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Principal Investigator: Philip Andrew Moresco
Organization: STATE UNIVERSITY NEW YORK STONY BROOK
Fiscal Year: 2024
Award: $53,974
Funding agency: National Cancer Institute
Project Summary/Abstract
Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer related deaths in the United
States, with a five-year survival rate of 7-8%. This poor survivability can be partially attributed to late detection,
however PDAC therapies have remained stagnant which amplifies the discordance between the survivability of
PDAC and other cancers. Immune checkpoint blockade (ICB) has revolutionized the treatment of certain malig-
nancies, but most carcinomas, including PDAC, remain refractory. The Fearon Lab has identified that the chem-
okine (C-X-C motif) ligand 12 (CXCL12) “coats” cancer cells in mouse models of PDAC, and that blocking the
interaction between CXCL12 and chemokine (C-X-C motif) receptor 4, the CXCL12 receptor, results in T cell
influx into cancer cell nests and response to ICB. Additionally, we find that CXCL12 is covalently linked to keratin
19 (KRT19) on the surface of cancer cells by the isopeptide bond forming enzyme transglutaminase 2 (TGM2),
and knockout of either Krt19 or Tgm2 results in T cell entry into cancer cell nests and response to ICB. This data
indicates that the CXCL12-KRT19 conjugate is a primary mediator of tumor immune suppression, however, our
understanding of the biochemical process of its formation, and kinetics of its formation and disassembly, are
lacking yet would provide insight for future studies attempting to regulate these processes. Therefore, I will de-
termine the glutamine of KRT19 required to covalently bond to CXCL12 in vivo and determine if its mutation
results in loss of CXCL12 coating and response to ICB. Additionally, I will quantify the rate of formation and
disassembly of the CXCL12 coat and characterize the accompanying changes to immune cell activity.
In Aim 1 I will generate the CXCL12-KRT19 conjugate using TGM2 in vitro and subject this complex to mass
spectrometry analysis to identify the crosslinked residues between CXCL12 and KRT19. I will generate recom-
binant KRT19 containing a Q to N mutation of the residue found to be crosslinked to CXCL12 and validate that
CXCL12 can no longer be linked to KRT19. I will then express the mutant KRT19 in mouse PDA 1242 cells and
validate that CXCL12 fails to become linked to cancer cells in vivo and determine if this failure results in suscep-
tibility to ICB. In Aim 2 I will inject Matrigel suspensions of 1242 cells into C57BL/6J mice to facilitate early tumor
harvesting and quantification of the rate of formation of the CXCL12 coat using immunofluorescent tissue stain-
ing. I will use a Doxycycline-Off KRT19 expression system to study the rate of CXCL12 coat disassembly and
perform immune cell profiling using RNA sequencing to determine how infiltrative T cells respond to coat disas-
sembly. This proposal will study how the CXCL12 coat forms, the rate of its formation and disassembly, and
highlight that modulating the CXCL12 coat reciprocally modifies anti-tumor immune activity. Additionally. this
fellowship will provide training for my longitudinal goal of studying translational tumor immunology through in-lab
training, workshops, meetings, and clinical work at Cold Spring Harbor Laboratory and Stony Brook University.
Terms: <170-kD Melanoma Membrane-Bound Gelatinase><4q Chemokine><Alpha Subunit Fibroblast Activation Protein><Amino Acids><Biochemical Process><Biochemistry><Biological Chemistry><Blocking Antibodies><C-X-C Chemokine Receptor Type 4><C-X-C Chemokines><CD184 Antigen><CK-19><CXC Chemokines><CXCL12><CXCL12 gene><CXCL12 protein><CXCR4 Receptors><Cancer Cause><Cancer Etiology><Cancers><Carcinoma><Cell Body><Cell Coat><Cell Line><CellLine><Cells><Cessation of life><Chemokine (C-X-C Motif) Ligand 12><Chemokine (C-X-C Motif) Receptor 4><Chemokine, CXC Motif, Receptor 4><Chemotactic Cytokines><Clinical><Clinical Research><Clinical Study><Complex><Cytokeratin 19><Data><Death><Detection><Diathesis><Disease susceptibility><Doxycycline><Educational workshop><Engraftment><Enzyme Gene><Enzymes><Epithelial cancer><FAP-Alpha><FAPalpha><Failure><Fellowship><Fibroblasts><Fusin><Future><Generalized Growth><Generations><Genetic Alteration><Genetic Change><Genetic defect><Gln><Glutamine><Glycocalyx><Goals><Growth><Harvest><Homologous Chemotactic Cytokines><Human><Immune><Immune Cell Activation><Immune system><Immunes><Immunosuppression><Immunosuppression Effect><Immunosuppressive Effect><In Vitro><Integral Membrane Serine Protease><Intercrines><Keratin-19><Kinetics><Knock-out><Knockout><Knowledge><L-Glutamine><LESTR Receptor><LPS-Associated Protein 3><Laboratories><Leukocyte-Derived Seven-Transmembrane Domain Receptor><Ligands><Link><Lipopolysaccharide-Associated Protein 3><Malignant Cell><Malignant Epithelial Neoplasms><Malignant Epithelial Tumors><Malignant Neoplasms><Malignant Pancreatic Neoplasm><Malignant Tumor><Malignant neoplasm of pancreas><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Mediating><Mediator><Mice><Mice Mammals><Modern Man><Murine><Mus><Mutation><Neuropeptide Y Receptor Y3><PBSF><PDA model><PDAC Model><Pancreas Cancer><Pancreas Ductal Adenocarcinoma><Pancreatic Cancer><Pancreatic Ductal Adenocarcinoma><Pre-B Cell Growth Stimulating Factor><Predisposition><Procedures><Process><Q Levoglutamide><Q. Levoglutamide><RNA Seq><RNA sequencing><RNAseq><Receptor Protein><Recombinants><Refractory><Repression><Resistance><Risk><SCYB12><SDF-1><SDF-1 Receptor><SDF-1A><SDF-1B><SDF-1alpha><SDF1><SDF1/PBSF Receptor CXCR4><SDF1A><SDF1B><SIS cytokines><Scanning><Sdf1 protein><Seprase><Site><Source><Spleen Seven-Transmembrane-Segment Receptor><Staining method><Stains><Strains Cell Lines><Stromal Cell-Derived Factor 1><Stromal Cell-Derived Factor 1 Receptor><Surface><Survival Rate><Susceptibility><Suspension substance><Suspensions><System><T-Cells><T-Lymphocyte><TGase II><TLSF-A><TLSF-B><TPAR1><Time><Tissue Growth><Tissue Stains><Training><Tumor Immunity><United States><Universities><Vibramycin><Work><Workshop><aPD-1><aPD1><adaptive immune response><alpha-6-Deoxyoxytetracycline><alpha-Chemokines><aminoacid><anti programmed cell death 1><anti-PD-1><anti-PD1><anti-programmed cell death protein 1><anti-tumor immunity><antiPD-1><antiPD1><antitumor immunity><cancer cell><cancer immunity><cancer immunology><cancer infiltrating T cells><check point blockade><checkpoint blockade><chemoattractant cytokine><chemokine><covalent bond><crosslink><cultured cell line><epithelial carcinoma><extracellular><fibroblast activation protein alpha><functional loss><genome mutation><hIRH><immune activation><immune check point blockade><immune checkpoint blockade><immune suppression><immune suppressive activity><immune suppressive function><immunosuppressive activity><immunosuppressive function><immunosuppressive response><in vivo><insight><keratin 12><lab experience><lab training><laboratory experience><laboratory training><liability to disease><malignancy><matrigel><meeting><meetings><mouse model><murine model><mutant><neoplasm immunology><neoplasm/cancer><ontogeny><pancreatic ductal adenocarcinoma model><pancreatic malignancy><pre-clinical><pre-clinical study><preclinical><preclinical study><prevent><preventing><receptor><resistant><response><stromal cell-derived factor-1alpha><subcutaneous><subdermal><surface expressed protease><thymus derived lymphocyte><tissue transglutaminase><tissue-type transglutaminase><transcriptome sequencing><transcriptomic sequencing><transglutaminase 2><transglutaminase C><transglutaminase II><transglutaminase TGM2><tumor><tumor growth><tumor immunology><tumor infiltrating T cells><vector><αPD-1><αPD1>