Ian S. Dunn

Role of the Mitogen-Activated Protein Kinase Signaling Pathway in the Regulation of Human Melanocytic Antigen Expression

Heterogeneous expression of melanocytic antigens occurs frequently in melanomas and represents a potent barrier to immunotherapy. We previously showed that coordinated losses of several melanocytic antigens are generally attributable to down-regulation of antigen gene expression rather than irreversible mutation. Treatment of melanoma cells with mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors blocks ERK activation and increases steady-state levels of mRNAs and corresponding protein expression for the melanocytic antigens Melan-A/MART-1, gp100, and tyrosinase. Although the degree of MEK inhibitor enhancement of antigen expression varied among different cell lines irrespective of their antigen expression status, all showed detectable responses. Notably, the antigen-enhancing effects of the MEK inhibitors could not be attributed to the master melanocytic regulator MITF-M. Because MAPK pathway activation via constitutively active mutant forms of BRAF is common in melanomas, correlation between BRAF function and antigen expression was investigated. No simple correlation of endogenous BRAF mutational status and antigen levels was observed, but transient overexpression of V600E BRAF increased ERK activation and reduced Melan-A/MART-1 levels in antigen-positive cell lines. These data indicate that whereas multiple factors may regulate antigen expression in melanomas, enhancement of MAPK signaling can act as a negative influence. Blocking such signaling with MEK inhibitors accordingly augments antigen levels, thereby enhancing Melan-A/MART-1–specific cytotoxic T-cell responses to antigen-negative cells following MEK inhibition treatment. Consequently, MAPK inhibition may assist targeting of melanomas for immunotherapy. (Mol Cancer Res 2006;4(10):779–92)

A Novel Autocrine Pathway of Tumor Escape from Immune Recognition: Melanoma Cell Lines Produce a Soluble Protein That Diminishes Expression of the Gene Encoding the Melanocyte Lineage Melan-A/MART-1 Antigen Through Down-Modulation of Its Promoter

We have observed that malignant melanoma cells produce a soluble protein factor(s), which down-regulates melanocyte lineage Melan-A/MART-1 Ag expression by melanoma cells with concomitant loss of recognition by Melan-A/MART-1-specific T cells. This down-modulation of Melan-A/MART-1 expression, which we refer to as “Ag silencing,” is mediated via its minimal promoter, whereas the promoter for the restricting Ag-presenting HLA-A2 molecule is not affected. Significantly, this Ag silencing is reversible, as removal of factor-containing supernatants from Melan-A/MART-1-expressing cells results in up-regulation of the promoter for the gene encoding this Ag, and renewed expression of the protein. We have evaluated over 20 known factors, none of which accounts for the Ag-silencing activity of the melanoma cell culture supernatants. The existence of this autocrine pathway provides an additional novel explanation for melanoma tumor progression in vivo in the presence of CTL specific for this melanocyte lineage Ag. These observations may have important implications for Melan-A/MART-1-specific CTL-mediated immunotherapy of melanoma tumors.

Tracking membrane and secretory immunoglobulin α heavy chain mRNA variation during B-cell differentiation by real-time quantitative polymerase chain reaction

Primary transcripts for all Ig heavy chain isotypes are alternatively processed to encode either secreted or membrane forms of the same antibody and, in plasma cells, a shift towards the secreted form occurs. In principle, measuring the relative quantities of secreted and membrane forms for a particular isotype could monitor B‐cell plasmacytoid differentiation. Ratios of α heavy chain mRNA secreted (αs) to membrane (αm) form were assessed by quantitative reverse transcriptase–polymerase chain reaction (RT‐PCR; TaqMan) using an IgA plasma cell line (NCI‐H929), a surface IgA+ line (Dakiki) and human tonsillar B cells. While NCI‐H929 cells showed the highest αs : αm ratio as expected, αs mRNA predominated for all unstimulated B cells and Dakiki cells. Treatment of B cells and Dakiki cells with IL‐2 and IL‐10 resulted in a further progression towards the αs form, correlating with increased human plasma cell antigen‐1 (HPC1) mRNA levels. However, α mRNA processing and HPC1 expression were independently regulated, as IFN‐γ treatment suppressed HPC1 levels while increasing αs : αm ratios. Cytokine‐mediated increases in the αs : αm ratio resulted from strongly enhanced levels of αs with relatively constant αm values. Differentiation‐related changes in mRNA processing can thus be tracked by automated quantitative PCR.

Quantifiable analysis of human immunoglobulin heavy chain class-switch recombination to all isotypes.

Somatic recombinational events, including the immunoglobulin heavy chain class-switch, are a normal feature of B-cell maturation. To enable comprehensive and sensitive class-switch analysis in ex vivo human B cells, we have developed multiple digestion-circularization PCR (DC-PCR) techniques for quantifiable detection of switching to all immunoglobulin isotypes. This technology was validated by extensive sequencing of PCR products, tests with control non-lymphoid cells and B-cell lines of known isotypic specificities, and by demonstrating DC-PCR selectivity in a model system. With tonsillar B-cell DNA, switching to gamma 3, gamma 1, alpha1, gamma 2, gamma 4 and alpha2 isotypes was reproducibly detectable among different individuals. Levels of epsilon switching were relatively low and usually required higher total amounts of template DNAs for detection. Quantitation of alpha1 class switching in a panel of human tonsillar whole B cells was performed by the internal-competitor approach, and showed a pattern consistent with previous studies on IgA+ tonsillar cells. We demonstrate that these assays can rapidly show germline status or specific switch rearrangements in B lymphoid cell lines.

Topoisomerase Inhibitors Modulate Expression of Melanocytic Antigens and Enhance T Cell Recognition of Tumor Cells

While there are many obstacles to immune destruction of autologous tumors, there is mounting evidence that tumor antigen recognition does occur. Unfortunately, immune recognition rarely controls clinically significant tumors. Even the most effective immune response will fail if tumors fail to express target antigens. Importantly, reduced tumor antigen expression often results from changes in gene regulation rather than irrevocable loss of genetic information. Such perturbations are often reversible by specific compounds or biological mediators, prompting a search for agents with improved antigen-enhancing properties. Some recent findings have suggested that certain conventional chemotherapeutic agents may have beneficial properties for cancer treatment beyond their direct cytotoxicities against tumor cells. Accordingly, we screened an important subset of these agents, topoisomerase inhibitors, for their effects on antigen levels in tumor cells. Our analyses demonstrate upregulation of antigen expression in a variety of melanoma cell lines and gliomas in response to nanomolar levels of certain specific topoisomerase inhibitors. To demonstrate the ability of CD8+ T cells to recognize tumors, we assayed cytokine secretion in T cells transfected with T cell receptors directed against Melan-A/MART-1 antigen. Three days of daunorubicin treatment resulted in enhanced antigen expression by tumor cells, in turn inducing co-cultured antigen-specific T cells to secrete Interleukin-2 and Interferon-γ. These results demonstrate that specific topoisomerase inhibitors can augment melanoma antigen production, suggesting that a combination of chemotherapy and immunotherapy may be of potential value in the treatment of otherwise insensitive cancers.

Heat Shock Protein-90 Inhibitors Enhance Antigen Expression on Melanomas and Increase T Cell Recognition of Tumor Cells

In an effort to enhance antigen-specific T cell recognition of cancer cells, we have examined numerous modulators of antigen-expression. In this report we demonstrate that twelve different Hsp90 inhibitors (iHsp90) share the ability to increase the expression of differentiation antigens and MHC Class I antigens. These iHsp90 are active in several molecular and cellular assays on a series of tumor cell lines, including eleven human melanomas, a murine B16 melanoma, and two human glioma-derived cell lines. Intra-cytoplasmic antibody staining showed that all of the tested iHsp90 increased expression of the melanocyte differentiation antigens Melan-A/MART-1, gp100, and TRP-2, as well as MHC Class I. The gliomas showed enhanced gp100 and MHC staining. Quantitative analysis of mRNA levels showed a parallel increase in message transcription, and a reporter assay shows induction of promoter activity for Melan-A/MART-1 gene. In addition, iHsp90 increased recognition of tumor cells by T cells specific for Melan-A/MART-1. In contrast to direct Hsp90 client proteins, the increased levels of full-length differentiation antigens that result from iHsp90 treatment are most likely the result of transcriptional activation of their encoding genes. In combination, these results suggest that iHsp90 improve recognition of tumor cells by T cells specific for a melanoma-associated antigen as a result of increasing the expressed intracellular antigen pool available for processing and presentation by MHC Class I, along with increased levels of MHC Class I itself. As these Hsp90 inhibitors do not interfere with T cell function, they could have potential for use in immunotherapy of cancer.

A Screening Assay to Identify Agents That Enhance T-Cell Recognition of Human Melanomas

Although a series of melanoma differentiation antigens for immunotherapeutic targeting has been described, heterogeneous expression of antigens such as Melan-A/MART-1 and gp100 results from a loss of antigenic expression in many late stage tumors. Antigen loss can represent a means for tumor escape from immune recognition, and a barrier to immunotherapy. However, since antigen-negative tumor phenotypes frequently result from reversible gene regulatory events, antigen enhancement represents a potential therapeutic opportunity. Accordingly, we have developed a cell-based assay to screen for compounds with the ability to enhance T-cell recognition of melanoma cells. This assay is dependent on augmentation of MelanA/MART-1 antigen presentation by a melanoma cell line (MU89). T-cell recognition is detected as interleukin-2 production by a Jurkat T cell transduced to express a T-cell receptor specific for an HLA-A2 restricted epitope of the Melan-A/MART-1 protein. This cellular assay was used to perform a pilot screen by using 480 compounds of known biological activity. From the initial proof-of-principle primary screen, eight compounds were identified as positive hits. A panel of secondary screens, including orthogonal assays, was used to validate the primary hits and eliminate false positives, and also to measure the comparative efficacy of the identified compounds. This cell-based assay, thus, yields consistent results applicable to the screening of larger libraries of compounds that can potentially reveal novel molecules which allow better recognition of treated tumors by T cells.

Targeted tumor therapy: turning the genome on its head

TriBiotica has combined molecular biology of cancer and proprietary chemistry to implement an innovative therapeutic approach to cancer treatment. Instead of developing immunological reagents (such as cells, antibodies or chimeric receptors) targeting antigens already present on tumor cells, the new technology produces novel structures that are not native to the tumor cell. The generation of novel molecules in a tumor-specific manner accordingly allows implementation of immunotherapeutic interventions that recognize and destroy tumor cells. Unlike immunotherapy protocols that are limited by availability of target antigens, TriBiotica’s technology utilizes the tumor’s unique transcriptome to force tumor cells to produce novel molecules that are either toxic by themselves, or allow immunotherapeutic destruction of the tumor cells without inducing toxicity against normal cells. As a proof of concept, we have used an HPV16 template from a cervical carcinoma cell to create a FLAG-tag that is uniquely assembled in HPV16 expressing tumor cells. Of the cervical cancer cell lines Caski, HeLa, and C33A, only Caski contains the HPV16 E6/E7 oncogene. Each of these lines was treated with templated assembly of bio-orthogonal compounds designed to produce FLAG tag peptide specifically in the presence of HPV16 E6/E7 mRNA. At 24 hours post-treatment, in situ production of FLAG tag in each tumor type was assayed by ELISA. The results demonstrate that FLAG tag is selectively produced in Caski cells harboring the target HPV16 template. To our knowledge, this represents the first oncogene-templated synthesis of a peptide tag within living cells. The approach offers the potential for producing a wide variety of biologically-active molecules specifically in targeted cells, leaving normal cells unaffected. This technology offers a platform that links next-generation sequencing technologies to identify genetic markers of cancers as potential targets in a vast array of tumors. Combined with genetic analyses, the therapy is adaptable to the ever-changing genetic landscape of tumors, as the same novel effector molecule can be assembled on an almost limitless array of target nucleic acid templates, so that it is possible to adapt the formation of the novel therapeutic product molecule to any target genetic templates arising from the outgrowth subsets of tumor cells. Combined with appropriate diagnostic testing, the TriBiotica technology has the potential to create a new paradigm for targeting of tumor cells that opens the door to the treatment of virtually any tumor type. The technology relies on identifying specific tumor genetic templates, and utilizing a proprietary technology that assembles novel epitopes present only in the tumor cells.