2001 — 2004 |
Zhou, Pengbo |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) R33Activity Code Description: The R33 award is to provide a second phase for the support for innovative exploratory and development research activities initiated under the R21 mechanism. Although only R21 awardees are generally eligible to apply for R33 support, specific program initiatives may establish eligibility criteria under which applications could be accepted from applicants demonstrating progress equivalent to that expected under R33. |
Protein Knockout Technology/Molecular Analysis of Cancer @ Weill Medical College of Cornell Univ
DESCRIPTION: (provided by applicant) The ubiquitin-proteasome pathway is a major cellular proteolysis machinery that selectively targets cellular proteins for degradation. Protein knockout, a new technology we recently developed, harnesses the specificity of the ubiquitin proteolytic system to direct the degradation of otherwise stable cellular proteins. The long-range goal of this research plan is to apply the protein knockout system as a rapid molecular analysis tool to decipher the function of cellular oncoproteins and to validate their potential use as drug targets. The objective of this application is to improve the efficiency and specificity of the protein knockout system and to assess its efficacy in the proteolytic removal of the overexpressed c-myc oncoprotein in established cell culture and animal models for leukemogenesis. The rationale is that recognition of specific cellular proteins by the engineered substrate receptors of the ubiquitination machinery allows for their degradation by the ubiquitin-proteasome pathway. The research plan has been formulated on the basis of strong preliminary data, and on recent studies by many laboratories including our own. We are uniquely prepared to undertake the proposed research, because we have strong preliminary data demonstrating the efficacy of the protein knockout technology in the selective degradation of stable cellular proteins. The objective of the application will be accomplished by pursuing the following specific aims: (1) R21 phase: To improve the efficiency, specificity and delivery of the protein knockout system. (2) R33 phase: To evaluate targeted c-myc degradation in the inhibition of oncogenic transformation in established cell culture systems. (3) R33 phase: To antagonize myc-mediated tumorigenecity by protein knockout in mouse models for leukemogenesis. The proposed work is innovative, because it capitalizes on the recently identified properties of the ubiquitination machinery by our group and by others. It is our expectation that this approach will offer an efficient means to downregulate the c-myc oncoprotein at the protein level and to inhibit c-myc-mediated neoplastic transformation. These results are significant, because they are expected to provide a comprehensive evaluation of the protein knockout technology as a simple and cost effective molecular analysis tool to elucidate the function of cancer-related proteins and genetic pathways, and to validate their potential use as targets for therapeutic intervention.
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1 |
2003 — 2013 |
Zhou, Pengbo |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Regulation of Nucleotide Excision Repair by Proteolysis @ Weill Medical College of Cornell Univ
DESCRIPTION (provided by applicant): Nucleotide excision repair (NER) plays an important role in maintaining genomic integrity through removing helix-distorting DNA damages caused by UV irradiation or chemical mutagens. Defects in NER underlie the human hereditary disease, xeroderma pigmentosum, which is characterized as sensitivity to ultraviolet light and a high incidence of skin cancer. Studies of NER have been focused on identifying components and the biochemistry of excision and repair reactions, while little is known about the regulatory mechanisms cells employ to control the NER activity. The Cullin 4A ubiquitination machinery has recently been shown to mediate ubiquitin-dependent proteolysis of the p48 subunit of damaged DNA binding proteins that are believed to participate in the initial DNA damage recognition step of NER. Our long-range goal is to understand how the ubiquitin-proteolytic pathway regulates NER, and to relate this understanding to human diseases associated with defective NER as well as the malfunctions of the ubiquitination machinery. The objective of this application is to understand the molecular basis, the regulatory pathways, and the functional significance of CUL-4A-mediated p48 degradation in controlling the damage-sensing step of nucleotide excision repair. The central hypothesis of the application is that the CUL-4A ubiquitination machinery controls the ability of the NER machinery to recognize and remove specific DNA damages through restricting the abundance of p48. The specific aims proposed are (1) To determine the molecular basis for CUL- 4A/DDB interactions and for subcellular distribution of DDB proteins. (2) To determine the functional significance of CUL-4A in DNA damage recognition and repair. (3) To assess the role of c-Abl in regulating p48 degradation and nucleotide excision repair. The results of this work will provide a new paradigm for the regulation of nucleotide excision repair by ubiquitin-dependent proteolysis, and generate a better understanding of the biochemical mechanisms controlling the intracellular distribution and abundance of DDB proteins. Completion of the proposed studies will also shed light on how abnormal activation of CUL- 4A contributes to tumor development.
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1 |
2006 — 2010 |
Zhou, Pengbo |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Ubiquitin-Proteolytic Control of Hoxa9 in Leukemogenesis @ Weill Medical Coll of Cornell Univ
The HOX homeodomain (HD) proteins are key regulators of hematopoiesis. Aberrant expression or chromosomal translocations involving certain HOX proteins, such as HOXA9, have been implicated in the pathogenesis of acute myeloid leukemia (AML). Our understanding of how the intracellular levels and activities of the HOX proteins are controlled during hematopoiesis is current limited to transcriptional regulation and signal transduction. Little isknown about the posttranslational control mechanisms that govern the abundance of the HOX hematopoietic regulators and the functional significance for such regulations. The long-term goal of this study is to understand how ubiquitin- dependent protein degradationregulates normal and malignant hematopoiesis. The central hypothesis of the application is that the cullin 4A (CUL-4A) ubiquitin-ligase controls hematopoietic development throughtargeted degradation of key hematopoietic regulators. The hypothesis has been formulated on the basis of strong preliminary data, which demonstratedthat CUL-4A targets HOXA9 for degradation, and regulates myeloid differentiationand maturation. Hematopoietic-specific knockout of the CUL-4A gene in mice led to an increased expansion of bone marrow progenitor cells and peripheral blood leukocytes. This proposal seeks to determine the biochemical mechanisms underlying the CUL-4A-dependent proteolytic control of HOXA9 and the chromosomal translocation- derived NUP98-HOXA9 fusion, and to elucidate the functional significance of CUL-4A in suppressing leukemic transformation. We are uniquely prepared to undertake the proposed research, since we have recently generateda CUL-4A-resistant HOXA9 mutant, and developed conditional CUL-4A knockout mice and CUL-4A siRNA to eliminate or modulate CUL-4A activity. We have also optimized lentiviral- and retroviral-based gene delivery systemsfor efficient transduction in primary hematopoietic stem and progenitor cells. We propose to combine the biochemical and molecular genetic approachesin Dr. Pengbo Zhou's lab and the expertise in ex vivo and in vivo hematopoieticanalysis in Dr. Malcolm Moore's lab to address the following specific aims: (1) to define the biochemical mechanisms underlying CUL~4A-dependent ubiquitination and degradation of HOXA9. (2) to elucidate the functional significance of HOXA9 degradation by CUL-4A in the pathogenesis of AML. (3) to determine the molecular basis for CUL-4A resistanceby the leukemogenic NUP98-HOXA9fusion and to assess the impact of CUL-4A ablation in the mouse model of NUP98- HOXA9-induced leukemia. Since little information is available regarding the roles of protein degradation during leukemogenesis, successful completion of this proposal will represent a significant advance in understanding a novel posttranslational mechanism that governs the functions of key hematopoietic regulators, and provide a frameworkfor future investigations of targeted protein degradation in normal and malignant hematopoiesis.
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1 |
2011 |
Zhou, Pengbo |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Development of High Throughput Assays to Identify Small Molecule Inhibitors of Th @ Weill Medical Coll of Cornell Univ
DESCRIPTION (provided by applicant): The CUL4A ubiquitin ligase gene is frequently amplified or overexpressed in a wide variety of tumor types, including breast cancer, hepatocellular carcinomas, mesotheliomas, and squamous cell carcinomas. While recent studies have identified the components of the multimeric CUL4A E3 ligase complex and several cellular targets, the role of CUL4A in tumorigenesis remains largely elusive. We and others showed that CUL4A plays an inhibitory role in nucleotide excision repair through targeted degradation of the DDB2 and XPC DNA damage sensors, as well as the G1/S DNA damage checkpoint protein p21. Importantly, our skin-specific CUL4A knockout mice are hyper- resistant to UV- and chemical carcinogen-induced skin cancer, and are otherwise healthy and display no abnormalities, suggesting that CUL4A may be an attractive target for cancer prevention. The primary objective is to develop and optimize a high- throughput screening assay to identify small molecule inhibitors of the CUL4A ubiquitin ligase from chemical libraries at the NIH Chemical Genomic Center (NCGC). This application is built upon the extensive biochemical, cell biological, and genetic characterization of the CUL4A ubiquitin ligase performed in Dr. Pengbo Zhou's lab during the last 10 years, and the comprehensive array of in vitro and cell-based assays as well as the mouse knockout models to examine CUL4A-DDB1 functions. The studies are complemented by the extensive high-throughput screening expertise of Dr. Yueming Li's group at Memorial Sloan-Kettering Cancer and Dr. Wei Zheng's group at the NIH Chemical Genomic Center. We are uniquely positioned to pursue the following two specific aims: (1) to develop and optimize an AlphaLISA(R)-based HTS assay for CUL4A- DDB1 interaction;(2) to configure secondary HTS assays required to evaluate the hit compounds. Successful completion of the proposed studies will set the stage for application of the CUL4A-DDB1(BPB) AlphaLISA(R) assay on the HTS platform to screen the NCGC 300K chemical libraries and identify small molecule inhibitors of the CUL4A- DDB1 ubiquitin ligase. These studies represent the first step towards evaluating the efficacy of pharmacological CUL4A inhibition as an effective approach for cancer prevention and intervention. PUBLIC HEALTH RELEVANCE: The CUL4A gene is frequently found amplified or overexpressed in a wide variety of tumor types, including breast cancer, hepatocellulat carcinomas, mesotheliomas and squamous cell carcinomas. Recent studies suggest that abrogation of CUL4A presents significant benefits in enhancing DNA repair and DNA damage response, and protects against carcinogenesis. However, there is currently no pharmacological agent available for blocking the CUL4A ubiquitin ligase activity. Our overall goal is to develop pharmacological inhibitors against the DDB-CUL4A (or CRL4) ubiquitin ligase as anticancer agents. Here we propose to take the critical first step to develop and optimize a high throughput screening assay and to establish secondary validation assays, with the aim of implementing a high throughput screening of large chemical libraries to identify small molecule inhibitors of the CUL4A ubiquitin ligase. This R21 project expected to set the stage for developing pharmacological agents that selectively target CUL4A as novel cancer prevention and/or therapeutic agents.
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0.976 |
2011 — 2015 |
Shin, Sandra Zhou, Pengbo |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Tumorigenic Role of the Cul4a Ubiquitin Ligase @ Weill Medical Coll of Cornell Univ
Abstract The CUL4A ubiquitin ligase gene is frequently amplified and/or overexpressed in breast cancer, hepatocellular carcinoma, mesothelioma and many other tumor types. CUL4A is also a direct transcriptional target of Wnt signaling, which is aberrantly activated in up to 63% of all breast cancers. Recent studies identified multiple substrates and cellular pathways that are subjected to ubiquitin-proteolytic control by CUL4A and are implicated in oncogenic transformation. However, the role of CUL4A in tumorigenesis remains unclear, and this knowledge gap is largely due to the lack of in vivo tumor models in which CUL4A expression can be manipulated in a temporal and tissue-specific manner during the course of tumor development. This proposal seeks to definitively determine the pathophysiological role of CUL4A in mammary tumorigenesis, and begins to dissect the specific ubiquitin-proteolytic events that underlie tumor initiation, maintenance, and resistance to camptothecin-type chemotherapy drugs. We are uniquely positioned to perform the proposed studies, as (1) we recently generated conditional CUL4A knockout mice that will overcome the limitations of unavailable experimental models, (2) we have at our disposal a well-established breast tumor bank with over 3,000 specimens, including over 250 triple negative breast cancer (TNBC) cases (CUL4A amplification was detected in ~20% TNBCs.), and patient clinico-pathological information, and (3) we discovered a novel function of the Cockayne syndrome A (CSA) DNA repair protein in CUL4 (CUL4A and its family member CUL4B)- dependent degradation of topoisomerase 1 (TOP1), implicating CUL4 dysregulation as a mechanism for resistance of tumor patients to TOP1-directed chemotherapy (e.g. camptothecin). We plan to test the hypothesis that CUL4A dysregulation promotes tumorigenesis and confers resistance to chemotherapy by pursuing two specific aims: (1) Determine the role and mechanistic basis of CUL4A in mammary tumor development and maintenance; (2) Delineate the mechanistic role of CUL4 dysregulation in tumor resistance to topoisomerase I inhibitor chemotherapy drugs. Successful completion of the proposed studies is anticipated to definitively establish the role of CUL4A- mediated ubiquitination in the pathogenesis of breast cancer, and shed light on the resistance of certain tumors to chemotherapy by camptothecin-type drugs. The evaluation of the efficacy of CUL4 inhibition using a combination of cell culture, mouse models and human breast cancer specimens is expected to facilitate the development of new and effective therapeutic strategies against breast cancer and other tumor types with dysregulated CUL4A expression, such as those with aberrant activation of Wnt signaling.
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0.976 |
2017 — 2021 |
Zhou, Pengbo |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Harnessing the Crl4 Ubiquitin Ligase For Antagonizing Colorectal Carcinogenesis @ Weill Medical Coll of Cornell Univ
Colorectal cancer (CRC) is the 4th most common cancer and the 2nd leading cause of cancer?related deaths in the United States. The American Cancer Society estimates that 134,490 people will be diagnosed and 49,190 will die from CRC in 2016. Although biologic agents have received much attention, the first-line chemotherapy for treatment of metastatic CRC remains based on a cytotoxic combination chemotherapy backbone of either FOLFIRI (folinic acid+5-FU+irinotecan) or FOLFOX (folinic acid+5-FU+oxilaplatin). It is noteworthy that only 18-25% of CRC patients respond favorably to irinotecan. Although the FDA-approved irinotecan has been used in clinic for 20 years, there is still no diagnostic biomarker to help oncologists decide which of the two regimens is more likely to be effective for a given CRC patient. Our studies suggest that the CULLIN 4B (CUL4B) ubiquitin ligase is a promising predictive biomarker, as well as a therapeutic target for tumor response to irinotecan. The CUL4B gene has been found amplified or overexpressed in a wide range of solid tumors, including colorectal, breast, lung, ovarian, and prostate cancers. Upon treatment with the camptothecin family of chemotherapy drugs, CUL4B, but not its paralog CUL4A, targets topoisomerase I (Top1) for degradation in cancer cells. As a result, CRCs with high levels of CUL4B expression induce excessive destruction of Top1, effectively attenuating the cytotoxicity of this chemotherapy drug. We hypothesize that excessive Top1 degradation by CUL4B is a major mechanism by which CRCs become refractory to irinotecan. Importantly, we showed that inactivation of CUL4B, but not CUL4A, effectively sensitized irinotecan-resistant CUL4Bhigh tumors to cytotoxic killing by Top1-directed chemotherapy drugs. This data suggest that CUL4B is an attractive target for intervention, potentially leading to sensitization of the 75-82% CRC patients who were previously resistant to treatment with a Top1-directed chemotherapeutic agent (e.g. FOLFIRI regimen). In three specific aims, we will (1) assess the value of CUL4B as a biomarker for treatment decisions of CRC using our unique collection of 572 well annotated CRC patient samples treated with irinotecan or FOLFIRI; (2) further develop and validate potent small molecule CUL4B inhibitors we identified via high throughput screening to increase their potency and pharmacological properties; (3) examine our lead CUL4B inhibitors that synergize with irinotecan to enhance its tumoricidal activity in vivo using CRC cell line xenografts, genetic WNT/APC-induced intestinal and colon adenomas, and patient-derived tumor xenograft (PDTX) models of CRC. We are uniquely prepared for both Laboratory-to-Clinic studies to develop a predictive biomarker for irinotecan-based chemotherapy that is immediately applicable for informing decision- making of CRC management in the clinic, and Clinic-to-Laboratory studies aimed at validating CUL4B as a feasible drug target in pre-clinical CRC models, and developing a new therapeutic agent to render CUL4Bhigh CRC patients, which represent approximately 75% of all CRCs, sensitive to irinotecan-based therapy.
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0.976 |
2018 — 2021 |
Zhou, Pengbo |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
G3bp1 Suppresses Spop Ubiquitin Ligase to Promote Prostate Tumorigenesis @ Weill Medical Coll of Cornell Univ
Prostate cancer (PCa) accounts for an estimated 26,120 deaths in 2016 representing the second-greatest cause of cancer death among men. Recent molecular characterization of PCa has revealed striking genomic heterogeneity and defined distinct molecular subclasses that may provide insight to the variable clinical course. Recurrent mutations in SPOP are the most common point mutations in prostate cancer, occurring in about 10% of patients across early and advanced disease. SPOP is a substrate receptor of the Cullin 3-based ubiquitin ligase, which recruits androgen receptor (AR), TRIM24 and SRC-3 and other key regulators for ubiquitination and degradation, thereby governing the threshold of AR transcription, DNA damage repair, and tumor suppression. SPOPmut defines a distinct molecular class of prostate cancer characterized by activation of AR signaling and impairment of DNA double strand break (DSB) repair with transcriptional response (gene set signature) similar to that of BRCA1 inactivation. We have interrogated the TCGA RNA-seq datasets on primary (untreated) prostate cancer to define a SPOPmut transcriptional signature of 213 differentially expressed genes, and validated in independent RNA-seq cohorts for significant enrichment of this gene set in multiple cohorts of SPOPmut cases. Surprisingly, we also detected primary prostate tumors without mutation of the SPOP gene, yet showing the same gene expression characteristics to the SPOP mutant subclass, indicating that other events can phenocopy SPOP mutations in these primary ?SPOPmut-like? PCa. To further understand the function and regulation of SPOP in prostate tumorigenesis, we performed tandem affinity purification coupled with mass spectrometry analysis, and identified G3BP1 as a novel SPOP- binding protein. G3BP1 is not a substrate of SPOP, but acts as a potent inhibitor of the SPOP ubiquitin ligase. This first-in-kind SPOP inhibitor revealed previously unrecognized means of SPOP inactivation that is independent of PCa-associated SPOP gene mutations. Importantly, we detected abnormally high levels of G3BP1 in PCa either with or without SPOP mutations. We hypothesize that dysregulation of G3BP1 defines a new subclass of prostate cancer with SPOPmut-like molecular signature, pathophysiological characteristics, and sensitivity to AR- and PARP targeting therapeutics. This application is built upon the unique and complementary strengths and resources of a team of investigators in prostate cancer genomics and pathology (Rubin), molecular classification, clinical management and precision therapy of PCa patients (Barbieri), computational genomics (Sboner), and cullin-based ubiquitin ligases (Zhou). In this proposal, we will 1) determine the clinical value of G3BP1 dysregulation in PCa classification and potential therapeutic implications, 2) determine the biochemical mechanisms underlying SPOP inhibition by G3BP1 and the roles of this newly discovered G3BP1-SPOP ubiquitin signaling pathway in AR transcription, DNA repair and prostate tumorigenesis, and 3) assess the susceptibility of G3BP1high PCa to AR- and PARP-targeting therapies.
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0.976 |