Kimberley Stannard

Antimetastatic Effects of Blocking PD-1 and the Adenosine A2A Receptor

Adenosine targeting is an attractive new approach to cancer treatment, but no clinical study has yet examined adenosine inhibition in oncology despite the safe clinical profile of adenosine A2A receptor inhibitors (A2ARi) in Parkinson disease. Metastasis is the main cause of cancer-related deaths worldwide, and therefore we have studied experimental and spontaneous mouse models of melanoma and breast cancer metastasis to demonstrate the efficacy and mechanism of a combination of A2ARi in combination with anti-PD-1 monoclonal antibody (mAb). This combination significantly reduces metastatic burden and prolongs the life of mice compared with either monotherapy alone. Importantly, the combination was only effective when the tumor expressed high levels of CD73, suggesting a tumor biomarker that at a minimum could be used to stratify patients that might receive this combination. The mechanism of the combination therapy was critically dependent on NK cells and IFNγ, and to a lesser extent, CD8(+) T cells and the effector molecule, perforin. Overall, these results provide a strong rationale to use A2ARi with anti-PD-1 mAb for the treatment of minimal residual and metastatic disease.

CIS is a potent checkpoint in NK cell–mediated tumor immunity

The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-15. We identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signaling in NK cells. Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-γ production and cytotoxicity toward tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, inhibiting its enzymatic activity and targeting JAK for proteasomal degradation. Cish−/− mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. Our data uncover a potent intracellular checkpoint in NK cell–mediated tumor immunity and suggest possibilities for new cancer immunotherapies directed at blocking CIS function.

Suppression of Metastases Using a New Lymphocyte Checkpoint Target for Cancer Immunotherapy

UNLABELLED CD96 has recently been shown as a negative regulator of mouse natural killer (NK)-cell activity, with Cd96(-/-)mice displaying hyperresponsive NK cells upon immune challenge. In this study, we have demonstrated that blocking CD96 with a monoclonal antibody inhibited experimental metastases in three different tumor models. The antimetastatic activity of anti-CD96 was dependent on NK cells, CD226 (DNAM-1), and IFNγ, but independent of activating Fc receptors. Anti-CD96 was more effective in combination with anti-CTLA-4, anti-PD-1, or doxorubicin chemotherapy. Blocking CD96 in Tigit(-/-)mice significantly reduced experimental and spontaneous metastases compared with its activity in wild-type mice. Co-blockade of CD96 and PD-1 potently inhibited lung metastases, with the combination increasing local NK-cell IFNγ production and infiltration. Overall, these data demonstrate that blocking CD96 is a new and complementary immunotherapeutic strategy to reduce tumor metastases. SIGNIFICANCE This article illustrates the antimetastatic activity and mechanism of action of an anti-CD96 antibody that inhibits the CD96-CD155 interaction and stimulates NK-cell function. Targeting host CD96 is shown to complement surgery and conventional immune checkpoint blockade.

Natural killer cells are essential for the ability of BRAF inhibitors to control BRAFV600E-mutant metastatic melanoma.

BRAF(V600E) is a major oncogenic mutation found in approximately 50% of human melanoma that confers constitutive activation of the MAPK pathway and increased melanoma growth. Inhibition of BRAF(V600E) by oncogene targeting therapy increases overall survival of patients with melanoma, but is unable to produce many durable responses. Adaptive drug resistance remains the main limitation to BRAF(V600E) inhibitor clinical efficacy and immune-based strategies could be useful to overcome disease relapse. Tumor microenvironment greatly differs between visceral metastasis and primary cutaneous melanoma, and the mechanisms involved in the antimetastatic efficacy of BRAF(V600E) inhibitors remain to be determined. To address this question, we developed a metastatic BRAF(V600E)-mutant melanoma cell line and demonstrated that the antimetastatic properties of BRAF inhibitor PLX4720 (a research analogue of vemurafenib) require host natural killer (NK) cells and perforin. Indeed, PLX4720 not only directly limited BRAF(V600E)-induced tumor cell proliferation, but also affected NK cell functions. We showed that PLX4720 increases the phosphorylation of ERK1/2, CD69 expression, and proliferation of mouse NK cells in vitro. NK cell frequencies were significantly enhanced by PLX4720 specifically in the lungs of mice with BRAF(V600E) lung metastases. Furthermore, PLX4720 also increased human NK cell pERK1/2, CD69 expression, and IFNγ release in the context of anti-NKp30 and IL2 stimulation. Overall, this study supports the idea that additional NK cell-based immunotherapy (by checkpoint blockade or agonists or cytokines) may combine well with BRAF(V600E) inhibitor therapy to promote more durable responses in melanoma.

Adenosine 2B Receptor Expression on Cancer Cells Promotes Metastasis

Adenosine plays an important role in inflammation and tumor development, progression, and responses to therapy. We show that an adenosine 2B receptor inhibitor (A2BRi) decreases both experimental and spontaneous metastasis and combines with chemotherapy or immune checkpoint inhibitors in mouse models of melanoma and triple-negative breast cancer (TNBC) metastasis. Decreased metastasis upon A2BR inhibition is independent of host A2BR and lymphocytes and myeloid cells. Knockdown of A2BR on mouse and human cancer cells reduces their metastasis in vivo and decreases their viability and colony-forming ability, while transiently delaying cell-cycle arrest in vitro The prometastatic activity of adenosine is partly tumor A2BR dependent and independent of host A2BR expression. In humans, TNBC cell lines express higher A2BR than luminal and Her2(+) breast cancer cell lines, and high expression of A2BR is associated with worse prognosis in TNBC. Collectively, high A2BR on mouse and human tumors promotes cancer metastasis and is an ideal candidate for therapeutic intervention. Cancer Res; 76(15); 4372-82. ©2016 AACR.

Co-inhibition of colony stimulating factor-1 receptor and BRAF oncogene in mouse models of BRAFV600E melanoma

ABSTRACT The presence of colony stimulating factor-1 (CSF1)/CSF1 receptor (CSF1R)-driven tumor-infiltrating macrophages and myeloid-derived suppressor cells (MDSCs) is shown to promote targeted therapy resistance. In this study, we demonstrate the superior effect of a combination of CSF1R inhibitor, PLX3397 and BRAF inhibitor, PLX4720, in suppressing primary and metastatic mouse BRAFV600E melanoma. Using flow cytometry to assess SM1WT1 melanoma-infiltrating leukocytes immediately post therapy, we found that PLX3397 reduced the recruitment of CD11b+ Gr1lo and CD11b+ Gr1int M2-like macrophages, but this was accompanied by an accumulation of CD11b+ Gr1hi cells. PDL1 expression on remaining myeloid cells potentially dampened the antitumor efficacy of PLX3397 and PLX4720 in combination, since PD1/PDL1 axis blockade improved outcome. We also reveal a role for PLX3397 in reducing tumor-infiltrating lymphocytes, and interestingly, this feature was rescued by the co-administration of PLX4720. Our findings, from three different mouse models of BRAF-mutated melanoma, support clinical approaches that co-target BRAF oncogene and CSF1R.

Co-blockade of immune checkpoints and adenosine A2A receptor suppresses metastasis

Immunosuppressive pathways active within the tumor microenvironment must be targeted in combination to sufficiently bolster antitumor immune defenses. Inhibition of A2A adenosine receptor signaling in combination with immune checkpoint blockade enhances CD8+ T and NK cell anti-metastatic activity. This results in reduced metastatic burden and improved survival in pre-clinical models.

Targeting cytokine signaling checkpoint CIS activates NK cells to protect from tumor initiation and metastasis

ABSTRACT The cytokine-induced SH2-containing protein CIS belongs to the suppressor of cytokine signaling (SOCS) protein family. Here, we show the critical role of CIS in suppressing natural killer (NK) cell control of tumor initiation and metastasis. Cish-deficient mice were highly resistant to methylcholanthrene-induced sarcoma formation and protected from lung metastasis of B16F10 melanoma and RM-1 prostate carcinoma cells. In contrast, the growth of primary subcutaneous tumors, including those expressing the foreign antigen OVA, was unchanged in Cish-deficient mice. The combination of Cish deficiency and relevant targeted and immuno-therapies such as combined BRAF and MEK inhibitors, immune checkpoint blockade antibodies, IL-2 and type I interferon revealed further improved control of metastasis. The data clearly indicate that targeting CIS promotes NK cell antitumor functions and CIS holds great promise as a novel target in NK cell immunotherapy.

A2AR Adenosine Signaling Suppresses Natural Killer Cell Maturation in the Tumor Microenvironment

Extracellular adenosine is a key immunosuppressive metabolite that restricts activation of cytotoxic lymphocytes and impairs antitumor immune responses. Here, we show that engagement of A2A adenosine receptor (A2AR) acts as a checkpoint that limits the maturation of natural killer (NK) cells. Both global and NK-cell-specific conditional deletion of A2AR enhanced proportions of terminally mature NK cells at homeostasis, following reconstitution, and in the tumor microenvironment. Notably, A2AR-deficient, terminally mature NK cells retained proliferative capacity and exhibited heightened reconstitution in competitive transfer assays. Moreover, targeting A2AR specifically on NK cells also improved tumor control and delayed tumor initiation. Taken together, our results establish A2AR-mediated adenosine signaling as an intrinsic negative regulator of NK-cell maturation and antitumor immune responses. On the basis of these findings, we propose that administering A2AR antagonists concurrently with NK cell-based therapies may heighten therapeutic benefits by augmenting NK cell-mediated antitumor immunity.Significance: Ablating adenosine signaling is found to promote natural killer cell maturation and antitumor immunity and reduce tumor growth. Cancer Res; 78(4); 1003-16. ©2017 AACR.

CD155 loss enhances tumor suppression via combined host and tumor-intrinsic mechanisms

Critical immune-suppressive pathways beyond programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) require greater attention. Nectins and nectin-like molecules might be promising targets for immunotherapy, since they play critical roles in cell proliferation and migration and exert immunomodulatory functions in pathophysiological conditions. Here, we show CD155 expression in both malignant cells and tumor-infiltrating myeloid cells in humans and mice. Cd155–/– mice displayed reduced tumor growth and metastasis via DNAM-1 upregulation and enhanced effector function of CD8+ T and NK cells, respectively. CD155-deleted tumor cells also displayed slower tumor growth and reduced metastases, demonstrating the importance of a tumor-intrinsic role of CD155. CD155 absence on host and tumor cells exerted an even greater inhibition of tumor growth and metastasis. Blockade of PD-1 or both PD-1 and CTLA4 was more effective in settings in which CD155 was limiting, suggesting the clinical potential of cotargeting PD-L1 and CD155 function.