NCI Director Dr. Andrew von Eschenbach Visits the CCR

n early April, NCI Director, Andrew von Eschenbach, MD, visited Building 41 on the NIH Campus as part of his ongoing efforts to learn more about the science being conducted at the CCR and to discuss its implications and impact in the field of cancer research. CCR scientists are committed to advancing the NCI mission of eliminating suffering and death due to cancer by 2015—a goal put forth by Dr. von Eschenbach. During his visit, several CCR investigators had an opportunity to discuss their progress in four specific areas: 1) live cell and genome imaging, 2) host genetics, inflammation, and therapy, 3) drug discovery, and 4) lymphoma diagnosis and treatment. A brief overview of the research presented during Dr. von Eschenbach’s visit follows.

Live Cell and Genome Imaging

Gordon Hager, PhD, of the Laboratory of Receptor Biology and Gene Expression, described recent developments in real-time live-cell imaging using a fluorescent molecule tag to visualize the location and movement of proteins within a living cell. Real-time imaging has proven to be an amazingly powerful tool for studying the proteins of interest to Dr. Hager’s lab: those that interact with chromatin and DNA to regulate gene expression. Using a technique called fluorescence recovery after photobleaching (FRAP), Dr. Hager was able to watch as tagged glucocorticoid receptor (GR) molecules interacted with their target DNA regulatory sites (Figure 1) and show that this lasted for only a few seconds, overturning a 40-year-old central tenet that these steroid receptors bind statically for extended periods as part of large, multiprotein transcription factor complexes. Dr. Hager concluded from these experiments that nuclear receptors are highly mobile on their gene targets, both in vitro and in vivo, calling this the “hit-and-run” hypothesis. GR interacts transiently and periodically with hormone-response elements during chromatin remodeling. By testing the inducibility of 30,000 mouse genes using microarrays and a dominant-negative mutant for remodeling protein, Dr. Hager—in collaboration with Paul Meltzer, MD, PhD, of the National Human Genome Research Institute—established that chromatin remodeling is a common feature central to regulation by nuclear receptors. The dynamic interaction of these molecules with their targets is also strongly ligand dependent, a critical observation because nuclear receptors are major targets in cancer therapy.

Figure 1. Green fluorescent protein (GFP)–glucocorticoid receptor bound to amplified promoter array: The interaction of GFP-tagged nuclear receptors can be observed on amplified natural gene targets in living cells. (Figure and legend courtesy of Gordon Hager, PhD)

Tom Misteli, PhD, of the Laboratory of Receptor Biology and Gene Expression, presented his ongoing work on the application of imaging techniques to study the human genome. The basis of this approach is the fundamental property of genomes that chromosomes, as well as single genes, occupy preferential positions within the nucleus; for example, in human lymphocyte nuclei, chromosome 18 is generally located at the periphery. There is good evidence that these positioning patterns are functionally relevant, and they are known to change during differentiation and development. Dr. Misteli’s laboratory has been able to show that nonrandom spatial proximity of genes facilitates the formation of the translocations in Burkitt’s lymphoma and other tumors, and his group is conducting experiments to determine positioning patterns during tumor formation and progression. To map and compare positioning patterns of multiple genes and chromosomes more efficiently and accurately, Dr. Misteli is using NCI’s extensive imaging and bioinformatics resources to develop a high-throughput automated image acquisition system. The objective is to acquire a large number of highly accurate genome images of normal and tumor cells. Genome positioning data will be combined with microarray data to correlate positioning with gene expression in normal, premalignant, and metastatic cells, as well as cancer stem cells. The findings can be applied to both basic discovery and potential diagnostic applications.

Host Genetics, Inflammation, and Therapy

Kent Hunter, PhD, of the Laboratory of Population Genetics, presented work on the characterization of metastasis susceptibility genes, an area that may explain the seemingly random appearances of metastases in the human population. Crossing transgenic mice that develop mammary tumors and pulmonary metastases with several normal inbred strains resulted in progeny with differing propensities to develop metastatic tumors. Analysis of high and low metastatic strains identified a polymorphism in the signal transduction protein Sipa1; inhibiting Sipa1 expression significantly reduced the incidence of metastasis in these mice, whereas its overexpression dramatically increased the ability of the tumors to metastasize. These results suggest that modulating the amount of Sipa1 in human tumor cells might have therapeutic effects. In collaboration with the research group of Hoda Anton-Culver, PhD, at the University of California at Irvine, Dr. Hunter identified Sipa1 polymorphisms associated with the development of human breast cancer. Because genetic polymorphisms are present in every cell in the body, Dr. Hunter is focusing on prospectively identifying patients at high risk for metastasis at, or potentially before, primary diagnosis. Using saliva from mouse models as a surrogate, it has been possible to predict with high sensitivity and specificity which animals will develop highly metastatic disease. Dr. Hunter concluded by mentioning that his lab is working to identify metastasis chemoprevention agents.

Lalage Wakefield, DPhil, of the Laboratory of Cell Regulation and Carcinogenesis, discussed the development of transforming growth factor-β (TGF-β) antagonists as novel therapeutic agents, noting that the NIH has more TGF-β expertise than any other institution in the world. Research conducted in her lab has shown that TGF-β switches from acting as a tumor suppressor early in cancer progression to functioning as a prometastatic factor later in the course of the disease. Treating transgenic mice that develop metastatic breast cancer with antibody-like TGF-β antagonists resulted in a significant reduction in the incidence of metastasis (Figure 2) without affecting primary tumor formation or causing any toxicity that might be expected from inhibiting TGF-β, such as autoimmunity, inflammation, or enhanced spontaneous tumorigenesis in other organs. Dr. Wakefield’s mechanistic analysis demonstrated that TGF-β antagonists act by enhancing endogenous immune surveillance against the tumor. TGF-β antagonists potentially could be combined with other therapeutic modalities to enhance their efficacy; for example, TGF-β seems to be largely responsible for fibrosis following radiation therapy. To facilitate preclinical development of TGF-β-antagonist therapeutic antibodies, Dr. Wakefield and colleagues developed a multi-investigator cooperative research and development agreement (CRADA) with Genzyme Corporation that will also support clinical trials to be conducted at the NCI.

Figure 2. Prolonged treatment with an antibody-like transforming growth factor-β (TGF-β) antagonist can suppress spontaneous metastasis in the MMTV-Neu transgenic mouse model of metastatic breast cancer. (Figure and legend courtesy of Lalage Wakefield, DPhil)

John Letterio, MD, also of the Laboratory of Cell Regulation and Carcinogenesis, described the research conducted in his laboratory through which he and other investigators are evaluating how alterations in signaling through the TGF-β pathway influence carcinogenesis. His lab has developed in vivo models in which components of the TGF-β pathway have been disrupted specifically in immune cells. His group is also developing and evaluating new therapeutic agents that target this pathway. Dr. Letterio described studies characterizing the TGF-β receptor-activated Smad3 protein as a key effector of the response to TGF-β in normal T cells, and as an important suppressor of leukemogenesis in humans. Smad3 expression is lost in leukemic cells of patients with pediatric acute T-cell lymphoblastic leukemia (T-ALL). Dr. Letterio next discussed an important, recently developed model in which deleting the gene encoding Smad4 specifically in T cells resulted in epithelial carcinomas throughout the alimentary tract (unpublished data). Normal epithelial cells produce TGF-β after exposure to environmental microorganisms; it is suspected that the loss of Smad4-dependent signaling in T cells leads to progressive activation of lymphocytes and production of Th2 cytokines, resulting in epithelial hyperplasia. Finally, Dr. Letterio described the chemopreventive activity of novel synthetic triterpenoids in a mouse model of inflammatory bowel disease that progresses to colon cancer. These agents (synthesized in the lab of NCI’s Eminent Scholar, Michael Sporn, MD), along with TGF-β antagonists, have great potential for clinical development.

Drug Discovery

Jeffrey Rubin MD, PhD, of the Laboratory of Cellular and Molecular Biology, who purified keratinocyte growth factor (KGF), summarized his basic and clinical research with this protein. Dr. Rubin explained that KGF, which is derived from mesenchymal cells, is a paracrine mediator of epithelial homeostasis with remarkable cytoprotective effects. Oral mucositis resulting from high-dose chemotherapy and radiation seemed a good setting in which to explore a therapeutic use for KGF. He discussed results from a phase III clinical trial sponsored by Amgen, Inc., in which patients with hematologic malignancies received radiation and chemotherapy followed by autologous peripheral blood progenitor cell transplants. Results showed that the patients who were also treated with KGF had a significant reduction in the incidence and duration of severe oral mucositis. Patients treated with KGF required less pain medication and less total parenteral nutrition to supplement oral intake. There was also a decrease in the incidence of febrile neutropenia, suggesting that reducing the damage to the mucosa decreases the incidence of infection. The U.S. Food and Drug Administration has approved KGF (palifermin) for this indication; favorable results in ongoing clinical trials in solid tumors could mean that approximately 100,000 patients per year may be treated with KGF. In his concluding remarks, Dr. Rubin noted that by increasing patients’ tolerance of aggressive therapies, KGF administration is widening the therapeutic window for existing treatments.

Phillip Dennis, MD, PhD, of the Cancer Therapeutics Branch, presented his work in using the Akt pathway as a target for the prevention and treatment of lung cancer. Akt is a proto-oncogene, and its activation is an early event in tobacco-related carcinogenesis. Two tobacco components, nicotine and the tobacco-specific carcinogen NNK, activate the Akt pathway in normal human lung epithelial cells, resulting in partial cell transformation and an altered apoptotic threshold. Cells that survive with DNA damage can ultimately develop into lung tumors. These studies have prompted extramural investigators to include Akt activation as a molecular end point in lung cancer prevention trials. Studies using tissue microarray analysis revealed that Akt was active (i.e., phosphorylated) in more than half of the different tumor types tested and that activation confers a poor prognosis in patients with stage I non-small cell lung cancer, a finding of vital importance, as increasing numbers of patients are being diagnosed with this disease in screening protocols. Patients diagnosed with tumors with active Akt can thus be stratified to receive more aggressive treatment. Dr. Dennis is also developing new Akt pathway inhibitors—such as several phosphatidylinositol ether lipid analogs (PIAs)—and is testing as potential Akt pathway inhibitors drugs previously approved for other indications. To validate the Akt pathway as a molecular target, clinical trials with Akt pathway inhibitors are being planned at the NCI in patients who have tumors in which Akt is highly active.

Lymphoma Diagnosis and Treatment

Louis Staudt, MD, PhD, of the Metabolism Branch, provided an overview of his work on the molecular diagnosis of lymphoid malignancies. His group has developed an accurate and reproducible single DNA microarray that can provide diagnostic and prognostic information for patients with cancer. Dr. Staudt and colleagues have also developed molecular predictors of outcome—length of survival or response to therapy—in diffuse lymphoma, mantle-cell lymphoma, follicular lymphoma, and chronic lymphocytic leukemia, based on gene expression profiling of diagnostic biopsies. This technology was also used to determine that diffuse large B-cell lymphomas (DLBCL) can be subclassified into three molecularly and clinically distinct diseases: germinal center B-cell–like (GCB) DLBCL, activated B-cell–like (ABC) DLBCL, and primary mediastinal B-cell lymphoma (PMBL). These subtypes arise from B cells at different stages of differentiation, use different oncogenic pathways, and exhibit distinct survival rates following treatment. The discovery of the lymphoma subtypes has also led to the identification of new molecular targets. Dr. Staudt’s group is currently attempting to identify additional molecular targets in lymphomas.

The clinical application of Dr. Staudt’s research was nicely illustrated by Wyndham Wilson, MD, PhD, of the Experimental Transplantation and Immunology Branch, who described targeted therapeutic approaches for lymphoma. With the addition of rituximab to standard therapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), the cure rate for DLBCL was raised from approximately 30% to 50%. To possibly increase the cure rate for DLBCL further, Dr. Wilson developed a novel, continuous-infusion regimen called Dose-Adjusted (DA)-EPOCH, based on a rational design that included optimizing the dosing schedule and pharmacokinetics through application of pharmacodynamic dose adjustment. In contrast to standard therapy with CHOP, this new regimen has proven to be effective against rapidly proliferating tumors. Dr. Wilson’s group has shown that the addition of rituximab to DA-EPOCH appears to overcome ABC DLBCL resistance, and preliminary results suggest that the cure of DLBCL has been increased from approximately 33% with CHOP to approximately 75% with DA-EPOCH–rituximab.

Elaine Jaffe, MD, of the Laboratory of Pathology, highlighted the role of the pathologist in lymphoma research, stating that “most of the insights into the pathogenesis of malignant lymphoma have followed on the heels of an accurate pathological description.” Knowledge of the pathogenesis of a disease (its molecular pathways) leads to the development of new diagnostic tools, and these tools help to further characterize the disease. This conceptual framework formed the basis for the development of the Revised European American Lymphoma (REAL) classification and of its descendant, the World Health Organization (WHO) classification—the first internationally accepted classification of lymphomas and leukemias. Dr. Jaffe was a contributor to both systems. Studies by Dr. Jaffe with follicular lymphoma (FL) show how pathogenetic insights can proceed from what is essentially a morphologic observation. She identified an in situ form of FL involving germinal centers. Her studies suggest that FL may arise from mature B cells in the germinal centers and not from pre-B cells in the bone marrow. Recent studies using laser-capture microdissection and reverse-phase protein microarray were conducted to compare the apoptotic pathways in Bcl-2–positive and Bcl-2–negative FL. Results show that Bcl X_L, an anti-apoptotic protein, was increased in Bcl-2–negative FL. The apoptotic pathways were otherwise very similar between the two FL groups, suggesting that all FLs use a common biochemical pathway.

Major Contributions

Dr. von Eschenbach’s visit provided an excellent opportunity to exchange ideas and discuss future directions. He asked insightful and thought-provoking questions that stimulated an excellent dialogue. The science that was presented clearly demonstrated how many results from intramural research are advancing NCI’s mission of eliminating death and suffering due to cancer. Imaging approaches are rapidly advancing our knowledge of fundamental cellular processes—knowledge that will be critical for preventing, diagnosing, detecting, and treating cancer. Furthermore, numerous molecular targets and drugs for those targets are being identified for either prevention or treatment of cancer, and critical targets are being recognized that have the potential for reducing cancer treatment-associated pain and suffering in thousands of individuals. Finally, this work is being conducted by numerous individuals who build on and extend the successes of their intramural colleagues and the extramural research community. The end result is outstanding science and innovative clinical approaches that are providing new hope for cancer patients and their families.

Robert H. Wiltrout, PhD
Director

Special thanks to L. Michelle Bennett, PhD; Jackie Lavigne, PhD, MPH; Gordon Hager, PhD; Tom Misteli, PhD; Kent Hunter, PhD; Lalage Wakefield, DPhil; John Letterio, MD; Jeffrey Rubin MD, PhD; Phillip Dennis, MD, PhD; Louis Staudt, MD, PhD; Wyndham Wilson, MD, PhD; and Elaine Jaffe, MD

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