David Frederick Stern - US grants

Many oncogenes act by disrupting pathways regulated by peptide growth factors. These oncogenes transform cells by causing inappropriate production of growth factors or by encoding mutated growth factor receptors. These mechanisms for transformation will be investigated using two different experimental systems. The neu oncogene will be studied to determine the mechanism of activation of an oncogenic growth factor receptor. A model autocrine system, in which cells are transformed because they constitutively produce EGF, will be used to develop methods for reverting transformation mediated by autocrine circuits and to identify genes that cooperate with the EGF gene in cell transformation. The neu oncogene encodes a protein designated p185 that is closely related to, but distinct from, the receptor for EGF. The product of the neu proto-oncogene is itself likely to be a growth factor receptor. Because normal and transforming forms of p185 differ by only a single amino acid replacement, this system is ideal for investigating the mechanism of oncogenic activation of a growth factor receptor. The effects of the activating mutation on activity and specificity of the p185 kinase, sites of phosphorylation of p185, and oligomerization will be examined. These studies will yield insights into the mechanism of signal transduction by the tyrosine kinase family of growth factor receptors. Cells expressing a transfected EGF gene at high levels are tumorigenic but differ from ras-transformed cells in being anchorage-dependent and growing only slowly as tumors. These partially transformed cells will be used to study multi-step carcinogenesis in which one of the affected genes induces production of EGF-like factors. Genes that are known to act synergistically with EGF in stimulating cell proliferation will be tested for the ability to cooperate with EGF in tumorigenesis. Transformation by the EGF expression plasmid is mediated by an extracellular autocrine circuit that can be interrupted with an anti-EGF monoclonal antibody. EGF-transformed cells will be used as a model therapeutic system for tumors mediated by autocrine circuits. These cells will be used to test the efficacy of anti-EGF antibodies, anti-EGF receptor antibodies, and EGF antagonists in blocking tumorigenicity.

[unreadable] DESCRIPTION (provided by applicant): The gene encoding the receptor tyrosine kinase (RTK) HER2/neu/ErbB2 is an important human breast cancer oncogene, and a validated therapeutic target. We hypothesize that optimal development and use of ErbB2-targeted therapeutics will require a thorough understanding of the normal biological functions of ErbB2 and its regulation by interactions with other ErbB family receptors. Aim 1 continues our analysis of normal functions of ErbB2 and ErbBS in mammary gland development. Aim 2 investigates a new hypothesis in which excessive signaling through ErbB2 drives genomic instability in breast cancer through actuation of DNA checkpoint signaling and selection for checkpoint bypass. Aim 3 will evaluate phosphorylation markers for measuring for ErbB and pathway activation in human cancer n the best-case setting of core biopsies. The significance of these studies extends well beyond breast cancer, since the Epidermal Growth Factor Receptor (EGFR) and ErbB2 are mutated or overexpressed in many types of adenocarcinoma. Since ErbBs are forefront targets for new RTK inhibitors in cancer treatment, success of our efforts to merge ErbB biology with tumor studies will pave the way for similar approaches to cancers caused by other RTKs. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The four members of the epidermal growth factor receptor (ErbB) family of hormone receptors are forefront targets for the new generation of FDA-approved rational cancer therapeutics, including Herceptin, Cetuximab, Tykerb, and Tarceva. The first receptor (EGF receptor) is a therapeutic target in two of the greatest human cancer killers: non-small cell lung carcinoma and colon carcinoma. The second receptor, HER2/neu/ErbB2, is a validated therapeutic target in breast cancer. In contrast, the role of the fourth receptor, ErbB4, is uncertain. There are four different forms of this receptor with different structures. Preliminary evidence indicates that each form of ErbB4 has unique functions, with some forms having opposite effects on processes related to cancer. We will determine what the functions of these four ErbB4 receptor forms are, and how this relates to the well-being of cancer patients expressing these forms. The results will have important implications for human cancer treatment, including whether ErbB4 is a reasonable therapeutic target, and may identify unique biological functions for ErbB4 that may be harnessed in cancer therapies. The work will reveal whether there are subsets of patients with strong ErbB4 activity that potentially would be harmed by EGFR and ErbB2-directed therapies. This work will also be important in other human diseases involving ErbB4. Neuregulin-1, one of the hormones that activates ErbB4, may have therapeutic potential in cardiovascular disease, and has been linked to schizophrenia and bipolar disorder. Presenilin-dependent cleavage of ErbB4, may be important in Alzheimer's Disease. Aim 1 will create mouse models for determining the differential effects of the ErbB4 forms on mammary development. This will reveal which of these forms affects which normal processes. Aim 2 will first use the mouse models from Aim 1 to evaluate positive and negative effects on development of breast cancer. Then, we will determine the clinical correlations of excessive expression of these individual ErbB4 isoforms in a set of nearly 1000 human breast cancers with sixteen years patient follow up information. This will directly test the impact of individual isoforms on patient prognosis. Aim 3 will evaluate unique features of cell regulation by ErbB4 forms that act in the cell nucleus. This affords ErbB4 the ability to reprogram major cell activities, some relevant to cancer. These experiments will provide a global view of these functions. PUBLIC HEALTH RELEVANCE: The four different forms of the hormone receptor ErbB4 potentially induce or antagonize cancer, depending on which form is expressed. The focus of this application is on determining the processes regulated by these individual forms, and how this impacts on development of breast cancer and other cancers. Besides the possibility that ErbB4 functions autonomously as an oncogene or tumor suppressor, ErbB4 forms complexes with the related EGF receptor and ErbB2, which are the targets for new drugs that are used in treatment of breast, colon, and lung cancers. Our findings will reveal whether ErbB4 tempers or augments their activities in cancer, with important therapeutic implications. ErbB4 is also important in cardiovascular and nervous systems. Neuregulins, which are hormones that bind and activate ErbB4, may be useful in promoting recovery from cardiac injury. These hormones have also been linked to familial schizophrenia and bipolar disorder. Similarly, polymorphisms in ErbB4 itself have been genetically linked to schizophrenia. Finally, ErbB4 is potentially an important biological target for presenilin cleavage in Alzheimer's disease.

[unreadable] DESCRIPTION (provided by applicant): DNA damage response networks are common targets for carcinogenic mutations and may be excellent targets for response-modifying drugs. The central core of these networks consists of phosphatidyl inositol 3-kinase-related kinases (PIKKs), e.g. human Atm and Atr, which activate downstream pathways in response to DNA damage. PIKKs phosphorylate components of complexes that are recruited to damageinduced foci, including the Mrell/Rad50/Nbsl complex, 53BP1, and Brcal, and activate effector kinases Chkl and Chk2 that amplify the signal and help convey it to effector proteins. In budding yeast, the PIKK Mecl (Atm/Atr ortholog) is coupled to protein kinase Rad53 (Chk2 ortholog) through Mecl-dependent phosphorylation of the "mediator" Rad9 (unrelated to human Rad9). Mammalian BRCA1, 53BP1, and now MDC1/NFBD1 are candidate Rad9 orthologs that resemble Rad9 in having twin carboxyl terminal BRCT domains. Like Rad9, they undergo PIKK-dependent phosphorylation, form damage-induced complexes with other checkpoint proteins and are recruited to sites of DNA damage. MDC1/NFBD1 is intimately involved in DNA damage response mechanisms. The PIKK-Mediator-Chk core of DNA damage signaling will be investigated using both the simpler yeast cascade Mecl to Rad9 to Rad53, and the more complex interactions of ATM/ATR in regulation of NFBD1. [unreadable] [unreadable] Aim 1. A combination of biochemical and molecular methods will be used to determine the mechanism [unreadable] through which the mediator Rad9 and the PIKK Mecl regulate Rad53. [unreadable] [unreadable] Aim 2.. siRNA oligonucleotide knockdowns will be used to identify unique and overlapping functions of BRCA1, 53BP1, and NFBD1 in DNA checkpoint responses. [unreadable] [unreadable] Aim 3. Molecular techniques will be used for functional analysis of NFBD1/MDC1 in the activities [unreadable] identified in Aim 2. [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Recent advances in understanding cancer, combined with unprecedented access to patient DNA sequence data, and new rational therapeutic approaches create the need for a revolution in Ph.D. training of scientists. We have developed a new Cancer Biology Training Program that will take advantage of the high caliber of biologists at Yale, the commitment to multidisciplinary training in the life sciences, and the close collaboration of Yale cancer biologists and clinicians fostered by the Yale Cancer Center (YCC) to provide a unique cancer-focused training experience intended to spawn the next generation of cancer scientific leaders. Training will cover the genetic and biological underpinnings of cancer, the pathway to development of new therapies based upon this knowledge, and the practical challenges in applying these new therapies in cancer clinics. Predoctoral trainees will be trained in foundational biological areas through course work in fundamental areas of biology and physiology and join the program in their second year. Postdoctoral fellows will join the program early in postdoctoral training. All trainees will be spend 90% of their time in laboratory research projects mentored by highly qualified faculty who are leaders in disciplines of cancer research including tumor virology, cancer immunobiology, cancer genetics and epigenetics, stem cell research, pharmacology, and signal transduction. The program will begin by training two new predoctoral and four new postdoctoral trainees each year, with funding for one of the predocs and three of the postdocs to be provided through this T32. For predoctoral trainees, the training experience will include three research rotations and didactic and seminar courses, and continue with qualifying examinations and written dissertation in a projected period of five years overall. Postdoctoral training includes participation in research seminars and talks. Cancer-specific training provided for all trainees by the program will include three formal courses: 1. a general survey class covering basic principles of cancer biology and genetics; 2. a seminar course in which selected topics will be analyzed and discussed in depth, and a clinically-oriented workshop that covers clinical trials, 3. patient treatment patterns and clinical questions for major diseases; and personalized cancer medicine based on tumor resequencing. Every trainee will have a clinical co-mentor to foster exposure to clinical concepts through tumor boards and clinics.

DEVELOPMENTAL RESEARCH PROGRAM: PROJECT SUMMARY The primary goal of the Yale SPORE in Skin Cancer Developmental Research Program (DRP) is to provide limited support (maximum of $50,000/year, typically for no more than two years) for a broad spectrum of innovative skin cancer pilot projects (involving research, resources, and technology development applicable to human skin cancer risk, prevention, diagnosis, prognosis, or treatment). These pilot projects must have promising translational research potential, with anticipation that they can eventually develop into, or be incorporated into, full projects with an unequivocal clinical translational component. Such projects could either be developed into independent external funding at a scope and scale equivalent to a NIH R01 grant, or alternatively augment or replace a Project in future cycles of the Yale SPORE in Skin Cancer. A total of at least $230,000/year will be used for Developmental Research Projects ($56,262 in direct costs and at least $173,738 in institutional matching funds guaranteed by the Yale Cancer Center and the Department of Dermatology). These funds will make it possible to support up to five DRP projects annually, ranging from $30,000 to $50,000 each. A second goal of this DRP is broaden the collective of Yale investigators who are actively engaged in research related to human cutaneous oncology. During its second five-year funding period, the YSPORE DRP has funded 22 different projects (20 related to melanoma, one to cutaneous T cell lymphoma, one to keratoacanthoma) involving 28 different investigators from 15 different departments and sections at Yale, and two outside institutions. Six of the projects involved multiple PIs and encompassed multidisciplinary activities.

CAREER ENHANCEMENT PROGRAM: PROJECT SUMMARY The Career Enhancement Program (CEP) of the Yale SPORE in Skin Cancer will develop a new cadre of investigative cutaneous oncologists and scientists committed to multidisciplinary studies investigating the relevance of biological discoveries in human skin cancer risk, prevention, diagnosis, prognosis or treatment, and enhance the careers of individuals who are already productively investigating this field. $56,262 per year is requested to support CEP, which will be matched with $56,108 from the Yale Cancer Center, to be used for the support of two faculty-level CEP Awards per year. Funding is for one year, but may be renewed for a second year. Potential candidates for these awards include promising junior faculty who are either appointed at Yale or are outside candidates or new recruits for Yale appointment. Alternatively, candidates may be established investigators, either currently at Yale or in the process of being recruited to Yale, with previous research focus in other arenas, but who will re-channel a significant portion of their focus to translational research in cutaneous oncology. All junior faculty awardees are paired with an established investigator in translational cutaneous oncology with a documented record of successful mentoring. During the second five-year funding cycle, YSPORE CEP funds were used to support the career development of seven different investigators including four women. Individuals supported to date have had diverse backgrounds and previous experience; 4 PhDs, 1 MBBS, and 2 MD/PhDs, with experience including an entry level lung carcinoma geneticist refocusing to melanoma research; established clinician scientists enabled to devote significant time to B cell anti- melanoma responses, and a junior clinician-scientist who discovered driver mutations in cutaneous T cell lymphoma.

PROJECT SUMMARY/ABSTRACT: Triple negative breast cancer (TNBC) is an aggressive and difficult to manage disease that disproportionately afflicts underrepresented minority populations. Tumor cellular heterogeneity is a major source of treatment resistance and resilience, but is poorly understood. Thus, better understanding of tumor heterogeneity will lead to development of therapeutic strategies to treat underrepresented minority TNBC patients. The objective of this proposal is to identify and characterize biomarkers of cell subsets that drive tumor resilience in African- American (AA) patients. The central hypothesis is that differential cell population heterogeneity contributes to disparate TNBC prevalence and treatment outcomes in AA and other underrepresented patient groups. The rationale for the proposed research is that better understanding of tumor cellular heterogeneity will result in new and innovative approaches to TNBC treatment for underrepresented minority TNBC patients. Therefore, the hypothesis will be tested by pursuing the following specific aims: Aim 1) Identification of phenotypic states in AA TNBC with single cell RNA-seq; Aim 2) Associations of candidate biomarker sets with AA TNBC, clinical variables, immune phenotypes, genomic information, and outcomes in a TNBC neoadjuvant trial. The proposed research is innovative, because of the novel approach to use single cell profiling to define and understand novel tumor cell lineages and microenvironment for AA TNBC tumors. The proposed research is significant, because it is expected to advance and expand our understanding of tumor heterogeneity and drug resistance in AA TNBC patients. Such knowledge is a critical foundation for the development of cancer therapies targeting vulnerabilities of tumor heterogeneity in AA TNBC patients.

CCM3 is one of mutated genes responsible for the human CCM disease, a pathological condition that affects the vasculature of the central nervous system and results in stroke, seizure and cerebral hemorrhage with a high prevalence. CCM consists of dilated and multiple capillary channels formed by a single layer of endothelium, lacking all other normal vessel wall elements. Patients with inherited autosomal dominant CCM carry loss of function mutations in one of three genes: CCM1, CCM2 and CCM3. Deletion in one of the three Ccm genes in vascular EC induces CCM lesions in mice. However, it is unknown why both humans and mice with CCM3 loss exhibit more severe phenotype than those with loss of CCM1 or CCM2. Our unexpected discovery of the involvement of CCM3 in EC exocytosis, prompt us to hypothesize that alteration in CCM3-regulated EC exocytosis contributes to the pathogenesis of the CCM disease. We propose the following two specific aims: 1) To determine the role of CCM3-regulated brain EC exocytosis in CCM disease phenotypes. We will establish mouse CCM models even closer to human CCM disease by creating brain EC-specific Ccm3 deletion, and determine therapeutic effects of Angpt2 neutralization antibodies in the new CCM3 mouse models. 2) To explore crosstalk of CCM3-mediated EC exocytosis with other pathways implicated in CCM formation. We will test if inhibition of exocytosis in ECs blocks RhoA-dependent EC stress fiber formation, TGF-?/Smad/BMP-mediated EndMT signaling, and MEKK3-ERK5-KLF4-mediated matrix remodeling. Conversely, test if gain- or loss-of-function of RhoA, TGF-? and MEKK3-ERK5-KLF4 signaling regulate EC exocytosis. In summary, the complementary approaches using genetic, cell biological and imaging analyses will facilitate our understanding of the molecular mechanisms and pathogenesis involved in acquisition of cerebral cavernous malformations, and help in defining new and more effective therapies. Our findings should benefit the general understanding of the regulatory mechanisms of exocytosis, which also occurs in other cardiovascular cells and ECs of other cardiovascular organs such as heart, lung and aortae. Therefore, our present study is of broad significance in cardiovascular research.