Adoptive T Cell Therapy Targeting Different Gene Products Reveals Diverse and Context-Dependent Immune Evasion in Melanoma.

Tumor immune escape limits durable responses to T cell therapy. Here, we examined how regulation and function of gene products that provide the target epitopes for CD8+ T cell anti-tumor immunity influence therapeutic efficacy and resistance. We used a CRISPR-Cas9-based method (CRISPitope) in syngeneic melanoma models to fuse the same model CD8+ T cell epitope to the C-termini of different endogenous gene products. Targeting melanosomal proteins or oncogenic CDK4R24C (Cyclin-dependent kinase 4) by adoptive cell transfer (ACT) of the same epitope-specific CD8+ T cells revealed diverse genetic and non-genetic immune escape mechanisms. ACT directed against melanosomal proteins, but not CDK4R24C, promoted melanoma dedifferentiation, and increased myeloid cell infiltration. CDK4R24C antigen persistence was associated with an interferon-high and T-cell-rich tumor microenvironment, allowing for immune checkpoint inhibition as salvage therapy. Thus, the choice of target antigen determines the phenotype and immune contexture of recurrent melanomas, with implications to the design of cancer immunotherapies.

Reactive Neutrophil Responses Dependent on the Receptor Tyrosine Kinase c-MET Limit Cancer Immunotherapy.

Inhibitors of the receptor tyrosine kinase c-MET are currently used in the clinic to target oncogenic signaling in tumor cells. We found that concomitant c-MET inhibition promoted adoptive T cell transfer and checkpoint immunotherapies in murine cancer models by increasing effector T cell infiltration in tumors. This therapeutic effect was independent of tumor cell-intrinsic c-MET dependence. Mechanistically, c-MET inhibition impaired the reactive mobilization and recruitment of neutrophils into tumors and draining lymph nodes in response to cytotoxic immunotherapies. In the absence of c-MET inhibition, neutrophils recruited to T cell-inflamed microenvironments rapidly acquired immunosuppressive properties, restraining T cell expansion and effector functions. In cancer patients, high serum levels of the c-MET ligand HGF correlated with increasing neutrophil counts and poor responses to checkpoint blockade therapies. Our findings reveal a role for the HGF/c-MET pathway in neutrophil recruitment and function and suggest that c-MET inhibitor co-treatment may improve responses to cancer immunotherapy in settings beyond c-MET-dependent tumors.

Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma.

Intermittent intense ultraviolet (UV) exposure represents an important aetiological factor in the development of malignant melanoma. The ability of UV radiation to cause tumour-initiating DNA mutations in melanocytes is now firmly established, but how the microenvironmental effects of UV radiation influence melanoma pathogenesis is not fully understood. Here we report that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression, independent of its tumour-initiating effects. UV irradiation enhanced the expansion of tumour cells along abluminal blood vessel surfaces and increased the number of lung metastases. This effect depended on the recruitment and activation of neutrophils, initiated by the release of high mobility group box 1 (HMGB1) from UV-damaged epidermal keratinocytes and driven by Toll-like receptor 4 (TLR4). The UV-induced neutrophilic inflammatory response stimulated angiogenesis and promoted the ability of melanoma cells to migrate towards endothelial cells and use selective motility cues on their surfaces. Our results not only reveal how UV irradiation of epidermal keratinocytes is sensed by the innate immune system, but also show that the resulting inflammatory response catalyses reciprocal melanoma-endothelial cell interactions leading to perivascular invasion, a phenomenon originally described as angiotropism in human melanomas by histopathologists. Angiotropism represents a hitherto underappreciated mechanism of metastasis that also increases the likelihood of intravasation and haematogenous dissemination. Consistent with our findings, ulcerated primary human melanomas with abundant neutrophils and reactive angiogenesis frequently show angiotropism and a high risk for metastases. Our work indicates that targeting the inflammation-induced phenotypic plasticity of melanoma cells and their association with endothelial cells represent rational strategies to specifically interfere with metastatic progression.

Amplification of N-Myc is associated with a T-cell-poor microenvironment in metastatic neuroblastoma restraining interferon pathway activity and chemokine expression.

Immune checkpoint inhibitors have significantly improved the treatment of several cancers. T-cell infiltration and the number of neoantigens caused by tumor-specific mutations are correlated to favorable responses in cancers with a high mutation load. Accordingly, checkpoint immunotherapy is thought to be less effective in tumors with low mutation frequencies such as neuroblastoma, a neuroendocrine tumor of early childhood with poor outcome of the high-risk disease group. However, spontaneous regressions and paraneoplastic syndromes seen in neuroblastoma patients suggest substantial immunogenicity. Using an integrative transcriptomic approach, we investigated the molecular characteristics of T-cell infiltration in primary neuroblastomas as an indicator of pre-existing immune responses and potential responsiveness to checkpoint inhibition. Here, we report that a T-cell-poor microenvironment in primary metastatic neuroblastomas is associated with genomic amplification of the MYCN (N-Myc) proto-oncogene. These tumors exhibited lower interferon pathway activity and chemokine expression in line with reduced immune cell infiltration. Importantly, we identified a global role for N-Myc in the suppression of interferon and pro-inflammatory pathways in human and murine neuroblastoma cell lines. N-Myc depletion potently enhanced targeted interferon pathway activation by a small molecule agonist of the cGAS-STING innate immune pathway. This promoted chemokine expression including Cxcl10 and T-cell recruitment in microfluidics migration assays. Hence, our data suggest N-Myc inhibition plus targeted IFN activation as adjuvant strategy to enforce cytotoxic T-cell recruitment in MYCN-amplified neuroblastomas.