Dichotomous effects of cellular expression of STAT3 on tumor growth of HNSCC.

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.

Loss of cancer cell STAT1 improves response to radiation therapy and promotes T cell activation in head and neck squamous cell carcinoma.

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.

Response to radiotherapy in pancreatic ductal adenocarcinoma is enhanced by inhibition of myeloid-derived suppressor cells using STAT3 anti-sense oligonucleotide.

Pancreatic ductal adenocarcinoma (PDAC) has a heterogeneous tumor microenvironment (TME) comprised of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages, neutrophils, regulatory T cells, and myofibroblasts. The precise mechanisms that regulate the composition of the TME and how they contribute to radiotherapy (RT) response remain poorly understood. In this study, we analyze changes in immune cell populations and circulating chemokines in patient samples and animal models of pancreatic cancer to characterize the immune response to radiotherapy. Further, we identify STAT3 as a key mediator of immunosuppression post-RT. We found granulocytic MDSCs (G-MDSCs) and neutrophils to be increased in response to RT in murine and human PDAC samples. We also found that RT-induced STAT3 phosphorylation correlated with increased MDSC infiltration and proliferation. Targeting STAT3 using an anti-sense oligonucleotide in combination with RT circumvented RT-induced MDSC infiltration, enhanced the proportion of effector T cells, and improved response to RT. In addition, STAT3 inhibition contributed to the remodeling of the PDAC extracellular matrix when combined with RT, resulting in decreased collagen deposition and fibrotic tissue formation. Collectively, our data provide evidence that targeting STAT3 in combination with RT can mitigate the pro-tumorigenic effects of RT and improve tumor response.

The effects of ephrinB2 signaling on proliferation and invasion in glioblastoma multiforme.

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.

A comparison of overall survival for patients with T4 larynx cancer treated with surgical versus organ-preservation approaches: A National Cancer Data Base analysis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Ephrin-B2 overexpression predicts for poor prognosis and response to therapy in solid tumors.

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.

Combined EphB2 receptor knockdown with radiation decreases cell viability and invasion in medulloblastoma.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Gene Therapies for Cancer: Strategies, Challenges and Successes.

Gene therapy, which involves replacement of a defective gene with a functional, healthy copy of that gene, is a potentially beneficial cancer treatment approach particularly over chemotherapy, which often lacks selectivity and can cause non-specific toxicity. Despite significant progress pre-clinically with respect to both enhanced targeting and expression in a tumor-selective manner several hurdles still prevent success in the clinic, including non-specific expression, low-efficiency delivery and biosafety. Various innovative genetic approaches are under development to reconstruct vectors/transgenes to make them safer and more effective. Utilizing cutting-edge delivery technologies, gene expression can now be targeted in a tissue- and organ-specific manner. With these advances, gene therapy is poised to become amenable for routine cancer therapy with potential to elevate this methodology as a first line therapy for neoplastic diseases. This review discusses recent advances in gene therapy and their impact on a pre-clinical and clinical level.

MDA-7/IL-24: multifunctional cancer killing cytokine.

First identified almost two decades ago as a novel gene differentially expressed in human melanoma cells induced to terminally differentiate, MDA-7/IL-24 has since shown great potential as an anti-cancer gene. MDA-7/IL24, a secreted protein of the IL-10 family, functions as a cytokine at normal physiological levels and is expressed in tissues of the immune system. At supra-physiological levels, MDA-7/IL-24 plays a prominent role in inhibiting tumor growth, invasion, metastasis and angiogenesis and was recently shown to target tumor stem/initiating cells for death. Much of the attention focused on MDA-7/IL-24 originated from the fact that it can selectively induce cell death in cancer cells without affecting normal cells. Thus, this gene originally shown to be associated with melanoma cell differentiation has now proven to be a multi-functional protein affecting a broad array of cancers. Moreover, MDA-7/IL-24 has proven efficacious in a Phase I/II clinical trial in humans with multiple advanced cancers. As research in the field progresses, we will unravel more of the functions of MDA-7/IL-24 and define novel ways to utilize MDA-7/IL-24 in the treatment of cancer.

Iron-catalyzed stereoselective C-H alkylation for simultaneous construction of C-N axial and C-central chirality.

The assembly of chiral molecules with multiple stereogenic elements is challenging, and, despite of indisputable advances, largely limited to toxic, cost-intensive and precious metal catalysts. In sharp contrast, we herein disclose a versatile C-H alkylation using a non-toxic, low-cost iron catalyst for the synthesis of substituted indoles with two chiral elements. The key for achieving excellent diastereo- and enantioselectivity was substitution on a chiral N-heterocyclic carbene ligand providing steric hindrance and extra represented by noncovalent interaction for the concomitant generation of C-N axial chirality and C-stereogenic center. Experimental and computational mechanistic studies have unraveled the origin of the catalytic efficacy and stereoselectivity.

The pro-tumoral and anti-tumoral roles of EphA4 on T regulatory cells and tumor associated macrophages during HNSCC tumor progression.

Head and Neck Squamous Cell Carcinoma (HNSCC) is a deadly cancer with poor response to targeted therapy, largely driven by an immunosuppressive tumor microenvironment (TME). Here we examine the immune-modulatory role of the receptor tyrosine kinase EphA4 in HNSCC progression. Within the TME, EphA4 is primarily expressed on regulatory T cells (Tregs) and macrophages. In contrast ephrinB2, an activating ligand of EphA4, is expressed in tumor blood vessels. Using genetically engineered mouse models, we show that EphA4 expressed in Tregs promotes tumor growth, whereas EphA4 expressed in monocytes inhibits tumor growth. In contrast, ephrinB2 knockout in blood vessels reduces both intratumoral Tregs and macrophages. A novel specific EphA4 inhibitor, APY-d3-PEG4, reverses the accelerated tumor growth we had previously reported with EphB4 cancer cell knockout. EphA4 knockout in macrophages not only enhanced their differentiation into M2 macrophage but also increased Treg suppressive activity. APY-d3-PEG4 reversed the accelerated growth seen in the EphA4 knockout of monocytes but conferred no additional benefit when EphA4 was knocked out on Tregs. Underscoring an EphA4-mediated interplay between Tregs and macrophages, we found that knockout of EphA4 in Tregs not only decreases their activation but also reduces tumor infiltration of pro-tumoral M2 macrophages. These data identify Tregs as a primary target of APY-d3-PEG4 and suggest a role for Tregs in regulating macrophage conversion. These data also support the possible anti-cancer therapeutic value of bispecific peptides or antibodies capable of promoting EphA4 blockade in Tregs but not macrophages. Significance: EphA4 in regulatory T cells has a pro-tumoral effect while EphA4 in macrophages plays an anti-tumoral role underscoring the necessity of developing biologically rational therapeutics.

Manipulating the EphB4-ephrinB2 axis to reduce metastasis in HNSCC.

The EphB4-ephrinB2 signaling axis has been heavily implicated in metastasis across numerous cancer types. Our emerging understanding of the dichotomous roles that EphB4 and ephrinB2 play in head and neck squamous cell carcinoma (HNSCC) poses a significant challenge to rational drug design. We find that EphB4 knockdown in cancer cells enhances metastasis in preclinical HNSCC models by augmenting immunosuppressive cells like T regulatory cells (Tregs) within the tumor microenvironment. EphB4 inhibition in cancer cells also amplifies their ability to metastasize through increased expression of genes associated with epithelial mesenchymal transition and hallmark pathways of metastasis. In contrast, vascular ephrinB2 knockout coupled with radiation therapy (RT) enhances anti-tumor immunity, reduces Treg accumulation into the tumor, and decreases metastasis. Notably, targeting the EphB4-ephrinB2 signaling axis with the engineered EphB4 ligands EFNB2-Fc-His and Fc-TNYL-RAW-GS reduces local tumor growth and distant metastasis in a preclinical model of HNSCC. Our data suggest that targeted inhibition of vascular ephrinB2 while avoiding inhibition of EphB4 in cancer cells could be a promising strategy to mitigate HNSCC metastasis.

Identification of HPV16 E1 and E2-specific T cells in the oropharyngeal cancer tumor microenvironment.

BACKGROUND: High-risk human papillomavirus (HPV) is a primary cause of an increasing number of oropharyngeal squamous cell carcinomas (OPSCCs). The viral etiology of these cancers provides the opportunity for antigen-directed therapies that are restricted in scope compared with cancers without viral components. However, specific virally-encoded epitopes and their corresponding immune responses are not fully defined. METHODS: To understand the OPSCC immune landscape, we conducted a comprehensive single-cell analysis of HPV16+ and HPV33+ primary tumors and metastatic lymph nodes. We used single-cell analysis with encoded peptide-human leukocyte antigen (HLA) tetramers to analyze HPV16+ and HPV33+ OPSCC tumors, characterizing the ex vivo cellular responses to HPV-derived antigens presented in major Class I and Class II HLA alleles. RESULTS: We identified robust cytotoxic T-cell responses to HPV16 proteins E1 and E2 that were shared across multiple patients, particularly in HLA-A*01:01 and HLA-B*08:01. Responses to E2 were associated with loss of E2 expression in at least one tumor, indicating the functional capacity of these E2-recognizing T cells and many of these interactions validated in a functional assay. Conversely, cellular responses to E6 and E7 were limited in quantity and cytotoxic capacity, and tumor E6 and E7 expression persisted. CONCLUSIONS: These data highlight antigenicity beyond HPV16 E6 and E7 and nominate candidates for antigen-directed therapies.

Targeting Treg-Expressed STAT3 Enhances NK-Mediated Surveillance of Metastasis and Improves Therapeutic Response in Pancreatic Adenocarcinoma.

PURPOSE: Metastasis remains a major hurdle in treating aggressive malignancies such as pancreatic ductal adenocarcinoma (PDAC). Improving response to treatment, therefore, requires a more detailed characterization of the cellular populations involved in controlling metastatic burden. EXPERIMENTAL DESIGN: PDAC patient tissue samples were subjected to RNA sequencing analysis to identify changes in immune infiltration following radiotherapy. Genetically engineered mouse strains in combination with orthotopic tumor models of PDAC were used to characterize disease progression. Flow cytometry was used to analyze tumor infiltrating, circulating, and nodal immune populations. RESULTS: We demonstrate that although radiotherapy increases the infiltration and activation of dendritic cells (DC), it also increases the infiltration of regulatory T cells (Treg) while failing to recruit natural killer (NK) and CD8 T cells in PDAC patient tissue samples. In murine orthotopic tumor models, we show that genetic and pharmacologic depletion of Tregs and NK cells enhances and attenuates response to radiotherapy, respectively. We further demonstrate that targeted inhibition of STAT3 on Tregs results in improved control of local and distant disease progression and enhanced NK-mediated immunosurveillance of metastasis. Moreover, combination treatment of STAT3 antisense oligonucleotide (ASO) and radiotherapy invigorated systemic immune activation and conferred a survival advantage in orthotopic and metastatic tumor models. Finally, we show the response to STAT3 ASO + radiotherapy treatment is dependent on NK and DC subsets. CONCLUSIONS: Our results suggest targeting Treg-mediated immunosuppression is a critical step in mediating a response to treatment, and identifying NK cells as not only a prognostic marker of improved survival, but also as an effector population that functions to combat metastasis.

EphB4 and ephrinB2 act in opposition in the head and neck tumor microenvironment.

Differential outcomes of EphB4-ephrinB2 signaling offers formidable challenge for the development of cancer therapeutics. Here, we interrogate the effects of targeting EphB4 and ephrinB2 in head and neck squamous cell carcinoma (HNSCC) and within its microenvironment using genetically engineered mice, recombinant constructs, pharmacologic agonists and antagonists. We observe that manipulating the EphB4 intracellular domain on cancer cells accelerates tumor growth and angiogenesis. EphB4 cancer cell loss also triggers compensatory upregulation of EphA4 and T regulatory cells (Tregs) influx and their targeting results in reversal of accelerated tumor growth mediated by EphB4 knockdown. EphrinB2 knockout on cancer cells and vasculature, on the other hand, results in maximal tumor reduction and vascular normalization. We report that EphB4 agonism provides no additional anti-tumoral benefit in the absence of ephrinB2. These results identify ephrinB2 as a tumor promoter and its receptor, EphB4, as a tumor suppressor in HNSCC, presenting opportunities for rational drug design.

A phase I/Ib trial and biological correlate analysis of neoadjuvant SBRT with single-dose durvalumab in HPV-unrelated locally advanced HNSCC.

Five-year survival for human papilloma virus-unrelated head and neck squamous cell carcinomas remain below 50%. We assessed the safety of administering combination hypofractionated stereotactic body radiation therapy with single-dose durvalumab (anti-PD-L1) neoadjuvantly (n = 21) ( NCT03635164 ). The primary endpoint of the study was safety, which was met. Secondary endpoints included radiographic, pathologic and objective response; locoregional control; progression-free survival; and overall survival. Among evaluable patients at an early median follow-up of 16 months (448 d or 64 weeks), overall survival was 80.1% with 95% confidence interval (95% CI) (62.0%, 100.0%), locoregional control and progression-free survival were 75.8% with 95% CI (57.5%, 99.8%), and major pathological response or complete response was 75% with 95% exact CI (51.6%, 100.0%). For patients treated with 24 Gy, 89% with 95% CI (57.1%, 100.0%) had MPR or CR. Using high-dimensional multi-omics and spatial data as well as biological correlatives, we show that responders had: (1) an increase in effector T cells; (2) a decrease in immunosuppressive cells; and (3) an increase in antigen presentation post-treatment.

Intermediates and mechanism in iron-catalyzed C-H methylation with trimethylaluminum.

A mechanistic study is performed on the reaction method for iron-catalyzed C-H methylation with AlMe3 reagent, previously proposed to involve cyclometalated iron(III) intermediates and an iron(III)/(I) reaction cycle. Detailed spectroscopic studies (57Fe Mössbauer, EPR) during catalysis and in stoichiometric reactions identify iron(II) complexes, including cyclometalated iron(II) intermediates, as the major iron species formed in situ under catalytic reaction conditions. Reaction studies identify a cyclometalated iron(II)-methyl species as the key intermediate leading to C-H methylated product upon reaction with oxidant, consistent with a previously proposed iron(II)/iron(III)/iron(I) reaction manifold for C-H arylation.

Complement C3a and C5a receptor blockade modulates regulatory T cell conversion in head and neck cancer.

BACKGROUND: Resistance to therapy is a major problem in treating head and neck squamous cell carcinomas (HNSCC). Complement system inhibition has been shown to reduce tumor growth, metastasis, and therapeutic resistance in other tumor models, but has yet to be explored in the context of HNSCC. Here, we tested the effects of complement inhibition and its therapeutic potential in HNSCC. METHODS: We conducted our studies using two Human Papilloma Virus (HPV)-negative HNSCC orthotopic mouse models. Complement C3aR and C5aR1 receptor antagonists were paired with radiation therapy (RT). Tumor growth was measured and immune populations from tumor, lymph node, and peripheral blood were compared among various treatment groups. Genetically engineered mouse models DEREG and C3-/- were used in addition to standard wild type models. Flow cytometry, clinical gene sets, and in vitro assays were used to evaluate the role complement receptor blockade has on the immunological makeup of the tumor microenvironment. RESULTS: In contrast to established literature, inhibition of complement C3a and C5a signaling using receptor antagonists accelerated tumor growth in multiple HNSCC cell lines and corresponded with increased frequency of regulatory T cell (Treg) populations. Local C3a and C5a signaling has importance for CD4 T cell homeostasis and eventual development into effector phenotypes. Interruption of this signaling axis drives a phenotypic conversion of CD4+ T cells into Tregs, characterized by enhanced expression of Foxp3. Depletion of Tregs reversed tumor growth, and combination of Treg depletion and C3a and C5a receptor inhibition decreased tumor growth below that of the control groups. Complete knockout of C3 does not harbor the expected effect on tumor growth, indicating a still undetermined compensatory mechanism. Dexamethasone is frequently prescribed to patients undergoing RT and inhibits complement activation. We report no deleterious effects associated with dexamethasone due to complement inhibition. CONCLUSIONS: Our data establish Tregs as a pro-tumorigenic driver during complement inhibition and provide evidence that targeted C3a and C5a receptor inhibition may add therapeutic advantage when coupled with anti-Treg therapy.

FLT3L Release by Natural Killer Cells Enhances Response to Radioimmunotherapy in Preclinical Models of HNSCC.

PURPOSE: Natural killer (NK) cells are type I innate lymphoid cells that are known for their role in killing virally infected cells or cancer cells through direct cytotoxicity. In addition to direct tumor cell killing, NK cells are known to play fundamental roles in the tumor microenvironment through secretion of key cytokines, such as FMS-like tyrosine kinase 3 ligand (FLT3L). Although radiotherapy is the mainstay treatment in most cancers, the role of radiotherapy on NK cells is not well characterized. EXPERIMENTAL DESIGN: This study combines radiation, immunotherapies, genetic mouse models, and antibody depletion experiments to identify the role of NK cells in overcoming resistance to radiotherapy in orthotopic models of head and neck squamous cell carcinoma. RESULTS: We have found that NK cells are a crucial component in the development of an antitumor response, as depleting them removes efficacy of the previously successful combination treatment of radiotherapy, anti-CD25, and anti-CD137. However, in the absence of NK cells, the effect can be rescued through treatment with FLT3L. But neither radiotherapy with FLT3L therapy alone nor radiotherapy with anti-NKG2A yields any meaningful tumor growth delay. We also identify a role for IL2 in activating NK cells to secrete FLT3L. This activity, we show, is mediated through CD122, the intermediate affinity IL2 receptor, and can be targeted with anti-CD25 therapy. CONCLUSIONS: These findings highlight the complexity of using radio-immunotherapies to activate NK cells within the tumor microenvironment, and the importance of NK cells in activating dendritic cells for increased tumor surveillance.

Targeting resistance to radiation-immunotherapy in cold HNSCCs by modulating the Treg-dendritic cell axis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.