2011 Medical Research Report

Dr. Seung Kim, Stanford University

I. Discovery of methods to regenerate insulin-producing b-cells for diabetes

Functional restoration of diseased solid organs is a broad goal motivating intensive effort in biomedical research. Replacement or regeneration of  pancreatic islets of Langerhans, endocrine organs that secrete insulin and glucagon, has emerged as a paradigm for organ restoration in recent years. Deficiency of insulin-producing islet β-cells underlies the pathogenesis of diabetes mellitus, a disease with devastating autoimmune (type 1) and pandemic (type 2) forms Islet replacement in diabetes is ultimately limited, however, by our inadequate understanding of mechanisms controlling islet formation and growth. Thus, islet replacement is a specific challenge to the consensus that knowledge about solid organ development and  expansion can be used to restore organ function in human diseases.

To meet this challenge, our group has created new approaches to produce, expand, and regenerate islets. Once viewed as incapable of significant proliferation, mature β-cells in the pancreas are now recognized to have a significant capacity to replicate, and thereby maintain β-cell mass. For this reason, expansion of islets in culture or in the pancreas may become a therapeutic option for diabetes. However, prior attempts to expand islets with mitogens have been bedeviled by the loss of key β-cell features, like insulin expression, that accompanies proliferation. Thus, it remains elusive how adult β-cells "remember" their differentiated fate while proliferating. Vigorous pancreatic β-cell replication in juvenile mice and humans declines with age, and elucidating the basis for this decay may reveal strategies for inducing β-cell expansion. In a recent paper scheduled for publication this autumn in the journal Nature (Chen et al 2011) that cites support from the Snyder Foundation, we describe how Platelet-derived growth factor receptor (Pdgfr) signaling controls expression of β-cell Enhancer of zeste homolog 2 (Ezh2) to control age-dependent β- cell proliferation in mouse and human islets. Discovery of a conserved signaling pathway controlling age-dependent β-cell proliferation suggests new strategies for-cell expansion. Reprints of this paper, which according to one reviewer 'should powerfully influence investigators in this field', will be forwarded when available.

II.  Creation of new human cells for discovering the basis of organ  development  

    In a study published earlier this year in the journal Cell Stem Cell, our group reported generation of a unique human ES cell line harboring an eGFP insertion in SOX17, and used this cell line to purify human SOX17-eGFP+ progeny and monitored of in vitro and in vivo endodermal differentiation. These studies have permitted novel conclusions and created unique tools for analyzing development of human endoderm, the tissue from which multiple organs including the pancreas derive. To accomplish these goals Dr. Pei Wang in my group learned to (1) grow and maintain human ES cell lines, (2) created targeting constructs for the SOX17 locus, (3) performed screens to isolate cell lines that successfully inserted the eGFP reporter gene by homologous recombination at the native SOX17 locus, (4) purifiedSOX17-eGFP+ cells resembling definitive endoderm by FACS from her cultures, (5) performed gene expression profiling and bioinformatics analyses of this hESC-derived definitive endoderm, (6) built new assays for assessing the developmental potential of purified hESC progeny, and (7) repeated steps 1-6 for human iPSC lines. We have had a tremendous response to this publication, with multiple requests for materials generated from that study. In addition, we wrote an invited Perspective for Cell Stem Cell, based largely on the findings and implications of this work. This review has been well-received by investigators in our field  
Recent publications  
1.  H. Chen, Xueying Gu, Yinghua Liu, Jing Wang, Stacey E. Wirt, Rita Bottino, Hubert Schorle, Julien Sage, and Seung K. Kim. PDGF signaling controls age-dependent proliferation in pancreatic b-cells. 2011. Nature 478:349-55.

2.  P. Wang, R. T. Rodriguez, J. Wang, A. Ghodasara, S.K. Kim. 2011. Targeting SOX17 in Human Embryonic Stem Cells Creates Unique Strategies for Isolating and Analyzing Developing Endoderm. Cell Stem Cell 8:335-46

3.  K.D. McKnight, P. Wang and S. K. Kim. 2010. Deconstructing pancreas development to reconstruct human islets from pluripotent stem cells.Cell Stem Cell 6:300-8.

4.  C. Benitez, W. Goodyer and S.K. Kim. 2012. Deconstructing pancreas developmental biology. In "Mammalian Development: Networks, Switches, and Morphogenetic Processes", Patrick Tam, W. James Nelson, and Janet Rossant, eds. Cold Spring Harbor Press.

Dr. Chih-Pin Liu, City of Hope, Beckman Research Institute

Role of regulatory T cells in the control of type 1 diabetes

Several studies indicate that regulatory T (Treg) cells play a critical role in preventing autoimmune disease. In autoimmune diseases, Treg cells lose their authority, so the immune cells begin attacking healthy tissues. For type 1 diabetes, the loss or decrease of functional Treg cells may contribute to early onset of inflammation in the islets of pancreas and pathogenic immune cells attack target cells in the islets that produce insulin,
    which helps the body turn sugar from food into energy. The resulting build-up of sugar in the blood can be lethal and lead to severe and life-threatening complications. In order to intervene or prevent these pathogenic processes and regain our body’s control  in insulin production, it is necessary to restore a functional population of Treg cells. Therefore,  the main purposes of our study are to determine if  
    1) we can improve the function of Treg cells, and (2) functional Treg cells can be isolated and expanded from patients with established type 1 diabetes.  
    (1) To identify genetic factors that may be involved in modulating Treg cell function, we performed genome-wide comparative gene expression analyses of Treg cells. From extensive analyses of our novel results, we noted unique increased expression of a protein called KIR3DL1 in these cells compared to other non-Treg cells. Further functional analyses of Treg cells showed that elevated levels of KIR3DL1 may have caused them to lose control of pathogenic immune cells. These studies suggest that KIR3DL1 contributes to the faulty Treg function and immune regulation leading to diabetes.  
    After we knocked down the Treg cells’ ability to produce KIR3DL1, Treg cells regained control of pathogenic immune cells and kept them from attacking insulin-producing cells, thus prevented diabetes development. Our results suggest that, by hindering the KIR3DL1protein production, we can help return the immune system to normal and alleviate diabetes or other autoimmune diseases. Therefore, we have identified a gene that may play a key role in modulating Treg cell function and their roles in controlling development of type 1 diabetes. Our on-going studies will continue to understand how this protein may affect the function of Treg cells and whether genetic modulation may reprogram Treg cells to become much better defender of our body to prevent diabetes development.  
    (2) In order to translate our findings obtained from studies using animal models to the clinics, the first step that must be taken in this challenging task is to determine if Treg cells can be taken from not only healthy individuals but also diabetic patients and multiplied in large enough numbers to be used in clinical treatment. This study will test methods for isolating Treg cells from healthy and diabetic human blood samples and growing them in the lab. This study will also use peripheral blood mononuclear cells as source of immune effector cells (cells that are responsible for induction of an immune response leading to destruction of the islet cells). We will determine whether a sufficient number of Treg cells can be isolated and expanded from diabetic patients, and whether the expanded human Treg cells can retain their function in suppressing pathogenic immune effector cells.    
    Preliminary results obtained from these studies provide exciting positive outcomes indicating that we are able to achieve this goal.  Our initial studies also suggest that human Treg cells expanded in vitro are functional in suppressing immune effector cells. Additional extensive studies involving more human subjects would further confirm these promising findings.  
    Therefore, in the past year, we have made significant progress in our exciting research on the role of Treg cells in the control of type 1 diabetes development. These studies suggest new and promising opportunities for developing therapeutic approaches to treat human type 1 diabetes by expanding a small population of human Treg cells and by altering their genetic program to improve their function in therapy.  
Qin, H., Wang, Z., Du, W., Lee, W.-H., Wu, X., Riggs, A.D., and Liu, C.-P. 2011. KIR3DL1 down regulation enhances inhibition of type 1 diabetes by autoantigen-specific regulatory T cells. Proc. Natl. Acad. Sci. USA. 108: 2016-2021.  

Dr. Phillip Kantoff, The Lank Center for Genitourinary Oncology, Dana-Farber Cancer Institute

Dana-Farber’s Lank Center for Genitourinary Oncology, led by Philip Kantoff, MD, provides compassionate care and the most effective therapies for patients with prostate, kidney, bladder and testicular cancers, as well as many other rare and common cancers.  Patient care at the Lank Center is fueled by ongoing discovery, and accordingly the Lank Center is home to a host of physician-researchers who are committed to conducting cutting-edge clinical, translational, and basic research.  
Through its generous support of the Hahn and Freedman Laboratories within the Lank Center, the H.L. Snyder Medical Foundation has been a strong ally in the Center's ultimate goal of developing effective treatments for patients with genitourinary cancers.  
Lank Center-wide research highlights from 2011 include significant developments toward the discovery of new biomarkers, targets and targeted therapies in prostate cancer, as well as progress in the identification of genes that drive metastasis in bladder cancer.  In the target discovery arena, prostate cancer research conducted in the Lank Center resulted in the publication of a seminal paper this past February (Nature. 2011 Feb 10;470(7333):214-20) which described the first whole genome sequencing analysis of human prostate cancers.  The authors of this study identified potentially new prostate cancer drug targets and oncogenes, gleaned valuable insights about prostate tumor biology, and also were able to form hypotheses about which cellular processes within the prostate are likely going awry during tumor formation.  In addition to discovering novel drug targets, an effort to identify actual drug like chemicals that bind to the known prostate cancer target TMPRSS2-Erg is being performed by Lank Center scientists, and has yielded two small molecule inhibitors that will be further studied for their clinical and investigational utility.  Another important area of prostate cancer research is learning how to distinguish indolent from aggressive cancers as soon as possible after diagnosis.  Drawing upon a large prostate tumor "biospecimen bank" at the Lank Center, researchers have designed a promising study to compare RNA sequences from prostate tumors of long term survivors with tumors from those who have died from this disease; sequencing and analysis of these samples are now underway.  In the field of bladder cancer research, Lank Center clinical scientists seek to further understand the genetic basis of metastasis.  They have thus far identified three chromosomal regions associated with metastasis in a cohort of 100 patients and are now homing in on pinpointing actual genes that underlie metastasis in bladder cancer.  
Drs. William Hahn and Matthew Freedman have each contributed to aspects of the above referenced investigations.  In addition, funding from the H. L. Snyder Medical Foundation has specifically enabled the progress made in the key projects described below.  

William Hahn, MD, PhD - Associate Professor of Medicine, Dana-Farber Cancer Institute/Harvard Medical School Co-Director, Center for Cancer Genome Discovery,      Dana-Farber Cancer Institute

Prostate cancer is the second most common cause of cancer death in men, and the majority of these deaths are in patients with metastatic castrate-resistant prostate cancer (CRPC). The androgen receptor (AR) signaling pathway is critical in patients with CRPC, as demonstrated by positive clinical responses to drugs that are able to decrease circulating androgens to below castrate levels  
    in some patients. suggesting that other, non-AR pathways for survival and growth are activated in their cancer cells. Over-activation of kinase proteins such as ERBB2, MAPK, PI3K/Akt, nd Src have been implicated in the development of castrate resistance, however, phase II clinical trials of Src inhibitors and similar compounds in metastatic CRPC have. However, a large proportion of patients do not respond to these therapies, shown only modest clinical benefits.  
    Dr. Hahn has hypothesized that other signaling pathways are involved in conferring castrate resistance, and his laboratory is thus pursuing an unbiased approach to identify novel pathways involved in CRPC, which also may serve as therapeutic targets in patients with CRPC.  
    Previous work in the Hahn Laboratory (Berger et al., 2004) resulted in the development of mouse models that harbor well differentiated tumors that are also androgen dependent, i.e., unable to grow in female or castrated mice. Support from the H.L. Snyder Medical Foundation has enabled Dr. Hahn to perform a high throughput, in vivo genetic screen to identify kinase proteins that permit these androgen dependent cells to form tumors in castrate animals. In the last year, Dr. Hahn has identified several such proteins.  The Hahn Laboratory is in the process of validating these kinases as targets for patients with metastatic prostate cancer.

Matthew Freedman, MD - Assistant Professor, Dana-Farber Cancer Institute and Harvard Medical School

One of the basic tenets of genetics is to understand the relationship between genotype and phenotype. Human geneticists endeavor to map the genetic determinants underlying traits, such as disease susceptibility. Over the past 5 years, genome wide association scans (GWAS) have enabled the identification of thousands of genetic variants (single nucleotide polymorphisms – SNPs) associated with hundreds of traits.  

Somewhat unexpectedly, genes underlying complex traits tend to map to non-protein coding regions of DNA. Since our understanding of the non-protein coding region of the genome is rudimentary compared to our understanding of the protein coding regions, elucidating disease mechanisms is a challenge. Given the rapidity with which GWAS studies are discovering regions of the chromosome associated with complex traits, identification of genes influenced by such chromosomal regions has become a bottleneck. Once a gene is identified, assays can then be performed to interrogate the function leading to a deeper understanding of disease mechanism.  
To date, GWAS have discovered 40 loci associated with the risk of developing prostate cancer. Similar to other traits, the majority of these loci are located outside of protein coding regions. Over the past year, the Freedman Laboratory has studied 12 of these loci. The Laboratory's approach is based on the observation that RNA transcript levels are under genetic control; that is, the number of alleles that an individual carries can influence RNA levels of certain genes.  
    With funding from the H. L. Snyder Medical Foundation, Dr. Freedman tested the correlation between risk allele status and RNA transcript abundance for each of the 12 risk variants. A total of 103 transcripts were evaluated in 662 prostate tissue samples from 483 individuals of varying ethnic backgrounds. In a pooled analysis across all groups, 4 risk variants were strongly associated with 5 transcripts in histologically normal tissue. In collaboration with Dr. Hahn, Dr. Freedman performed functional studies demonstrating that suppression of the expression of the genes NUDT11, HNF1B, and SLC22A3 influence cellular phenotypes associated with tumor related properties in prostate cancer cell lines. Together, these observations suggest that NUDT11, HNF1B and SLC22A3 contribute to human prostate cancer pathogenesis. Ultimately, the annotation of risk loci and the connection with their target genes will inform the biology of prostate cancer and may reveal opportunities to more rationally intervene in treatment and prevention.

Dr. Judy Shizuru, Stanford University - Hematologist Internal Medicine Physician Hematologist / Oncologist, Medical Oncologist

The Shizuru laboratory continues their work on bone marrow transplantation (BMT) to treat many life-threatening disorders.  Diseases currently cured by BMT include various cancers as well as non-cancerous states such as childhood defects in blood formation (sickle cell anemia, severe combined immune deficiency [i.e., bubble boy disease]).  BMT has the potential to cure many others. While this procedure effectively treats many adults, children and babies, it remains imperfect.  Patients can die from the procedure itself or the transplant may fail to cure their disorder. 
    The dangers of the procedure have prevented its use in treating diseases such as childhood diabetes or multiple sclerosis. The Shizuru laboratory strives to make the procedure safer and more effective for the many tens of thousands of patients that undergo this procedure yearly.  
    The approach they have taken is to utilize a cell separation technology developed at Stanford University to select out only the blood forming stem cells from the marrow grafts, and exclude other cells from the graft that can cause complications.  In the past year, a post-doctoral fellow (Dr. Antonia Mueller) funded by the Snyder Foundation showed in a mouse model that contrary to what many scientist believe, transplantation of purified blood forming stem cells from a donor to a recipient resulted in better regeneration of the immune system than standard bone marrow grafts.  When pure stem cells were transplanted many more new functional lymphocytes were generated in recipient mice compared to those that received standard grafts containing mixed cell populations.  This study will open the way to changing how transplantations are performed between a donor and recipient in the future.  The implications of the study are that grafts of pure blood forming stem cells are safer and perhaps even more effective in regenerating a working immune system after transplant compared with standard bone marrow grafts, the latter which have been used for decades.  
    In addition to their work in mice, Drs. Mueller and Shizuru set out to determine the fate of women with metastatic breast cancer who were transplanted at Stanford in the late 1990’s with their own cancer-free purified blood forming stem cells.  They found that the Stanford patients had much better long-term outcomes than expected as compared to metastatic breast cancer patients that received transplantations outside of this study or those treated in any other way.  This follow-up study, which was published in 2011, is controversial because many believe that transplants for breast cancer “do not work”.  As a result of this thinking, transplants are no longer offered for this disease.  However, the unique feature of the Stanford study was that the patients received grafts that were cleaned free of cancer cells by the their cell separation technology.  The profound difference in longevity of this patient group as compared to current expectations of survival of women with this disease has convinced the Stanford team to open a new study for metastatic breast cancer.    

Dr. Berl R. Oakley - University of Kansas / BioScience Department / Irving S. Johnson Distinguished Professor

Background: One of the earliest signs of Alzheimer’s disease is memory loss, followed by difficulties in planning, problem
solving, speech and vision that ultimately result in an inability to function in daily life. As these symptoms appear and become worse over time, they are accompanied by a change in the brain. Many of the brain cells, or neurons, stop functioning properly and begin to die.  

While  we still do not know with complete certainty what causes the cells to malfunction, there is an accumulation  of abnormal  proteinsthat coincides with the areas of the brain that are damaged .  
One of the abnormal proteins is named “tau” and it helps neurons maintain their shape and function. In Alzheimer’s disease, tau begins to change its shape and aggregate into fibers that resemble spider silk, which are as strong as, or stronger than, many industrial fibers. There is considerable evidence that tau aggregates or tangles are an important component of Alzheimer’s pathology and there are a number of other neurological disorders associated with tau aggregation. Our goal is to find drugs that prevent the formation of tau fibers and aggregates or even dissolve them. We have found that the fungus Aspergillus nidulans produces a number of compounds that are members of, or related to, a class of compounds, the anthraquinones, some of which inhibit tau aggregation and break down pre-formed aggregates in vitro. There are three labs involved in this effort. The Oakley lab at the University of Kansas is creating strains of Aspergillus nidulans that accumulate compounds predicted to inhibit tau aggregation. The lab of Dr. Clay Wang at the University of Southern California is identifying and purifying the compounds and the lab of Dr. Chris Gamblin at the University of Kansas is testing the purified compounds for their ability to inhibit tau aggregation or to disassemble tau aggregates.

    Since the last report in May, the Oakley lab has created a set of deletions in the genes of the sterigmatocystin gene cluster. Sterigmatocystin is a serious toxin and would not be usable as an anti-tau compound but the sterigmatocystin biosynthetic pathway is very long and many intermediate compounds, that may be active against tau, are produced on the way to the final product. Normally each compound is converted rapidly to the next compound in the pathway and the intermediates do not accumulate in useful amounts. We wished to devise a way to accumulate potentially useful intermediates. Enzymes encoded by a cluster of genes catalyze the steps of the sterigmatocystin biosynthetic pathway. By individually deleting genes in the cluster, we hoped to be able to block the pathway at specific points such that intermediates accumulate in useful quantities. Dr. Ruth Entwistle, supported by this grant, has deleted 20 genes in the sterigmatocystin gene cluster and sent the deletion strains to the Wang lab for analysis. As hoped and anticipated, many of the strains accumulate intermediates. Dr. Wang’s lab will next scale up production to produce compounds in sufficient quantities to allow them to be used for screening  
    As of our last report in May, Dr. Wang’s lab had purified ten compounds from strains sent to them by the Oakley lab. Dr. Gamblin had tested them for efficacy against tau aggregation in vitro using electron microscopy to visualize tau fibers and aggregates. Six of the compounds significantly altered tau fiber formation and three significantly reduced the overall amount of fiber formation. One compound, chrysophanol, consistently resulted in greatly diminished tau aggregation. Dr. Gamblin has been developing another assay to verify these data and to allow more rapid and easily quantified screening. So far this assay has been difficult to standardize and making this assay robust and reliable is a priority in coming months  
    Goals for the coming year  
Oakley lab—create additional strains that will produce potential anti-tau compounds.
Wang lab—analyze strains produced by the Oakley lab and produce assayable quantities of compounds.
Gamblin lab—perfect assay for anti-tau compounds and screen additional compounds  

Dr. Karl Deisseroth - Howard Hughes Medical Institute - Department of Bioengineering
Department of Psychiatry and Behavioral Sciences
Stanford University      

Karl Deisseroth was elected to the National Academy of Science in October. His research continues at a rapid pace. There are 40 scientists working in his laboratory, and approximately 2000 labs worldwide using the optogenetic techniques he originated just 7 years ago. Optogenetics is a technology that allows targeted, fast control of precisely defined events in biological systems as complex as freely moving mammals. By  
    delivering optical control at the speed (millisecond- scale) and with the precision (cell type–specific) required for biological processing, optogenetic approaches have opened new landscapes for the study of biology, both in health and disease. The challenges faced today in the study of diverse intact biological systems conceptually parallel the challenge faced by neuroanatomy more than a hundred years ago, with the common theme being the need to link information across spatial scales. At that time in history, microscopy had defined small cellular elements of the brain and large architectonic divisions, but the broad mesoscale of cellular connections in intact neuronal circuitry was largely inaccessible until Ramón y Cajal and his students and colleagues used the Golgi technology to systematically map local circuit relationships with cellular precision yet still within the intact system. Inferences from even these descriptive anatomical methods still reverberate through neuroscience. Optogenetics has targeted the analogous need for causal control of defined small-scale events occurring in specified cellular populations while these populations still remain embedded and functioning within larger intact-tissue systems, at appropriate spatial and temporal resolution and under normal or pathological conditions.  
    Since the H. L. Snyder Medical Foundation started supporting Dr. Deisseroth research in 2004, he has had 68 publications. Moreover, his Optogenetics method was awarded “Method of the Year” in 2010.  
    For an excellent presentation of what his work entails, see the      You Tube presentation at the website: