Ayman Oweida

Purpose: Radiotherapy (RT) can transform the immune landscape and render poorly immunogenic tumors sensitive to PD-L1 inhibition. Here, we established that the response to combined RT and PD-L1 inhibition is transient and investigated mechanisms of resistance.Experimental Design: Mechanisms of resistance to RT and PD-L1 blockade were investigated in orthotopic murine head and neck squamous cell carcinoma (HNSCC) tumors using mass cytometry and whole-genome sequencing. Mice were treated with anti-PD-L1 or anti-TIM-3 alone and in combination with and without RT. Tumor growth and survival were assessed. Flow cytometry was used to assess phenotypic and functional changes in intratumoral T-cell populations. Depletion of regulatory T cells (Treg) was performed using anti-CD25 antibody.Results: We show that the immune checkpoint receptor, TIM-3, is upregulated on CD8 T cells and Tregs in tumors treated with RT and PD-L1 blockade. Treatment with anti-TIM-3 concurrently with anti-PD-L1 and RT led to significant tumor growth delay, enhanced T-cell cytotoxicity, decreased Tregs, and improved survival in orthotopic models of HNSCC. Despite this treatment combination, the response was not durable, and analysis of relapsed tumors revealed resurgence of Tregs. Targeted Treg depletion, however, restored antitumor immunity in mice treated with RT and dual immune checkpoint blockade and resulted in tumor rejection and induction of immunologic memory.Conclusions: These data reveal multiple layers of immune regulation that can promote tumorigenesis and the therapeutic potential of sequential targeting to overcome tumor resistance mechanisms. We propose that targeted Treg inhibitors may be critical for achieving durable tumor response with combined radiotherapy and immunotherapy. Clin Cancer Res; 24(21); 5368-80. ©2018 AACR.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has recently emerged in China and caused a disease called coronavirus disease 2019 (COVID-19). The virus quickly spread around the world, causing a sustained global outbreak. Although SARS-CoV-2, and other coronaviruses, SARS-CoV and Middle East respiratory syndrome CoV (MERS-CoV) are highly similar genetically and at the protein production level, there are significant differences between them. Research has shown that the structural spike (S) protein plays an important role in the evolution and transmission of SARS-CoV-2. So far, studies have shown that various genes encoding primarily for elements of S protein undergo frequent mutation. We have performed an in-depth review of the literature covering the structural and mutational aspects of S protein in the context of SARS-CoV-2, and compared them with those of SARS-CoV and MERS-CoV. Our analytical approach consisted in an initial genome and transcriptome analysis, followed by primary, secondary and tertiary protein structure analysis. Additionally, we investigated the potential effects of these differences on the S protein binding and interactions to angiotensin-converting enzyme 2 (ACE2), and we established, after extensive analysis of previous research articles, that SARS-CoV-2 and SARS-CoV use different ends/regions in S protein receptor-binding motif (RBM) and different types of interactions for their chief binding with ACE2. These differences may have significant implications on pathogenesis, entry and ability to infect intermediate hosts for these coronaviruses. This review comprehensively addresses in detail the variations in S protein, its receptor-binding characteristics and detailed structural interactions, the process of cleavage involved in priming, as well as other differences between coronaviruses.

Abstract Background Radioresistance represents a major problem in the treatment of head and neck cancer (HNC) patients. To improve response, understanding tumor microenvironmental factors that contribute to radiation resistance is important. Regulatory T cells (Tregs) are enriched in numerous cancers and can dampen the response to radiation by creating an immune-inhibitory microenvironment. The purpose of this study was to investigate mechanisms of Treg modulation by radiation in HNC. Methods We utilized an orthotopic mouse model of HNC. Anti-CD25 was used for Treg depletion. Image-guided radiation was delivered to a dose of 10 Gy. Flow cytometry was used to analyze abundance and function of intratumoral immune cells. Enzyme-linked immunosorbent assay was performed to assess secreted factors. For immune-modulating therapies, anti–PD-L1, anti-CTLA-4, and STAT3 antisense oligonucleotide (ASO) were used. All statistical tests were two-sided. Results Treatment with anti-CD25 and radiation led to tumor eradication (57.1%, n = 4 of 7 mice), enhanced T-cell cytotoxicity compared with RT alone (CD4 effector T cells [Teff]: RT group mean = 5.37 [ 0.58] vs RT + αCD25 group mean =10.71 [0.67], P = .005; CD8 Teff: RT group mean = 9.98 [0.81] vs RT + αCD25 group mean =16.88 [2.49], P = .01) and induced tumor antigen-specific memory response (100.0%, n = 4 mice). In contrast, radiation alone or when combined with anti-CTLA4 did not lead to durable tumor control (0.0%, n = 7 mice). STAT3 inhibition in combination with radiation, but not as a single agent, improved tumor growth delay, decreased Tregs, myeloid-derived suppressor cells, and M2 macrophages and enhanced effector T cells and M1 macrophages. Experiments in nude mice inhibited the benefit of STAT3 ASO and radiation. Conclusion We propose that STAT3 inhibition is a viable and potent therapeutic target against Tregs. Our data support the design of clinical trials integrating STAT3 ASO in the standard of care for cancer patients receiving radiation.

Immunotherapy clinical trials targeting the programmed-death ligand axis (PD-1/PD-L1) show that most head and neck squamous cell carcinoma (HNSCC) patients are resistant to PD-1/PD-L1 inhibition. We investigated whether local radiation to the tumor can transform the immune landscape and render poorly immunogenic HNSCC tumors sensitive to PD-L1 inhibition. We used the first novel orthotopic model of HNSCC with genetically distinct murine cell lines. Tumors were resistant to PD-L1 checkpoint blockade, harbored minimal PD-L1 expression and tumor infiltrating lymphocytes at baseline, and were resistant to radiotherapy. The combination of radiation and PD-L1 inhibition significantly enhanced tumor control and improved survival. This was mediated in part through upregulation of PD-L1 on tumor cells and increased T-cell infiltration after RT, resulting in a highly inflamed tumor. Depletion of both CD4 and CD8 T-cells completely abrogated the effect of anti PD-L1 with radiation on tumor growth. Our findings provide evidence that radiation to the tumor can induce sensitivity to PD-L1 checkpoint blockade in orthotopic models of HNSCC. These findings have direct relevance to high risk HNSCC patients with poorly immunogenic tumors and who may benefit from combined radiation and checkpoint blockade.

Radiation therapy has been used for many years to treat tumors based on its DNA-damage-mediated ability to kill cells. More recently, RT has been shown to exert beneficial modulatory effects on immune responses, such as triggering immunogenic cell death, enhancing antigen presentation, and activating cytotoxic T-cells. Consequently, combining radiation therapy with immunotherapy represents an important area of research. Thus far, immune-checkpoint inhibitors targeting programmed death-ligand 1 (PD-L1), programmed cell death protein 1 (PD-1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have been the focus of many research studies and clinical trials. The available data suggest that such immunotherapies are enhanced when combined with radiation therapy. However, treatment resistance, intrinsic or acquired, is still prevalent. Various theories as to how to enhance these combination therapies to overcome treatment resistance have been proposed. In this review, we focus on the principles surrounding radiation therapy’s positive and negative effects on the tumor microenvironment. We explore mechanisms underlying RT’s synergistic and antagonistic effects on immune responses and provide a base of knowledge for radio-immunology combination therapies to overcome treatment resistance. We provide evidence for targeting regulatory T-cells, tumor-associated macrophages, and cancer-associated fibroblasts in combination radio-immunotherapies to improve cancer treatment.

Abstract Stromal fibrosis activates prosurvival and proepithelial-to-mesenchymal transition (EMT) pathways in pancreatic ductal adenocarcinoma (PDAC). In patient tumors treated with neoadjuvant stereotactic body radiation therapy (SBRT), we found upregulation of fibrosis, extracellular matrix (ECM), and EMT gene signatures, which can drive therapeutic resistance and tumor invasion. Molecular, functional, and translational analysis identified two cell-surface proteins, a disintegrin and metalloprotease 10 (ADAM10) and ephrinB2, as drivers of fibrosis and tumor progression after radiation therapy (RT). RT resulted in increased ADAM10 expression in tumor cells, leading to cleavage of ephrinB2, which was also detected in plasma. Pharmacologic or genetic targeting of ADAM10 decreased RT-induced fibrosis and tissue tension, tumor cell migration, and invasion, sensitizing orthotopic tumors to radiation killing and prolonging mouse survival. Inhibition of ADAM10 and genetic ablation of ephrinB2 in fibroblasts reduced the metastatic potential of tumor cells after RT. Stimulation of tumor cells with ephrinB2 FC protein reversed the reduction in tumor cell invasion with ADAM10 ablation. These findings represent a model of PDAC adaptation that explains resistance and metastasis after RT and identifies a targetable pathway to enhance RT efficacy. Significance: Targeting a previously unidentified adaptive resistance mechanism to radiation therapy in PDAC tumors in combination with radiation therapy could increase survival of the 40% of PDAC patients with locally advanced disease. See related commentary by Garcia Garcia et al., p. 3158

Abstract Purpose: Our goal was to evaluate the safety and toxicity of combining a PARP inhibitor, olaparib, with cetuximab and fractionated intensity-modulated radiotherapy for patients with locally advanced head and neck cancer and heavy smoking histories. Patients and Methods: Patients with ≥10 packs/year history of smoking were treated with olaparib at doses ranging from 25–200 mg orally twice daily beginning approximately 10 days prior to initiation of and with concurrent radiation (69.3 Gy in 33 fractions) using a time-to-event continual reassessment method model. Cetuximab was administered starting approximately 5 days prior to radiation per standard of care. Results: A total of 16 patients were entered onto the study, with 15 evaluable for acute toxicity. The most common treatment-related grade 3–4 side effects were radiation dermatitis and mucositis (38% and 69%, respectively). The MTD was determined to be 50 mg orally twice daily, but the recommended phase II dose was deemed to be 25 mg orally twice daily. At a median follow-up of 26 months, the actuarial median overall survival was 37 months, but was not reached for other endpoints. Two-year overall survival, progression-free survival, local control, and distant control rates were 72%, 63%, 72%, and 79%, respectively. Patients who continued to smoke during therapy experienced higher recurrence rates. MYC and KMT2A were identified as potential correlatives of response on gene amplification and mutational analysis. Conclusions: Olaparib at 25 mg orally twice daily with concurrent cetuximab and radiation was well tolerated with reduced dermatitis within the radiation field. Response rates were promising for this high-risk population. Clin Cancer Res; 24(20); 4949–59. ©2018 AACR.

Numerous trials combining radiation therapy (RT) and immunotherapy in head and neck squamous cell carcinoma (HNSCC) are failing. Using preclinical immune cold models of HNSCC resistant to RT-immune checkpoint inhibitors, we investigate therapeutic approaches of overcoming such resistance by examining the differential microenvironmental response to RT.We subjected two HPV-negative orthotopic mouse models of HNSCC to combination RT, regulatory T cells (Treg) depletion, and/or CD137 agonism. Tumor growth was measured and intratumorous and lymph node immune populations were compared among treatment groups. Human gene sets, genetically engineered mouse models DEREG and BATF3-/-, flow and time-of-flight cytometry, RNA-Seq, Treg adoptive transfer studies, and in vitro experiments were used to further evaluate the role of dendritic cells (DCs) and Tregs in these treatments.In MOC2 orthotopic tumors, we find no therapeutic benefit to targeting classically defined immunosuppressive myeloids, which increase with RT. In these radioresistant tumors, supplementing combination RT and Treg depletion with anti-CD137 agonism stimulates CD103+ DC activation in tumor-draining lymph nodes as characterized by increases in CD80+ and CCR7+ DCs, resulting in a CD8 T cell-dependent response. Simultaneously, Tregs are reprogrammed to an effector phenotype demonstrated by increases in interferonγ+, tumor necrosis factorα+, PI3K+, pAKT+ and Eomes+ populations as well as decreases in CTLA4+ and NRP-1+ populations. Tumor eradication is observed when RT is increased to an 8 Gy x 5 hypofractionated regimen and combined with anti-CD25+ anti-CD137 treatment. In a human gene set from oral squamous cell carcinoma tumors, high Treg number is associated with earlier recurrence.Regulating Treg functionality and DC activation status within the lymph node is critical for generating a T cell effector response in these highly radioresistant tumors. These findings underscore the plasticity of Tregs and represent a new therapeutic opportunity for reprogramming the tumor microenvironment in HNSCCs resistant to conventional radioimmunotherapy approaches.

Although laryngectomy is the treatment of choice for patients with T4 larynx cancer, many patients are unable or unwilling to undergo laryngectomy and instead pursue larynx-preservation strategies combining radiotherapy (RT) and chemotherapy. Herein, the authors analyzed the National Cancer Data Base to evaluate overall survival (OS) between patients treated with surgical and organ-preserving modalities.The National Cancer Data Base was queried for patients diagnosed from 2004 through 2012 with T4M0 laryngeal cancer who underwent either laryngectomy (surgery) with adjuvant RT (SRT), chemotherapy starting concurrently within 7 days of RT (CCRT), or multiagent induction chemotherapy starting 43 to 98 days before RT (ICRT). Multivariate analysis and propensity score matching were used to explore the association between the intervention and OS. Recursive partitioning analysis was performed to identify groups benefiting from particular modalities.A total of 1559 patients who underwent SRT, 1597 patients who underwent CCRT, and 386 patients who underwent ICRT were included. Adjusting for covariates, CCRT was found to be associated with inferior OS compared with SRT (hazard ratio [HR], 1.55; 95% confidence interval [95% CI], 1.41-1.70 [P<.01]) and with ICRT (HR, 1.25; 95% CI, 1.07-1.45 [P<.01]). OS among the patients treated with SRT did not appear to differ significantly from that of the ICRT cohort (HR, 0.87; 95% CI, 0.73-1.03 [P = 0.10]), a finding confirmed with propensity score matching. Recursive partitioning analysis identified no subset of patients that derived an OS benefit from either approach over the other.OS among patients undergoing SRT was found to be superior to that of patients treated with CCRT but did not significantly differ from that of those undergoing ICRT. Because these intriguing findings require validation, SRT should remain the standard of care for patients with this disease. However, organ preservation with ICRT may be a reasonable alternative in certain patients. Cancer 2017;123:600-608. © 2016 American Cancer Society.

Abstract Identifying targets present in the tumor microenvironment that contribute to immune evasion has become an important area of research. In this study, we identified EphB4–ephrin-B2 signaling as a regulator of both innate and adaptive components of the immune system. EphB4 belongs to receptor tyrosine kinase family that interacts with ephrin-B2 ligand at sites of cell–cell contact, resulting in bidirectional signaling. We found that EphB4–ephrin-B2 inhibition alone or in combination with radiation (RT) reduced intratumoral regulatory T cells (Tregs) and increased activation of both CD8+ and CD4+Foxp3− T cells compared with the control group in an orthotopic head and neck squamous cell carcinoma (HNSCC) model. We also compared the effect of EphB4–ephrin-B2 inhibition combined with RT with combined anti-PDL1 and RT and observed similar tumor growth suppression, particularly at early time-points. A patient-derived xenograft model showed reduction of tumor-associated M2 macrophages and favored polarization towards an antitumoral M1 phenotype following EphB4–ephrin-B2 inhibition with RT. In vitro, EphB4 signaling inhibition decreased Ki67-expressing Tregs and Treg activation compared with the control group. Overall, our study is the first to implicate the role of EphB4–ephrin-B2 in tumor immune response. Moreover, our findings suggest that EphB4–ephrin-B2 inhibition combined with RT represents a potential alternative for patients with HNSCC and could be particularly beneficial for patients who are ineligible to receive or cannot tolerate anti-PDL1 therapy. Significance: These findings present EphB4–ephrin-B2 inhibition as an alternative to anti-PDL1 therapeutics that can be used in combination with radiation to induce an effective antitumor immune response in patients with HNSCC.

Ephrin B2 is variably expressed on tumor cells and its blockade has been shown to inhibit angiogenesis in animal models of pancreatic, colorectal, lung and head, and neck squamous cell carcinomas. However, the implications of ephrinB2 expression in cancer patients have remained elusive. In this study, we analyzed the cancer genome atlas (TCGA) for ephrinB2 expression. We report significant correlations between EFNB2 expression, overall survival and disease‐free survival in head and neck squamous cell carcinoma (HNSCC, n = 519), pancreatic adenocarcinoma ( n = 186), and bladder urothelial carcinoma ( n = 410). In HNSCC patients, high‐EFNB2 mRNA expression was associated with tumor HPV negativity, oral cavity location, alcohol intake, higher TP53 mutation, and EGFR amplification. EphrinB2 overexpression also correlated with worse response to chemotherapy and radiotherapy. The therapeutic potential of blocking ephrinB2 was validated in HNSCC patient‐derived tumor xenografts and showed significant improvement in survival and tumor growth delay. Our data shows that ephrinB2 overexpression can serve as a critical biomarker for patient prognosis and response to therapy. These results should guide design of future clinical trials exploring EphrinB2 inhibition in cancer patients. © 2016 Wiley Periodicals, Inc.

Patients with melanoma brain metastases (MBM) have been excluded from trials evaluating immunotherapy in melanoma. As such, immunotherapy's role in MBM is poorly understood, particularly in combination with radiotherapy.The National Cancer Database was queried for patients with MBM receiving brain radiotherapy. They were classified according to immunotherapy receipt. Multivariate Cox regression was performed to identify factors associated with survival.Among 1287 patients, 185 received immunotherapy. Factors associated with improved survival included younger age, academic facility, lower extracranial disease burden, stereotactic radiotherapy, chemotherapy, and immunotherapy.Adding immunotherapy to radiotherapy for MBM is associated with improved survival.

Toll-like receptors (TLRs) are indispensable for the activation, maintenance and halting of immune responses. TLRs can mediate inflammation by recognizing molecular patterns in microbes (pathogen-associated molecular patterns: PAMPs) and endogenous ligands (danger-associated molecular patterns: DAMPs) released by injured or dead cells. For this reason, TLR ligands have attracted much attention in recent years in many cancer vaccines, alone or in combination with immunotherapy, chemotherapy and radiotherapy (RT). TLRs have been shown to play controversial roles in cancer, depending on various factors that can mediate tumor progression or apoptosis. Several TLR agonists have reached clinical trials and are being evaluated in combination with standard of care therapies, including RT. Despite their prolific and central role in mediating immune responses, the role of TLRs in cancer, particularly in response to radiation, remains poorly understood. Radiation is recognized as either a direct stimulant of TLR pathways, or indirectly through the damage it causes to target cells that subsequently activate TLRs. These effects can mediate pro-tumoral and anti-tumoral effects depending on various factors such as radiation dose and fractionation, as well as host genomic features. In this review, we examine how TLR signaling affects tumor response to RT, and we provide a framework for the design of TLR-based therapies with RT.

Abstract Background Epidermal growth factor receptor (EGFR) is frequently amplified or overexpressed in head and neck squamous cell carcinoma (HNSCC) and is a clinically validated target for the therapeutic antibody, cetuximab, in the management of this cancer. The degree of response to EGFR inhibitors measured by tumor shrinkage varies widely among HNSCC patients, and the biological mechanisms that underlie therapeutic heterogeneity amongst HNSCC patients remain ill-defined. Methods EGFR-dependent human and murine HNSCC cell lines were treated with the EGFR/ERBB inhibitors, gefitinib and AZD8931, and submitted to RNAseq, GSEA, and qRT-PCR. Conditioned media was analyzed by ELISA and Luminex assays. Murine HNSCC tumors were stained for T cell markers by immunofluorescence. Primary HSNCC patient specimens treated with single agent cetuximab were stained with Vectra multispectral immunofluorescence. Results The transcriptional reprogramming response to EGFR/ERBB-specific TKIs was measured in a panel of EGFR-dependent human HNSCC cell lines and interferon (IFN) α and γ responses identified as top-ranked TKI-induced pathways. Despite similar drug sensitivity, responses among 7 cell lines varied quantitatively and qualitatively, especially regarding the induced chemokine and cytokine profiles. Of note, the anti-tumorigenic chemokine, CXCL10, and the pro-tumorigenic factor, IL6, exhibited wide-ranging and non-overlapping induction. Similarly, AZD8931 exerted potent growth inhibition, IFNα/IFNγ pathway activation, and CXCL10 induction in murine B4B8 HNSCC cells. AZD8931 treatment of immune-competent mice bearing orthotopic B4B8 tumors increased CD8 + T cell content and the therapeutic response was abrogated in nu/nu mice relative to BALB/c mice. Finally, Vectra 3.0 analysis of HNSCC patient tumors prior to and after 3–4 weeks of single agent cetuximab treatment revealed increased CD8 + T cell content in specimens from patients exhibiting a therapeutic response relative to non-responders. Conclusions The findings reveal heterogeneous, tumor cell-intrinsic, EGFR/ERBB inhibitor-induced IFN pathway activation in HNSCC and suggest that individual tumor responses to oncogene-targeted agents are a sum of direct growth inhibitory effects and variably-induced participation of host immune cells.

Abstract Purpose To determine the contribution of blood‐derived macrophages to the signal loss observed in MR images of inflammatory lesions in experimental autoimmune encephalomyelitis (EAE). Materials and Methods A relapsing‐remitting form of EAE was induced in transgenic mice that express enhanced green fluorescent protein (EGFP) specifically in hematopoietic cells of the myelomonocytic lineage. Animals were injected with Feridex, a superparamagnetic iron oxide (SPIO) nanoparticle, 24 hours prior to in vivo MRI. MRI was performed using a 1.5T whole‐body scanner; a high‐performance, custom‐built gradient coil insert; and a 3D steady‐state free precession (SSFP) imaging pulse sequence. Comparisons were made between MR images and corresponding anti‐GFP and Perl's Prussian blue (PPB)‐stained brain sections. Results MR images revealed the presence of discrete regions of signal loss throughout the brains of EAE animals that were administered Feridex. Histological staining showed that regions of signal loss on MR images corresponded anatomically with regions of PPB‐ and GFP‐positive cells. Conclusion This experiment provides the first direct evidence that macrophages of hematogenous origin are labeled with SPIO after intravenous administration of Feridex, and contribute to the regions of signal loss detected in MR images of EAE brain. J. Magn. Reson. Imaging 2007;26:144–151. © 2007 Wiley‐Liss, Inc.

Purpose: The clinical success of targeted therapies such as cetuximab and radiotherapy (RT) is hampered by the low response rates and development of therapeutic resistance. In the current study, we investigated the involvement of EphB4-ephrin-B2 protumorigenic signaling in mediating resistance to EGFR inhibition and RT in head and neck cancers.Experimental Design: We used patient-derived xenograft (PDX) models of head and neck squamous cell carcinoma (HNSCC) and HNSCC cell lines to test our hypothesis. Tumor tissues were subjected to PhosphoRTK array, and Western blotting to detect changes in EphB4-ephrin-B2 targets. mRNA sequencing and microarray data analysis were performed on PDX tumors and HNSCC cell lines, respectively, to determine differences in gene expression of molecules involved in tumor cell growth, proliferation, and survival pathways. Effects on cell growth were determined by MTT assay on HNSCC cells downregulated for EphB4/ephrin-B2 expression, with and without EGFR inhibitor and radiation.Results: Our data from locally advanced HNSCC patients treated with standard-of-care definitive chemo-RT show elevated EphB4 and ephrin-B2 levels after failure of treatment. We observed significant response toward cetuximab and RT following EphB4-ephrin-B2 inhibition, resulting in improved survival in tumor-bearing mice. Tumor growth inhibition was accompanied by a decrease in the levels of proliferation and prosurvival molecules and increased apoptosis.Conclusions: Our findings underscore the importance of adopting rational drug combinations to enhance therapeutic effect. Our study documenting enhanced response of HNSCC to cetuximab-RT with EphB4-ephrin-B2 blockade has the potential to translate into the clinic to benefit this patient population. Clin Cancer Res; 24(18); 4539-50. ©2018 AACR.

Squamous cell carcinoma of the head and neck (HNSCC) represents an array of disease processes with a generally unfavorable prognosis. Inflammation plays an important role in tumor development and response to therapy. We performed a retrospective analysis of HNSCC patients to explore the relationship of the lymphocyte and neutrophil counts, the neutrophil-to-lymphocyte ratio (NLR) overall survival (OS), cancer-specific survival (CSS), local control (LC) and distant control (DC).All patients received definitive treatment for cancers of the oropharynx or larynx between 2006-2015. Neutrophil and lymphocyte counts were collected pre-, during-, and post-treatment. The correlations of patient, tumor, and biological factors to OS, CSS, LC and DC were assessed.196 patients met our inclusion criteria; 171 patients were Stage III or IV. Median follow-up was 2.7 years. A higher neutrophil count at all treatment time points was predictive of poor OS with the pre-treatment neutrophil count and overall neutrophil nadir additionally predictive of DC. Higher pre-treatment and overall NLR correlated to worse OS and DC, respectively.A higher pre-treatment neutrophil count correlates to poor OS, CSS and DC. Lymphocyte counts were not found to impact survival or tumor control. Higher pre-treatment NLR is prognostic of poor OS.

Abstract Purpose: SMAD4 loss causes genomic instability and the initiation/progression of head and neck squamous cell carcinoma (HNSCC). Here, we study whether SMAD4 loss sensitizes HNSCCs to olaparib (PARP inhibitor) in combination with radiotherapy (RT). Experimental Design: We analyzed HNSCC The Cancer Genome Atlas data for SMAD4 expression in association with FANC/BRCA family gene expression. Human HNSCC cell lines were screened for sensitivity to olaparib. Isogenic HNSCC cell lines were generated to restore or reduce SMAD4 expression and treated with olaparib, radiation, or the combination. HNSCC pretreatment specimens from a phase I trial investigating olaparib were analyzed. Results: SMAD4 levels correlated with levels of FANC/BRCA genes in HNSCC. HNSCC cell lines with SMAD4 homozygous deletion were sensitive to olaparib. In vivo, olaparib or RT monotherapy reduced tumor volumes in SMAD4-mutant but not SMAD4-positive tumors. Olaparib with RT dual therapy sustained tumor volume reduction in SMAD4-deficient (mutant or knockdown) xenografts, which exhibited increased DNA damage and cell death compared with vehicle-treated tumors. In vitro, olaparib alone or in combination with radiation caused lower clonogenic survival, more DNA damage–associated cell death, and less proliferation in SMAD4-deficient cells than in SMAD4-positive (endogenous SMAD4 or transduced SMAD4) cells. Applicable to clinic, 5 out of 6 SMAD4-negative HNSCCs and 4 out of 8 SMAD4-positive HNSCCs responded to a standard treatment plus olaparib in a phase I clinical trial, and SMAD4 protein levels inversely correlated with DNA damage. Conclusions: SMAD4 levels are causal in determining sensitivity to PARP inhibition in combination with RT in HNSCCs.

Abstract Members of the Eph family of receptor tyrosine kinases have been implicated in a wide array of human cancers. The EphB4 receptor is ubiquitously expressed in head and neck squamous cell carcinoma (HNSCC) and has been shown to impart tumorigenic and invasive characteristics to these cancers. In this study, we investigated whether EphB4 receptor targeting can enhance the radiosensitization of HNSCC. Our data show that EphB4 is expressed at high to moderate levels in HNSCC cell lines and patient-derived xenograft (PDX) tumors. We observed decreased survival fractions in HNSCC cells following EphB4 knockdown in clonogenic assays. An enhanced G2 cell cycle arrest with activation of DNA damage response pathway and increased apoptosis was evident in HNSCC cells following combined EphB4 downregulation and radiation compared to EphB4 knockdown and radiation alone. Data using HNSCC PDX models showed significant reduction in tumor volume and enhanced delay in tumor regrowth following sEphB4-HSA administration with radiation compared to single agent treatment. sEphB4-HSA is a protein known to block the interaction between the EphB4 receptor and its ephrin-B2 ligand. Overall, our findings emphasize the therapeutic relevance of EphB4 targeting as a radiosensitizer that can be exploited for the treatment of human head and neck carcinomas.

Abstract Purpose: A driving factor in pancreatic ductal adenocarcinoma (PDAC) treatment resistance is the tumor microenvironment, which is highly immunosuppressive. One potent immunologic adjuvant is radiotherapy. Radiation, however, has also been shown to induce immunosuppressive factors, which can contribute to tumor progression and formation of fibrotic tumor stroma. To capitalize on the immunogenic effects of radiation and obtain a durable tumor response, radiation must be rationally combined with targeted therapies to mitigate the influx of immunosuppressive cells and fibrosis. One such target is ephrinB2, which is overexpressed in PDAC and correlates negatively with prognosis. Experimental Design: On the basis of previous studies of ephrinB2 ligand-EphB4 receptor signaling, we hypothesized that inhibition of ephrinB2-EphB4 combined with radiation can regulate the microenvironment response postradiation, leading to increased tumor control in PDAC. This hypothesis was explored using both cell lines and in vivo human and mouse tumor models. Results: Our data show this treatment regimen significantly reduces regulatory T-cell, macrophage, and neutrophil infiltration and stromal fibrosis, enhances effector T-cell activation, and decreases tumor growth. Furthermore, our data show that depletion of regulatory T cells in combination with radiation reduces tumor growth and fibrosis. Conclusions: These are the first findings to suggest that in PDAC, ephrinB2–EphB4 interaction has a profibrotic, protumorigenic role, presenting a novel and promising therapeutic target.

The ability to visualize cell infiltration in experimental auto-immune encephalomyelitis (EAE), a well-known animal model for multiple sclerosis in humans, was investigated using a clinical 1.5-T magnetic resonance imaging (MRI) scanner, a custom-built, high-strength gradient coil insert, a 3-D fast imaging employing steady-state acquisition (FIESTA) imaging sequence and a superparamagnetic iron oxide (SPIO) contrast agent. An "active labeling" approach was used with SPIO administered intravenously during inflammation in EAE. Our results show that small, discrete regions of signal void corresponding to iron accumulation in EAE brain can be detected using FIESTA at 1.5 T. This work provides early evidence that cellular abnormalities that are the basis of diseases can be probed using cellular MRI and supports our earlier work which indicates that tracking of iron-labeled cells will be possible using clinical MR scanners.

Anaplastic thyroid cancer (ATC) is an aggressive and highly lethal disease with poor outcomes and resistance to therapy. Despite multimodality treatment, including radiation therapy and chemotherapy, response rates remain <15%, with a median time to progression of less than three months. Recent advances in radiotherapy (RT) delivery and gene-expression profiling may help guide patient selection for personalized therapy. The purpose of this study was to characterize the response to radiation in a panel of ATC cell lines and to test alternative RT fractionation schedules for overcoming radioresistance.The cellular response to radiation was characterized based on clonogenic assays. Radiation response was correlated with microarray gene-expression data. Hypofractionated and conventional RT was tested in an orthotopic ATC tumor model, and tumor growth was assayed locally and distantly with in vivo and ex vivo bioluminescence imaging.A spectrum of radiosensitivities was observed in ATC cell lines. Radioresistant cell lines had higher levels of CXCR4 compared to radiosensitive cell lines. Compared to conventionally fractionated RT, hypofractionated RT resulted in significantly improved tumor growth delay, decreased regional and distant metastases, and improved overall survival.The findings demonstrate the heterogeneity of response to radiation in ATC tumors and the superiority of hypofractionated RT in improving local control, metastatic spread, and survival in preclinical models. These data support the design of clinical trials targeting radioresistant pathways in combination with hypofractionated RT.

Medulloblastoma is one of the most common types of pediatric brain tumor characterized by the subpopulation of cells that exhibit high invasive potential and radioresistant properties. In addition, dysregulated function and signaling by Eph family of receptors have been shown to impart pro-tumorigenic characteristics in this brain malignancy. In the current study, we investigated whether EphB2 knockdown in combination with radiation can alter invasiveness and decrease medulloblastoma tumor growth or viability in vitro.The expression of EphB2 receptor was analyzed by immunohistochemistry and Western blotting. Microarray analysis and mRNA analysis was performed on medulloblastoma patient datasets and compared to the normal cerebellum. The radiosensitization effect following EphB2 knockdown was determined by clonogenic assay in human medulloblastoma cells. Effects of EphB2-siRNA in absence or presence of radiation on cell cycle distribution, cell viability, and invasion were analyzed by flow cytometry, MTT assay, trypan blue exclusion assay, xcelligence system, and Western blotting.We observed that EphB2 is expressed in both medulloblastoma cell lines and patient samples and its downregulation sensitized these cells to radiation as evident by decreased clonogenic survival fractions. EphB2 expression was also high across different medulloblastoma subgroups compared to normal cerebellum. The radiosensitization effect observed following EphB2 knockdown was in part mediated by enhanced G2/M cell cycle arrest. We also found that the combined approach of EphB2 knockdown and radiation exposure significantly reduced overall cell viability in medulloblastoma cells compared to control groups. Similar results were obtained in the xcelligence-based invasion assay. Western blot analysis also demonstrated changes in the protein expression of cell proliferation, cell survival, and invasion molecules in the combination group versus others.Overall, our findings indicate that specific targeting of EphB2 receptor in combination with radiation may serve as an effective therapeutic strategy in medulloblastoma. Future studies are warranted to test the efficacy of this approach in in vivo preclinical models.

Extracapsular extension (ECE) in regional lymph nodes and positive surgical margins (PSM) are considered high-risk adverse pathologic features in patients with oropharyngeal squamous cell carcinoma (OPSCC) that each constitute an indication for postoperative adjuvant chemoradiation. We identify pre-operative clinical factors that can predict post-operative ECE and/or PSM and create a nomogram to help clinical decision making.Adult patients with non-metastatic OPSCC with initial surgical treatment and confirmed HPV status diagnosed between 2010 and 2014 were selected from the National Cancer Database. Clinical staging was modified to American Joint Committee on Cancer 8th edition parameters. Logistic regression was used for multivariate analysis to identify predictors of pathologic ECE and/or PSM.5065 patients were included. 47.5% of the 3336 HPV-positive (HPV+) patients had ECE/PSM. 40.4% of the 1729 HPV-negative (HPV-) patients with had ECE/PSM. A model was built that included age, clinical ECE, tumor grade, and clinical T and N staging for HPV+ patients. Increasing N-classification was highly predictive of pathologic ECE and/or PSM (N1 OR = 3.6, N2 OR = 7.0, N3 OR = 11.2, p < 0.01). Clinical ECE (OR = 4.1, p < 0.01), tumor grade (ORs 2.2-4.4 with p < 0.05), and increasing clinical T-classification (ORs 1.2-1.8, p < 0.05) were also associated with ECE and/or PSM. A similar model was built for HPV- with similar predictive capability. Two internally validated nomograms were designed that demonstrated good discrimination (HPV+ AUC = 0.66, 95% CI: 0.64-0.68, and HPV- AUC = 0.70, 95% CI: 0.67-0.72) and good calibration (goodness-of-fit statistic of HPV+ 6.32, p = 0.61 and HPV- 11.66, p = 0.17).These are the first nomograms designed to help predict ECE or PSM for both HPV+ and HPV- OPSCC. The nomograms can facilitate shared decision-making between clinicians and patients as they consider upfront treatment selection for OPSCC.

Abstract The aggressive nature of glioblastoma multiforme (GBM) may be attributed to the dysregulation of pathways driving both proliferation and invasion. EphrinB2, a membrane‐bound ligand for some of the Eph receptors, has emerged as a critical target regulating these pathways. In this study, we investigated the role of ephrinB2 in regulating proliferation and invasion in GBM using intracranial and subcutaneous xenograft models. The Cancer Genome Atlas analysis suggested high transcript and low methylation levels of ephrinB2 as poor prognostic indicators in GBM, consistent with its role as an oncogene. EphrinB2 knockdown, however, increased tumor growth, an effect that was reversed by ephrinB2 Fc protein. This was associated with EphB4 receptor activation, consistent with the data showing a significant decrease in tumor growth with ephrinB2 overexpression. Mechanistic analyses showed that ephrinB2 knockdown has anti‐invasive but pro‐proliferative effects in GBM. EphB4 stimulation following ephrinB2 Fc treatment in ephrinB2 knockdown tumors was shown to impart strong anti‐proliferative and anti‐invasive effects, which correlated with decrease in PCNA, p‐ERK, vimentin, Snail, Fak, and increase in the E‐cadherin levels. Overall, our study suggests that ephrinB2 cannot be used as a sole therapeutic target. Concomitant inhibition of ephrinB2 signaling with EphB4 activation is required to achieve maximal therapeutic benefit in GBM.

Treatment for pediatric posterior fossa group A (PFA) ependymoma with gain of chromosome 1q (1q+) has not improved over the past decade owing partially to lack of clinically relevant models. We described the first 2 1q+ PFA cell lines, which have significantly enhanced our understanding of PFA tumor biology and provided a tool to identify specific 1q+ PFA therapies. However, cell lines do not accurately replicate the tumor microenvironment. Our present goal is to establish patient-derived xenograft (PDX) mouse models.Disaggregated tumors from 2 1q+ PFA patients were injected into the flanks of NSG mice. Flank tumors were then transplanted into the fourth ventricle or lateral ventricle of NSG mice. Characterization of intracranial tumors was performed using imaging, histology, and bioinformatics.MAF-811_XC and MAF-928_XC established intracranially within the fourth ventricle and retained histological, methylomic, and transcriptomic features of primary patient tumors. We tested the feasibility of treating PDX mice with fractionated radiation or chemotherapy. Mice tolerated radiation despite significant tumor burden, and follow-up imaging confirmed radiation can reduce tumor size. Treatment with fluorouracil reduced tumor size but did not appear to prolong survival.MAF-811_XC and MAF-928_XC are novel, authentic, and reliable models for studying 1q+ PFA in vivo. Given the successful response to radiation, these models will be advantageous for testing clinically relevant combination therapies to develop future clinical trials for this high-risk subgroup of pediatric ependymoma.

STAT3 signaling has been shown to regulate cellular function and cytokine production in the tumor microenvironment (TME). Within the head and neck squamous cell carcinoma (HNSCC) TME, we previously showed that therapeutic targeting of STAT3 in combination with radiation resulted in improved tumor growth delay. However, given the independent regulatory effects STAT3 has on anti-tumor immunity, we aimed to decipher the effects of individually targeting STAT3 in the cancer cell, regulatory T cells (Tregs), and natural killer (NK) cell compartments in driving tumor growth and resistance to therapy in HNSCCs. We utilized a CRISPR knockout system for genetic deletion of STAT3 within the cancer cell as well as two genetic knockout mouse models, FoxP3-Cre/STAT3 fl and NKp46-Cre/STAT3 fl, for Tregs and NK cell targeting, respectively. Our data revealed differences in development of resistance to treatment with STAT3 CRISPR knockout in the cancer cell, driven by differential recruitment of immune cells. Knockout of STAT3 in Tregs overcomes this resistance and results in Treg reprogramming and recruitment and activation of antigen-presenting cells. In contrast, knockout of STAT3 in the NK cell compartment results in NK cell inactivation and acceleration of tumor growth. These data underscore the complex interplay between the cancer cell and the immune TME and carry significant implications for drug targeting and design of combination approaches in HNSCCs. STAT3 signaling has been shown to regulate cellular function and cytokine production in the tumor microenvironment (TME). Within the head and neck squamous cell carcinoma (HNSCC) TME, we previously showed that therapeutic targeting of STAT3 in combination with radiation resulted in improved tumor growth delay. However, given the independent regulatory effects STAT3 has on anti-tumor immunity, we aimed to decipher the effects of individually targeting STAT3 in the cancer cell, regulatory T cells (Tregs), and natural killer (NK) cell compartments in driving tumor growth and resistance to therapy in HNSCCs. We utilized a CRISPR knockout system for genetic deletion of STAT3 within the cancer cell as well as two genetic knockout mouse models, FoxP3-Cre/STAT3 fl and NKp46-Cre/STAT3 fl, for Tregs and NK cell targeting, respectively. Our data revealed differences in development of resistance to treatment with STAT3 CRISPR knockout in the cancer cell, driven by differential recruitment of immune cells. Knockout of STAT3 in Tregs overcomes this resistance and results in Treg reprogramming and recruitment and activation of antigen-presenting cells. In contrast, knockout of STAT3 in the NK cell compartment results in NK cell inactivation and acceleration of tumor growth. These data underscore the complex interplay between the cancer cell and the immune TME and carry significant implications for drug targeting and design of combination approaches in HNSCCs.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Abstract Eph proteins have emerged as critical drivers affecting tumor growth and progression in human malignancies. Our The Cancer Genome Atlas (TCGA) data analysis showed that EphB3, a receptor tyrosine kinase, is frequently coamplified with PIK3CA in head and neck squamous cell carcinoma (HNSCC). We therefore hypothesized that EphB3 amplification plays a protumorigenic role in HNSCC and that EphB3 and PIK3CA are cooperating oncogenes that contribute toward its pathogenesis. This hypothesis was not experimentally supported, because EphB3 knockdown failed to alter HNSCC tumor cell growth in vitro or in vivo with an orthotopic model. However, responsiveness of EphB3 knockdown tumors to the PI3K inhibitor, BKM120, was significantly decreased in terms of both tumor growth delay and survival. This is correlated with an increase in prosurvival proteins, S6 and BcL-XL, in the EphB3 shRNA tumors treated with BKM120 compared with controls. We further observed that EphB3 knockdown resulted in increased migration in vitro and increased EMT gene signature in vivo. To explain these results, we examined EphB3 phosphorylation levels in HNSCC at baseline. Although total EphB3 levels were high, we found low phospho-EphB3 levels in HNSCCs. Forced EphB3 phosphorylation with an ephrin-B2–Fc fusion protein resulted in decreased HNSCC migration and cell growth, and enhanced response to BKM120 in vitro. These data collectively indicate that progression of HNSCC selects for low/inhibited EphB3 activity to enhance their survival and migratory abilities and decrease response to PI3K signaling. Therefore, strategies focused on activating EphB3 might be helpful to inhibit tumor growth and enhance sensitivity to PI3K inhibitors in HNSCC. Mol Cancer Ther; 17(9); 2049–59. ©2018 AACR.

Stereotactic ablative radiotherapy (SABR) has emerged as a highly promising treatment for medically inoperable early-stage non-small cell lung cancer patients. Treatment outcomes after SABR have been excellent compared to conventional fractionated radiotherapy (CFRT). However, the biological determinants of the response to ablative doses of radiation remain poorly characterized. Furthermore, there's little data on the cellular and molecular response of genetically distinct NSCLC subtypes to radiation. We assessed the response of 3 genetically distinct lung adenocarcinoma cell lines to ablative and fractionated ionizing radiation (AIR and FIR). We studied clonogenic survival, cell proliferation, migration, invasion, apoptosis and senescence. We also investigated the effect of AIR and FIR on the expression of pro-invasive proteins, epithelial-to-mesenchymal transition (EMT), extracellular signal-regulated kinases (ERK1/2) and the transmembrane receptor cMET. Our findings reveal that AIR significantly reduced cell proliferation and clonogenic survival compared to FIR in A549 cells only. This differential response was not observed in HCC827 or H1975 cells. AIR significantly enhanced the invasiveness of A549 cells, but not HCC827 or H1975 cells compared to FIR. Molecular analysis of pathways involved in cell proliferation and invasion revealed that AIR significantly reduced phosphorylation of ERK1/2 and upregulated cMET expression in A549 cells. Our results show a differential proliferative and invasive response to AIR that is dependent on genetic subtype and independent of intrinsic radioresistance. Further examination of these findings in a larger panel of NSCLC cell lines and in pre-clinical models is warranted for identification of biomarkers of tumor response to AIR.

Stereotactic ablative radiotherapy (SABR) is the main treatment for inoperable early-stage non-small cell lung cancer (NSCLC).Despite the widespread use of SABR, the biological determinants of response to SABR remain poorly investigated.We developed an orthotopic NSCLC animal model to study the response to clinicallyrelevant doses of SABR.Image-guided intra-thoracic injection of NSCLC cells was performed in the right lung of nude rats.A highly conformal dose of 34 Gy was delivered in a single fraction using clinical photon energies.Animals were sacrificed 10-60 days post treatment.Lung tumors were assessed for tumor differentiation, proliferation and invasiveness.An analysis of 770 cancer-related genes was performed on tumorderived cell lines from treated animals at early and late time points after SABR.The majority of animals receiving SABR demonstrated complete response (67%), while 33% demonstrated local failure.50% of animals with complete response failed distantly.Analysis of cancer-related genes revealed significant differences between tumors treated with SABR and untreated tumors.SABR significantly modulated expression of genes involved in adhesion, migration and angiogenesis.In particular, interleukin-8 (IL8) which plays a critical role in promoting tumor invasion was found to be secreted at high levels after SABR.In vitro invasion assays confirmed SABRinduced invasion and demonstrated induction of IL-8 secretion in multiple NSCLC cell lines.Our findings underscore the importance of developing targeted therapies that can circumvent the pro-invasive effects of SABR in NSCLC.

Resistance to radiation therapy (RT) remains an obstacle in HPV-negative head and neck squamous cell carcinomas (HNSCCs)-even with a combined RT-immunotherapy approach. Jak-Stat proteins have long been studied for both their immune regulatory role in the host immune response as well as their cancer cell signaling role in shaping the tumor microenvironment (TME). Here, we identify STAT1 as a mediator of radioresistance in HPV-negative preclinical mouse models of HNSCC, by which knockout of STAT1 in the cancer cell (STAT1 KO)-but not in the host-resulted in decreased tumor growth alongside increased immune activation. We show that RT increases STAT1/pSTAT1 expression, which may act as a marker of radioresistance. Whereas RT increased JAK-STAT and interferon (IFN) signaling, transcriptomic analysis revealed that STAT1 KO in the cancer cell resulted in decreased expression of IFN-associated genes of resistance. In vitro experiments showed that STAT1 KO increased T cell chemoattraction and decreased baseline growth. These results indicate that STAT1 may serve a tumor-promoting role in the cancer cell and will inform biomarker development and treatment regimens for HNSCC incorporating RT.

Objective Management of tracheal squamous cell carcinoma (TSCC) has been complicated by the lack of prognostic data and staging. We describe the epidemiology of TSCC and current treatment approaches. Methods Five hundred thirty‐two adult patients with primary TSCC from 2004 to 2012 in the National Cancer Database were identified. Demographic, clinical factors, and 5‐year overall survival were analyzed. Staging was classified as localized, regional extension, and distant spread. Treatment modality was defined as “no treatment (NT),” “limited surgery (LS),” “curative surgery (CS),” “LS with any adjuvant therapy (AT) (LS+AT),” “CS with AT (CS+AT),” “radiation therapy (RT),” or “chemoradiation (CRT).” Results Overall survival was 25%. Majority of cases were males, white, and occurred in sixth/seventh decades. Twenty‐six percent of cases received CRT, 20% underwent LS+AT or CS+AT, 20% underwent LS or CS only, and 17% underwent RT alone. On multivariate analysis, CS (HR 0.42, 95% CI: 0.26–0.69), CS+AT (HR 0.44, 95% CI: 0.36–0.77), CRT (HR 0.48, 95% CI: 0.35–0.67), and RT (HR, 0.66 95% CI: 0.46–0.94) were associated with decreased likelihood of death compared to NT. Elderly patients and those with poor performance status had worse outcomes even on multivariate analysis. Conclusions TSCC is increasingly treated with surgery and systemic therapy in addition to RT, with improved survival outcomes. CS, CS+AT, CRT, or RT provided improved survival advantage in patients with variable levels of improvement based on the extent of the disease. Prospective trials would help differentiate survival advantages between treatment modalities. Patients’ goals of care, comorbidities, and age should be considered when deciding appropriate treatment recommendations. Level of Evidence NA Laryngoscope , 130:405–412, 2020

70 Background: Head and neck tumors are highly enriched in regulatory T cells which dampen the response to radiotherapy by creating an immune-inhibitory microenvironment. We explored mechanisms of Treg infiltration and assessed their modulation by RT in murine models of HNSCC. Methods: Mechanisms of Treg infiltration were investigated in murine HNSCC tumors using whole genome sequencing and flow cytometry. Mice were treated with anti-CTLA-4, anti-CD-25 and/or anti-PD-L1 alone and in combination with RT. Tumor growth and survival were assessed. Flow cytometry was used to assess phenotypic and functional changes in intratumoral T cell populations. Multiplex ELISA was performed for assessment of cytokines. RNA Sequencing was performed to interrogate mechanisms of response and resistance to treatment. Results: Treatment with anti-CD-25 concurrently with RT led to significant tumor growth delay, enhanced T cell cytotoxicity, decreased Tregs and improved survival. In contrast CTLA-4 blockade did not affect tumor growth or survival. Treg depletion induced an influx of CD8 and CD4 T cells when combined with RT. In addition, Treg depletion in combination with RT transformed myeloid populations decreasing M2 macrophages and MDSCs and increasing M1 macrophages. Mechanistically, tumors secrete CCL20, a potent Treg chemoattractant responsible for creating a highly immunuosuppressive tumor microenvironment and potentially responsible for treatment resistance. Conclusions: These data reveal a critical role for regulatory T cells in mediating resistance to RT. Targeted depletion of Tregs represents an important mechanism of sensitizing tumors to RT. Our data support the design of clinical trials integrating targeted Treg inhibitors in the standard of care for cancer patients receiving RT.

&lt;p&gt;qPCR analysis shows elevated expression of EphB4 on intratumoral macrophages harvested from Ly2 tumors compared to ephrin-B2.&lt;/p&gt;

&lt;p&gt;(A) ELISA assay measuring the concentration of TNYL-RAW-Fc protein in plasma 7 days after hydrodynamic injection of the TNYL-RAW-Fc plasmid. Data represent mean {plus minus} SD from measurements using different mice (n=2). (B) Inhibition of EphB4-ephrin-B2 signaling by TNYL-RAW-Fc does not affect immune cell populations in the circulation of Ly2 tumor-bearing mice.&lt;/p&gt;

&lt;p&gt;IHC staining confirming expression of ephrin-B2 in areas stained with fibroblast-expressing α-Smooth muscle actin (α-SMA)&lt;/p&gt;

&lt;p&gt;Systemic depletion of CD8+ T cells results in increased tumor growth in Ly2 tumors treated with TNYL-RAW-Fc (A). Confirmation of CD8+ T cell depletion by flow cytometry (B). Student's t-test or one-way ANOVA was used to calculate the significance of the differences between the groups.&lt;/p&gt;

&lt;p&gt;IHC staining confirming expression of ephrin-B2 in areas stained with fibroblast-expressing α-Smooth muscle actin (α-SMA)&lt;/p&gt;

&lt;p&gt;Mice implanted with PDX tumors show significantly reduced uptake of supra-paramagnetic iron oxide nanoparticles (SPIO) in the combined sEphB4-HSA and RT group compared to the groups treated with RT alone or with sEphB4-HSA alone in DCE-MRI imaging (A-B). Delta T2 represents magnitude of decrease in T2 signal. One-way ANOVA was used to calculate the significance of the differences between groups. CIBERSORT analysis of a HNSCC TCGA dataset shows that patients with low M2:M1 ratio have improved overall survival (C) and disease-free survival (D). Analysis of HNSCC TCGA showing correlation between M2-related marker-HRH1 and ephrin-B2 (EFNB2) (E), EphB4 (F).&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;Identifying targets present in the tumor microenvironment that contribute to immune evasion has become an important area of research. In this study, we identified EphB4–ephrin-B2 signaling as a regulator of both innate and adaptive components of the immune system. EphB4 belongs to receptor tyrosine kinase family that interacts with ephrin-B2 ligand at sites of cell–cell contact, resulting in bidirectional signaling. We found that EphB4–ephrin-B2 inhibition alone or in combination with radiation (RT) reduced intratumoral regulatory T cells (Tregs) and increased activation of both CD8&lt;sup&gt;+&lt;/sup&gt; and CD4&lt;sup&gt;+&lt;/sup&gt;Foxp3&lt;sup&gt;−&lt;/sup&gt; T cells compared with the control group in an orthotopic head and neck squamous cell carcinoma (HNSCC) model. We also compared the effect of EphB4–ephrin-B2 inhibition combined with RT with combined anti-PDL1 and RT and observed similar tumor growth suppression, particularly at early time-points. A patient-derived xenograft model showed reduction of tumor-associated M2 macrophages and favored polarization towards an antitumoral M1 phenotype following EphB4–ephrin-B2 inhibition with RT. &lt;i&gt;In vitro&lt;/i&gt;, EphB4 signaling inhibition decreased Ki67-expressing Tregs and Treg activation compared with the control group. Overall, our study is the first to implicate the role of EphB4–ephrin-B2 in tumor immune response. Moreover, our findings suggest that EphB4–ephrin-B2 inhibition combined with RT represents a potential alternative for patients with HNSCC and could be particularly beneficial for patients who are ineligible to receive or cannot tolerate anti-PDL1 therapy.&lt;/p&gt;Significance:&lt;p&gt;These findings present EphB4–ephrin-B2 inhibition as an alternative to anti-PDL1 therapeutics that can be used in combination with radiation to induce an effective antitumor immune response in patients with HNSCC.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Identifying targets present in the tumor microenvironment that contribute to immune evasion has become an important area of research. In this study, we identified EphB4–ephrin-B2 signaling as a regulator of both innate and adaptive components of the immune system. EphB4 belongs to receptor tyrosine kinase family that interacts with ephrin-B2 ligand at sites of cell–cell contact, resulting in bidirectional signaling. We found that EphB4–ephrin-B2 inhibition alone or in combination with radiation (RT) reduced intratumoral regulatory T cells (Tregs) and increased activation of both CD8&lt;sup&gt;+&lt;/sup&gt; and CD4&lt;sup&gt;+&lt;/sup&gt;Foxp3&lt;sup&gt;−&lt;/sup&gt; T cells compared with the control group in an orthotopic head and neck squamous cell carcinoma (HNSCC) model. We also compared the effect of EphB4–ephrin-B2 inhibition combined with RT with combined anti-PDL1 and RT and observed similar tumor growth suppression, particularly at early time-points. A patient-derived xenograft model showed reduction of tumor-associated M2 macrophages and favored polarization towards an antitumoral M1 phenotype following EphB4–ephrin-B2 inhibition with RT. &lt;i&gt;In vitro&lt;/i&gt;, EphB4 signaling inhibition decreased Ki67-expressing Tregs and Treg activation compared with the control group. Overall, our study is the first to implicate the role of EphB4–ephrin-B2 in tumor immune response. Moreover, our findings suggest that EphB4–ephrin-B2 inhibition combined with RT represents a potential alternative for patients with HNSCC and could be particularly beneficial for patients who are ineligible to receive or cannot tolerate anti-PDL1 therapy.&lt;/p&gt;Significance:&lt;p&gt;These findings present EphB4–ephrin-B2 inhibition as an alternative to anti-PDL1 therapeutics that can be used in combination with radiation to induce an effective antitumor immune response in patients with HNSCC.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Total number of CD4+T cells remain unchanged following Control-Fc and ephrin-B2-Fc (20 μg/ml) treatment of T cells (A). Correlation analysis was also performed between TGF-β and EFNB2, EphB4 on R2 analysis platform (B-C). Alterations of cell growth and survival markers in western blots performed using lysates of T cells treated with ephrin-B2-Fc and control-Fc (D). These samples were run on the same gel.&lt;/p&gt;

&lt;p&gt;Total number of CD4+T cells remain unchanged following Control-Fc and ephrin-B2-Fc (20 μg/ml) treatment of T cells (A). Correlation analysis was also performed between TGF-β and EFNB2, EphB4 on R2 analysis platform (B-C). Alterations of cell growth and survival markers in western blots performed using lysates of T cells treated with ephrin-B2-Fc and control-Fc (D). These samples were run on the same gel.&lt;/p&gt;

&lt;p&gt;qPCR analysis shows elevated expression of EphB4 on intratumoral macrophages harvested from Ly2 tumors compared to ephrin-B2.&lt;/p&gt;

&lt;p&gt;Immunofluorescence staining showing co-expression of EphB4 and pan-keratin in Ly2 tumors.&lt;/p&gt;

&lt;p&gt;Blockade of EphB4-ephrin-B2 interaction combined with RT suppresses tumor growth in HNSCC tumors&lt;/p&gt;

&lt;p&gt;(A) ELISA assay measuring the concentration of TNYL-RAW-Fc protein in plasma 7 days after hydrodynamic injection of the TNYL-RAW-Fc plasmid. Data represent mean {plus minus} SD from measurements using different mice (n=2). (B) Inhibition of EphB4-ephrin-B2 signaling by TNYL-RAW-Fc does not affect immune cell populations in the circulation of Ly2 tumor-bearing mice.&lt;/p&gt;

&lt;p&gt;Systemic depletion of CD8+ T cells results in increased tumor growth in Ly2 tumors treated with TNYL-RAW-Fc (A). Confirmation of CD8+ T cell depletion by flow cytometry (B). Student's t-test or one-way ANOVA was used to calculate the significance of the differences between the groups.&lt;/p&gt;

&lt;p&gt;Immunofluorescence staining showing co-expression of EphB4 and pan-keratin in Ly2 tumors.&lt;/p&gt;

&lt;p&gt;Mice implanted with PDX tumors show significantly reduced uptake of supra-paramagnetic iron oxide nanoparticles (SPIO) in the combined sEphB4-HSA and RT group compared to the groups treated with RT alone or with sEphB4-HSA alone in DCE-MRI imaging (A-B). Delta T2 represents magnitude of decrease in T2 signal. One-way ANOVA was used to calculate the significance of the differences between groups. CIBERSORT analysis of a HNSCC TCGA dataset shows that patients with low M2:M1 ratio have improved overall survival (C) and disease-free survival (D). Analysis of HNSCC TCGA showing correlation between M2-related marker-HRH1 and ephrin-B2 (EFNB2) (E), EphB4 (F).&lt;/p&gt;

&lt;p&gt;Blockade of EphB4-ephrin-B2 interaction combined with RT suppresses tumor growth in HNSCC tumors&lt;/p&gt;

&lt;p&gt;Supplementary Figure S3 demonstrates doxycyline-dependent modulation of SMAD4&lt;/p&gt;

&lt;p&gt;Supplementary Figure S6 demonstrates the influence of SMAD4 status on DNA damage response to RT + olaparib&lt;/p&gt;

&lt;p&gt;Supplementary Figure S4 demonstrates the influence of SMAD4 expression in CAL27 xenograft growth&lt;/p&gt;

&lt;p&gt;Supplementary Figure S5 demonstrates the influence of SMAD4 status on proliferation (with and without treatment)&lt;/p&gt;

&lt;p&gt;Supplementary Figure S1 demonstrates the correlation between SMAD4 levels and FANC/BRCA family genes (in TCGA analyses)&lt;/p&gt;

&lt;p&gt;Supplementary Table S1 lists reagents used&lt;/p&gt;

&lt;p&gt;Supplementary Figure S2 demonstrates the enrichment of Fanoni Anemia pathway genes as a function of SMAD4 levels (in TCGA analyses)&lt;/p&gt;

&lt;p&gt;Levels of EGFR expression in HNSCC PDX tumors&lt;/p&gt;

&lt;p&gt;Blockade of EphB4-ephrin-B2 enhances response to EGFR inhibitor (cetuximab) and RT resulting in significant delay in tumor growth and enhanced survival in HNSCC PDX models. Tumor growth volumes are displayed in CUHN013 (A) and CUHN004 (B) tumors including cisplatin-RT treated groups in the absence and presence of sEphB4-HSA. Cisplatin-RT treated groups did not show any significant changes in tumor growth in the absence and presence of sEphB4-HSA in CUHN013 tumors. Survival graphs are shown for CUHN013 (C) and CUHN004 (D) tumors.&lt;/p&gt;

&lt;p&gt;Supplementary Figure S1 demonstrates the correlation between SMAD4 levels and FANC/BRCA family genes (in TCGA analyses)&lt;/p&gt;

&lt;p&gt;Supplementary Figure S6 demonstrates the influence of SMAD4 status on DNA damage response to RT + olaparib&lt;/p&gt;

&lt;p&gt;Supplementary Figure S5 demonstrates the influence of SMAD4 status on proliferation (with and without treatment)&lt;/p&gt;

&lt;p&gt;Schema Utilizing Time-to-Event Continual Reassessment Method (TITE-CRM) Design&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; The clinical success of targeted therapies such as cetuximab and radiotherapy (RT) is hampered by the low response rates and development of therapeutic resistance. In the current study, we investigated the involvement of EphB4–ephrin-B2 protumorigenic signaling in mediating resistance to EGFR inhibition and RT in head and neck cancers.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; We used patient-derived xenograft (PDX) models of head and neck squamous cell carcinoma (HNSCC) and HNSCC cell lines to test our hypothesis. Tumor tissues were subjected to PhosphoRTK array, and Western blotting to detect changes in EphB4–ephrin-B2 targets. mRNA sequencing and microarray data analysis were performed on PDX tumors and HNSCC cell lines, respectively, to determine differences in gene expression of molecules involved in tumor cell growth, proliferation, and survival pathways. Effects on cell growth were determined by MTT assay on HNSCC cells downregulated for EphB4/ephrin-B2 expression, with and without EGFR inhibitor and radiation.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; Our data from locally advanced HNSCC patients treated with standard-of-care definitive chemo-RT show elevated EphB4 and ephrin-B2 levels after failure of treatment. We observed significant response toward cetuximab and RT following EphB4–ephrin-B2 inhibition, resulting in improved survival in tumor-bearing mice. Tumor growth inhibition was accompanied by a decrease in the levels of proliferation and prosurvival molecules and increased apoptosis.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Our findings underscore the importance of adopting rational drug combinations to enhance therapeutic effect. Our study documenting enhanced response of HNSCC to cetuximab-RT with EphB4–ephrin-B2 blockade has the potential to translate into the clinic to benefit this patient population. &lt;i&gt;Clin Cancer Res; 24(18); 4539–50. ©2018 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Our goal was to evaluate the safety and toxicity of combining a PARP inhibitor, olaparib, with cetuximab and fractionated intensity-modulated radiotherapy for patients with locally advanced head and neck cancer and heavy smoking histories.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Patients and Methods:&lt;/b&gt; Patients with ≥10 packs/year history of smoking were treated with olaparib at doses ranging from 25–200 mg orally twice daily beginning approximately 10 days prior to initiation of and with concurrent radiation (69.3 Gy in 33 fractions) using a time-to-event continual reassessment method model. Cetuximab was administered starting approximately 5 days prior to radiation per standard of care.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; A total of 16 patients were entered onto the study, with 15 evaluable for acute toxicity. The most common treatment-related grade 3–4 side effects were radiation dermatitis and mucositis (38% and 69%, respectively). The MTD was determined to be 50 mg orally twice daily, but the recommended phase II dose was deemed to be 25 mg orally twice daily. At a median follow-up of 26 months, the actuarial median overall survival was 37 months, but was not reached for other endpoints. Two-year overall survival, progression-free survival, local control, and distant control rates were 72%, 63%, 72%, and 79%, respectively. Patients who continued to smoke during therapy experienced higher recurrence rates. &lt;i&gt;MYC&lt;/i&gt; and &lt;i&gt;KMT2A&lt;/i&gt; were identified as potential correlatives of response on gene amplification and mutational analysis.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Olaparib at 25 mg orally twice daily with concurrent cetuximab and radiation was well tolerated with reduced dermatitis within the radiation field. Response rates were promising for this high-risk population. &lt;i&gt;Clin Cancer Res; 24(20); 4949–59. ©2018 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Radiotherapy (RT) can transform the immune landscape and render poorly immunogenic tumors sensitive to PD-L1 inhibition. Here, we established that the response to combined RT and PD-L1 inhibition is transient and investigated mechanisms of resistance.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; Mechanisms of resistance to RT and PD-L1 blockade were investigated in orthotopic murine head and neck squamous cell carcinoma (HNSCC) tumors using mass cytometry and whole-genome sequencing. Mice were treated with anti–PD-L1 or anti–TIM-3 alone and in combination with and without RT. Tumor growth and survival were assessed. Flow cytometry was used to assess phenotypic and functional changes in intratumoral T-cell populations. Depletion of regulatory T cells (Treg) was performed using anti-CD25 antibody.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; We show that the immune checkpoint receptor, TIM-3, is upregulated on CD8 T cells and Tregs in tumors treated with RT and PD-L1 blockade. Treatment with anti–TIM-3 concurrently with anti–PD-L1 and RT led to significant tumor growth delay, enhanced T-cell cytotoxicity, decreased Tregs, and improved survival in orthotopic models of HNSCC. Despite this treatment combination, the response was not durable, and analysis of relapsed tumors revealed resurgence of Tregs. Targeted Treg depletion, however, restored antitumor immunity in mice treated with RT and dual immune checkpoint blockade and resulted in tumor rejection and induction of immunologic memory.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; These data reveal multiple layers of immune regulation that can promote tumorigenesis and the therapeutic potential of sequential targeting to overcome tumor resistance mechanisms. We propose that targeted Treg inhibitors may be critical for achieving durable tumor response with combined radiotherapy and immunotherapy. &lt;i&gt;Clin Cancer Res; 24(21); 5368–80. ©2018 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; The clinical success of targeted therapies such as cetuximab and radiotherapy (RT) is hampered by the low response rates and development of therapeutic resistance. In the current study, we investigated the involvement of EphB4–ephrin-B2 protumorigenic signaling in mediating resistance to EGFR inhibition and RT in head and neck cancers.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; We used patient-derived xenograft (PDX) models of head and neck squamous cell carcinoma (HNSCC) and HNSCC cell lines to test our hypothesis. Tumor tissues were subjected to PhosphoRTK array, and Western blotting to detect changes in EphB4–ephrin-B2 targets. mRNA sequencing and microarray data analysis were performed on PDX tumors and HNSCC cell lines, respectively, to determine differences in gene expression of molecules involved in tumor cell growth, proliferation, and survival pathways. Effects on cell growth were determined by MTT assay on HNSCC cells downregulated for EphB4/ephrin-B2 expression, with and without EGFR inhibitor and radiation.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; Our data from locally advanced HNSCC patients treated with standard-of-care definitive chemo-RT show elevated EphB4 and ephrin-B2 levels after failure of treatment. We observed significant response toward cetuximab and RT following EphB4–ephrin-B2 inhibition, resulting in improved survival in tumor-bearing mice. Tumor growth inhibition was accompanied by a decrease in the levels of proliferation and prosurvival molecules and increased apoptosis.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Our findings underscore the importance of adopting rational drug combinations to enhance therapeutic effect. Our study documenting enhanced response of HNSCC to cetuximab-RT with EphB4–ephrin-B2 blockade has the potential to translate into the clinic to benefit this patient population. &lt;i&gt;Clin Cancer Res; 24(18); 4539–50. ©2018 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;&lt;i&gt;SMAD4&lt;/i&gt; loss causes genomic instability and the initiation/progression of head and neck squamous cell carcinoma (HNSCC). Here, we study whether &lt;i&gt;SMAD4&lt;/i&gt; loss sensitizes HNSCCs to olaparib (PARP inhibitor) in combination with radiotherapy (RT).&lt;/p&gt;Experimental Design:&lt;p&gt;We analyzed HNSCC The Cancer Genome Atlas data for &lt;i&gt;SMAD4&lt;/i&gt; expression in association with &lt;i&gt;FANC/BRCA&lt;/i&gt; family gene expression. Human HNSCC cell lines were screened for sensitivity to olaparib. Isogenic HNSCC cell lines were generated to restore or reduce SMAD4 expression and treated with olaparib, radiation, or the combination. HNSCC pretreatment specimens from a phase I trial investigating olaparib were analyzed.&lt;/p&gt;Results:&lt;p&gt;&lt;i&gt;SMAD4&lt;/i&gt; levels correlated with levels of &lt;i&gt;FANC/BRCA&lt;/i&gt; genes in HNSCC. HNSCC cell lines with &lt;i&gt;SMAD4&lt;/i&gt; homozygous deletion were sensitive to olaparib. &lt;i&gt;In vivo&lt;/i&gt;, olaparib or RT monotherapy reduced tumor volumes in &lt;i&gt;SMAD4&lt;/i&gt;-mutant but not &lt;i&gt;SMAD4&lt;/i&gt;-positive tumors. Olaparib with RT dual therapy sustained tumor volume reduction in &lt;i&gt;SMAD4&lt;/i&gt;-deficient (mutant or knockdown) xenografts, which exhibited increased DNA damage and cell death compared with vehicle-treated tumors. &lt;i&gt;In vitro&lt;/i&gt;, olaparib alone or in combination with radiation caused lower clonogenic survival, more DNA damage–associated cell death, and less proliferation in &lt;i&gt;SMAD4&lt;/i&gt;-deficient cells than in &lt;i&gt;SMAD4&lt;/i&gt;-positive (endogenous SMAD4 or transduced SMAD4) cells. Applicable to clinic, 5 out of 6 SMAD4-negative HNSCCs and 4 out of 8 SMAD4-positive HNSCCs responded to a standard treatment plus olaparib in a phase I clinical trial, and SMAD4 protein levels inversely correlated with DNA damage.&lt;/p&gt;Conclusions:&lt;p&gt;&lt;i&gt;SMAD4&lt;/i&gt; levels are causal in determining sensitivity to PARP inhibition in combination with RT in HNSCCs.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Radiotherapy (RT) can transform the immune landscape and render poorly immunogenic tumors sensitive to PD-L1 inhibition. Here, we established that the response to combined RT and PD-L1 inhibition is transient and investigated mechanisms of resistance.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; Mechanisms of resistance to RT and PD-L1 blockade were investigated in orthotopic murine head and neck squamous cell carcinoma (HNSCC) tumors using mass cytometry and whole-genome sequencing. Mice were treated with anti–PD-L1 or anti–TIM-3 alone and in combination with and without RT. Tumor growth and survival were assessed. Flow cytometry was used to assess phenotypic and functional changes in intratumoral T-cell populations. Depletion of regulatory T cells (Treg) was performed using anti-CD25 antibody.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; We show that the immune checkpoint receptor, TIM-3, is upregulated on CD8 T cells and Tregs in tumors treated with RT and PD-L1 blockade. Treatment with anti–TIM-3 concurrently with anti–PD-L1 and RT led to significant tumor growth delay, enhanced T-cell cytotoxicity, decreased Tregs, and improved survival in orthotopic models of HNSCC. Despite this treatment combination, the response was not durable, and analysis of relapsed tumors revealed resurgence of Tregs. Targeted Treg depletion, however, restored antitumor immunity in mice treated with RT and dual immune checkpoint blockade and resulted in tumor rejection and induction of immunologic memory.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; These data reveal multiple layers of immune regulation that can promote tumorigenesis and the therapeutic potential of sequential targeting to overcome tumor resistance mechanisms. We propose that targeted Treg inhibitors may be critical for achieving durable tumor response with combined radiotherapy and immunotherapy. &lt;i&gt;Clin Cancer Res; 24(21); 5368–80. ©2018 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Our goal was to evaluate the safety and toxicity of combining a PARP inhibitor, olaparib, with cetuximab and fractionated intensity-modulated radiotherapy for patients with locally advanced head and neck cancer and heavy smoking histories.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Patients and Methods:&lt;/b&gt; Patients with ≥10 packs/year history of smoking were treated with olaparib at doses ranging from 25–200 mg orally twice daily beginning approximately 10 days prior to initiation of and with concurrent radiation (69.3 Gy in 33 fractions) using a time-to-event continual reassessment method model. Cetuximab was administered starting approximately 5 days prior to radiation per standard of care.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; A total of 16 patients were entered onto the study, with 15 evaluable for acute toxicity. The most common treatment-related grade 3–4 side effects were radiation dermatitis and mucositis (38% and 69%, respectively). The MTD was determined to be 50 mg orally twice daily, but the recommended phase II dose was deemed to be 25 mg orally twice daily. At a median follow-up of 26 months, the actuarial median overall survival was 37 months, but was not reached for other endpoints. Two-year overall survival, progression-free survival, local control, and distant control rates were 72%, 63%, 72%, and 79%, respectively. Patients who continued to smoke during therapy experienced higher recurrence rates. &lt;i&gt;MYC&lt;/i&gt; and &lt;i&gt;KMT2A&lt;/i&gt; were identified as potential correlatives of response on gene amplification and mutational analysis.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Olaparib at 25 mg orally twice daily with concurrent cetuximab and radiation was well tolerated with reduced dermatitis within the radiation field. Response rates were promising for this high-risk population. &lt;i&gt;Clin Cancer Res; 24(20); 4949–59. ©2018 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;&lt;i&gt;SMAD4&lt;/i&gt; loss causes genomic instability and the initiation/progression of head and neck squamous cell carcinoma (HNSCC). Here, we study whether &lt;i&gt;SMAD4&lt;/i&gt; loss sensitizes HNSCCs to olaparib (PARP inhibitor) in combination with radiotherapy (RT).&lt;/p&gt;Experimental Design:&lt;p&gt;We analyzed HNSCC The Cancer Genome Atlas data for &lt;i&gt;SMAD4&lt;/i&gt; expression in association with &lt;i&gt;FANC/BRCA&lt;/i&gt; family gene expression. Human HNSCC cell lines were screened for sensitivity to olaparib. Isogenic HNSCC cell lines were generated to restore or reduce SMAD4 expression and treated with olaparib, radiation, or the combination. HNSCC pretreatment specimens from a phase I trial investigating olaparib were analyzed.&lt;/p&gt;Results:&lt;p&gt;&lt;i&gt;SMAD4&lt;/i&gt; levels correlated with levels of &lt;i&gt;FANC/BRCA&lt;/i&gt; genes in HNSCC. HNSCC cell lines with &lt;i&gt;SMAD4&lt;/i&gt; homozygous deletion were sensitive to olaparib. &lt;i&gt;In vivo&lt;/i&gt;, olaparib or RT monotherapy reduced tumor volumes in &lt;i&gt;SMAD4&lt;/i&gt;-mutant but not &lt;i&gt;SMAD4&lt;/i&gt;-positive tumors. Olaparib with RT dual therapy sustained tumor volume reduction in &lt;i&gt;SMAD4&lt;/i&gt;-deficient (mutant or knockdown) xenografts, which exhibited increased DNA damage and cell death compared with vehicle-treated tumors. &lt;i&gt;In vitro&lt;/i&gt;, olaparib alone or in combination with radiation caused lower clonogenic survival, more DNA damage–associated cell death, and less proliferation in &lt;i&gt;SMAD4&lt;/i&gt;-deficient cells than in &lt;i&gt;SMAD4&lt;/i&gt;-positive (endogenous SMAD4 or transduced SMAD4) cells. Applicable to clinic, 5 out of 6 SMAD4-negative HNSCCs and 4 out of 8 SMAD4-positive HNSCCs responded to a standard treatment plus olaparib in a phase I clinical trial, and SMAD4 protein levels inversely correlated with DNA damage.&lt;/p&gt;Conclusions:&lt;p&gt;&lt;i&gt;SMAD4&lt;/i&gt; levels are causal in determining sensitivity to PARP inhibition in combination with RT in HNSCCs.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Supplementary Table 1 â€" Effect of varying RT regimen and dose on survival when combined with anti PD-L1 in orthotopic Balb/c mice harboring LY2 tumors; Supplementary Table 2A â€" Effect of varying combinations of RT and dual immune checkpoint on survival in LY2 mice; Supplementary Figure 1 â€" Cibersort analysis of RNAseq data obtained from orthotopic LY2, MOC1 and MOC2 tumors. Supplementary Figure 2 â€" Tumor growth curves for (A) LY2 and (B) MOC2 tumors treated with anti-PD-L1 and anti-TIM-3 alone and in combination with RT; Supplementary Figure 3 â€" Analysis of relative abundance of mRNA transcript levels of genes involved in Treg differentiation and homing and effector T cell function from LY2 tumors harvested 1-week and 3-weeks after initiation of RT and anti-PD-L1 treatment; Supplementary Figure 4 â€" Analysis of patient samples with the Vectra immuno-fluorescence platform. Two patients with similar disease course but different outcomes were selected for analysis of immune cell profile (CD3, CD8, CD4, FoxP3, DAPI).&lt;/p&gt;

&lt;p&gt;Supplementary Figure S4 demonstrates the influence of SMAD4 expression in CAL27 xenograft growth&lt;/p&gt;

&lt;p&gt;Supplementary Figure S3 demonstrates doxycyline-dependent modulation of SMAD4&lt;/p&gt;

&lt;p&gt;Supplementary Figure S2 demonstrates the enrichment of Fanoni Anemia pathway genes as a function of SMAD4 levels (in TCGA analyses)&lt;/p&gt;

&lt;p&gt;Supplementary Table S1 lists reagents used&lt;/p&gt;

&lt;p&gt;Schema Utilizing Time-to-Event Continual Reassessment Method (TITE-CRM) Design&lt;/p&gt;

&lt;p&gt;Blockade of EphB4-ephrin-B2 enhances response to EGFR inhibitor (cetuximab) and RT resulting in significant delay in tumor growth and enhanced survival in HNSCC PDX models. Tumor growth volumes are displayed in CUHN013 (A) and CUHN004 (B) tumors including cisplatin-RT treated groups in the absence and presence of sEphB4-HSA. Cisplatin-RT treated groups did not show any significant changes in tumor growth in the absence and presence of sEphB4-HSA in CUHN013 tumors. Survival graphs are shown for CUHN013 (C) and CUHN004 (D) tumors.&lt;/p&gt;

&lt;p&gt;Levels of EGFR expression in HNSCC PDX tumors&lt;/p&gt;

&lt;p&gt;Supplementary Table 1 â€" Effect of varying RT regimen and dose on survival when combined with anti PD-L1 in orthotopic Balb/c mice harboring LY2 tumors; Supplementary Table 2A â€" Effect of varying combinations of RT and dual immune checkpoint on survival in LY2 mice; Supplementary Figure 1 â€" Cibersort analysis of RNAseq data obtained from orthotopic LY2, MOC1 and MOC2 tumors. Supplementary Figure 2 â€" Tumor growth curves for (A) LY2 and (B) MOC2 tumors treated with anti-PD-L1 and anti-TIM-3 alone and in combination with RT; Supplementary Figure 3 â€" Analysis of relative abundance of mRNA transcript levels of genes involved in Treg differentiation and homing and effector T cell function from LY2 tumors harvested 1-week and 3-weeks after initiation of RT and anti-PD-L1 treatment; Supplementary Figure 4 â€" Analysis of patient samples with the Vectra immuno-fluorescence platform. Two patients with similar disease course but different outcomes were selected for analysis of immune cell profile (CD3, CD8, CD4, FoxP3, DAPI).&lt;/p&gt;

&lt;p&gt;Microarray analysis showing EphB3 levels in different tumor cell lines&lt;/p&gt;

&lt;p&gt;EphB3 knockdown does not affect cell viability but enhances cell migration in LY2 cell line.&lt;/p&gt;

&lt;p&gt;EphB3 and PI3 Kinase p110α are variably expressed in human HNSCC cells.&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;Eph proteins have emerged as critical drivers affecting tumor growth and progression in human malignancies. Our The Cancer Genome Atlas (TCGA) data analysis showed that EphB3, a receptor tyrosine kinase, is frequently coamplified with PIK3CA in head and neck squamous cell carcinoma (HNSCC). We therefore hypothesized that EphB3 amplification plays a protumorigenic role in HNSCC and that &lt;i&gt;EphB3&lt;/i&gt; and &lt;i&gt;PIK3CA&lt;/i&gt; are cooperating oncogenes that contribute toward its pathogenesis. This hypothesis was not experimentally supported, because EphB3 knockdown failed to alter HNSCC tumor cell growth &lt;i&gt;in vitro&lt;/i&gt; or &lt;i&gt;in vivo&lt;/i&gt; with an orthotopic model. However, responsiveness of EphB3 knockdown tumors to the PI3K inhibitor, BKM120, was significantly decreased in terms of both tumor growth delay and survival. This is correlated with an increase in prosurvival proteins, S6 and BcL-XL, in the EphB3 shRNA tumors treated with BKM120 compared with controls. We further observed that EphB3 knockdown resulted in increased migration &lt;i&gt;in vitro&lt;/i&gt; and increased &lt;i&gt;EMT&lt;/i&gt; gene signature &lt;i&gt;in vivo&lt;/i&gt;. To explain these results, we examined EphB3 phosphorylation levels in HNSCC at baseline. Although total EphB3 levels were high, we found low phospho-EphB3 levels in HNSCCs. Forced EphB3 phosphorylation with an ephrin-B2–Fc fusion protein resulted in decreased HNSCC migration and cell growth, and enhanced response to BKM120 &lt;i&gt;in vitro&lt;/i&gt;. These data collectively indicate that progression of HNSCC selects for low/inhibited EphB3 activity to enhance their survival and migratory abilities and decrease response to PI3K signaling. Therefore, strategies focused on activating EphB3 might be helpful to inhibit tumor growth and enhance sensitivity to PI3K inhibitors in HNSCC. &lt;i&gt;Mol Cancer Ther; 17(9); 2049–59. ©2018 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;EphB3 and PI3 Kinase p110α are variably expressed in human HNSCC cells.&lt;/p&gt;

&lt;p&gt;EphB3 knockdown does not affect cell viability but enhances cell migration in LY2 cell line.&lt;/p&gt;

&lt;p&gt;Microarray analysis showing EphB3 levels in different tumor cell lines&lt;/p&gt;

Diffuse midline glioma (DMG) is an aggressive pediatric central nervous system tumor with strong metastatic potential. As localized treatment of the primary tumor improves, metastatic disease is becoming a more important factor in treatment. We hypothesized that we could model craniospinal irradiation (CSI) through a DMG patient-derived xenograft (PDX) model and that CSI would limit metastatic tumor.We used a BT245 murine orthotopic DMG PDX model for this work. We developed a protocol and specialized platform to deliver craniospinal irradiation (CSI) (4 Gy x2 days) with a pontine boost (4 Gy x2 days) and compared metastatic disease by pathology, bioluminescence, and MRI to mice treated with focal radiation only (4 Gy x4 days) or no radiation.Mice receiving CSI plus boost showed minimal spinal and brain leptomeningeal metastatic disease by bioluminescence, MRI, and pathology compared to mice receiving radiation to the pons only or no radiation.In a DMG PDX model, CSI+boost minimizes tumor dissemination compared to focal radiation. By expanding effective DMG treatment to the entire neuraxis, CSI has potential as a key component to combination, multimodality treatment for DMG designed to achieve long-term survival once novel therapies definitively demonstrate improved local control.

<h3>Background</h3> STING agonists have become an attractive immunomodulatory approach to activate the cGAS-STING pathway for turning cold tumors hot and thereby improving the efficacy of Immune Checkpoint Inhibitors (ICI). However, considering their modest clinical efficacy, there is a substantial need for other approaches to activating the cGAS-STING pathway for cancer immunotherapy. Inhibition of the enzyme ENPP1, a negative regulator of the cGAS-STING pathway is one such approach. ENPP1 hydrolyses 2′3′- cGAMP (endogenous STING agonist) and negatively regulates STING dependent immune activation. Several tumors like human astrocyte tumors and TNBC like 4T1 and MDA-MB-231 have high ENPP1 expression which plays a key role in tumor progression and blocks T cell infiltration. ENPP1 not only abolishes the cGAS-STING mediated immune activation but also produces adenosine, an immune suppressor which promotes cell migration. AVA-NP-695 is a highly potent orally available small molecule ENPP1 inhibitor being developed for cancer immunotherapy. <h3>Methods</h3> Inhibition potencies of AVA-NP-695 were confirmed by enzymatic assays using substrates like <i>p</i>-Nitrophenyl-5′-TMP, 2’3’-cGAMP and ATP. The efficacy of AVA-NP-695 was demonstrated in 4T1 Tumor bearing BALB/c mice as monotherapy and in combination with anti-PD-L1, Olaparib and Paclitaxel. Efficacy of AVA-NP-695 in combination with radiation was also evaluated in ENPP1 overexpressing breast tumors. Finally, combination of PSMA-RLT with AVA-NP-695 was evaluated for treating prostate cancer. <h3>Results</h3> We demonstrate that AVA-NP-695, a selective and potent small molecule ENPP1 inhibitor showed no adverse effects at 1000 mg/kg BID in 14 day repeated dose toxicity in BALB/c mice, thereby demonstrating an excellent therapeutic window. Results from <i>in-vivo</i> studies have shown superior tumor growth inhibition (TGI) and impact on metastasis by AVA-NP-695 (6 mg/kg BID) compared to Olaparib and Anti-PD1 in a syngeneic 4T1 breast cancer mouse model. Subsequently, combination of AVA-NP-695 with Anti-PDL1, Olaparib and Paclitaxel demonstrated encouraging combinatorial efficacy of AVA-NP-695 along with Paclitaxel. Monotherapeutic arm for Paclitaxel and AVA-NP-695 depicted 40% and 44% TGI respectively; however their combined treatment resulted in ~60% TGI. Additionally, the AVA-NP-695 treatment alone showed 50% enhanced mean survival time followed by 68%, 68% and 72% when given in combination with anti-PD-L1, Olaparib and Paclitaxel respectively. Finally, combination of AVA-NP-695 with radiation (Fractionated dose and Targeted Radionuclide) demonstrated substantial tumor growth reduction across various tumor models. <h3>Conclusions</h3> The potent anti-tumor efficacy of AVA-NP-695 both as monotherapy and combination along with its safety profile provides a strong rationale for the therapeutic potential of AVA-NP-695 against solids tumors, particularly breast cancer. <h3>Ethics Approval</h3> All animal studies involved obtained the prerequisite ethics committee(s) approval.

&lt;p&gt;Supplementary figure&lt;/p&gt;

&lt;p&gt;Supplementary figure&lt;/p&gt;

&lt;p&gt;Supplementary figure&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;Stromal fibrosis activates prosurvival and proepithelial-to-mesenchymal transition (EMT) pathways in pancreatic ductal adenocarcinoma (PDAC). In patient tumors treated with neoadjuvant stereotactic body radiation therapy (SBRT), we found upregulation of fibrosis, extracellular matrix (ECM), and EMT gene signatures, which can drive therapeutic resistance and tumor invasion. Molecular, functional, and translational analysis identified two cell-surface proteins, a disintegrin and metalloprotease 10 (ADAM10) and ephrinB2, as drivers of fibrosis and tumor progression after radiation therapy (RT). RT resulted in increased ADAM10 expression in tumor cells, leading to cleavage of ephrinB2, which was also detected in plasma. Pharmacologic or genetic targeting of ADAM10 decreased RT-induced fibrosis and tissue tension, tumor cell migration, and invasion, sensitizing orthotopic tumors to radiation killing and prolonging mouse survival. Inhibition of ADAM10 and genetic ablation of ephrinB2 in fibroblasts reduced the metastatic potential of tumor cells after RT. Stimulation of tumor cells with ephrinB2 FC protein reversed the reduction in tumor cell invasion with ADAM10 ablation. These findings represent a model of PDAC adaptation that explains resistance and metastasis after RT and identifies a targetable pathway to enhance RT efficacy.&lt;/p&gt;Significance:&lt;p&gt;Targeting a previously unidentified adaptive resistance mechanism to radiation therapy in PDAC tumors in combination with radiation therapy could increase survival of the 40% of PDAC patients with locally advanced disease.&lt;/p&gt;&lt;p&gt;&lt;i&gt;See related commentary by Garcia Garcia et al., p. 3158&lt;/i&gt;&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Supplementary Tables 1-3.&lt;/p&gt;

&lt;p&gt;Supplementary Tables 1-3.&lt;/p&gt;