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Summary: Type I interferon (IFN-I) and IFN-γ foster antitumor immunity by facilitating T cell responses. Paradoxically, IFNs may promote T cell exhaustion by activating immune checkpoints. The downstream regulators of these disparate responses are incompletely understood. Here, we describe how interferon regulatory factor 1 (IRF1) orchestrates these opposing effects of IFNs. IRF1 expression in tumor cells blocks Toll-like receptor- and IFN-I-dependent host antitumor immunity by preventing interferon-stimulated gene (ISG) and effector programs in immune cells. In contrast, expression of IRF1 in the host is required for antitumor immunity. Mechanistically, IRF1 binds distinctly or together with STAT1 at promoters of immunosuppressive but not immunostimulatory ISGs in tumor cells. Overexpression of programmed cell death ligand 1 (PD-L1) in Irf1−/− tumors only partially restores tumor growth, suggesting multifactorial effects of IRF1 on antitumor immunity. Thus, we identify that IRF1 expression in tumor cells opposes host IFN-I- and IRF1-dependent antitumor immunity to facilitate immune escape and tumor growth.

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Heart disease is a significant adverse event caused by radiotherapy for some cancers. Identifying the origins of radiogenic heart disease will allow therapies to be developed. Previous studies showed non-targeted effects manifest as fibrosis in the non-irradiated heart after 120 days following targeted X-irradiation of the kidneys with 10 Gy in WAG/RijCmcr rats. To demonstrate the involvement of T cells in driving pathophysiological responses in the out-of-field heart, and to characterize the timing of immune cell engagement, we created and validated a T cell knock downrat on the WAG genetic backgrou nd. Irradiation of the kidneys with 10 Gy of X-rays in wild-type rats resulted in infiltration of T cells, natural killer cells, and macrophages after 120 days, and none of these after 40 days, suggesting immune cell engagement is a late response. The radiation nephropathy and cardiac fibrosis that resulted in these animals after 120 days was significantly decreased in irradiated T cell depleted rats. We conclude that T cells function as an effector cell in communicating signals from the irradiated kidneys which cause pathologic remodeling of non-targeted heart.

Abstract Background We previously reported the results of a multicentric prospective randomized trial of chemo-refractory metastatic breast cancer patients testing the efficacy of two doses of TGFβ blockade during radiotherapy. Despite a lack of objective responses to the combination, patients who received a higher dose of TGFβ blocking antibody fresolimumab had a better overall survival when compared to those assigned to lower dose (hazard ratio of 2.73, p = 0.039). They also demonstrated an improved peripheral blood mononuclear cell (PBMC) counts and increase in the CD8 central memory pool. We performed additional analysis on residual PBMC using single cell network profiling (SCNP). Methods The original trial randomized metastatic breast cancer patients to either 1 or 10 mg/kg of fresolimumab, every 3 weeks for 5 cycles, combined with radiotherapy to a metastatic site at week 1 and 7 (22.5 Gy given in 3 doses of 7.5 Gy). Trial immune monitoring results were previously reported. In 15 patients with available residual blood samples, additional functional studies were performed, and compared with data obtained in parallel from seven healthy female donors (HD): SCNP was applied to analyze T cell receptor (TCR) modulated signaling via CD3 and CD28 crosslinking and measurement of evoked phosphorylation of AKT and ERK in CD4 and CD8 T cell subsets defined by PD-1 expression. Results At baseline, a significantly higher level of expression (p

Abstract Background Various proinflammatory cytokines can be detected within the melanoma tumor microenvironment. Interleukin 32 (IL32) is produced by T cells, NK cells and monocytes/macrophages, but also by a subset of melanoma cells. We sought to better understand the biology of IL32 in human melanoma. Methods We analyzed RNA sequencing data from 53 in-house established human melanoma cell lines and 479 melanoma tumors from The Cancer Genome Atlas dataset. We evaluated global gene expression patterns associated with IL32 expression. We also evaluated the impact of proinflammatory molecules TNFα and IFNγ on IL32 expression and dedifferentiation in melanoma cell lines in vitro. In order to study the transcriptional regulation of IL32 in these cell lines, we cloned up to 10.5 kb of the 5′ upstream region of the human IL32 gene into a luciferase reporter vector. Results A significant proportion of established human melanoma cell lines express IL32, with its expression being highly correlated with a dedifferentiation genetic signature (high AXL/low MITF). Non IL32-expressing differentiated melanoma cell lines exposed to TNFα or IFNγ can be induced to express the three predominant isoforms (α, β, γ) of IL32. Cis-acting elements within this 5′ upstream region of the human IL32 gene appear to govern both induced and constitutive gene expression. In the tumor microenvironment, IL32 expression is highly correlated with genes related to T cell infiltration, and also positively correlates with high AXL/low MITF dedifferentiated gene signature. Conclusions Expression of IL32 in human melanoma can be induced by TNFα or IFNγ and correlates with a treatment-resistant dedifferentiated genetic signature. Constitutive and induced expression are regulated, in part, by cis-acting sequences within the 5′ upstream region.

Nuclear factor erythroid 2-related factor 2 (NRF2) is recognized as a master transcription factor that regulates expression of numerous detoxifying and antioxidant cytoprotective genes. In fact, models of NRF2 deficiency indicate roles not only in redox regulation, but also in metabolism, inflammatory/autoimmune disease, cancer, and radioresistancy. Since ionizing radiation (IR) generates reactive oxygen species (ROS), it is not surprising it activates NRF2 pathways. However, unexpectedly, activation is often delayed for many days after the initial ROS burst. Here, we demonstrate that, as assayed by γ-H2AX staining, rapid DNA double strand break (DSB) formation by IR in primary mouse Nrf2–/– MEFs was not affected by loss of NRF2, and neither was DSB repair to any great extent. In spite of this, basal and IR-induced transformation was greatly enhanced, suggesting that NRF2 protects against late IR-induced genomic instability, at least in murine MEFs. Another possible IR- and NRF2-related event that could be altered is inflammation and NRF2 deficiency increased IR-induced NF-κB pro-inflammatory responses mostly late after exposure. The proclivity of NRF2 to restrain inflammation is also reflected in the reprogramming of tumor antigen-specific lymphocyte responses in mice where Nrf2 k.o. switches Th2 responses to Th1 polarity. Delayed NRF2 responses to IR may be critical for the immune transition from prooxidant inflammation to antioxidant healing as well as in driving cellular radioresistance and survival. Targeting NRF2 to reprogram immunity could be of considerable therapeutic benefit in radiation and immunotherapy.

We previously reported several vignettes on types and classes of drugs able to mitigate acute and, in at least one case, late radiation syndromes in mice. Most of these had emerged from high throughput screening (HTS) of bioactive and chemical drug libraries using ionizing radiation-induced lymphocytic apoptosis as a readout. Here we report the full analysis of the HTS screen of libraries with 85,000 small molecule chemicals that identified 220 “hits.” Most of these hits could be allocated by maximal common substructure analysis to one of 11 clusters each containing at least three active compounds. Further screening validated 23 compounds as being most active; 15 of these were cherry-picked based on drug availability and tested for their ability to mitigate acute hematopoietic radiation syndrome (H-ARS) in mice. Of these, five bore a 4-nitrophenylsulfonamide motif while 4 had a quinoline scaffold. All but two of the 15 significantly (p < 0.05) mitigated H-ARS in mice. We had previously reported that the lead 4-(nitrophenylsulfonyl)-4-phenylpiperazine compound (NPSP512), was active in mitigating multiple acute and late radiation syndromes in mice of more than one sex and strain. Unfortunately, the formulation of this drug had to be changed for regulatory reasons and we report here on the synthesis and testing of active analogs of NPSP512 (QS1 and 52A1) that have increased solubility in water and in vivo bioavailability while retaining mitigator activity against H-ARS (p < 0.0001) and other radiation syndromes. The lead quinoline 057 was also active in multiple murine models of radiation damage. Taken together, HTS of a total of 150,000 bioactive or chemical substances, combined with maximal common substructure analysis has resulted in the discovery of diverse groups of compounds that can mitigate H-ARS and at least some of which can mitigate multiple radiation syndromes when given starting 24 h after exposure. We discuss what is known about how these agents might work, and the importance of formulation and bioavailability.

Ionizing radiation interacts with the immune system in many ways with a multiplicity that mirrors the complexity of the immune system itself: namely the need to maintain a delicate balance between different compartments, cells and soluble factors that work collectively to protect, maintain, and restore tissue function in the face of severe challenges including radiation damage. The cytotoxic effects of high dose radiation are less relevant after low dose exposure, where subtle quantitative and functional effects predominate that may go unnoticed until late after exposure or after a second challenge reveals or exacerbates the effects. For example, low doses may permanently alter immune fitness and therefore accelerate immune senescence and pave the way for a wide spectrum of possible pathophysiological events, including early-onset of age-related degenerative disorders and cancer. By contrast, the so called low dose radiation therapy displays beneficial, anti-inflammatory and pain relieving properties in chronic inflammatory and degenerative diseases. In this review, epidemiological, clinical and experimental data regarding the effects of low-dose radiation on the homeostasis and functional integrity of immune cells will be discussed, as will be the role of immune-mediated mechanisms in the systemic manifestation of localized exposures such as inflammatory reactions. The central conclusion is that ionizing radiation fundamentally and durably reshapes the immune system. Further, the importance of discovery of immunological pathways for modifying radiation resilience amongst other research directions in this field is implied.

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.

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.

Glioblastoma stem cells (GSC) are a significant cell model for explaining brain tumor recurrence. However, mechanisms underlying their radiochemoresistance remain obscure. Here we show that most clonogenic cells in GSC cultures are sensitive to radiation treatment (RT) with or without temozolomide (TMZ). Only a few single cells survive treatment and regain their self-repopulating capacity. Cells re-populated from treatment-resistant GSC clones contain more clonogenic cells compared to those grown from treatment-sensitive GSC clones, and repeated treatment cycles rapidly enriched clonogenic survival. When compared to sensitive clones, resistant clones exhibited slower tumor development in animals. Upregulated genes identified in resistant clones via comparative expression microarray analysis characterized cells under metabolic stress, including blocked glucose uptake, impaired insulin/Akt signaling, enhanced lipid catabolism and oxidative stress, and suppressed growth and inflammation. Moreover, many upregulated genes highlighted maintenance and repair activities, including detoxifying lipid peroxidation products, activating lysosomal autophagy/ubiquitin-proteasome pathways, and enhancing telomere maintenance and DNA repair, closely resembling the anti-aging effects of caloric/glucose restriction (CR/GR), a nutritional intervention that is known to increase lifespan and stress resistance in model organisms. Although treatment-introduced genetic mutations were detected in resistant clones, all resistant and sensitive clones were subclassified to either proneural (PN) or mesenchymal (MES) glioblastoma subtype based on their expression profiles. Functional assays demonstrated the association of treatment resistance with energy stress, including reduced glucose uptake, fatty acid oxidation (FAO)-dependent ATP maintenance, elevated reactive oxygen species (ROS) production and autophagic activity, and increased AMPK activity and NAD(+) levels accompanied by upregulated mRNA levels of SIRT1/PGC-1α axis and DNA repair genes. These data support the view that treatment resistance may arise from quiescent GSC exhibiting a GR-like phenotype, and suggest that targeting stress response pathways of resistant GSC may provide a novel strategy in combination with standard treatment for glioblastoma.

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.

Stereotactic radiation approaches are gaining more popularity for the treatment of intracranial as well as extracranial tumors in organs such as the liver and lung. Technology, rather than biology, is driving the rapid adoption of stereotactic body radiation therapy (SBRT), also known as stereotactic ablative radiotherapy (SABR), in the clinic due to advances in precise positioning and targeting. Dramatic improvements in tumor control have been demonstrated; however, our knowledge of normal tissue biology response mechanisms to large fraction sizes is lacking. Herein, we will discuss how SABR can induce cellular expression of MHC I, adhesion molecules, costimulatory molecules, heat shock proteins, inflammatory mediators, immunomodulatory cytokines, and death receptors to enhance antitumor immune responses.