Christine Wagner

Targeting CD20 in Melanoma Patients at High Risk of Disease Recurrence

Melanomas contain distinct cell subpopulations. Several of these subpopulations, including one expressing CD20, may harbor stem cell-like or tumor-initiating characteristics. We hypothesized that patients at high risk of disease recurrence could benefit from an adjuvant anti-CD20 therapy. Therefore, we initiated a small pilot trial to study the effect of the anti-CD20 antibody rituximab in a group of melanoma patients with stage IV metastatic disease who had been rendered without evident disease by way of surgery, chemotherapy and/or radiation therapy. The major objective was safety, while secondary objectives were description of recurrence-free intervals (RFI) and overall survival (OS). Nine patients received rituximab at 375 mg/m(2) qw for 4 weeks followed by a maintenance therapy every 8 weeks. Treatment was discontinued after 2 years or with disease recurrence. Treatment was well tolerated. After a median observation of 42 months, the median neither of RFI nor of OS has been reached. Despite therapy that ended after 2 years, six out of nine patients are still alive and five of them are recurrence-free. Though the patient number is too small for definitive conclusions, our data may represent a first example of the potential therapeutic value of targeting CD20(+) cell populations-at least for a subset of patients.

Inhibition of CRM1-Mediated Nucleocytoplasmic Transport: Triggering Human Melanoma Cell Apoptosis by Perturbing Multiple Cellular Pathways

Development of multiple drug resistance mechanisms in melanomas necessitates the identification of new drug targets, which when inhibited could impact multiple cellular pathways, thus circumventing potential resistance. By performing complementary DNA microarray analysis, we identified four key components of the nucleocytoplasmic transport machinery-CRM1, RAN (RAN-GTPase), RANGAP1, and RANBP1-to be overexpressed in human melanoma metastases. Chromosome region maintenance 1 (CRM1) inhibition induced a marked depletion of prosurvival/cytoplasmic extracellular signal-regulated kinase 1/2 (Erk1/2) and p90 ribosomal S6 kinase1 and elicited persistent Erk-signaling hyperactivation. Consistently, CRM1 inhibition inflicted extensive apoptosis in melanoma cells while sparing nontransformed melanocytes and primary lung fibroblasts. Apoptosis required both the intrinsic and extrinsic apoptotic pathways and was associated with a nuclear entrapment and downregulation of the antiapoptotic CRM1 target protein, Survivin. Apoptosis was preceded by a G1 cell-cycle arrest, and even though CRM1 inhibition mediated marked p53 and p21 induction in wild-type p53 melanoma cells, the latters silencing or inactivation failed to alleviate apoptosis. Notably, CRM1 inhibition induced cell line-specific, G1 to S progression-retarding changes in the expression of multiple cell-cycle regulatory proteins, thus potentially explaining p53 dispensability. We propose CRM1 as a potential therapeutic target in human melanoma, whose inhibition induces loss of prosurvival/cytoplasmic Erk1/2, mediates persistent Erk hyperactivation, and initiates a multitude of cell context-dependent molecular events to trigger G1 arrest followed by massive apoptosis.

MTSS1 is a metastasis driver in a subset of human melanomas

In cancers with a highly altered genome, distinct genetic alterations drive subsets rather than the majority of individual tumours. Here we use a sequential search across human tumour samples for transcript outlier data points with associated gene copy number variations that correlate with patients survival to identify genes with pro-invasive functionality. Employing loss and gain of function approaches in vitro and in vivo, we show that one such gene, MTSS1, promotes the ability of melanocytic cells to metastasize and engages actin dynamics via Rho-GTPases and cofilin in this process. Indeed, high MTSS1 expression defines a subgroup of primary melanomas with unfavourable prognosis. These data underscore the biological, clinical and potential therapeutic implications of molecular subsets within genetically complex cancers.

Analysis of Pmel17/gp100 expression in primary human tissue specimens: implications for melanoma immuno- and gene-therapy

Abstract Pmel17/gp100-encoded tumor-associated antigens are recognized by cytotoxic T lymphocytes in melanoma patients and may represent attractive target antigens for immuno- and gene-therapeutic strategies. An important prerequisite for identifcation and monitoring of melanoma pateints that could potentially benefit from Pmel17/gpl00-based immuno- and gene-therapies is the detailed knowledge of Pmel17/gpl00 expression in vivo. Immunophenotyping is considerably hampered by the different immunoreactivities of Pmel17/gpl00-reactive antibodies. Therefore, we analyzed an extended series of different primary normal and malignant human tumor specimens for Pmel17/gpl00 expression at the mRNA level. Transcripts were detectable in all malignant melanoma tissue specimens representing all stages of tumor progression, with significant levels even in early and amelanotic melanoma lesions. In contrast, normal melanocytes exhibited significantly less Pmel17/gpl00 mRNA in vivo, as determined by comparative in situ hybridization. Tissue specimens from the retina and substantia nigra also contained Pmel17/gpl00 mRNA, whereas other normal and malignant human tissues were negative. As determined by comparative in situ hybridisation and HMB-45 immunostaining, even tumor tissue lacking Pmel17/gpl00 immunoreactivity contained Pmel17/gp100 transcripts. Our results indicate a melanocytic-cell-lineage-restricted expression of Pmel17/gpl00 with significant transcript levels in all stages of melanoma progression, including early and amelanotic melanoma lesions, and a significantly differential expression between melanoma cells and normal melanocytes in vivo. Owing to its higher sensitivity, phenotyping of individual tumor specimens by mRNA expression analysis seems to be more valuable than phenotyping by immunostaining.

Automatic Identification of Diagnostic Significant Regions in Confocal Laser Scanning Microscopy of Melanocytic Skin Tumors

OBJECTIVES Confocal laser scanning microscopy (CLSM) is used for quick medical checkups. The aim of this study is to check the discrimination power of texture features for the automatic identification of diagnostic significant regions in CLSM views of skin lesions. METHODS In tissue counter analysis (TCA) the images are dissected in equal square elements, where different classes of features are calculated out. Features defined in the spatial domain are based on histogram (grey level distribution) and co-occurrence matrix (grey level combinations). The features defined in the frequency domain are based on spectral properties of the wavelet Daubechie 4 transform (texture exploration at different scales) and the Fourier transform (global texture properties are localized in the spectrum). Hundred cases of benign common nevi and malignant melanoma were used as the study set. Classification was done with CART (Classification and Regression Trees) analysis which splits the set of square elements into homogenous terminal nodes and generates a set of splitting rules. RESULTS Features based on the wavelet transform provide the best results with 96.0% of correctly classified elements from benign common nevi and 97.0% from malignant melanoma. The classification results are relocated to the images by use of the splitting rules as diagnostic aid. The discriminated square elements are highlighted in the images, showing tissue with features in good accordance with typical diagnostic CLSM features. CONCLUSION Square elements with more than 80% of discrimination power enable the identification of diagnostic highly significant parts in confocal microscopic views of malignant melanoma.

Dual Suppression of the Cyclin-Dependent Kinase Inhibitors CDKN2C and CDKN1A in Human Melanoma

Resistance to BRAFV600E inhibitors is associated with reactivation of mitogen-activated protein kinase (MAPK) signaling at different levels in melanoma. To identify downstream effectors of MAPK signaling that could be used as potential additional therapeutic targets for BRAFV600E inhibitors, we used hTERT/CDK4R24C/p53DD-immortalized primary human melanocytes genetically modified to ectopically express BRAF V600E or NRAS G12D and observed induction of the AP-1 transcription factor family member c-Jun. Using a dominant negative approach, in vitro cell proliferation assays, western blots, and flow cytometry showed that MAPK signaling via BRAFV600E promotes melanoma cell proliferation at G1 through AP-1-mediated negative regulation of the INK4 family member, cyclin-dependent kinase inhibitor 2C (CDKN2C), and the CIP/KIP family member, cyclin-dependent kinase inhibitor 1A (CDKN1A). These effects were antagonized by pharmacological inhibition of CDKN2C and CDKN1A targets CDK2 and CDK4 in vitro. In contrast to BRAF V600E or NRAS G12D-expressing melanocytes, melanoma cells have an inherent resistance to suppression of AP-1 activity by BRAFV600E- or MEK-inhibitors. Here, CDK2/4 inhibition statistically significantly augmented the effects of BRAFV600E- or MEK-inhibitors on melanoma cell viability in vitro and growth in athymic nude Foxn1 nu mice (P = .03 when mean tumor volume at day 13 was compared for BRAFV600E inhibitor vs BRAFV600E inhibitor plus CDK2/4 inhibition; P = .02 when mean tumor volume was compared for MEK inhibitor vs MEK inhibitor plus CDK2/4 inhibition; P values were calculated by a two-sided Welch t test; n = 4–8 mice per group).

Polo-Like Kinase 1 Is a Potential Therapeutic Target in Human Melanoma

Exploration of the human melanoma cell-cycle pathway can lead to identification of new therapeutic targets. By gene set enrichment analysis, we identified the cell-cycle pathway and its member polo-like kinase 1 (Plk-1) to be significantly overexpressed in primary melanomas and in melanoma metastases. In vitro expression of Plk-1 was peaked at the G2/M phase of the cell cycle. Plk-1 knockdown/inhibition led to induction of apoptosis, which was caspase-3/8-dependent and p53-independent, and involved BID and Bcl-2 proteins. Comparative genomic hybridization/single-nucleotide polymorphism arrays showed no genetic alteration in the Plk-1 locus. Previous suggestions and significant enrichment of the mitogen-activated protein kinase (MAPK) signaling pathway pointed to potential regulation of Plk-1 by MAPK signaling. Inhibition of this pathway resulted in decreased Plk-1 expression as a consequence of G1 cell-cycle arrest rather than direct regulation of Plk-1. Inhibition of MAPK and Plk-1 had an additive effect on reduced cell viability. This study shows that in human melanoma, Plk-1 expression is dynamically regulated during the cell cycle, knockdown of Plk-1 leads to apoptotic cell death, and that a combination of Plk-1 and MAPK inhibition has an additive effect on melanoma cell viability. We conclude that combined inhibition of Plk-1 and MAPK could be a potentially attractive strategy in melanoma therapy.

NVP-LDE225, a Potent and Selective SMOOTHENED Antagonist Reduces Melanoma Growth In Vitro and In Vivo

Melanoma is one of the most aggressive cancers and its incidence is increasing worldwide. So far there are no curable therapies especially after metastasis. Due to frequent mutations in members of the mitogen-activated protein kinase (MAPK) signaling pathway, this pathway is constitutively active in melanoma. It has been shown that the SONIC HEDGEHOG (SHH)-GLI and MAPK signaling pathway regulate cell growth in many tumors including melanoma and interact with each other in the regulation of cell proliferation and survival. Here we show that the SHH-GLI pathway is active in human melanoma cell lines as they express downstream target of this pathway GLI1. Expression of GLI1 was significantly higher in human primary melanoma tissues harboring BRAFV600E mutation than those with wild type BRAF. Pharmacologic inhibition of BRAFV600E in human melanoma cell lines resulted in decreased expression of GLI1 thus demonstrating interaction of SHH-GLI and MAPK pathways. Inhibition of SHH-GLI pathway by the novel small molecule inhibitor of smoothened NVP-LDE225 was followed by inhibition of cell growth and induction of apoptosis in human melanoma cell lines, interestingly with both BRAFV600E and BRAFWild Type status. NVP-LDE225 was potent in reducing cell proliferation and inducing tumor growth arrest in vitro and in vivo, respectively and these effects were superior to the natural compound cyclopamine. Finally, we conclude that inhibition of SHH-GLI signaling pathway in human melanoma by the specific smoothened inhibitor NVP-LDE225 could have potential therapeutic application in human melanoma even in the absence of BRAFV600E mutation and warrants further investigations.

Comprehensive comparative and semiquantitative proteome of a very low number of native and matched epstein-barr-virus-transformed B lymphocytes infiltrating human melanoma.

Melanoma, the deadliest form of skin cancer, is highly immunogenic and frequently infiltrated with immune cells including B cells. The role of tumor-infiltrating B cells (TIBCs) in melanoma is as yet unresolved, possibly due to technical challenges in obtaining TIBCs in sufficient quantity for extensive studies and due to the limited life span of B cells in vitro. A comprehensive workflow has thus been developed for successful isolation and proteomic analysis of a low number of TIBCs from fresh, human melanoma tissue. In addition, we generated in vitro-proliferating TIBC cultures using simultaneous stimulation with Epstein-Barr virus (EBV) and the TLR9 ligand CpG-oligodesoxynucleotide (CpG ODN). The FASP method and iTRAQ labeling were utilized to obtain a comparative, semiquantitative proteome to assess EBV-induced changes in TIBCs. By using as few as 100 000 B cells (∼5 μg protein)/sample for our proteomic study, a total number of 6507 proteins were identified. EBV-induced changes in TIBCs are similar to those already reported for peripheral B cells and largely involve changes in cell cycle proliferation, apoptosis, and interferon response, while most of the proteins were not significantly altered. This study provides an essential, further step toward detailed characterization of TIBCs including functional in vitro analysis.

TLR9-targeted CpG immunostimulatory treatment of metastatic melanoma: A phase II trial with CpG 7909 (ProMune)

7513 Background: Malignant melanoma is widely accepted as an immunologically responsive tumor, but large recently reported chemobiotherapy trials have been disappointing and prognosis is poor. The new synthetic oligodeoxynucleotide CpG 7909 activates plasmacytoid dendritic cells (pDC) and B cells through specific interaction with Toll-like receptor 9 (TLR9) and is a strong activator of both innate and specific immunity. CPG 7909 crossreacts with mouse TLR9 and has shown impressive antitumor activity in preclinical tumor models when used as monotherapy. METHODS Patients with confirmed metastatic melanoma (stage IV without CNS metastases) were enrolled into an open-label, multicenter, single arm study. Pts received 6 mg CPG 7909 weekly by SC injection for 24 weeks or until disease progression in an outpatient setting. Disease status was assessed at screening and weeks 8, 16, and 24 according to RECIST. RESULTS 20 pts (5 female, 15 male, aged between 37 and 74) were enrolled. Two pts achieved a confirmed partial response (PR) and one has maintained this response with continuing therapy for over 13 months. Three pts achieved stable disease (SD). CPG 7909 was well tolerated. Adverse events included transient injection site reactions (erythema, swelling, induration), fever and arthralgias. Hematological and non-hematological toxicities were limited, transient and did not result in any withdrawals. Phenotyping of PBMC revealed activation of pDC under CPG 7909 therapy consistent with the mode of action and pts exhibiting PR or SD could be distinguished from non-R pts by differential dynamics in NK cell-cytotoxicity (1.6-fold increase vs. 1.7-fold decrease in lytic units, p<0.05) during the first 8 weeks of treatment. CONCLUSIONS CPG 7909 exerts anti-tumor activity in pts with metastatic melanoma, can be administered safely and induces a phenotypic signature in PBMC associated with exposure and, possibly, response to therapy. A randomised phase II/III trial has been initiated to compare efficacy and safety of two dose levels of CPG 7909, CPG 7909 in combination with DTIC, and DTIC alone. [Table: see text].