Ellen Moore

Established T Cell–Inflamed Tumors Rejected after Adaptive Resistance Was Reversed by Combination STING Activation and PD-1 Pathway Blockade

Many patients with head and neck squamous cell carcinomas do not respond to current immunotherapies. Antitumor responses, with protective memory and control of distant tumors, developed in mouse models after treatment with PD-L1 mAb and synthetic cyclic dinucleotides. Patients with head and neck squamous cell carcinoma harbor T cell–inflamed and non–T cell–inflamed tumors. Despite this, only 20% of patients respond to checkpoint inhibitor immunotherapy. Lack of induction of innate immunity through pattern-recognition receptors, such as the stimulator of interferon (IFN) genes (STING) receptor, may represent a significant barrier to the development of effective antitumor immunity. Here, we demonstrate robust control of a T cell–inflamed (MOC1), but not non–T cell–inflamed (MOC2), model of head and neck cancer by activation of the STING pathway with the synthetic cyclic dinucleotide RP,RP dithio-c-di-GMP. Rejection or durable tumor control of MOC1 tumors was dependent upon a functional STING receptor and CD8 T lymphocytes. STING activation resulted in increased tumor microenvironment type 1 and type 2 IFN and greater expression of PD-1 pathway components in vivo. Established MOC1 tumors were rejected and distant tumors abscopally controlled, after adaptive immune resistance had been reversed by the addition of PD-L1 mAb. These findings suggest that PD-1 pathway blockade may reverse adaptive immune resistance following cyclic dinucleotide treatment, enhancing both local and systemic antitumor immunity. Cancer Immunol Res; 4(12); 1061–71. ©2016 AACR.

Anti-PD-L1 efficacy can be enhanced by inhibition of myeloid derived suppressor cells with a selective inhibitor of PI3Kδ/γ

Checkpoint inhibitors are relatively inefficacious in head and neck cancers, despite an abundance of genetic alterations and a T-cell-inflamed phenotype. One significant barrier to efficacy may be the recruitment of myeloid-derived suppressor cells (MDSC) into the tumor microenvironment. Here we demonstrate functional inhibition of MDSC with IPI-145, an inhibitor of PI3Kδ and PI3Kγ isoforms, which enhances responses to PD-L1 blockade. Combination therapy induced CD8+ T lymphocyte-dependent primary tumor growth delay and prolonged survival only in T-cell-inflamed tumor models of head and neck cancers. However, higher doses of IPI-145 reversed the observed enhancement of anti-PD-L1 efficacy due to off-target suppression of the activity of tumor-infiltrating T lymphocytes. Together, our results offer a preclinical proof of concept for the low-dose use of isoform-specific PI3Kδ/γ inhibitors to suppress MDSC to enhance responses to immune checkpoint blockade. Cancer Res; 77(10); 2607-19. ©2017 AACR.

mTOR and MEK1/2 inhibition differentially modulate tumor growth and the immune microenvironment in syngeneic models of oral cavity cancer

We investigated the effects of mTOR and MEK1/2 inhibition on tumor growth and the tumor microenvironment in immunogenic and poorly immunogenic models of murine oral cancer. In vitro, rapamycin and PD901 inhibited signaling through expected downstream targets, but only PD901 reduced viability and altered function of MOC cells. Following transplantation of MOC cells into immune-competent mice, effects on both cancer and infiltrating immune cells were characterized following rapamycin and/or PD901 treatment for 21 days. In vivo, both rapamycin and PD901 inhibition reduced primary growth of established MOC tumors on treatment. Following withdrawal of PD901, rapid rebound of tumor growth limited survival, whereas durable tumor control was observed following rapamycin treatment in immunogenic MOC1 tumors despite more robust inhibition of oncogenic signaling by PD901. Characterization of the immune microenvironment revealed diminished infiltration and activation of antigen-specific CD8+ T-cells and other immune cells following PD901 but not rapamycin in immunogenic tumors. Subsequent in vitro T-cell assays validated robust inhibition of T-cell expansion and activation following MEK inhibition compared to mTOR inhibition. CD8 cell depletion abrogated rapamycin-induced primary tumor growth inhibition in MOC1 mice. These data have critical implications in the design of combination targeted and immune therapies in oral cancer.

Resistance to CTLA-4 checkpoint inhibition reversed through selective elimination of granulocytic myeloid cells

Purpose Local immunosuppression remains a critical problem that limits clinically meaningful response to checkpoint inhibition in patients with head and neck cancer. Here, we assessed the impact of MDSC elimination on responses to CTLA-4 checkpoint inhibition. Experimental Design Murine syngeneic carcinoma immune infiltrates were characterized by flow cytometry. Granulocytic MDSCs (gMDSCs) were depleted and T-lymphocyte antigen-specific responses were measured. Tumor-bearing mice were treated with MDSC depletion and CTLA-4 checkpoint blockade. Immune signatures within the human HNSCC datasets from The Cancer Genome Atlas (TCGA) were analyzed and differentially expressed genes from sorted human peripheral MDSCs were examined. Results gMDSCs accumulated with tumor progression and correlated with depletion of effector immune cells. Selective depletion of gMDSC restored tumor and draining lymph node antigen-specific T-lymphocyte responses lost with tumor progression. A subset of T-cell inflamed tumors responded to CTLA-4 mAb alone, but the addition of gMDSC depletion induced CD8 T-lymphocyte-dependent rejection of established tumors in all treated mice that resulted in immunologic memory. MDSCs differentially expressed chemokine receptors. Analysis of the head and neck cancer TCGA cohort revealed high CTLA-4 and MDSC-related chemokine and an MDSC-rich gene expression profile with a T-cell inflamed phenotype in > 60% of patients. CXCR2 and CSF1R expression was validated on sorted peripheral blood MDSCs from HNSCC patients. Conclusions MDSCs are a major contributor to local immunosuppression that limits responses to checkpoint inhibition in head and neck cancer. Limitation of MDSC recruitment or function represents a rational strategy to enhance responses to CTLA-4-based checkpoint inhibition in these patients.PURPOSE Local immunosuppression remains a critical problem that limits clinically meaningful response to checkpoint inhibition in patients with head and neck cancer. Here, we assessed the impact of MDSC elimination on responses to CTLA-4 checkpoint inhibition. EXPERIMENTAL DESIGN Murine syngeneic carcinoma immune infiltrates were characterized by flow cytometry. Granulocytic MDSCs (gMDSCs) were depleted and T-lymphocyte antigen-specific responses were measured. Tumor-bearing mice were treated with MDSC depletion and CTLA-4 checkpoint blockade. Immune signatures within the human HNSCC datasets from The Cancer Genome Atlas (TCGA) were analyzed and differentially expressed genes from sorted human peripheral MDSCs were examined. RESULTS gMDSCs accumulated with tumor progression and correlated with depletion of effector immune cells. Selective depletion of gMDSC restored tumor and draining lymph node antigen-specific T-lymphocyte responses lost with tumor progression. A subset of T-cell inflamed tumors responded to CTLA-4 mAb alone, but the addition of gMDSC depletion induced CD8 T-lymphocyte-dependent rejection of established tumors in all treated mice that resulted in immunologic memory. MDSCs differentially expressed chemokine receptors. Analysis of the head and neck cancer TCGA cohort revealed high CTLA-4 and MDSC-related chemokine and an MDSC-rich gene expression profile with a T-cell inflamed phenotype in > 60% of patients. CXCR2 and CSF1R expression was validated on sorted peripheral blood MDSCs from HNSCC patients. CONCLUSIONS MDSCs are a major contributor to local immunosuppression that limits responses to checkpoint inhibition in head and neck cancer. Limitation of MDSC recruitment or function represents a rational strategy to enhance responses to CTLA-4-based checkpoint inhibition in these patients.

Enhanced Tumor Control with Combination mTOR and PD-L1 Inhibition in Syngeneic Oral Cavity Cancers

Inhibition of mTOR is felt to be systemically immunosuppressive. However, the antitumor immunity induced by checkpoint inhibition in an immunogenic model of oral cavity cancer was enhanced by the mTOR inhibitor rapamycin via a T cell–dependent mechanism. Significant subsets of patients with oral cancer fail to respond to single-agent programmed death (PD) blockade. Syngeneic models of oral cancer were used to determine if blocking oncogenic signaling improved in vivo responses to PD-L1 monoclonal antibody (mAb). Anti–PD-L1 enhanced durable primary tumor control and survival when combined with mTOR (rapamycin), but not in combination with MEK inhibition (PD901) in immunogenic MOC1 tumors. Conversely, PD-L1 mAb did not enhance tumor control in poorly immunogenic MOC2 tumors. Rapamycin enhanced expansion of peripheral antigen-specific CD8 T cells and IFNγ production following ex vivo antigen stimulation. More CD8 T cells infiltrated and were activated after PD-L1 mAb treatment in mice with immunogenic MOC1 tumors, which were stable or increased by the addition of rapamycin, but suppressed when PD901 was added. Rapamycin increased IFNγ production capacity in peripheral and tumor-infiltrating CD8 T cells. In vivo antibody depletion revealed a CD8 T-cell–dependent, and not NK cell–dependent mechanism of tumor growth inhibition after treatment with rapamycin and PD-L1 mAb, ruling out significant effects from NK cell–mediated antibody-dependent cellular cytotoxicity. Rapamycin also enhanced IFNγ or PD-L1 mAb treatment–associated induction of MHC class I expression on MOC1 tumor cells, an effect abrogated by depleting infiltrating CD8 T cells from the tumor microenvironment. These data conflict with traditional views of rapamycin as a universal immunosuppressant, and when combined with evidence of enhanced antitumor activity with the combination of rapamycin and PD-L1 mAb, suggest that this treatment combination deserves careful evaluation in the clinical setting. Cancer Immunol Res; 4(7); 611–20. ©2016 AACR.

Cisplatin Alters Antitumor Immunity and Synergizes with PD-1/PD-L1 Inhibition in Head and Neck Squamous Cell Carcinoma

The combination of cisplatin chemotherapy with anti–PD-1/PD-L1 immunotherapy is under investigation in clinical trials. Optimal doses of cisplatin were found to enhance the antitumor immune response, which was further improved by adding anti–PD-1/PD-L1 immunotherapy. Head and neck squamous cell carcinoma (HNSCC) has been treated for decades with cisplatin chemotherapy, and anti–PD-1 immunotherapy has recently been approved for the treatment of this disease. However, preclinical studies of how antitumor immunity in HNSCC is affected by cisplatin alone or in combination with immunotherapies are lacking. Here, we show that sublethal doses of cisplatin may enhance antigen presentation and T-cell killing in vitro, though cisplatin also upregulates tumor cell expression of PD-L1 and may impair T-cell function at higher doses. In a syngeneic mouse model of HNSCC, concurrent use of cisplatin and anti–PD-L1/PD-1 delayed tumor growth and enhanced survival without significantly reducing the number or function of tumor-infiltrating immune cells or increasing cisplatin-induced toxicities. These results suggest that moderate doses of cisplatin may enhance antitumor immunity by mechanisms other than direct tumor cell killing, which may be further enhanced by anti–PD-L1/PD-1 therapy. Cancer Immunol Res; 5(12); 1141–51. ©2017 AACR.

Dose-dependent enhancement of T-lymphocyte priming and CTL lysis following ionizing radiation in an engineered model of oral cancer

OBJECTIVES Determine if direct tumor cell cytotoxicity, antigen release, and susceptibility to T-lymphocyte killing following radiation treatment is dose-dependent. MATERIALS AND METHODS Mouse oral cancer cells were engineered to express full-length ovalbumin as a model antigen. Tumor antigen release with uptake and cross presentation of antigen by antigen presenting cells with subsequent priming and expansion of antigen-specific T-lymphocytes following radiation was modeled in vitro and in vivo. T-lymphocyte mediated killing was measured following radiation treatment using a novel impedance-based cytotoxicity assay. RESULTS Radiation treatment induced dose-dependent induction of executioner caspase activity and apoptosis in MOC1 cells. In vitro modeling of antigen release and T-lymphocyte priming demonstrated enhanced proliferation of OT-1 T-lymphocytes with 8Gy treatment of MOC1ova cells compared to 2Gy. This was validated in vivo following treatment of established MOC1ova tumors and adoptive transfer of antigen-specific T-lymphocytes. Using a novel impedance-based cytotoxicity assay, 8Gy enhanced tumor cell susceptibility to T-lymphocyte killing to a greater degree than 2Gy. CONCLUSION In the context of using clinically-relevant doses of radiation treatment as an adjuvant for immunotherapy, 8Gy is superior to 2Gy for induction of antigen-specific immune responses and enhancing tumor cell susceptibility to T-lymphocyte killing. These findings have significant implications for the design of trials combining radiation and immunotherapy.

Inhibition of WEE1 kinase and cell cycle checkpoint activation sensitizes head and neck cancers to natural killer cell therapies

BackgroundNatural killer (NK) cells recognize and lyse target tumor cells in an MHC-unrestricted fashion and complement antigen- and MHC-restricted killing by T-lymphocytes. NK cells and T-lymphocytes mediate early killing of targets through a common granzyme B-dependent mechanism. Tumor cell resistance to granzyme B and how this alters NK cell killing is not clearly defined.MethodsTumor cell sensitivity to cultured murine KIL and human high affinity NK (haNK) cells in the presence or absence of AZD1775, a small molecule inhibitor of WEE1 kinase, was assessed via real time impedance analysis. Mechanisms of enhanced sensitivity to NK lysis were determined and in vivo validation via adoptive transfer of KIL cells into syngeneic mice was performed.ResultsCultured murine KIL cells lyse murine oral cancer 2 (MOC2) cell targets more efficiently than freshly isolated peripheral murine NK cells. MOC2 sensitivity to granzyme B-dependent KIL cell lysis was enhanced by inhibition of WEE1 kinase, reversing G2/M cell cycle checkpoint activation and resulting in enhanced DNA damage and apoptosis. Treatment of MOC2 tumor-bearing wild-type C57BL/6 mice with AZD1775 and adoptively transferred KIL cells resulted in enhanced tumor growth control and survival over controls or either treatment alone. Validating these findings in human models, WEE1 kinase inhibition sensitized two human head and neck cancer cell lines to direct lysis by haNK cells. Further, WEE1 kinase inhibition sensitized these cell lines to antibody-dependent cell-mediated cytotoxicity when combined with the anti-PD-L1 IgG1 mAb Avelumab.ConclusionsTumor cell resistance to granzyme B-induced cell death can be reversed through inhibition of WEE1 kinase as AZD1775 sensitized both murine and human head and neck cancer cells to NK lysis. These data provide the pre-clinical rationale for the combination of small molecules that reverse cell cycle checkpoint activation and NK cellular therapies.

Nanocomplex-based TP53 gene therapy promotes anti-tumor immunity through TP53- and STING-dependent mechanisms

ABSTRACT Loss or mutation of TP53 has been linked to alterations in anti-tumor immunity as well as dysregulation of cell cycle and apoptosis. We explored immunologic effects and mechanisms following restoration of wild-type human TP53 cDNA in murine oral cancer cells using the therapeutic nanocomplex scL-53. We demonstrated scL-53 induces dose-dependent expression of TP53 and induction of apoptosis and immunogenic cell death. We further demonstrated both TP53-dependent and independent induction of tumor cell immunogenicity through the use of blocking mAbs, nanocomplex loaded with DNA plasmid with or without TP53 cDNA, empty nanocomplex and siRNA knockdown techniques. TP53-independent immune modulation was observed following treatment with nanocomplex loaded with DNA plasmid lacking TP53 cDNA and abrogated in STING-deficient tumor cells, supporting the presence of a cytoplasmic DNA sensing, STING-dependent type-I IFN response. Cooperatively, TP53- and STING-dependent alterations sensitized tumor cells to CTL-mediated lysis, which was further enhanced following reversal of adaptive immune resistance with PD-1 mAb. In vivo, combination scL-53 and PD-1 mAb resulted in growth control or rejection of established tumors that was abrogated in mice depleted of CD8+ cells or in STING deficient mice. Cumulatively, this work demonstrates 1) a direct anti-tumor effects of functional TP53; 2) non-redundant TP53- and STING-dependent induction of tumor cell immunogenicity following scL-53 treatment; and 3) that adaptive immune resistance following scL-53 treatment can be reversed with PD-based immune checkpoint blockade, resulting in the rejection or control of syngeneic murine tumors. These data strongly support the clinical combination of scL-53 and immune checkpoint blockade.

WEE1 kinase inhibition reverses G2/M cell cycle checkpoint activation to sensitize cancer cells to immunotherapy

ABSTRACT Intrinsic resistance to cytotoxic T-lymphocyte (CTL) killing limits responses to immune activating anti-cancer therapies. Here, we established that activation of the G2/M cell cycle checkpoint results in tumor cell cycle pause and protection from granzyme B-induced cell death. This was reversed with WEE1 kinase inhibition, leading to enhanced CTL killing of antigen-positive tumor cells. Similarly, but at a later time point, cell cycle pause following TNFα exposure was reversed with WEE1 kinase inhibition, leading to CTL transmembrane TNFα-dependent induction of apoptosis and necroptosis in bystander antigen-negative tumor cells. Results were reproducible in models of oral cavity carcinoma, melanoma and colon adenocarcinoma harboring variable Tp53 genomic alterations. WEE1 kinase inhibition sensitized tumors to PD-1 mAb immune checkpoint blockade in vivo, resulting in CD8+-dependent rejection of established tumors harboring antigen-positive or mixed antigen-positive and negative tumor cells. Together, these data describe activation of the G2/M cell cycle checkpoint in response to early and late CTL products as a mechanism of resistance to CTL killing, and provide pre-clinical rationale for the clinical combination of agents that inhibit cell cycle checkpoints and activate anti-tumor immunity.