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Principal Investigator: JAMES A WELLS
Organization: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
Fiscal Year: 2024
Award: $345,184
Funding agency: National Cancer Institute
Project Summary
Extracellular proteolysis is a hallmark of cancer. Proteolysis has major functional consequences for
remodeling cell surface proteins and matrix including receptor activation and shedding of receptor
extracellular domains. Of the more than 600 human proteases, over half are on the cell surface or
secreted reflecting their significance for processing the extracellular space. Yet we understand very
little about the targets for cell surface proteolysis (CSP), the proteases responsible, and resulting neo-
cleaved products that could serve as new biomarkers or drug targets.
What has been missing are robust technologies for unbiased identification of the CSP targets,
their specific sites of cleavage, the proteases responsible for promoting disease, and the generation of
neo-epitope specific antibodies. My group has engineered a novel peptide ligase, called subtiligase,
and shown it robustly labels sites of proteolysis of soluble proteins. Our preliminary data show that this
powerful tool can be used to determine the targets and precise sites of cleavage for CSP. We propose
this novel CSP technology can be used to determine CSP signatures in cancer. We will initially focus
on KRAS, the most dominant oncogene, especially in pancreatic cancer. We will determine KRAS-
induced proteolysis in isogenic pancreatic cell lines, with and without mutant KRAS, grown in hypoxic
and normoxic conditions in the context of two- and three-dimensional culture. We will identify the
surface proteases up-regulated using cell surface capture (CSC) proteomics and confirm their activities
using CRISPRi or small molecule inhibitors. We will characterize their specificities using new substrate
phage technology coupled with NGS and match these to neo-epitopes we discover. Lastly, we will
further exploit differential antibody phage display selection technology, and generate antibodies specific
to the cleaved forms of various identified targets, including CDCP1. We will develop important
technologies for new neo-epitope biomarkers and potential immune-oncology reagents for the
characterization of these important cleavage events.
Impact: Our studies will provide robust protein engineering-based technologies for unbiased
insights into how CSP remodels cells, the proteases responsible, and actionable oncogene specific
neo-epitopes to generate antibodies as potential biomarkers and therapeutics. We believe these
approaches will be generally applicable to characterizing proteolysis in other solid tumor cancers.
Terms: <2-dimensional><3-D><3-Dimensional><3D><Antibodies><Antibody Affinity><Bacteriophages><Binding><Biological Markers><Breast Cancer><C-K-RAS><CRISPR interference><CRISPR-dCas9-mediated repression><CRISPR/dCas9 interference><CRISPR/dCas9-mediated transcriptional inhibition><CRISPRi><Cancer Genes><Cancer cell line><Cancer-Promoting Gene><Cancers><Cell Body><Cell Line><Cell Surface Proteins><Cell model><Cell secretion><Cell surface><CellLine><Cells><Cellular Secretion><Cellular model><Clustered Regularly Interspaced Short Palindromic Repeats interference><Coupled><Data><Disease><Disorder><Drug Targeting><EC 3.4.21.34><Engineering><Environmental Factor><Environmental Risk Factor><Esteroproteases><Event><External Domain><Extracellular Domain><Extracellular Space><Generations><Human><Hypoxia><Hypoxic><Immunooncology><Intercellular Space><K-RAS2A><K-RAS2B><K-Ras><K-Ras 2A><K-Ras-2 Oncogene><KRAS><KRAS(G12D)><KRAS2><KRAS2 gene><KRASG12D><Kallikrein 3><Ki-RAS><Label><Libraries><Ligase><Ligase Gene><Malignant Breast Neoplasm><Malignant Cell><Malignant Neoplasms><Malignant Pancreatic Neoplasm><Malignant Tumor><Malignant Tumor of the Prostate><Malignant neoplasm of pancreas><Malignant neoplasm of prostate><Malignant prostatic tumor><Measures><Modern Man><Molecular Interaction><Oncogene K-Ras><Oncogenes><Oncogenic><Oxygen Deficiency><P-30 Antigen><Pancreas Cancer><Pancreas Ductal Adenocarcinoma><Pancreatic Cancer><Pancreatic Ductal Adenocarcinoma><Pancreatic duct><Peptidases><Peptide Hydrolases><Peptides><Phage Display><Phages><Plasma Kallikrein Precursor><Plasma Prekallikrein><Prostate CA><Prostate Cancer><Prostate Specific Antigen Preproprotein><Prostate malignancy><Prostate-Specific Antigen><Prostatic Cancer><Protease Gene><Proteases><Protein Cleavage><Protein Engineering><Proteinases><Proteins><Proteolysis><Proteolytic Enzymes><Proteome><Proteomics><RASK2><Reagent><Receptor Activation><Receptor Protein><Semenogelase><Seminin><Site><Solid Neoplasm><Solid Tumor><Specificity><Strains Cell Lines><Surface><Synthetases><Technology><Therapeutic><Tissue Sample><Transforming Genes><U-PA><U-Plasminogen Activator><Urinary Plasminogen Activator><Urokinase><Urokinase Plasminogen Activator><Urokinase-Type Plasminogen Activator><Wirsung canal><antigen antibody affinity><bacterial virus><bio-markers><biologic marker><biomarker><cancer biomarkers><cancer cell><cancer markers><cancer microenvironment><cultured cell line><design><designing><environmental risk><experiment><experimental research><experimental study><experiments><extracellular><gamma-Seminoprotein><genetic protein engineering><hK3 Kallikrein><immune-oncology><immuno oncology><immunology oncology><inhibitor><insight><knock-down><knockdown><malignancy><malignant breast tumor><mutant><neo-antigen><neo-epitopes><neoantigens><neoepitopes><neoplasm/cancer><new technology><novel><novel technologies><oncoimmunology><pancreatic cancer model><pancreatic cell line><pancreatic malignancy><pancreatic tumor model><potential biological marker><potential biomarker><protein design><receptor><repressing CRISPR-dCas9 system><small molecular inhibitor><small molecule inhibitor><subtiligase><three dimensional><tool><tumor><tumor microenvironment><two-dimensional><v-Ki-RAS2 Kirsten Rat Sarcoma 2 Viral Oncogene Homolog>