Expression Of Melanocyte Differentiation Antigens Research Articles

Syngeneic B16-F1 cells are more efficient than allogeneic Cloudman cells as antigen source in DC-based vaccination in the B16-F1 murine melanoma model

A major obstacle to obtaining relevant results in cancer vaccination has been the lack of identification of immunogenic antigens. Dendritic cell (DC)-based cancer vaccines used preventively may afford protection against tumor inoculation, but the effect of antigen choice on anti-tumor protection is not clear. When using irradiated syngeneic tumor cells to load DCs, tumor self-antigens are provided, including tumor-associated antigens (TAAs) and neoantigens generated by tumor mutations. On the other hand, allogeneic tumor cells could only supply shared TAAs. To assess the advantages of each source in protective vaccination, we analyzed in C57BL/6 mice the effect of loading DCs with irradiated syngeneic B16-F1 or allogeneic Cloudman melanoma cells; both cell lines were characterized by whole exome sequencing and RNAseq. Tumor cell components from the two irradiated cell lines were efficiently internalized by DCs, and transported to MHC-class II positive tubulovesicular compartments (MIICs). DCs loaded with allogeneic irradiated Cloudman cells (DC-ApoNecALLO) induced a partially effective anti-melanoma protection, although Cloudman and B16-F1 cells share the expression of melanocyte differentiation antigens (MDAs), cancer-testis antigens (CTAs) and other TAAs. DCs loaded with syngeneic B16-F1 cells (DC-ApoNecSYN) established a more potent and long-lasting protection and induced a humoral anti-B16F1 response, thus suggesting that neoepitopes are needed for inducing long-lasting protection.

NFATc2 is an intrinsic regulator of melanoma dedifferentiation.

Melanoma dedifferentiation, characterized by the loss of MITF and MITF regulated genes and by upregulation of stemness markers as CD271, is implicated in resistance to chemotherapy, target therapy and immunotherapy. The identification of intrinsic mechanisms fostering melanoma dedifferentiation may provide actionable therapeutic targets to improve current treatments. Here, we identify NFATc2 transcription factor as an intrinsic regulator of human melanoma dedifferentiation. In panels of melanoma cell lines, NFATc2 expression correlated inversely with MITF at both mRNA and protein levels. NFATc2(+/Hi) melanoma cell lines were CD271(+) and deficient for expression of melanocyte differentiation antigens (MDAs) MART-1, gp100, tyrosinase and of GPNMB, PGC1-α and Rab27a, all regulated by MITF. Targeting of NFATc2 by small interfering RNA, short hairpin RNA and by an NFATc2 inhibitor upregulated MITF, MDAs, GPNMB, PGC-1α, tyrosinase activity and pigmentation and suppressed CD271. Mechanistically, we found that NFATc2 controls melanoma dedifferentiation by inducing expression in neoplastic cells of membrane-bound tumor necrosis factor-α (mTNF-α) and that melanoma-expressed TNF-α regulates a c-myc-Brn2 axis. Specifically, NFATc2, mTNF-α and expression of TNF receptors were significantly correlated in panels of cell lines. NFATc2 silencing suppressed TNF-α expression, and neutralization of melanoma-expressed TNF-α promoted melanoma differentiation. Moreover, silencing of NFATc2 and TNF-α neutralization downmodulated c-myc and POU3F2/Brn2. Brn2 was strongly expressed in NFATc2(+/Hi) MITF(Lo) cell lines and its silencing upregulated MITF. Targeting of c-myc, by silencing or by a c-myc inhibitor, suppressed Brn2 and upregulated MITF and MART-1 in melanoma cells. The relevance of NFATc2-dependent melanoma dedifferentiation for immune escape was shown by cytolytic T-cell assays. NFATc2(Hi) MITF(Lo) MDA(Lo) HLA-A2.1(+) melanoma cells were poorly recognized by MDA-specific and HLA-A2-restricted CTL lines, but NFATc2 targeting significantly increased CTL-mediated tumor recognition. Taken together, these results suggest that the expression of NFATc2 promotes melanoma dedifferentiation and immune escape.

Desmoplastic Melanoma With Sarcomatoid Dedifferentiation

Desmoplastic melanoma (DM) is a variant of melanoma, which typically affects chronically sun-damaged skin of elderly patients. Pure DM displays a low density of fusiform melanocytes in a collagen-rich matrix. In mixed DM, tumor cell density is higher, and parts of the tumor lack abundant stromal fibrosis. Both pure and mixed DMs usually express S100 protein homogenously. We report herein an unusual biphenotypic tumor characterized by the association of a pure DM with an undifferentiated solid spindle cell nodule. It occurred on the scalp of a 66-year-old man. A biopsy of the undifferentiated spindle cell nodule was initially interpreted at a commercial laboratory as atypical fibroxanthoma. The pure DM was seen only in the excisional specimen. All cells of the pure DM stained for S100 protein and SOX10. The adjacent solid sarcomatoid spindle cell nodule lacked expression of S100 protein, SOX10, as well as melan-A, gp100, and microphthalmia-associated transcription factor in >95% of its tumor cells. Although focal expression of melanocyte differentiation antigens in the solid tumor component made us favor a combined DM with sarcomatoid dedifferentiation, we also considered the possibility of a collision scenario, that is, a pleomorphic dermal sarcoma incidentally colliding with a DM. To further assess a possible relationship of the sarcomatoid nodule with the DM, we performed next-generation sequencing analysis on each component separately. The analysis revealed shared chromosomal copy number changes and a high number of common mutations, thereby supporting the concept of a DM with a dedifferentiated sarcomatoid component. An interesting finding is the presence of mutations of the neurofibromin 1 (NF1) gene in both tumor components.

Abstract 958: Treatment with a selective inhibitor of BRAFV600E increases melanocyte antigen expression and CD8 T cell infiltrate in tumors of patients with metastatic melanoma

Abstract Targeted therapy against oncogenic BRAF (BRAFV600E) is a promising new therapeutic approach for the treatment of melanoma with up to 80% of patients with stage IV melanoma responding to treatment. Despite these results, the median duration of response is only 8.5 months. Investigators propose combining this therapy with other targeted agents to address redundancy and signaling through different oncogenic pathways in hopes to improve the durability of response. An alternate approach is to combine BRAF-targeted agents with immunotherapy based on evidence that oncogenic BRAF contributes to immune escape. We recently reported data supporting this hypothesis, showing that treatment of melanoma cell lines with MEK or selective BRAFV600E inhibition results in increased expression of melanocyte differentiation antigens (MDAs) which is associated with enhanced recognition by antigen-specific T cells. The purpose of these studies was to validate these findings by assaying expression of MDAs in patients with metastatic melanoma treated with BRAFV600E inhibition. We also sought to test immune cell infiltrate in tumors to analyze host immune response. Patients with metastatic melanoma undergoing treatment with a selective BRAFV600E inhibitor were consented on a tissue procurement protocol approved through the IRB at the Massachusetts General Hospital. Patients underwent treatment with the BRAFV600E inhibitor (PLX4032) per protocol (960 mg given twice daily). Tumor biopsies and blood draws were performed pre-treatment and 10-14 days after initiation of treatment. RNA was harvested and expression of MDAs was assayed by quantitative RT-PCR. The level of MITF, the master transcriptional regulator of melanocyte which controls MDA expression, was also assayed. Immunohistochemistry was performed on formalin-fixed, paraffin embedded tissue for MDAs, as well as CD4 and CD8. The effect of BRAFV600E inhibition on T lymphocyte function was analyzed by assaying fold-expansion and viability in blood samples collected before and during treatment. Treatment with a selective inhibitor of BRAFV600E resulted in significantly increased levels of MDAs (up to 95-fold) which was associated with higher levels of MITF. In addition, CD8 T cell infiltrate also increased after treatment. Of note, T cell function was preserved after treatment with a specific inhibitor of BRAFV600E. These data provide in vivo correlates to our in vitro findings that selective BRAFV600E inhibition enhances MDA expression in melanoma and suggest a possible improved host immune response. The studies provide further support for potential synergy between BRAF-targeted therapy and immunotherapy in the treatment of melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 958. doi:10.1158/1538-7445.AM2011-958

Selective BRAFV600E Inhibition Enhances T-Cell Recognition of Melanoma without Affecting Lymphocyte Function

Targeted therapy against the BRAF/mitogen-activated protein kinase (MAPK) pathway is a promising new therapeutic approach for the treatment of melanoma. Treatment with selective BRAF inhibitors results in a high initial response rate but limited duration of response. To counter this, investigators propose combining this therapy with other targeted agents, addressing the issue of redundancy and signaling through different oncogenic pathways. An alternative approach is combining BRAF/MAPK-targeted agents with immunotherapy. Preliminary evidence suggests that oncogenic BRAF (BRAF(V600E)) contributes to immune escape and that blocking its activity via MAPK pathway inhibition leads to increased expression of melanocyte differentiation antigens (MDA). Recognition of MDAs is a critical component of the immunologic response to melanoma, and several forms of immunotherapy capitalize on this recognition. Among the various approaches to inhibiting BRAF/MAPK, broad MAPK pathway inhibition may have deleterious effects on T lymphocyte function. Here, we corroborate the role of oncogenic BRAF in immune evasion by melanoma cells through suppression of MDAs. We show that inhibition of the MAPK pathway with MAPK/extracellular signal-regulated kinase kinase (MEK) inhibitors or a specific inhibitor of BRAF(V600E) in melanoma cell lines and tumor digests results in increased levels of MDAs, which is associated with improved recognition by antigen-specific T lymphocytes. However, treatment with MEK inhibitors impairs T lymphocyte function, whereas T-cell function is preserved after treatment with a specific inhibitor of BRAF(V600E). These findings suggest that immune evasion of melanomas mediated by oncogenic BRAF may be reversed by targeted BRAF inhibition without compromising T-cell function. These findings have important implications for combined kinase-targeted therapy plus immunotherapy for melanoma.

Abstract 1970: Inhibition of BRAF-V600E increases melanocyte antigen expression and antigen-specific recognition in melanoma without altering T cell function

Abstract Given the high prevalence of BRAFV600E mutations in melanoma, targeted therapy against BRAF, or its mutated form, is a promising new therapeutic approach for patients with melanoma. Preliminary evidence suggests that BRAFV600E contributes to immune escape, and that blocking its activity via MAPK pathway inhibition leads to increased expression of melanocyte differentiation antigens, supporting the potential use of combination targeted therapy and immunotherapy. However, indiscriminate MAPK pathway inhibition may have deleterious effects on the immune system and lead to T cell dysfunction. The purpose of these studies was to explore the role of oncogenic BRAF in immune evasion by melanoma cells through its suppression of melanocyte differentiation antigens (MDAs). Furthermore, we sought to evaluate the effect of MAPK pathway inhibition on T lymphocyte function as a basis for combined targeted therapy and immunotherapy. Melanoma cell lines were cultured with MEK inhibitors (U0126, PD0325901) or a specific inhibitor of BRAFV600E (PLX4720) and expression of MDAs was assayed at the mRNA and protein level. The level of micro-opthalmia transcription factor (MITF), a transcriptional regulator of MDAs, was also assayed. Untreated and treated melanoma cell lines were co-cultured with antigen-specific T lymphocytes (generated by transduction of lymphocytes with MART-1- and gp100-specific T cell receptors) and reactivity was measured via interferon gamma release. The effect of MAPK pathway inhibition on T lymphocyte function was analyzed by assaying fold-expansion after OKT-3 & IL-2 stimulation as well as antigen-specific interferon gamma release. Inhibition of MAPK pathway in melanoma cell lines resulted in increased levels of MDAs which was associated with improved antigen-specific recognition by MART-1- and gp100-specific T lymphocytes. This was associated with higher intracellular levels of MITF. Treatment with MEK inhibitors resulted in impaired T lymphocyte function, as assayed by T cell expansion and antigen-specific interferon gamma release, whereas T cell function was preserved after treatment with a specific inhibitor of BRAFV600E. These findings suggest that oncogenic BRAF contributes to immune evasion by transcriptional repression of melanocyte antigen expression through MITF, which may be reversed by targeted MAPK pathway inhibition. Selective inhibition of BRAFV600E enhances MDA expression while preserving T cell function, whereas MEK inhibition has deleterious effects on T cell function. These data have important implications for the design of clinical trials using combined targeted therapy and immunotherapy for melanoma as well as other cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1970.

The SCF/KIT Pathway Plays a Critical Role in the Control of Normal Human Melanocyte Homeostasis

During development, the interaction of stem cell factor (SCF) with its receptor, KIT, is critical for the survival of melanocytes. Limited in vivo human studies have suggested a possible activating role of SCF on adult human melanocytes. In order to study the impact of this pathway on normal melanocyte homeostasis, human skin xenografts were treated with serial injections of recombinant human SCF or a KIT-inhibitory antibody (K44.2). On histologic evaluation, SCF injection increased, whereas KIT inhibition decreased the number, size, and dendricity of melanocytes. Immunohistochemical expression of melanocyte differentiation antigens, including tyrosinase-related-protein-1 and gp100/pmel17, was markedly increased by treatment with SCF, and decreased by K44.2 treatment. The number of Ki67-positive melanocytes was increased in the SCF-treated tissue, suggesting a direct proliferative effect of SCF; conversely, treatment with K44.2 resulted in melanocyte loss, which did not appear reversible with prolonged treatment. These findings demonstrate that the SCF/KIT pathway remains critical in adult human skin, and that pharmacologic modulation of this single pathway can control cutaneous melanocyte homeostasis.

Surface antigens of melanocytes and melanomas. Markers of melanocyte differentiation and melanoma subsets.

The surface antigens of melanocytes from newborn and adult skin have been analyzed with monoclonal antibodies detecting cell surface antigens of malignant melanoma. Antigenic markers that distinguish early, intermediate, and mature stages in melanocyte differentiation have been defined. The characteristics of the normal melanocyte precursor have been inferred from the features of melanomas that express early markers of melanocyte differentiation. A rudimentary surface antigen map of cells undergoing melanocyte differentiation and a new classification of melanomas based on the expression of melanocyte differentiation antigens are proposed.