Els M. E. Verdegaal

High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4 + T cells in human melanoma

Tumor-specific neo-antigens that arise as a consequence of mutations are thought to be important for the therapeutic efficacy of cancer immunotherapies. Accumulating evidence suggests that neo-antigens may be commonly recognized by intratumoral CD8+ T cells, but it is unclear whether neo-antigen–specific CD4+ T cells also frequently reside within human tumors. In view of the accepted role of tumor-specific CD4+ T-cell responses in tumor control, we addressed whether neo-antigen–specific CD4+ T-cell reactivity is a common property in human melanoma.

Neoantigen landscape dynamics during human melanoma–T cell interactions

Recognition of neoantigens that are formed as a consequence of DNA damage is likely to form a major driving force behind the clinical activity of cancer immunotherapies such as T-cell checkpoint blockade and adoptive T-cell therapy. Therefore, strategies to selectively enhance T-cell reactivity against genetically defined neoantigens are currently under development. In mouse models, T-cell pressure can sculpt the antigenicity of tumours, resulting in the emergence of tumours that lack defined mutant antigens. However, whether the T-cell-recognized neoantigen repertoire in human cancers is constant over time is unclear. Here we analyse the stability of neoantigen-specific T-cell responses and the antigens they recognize in two patients with stage IV melanoma treated by adoptive T-cell transfer. The T-cell-recognized neoantigens can be selectively lost from the tumour cell population, either by overall reduced expression of the genes or loss of the mutant alleles. Notably, loss of expression of T-cell-recognized neoantigens was accompanied by development of neoantigen-specific T-cell reactivity in tumour-infiltrating lymphocytes. These data demonstrate the dynamic interactions between cancer cells and T cells, which suggest that T cells mediate neoantigen immunoediting, and indicate that the therapeutic induction of broad neoantigen-specific T-cell responses should be used to avoid tumour resistance.

Successful treatment of metastatic melanoma by adoptive transfer of blood-derived polyclonal tumor-specific CD4+ and CD8+ T cells in combination with low-dose interferon-alpha

A phase I/II study was conducted to test the feasibility and safety of the adoptive transfer of tumor-reactive T cells and daily injections of interferon-alpha (IFNα) in metastatic melanoma patients with progressive disease. Autologous melanoma cell lines were established to generate tumor-specific T cells by autologous mixed lymphocyte tumor cell cultures using peripheral blood lymphocytes. Ten patients were treated with on average 259 (range 38–474) million T cells per infusion to a maximum of six infusions, and clinical response was evaluated according to the response evaluation criteria in solid tumors (RECIST). Five patients showed clinical benefit from this treatment, including one complete regression, one partial response, and three patients with stable disease. No treatment-related serious adverse events were observed, except for the appearance of necrotic-like fingertips in one patient. An IFNα-related transient leucopenia was detected in 6 patients, including all responders. One responding patient displayed vitiligo. The infused T-cell batches consisted of tumor-reactive polyclonal CD8+ and/or CD4+ T cells. Clinical reactivity correlated with the functional properties of the infused tumor-specific T cells, including their in vitro expansion rate and the secretion of mainly Th1 cytokines as opposed to Th2 cytokines. Our study shows that relatively low doses of T cells and low-dose IFNα can lead to successful treatment of metastatic melanoma and reveals a number of parameters potentially associated with this success.

Quantitative expression profiling of G‐protein‐coupled receptors (GPCRs) in metastatic melanoma: the constitutively active orphan GPCR GPR18 as novel drug target

G‐protein‐coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. Utilizing a robust quantitative PCR array, we quantified expression of 94 human GPCRs, including 75 orphan GPCRs and 19 chemokine receptors, and 36 chemokine ligands, in 40 melanoma metastases from different individuals and benign nevi. Inter‐metastatic site comparison revealed that orphan GPR174 and CCL28 are statistically significantly overexpressed in subcutaneous metastases, while P2RY5 is overexpressed in brain metastases. Comparison between metastases (all three metastatic sites) and benign nevi revealed that 16 genes, including six orphan receptors (GPR18, GPR34, GPR119, GPR160, GPR183 and P2RY10) and chemokine receptors CCR5, CXCR4, and CXCR6, were statistically significantly differentially expressed. Subsequent functional experiments in yeast and melanoma cells indicate that GPR18, the most abundantly overexpressed orphan GPCR in all melanoma metastases, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival. GPR18 and five other orphan GPCRs with yet unknown biological function may be considered potential novel anticancer targets in metastatic melanoma.

Genome-wide promoter methylation analysis identifies epigenetic silencing of MAPK13 in primary cutaneous melanoma.

The involvement of epigenetic alterations in the pathogenesis of melanoma is increasingly recognized. Here, we performed genome‐wide DNA methylation analysis of primary cutaneous melanoma and benign melanocytic nevus interrogating 14 495 genes using BeadChip technology. This genome‐wide view of promoter methylation in primary cutaneous melanoma revealed an array of recurrent DNA methylation alterations with potential diagnostic applications. Among 106 frequently hypermethylated genes, there were many novel methylation targets and tumor suppressor genes. Highly recurrent methylation of the HOXA9, MAPK13, CDH11, PLEKHG6, PPP1R3C, and CLDN11 genes was established. Promoter methylation of MAPK13, encoding p38δ, was present in 67% of primary and 85% of metastatic melanomas. Restoration of MAPK13 expression in melanoma cells exhibiting epigenetic silencing of this gene reduced proliferation, indicative of tumor suppressive functions. This study demonstrates that DNA methylation alterations are widespread in melanoma and suggests that epigenetic silencing of MAPK13 contributes to melanoma progression.

Inhibition of the type I immune responses of human monocytes by IFN-α and IFN-β.

Interleukin-12 (IL-12), IL-23 and interferon-γ (IFN-γ) are pivotal cytokines acting in concert with tumor necrosis factor (TNF) and IL-1β to shape type I immune responses against bacterial pathogens. Recently, several groups reported that type I immunity can be inhibited by IFN-α/β. Here we show the extent of the inhibitory effects of IFN-α and IFN-β on the responsiveness of human monocytes to Toll like receptor-ligands and IFN-γ. Both IFN-α and IFN-β strongly reduced the production of IL-12p40, IL-1β and TNF and the IFN-γ induced CD54 and CD64 expression. High IFN-γ concentrations could not counterbalance the inhibitions and IFN-α still inhibited monocytes 24h after stimulation in vitro as well as in vivo in patients undergoing IFN-α treatment. Next, we explored the mechanism of inhibition. We confirm that IFN-α/β interferes with the IFN-γR1 expression, by studying the kinetics of IFN-γR1 downregulation. However, IFN-γR1 downregulation occurred only after two hours of IFN-α/β stimulation and was transient, which cannot explain the IFN-γ unresponsiveness observed directly and late after IFN-α/β stimulation. Additional experiments indeed indicate that other mechanisms are involved. IFN-α may interfere with IFN-γ-elicited phosphorylation of signal transducer and activator of transcription 1 (STAT1). IFN-α may also activate methyltransferases which in turn reduce, at least partly, the TNF and IL-1β production and CD54 expression. IFN-α also induces the protein inhibitor of activated STAT1 (PIAS1). In conclusion, IFN-α and IFN-β strongly inhibit the IFN-γ responsiveness and the production of type I cytokines of monocytes, probably via various mechanisms. Our findings indicate that IFN-α/β play a significant role in the immunopathogenesis of bacterial infections, for example Mycobacterium tuberculosis infection.

Antagonistic TSC22D1 variants control BRAF(E600)-induced senescence

Oncogene‐induced cellular senescence (OIS) is an increasingly recognized tumour suppressor mechanism that confines the outgrowth of neoplastic cells in vivo. It relies on a complex signalling network, but only few components have been identified so far. Gene‐expression profiling revealed a >100‐fold increase in the levels of the transcription factor and putative tumour suppressor gene TGFβ‐stimulated clone 22 (TSC22D1) in BRAFE600‐induced senescence, in both human fibroblasts and melanocytes. Only the short TSC22D1 transcript was upregulated, whereas the abundance of the large protein variant was suppressed by proteasomal degradation. The TSC22D1 protein variants, in complex with their dimerization partner TSC22 homologue gene 1 (THG1), exerted opposing functions, as selective depletion of the short form, or conversely, overexpression of the large variant, resulted in abrogation of OIS. This was accompanied by the suppression of several inflammatory factors and p15INK4B, with TSC22D1 acting as a critical effector of C/EBPβ. Our results demonstrate that the differential regulation of antagonistic TSC22D1 variants is required for the establishment of OIS and suggest distinct contributions of TSC22 family members to the progression of BRAFE600‐driven neoplasia.

Cooperative induction of apoptosis in NRAS mutant melanoma by inhibition of MEK and ROCK

No effective targeted therapy is currently available for NRAS mutant melanoma. Experimental MEK inhibition is rather toxic and has only limited efficacy in clinical trials. At least in part, this is caused by the emergence of drug resistance, which is commonly seen for single agent treatment and shortens clinical responses. Therefore, there is a dire need to identify effective companion drug targets for NRAS mutant melanoma. Here, we show that at concentrations where single drugs had little effect, ROCK inhibitors GSK269962A or Fasudil, in combination with either MEK inhibitor GSK1120212 (Trametinib) or ERK inhibitor SCH772984 cooperatively caused proliferation inhibition and cell death in vitro. Simultaneous inhibition of MEK and ROCK caused induction of BimEL, PARP, and Puma, and hence apoptosis. In vivo, MEK and ROCK inhibition suppressed growth of established tumors. Our findings warrant clinical investigation of the effectiveness of combinatorial targeting of MAPK/ERK and ROCK in NRAS mutant melanoma.

Adoptive cell therapy: a highly successful individualized therapy for melanoma with great potential for other malignancies

Adoptive cell therapy (ACT) by infusion of autologous or redirected tumor-specific T-cells has had a major impact on the treatment of several metastasized malignancies that were until now hardly treatable. Recent findings provide a more profound knowledge on the underlying mechanisms of success and allow the optimization of the ACT protocol with respect to (1) the treatment related side-effects, (2) the quality and specificity of infused T-cells, and (3) the immunosuppressive phenotype of the tumor environment. In this review, the results and insights in the success of ACT as well as the possibilities to improve ACT and its exploitation as treatment option for various metastatic cancer types, will be discussed.

Preclinical evaluation of DISC-GMCSF for the treatment of breast carcinoma

DISC‐hGMCSF is a gH‐deleted HSV‐2 based vector expressing human GM‐CSF that has entered clinical trials for the therapy of metastatic melanoma. To determine whether this product also has potential to treat breast carcinoma, a series of in vitro and in vivo studies were made.