Natural Killer Cell Dysfunction In Human Bladder Cancer Is Caused By Tissue-Specific Suppression of SLAMF6 Signaling.

NK cells are innate lymphocytes critical for surveillance of viruses and tumors, however the mechanisms underlying NK cell dysfunction in cancer are incompletely understood. We assessed the effector function of NK cells from bladder cancer patients and found severe dysfunction in NK cells derived from tumors versus peripheral blood. While both peripheral and tumor-infiltrating NK cells exhibited conserved patterns of inhibitory receptor over-expression, this did not explain the observed defects in NK surveillance in bladder tumors. Rather, TME-specific TGF-ß and metabolic perturbations such as hypoxia directly suppressed NK cell function. Specifically, an oxygen-dependent reduction in signaling through SLAMF6 was mechanistically responsible for poor NK cell function, as tumor-infiltrating NK cells cultured ex vivo under normoxic conditions exhibited complete restoration of function, while deletion of SLAMF6 abrogated NK cell cytolytic function even under normoxic conditions. Collectively, this work highlights the role of tissue-specific factors in dictating NK cell function, and implicates SLAMF6 signaling as a rational target for immuno-modulation to improve NK cell function in bladder cancer.

Structural basis for self-discrimination by neoantigen-specific TCRs.

T cell receptors (TCR) are pivotal in mediating tumour cell cytolysis via recognition of mutation-derived tumour neoantigens (neoAgs) presented by major histocompatibility class-I (MHC-I). Understanding the factors governing the emergence of neoAg from somatic mutations is a major focus of current research. However, the structural and cellular determinants controlling TCR recognition of neoAgs remain poorly understood. This study describes the multi-level analysis of a model neoAg from the B16F10 murine melanoma, H2-Db/Hsf2 p.K72N68-76, as well as its cognate TCR 47BE7. Through cellular, molecular and structural studies we demonstrate that the p.K72N mutation enhances H2-Db binding, thereby improving cell surface presentation and stabilizing the TCR 47BE7 epitope. Furthermore, TCR 47BE7 exhibited high functional avidity and selectivity, attributable to a broad, stringent, binding interface enabling recognition of native B16F10 despite low antigen density. Our findings provide insight into the generation of anchor-residue modified neoAg, and emphasize the value of molecular and structural investigations of neoAg in diverse MHC-I contexts for advancing the understanding of neoAg immunogenicity.

Combination oncolytic virus, radiation therapy, and immune checkpoint inhibitor treatment in anti-PD-1-refractory cancer.

BACKGROUND: Immunotherapies are becoming front-line treatments for many advanced cancers, and combinations of two or more therapies are beginning to be investigated. Based on their individual antitumor capabilities, we sought to determine whether combination oncolytic virus (OV) and radiation therapy (RT) may improve cancer outcomes. METHODS: To investigate the activity of this combination therapy, we used in vitro mouse and human cancer cell lines as well as a mouse model of skin cancer. After initial results, we further included immune checkpoint blockade, whose addition constituted a triple combination immunotherapy. RESULTS: Our findings demonstrate that OV and RT reduce tumor growth via conversion of immunologically 'cold' tumors to 'hot', via a CD8+ T cell-dependent and IL-1α-dependent mechanism that is associated with increased PD-1/PD-L1 expression, and the triple combination of OV, RT, and PD-1 checkpoint inhibition impedes tumor growth and prolongs survival. Further, we describe the response of a PD-1-refractory patient with cutaneous squamous cell carcinoma who received the triple combination of OV, RT, and immune checkpoint inhibitor (ICI), and went on to experience unexpected, prolonged control and survival. He remains off-treatment and is without evidence of progression for >44 months since study entry. CONCLUSIONS: Effective systemic antitumor immune response is rarely elicited by a single therapy. In a skin cancer mouse model, we demonstrate improved outcomes with combination OV, RT, and ICI treatment, which is associated with mechanisms involving augmented CD8+ T cell infiltration and IL-1α expression. We report tumor reduction and prolonged survival of a patient with skin cancer treated with combination OV, RT, and ICI. Overall, our data provide strong rationale for combining OV, RT, and ICI for treatment of patients with ICI-refractory skin and potentially other cancers.

TREM2 macrophages drive NK cell paucity and dysfunction in lung cancer.

Natural killer (NK) cells are commonly reduced in human tumors, enabling many to evade surveillance. Here, we sought to identify cues that alter NK cell activity in tumors. We found that, in human lung cancer, the presence of NK cells inversely correlated with that of monocyte-derived macrophages (mo-macs). In a murine model of lung adenocarcinoma, we show that engulfment of tumor debris by mo-macs triggers a pro-tumorigenic program governed by triggering receptor expressed on myeloid cells 2 (TREM2). Genetic deletion of Trem2 rescued NK cell accumulation and enabled an NK cell-mediated regression of lung tumors. TREM2+ mo-macs reduced NK cell activity by modulating interleukin (IL)-18/IL-18BP decoy interactions and IL-15 production. Notably, TREM2 blockade synergized with an NK cell-activating agent to further inhibit tumor growth. Altogether, our findings identify a new axis, in which TREM2+ mo-macs suppress NK cell accumulation and cytolytic activity. Dual targeting of macrophages and NK cells represents a new strategy to boost antitumor immunity.

Structural Basis for Self-Discrimination by Neoantigen-Specific TCRs.

Physical interactions between T cell receptors (TCRs) and mutation-derived tumour neoantigens (neoAg) presented by major histocompatibility class-I (MHC-I) enable sensitive and specific cytolysis of tumour cells. Adoptive transfer of neoAg-reactive T cells in patients is correlated with response to immunotherapy; however, the structural and cellular mechanisms of neoAg recognition remain poorly understood. We have identified multiple cognate neoAg:TCRs from B16F10, a common murine implantable tumour model of melanoma. We identified a high affinity TCR targeting H2-Db-restricted Hsf2K72N that conferred specific recognition of B16F10 in vitro and in vivo. Structural characterization of the peptide-MHC (pMHC) binary and pMHC:TCR ternary complexes yielded high-resolution crystal structures, revealing the formation of a solvent-exposed hydrophobic arch in H2-Db that enables multiple intermolecular contacts between pMHC and the TCR. These features of structural stability strikingly mimic that of a previously published influenza peptide-H2-Db complex and its corresponding TCR, suggesting that there are shared structural motifs between neoantigens and viral peptides that explain their shared immunogenicity.

Gut Microbial Shifts Indicate Melanoma Presence and Bacterial Interactions in a Murine Model.

Through a multitude of studies, the gut microbiota has been recognized as a significant influencer of both homeostasis and pathophysiology. Certain microbial taxa can even affect treatments such as cancer immunotherapies, including the immune checkpoint blockade. These taxa can impact such processes both individually as well as collectively through mechanisms from quorum sensing to metabolite production. Due to this overarching presence of the gut microbiota in many physiological processes distal to the GI tract, we hypothesized that mice bearing tumors at extraintestinal sites would display a distinct intestinal microbial signature from non-tumor-bearing mice, and that such a signature would involve taxa that collectively shift with tumor presence. Microbial OTUs were determined from 16S rRNA genes isolated from the fecal samples of C57BL/6 mice challenged with either B16-F10 melanoma cells or PBS control and analyzed using QIIME. Relative proportions of bacteria were determined for each mouse and, using machine-learning approaches, significantly altered taxa and co-occurrence patterns between tumor- and non-tumor-bearing mice were found. Mice with a tumor had elevated proportions of Ruminococcaceae, Peptococcaceae.g_rc4.4, and Christensenellaceae, as well as significant information gains and ReliefF weights for Bacteroidales.f__S24.7, Ruminococcaceae, Clostridiales, and Erysipelotrichaceae. Bacteroidales.f__S24.7, Ruminococcaceae, and Clostridiales were also implicated through shifting co-occurrences and PCA values. Using these seven taxa as a melanoma signature, a neural network reached an 80% tumor detection accuracy in a 10-fold stratified random sampling validation. These results indicated gut microbial proportions as a biosensor for tumor detection, and that shifting co-occurrences could be used to reveal relevant taxa.

Uncloaking Breast Tumor Neoantigens with Radiation.

Currently, immunotherapy is not effective for all cancer patients, partly due to an inability to mount sufficient tumor-specific T cell responses. Here, we highlight a recent study by Lhuillier et al. demonstrating that radiation potentiates neoantigen expression, eliciting CD4+ and CD8+ T cell responses and tumor growth control in mice.

Intratumoral injection of the seasonal flu shot converts immunologically cold tumors to hot and serves as an immunotherapy for cancer.

Reprogramming the tumor microenvironment to increase immune-mediated responses is currently of intense interest. Patients with immune-infiltrated "hot" tumors demonstrate higher treatment response rates and improved survival. However, only the minority of tumors are hot, and a limited proportion of patients benefit from immunotherapies. Innovative approaches that make tumors hot can have immediate impact particularly if they repurpose drugs with additional cancer-unrelated benefits. The seasonal influenza vaccine is recommended for all persons over 6 mo without prohibitive contraindications, including most cancer patients. Here, we report that unadjuvanted seasonal influenza vaccination via intratumoral, but not intramuscular, injection converts "cold" tumors to hot, generates systemic CD8+ T cell-mediated antitumor immunity, and sensitizes resistant tumors to checkpoint blockade. Importantly, intratumoral vaccination also provides protection against subsequent active influenza virus lung infection. Surprisingly, a squalene-based adjuvanted vaccine maintains intratumoral regulatory B cells and fails to improve antitumor responses, even while protecting against active influenza virus lung infection. Adjuvant removal, B cell depletion, or IL-10 blockade recovers its antitumor effectiveness. Our findings propose that antipathogen vaccines may be utilized for both infection prevention and repurposing as a cancer immunotherapy.

Safety and enhanced immunostimulatory activity of the DRD2 antagonist ONC201 in advanced solid tumor patients with weekly oral administration.

BACKGROUND: ONC201 is a small molecule antagonist of DRD2, a G protein-coupled receptor overexpressed in several malignancies, that has prolonged antitumor efficacy and immunomodulatory properties in preclinical models. The first-in-human trial of ONC201 previously established a recommended phase II dose (RP2D) of 625 mg once every three weeks. Here, we report the results of a phase I study that evaluated the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of weekly ONC201. METHODS: Patients ≥ 18 years old with an advanced solid tumor refractory to standard treatment were enrolled. Dose escalation proceeded with a 3 + 3 design from 375 mg to 625 mg of ONC201. One cycle, also the dose-limiting toxicity (DLT) window, was 21 days. The primary endpoint was to determine the RP2D of weekly ONC201, which was confirmed in an 11-patient dose expansion cohort. RESULTS: Twenty patients were enrolled: three at 375 mg and 17 at 625 mg of ONC201. The RP2D was defined as 625 mg with no DLT, treatment discontinuation, or dose modifications due to drug-related toxicity. PK profiles were consistent with every-three-week dosing and similar between the first and fourth dose. Serum prolactin and caspase-cleaved cytokeratin-18 induction were detected, along with intratumoral integrated stress response activation and infiltration of granzyme B+ Natural Killer cells. Induction of immune cytokines and effectors was higher in patients who received ONC201 once weekly versus once every three weeks. Stable disease of > 6 months was observed in several prostate and endometrial cancer patients. CONCLUSIONS: Weekly, oral ONC201 is well-tolerated and results in enhanced immunostimulatory activity that warrants further investigation. TRIAL REGISTRATION: NCT02250781 (Oral ONC201 in Treating Patients With Advanced Solid Tumors), NCT02324621 (Continuation of Oral ONC201 in Treating Patients With Advanced Solid Tumors).

Novel bone morphogenetic protein receptor inhibitor JL5 suppresses tumor cell survival signaling and induces regression of human lung cancer.

BMP receptor inhibitors induce death of cancer cells through the downregulation of antiapoptotic proteins XIAP, pTAK1, and Id1-Id3. However, the current most potent BMP receptor inhibitor, DMH2, does not downregulate BMP signaling in vivo because of metabolic instability and poor pharmacokinetics. Here we identified the site of metabolic instability of DMH2 and designed a novel BMP receptor inhibitor, JL5. We show that JL5 has a greater volume of distribution and suppresses the expression of Id1 and pTak1 in tumor xenografts. Moreover, we demonstrate JL5-induced tumor cell death and tumor regression in xenograft mouse models without immune cells and humanized with adoptively transferred human immune cells. In humanized mice, JL5 additionally induces the infiltration of immune cells within the tumor microenvironment. Our studies show that the BMP signaling pathway is targetable in vivo and BMP receptor inhibitors can be developed as a therapeutic to treat cancer patients.

Dose intensification of TRAIL-inducing ONC201 inhibits metastasis and promotes intratumoral NK cell recruitment.

ONC201 is a first-in-class, orally active antitumor agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. ONC201 has demonstrated safety and preliminary efficacy in a first-in-human trial in which patients were dosed every 3 weeks. We hypothesized that dose intensification of ONC201 may impact antitumor efficacy. We discovered that ONC201 exerts dose- and schedule-dependent effects on tumor progression and cell death signaling in vivo. With dose intensification, we note a potent anti-metastasis effect and inhibition of cancer cell migration and invasion. Our preclinical results prompted a change in ONC201 dosing in all open clinical trials. We observed accumulation of activated NK+ and CD3+ cells within ONC201-treated tumors and that NK cell depletion inhibits ONC201 efficacy in vivo, including against TRAIL/ONC201-resistant Bax-/- tumors. Immunocompetent NCR1-GFP mice, in which NK cells express GFP, demonstrated GFP+ NK cell infiltration of syngeneic MC38 colorectal tumors. Activation of primary human NK cells and increased degranulation occurred in response to ONC201. Coculture experiments identified a role for TRAIL in human NK-mediated antitumor cytotoxicity. Preclinical results indicate the potential utility for ONC201 plus anti-PD-1 therapy. We observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients after ONC201 treatment. The results offer what we believe to be a unique pathway of immune stimulation for cancer therapy.

Infection: a Cause of and Cure for Cancer.

PURPOSE OF REVIEW: This article provides a brief overview of the role that infections play in cancer emergence and cancer treatment. RECENT FINDINGS: A select number of pathogens have been reported to increase the incidence of specific cancers (directly through altering gene expression or indirectly through inducing chronic inflammation). These have been referred to as oncogenic pathogens. Conversely, a subset of pathogens has been demonstrated to preferentially cause lysis of tumor cells, leading to tumor regression and improved anti-tumor immunity. These have been termed oncolytic pathogens. However, the contribution of non-oncogenic, non-oncolytic pathogens to both tumor growth and regression is likewise being increasingly recognized. SUMMARY: Pathogens have both the ability to cause and cure cancer. However, the mechanisms underlying these pathogen-mediated outcomes are not fully understood. With the recent emergence of interest in the immunotherapy of cancer, it is important that future studies focus specifically on preventing the negative effects of oncogenic infections, deconstructing the positive role of oncolytic pathogens, and finally providing insight into the dual roles of non-oncolytic, non-oncogenic pathogens so that anti-pathogen immune responses can be harnessed as a transformative means to treat cancer.

Non-oncogenic Acute Viral Infections Disrupt Anti-cancer Responses and Lead to Accelerated Cancer-Specific Host Death.

In light of increased cancer prevalence and cancer-specific deaths in patients with infections, we investigated whether infections alter anti-tumor immune responses. We report that acute influenza infection of the lung promotes distal melanoma growth in the dermis and leads to accelerated cancer-specific host death. Furthermore, we show that during influenza infection, anti-melanoma CD8+ T cells are shunted from the tumor to the infection site, where they express high levels of the inhibitory receptor programmed cell death protein 1 (PD-1). Immunotherapy to block PD-1 reverses this loss of anti-tumor CD8+ T cells from the tumor and decreases infection-induced tumor growth. Our findings show that acute non-oncogenic infection can promote cancer growth, raising concerns regarding acute viral illness sequelae. They also suggest an unexpected role for PD-1 blockade in cancer immunotherapy and provide insight into the immune response when faced with concomitant challenges.