|T H E N I H C A T A L Y S T||J U L Y A U G U S T 2006|
|P E O P L E|
Alan DeCherney received his M.D. degree from Temple University School of Medicine, Philadelphia, in 1967. He received additional training at the Lister Institute in London, the University of Pittsburgh, and the University of Pennsylvania in Philadelphia and held high-ranking academic positions at Yale University School of Medicine in New Haven, Tufts University School of Medicine in Boston; and the David Geffen School of Medicine, University of california at Los Angeles, before joining NIH in 2006 as chief of the Reproductive Biology and Medicine Branch, NICHD. He is also a member of the Institute of Medicine of the National Academies.
I am in the process of establishing the Section on Implantation and Oocyte Physiology within the Reproductive Biology and Medicine Branch. This section will conduct patient-oriented research in such reproductive problem areas as infertility, recurrent pregnancy wastage, and failed treatment modalities.
A major effort will be to serve as a "court of last resort" for patients with a history of multiple fertility treatment failures. In addition to the intensive evaluation of these patients, the section will also explore why, in the absence of fertility problems, the rate of loss of fertilized eggs is so high. Among fertile couples seeking to become pregnant, 80 percent of ovulated eggs are fertilized, yet only 20 percent of these result in live birthsalmost half are lost between conception and the next menstrual period, and another 20 percent end in later spontaneous abortions.
We have plans to establish a Center of Excellence for Recurrent Pregnancy Loss that will focus on basic immunologic and clinical investigation. Two tenure-track investigators will be recruited to conduct research concentrating on implantation and oocyte physiology.
There are also plans to expand existing branch programs in endometriosis, fibroids, premature ovarian failure, receptor physiology, endocrine diseases such as Cushing's syndrome, and neuroendocrinology.
received his M.D. and Ph.D. degrees from New York University School of Medicine
in 1991 and 1992, respectively. He completed an internal medicine residency
and medical oncology fellowship, and, with Michael Kastan, a postdoctoral fellowship
focusing on molecular control of apoptosis, all at Johns Hopkins University
in Baltimore. He joined NCI in 1999 as an investigator in the Developmental
Therapeutics Department and is now a senior investigator and leader of the Signal
Transduction Section in the Medical
Oncology Branch, NCI.
Lung cancer is the number-one cause of cancer-related death across the world and is most commonly associated with smoking. My work has focused on signal transduction pathways that promote lung tumorigenesis and the therapeutic resistance of established cancers.
Although my group has investigated many signaling pathways that contribute to the resistance of lung cancer cells to therapy, we are currently focused on one pathway, the PI3K/Akt/mTOR pathway.
Our body of work over the past few years has established that activation of the PI3K/Akt/mTOR pathway is important at the earliest and latest stages of lung cancer.
For example, tobacco components activate this pathway and promote a partially transformed phenotype in normal human epithelial cells. Increased activation of the pathway occurs with phenotypic progression of preneoplastic lesions. Moreover, inhibitors of the pathway such as the mTOR inhibitor rapamycin prevent tobacco carcinogeninduced lung tumors in two murine model systems.
Most lung cancer cell lines have constitutive activation of the pathway and depend on the pathway for survival; similarly, agents that inhibit the pathway also cause the death of lung cancer cells and increase the efficacy of chemotherapy or radiation therapy.
Most recently, we showed that Akt activation is indicative of a poor prognosis for all stages of lung cancer patients, but especially those with Stage 1 disease and/or tumors of less than 5 cm.
Our current studies focus on the mechanisms by which tobacco components signal through nicotinic receptors to the PI3K/Akt/mTOR pathway; to that end, we are creating new transgenic and knockout mouse models that will allow us to dissect the role of individual pathway components in tobacco-induced tumorigenesis.
We are also working on two approaches to develop inhibitors of the PI3K/Akt/mTOR pathway in lung cancer. First, we have used molecular modeling to guide the synthesis and characterization of lipid-based inhibitors of Akt called phosphatidylinositol ether lipid analogues (PIAs). We have identified the spectrum of activity of PIAs and molecular correlates of response to PIAs; we have also performed microarray analyses to identify changes in gene expression that are associated with PIA administration and have identified several genes that could serve as biomarkers in clinical trials. Indeed, PIAs have been identified as candidates for limited exploratory human trials to test the effects of low doses (so-called Phase 0 trials).
The second approach to inhibit the pathway is to test off-the-shelf drugs that are FDA approved for other indicationsthis approach could expedite the drug-development process.
We recently identified HIV protease inhibitors (HIV PI) as inhibitors of Akt that have a wide spectrum of activity and exert dose-dependent toxicity on all cell lines in the NCI60 cell line panel. A Phase I dose-escalation trial of the HIV PI nelfinavir in cancer patients is planned.
We perform all of our preclinical studies with an eye toward clinical translation to new therapies for lung cancer patients. Inhibitors of the PI3K/Akt/mTOR pathway hold the promise of clinical benefit for those at risk of developing lung cancer and forlung cancer patients who need better therapeutic options.
Steven Hou received his Ph.D. from the University of Chicago in 1994 and did his postdoctoral research in the laboratory of Norbert Perrimon at Harvard Medical School in Boston. He was recruited to the Laboratory of Immunobiology, NCI, in September 1997 as a tenure-track investigator and is currently a senior principal investigator at the Mouse Cancer Genetics Program, NCI.
My group has played a major role in developing the Drosophila model of the JAK/STAT and JNK/JUN signal-transduction pathways and is currently focused on these pathways' functions in stem cell regulation and animal aging in model organisms.
My research at NCI has three stages:
n Developing the Drosophila model of the JAK/STAT and JNK/JUN signal- transduction pathways
I identified the Drosophila stat and jun genesmutations in the course of my postdoctoral research. During the first few years at NCI, my group concentrated on identifying components of the JAK/STAT and JNK/JUN signal-transduction pathways.
We conducted a large-scale transposon P-elementmediated gene disruption screen, which enabled us to identify 900 different gene mutations (Genetics 163:195201, 2003). From that screen, we identified a receptor for the JAK/STAT signal-transduction pathway (Genes Dev. 16:388398, 2002); we also found that the JAK/STAT pathway and cyclin D/Cdk4 cooperatively regulate tumor development in the fly blood and eye (Dev. Cell 4:179190, 2003). In the JNK/JUN signal-transduction pathway, we cloned a new multidomain scaffolding protein (Mol. Cell. Biol. 22:17921803, 2002).
n Elucidating functions of the JAK/STAT and JNK/JUN signal-transduction pathways in stem cell regulation and animal aging
In a genetic screen for mutations that interact with the JAK/STAT signal-transduction pathway in regulating male germ-line stem cell (GSC) fates, we identified a small GTPase Rap guanine nucleotide exchange factor (Gef26) from our library of P-element mutations.
We demonstrated that the Rap-GEF/Rap signaling controls stem cell anchoring to the niche through regulating DE-cadherinmediated cell adhesion (Dev. Cell 10: 117126, 2006).
We also found that the Drosophila homologue of the Birt-Hogg-Dubé (BHD) syndrome tumor suppressor functions downstream of the JAK/STAT and Dpp/TGF-b signal transduction pathways and regulates male GSC maintenance (Oncogene Apr 24, 2006, Epub ahead of print).
These findings suggest that the BHD protein may regulate tumorigenesis through modulating stem cells in humans.
The JNK/JUN signal-transduction pathway regulates stress response and lifespan in the fly. We screened the P-element mutants, either generated by us or obtained from the public stock centers, and identified 40 long-lived mutants.
We are currently exploring the molecular mechanism of how these new mutants and the JNK signaling pathway cooperatively regulate fly lifespan.
n Exploring stem cell regulation and animal aging in mice
We are applying knowledge gained from the Drosophila systems to study stem cell regulation and animal aging in the mouse system.
There are two mouse orthologs of Gef26RapGEF1 and RapGEF2. We are in the process of generating the conditional knockout mice of the RapGEF1 and RapGEF2 genes.
We are also developing cell-labeling systems to specifically label stem cells and cancer stem cells in mice.
The powerful genetic manipulations available in Drosophila enable us to dissect the molecular mechanism of stem cell regulation and animal aging. Extending the findings in the fly system to the mouse system will enable us to develop better human disease models.
Our studies using both systems will not only lead to an enhanced biological understanding of stem cell regulation and animal aging but may also provide new targets for treating relevant human diseases.
Stan Lipkowitz received his M.D. and Ph.D. degrees from Weill Medical College of Cornell University in New York in 1984. After clinical training in internal medicine at The New York Hospital, he came to NCI as a medical oncology fellow. After completing a postdoctoral fellowship in the laboratory of Ilan Kirsch, he joined the Genetics Branch as a tenure-track investigator in 1997. In 2003, he moved to the Laboratory of Cellular and Molecular Biology, where he is currently a senior investigator.
My laboratory studies signal-transduction pathways that regulate growth and programmed cell death in epithelial cancer cells, with a focus on breast and ovarian cancer. We have three projects:
1) The function of Cbl proteins. Human epithelial malignancies frequently display deregulated tyrosine kinase activity. Understanding the mechanisms that regulate signaling by these kinases should uncover new ways to inhibit cancer cell growth. We are investigating the function of Cbl proteins, a family of proteins that regulate tyrosine kinase activity. Cbl proteins belong to the RING finger class of ubiquitin protein ligases (E3s) and function as E3s for activated tyrosine kinases.
My group cloned two of the three mammalian Cbl genes. We have focused primarily on the activated epidermal growth factor receptor (EGFR) as a model substrate for Cbl proteins and have shown that all mammalian Cbl proteins mediate ubiquitination and degradation of the activated EGFR. Furthermore, the Cbl proteins, as well as other components of the signaling complex, are degraded upon activation of the EGFR. Thus, Cbl proteins mediate degradation of the active EGFR signaling complex.
In collaboration with Allan Weissman, of the Laboratory of Protein Dynamics and Signaling, NCI, we have demonstrated that Nedd4 and Itch, two HECT E3s, target Cbl proteins for degradation. Thus, there is likely to be a network of regulation of E3s by other E3s.
Ongoing work is focused on understanding the biochemical and physiologic functions of the three mammalian Cbl proteins in epithelial cells and elucidating the differences in their specificity and/or function.
2) The function of death receptors in epithelial cancer cells. Cancer cells avoid apoptosis by a variety of genetic and epigenetic mechanisms. We are investigating the induction of apoptosis by activation of death receptors for the ligand TRAIL in breast and ovarian cancer cells. Our goal is to selectively trigger apoptosis in the cancer cells.
My group has shown that most breast and ovarian cancer cell lines are resistant to the induction of apoptosis by TRAIL, the ligand for the death receptors DR4 and DR5. We have demonstrated that resistance to TRAIL-induced apoptosis can be overcome by co-incubation of the cells with chemotherapeutic agents, semisynthetic retinoids (such as 4HPR), or molecularly targeted agents (such as EGFR or ErbB-2 inhibitors).
These observations are particularly important because agonists for the TRAIL receptors are being tested in patients with cancer. Our results suggest that many cancers will be resistant to these agents when they are used alone. Predicting and overcoming this resistance will be essential to the clinical success of these agents.
Our current work utilizes biochemical and genetic approaches to identify mechanisms that regulate the induction of death by TRAIL ligand in breast and ovarian cancer cells.
3) The assessment of molecular effects of targeted therapy in cancer patients. It is critical to assess the action of new therapeutic agents on the predicted targets in the tumor in order to correlate the molecular function of these agents with clinical outcomes.
In collaboration with Sandra Swain, of the NCI Medical Oncology Branch, we investigated the biochemical consequences of EGFR inhibition in breast cancer patients treated with the small-molecule EGFR inhibitor erlotinib. We were able to demonstrate effects of the inhibitor on EGFR signaling in biopsies of both surrogate and tumor tissue that expressed EGFR.
Using this pilot study as a template for the design of future studies, we will assess the biochemical effects of other molecularly targeted agents in breast cancer patientsparticularly those that are relevant to our own research, such as EGFR inhibitors and agents that activate the TRAIL receptors.
Daniel Masison received his Ph.D. in biomedical sciences from the University of Massachusetts Medical Center, Worcester, in 1993, after which he joined the Laboratory of Biochemistry and Genetics, NIDDK, as a postdoctoral fellow. He became a tenure-track investigator in 1998 and is currently a senior investigator in that lab.
My research focuses on how protein chaperones and their co-chaperone partners affect propagation of the yeast [PSI] prion, an infectious amyloid form of a cytosolic protein. Amyloid is a fibrous protein aggregate that self-assembles like a crystal, converting the soluble protein into the non-native amyloid form as it joins the fiber. Amyloid accumulation is associated with tissue pathology in many disorders, including type 2 diabetes, Alzheimer's disease, and prion diseases. Prion diseases are the only infectious amylodoses. What makes them infectious is unknown.
Prion particles, or "seeds," must replicate to be infectious or maintained in a growing yeast population. My isolation of an Hsp70 mutant that impairs [PSI] propagation led to our discovery that Hsp70 influences this replication and, thus, prion infectivity.
Hsp70 is a ubiquitous and essential chaperone that helps proteins adopt and maintain their native conformations. Because Hsp70 is important in many processes during which proteins are incompletely folded, such as translation, its activity is highly regulated by many co-chaperones. We found that modifying such co-chaperones could alter prion propagation by affecting Hsp70 activity in defined wayswhich revealed how the mutant Hsp70's reaction cycle was altered.
Although the mutant Hsp70 is incompatible with prion propagation, it has no overt effect on cell growth or stress protection, pointing to Hsp70 as a target for therapeutic treatment of amyloidoses. To this end, we are looking to identify compounds that alter Hsp70 function in a way similar to the mutation.
We also discovered that the small molecule guanidine, known for over 20 years to be a potent yeast prion-curing agent, acts specifically by inactivating Hsp104, another chaperone important for yeast prion replication. Hsp70 and its co-chaperones are components of the Hsp104 chaperone machinery, and our continuing studies are uncovering how this machinery acts to influence amyloid-forming and amyloid-eliminating processes as well as cell growth and stress protection.
Hsp70 is also a component of the Hsp90 chaperone machinery, which assists folding of many "client" proteins, in particular signaling and transcription factors. Our studies identified novel functions for several Hsp90 co-chaperones in the regulation of Hsp70 and Hsp90. In addition to uncovering differences in chaperone activities for cellular and prion functions, this work identified the Hsp90 co-chaperones as factors involved in yeast prion propagation and provided the first functional evidence for some of their specific activities in vivo.
We also developed yeast systems wherein we can replace various chaperone components with those from any species. We found that mammalian counterparts supported growth and prion propagation, which demonstrates their utility as models for studying human chaperone/co-chaperone functions and for screening for compounds effective against them.
Using one such system, we discovered functional distinctions between the nearly identical constitutive and stress-inducible mammalian Hsp70s, which implies that optimal stress protection requires a function lacking in Hsp70 isoforms expressed during non-stress conditions.
Our ability to identify chaperone defects that do not affect cell growth and metabolism is allowing us to make significant innovative contributions to the understanding of the functions of the chaperones and their co-chaperones.
Although we are primarily focused on how protein chaperones interact with each other and with amyloid to better define how they affect amyloid propagation at a molecular level, our studies are also helping us understand how chaperones function both independently and as collaborators in general aspects of protein folding.
John Tisdale received his M.D. degree from the Medical University of South Carolina in Charleston in 1990. He completed an internal medicine residency at Vanderbilt University Medical Center in Nashville and then trained in hematology in the Hematology Branch, NHLBI, where he served as a postdoctoral fellow under the mentorship of Cynthia Dunbar. He joined the Molecular and Clinical Hematology Branch of NIDDK in 1998 and is currently a senior investigator in that lab.
The description of sickle cell anemia (SCA) as a "molecular disease" by Linus Pauling over a half-century ago generated hope for a new era of molecular medicine. The defect was later traced to a single substitution at the sixth position of the b-globin chain of the hemoglobin (Hb) tetramer, resulting in an abnormal Hb among the erythroid progeny of hematopoietic stem cells (HSCs).
Our group focuses on HSC-based therapeutic approaches through the development of methods for transplantation of normal donor-derived HSCs or genetically modified patient-derived HSCs.
Though the curative potential of allogeneic HSC transplantation has been established in a select group of children with SCA, procedural toxicities limit this approach.
The development of conditioning regimens for graft-specific tolerance in the absence of conventional bone marrow-ablative chemoradiotherapy may allow extension of this approach to adults.
We and others have demonstrated the ability to achieve engraftment of allogeneic HSCs without the need for toxic ablative conditioning, yet full engraftment using intensive immunosuppression appeared to result from a donor T cellmediated immune response and was associated with significant complications.
We therefore sought to develop a transplantation regimen for adults with SCA for which engraftment does not depend on such alloreactivity. We explored low-dose radiation and the immunosuppressant rapamycin to induce tolerance in vivo in a murine HSC transplantation model and achieved phenotypic correction in a murine model of SCA, even with only moderate donor engraftment (mixed hematopoietic chimerism).
Based on these findings, we initiated a clinical trial of this novel transplantation approach in adults with severe SCA; initial results in the first three patients are encouraging, and our data support mixed hematopoietic chimerism as a reasonable goal for HSC transplantation in SCA.
For those lacking a suitable sibling matched donor, the permanent integration of potentially therapeutic genes into primary autologous HSCs using retroviral vectors remains a viable alternative.
Despite successful high-level gene transfer to murine HSCs and human progenitors in vitro, poor transgene expression and extremely low gene-transfer efficiency were observed in early human clinical trials. Our team and others have made significant progress over the past decade, with marking levels of 10 percent or higher at the HSC level now attainable in large animals.
Given the toxicity of myeloablative irradiation currently ued in our animal models, we have focused our recent efforts on determining the degree of host conditioning required to achieve moderate-level engraftment of genetically modified cells.
Until recently, the desired attainment of erythroid-specific expression of the transferred globin gene had been problematic. Lentiviral vector systems, however, now permit the incorporation of large-globin locus control region elements in viral vectorsa development that enabled Michel Sadelain and his colleagues at the Memorial Sloan-Kettering Cancer Center in New York to achieve for the first time regulated human b-globin expression sufficient to revert the phenotype in a murine model of b-thalassemia.
In collaboration with the Sadelain group, we have now established a preclinical, large-animal model for lentiviral globin gene transfer.
Using a VSV-G pseudotyped, modified HIV-1based vector, high gene-transfer rates to HSCs are achievable, with human b-globin expression of greater than 50 percent among erythroid progeny generated in vitro.
Transplantation studies in two rhesus macaques demonstrated human b-globin expression at greater than 10 percent early post-transplantation, with stabilization, albeit at lower levels, long-term.
Follow-up of these and other animals with clonal tracking of HSC progeny by integration site analysis will permit assessment of the safety of this approach, and the model will be used to support eventual clinical application in disorders of globin synthesis.
Nan-ping Weng graduated from Shanghai Medical College, Fudan University (former Shanghai First Medical College), Shanghai, China, in 1984. He received his Ph.D. in immunology from Baylor College of Medicine, Houston, in 1993 and did his postdoctoral training at the Experimental Immunology Branch, NCI. In 1997, he became a tenure-track investigator in the Laboratory of Immunology, NIA, where he is currently a senior investigator in the Lymphocyte Differentiation Unit.
Immunological memory, a hallmark of immune response, is characterized by a rapid and robust response to subsequent encounters of a previously experienced antigen.
This memory, housed in long-lived T and B lymphocytes, serves as the physiological basis for vaccination and immunization. Despite advances in the field, the molecular mechanisms underlying immunological memory have only begun to be understood.
My laboratory seeks to elucidate the mechanisms of memory T cell generation, response, and aging. Specifically, we have focused our efforts on three areas: 1) identifying and characterizing differentially expressed genes in memory T cells, 2) determining the epigenetic basis for differential gene expression in memory T cells, and 3) exploring the molecular and cellular alterations of memory T cells with aging.
The pattern of gene expression and silencing defines the cellular characteristics and functions. Thus, one of our major research goals is to identify genes that are differentially expressed in memory T cells and to further characterize the roles of those genes in the generation, function, and homeostasis of memory T cells.
Using DNA microarray technology, we have analyzed and compared gene-expression profiles of human and mouse naïve and memory CD4 T cells and human naïve and memory CD8 T cells.
We have identified dozens of genes that are differentially expressed in memory CD4 and CD8 T cells in both human and mouse.
We have used mouse models that lack expression of these differentially expressed genes to further examine their role in memory T cell formation and response.
Prospecting for the molecular basis of differential gene expression in memory T cells, we investigated the contribution of chromatin structureparticularly the modification of histonein the regulation of gene expression in memory T cells. We found that acetylation levels of histone H3 lysine 9 (H3K9) are higher in memory CD8 T cells than in naïve cells in both resting and activated states.
Furthermore, we found that higher H3K9 acetylation levels were detected in resting memory cells, prior to their activation, for those genes that were differentially expressed after activationindicating that hyperacetylation of histone H3K9 may play a role in the selective and rapid gene expression of memory CD8 T cells.
We are now extending analysis of histone H3K9 acetylation and other histone modifications to a genome-wide scale in parallel with genome-wide gene expression analysis. We hope such analyses will provide a genome-wide account of histone modification and gene expression in memory T cells.
Immune functions decline with age, with a resulting increase in infection-related morbidity and mortality in the elderly. We are interested in age-associated changes of memory T cell function and replicative lifespan.
It is known that short telomeres curtail cellular replication; not known is the in vivo rate of telomere attrition and whether in vivo T cell telomerase activity declines with age. A major research goal in my laboratory is to understand the role of telomere length and telomerase activity in memory T cell function and replicative lifespan and to investigate age-related changes in their regulation.
Currently, we are using human primary T cells (normal cells directly isolated from blood, as opposed to T cell lines) for molecular and cellular analyses and longitudinal studies to elucidate the changes of T cell function with aging.
We anticipate that knowledge derived from these experiments will further our understanding of the mechanisms of memory T cell formation, response, and age-associated functional decline. This understanding is essential for the rational design of vaccines to protect against infectious diseases and to develop strategies to combat cancer and autoimmune diseases in both the general and elderly populations.