AACR Anna D. Barker Fellowships in Basic Cancer Research foster basic research by scientists at the beginning of their careers in the cancer field. They are open to postdoctoral and clinical research fellows at an academic facility, teaching hospital or research institution who will be in the first, second or third year of their postdoctoral training at the start of the fellowship term. Fellowships support the salary and benefits of the fellow, with partial funds permitted to be designated to direct research expenses.
2011 GRANTEE
Daniel Schramek, Dr. Rer. Nat.Postdoctoral Fellow, The Rockefeller University, New York City, NY
In vivo RNAi Screen to Identify Regulators of Skin Tumorigenesis
"The identification of genes which are causally linked to human tumorigenesis has greatly improved our understanding of cancer biology and has often guided therapeutic approaches. However, many known oncogenes such as Ras family proteins are hard to target therapeutically, despite the fact that they are mutated in 30 percent of cancers and cancer cells critically depend on mutant Ras (Ras addiction). Due to the oncogenic transformation, cancer cells might also develop secondary dependencies on genes, which are themselves not oncogenes, but are required for viability and proliferation of such cancer cells. Such dependencies might, therefore, open a new therapeutic strategy."
"The ability to genetically manipulate cells using RNA interference has enabled genome-wide loss-of-function screens in cultured tumor cell lines and the identification of such “synthetic lethal interactions”. RNAi was also shown to be a powerful tool in intact animals and even allows for inducible and reversible loss-of-function analysis of single genes. I want to establish techniques to perform such an in vivo RNAi approach not just for a single gene but rather for the whole genome during an oncogenic transformation of a solid tumor within an intact mouse. To dissect the underlying processes involved, I want to target the RNAi to a particular epithelial cell type. However, my ultimate goal is to identify not only cell intrinsic genetic interactions within a given oncogenic network but essential extrinsic factors such as stroma interactions, neo-vascularisation, autocrine, paracrine as well as endocrine factors etc. that impact on the phenotypic manifestations that occur as a consequence of the alterations within the designated epithelial cell. In elucidating this critical pathway in tumor formation, I might also be able to answer some of the unresolved questions of cancer stem cell biology: Are there cancer stem cells (CSC)? How stringent/reversible are CSC → cancer cell transitions? Do differentiated cancer cells regulate CSC? Do CSC determine the kind of tumors? Do markers of normal stem cells mark CSC?"
"I have chosen to use mouse skin as a model system for study, since it offers multiple required features for this project: It is readily accessible for genetic manipulation, imaging and chemical treatments. In addition, the different epidermal lineages are spatially and temporally well-defined (markers for differentiation as well as for stemness), and established Cre lines allow inducible genetic manipulation as well as linage tracing and monitoring early changes. Moreover, adult stem cells for the hair follicle and interfollicular epidermis are well-characterized and can be cultured without losing stemness. Finally, recent technologies make it possible to specifically and efficiently target RNAi to the skin epithelium in vivo, fulfilling the criteria I delineated above."
"Non-melanoma skin cancers are the most common cancers worldwide and can be used as paradigm for many other life-threatening carcinomas such as those of the breast, the pancreas or the lung. Skin cancer affects approximately 3.5 million Americans every year, which is twice the number of all other cases of human cancers (Rogers et al.). Although the mortality is low, the life-time risk of developing non-melanoma skin cancer is approximately 30 per cent and skin cancer, therefore, constitutes a huge money burden on the public health system/Medicare (Bickers et al., 2006). Basal cell carcinoma (BCC) is the most common type of skin cancer making up about 75 percent of all reported cases, whereas squamous cell carcinomas (SCC) is the second most abundant skin cancer making up about 20 percent of all cases."
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2010 GRANTEE
Uddhav K. Shigdel, Ph.D.Postdoctoral Fellow, Harvard University, Cambridge, MA
Crystallization of Type IIA Topoisomerase Using Covalent Cross-Linking
"During cell division, segregation of the newly replicated chromosomes and proper assortment to the two daughter cells is a crucial event. Defects in chromosome segregation may promote the aneuploidy and genomic instability so commonly associated with genetic disease and cancer. A highly conserved class of proteins, type II topoisomerases (TOP2) act to circumvent the problems associated with chromosome catenation that arise during replication. These proteins introduce transient double-strand breaks in DNA, allowing passage of one duplex strand through another; the mechanism of this reaction involves a short-lived intermediate having the enzyme covalently linked to the DNA substrate. The ordinarily fleeting nature of the reaction intermediates has precluded their structural elucidation and, therefore, the structural basis of TOP2 action is poorly understood. TOP2 are indispensable for chromosome segregation in organisms ranging from simple bacteria to yeast to humans, and these enzymes are conserved in all known free-standing organisms.
"Taking advantage of cells’ dependency on topoisomerase, among various treatments, inhibition of TOP2 has shown to be an effective means of treating various cancers. Commonly prescribed TOP2 poisons like anthracyclines used for the treatment of breast cancer are believed to prolong the lifetime of TOP2-DNA cleavage complexes deleterious to the cell. Amid its extensive use as an active anti-breast cancer drug, an important side effect of using TOP2 poisons is the formation of secondary malignancies. Moreover, use of anthracycline has been attributed to cardiotoxicity. It is suggested that TOP2b (humans have two TOP2 isoforms termed a and b) might be the isoform, targeted by TOP2-targeting drugs, that is causing secondary malignancies as well as cardiotoxicity. Hence there might be considerable benefit in developing TOP2-targeting drugs that are specific for the TOP2a isoform that might reduce untoward effects in humans. None of the TOP2 targeting drugs that are clinically used were identified on the basis of their activity against TOP2 but rather on the basis of their anti-tumor activity. Thus currently used TOP2-targeting agents have poor potency (Ki of etoposide for TOP2 is in the range of 5-20mM) compared to other enzyme inhibitors which might be contributing to their toxic effects.
"Considering the side effects and the poor potency, the need for better TOP2-targeting drugs is immediate. In order to identify TOP2-targeting drugs that possess maximum efficacy and minimum secondary malignancy and other toxicities, determination of a ternary complex between drug, protein and DNA is a must. Crystal structure of the breakage reunion domain of TOP2 bound to DNA has been solved, but there is not a single three-dimensional structure of a drug-DNA-TOP2 ternary complex that would be valuable in understanding drug action. Despite the important role of this protein in cellular function - in particular, chromosome segregation whose dysfunction can cause cancer - little is known about the structural aspects of the elementary chemical reactions these enzymes perform during DNA cleavage and ligation. To understand how drugs such as anthracyclines interact with DNA-TOP2 as well as the elementary chemical reactions TOP2 perform during the DNA-gate opening and closing, I propose to use mechanism-based and structure-based crosslinking methods to covalently trap TOP2 at a state representing the covalent enzyme-DNA intermediate in presence of TOP2-interacting drugs. The resulting complexes will be characterized structurally. The structure of TOP2 bound to DNA will help elucidate the molecular mechanism employed by these fascinating enzymes, and will also guide us one step closer in designing better drugs that possess maximum efficacy and minimum secondary malignancy and other toxicities that will cure various cancers including breast cancer.
"Cancer as a disease is universal; it cares neither about nationality nor race. It takes thousands of lives every year; thus a collective effort from all areas of science is required to curb its metastasis. Coming from a country like Nepal where majority of people do not even know what cancer is, receiving the
Anna D. Barker Fellowship in Basic Cancer Research from the American Association for Cancer Research is truly an honor. To be part of the AACR community is of utmost significance to further augment my cancer biology knowledge for a future career in academia."
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