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Supplementary figures 1-4, supplementary table 1

Supplementary Table S1 and 6 Supplementary Figures, S1-6. Table S1 - Patient samples from the PredictR-HNC study. Figure S1 - CCT244747 and paclitaxel IC50 determination and analysis of CCT244747 combination therapies. Figure S2 - Investigation of triple therapies using low concentrations of CCT244747 and paclitaxel. Figure S3 - Cell morphology 48 hours after treatment. HN5 cells exhibit an early increase in the S phase population. Figure S4 - High CHK1 and p-CHK1 cytoplasmic staining correlate with recurring HPV+ HNSCC tumours. Figure S5 - High CHK1, p-CHK1 and p-ATM expression correlate with recurring tumours. Figure S6 - The HPV+ recurring cell line SCC090 is radioresistant, exhibits strong CHK1 activation after exposure to radiation, and is highly sensitive to triple therapy.

Purpose:ATR inhibitors (ATRi) are in early phase clinical trials and have been shown to sensitize to chemotherapy and radiotherapy preclinically. Limited data have been published about the effect of these drugs on the tumor microenvironment.Experimental Design: We used an immunocompetent mouse model of HPV-driven malignancies to investigate the ATR inhibitor AZD6738 in combination with fractionated radiation (RT). Gene expression analysis and flow cytometry were performed posttherapy.Results:Significant radiosensitization to RT by ATRi was observed alongside a marked increase in immune cell infiltration. We identified increased numbers of CD3+ and NK cells, but most of this infiltrate was composed of myeloid cells. ATRi plus radiation produced a gene expression signature matching a type I/II IFN response, with upregulation of genes playing a role in nucleic acid sensing. Increased MHC I levels were observed on tumor cells, with transcript-level data indicating increased antigen processing and presentation within the tumor. Significant modulation of cytokine gene expression (particularly CCL2, CCL5, and CXCL10) was found in vivo, with in vitro data indicating CCL3, CCL5, and CXCL10 are produced from tumor cells after ATRi + RT.Conclusions:We show that DNA damage by ATRi and RT leads to an IFN response through activation of nucleic acid–sensing pathways. This triggers increased antigen presentation and innate immune cell infiltration. Further understanding of the effect of this combination on the immune response may allow modulation of these effects to maximize tumor control through antitumor immunity.

Supplementary figures and tables

Purpose:The prevention and treatment of metastatic sarcoma are areas of significant unmet need. Immune checkpoint inhibitor monotherapy has shown little activity in sarcoma and there is great interest in identifying novel treatment combinations that may augment responses. In vitro and in vivo, we investigated the potential for an oncolytic vaccinia virus (GLV-1h68) delivered using isolated limb perfusion (ILP) to promote antitumor immune responses and augment response to PD-1 blockade in sarcoma.Experimental Design: In an established animal model of extremity sarcoma, we evaluated the potential of locoregional delivery of a vaccinia virus (GLV-1h68) alongside biochemotherapy (melphalan/TNFα) in ILP. Complementary in vitro assays for markers of immunogenic cell death were performed in sarcoma cell lines.Results:PD-1 monotherapy had minimal efficacy in vivo, mimicking the clinical scenario. Pretreatment with GLV-1h68 delivered by ILP (viral ILP) significantly improved responses. Furthermore, when performed prior to surgery and radiotherapy, viral ILP and PD-1 blockade prevented both local and distant relapse, curing a previously treatment-refractory model. Enhanced therapy was associated with marked modulation of the tumor microenvironment, with an increase in the number and penetrance of intratumoral CD8+ T cells and expansion and activation of dendritic cells. GLV-1h68 was capable of inducing markers of immunogenic cell death in human sarcoma cell lines.Conclusions:Viral ILP augments the response to PD-1 blockade, transforming this locoregional therapy into a potentially effective systemic treatment for sarcoma and warrants translational evaluation.

Supplementary tables and figures

Cytoplasmic pattern recognition receptors (PRRs) for double-stranded RNA (RIG-I/MDA5) are key mediators of anti-viral responses. PRR agonists, such as dsRNA oncolytic Reovirus type 3 Dearing (Rt3D), potently activate RNA sensors. We used an unbiased cytotoxicity screen to reveal synergistic drug-virotherapy combinations and found potent effects of Rt3D combined with the CDK4/6 inhibitor, palbociclib. The combination augmented oncolytic virus-induced endoplasmic reticulum (ER) stress/unfolded protein response (UPR) and the expression and activation/signaling of RNA sensors. Combined Rt3D-palbociclib treatment potently increased interferon production and signaling, and knockdown studies implicated key UPR proteins and the RNA sensor, RIG-I, as essential to the phenotype observed. Further experiments, using canonical RIG-I agonists and an ER stress inducer, thapsigargin, confirmed cross-talk between RNA sensing and ER stress pathways that augmented cancer cell death and interferon production. Combined Rt3D-palbociclib also increased innate immune activation within tumour cells and IFN-induced HLA expression. Analysis of the immunopeptidome revealed changes to HLA-captured peptides with Rt3D-palbociclib, including altered expression of peptides from cancer/testis antigens (CTA) and endogenous retroviral elements (ERVs). Our findings highlight cross-talk between UPR signaling and RNA-mediated PRR activation as a means of enhancing anti-cancer efficacy with potential pro-immunogenic consequences. This has implications for future clinical development of PRR agonists and oncolytic viruses, and broadens the therapeutic remit of CDK4/6 inhibitors to include roles as both ER stress and dsRNA PRR sensitizers.

The development of immune checkpoint inhibitors (ICIs) is revolutionizing the way we think about cancer treatment. Even so, for most types of cancer, only a minority of patients currently benefit from ICI therapies. Intrinsic and acquired resistance to ICIs has focused research towards new combination therapy approaches that seek to increase response rates, the depth of remission and the durability of benefit. In this Review, we describe how radiotherapy, through its immunomodulating effects, represents a promising combination partner with ICIs. We describe how recent research on DNA damage response (DDR) inhibitors in combination with radiotherapy may be used to augment this approach. Radiotherapy can kill cancer cells while simultaneously triggering the release of pro-inflammatory mediators and increasing tumour-infiltrating immune cells - phenomena often described colloquially as turning immunologically 'cold' tumours 'hot'. Here, we focus on new developments illustrating the key role of tumour cell-autonomous signalling after radiotherapy. Radiotherapy-induced tumour cell micronuclei activate cytosolic nucleic acid sensor pathways, such as cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING), and propagation of the resulting inflammatory signals remodels the immune contexture of the tumour microenvironment. In parallel, radiation can impact immunosurveillance by modulating neoantigen expression. Finally, we highlight how tumour cell-autonomous mechanisms might be exploited by combining DDR inhibitors, ICIs and radiotherapy.

Introduction: Immune checkpoint inhibitors (ICI) have dramatically improved the outcome for cancer patients across multiple tumor types. However the response rates to ICI monotherapy remain relativ...

Purpose: The prevention and treatment of metastatic sarcoma are areas of significant unmet need. Immune checkpoint inhibitor monotherapy has shown little activity in sarcoma and there is great interest in identifying novel treatment combinations that may augment responses. In vitro and in vivo, we investigated the potential for an oncolytic vaccinia virus (GLV-1h68) delivered using isolated limb perfusion (ILP) to promote antitumor immune responses and augment response to PD-1 blockade in sarcoma. Experimental Design: In an established animal model of extremity sarcoma, we evaluated the potential of locoregional delivery of a vaccinia virus (GLV-1h68) alongside biochemotherapy (melphalan/TNFα) in ILP. Complementary in vitro assays for markers of immunogenic cell death were performed in sarcoma cell lines. Results: PD-1 monotherapy had minimal efficacy in vivo, mimicking the clinical scenario. Pretreatment with GLV-1h68 delivered by ILP (viral ILP) significantly improved responses. Furthermore, when performed prior to surgery and radiotherapy, viral ILP and PD-1 blockade prevented both local and distant relapse, curing a previously treatment-refractory model. Enhanced therapy was associated with marked modulation of the tumor microenvironment, with an increase in the number and penetrance of intratumoral CD8+ T cells and expansion and activation of dendritic cells. GLV-1h68 was capable of inducing markers of immunogenic cell death in human sarcoma cell lines. Conclusions: Viral ILP augments the response to PD-1 blockade, transforming this locoregional therapy into a potentially effective systemic treatment for sarcoma and warrants translational evaluation.

The ability to chemically modify ionic liquids (ILs) has led to an expansion in interest in their use in a diversity of applications, not least as antimicrobials and biocides. Relatively little is known about cytotoxicity mechanisms of ILs in comparison to other biocides currently in widespread use, as well as their practical significance for the ecological environment and human health. Using NCTC 2544 and HaCat human keratinocyte cells, this study aimed to characterize cytotoxicity rates and mechanisms of a range of ILs. Using both lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) based cytotoxicity assays, it was confirmed that at biocide-relevant concentrations, ILs with longer alkyl chains exhibited greater biocidal activity than those with shorter alkyl chains, with comparable activity to the commonly used biocides chlorhexidine, benzalkonium chloride and cetylpyridinium chloride, at relevant in-use biocide concentrations. Mode of cell death, measured using fluorescence-activated cell sorting (FACS) and caspase 3/7 activity, determined necrosis to be the primary cytotoxic mechanism at higher concentrations of the biocides stated above, and with ILs [C14MIM]Cl and [C14quin]Br, with apoptosis observed at borderline necrotic concentrations. Perhaps most interestingly, modification of anion had a significant effect on cytotoxicity. The use of N[SO2CF3] as an anion to [C16MIM] attenuated cytotoxicity 10-fold in comparison to other anions, suggesting cytotoxicity may also be a tuneable property when using ILs as biocides.

This chapter deals with versions of Dante’s Commedia in English, confining itself to translations of either the whole poem or of at least one cantica. It notes that English translations of the Commedia appear later than those of other European languages, and that it is only after 1900 that English versions start to outnumber those in French. It charts the history of these translations over two centuries from Henry Boyd’s version in 1802 to Clive James’s translation in 2013 and to Alasdair Gray’s Inferno of 2018. The chapter also offers an analysis of a number of excerpts across all three cantiche, illustrating the adequacy or otherwise of the translations, and commenting on metrical solutions. The statistics are striking: since 1850 there have been an average of thirty-three English versions of the poem or of one cantica every half century, and even in the nineteen years since the millennium there have been twenty-seven such versions, so the enthusiasm of anglophone writers and readers for Dante even seems to be increasing.

Systemic relapse, after treatment of a localised primary tumour with neo-adjuvant radiotherapy and surgery, is the major cause of disease related mortality in patients with sarcoma. As with other cancers, many sarcoma patients derive no benefit from anti-PD-1 treatment. Combining radiotherapy and immunotherapy is under investigation as a means to improve response rates and control metastatic disease. Here, we use a retrospective cohort of sarcoma patients, treated with neoadjuvant radiotherapy, and TCGA data to explore patient stratification for immunotherapy and therapeutic targets of relevance to sarcoma. We show a group of patients with immune-hot undifferentiated pleomorphic sarcoma as one of the highest-ranking candidates for emerging 4-1BB targeting agents. A binary hot/cold classification method indicates 4-1BB-high hot sarcomas share many characteristics with immunotherapy responsive cancers of other pathologies. Hot tumours in sarcoma are however substantially less prevalent. Patient stratification, of intense interest for immunotherapies, is therefore even more important in sarcoma.

BackgroundThe aggressive clinical behavior of poorly differentiated and anaplastic thyroid cancers (PDTC and ATC) has proven challenging to treat, and survival beyond a few months from diagnosis is rare. Although 30%–60% of these tumors contain mutations in the BRAF gene, inhibitors designed specifically to target oncogenic BRAF have shown limited and only short-lasting therapeutic benefits as single agents, thus highlighting the need for improved treatment strategies, including novel combinations.MethodsUsing a BRAFV600E-driven mouse model of ATC, we investigated the therapeutic efficacy of the combination of BRAF inhibition and oncolytic herpes simplex virus (oHSV). Analyses of samples from tumor-bearing mice were performed to immunologically characterize the effects of different treatments. These immune data were used to inform the incorporation of immune checkpoint inhibitors into triple combination therapies.ResultsWe characterized the immune landscape in vivo following BRAF inhibitor treatment and detected only modest immune changes. We, therefore, hypothesized that the addition of oncolytic virotherapy to BRAF inhibition in thyroid cancer would create a more favorable tumor immune microenvironment, boost the inflammatory status of tumors and improve BRAF inhibitor therapy. First, we showed that thyroid cancer cells were susceptible to infection with oHSV and that this process was associated with activation of the immune tumor microenvironment in vivo. Next, we showed improved therapeutic responses when combining oHSV and BRAF inhibition in vivo, although no synergistic effects were seen in vitro, further confirming that the dominant effect of oHSV in this context was likely immune-mediated. Importantly, both gene and protein expression data revealed an increase in activation of T cells and natural killer (NK) cells in the tumor in combination-treated samples. The benefit of combination oHSV and BRAF inhibitor therapy was abrogated when T cells or NK cells were depleted in vivo. In addition, we showed upregulation of PD-L1 and CTLA-4 following combined treatment and demonstrated that blockade of the PD-1/PD-L1 axis or CTLA-4 further improved combination therapy.ConclusionsThe combination of oHSV and BRAF inhibition significantly improved survival in a mouse model of ATC by enhancing immune-mediated antitumor effects, and triple combination therapies, including either PD-1 or CTLA-4 blockade, further improved therapy.

The development of immune checkpoint inhibitors (ICIs) is revolutionizing the way we think about cancer treatment. Even so, for most types of cancer, only a minority of patients currently benefit from ICI therapies. Intrinsic and acquired resistance to ICIs has focused research towards new combination therapy approaches that seek to increase response rates, the depth of remission and the durability of benefit. In this Review, we describe how radiotherapy, through its immunomodulating effects, represents a promising combination partner with ICIs. We describe how recent research on DNA damage response (DDR) inhibitors in combination with radiotherapy may be used to augment this approach. Radiotherapy can kill cancer cells while simultaneously triggering the release of pro-inflammatory mediators and increasing tumour-infiltrating immune cells - phenomena often described colloquially as turning immunologically 'cold' tumours 'hot'. Here, we focus on new developments illustrating the key role of tumour cell-autonomous signalling after radiotherapy. Radiotherapy-induced tumour cell micronuclei activate cytosolic nucleic acid sensor pathways, such as cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING), and propagation of the resulting inflammatory signals remodels the immune contexture of the tumour microenvironment. In parallel, radiation can impact immunosurveillance by modulating neoantigen expression. Finally, we highlight how tumour cell-autonomous mechanisms might be exploited by combining DDR inhibitors, ICIs and radiotherapy.

BackgroundCombination herpes simplex virus (HSV) oncolytic virotherapy and BRAF inhibitors (BRAFi) represent promising immunogenic treatments for BRAF mutant melanoma, but an improved understanding of the immunobiology of combinations is needed to improve on the benefit of immune checkpoint inhibitors (ICI).MethodsUsing a BRAFV600E-driven murine melanoma model, we tested the immunogenicity of HSV/BRAFi in immunocompetent C57BL mice. In addition to standard FACS analysis, we used the ‘Timer of Cell Kinetics and Activity’ system, which can analyze the temporal dynamics of different T cell subsets. This immune data was used to inform the selection of ICI for triple combination therapy, the effects of which were then further characterized using transcriptomics.ResultsAdding BRAFi treatment to HSV improved anti-tumor effects in vivo but not in vitro. Immune characterization showed HSV or dual therapy led to fewer intratumoral Treg, although with a more activated phenotype, together with more effector CD8 +T cells. Tocky analysis further showed that HSV/BRAFi dual treatment reduced the Tocky signal (reflecting engagement with cognate antigen), in both Treg and conventional subsets of CD4+, but not in CD8 +cells. However, a higher percentage of Treg than of conventional CD4 +maintained frequent engagement with antigens on treatment, reflecting a predominance of suppressive over effector function within the CD4 +compartment. The only T cell subset which correlated with a reduction in tumor growth was within Tocky signal positive conventional CD4+, supporting their therapeutic role. Targeting CD25 high, antigen-engaged Treg with a depleting anti-CD25 ICI, achieved complete cures in 100% of mice with triple therapy. Transcriptomic analysis confirmed reduction in Foxp3 on addition of anti-CD25 to HSV/BRAFi, as well as increases in expression of genes reflecting interferon signaling and cytotoxic activity.ConclusionsCombination HSV/BRAFi is an immunogenic therapy for BRAF mutant melanoma, but cannot fully control tumors. Dual therapy results in changes in T cell dynamics within tumors, with relatively maintained antigen signaling in Treg compared with conv CD4+. Antigen-engaged CD4 +effectors correlate with tumor growth control, and depletion of Treg by addition of an anti-CD25 ICI, releasing suppression of conventional CD4 +effectors by Treg, enhances survival and activates immune signaling within tumors.

BackgroundDespite therapeutic gains from immune checkpoint inhibitors (ICI) in many tumor types, new strategies are needed to extend treatment benefits, especially in patients failing to mount effective antitumor T-cell responses. Radiation and drug therapies can profoundly affect the tumor immune microenvironment. Here, we aimed to identify immunotherapies to increase the antitumor response conferred by combined ataxia telangiectasia and Rad3-related kinase inhibition and radiotherapy.MethodsUsing the human papillomavirus (HPV)-negative murine oral squamous cell carcinoma model, MOC2, we assessed the nature of the antitumor response following ataxia telangiectasia and Rad3-related inhibitor (ATRi)/radiotherapy (RT) by performing RNA sequencing and detailed flow cytometry analyses in tumors. The benefit of immunotherapies based on T cell immunoreceptor with Ig and ITIM domains (TIGIT) and Programmed cell death protein 1 (PD-1) immune checkpoint blockade following ATRi/RT treatment was assessed in the MOC2 model and confirmed in another HPV-negative murine oral squamous cell carcinoma model called SCC7. Finally, immune profiling was performed by flow cytometry on blood samples in patients with head and neck squamous cell carcinoma enrolled in the PATRIOT clinical trial of combined ATRi/RT.ResultsATRi enhances radiotherapy-induced inflammation in the tumor microenvironment, with natural killer (NK) cells playing a central role in maximizing treatment efficacy. We demonstrated that antitumor activity of NK cells can be further boosted with ICI targeting TIGIT and PD-1. Analyses of clinical samples from patients receiving ATRi (ceralasertib) confirm the translational potential of our preclinical studies.ConclusionThis work delineates a previously unrecognized role for NK cells in the antitumor immune response to radiotherapy that can be augmented by small-molecule DNA damage–response inhibitors and immune checkpoint blockade.

Cytoplasmic nucleic acid sensors for double-stranded (ds) RNA (RIG-I/MDA5) and DNA (cGAS-STING) are pattern recognition receptors (PRRs) key to intracellular anti-viral responses. Recent research has highlighted roles for PRR agonists, including oncolytic virotherapy agents, in anti-tumor immunotherapy. Reovirus type 3 Dearing (Rt3D) is an oncolytic dsRNA virus with limited single-agent activity in clinical studies, but potential for use in combination regimens. We sought synergistic drug-virotherapy combinations using an unbiased screening approach that highlighted the CDK4/6 inhibitor, palbociclib, as a leading hit. We found that, when combined with Rt3D, palbociclib augmented oncolytic virus-induced endoplasmic reticulum (ER) stress/unfolded protein response (UPR) signaling. Combined Rt3D-palbociclib treatment potently increased interferon signaling and endogenous retroviral transcripts. Knockdown (siRNA) studies indicated key UPR proteins and the RNA sensor, RIG-I, were essential to the phenotype observed. Mechanistically independent experiments, using canonical RIG-I agonists and the ER stress inducer (thapsigargin), confirmed cross-talk between RNA sensing and ER stress pathways that augment cancer cell death and interferon production. Combined Rt3D-palbociclib increased innate immune activation and effector function. Our findings demonstrate that UPR signaling and innate immune RNA sensor crosstalk can be exploited to enhance anti-cancer efficacy with pro-immunogenic consequences. This has implications for future clinical development of PRR agonists and oncolytic viruses, as well as broadening the therapeutic remit of CDK4/6 inhibitors to include their role as ER stress sensitizers. Citation Format: Victoria Roulstone, Joan Kyula, Richard Elliot, Christopher J. Lord, Nik Matthews, Vicki Jennings, Harriet Whittock, David Mansfield, Jyoti Choudhary, James Wright, Lu Yu, Alan Melcher, Richard Vile, Matt Coffey, Martin McLaughlin, Kevin Harrington. Mechanisms of therapeutic synergy between pattern recognition response agonists and cdk4 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1960.

Reovirus (RT3D) is a naturally occurring double-stranded RNA oncolytic virus that has shown preclinical efficacy in a wide range of tumor types. Early phase clinical studies have shown that this agent has modest monotherapy efficacy and can safely be combined with cytotoxic chemotherapy regimens. In the current studies, we used a high-throughput drug screen approach of different targeted therapeutic agents with the aim of looking for potential viral sensitizers that could enhance RT3D tumor killing. BMN-673 (talazoparib), a clinically approved poly(ADP)-ribose polymerase 1 (PARP-1) inhibitor was identified as a top hit and found to sensitize profoundly to RT3D both in vitro and in vivo in human xenograft tumors in a nude mouse model. We found that RT3D activated cellular PARP1 and was associated with PARylation of cellular proteins, including components of the DISC-associated cell death machinery. Combined treatment with RT3D and talazoparib enhanced extrinsic apoptosis (amplified by autocrine/paracrine TNF-α and TRAIL signaling), NF-κB pathway activity and pro-inflammatory cytokine production (CCL5/RANTES, CXCL8/IL8, CXCL1/GRO and CXCL10/IP10). Signaling was shown to be dependent on nucleic acid sensing mechanisms mediated by RIG-I and TLR3. We also found anti-tumour efficacy in an immunocompetent mouse model and this correlated with an increase in an immune response following combination treatment of RT3D and talazoparib. Our data provide a strong rationale for the combination of oncolytic RT3D with PARP1 inhibitors to exploit immunogenic response in cancer treatment. Citation Format: Joan N. Kyula, Victoria Roulstone, Richard Elliott, Harriet Whittock, Galabina Bozhanova, Martin McLaughlin, Malin Pedersen, Dragomir Krastev, Stephen Pettitt, Arnaud Legrand, Tencho Tenev, James Wright, Lu Yu, Jyoti Choudhary, Pascal Meier, Christopher J. Lord, Alan Melcher, Grey Wilkinson, Matt Coffey, Kevin J. Harrington. Talazoparib interacts with oncolytic reovirus to enhance death-inducing signaling complex (DISC)-mediated apoptosis and immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1932.

The year 1974, the 500th anniversary of Ariosto’s birth, inaugurated an unprecedented upsurge of interest in the Orlando Furioso both in Italy and in English-speaking countries. In the UK Peter Brand published the first post-war English monograph on the poet. The same year also saw the publication of Guido Waldman’s prose translation of the poem for Oxford University Press, while Barbara Reynolds’ two-volume verse translation for Penguin appeared in 1973 and 1977. This chapter asks what sort of Furioso do British readers encounter in these two twentieth-century translations? There is as yet no substantial comparison of these two versions, so this essay attempts to fill that gap by sampling the renderings of some problematic erotic episodes from Ariosto’s epic. What emerges is a range of translation errors, omissions and euphemisms but also some intelligent, felicitous solutions that combine intertextual allusions to British culture, the British imaginary and its literary traditions.

PURPOSE: ATR inhibitors (ATRi) are in early phase clinical trials and have been shown to sensitise to chemotherapy and radiotherapy preclinically. Limited data have been published about the effect of these drugs on the tumor microenvironment. EXPERIMENTAL DESIGN: We used an immunocompetent mouse model of HPV-driven malignancies to investigate the ATR inhibitor AZD6738 in combination with fractionated radiation (RT). Gene expression analysis and flow cytometry were performed post-therapy. RESULTS: Significant radiosensitization to RT by ATRi was observed alongside a marked increase in immune cell infiltration. We identified increased numbers of CD3+ and NK cells but most of this infiltrate was composed of myeloid cells. ATRi plus radiation produced a gene expression signature matching a type I/II interferon response with upregulation of genes playing a role in nucleic acid sensing. Increased MHC I levels were observed on tumor cells, with transcript-level data indicating increased antigen processing and presentation within the tumor. Significant modulation of cytokine gene expression (particularly CCL2, CCL5 and CXCL10) was found in vivo, with in vitro data indicating CCL3, CCL5 and CXCL10 are produced from tumor cells after ATRi + RT. CONCLUSIONS: We show that DNA damage by ATRi and RT leads to an interferon response through activation of nucleic acid sensing pathways. This triggers increased antigen presentation and innate immune cell infiltration. Further understanding of the effect of this combination on the immune response may allow modulation of these effects to maximise tumor control through anti-tumor immunity.

Improvements in cancer survival mean that long-term toxicities are being increasingly recognised that contribute to the morbidity of cancer survivorship. Late adverse effects (LAEs) in normal tissues after radiotherapy (RT) are characterized by vascular dysfunction and fibrosis leading to volume loss and tissue contracture. For example, irradiation of free flaps used for immediate breast reconstruction after mastectomy results in LAEs that distort the reconstructed breast, necessitating salvage reconstruction long after cancer treatment is complete. We evaluated the efficacy of lentiviral superoxide dismutase 2 (LVSOD2) and connective tissue growth factor (CTGF) shRNA in reducing the severity of LAEs using a novel animal model of free flap LAEs. Vectors were delivered by intra-arterial injection, ex vivo, to target the vascular compartment. LVSOD2 and LVshCTGF monotherapy prior to irradiation resulted in significant preservation of flap volume or reduction in skin contracture, respectively. Flaps transduced with combination therapy experienced significant improvements in both volume loss and skin contracture. Both therapies significantly reduced the fibrotic burden after irradiation. LAEs were associated with impaired vascular perfusion, loss of endothelial permeability and stromal hypoxia that were further reversed in the described treatment model. Using a model of tumour recurrence within transduced flaps we showed that SOD2 over-expression in normal tissues did not compromise the cytotoxic efficacy of RT against tumour cells but instead, paradoxically, appeared to enhance it. LVSOD2 and LVshCTGF combination therapy by targeted, intravascular delivery reduced LAE severities in normal tissues without compromising the efficacy of RT and warrants translational evaluation as a free flap-targeted gene therapy.

Purpose Radiotherapy (RT) is used frequently in patients with melanoma, but results are suboptimal because the disease is often radioresistant. This may be due to constitutive activation of MAPK pathway signalling through mutations involving RAS/RAF. Thus, we studied whether trametinib, a potent and selective allosteric inhibitor of MEK1/2 could improve the efficacy of RT. Methods and materials Clonogenic survival assays were performed in human BRAF-mutant (A375), NRAS-mutant (D04, WM1631), KRAS-mutant (WM1791c) and wild-type (PMWK) melanoma cell lines. The effects of trametinib with and without radiation on protein levels of MEK effectors were measured by immunoblot analyses. Cell cycle effects, DNA damage repair, mitotic catastrophe and senescence were measured using flow cytometry, γH2Ax staining, nuclear fragmentation and β-galactosidase staining, respectively. Additionally, athymic mice with D04 flank tumours were treated with fractionated RT after gavage with trametinib and monitored for tumour growth. Results All cell lines, except PMWK, exhibited enhanced cytotoxicity when RT was combined with trametinib compared to either agent alone. Sensitiser enhancement ratios were 1.70, 1.32, 1.10, and 1.70 for A375, D04, WM1361 and WM1791c, respectively. Trametinib efficiently blocked RT-induced phosphorylation of ERK at nanomolar concentrations. Increased radiosensitivity correlated with prolonged G1 arrest and reduction in the radioresistant S phase up to 48h following RT. A larger population of senescence-activated β-galactosidase-positive cells was seen in the trametinib pretreated group, and this correlated with activation of two of the major mediators of induced senescence, p53 and pRb. Mice receiving the combination treatment (trametinib 1mg/kg and RT over 3days) showed a reduced mean tumour volume compared with mice receiving trametinib alone ( p =0.016), or RT alone ( p =0.047). No overt signs of drug toxicity were observed. Conclusion Trametinib radiosensitised RAS-/RAF-mutated melanoma cells by inducing prolonged G1 arrest and premature senescence. In this pre-clinical study we demonstrate that combining trametinib and RT is well tolerated, and reduces tumour growth in vivo .