions This study illustrates a novel approach to improve the efficacy of breast cancer radiation therapy by blocking endosomal pathways, which enhances radiation-induced cell death within the field and drives antitumor immunity to assist therapeutic cure. The study illuminates and merges seemingly disparate concepts regarding the importance of autophagy in cancer therapy.

Cells often autofluoresce in response to UV radiation excitation and this can reflect critical aspects of cellular metabolism. Here we report that many different human and murine cell types respond to ionizing radiation with a striking rise in autofluorescence that is dependent on dose and time. There was a highly reproducible fluorescent shift at various wavelengths, which was mirrored by an equally reproducible rise in the vital intracellular metabolic co-factors FAD and NADH. It appears that mitochondria, metabolism and Ca(2+) homeostasis are important for this to occur as cells without mitochondria or cells unable to alter calcium levels did not behave in this way. We believe these radiation-induced changes are of biological importance and that autofluorescence may even provide us with a tool to monitor radiation responses in the clinic.

Background: Squamous cell carcinoma of the head and neck (SCCHN) is a radiation-sensitive disease. Standard frontline treatment of locally advanced SCCHN entails concurrent chemotherapy with radiation. Treatment of relapsed or metastatic SCCHN often utilizes re-irradiation to the primary tumor for local control. However, there is no standard systemic treatment for relapsed or metastatic SCCHN in combination with radiation. Many SCCHN tumors display alterations in expression of DNA repair genes. To this end, we evaluated the effect of talazoparib (BMN-673), a potent poly-ADP ribose polymerase (PARP) inhibitor, alone or in combination with radiation, in vitro and in vivo. Methods: The 50% inhibitory concentration (IC50) of talazoparib was determined in 30 SCCHN cell lines in vitro. Using clonogenic assays, the effects of 0, 10 or 100nM talazoparib, alone or in combination with a single fraction of 4Gray radiation were determined in four cell lines (two sensitive and two resistant to talazoparib). Effects on DNA damage of talazoparib, alone or in combination with radiation were also determined by flow cytometric analysis of Gamma-H2A.X. Finally, the FaDu head and neck cells were implanted onto female J/nude. After tumors were established, mice were treated with talazoparib for 5 days and a total of 20 Gray radiation, alone or in combination, and effects on body weight and tumor size were determined. Results: Among 30 SCCHN cell lines treated with talazoparib alone, 18 displayed sensitivity with 50% inhibitory concentration (IC50) of 1μM or less. In clonogenic assays, addition of radiation to BMN673 in two sensitive (UMSCC-6 and UMSCC-38) and two resistant (UMSCC-5 and UMSCC-12) cell lines resulted in synergistic cytotoxicity at doses ranging from 0-100nM BMN673. Increased gamma-H2A.X was seen when cell lines were treated with talazoparib, radiation, or the combination, compared to controls. Combining radiation with talazoparib resulted in a transient decrease in body weight in J/nude mice, but resulted in statistically significant and prolonged decrease in tumor volume. These anti-tumor effects were sustained out to 60 days post inoculation. Conclusions: The PARP inhibitor talazoparib with radiation synergistically inhibited tumor growth in vitro and in vivo in SCCHN cell lines. Further evaluation of this combination is underway. Citation Format: Deborah JL Wong, David D. Shin, Josephine Ratikan, Meenal Chalukya, Kanthinh Manivong, Leonard Post, Dörthe Schaue, Richard S. Finn, Yuqiao (Jerry) Shen, William McBride, Dennis J. Slamon. Potent anti-tumor activity of talazoparib (BMN673) in combination with radiation for squamous cell carcinoma of the head and neck. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1795. doi:10.1158/1538-7445.AM2015-1795

Non-genotoxic small molecules of low systemic toxicity that can potentiate the effects of radiation could be valuable adjuvant therapeutics for solid tumors. Pyrrole-imidazole (Py-Im) polyamides are synthetic oligomers that bind the minor groove of DNA non-covalently with programmable sequence recognition and high affinity. We reported that a polyamide targeted to the DNA sequence 5’-WGWWCW-3’ (W = A/T) suppressed growth of prostate tumor xenografts. In cell culture, this polyamide interferes with RNA Polymerase II activity and results in a dose-dependent depletion of the RNA Polymerase II large subunit. We hypothesized that high-affinity DNA binding Py-Im polyamides may enhance the effects of ionizing radiation.

Background and purpose: Current treatment of head and neck cancer generally involves a combination of surgery, radiation and chemotherapy. Clinical outcomes for many patients remain suboptimal, and novel therapeutic strategies are needed. Targeted inhibition of poly(ADP-Ribose) polymerase (PARP), a key DNA repair enzyme, has been actively investigated in cancer medicine. BMN 673 is a potent orally active inhibitor of PARP, currently in phase I clinical trials in solid and hematologic malignancies. We hypothesized BMN 673 might have single agent activity in a subset of head and neck cancers and could significantly augment the effects of radiation in this disease. To test this strategy, we performed preclinical studies to evaluate single agent activity of BMN 673 in a large panel of human head and neck cell lines. We also aimed to define interactions between BMN 673 and radiation in these models. Materials and methods: Human head and neck cell lines (N=32) were treated with single-agent BMN 673 for 5 days, and in vitro growth inhibition assays were performed. Further, four cell lines were studied in colony formation assays by seeding and treating them with BMN 673 at 1, 10, or 100 nM (or vehicle) for 24 hours, followed by mock or 4 Gy irradiation (300kV, 10mA). Cells were incubated for 14 or 20 days and stained with crystal violet for colony enumeration. Each experiment was performed in triplicates. Survival fraction was calculated: (number of colonies for treated cells/number of cells plated)/(number of colonies for corresponding control/number of cells plated). Results: A gradation of sensitivity was observed with BMN 673 monotherapy: IC 50 ranges from 0.1 to 10 μM. Radiation alone reduced colony forming ability of the cells by 26% in UMSCC-5 and by approximately 65% in other three cell lines (UMSCC-6, 12, and 38). These cells exhibited enhanced cytotoxicity with addition of BMN 673 to radiation in a dose-dependent manner. Addition of 100nM of BMN 673 resulted in 80% reduction in the number of surviving colonies in the relatively radio-resistant UMSCC-5 and in 88-100% reduction in other cell lines. All changes were statistically significant. Conclusions: In vitro assays demonstrate human head and neck cancer cells exhibit relative growth sensitivity to BMN 673 monotherapy. In addition, radiation-induced cytotoxicity is augmented by the addition of BMN 673 to radiation in a dose-dependent manner. Notably, one relatively radio-resistant line was rendered radio-sensitive with the addition of BMN 673 to radiation. These data support a role of PARP inhibition as monotherapy and/or in combination with radiotherapy, representing a potential novel translational therapeutic strategy in head and neck cancers that warrant clinical testing. Citation Format: David D. Shin, Josephine Ratikan, Kanthinh Manivong, Meenal Chalukya, Leonard Post, Yuqiao (Jerry) Shen, William McBride, Dörthe Schaue, Dennis J. Slamon, Richard S. Finn. The poly(ADP-ribose) polymerase inhibitor BMN 673 has single agent activity and augments cytotoxicity of radiation in human head and neck tumor cell line in vitro: a novel strategy for radiosensitization in head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1595. doi:10.1158/1538-7445.AM2013-1595