Induction of an Antitumor Immune Response in Patients with Melanoma Using the Antimicrobial Peptide LL37
Richard E. Royal, M.D.
Patrick Hwu, M.D.
Tumors are potentially immunogenic. However, they fail to spontaneously induce immune responses capable of rejecting tumors. A major reason for this is that the tumor microenvironment lacks adequate innate immune activation required to initiate strong adaptive antitumor immunity. Plasmacytoid dendritic cells (pDCs) are highly specialized in that they sense microbial nucleic acids via intracellular Toll-like receptors. During viral infection, pDCs accumulate in infected tissues and are activated by viral nucleic acids to produce large amounts of type I interferons (IFNs) and generate protective immunity against the virus by activating myeloid dendritic cells, T cells, and natural killer cells. Tumors also contain pDCs but do not provide molecular signals to activate pDCs, although tumors contain self-DNA released in the extracellular environment at high concentrations as a result of increased turnover of tumor cells. pDCs, though activated by viral nucleic acids, clearly are normally not able to sense tumor-derived DNA and are thus unable to initiate strong innate immune responses. We recently found that pDCs can, in fact, sense and respond to self-DNA when combined with an endogenous peptide called LL37. LL37 can bind to self-DNA fragments released by dying cells to form aggregates and condensed structures that are delivered to and retained within early endosomes of pDCs. In these intracellular compartments, LL37/self-DNA can interact with Toll-like receptor 9 to trigger robust type I IFN production similarly to viral DNA. Because tumors release large amounts of self-DNA and contain pDCs but do not express LL37, our hypothesis for the proposed study described herein is that exogenous LL37 can be used to target tumor-derived self-DNA and convert it into a “danger signal” that triggers pDC activation and type I IFN production at the tumor site in patients with melanoma. This then induces T-cell--mediated immunity against melanoma by using the same mechanism by which anti-viral-immune responses are induced. Therefore, we will pursue the following: Specific Aim 1: Determine the mechanism of anti-tumor immune responses induced by intratumoral LL37 injection in mouse tumor models; Specific Aim 2: Evaluate anti-tumor immune responses and clinical efficacy of intratumoral LL37 injection in patients with melanoma; Specific Aim 3: Improve anti-tumor immune responses and efficacy of intratumoral LL37 injection in mouse tumor models. These studies may lead to principles in cancer immunotherapy that may be widely applicable to other cancers.
Effects of a Novel Phospho-STAT3 Inhibitor for Treatment of Stage IV Melanoma Patients, Including Those With CNS Metastasis
Amy Heimberger, M.D.
Waldemar Priebe, Ph.D.
Timothy Madden, PharmD
Melanoma is a common and deadly tumor that frequently metastasizes to the central nervous system (CNS), resulting in a median survival duration of 3.7 month and refractoriness to conventional therapy. A key transcriptional factor, signal transducer and activator of transcription (STAT)-3, drives the components of melanoma and is over expressed in 80% of melanoma CNS metastases,. Phosphorylated STAT-3 propagates tumorigenesis by enhancing proliferation, angiogenesis, invasion, metastasis, and immunosuppression. We developed WP1066, a potent orally administered inhibitor of STAT-3 that displays marked efficacy against established intracerebral melanoma in vivo. The mechanism of WP1066’s activity is a combination of direct melanoma cytotoxicity and immune cytotoxic clearance. In this proposed study, we will further clarify the mechanisms of activity and targets of WP1066 using murine melanoma models of metastasis to identify biomarkers and potential selection criteria for a Phase I/II clinical trial. This study will focus on whether over expression of phosphorylated STAT3 in the parenchymal tumors of patients with stage IV melanoma is predictive of CNS metastasis and whether this over expression is an independent predictor of survival. We have demonstrated that a significant mechanism of WP1066’s activity is enhancement of CD8+ T-cell effector responses and inhibition of regulatory T cells. Immunotherapy with both interferon-alpha and interleukin-2 is used for melanoma, but the response rates remain low. We will further determine whether WP1066 can markedly improve these immunotherapy modalities in in vivo murine model systems. These studies will lay the groundwork for a potential clinical trial of WP1066 as an immunomodulator. These following specific aims are the basis for conducting these studies: 1) Evaluate mechanisms of activity of WP1066 and identify biological markers for a clinical trial of WP1066; 2) Evaluate the natural history of p-STAT-3 expression in patients with melanoma and determine whether WP1066 can impact these processes in in vivo murine models to clarify clinical trial design; 3) Establish whether reversing immune suppression with WP1066 is synergistic with other immunotherapies for melanoma. These translational studies will bring to the forefront a novel therapeutic that has real potential to improve the survival of patients with melanoma having CNS metastatic disease, a major unmet clinical need.
Prognostic Significance and Targeting of an iNOS-Associated Human Melanoma Inflammatory Signature
Elizabeth A. Grimm, Ph.D.
Suhendan Ekmekcioglu, Ph.D.
Victor G. Prieto, M.D., Ph.D.
Kevin Kim, M.D.
Julie Ellerhorst, M.D., Ph.D.
Victoria Greene, M.D.
In our 2004-2009 SPORE, we studied the prognostic significance of inducible nitric oxide synthase (NOS) protein levels in both primary and metastatic melanoma, as well as the genetic and pharmacologic regulation of its constitutive expression. The results confirmed that iNOS protein is readily detectable in melanoma cell cytoplasm in the majority of patients, and the quantity, as detected by immunohistochemistry (IHC), provides prognostic information by identifying patients with poor survival in both univariate and multivariate analysis (p<0.001), independently of AJCC staging and associated prognostic criteria. Melanomas are recognized to be heterogeneous based on etiology and somatic mutational status and have aberrant expression of inflammatory genes and proteins. We aim to expand the testing of iNOS protein expression as a clinically useful prognostic marker and propose that iNOS represents a “node” of an identifiable melanoma inflammatory and oxidative stress network. We further hypothesize that a “signature of poor prognosis” for melanoma can be generated by directly testing tumors for expression of iNOS-related inflammatory markers.
Testing for association of iNOS protein expression and levels with both genetic alterations (mutations of BRAF and NRAS) and mitogen-activated protein kinase (MAPK) pathway activation was also performed in a large series of primary cutaneous tumor biopsies. We report that active MAPK can drive iNOS expression and that inhibition of MAPK and/or BRAF inhibits iNOS protein expression. iNOS protein in melanoma was also found to be sensitive to down regulation by pharmacologic agents, possibly for therapeutic advantage. Pharmacologic inhibition of iNOS or inhibition of its product, nitric oxide (NO), restores chemosensitivity in iNOS-positive melanoma cell lines. Building on these results, three new specific aims are now proposed:
1) identify iNOS-related inflammatory marker genes expressed in melanoma, as part of a proposed signature 2) determine which candidate marker proteins can be identified in melanoma tumors by standard immunohistochemistry and whether their presence adds value to the iNOS survival prediction model, and 3) test the inflammatory stress pathway as a target for melanoma patient therapy in a Phase I/II trial using an anti-inflammatory drug that inhibits expression of iNOS and other inflammatory mediators.
Targeting Angiogenesis and the Tumor Microenvironment Utilizing siRNA Nanoliposomes as a New Modality for Treatment of Melanoma
Menashe Bar-Eli, Ph.D.
Gabriel Lopez-Berestein, M.D.
Wen-Jen Hwu, M.D., Ph.D.
Chaan Ng, M.D.
Melanoma is a potentially lethal neoplasm with a propensity for acquiring the metastatic phenotype. The molecular basis for this acquisition is not very well defined and few treatment modalities are available for this neoplasm at advanced stages. Recently, we reported that one of the genes that stands out as differentially expressed in highly metastatic melanoma cells as compared to non-metastatic cells, is the thrombin receptor PAR-1, which promotes metastases through multiple mechanisms including angiogenesis, cell signaling, and adhesion, in large part via upregulation of IL-8. Our preliminary data indicated that PAR-1 silencing by short hairpin RNA (shRNA) in metastatic melanoma cells inhibited their growth and metastatic potential in vivo. In this proposed study, we will advance this work to the translational phase by pursuing inhibition of PAR-1 as well as the presumed less toxic IL-8 via siRNA encapsulated in neutral liposomes to induce tumor regression in model systems. In addition, results from our laboratory have demonstrated that expression of the angiogenic factor interleukin 8 (IL-8), which is itself upregulated through PAR-1 signaling, correlates with the metastatic potential of melanoma cells. Moreover, using a fully human neutralizing antibody against IL-8 (ABX-IL8 which is not available for clinical use), we were able to inhibit tumor growth and metastasis of melanoma in vivo. Our hypothesis is that intravenous administration of IL-8 small interfering RNA (siRNA) packaged in neutral liposomes will cause downregulation of IL-8 in vivo, thereby inhibiting melanoma growth and metastasis.
We will also evaluate the therapeutic strategy in melanoma patients. To these ends, we propose the following Specific Aims:
Specific Aim 1: To evaluate the in vivo effects of PAR-1 siRNA packaged in neutral liposomes as a possible therapeutic modality for advanced melanoma alone and/or in combination with chemotherapy.
Specific Aim 2: To evaluate the in vivo effects of IL-8 siRNA packaged in neutral liposomes as a new therapeutic modality for advanced melanoma alone and/or in combination with chemotherapy.
Specific Aim 3: To evaluate the safety and efficacy of IL-8 siRNA packaged in neutral liposomes
(IL-8 siRNA-DOPC) in a Phase I clinical trial.
This is the first attempt at using nanotechnology as a therapeutic modality for advanced melanoma.
Genetic Mechanisms of Immunity and Inflammation in Melanoma Progression: Development of an Integrated Risk Model
Jeffrey E. Lee, M.D.
Qingyi Wei, M.D., Ph.D.
Chris Amos, Ph.D.
John D. Reveille, M.D.
Yuling Wang, Ph.D.
Melanoma has the fastest growing incidence among all cancers in the United States; researchers estimate there were over 62,000 new melanoma cases in 2008. Most patients with melanoma present with early-stage disease and are cured with surgery alone. However, despite their overall good outcome, more than 15% of melanoma patients will suffer a recurrence. Standard clinical features (tumor thickness, ulceration, sentinel lymph node status) cannot completely predict which patients will recur. For those who do recur, current therapies are effective in only a minority. Thus, identifying more effective biologic markers to select high-risk patients for adjuvant therapies, identify those who will respond to treatment, elucidate mechanisms of recurrence, and suggest novel therapies is a necessity. One important way to identify relevant biologic markers is to examine the relationship of human genetic variation (genetic polymorphisms) to disease recurrence and progression, and an important potential mechanism regulating melanoma recurrence and progression is variation in the immune and inflammatory response to melanoma. Our recent investigations have identified specific polymorphisms in human leukocyte antigen (HLA) class II and transforming growth factor-β1 (TGF-β1) genes as markers of prognosis in early-stage melanoma patients. HLA class II polymorphisms can regulate melanoma immune responses by differential binding of peptide antigens, whereas TGF-β1 polymorphisms can regulate tumor growth and metastasis by differential expression of TGF-β1 and immunomodulation. We hypothesize that genetic polymorphisms in these and other immune and inflammatory genes influence host response to melanoma and thereby melanoma progression. We propose a coordinated investigation of our most promising and mechanistically related polymorphisms in a large cohort of patients with melanoma (Aim1) together with a genome-wide analysis to identify candidate loci most strongly linked with melanoma progression (Aim 2). We will use this information to develop an integrated and iterative risk model of melanoma progression incorporating clinical, histopathologic, serologic, and genetic information from more than 2000 patients with melanoma (Aim 3).
Administration, Evaluation, and Planning
Elizabeth A. Grimm, Ph.D.
Jeffrey E. Lee, M.D.
Jeffrey E. Gershenwald, M.D.
Patrick Hwu, M.D.
The Administrative Planning and Evaluation Core, Core A, is critical to the success of the SPORE. Dr. Elizabeth Grimm is the overall Core Director, and is assisted by three Co-Directors, including the founding Co-Director, Dr. Jeffrey E. Lee, who will chair the Executive Committee meetings which will be composed of all Co-PIs and one leader from each Project and Core. Two new co-directors include Patrick Hwu, M.D., as director of the Clinical Trials Resource which for now will be incorporated as part of Core A; and Jeffrey E. Gershenwald, M.D., as overall director of patient informatics and tumor acquisition and distribution. Our Executive Committee will meet monthly to review scientific progress and fiscal status and will help surmount problems or barriers to assure that all goals are met within a realistic time-frame and within the budget constraints. This Administrative Core will schedule all meetings with investigators and assure optimum communication with investigators at MD Anderson Cancer Center and elsewhere. All SPORE investigators will participate in weekly melanoma seminars and regularly report progress to the Internal Advisory Committee. An External Advisory Committee will evaluate status and progress of all SPORE research programs by annual meetings either in Houston or by web/video or teleconference. Specific responsibilities of Core A include
• Compliance with all general, governmental and NCI regulations and requirements.
• Communication and consultation with the NCI in preparation of all required reports and publications.
• Coordination of data control quality assurance issues
• Oversight and support for the Informatics, Tissue Resource and Pathology Core B.
• Optimization of clinical trial efforts by a Clinical Trials Management Resource in this Core A.
• Maintenance of fiscal and budgetary functions, and managing flexibility in funding.
• Convene SPORE Administrative meetings, including the Executive Committee, the Internal and External Advisory Committees, weekly scientific meetings, quarterly research meetings, lectures, and symposia.
• Administration of the Developmental Research and Career Development Programs.
• Coordination and Funding of Patient Advocate activities.
• Establishment and monitoring of policies for recruitment of women and minorities of this program.
• Coordination with other SPORE sites to promote and maintain communication and integration through sponsoring and/or attending a yearly Skin SPORE conference, and also through sharing and distribution of materials, electronic communications, and evaluation of progress reports.
Informatics, Tissue Resource and Pathology Core
Jeffrey E. Gershenwald, M.D.
Victor G. Prieto, M.D., Ph.D.
Michael Davies, M.D., Ph.D.
Effective procurement and utilization of well-characterized tissue is essential to perform meaningful translational research. The Informatics, Tissue Resource, and Pathology Core (Core B) will work with each of the SPORE projects and with the Administrative and Biostatistics Cores to ensure maximum efficiency in the use of tissue for translational research directed at improving the prevention, detection, and therapy of melanoma. Core B will continue to provide investigators at The University of Texas MD Anderson Cancer Center and other collaborating institutions with well-characterized, high-quality tissue, peripheral blood lymphocytes, plasma, and serum samples from patients with melanoma treated at MD Anderson Cancer Center whose clinical, pathologic treatment, follow-up, and recurrence data are maintained in a comprehensive relational database. Standardized procedures for procurement, processing, storage, quality control, histopathologic evaluation and distribution of samples will ensure optimal utilization and distribution of limited tissue samples according to the guidelines established by the Tissue Acquisition and Distribution Committee. A fully customized relational Core database tracks all samples from patient consent, to tissue acquisition and distribution of tissue and blood components to SPORE Projects. This system contains comprehensive clinical information on all patients as well as relevant histopathologic characteristics for all samples and provides information on sample availability for future distribution through an NCI-sponsored tissue network. As over 1200 new patients with melanoma are seen and treated at MD Anderson Cancer Center per year, this Core will be one of the largest available resources for translational research. The close relationship with the Biostatistics Core will allow an efficient analysis of the data produced by the different SPORE projects. Leadership for the Core is shared by a clinical investigator with expertise in melanoma patient care, database design and utilization and analysis of melanoma prognostic factors, and by a dermatopathologist with expertise in melanoma, including histopathologic evaluation, molecular analysis and quality-control procedures; both Co-leaders have expertise in melanoma specimen acquisition and processing. This shared leadership ensures maximal utilization of samples without compromising patient care. This centralized, comprehensive Core will contribute significantly to the success of the multidisciplinary and translational research projects outlined in this proposal.
Biostatistics and Bioinformatics Core
Donald Berry, Ph.D.
The Biostatistics and Bioinformatics Core for the University of Texas MD Anderson Cancer Center SPORE in Melanoma will serve multiple needs for the planning and conduct of the SPORE's translational research. Based on a strong track record for providing biostatistical support for translational research, the Core will be a comprehensive, multi-lateral resource for designing clinical and basic science experiments, performing statistical analyses, developing innovative statistical methodology, and publishing the research results generated from this Melanoma SPORE.
For this proposed competitive renewal, we plan to continue and to expand the role of the Core in supporting the Melanoma SPORE. We will incorporate sound experimental design principles within each project that will increase the clarity and enhance interpretability of study results. Each project will be provided with tailored analyses, accompanied by novel statistical development as necessary, to reveal apparent and hidden relationships among data. The Core will collaborate with all project investigators to facilitate the timely publication of Melanoma SPORE research.
The main objectives of the Biostatistics and Bioinformatics Core are:
1. To provide guidance for the statistical design and sample size and power calculations for each project.
2. To provide all statistical data analysis including descriptive statistical analysis, hypothesis testing, estimation, and modeling of prospectively generated data.
3. To ascertain that conclusions drawn from all projects are based on well-designed experiments and appropriate interpretation of results.
4. To develop and adapt innovative statistical methods pertinent to translational melanoma studies.
5. To generate statistical reports for all projects.
6. To collaborate and assist all project investigators in the publication of scientific results.