DNA-damaging chemotherapy and radiation therapy are integrated into the treatment paradigm of the majority of cancer patients. Recently, immunotherapy that targets the immunosuppressive interaction between programmed death 1 (PD-1) and its ligand PD-L1 has been approved for malignancies including non-small cell lung cancer, melanoma, and head and neck squamous cell carcinoma. ATR is a DNA damage-signaling kinase activated at damaged replication forks, and ATR kinase inhibitors potentiate the cytotoxicity of DNA-damaging chemotherapies. We show here that the ATR kinase inhibitor AZD6738 combines with conformal radiation therapy to attenuate radiation-induced CD8+ T cell exhaustion and potentiate CD8+ T cell activity in mouse models of Kras-mutant cancer. Mechanistically, AZD6738 blocks radiation-induced PD-L1 upregulation on tumor cells and dramatically decreases the number of tumor-infiltrating Tregs. Remarkably, AZD6738 combines with conformal radiation therapy to generate immunologic memory in complete responder mice. Our work raises the possibility that a single pharmacologic agent may enhance the cytotoxic effects of radiation while concurrently potentiating radiation-induced antitumor immune responses.

Radiation therapy (RT) can induce upregulation of programmed death ligand 1 (PD-L1) on tumor cells or myeloid cells, which may affect response to PD-1-based immunotherapy. PD-L1 upregulation during RT is a dynamic process that has been difficult to monitor during treatment. The aim of this study was to evaluate the RT-induced PD-L1 upregulation in the tumor and its microenvironment using immunoPET/CT imaging of two syngeneic murine tumor models (HPV+ head and neck squamous cell carcinoma (HNSCC) or B16F10 melanoma). Tumors were established in two locations per mouse (neck and flank), and fractionated RT (2 Gy × 4 or 2 Gy × 10) was delivered only to the neck tumor, alone or during anti-PD-1 mAb immunotherapy. PD-L1 expression was measured by PET/CT imaging using Zr-89 labeled anti-mouse PD-L1 mAb, and results were validated by flow cytometry. PET/CT imaging demonstrated significantly increased tracer uptake in irradiated neck tumors compared with non-irradiated flank tumors. analysis by biodistribution and flow cytometry validated PD-L1 upregulation specifically in irradiated tumors. In the HNSCC model, RT-induced PD-L1 upregulation was only observed after 2 Gy × 10 fractionated RT, while in the B16F10 model upregulation of PD-L1 occurred after 2 Gy × 4 fractionated RT. Fractionated RT, but not anti-PD-1 therapy, upregulated PD-L1 expression on tumor and infiltrating inflammatory cells in murine models, which could be non-invasively monitored by immunoPET/CT imaging using Zr-89 labeled anti-mouse PD-L1 mAb, and differentially identified anti-PD-1 responsive as well as selectively irradiated tumors .

The response rate of patients with head and neck squamous cell carcinoma (HNSCC) to cetuximab therapy is only 15% to 20%, despite frequent EGFR overexpression. Because immunosuppression is common in HNSCC, we hypothesized that adding a proinflammatory TLR8 agonist to cetuximab therapy might result in enhanced T-lymphocyte stimulation and anti-EGFR-specific priming. Fourteen patients with previously untreated HNSCC were enrolled in this neoadjuvant trial and treated preoperatively with 3 to 4 weekly doses of motolimod (2.5 mg/m) and cetuximab. Correlative tumor and peripheral blood specimens were obtained at baseline and at the time of surgical resection and analyzed for immune biomarker changes. Preclinical studies were also performed to assess the effect of cetuximab plus motolimod on myeloid cells. TLR8 stimulation skewed monocytes toward an M1 phenotype and reversed myeloid-derived suppressor cell (MDSC) suppression of T-cell proliferation These data were validated in a prospective phase Ib neoadjuvant trial, in which fewer MDSC and increased M1 monocyte infiltration were found in tumor-infiltrating lymphocytes. Motolimod plus cetuximab also decreased induction of Treg and reduced markers of suppression, including CTLA-4, CD73, and membrane-bound TGFβ. Significantly increased circulating EGFR-specific T cells were observed, concomitant with enhanced CD8 T-cell infiltration into tumors. These T cells manifested increased T-cell receptor (TCR) clonality, upregulation of the costimulatory receptor CD27, and downregulation of inhibitory receptor TIGIT. Enhanced inflammatory stimulation in the tumor microenvironment using a TLR agonist overcomes suppressive myeloid and regulatory cells, enhancing the cellular antitumor immune response by therapeutic mAb in HNSCC. .

In patients with an incomplete response after treatment of head and neck squamous cell carcinoma, the negative predictive value of the first posttreatment FDG-PET/CT was 85%, which is lower than the 91% negative predictive value of FDG-PET/CT in patients with an initial complete response. Patients with an incomplete response (NI-RADS 2) should undergo more frequent clinical and imaging surveillance than patients with an initial complete response (NI-RADS 1).

We show that ATM kinase inhibition using AZ31 prior to 9 or 9.25 Gy total body irradiation (TBI) reduced median time to moribund in mice to 8 days. ATR kinase inhibition using AZD6738 prior to TBI did not reduce median time to moribund. The striking finding associated with ATM inhibition prior to TBI was increased crypt loss within the intestine epithelium. ATM inhibition reduced upregulation of p21, an inhibitor of cyclin-dependent kinases, and blocked G1 arrest after TBI thereby increasing the number of S phase cells in crypts in wild-type but not Cdkn1a(p21CIP/WAF1)−/− mice. In contrast, ATR inhibition increased upregulation of p21 after TBI. Thus, ATM activity is essential for p21-dependent arrest while ATR inhibition may potentiate arrest in crypt cells after TBI. Nevertheless, ATM inhibition reduced median time to moribund in Cdkn1a(p21CIP/WAF1)−/− mice after TBI. ATM inhibition also increased cell death in crypts at 4 h in Cdkn1a(p21CIP/WAF1)−/−, earlier than at 24 h in wild-type mice after TBI. In contrast, ATR inhibition decreased cell death in crypts in Cdkn1a(p21CIP/WAF1)−/− mice at 4 h after TBI. We conclude that ATM activity is essential for p21-dependent and p21-independent mechanisms that radioprotect intestinal crypts and that ATM inhibition promotes GI syndrome after TBI.

Inhibitors of the DNA damage signaling kinase ATR increase tumor cell killing by chemotherapies that target DNA replication forks but also kill rapidly proliferating immune cells including activated T cells. Nevertheless, ATR inhibitor (ATRi) and radiotherapy (RT) can be combined to generate CD8 + T cell–dependent antitumor responses in mouse models. To determine the optimal schedule of ATRi and RT, we determined the impact of short-course versus prolonged daily treatment with AZD6738 (ATRi) on responses to RT (days 1–2). Short-course ATRi (days 1–3) plus RT caused expansion of tumor antigen–specific, effector CD8 + T cells in the tumor-draining lymph node (DLN) at 1 week after RT. This was preceded by acute decreases in proliferating tumor-infiltrating and peripheral T cells and a rapid proliferative rebound after ATRi cessation, increased inflammatory signaling (IFN-β, chemokines, particularly CXCL10) in tumors, and an accumulation of inflammatory cells in the DLN. In contrast, prolonged ATRi (days 1–9) prevented the expansion of tumor antigen–specific, effector CD8 + T cells in the DLN, and entirely abolished the therapeutic benefit of short-course ATRi with RT and anti–PD-L1. Our data argue that ATRi cessation is essential to allow CD8 + T cell responses to both RT and immune checkpoint inhibitors.

Purpose: Concurrent radiotherapy with cetuximab, an anti-EGFR mAb, is a standard treatment for locally advanced head and neck squamous carcinoma (HNSCC). Cytotoxic T lymphocyte antigen-4–positive (CTLA-4+) regulatory T cells (Treg) dampen cellular immunity and correlate negatively with clinical outcomes. This phase I study added ipilimumab, an anti–CTLA-4 mAb, to cetuximab-radiotherapy. Patients and Methods: A (3 + 3) design was used to establish the recommended phase II dose (RP2D) of ipilimumab, added at week 5 for four, every-3-week doses to fixed, standard cetuximab-radiotherapy. Eligible subjects had stage III to IVb, high-risk [human papillomavirus–negative (HPV−)] or intermediate-risk HPV-positive (HPV+)] HNSCC. Dose-limiting toxicity (DLT) was defined as any grade 4 adverse event (AE) except in-field radiation dermatitis or immune-related (ir) AE requiring ≥2 weeks of systemic steroids. Baseline tumor and serial blood specimens were collected for immune correlatives. Results: From July 2013 to May 2016, 18 patients enrolled. Two of 6 in cohort 1 (ipilimumab 3 mg/kg) experienced grade 3 dermatologic DLTs, triggering deescalation of ipilimumab to 1 mg/kg. Dose level -1 was expanded to N  =  12 without DLT. irAE included: grade 1, 2, and 3 dermatitis (2, 1, and 3 cases), grade 4 colitis (1), and grade 1 hyperthyroidism (1). Three-year disease-free survival (DFS) and overall survival were 72% [90% confidence interval (CI), 57–92] and 72% (90% CI, 56–92). High expression of coinhibitory receptors PD1/LAG3/CD39 on baseline tumor-infiltrating Treg was associated with worse DFS (HR = 5.6; 95% CI, 0.83–37.8; P = 0.08). Conclusions: The RP2D for ipilimumab plus standard cetuximab–radiotherapy is 1 mg/kg in weeks 5, 8, 11, and 14. The regimen is tolerable and yields acceptable survival without cytotoxic chemotherapy.

Objectives Data on the efficacy of including definitive local therapy to the primary site for head and neck squamous cell carcinoma (HNSCC) patients with synchronous distant metastasis are lacking. In multiple different solid tumor types, there has been benefit when using systemic therapy followed by local consolidative therapy (stereotactic ablative radiotherapy or surgery) directed at metastases. We proposed to retrospectively evaluate patients at our institution that received definitive treatment to the primary. Methods Single institution retrospective study evaluating 40 patients with metastatic HNSCC treated with definitive surgery (55%) or chemoradiation (45%) to the primary site from 2000 to 2020. The major endpoints were overall survival (OS) and progression‐free survival (PFS) for the total population and multiple sub‐groups. Some variables were evaluated with multiple covariates Cox model. Results The median PFS was 8.6 months (95% CI, 6.4–11.6), and OS was 14.2 months (95% CI, 10.9–27.5). In 28% of patients that received induction therapy, there was a twofold increase in median overall survival to 27.5 months. In the 33% of patients that received anti‐PD‐1 mAb as part of their treatment course, the median OS was significantly increased to 41.7 months (95% CI, 8.7‐NR) versus 12.1 months (95% CI, 8.4–14.4) with a 5‐year OS of 39%. Multivariate analysis for OS showed significance for age at diagnosis, use of IO, and number of metastatic sites. Conclusion We observed impressive survival outcomes in metastatic HNSCC patients treated with definitive local therapy to the primary site in addition to induction and/or immunotherapy. Further study is warranted. Level of Evidence: 3.