Proteolytic pathways are prominent in virtually all human diseases and are common hallmarks of basic cellular functions ranging from differentiation to death. The long-term goal of this proposal is to develop a novel, rapid, quantitative platform to monitor the proteolytic activity associated with apoptosis in patients undergoing chemotherapy for hematologic malignancies. We hypothesize that products of caspase proteolysis are released from dying apoptotic cancer cells into the circulation and that a subset of these markers will be cell type-specific, and therefore malignancy-specific. A serum biomarker approach to monitoring chemotherapy represents a paradigm shift in clinical management of cancer, which mostly relies upon radiographic imaging and laboratory tests. Because these studies are typically performed weeks to months after the start of therapy, unnecessary or ineffective chemotherapy is often administered. Since current methods to identify, detect, and quantify proteolytic fragments are cumbersome, low-throughput, and costly, we propose to develop a platform that involves a hypothesis-driven discovery method, novel neo-epitope targeted antibodies, and a new multiplexed quantification method. To identify these apoptotic biomarkers, we will develop targeted mass spectrometric methods for identifying proteolysis products created by caspase activity upon induction of apoptosis in cell culture models of hematologic malignancies and plasma of patients undergoing chemotherapy for these cancers (Aim 1). This approach permits an unprecedented large-scale targeted way to detect and stratify apoptotic biomarkers in serum that can be used to monitor treatment of patients undergoing chemotherapy. However, the current lack of reliable and high-throughput methods to measure proteolytically- derived biomarker levels in plasma prevents such a real-time strategy and thus, we will develop antibodies specifically directed against the neo-epitopes of proteins cleaved by caspases during apoptosis (Aim 2). The phage-based methods developed here will enable quantification of apoptotic biomarkers in a multi-parameter fashion and transform the laborious and often uncertain process of generating monoclonal antibodies with high affinities and specificities into a well-defined, cheap, and renewable one. Finally, we will develop unique methods for biomarker quantification, including a novel highly multiplexed phage and next generation sequencing assay (PHANGS) (Aim 3). Application of these methods in a pilot study to correlate biomarker levels to treatment response in patients with diffuse large B-cell lymphoma and multiple myeloma will provide essential groundwork to further biomarker validation studies. These approaches and methods have vast biomedical applications, including biological imaging and detection of proteolysis in single cells and animals as well as novel therapeutic uses of neo-epitope antibodies. Furthermore, while focused on hematologic malignancies, these tools and technologies will significantly elucidate the crucial role of proteolysis in a spectrum of human diseases, such as cancer, infectious disease, and neurodegeneration.

Public Health Relevance

The proposed study will result in the development of a serum biomarker approach to permit rapid and real-time monitoring of chemotherapeutic response and efficacy that will represent a significant advance in clinical management of cancer. Furthermore, the novel technologies developed here will significantly advance the study of proteolysis in other human diseases.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA154802-04
Application #
8608498
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Jessup, John M
Project Start
2011-02-01
Project End
2015-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
4
Fiscal Year
2014
Total Cost
$288,529
Indirect Cost
$101,779
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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Wiita, Arun P; Seaman, Julia E; Wells, James A (2014) Global analysis of cellular proteolysis by selective enzymatic labeling of protein N-termini. Methods Enzymol 544:327-58
Wiita, Arun P; Hsu, Gerald W; Lu, Chuanyi M et al. (2014) Circulating proteolytic signatures of chemotherapy-induced cell death in humans discovered by N-terminal labeling. Proc Natl Acad Sci U S A 111:7594-9
Koerber, James T; Thomsen, Nathan D; Hannigan, Brett T et al. (2013) Nature-inspired design of motif-specific antibody scaffolds. Nat Biotechnol 31:916-21