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Principal Investigator: CHRISTOPHER J. BAKKENIST
Organization: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
Fiscal Year: 2024
Award: $69,960
Funding agency: National Cancer Institute
PARENT GRANT – ABSTRACT (as per original submission)
The overarching goal of our laboratory is to determine how DNA damage response inhibitors (DDRi) can be used
to potentiate cancer cell killing while concurrently increasing anti-tumor immune responses after radiation therapy
(XRT). The DNA Damage Response (DDR) is a signaling system that integrates DNA repair pathways and the
cell cycle to safeguard genome stability. In addition to activating cell cycle checkpoints and DNA repair in cells
treated with XRT, the DDR limits origin firing and delays cell cycle transitions in unstressed cells. While cyclin-
dependent kinases are cell cycle accelerators, DDR kinases are cell cycle brakes and, in this analogy, DDRi
disable the brakes, causing unchecked acceleration. Here we will determine how the DDR is rewired in CD8+ T
cells to accommodate massive and concomitant DNA replication and transcription in S phase. We will also
determine the impact of DDRi in cancer and immune cells. We hypothesize that ATR kinase inhibitors induce
origin firing that causes ribonucleosides to be mis-incorporated into the genome, and that this generates
chimeric RNA-DNA fragments and type I IFN-dependent immunologic memory after XRT. To test our
hypothesis in cancer and immune cells, we have generated an innovative transplantable model of cancer. The
Mcm4Chaos3/Chaos3 mouse carries a mutation in Mcm4 that destabilizes the replicative helicase. Cells derived
from Mcm4Chaos3/Chaos3 mice have a 60% reduction in origin licensing. We have generated
Mcm4Chaos3/Chaos3 B16 cancer cells that can be transplanted into Mcm4wt/wt and Mcm4Chaos3/Chaos3
mice. This will allow us to separate the function of ATR that limits origin firing from that which mediates the repair
of replication forks in cancer and immune cells. In Aim 1, we will define cell cycle kinetics and determine how
ATR inhibitors induce DNA damage in immune and cancer cells in vitro. In Aim 2, we will define cell cycle kinetics
and determine whether ATR inhibitors induce DNA damage in immune cells and type 1 interferons in vivo. In Aim
3, we will determine whether ATR inhibitors combine with XRT to generate durable responses and immunologic
memory through effects on immune and/or cancer cells. Successful completion of this project will define how the
DDR is rewired in CD8+ T cells to accelerate cell cycle transitions and accommodate massive and concomitant
DNA replication and transcription in S phase which, accounts for ~70% of the cell cycle as G1 is abridged. These
studies are highly significant as the objective of checkpoint blockade and adoptive T cell transfer is to induce
rapid division in CD8+ T cells. Successful completion of this project will identify combinations and sequences of
DDRi that potentiate cancer cell killing while concurrently increasing anti-tumor immune responses in mouse
models of cancer treated with XRT. These studies are highly significant as we use DDRi that are currently in 115
clinical trials and XRT which is used to treat >50% of cancer patients, >60% with curative intent.
R01 Diversity Supplement
The goals of the current proposed research plan integrate with Aim 3 of the parent R01. The goal of the diversity
supplement is to determine the role of ATR signaling in T cell memory development. Previous findings in knockout
mouse models suggest that upstream and downstream signaling, by ETAA1 and DCK respectively, of ATR is
necessary for memory development. In addition, previous laboratory findings have shown that use of ATRi will
radiotherapy modulates the immunologic memory. Short-term ATRi treatments were found to potentiate memory
responses, while long-term use of this drug blunted immune memory development when combined with
radiotherapy. We hypothesize that ETAA1-dependent, ATR phosphorylation of DCK is essential for T cell
memory development and that ATRi can be used to potentiate memory. We will complete three aims in
order to determine this. Aim 1 will Determine whether ETAA1-ATR signaling is an intrinsic regulator of T cell
activation that impacts the development of T cell memory. Aim 2 will Determine whether DCK is an intrinsic
regulator of T cell activation that impacts the development of T cell memory. Aim 3 will Determine whether ETAA1-
ATR-DCK signaling is necessary for immunological memory responses to occur upon rechallenge. Successful
completion of these Aims will determine whether ETAA1-ATR-DCK activity in CD8+ T cells promotes memory
and whether this signaling axis can be targeted with pharmacologics to mitigate autoimmune diseases and
potentiate immune responses to infection and cancer. Successful completion of this proposal will directly prove
whether ATRi can potentiate the acquisition of immunologic memory and identify the optimal schedule of ATRi +
IR for clinical trials.
Terms: <(IFN) α><(IFN)-α><(IFN)α><Abscission><Acceleration><Affect><Alferon><Anabolism><Animals><Antigen Presentation><Antigens><Autoimmune Diseases><Base Pairing><Binding><Breeding><CD28><CD28 gene><CD3><CD3 Antigens><CD3 Complex><CD3 molecule><CD8 Cell><CD8 T cells><CD8 lymphocyte><CD8+ T cell><CD8+ T-Lymphocyte><CD8-Positive Lymphocytes><CD8-Positive T-Lymphocytes><Cancer Model><Cancer Patient><CancerModel><Cancers><Cell Body><Cell Communication and Signaling><Cell Count><Cell Cycle><Cell Cycle Checkpoint><Cell Cycle Kinetics><Cell Death><Cell Division Cycle><Cell Growth in Number><Cell Kinetics><Cell Multiplication><Cell Number><Cell Proliferation><Cell Signaling><Cell division><Cells><Cellular Expansion><Cellular Growth><Cellular Proliferation><Chemotherapy and Radiation><Chemotherapy and/or radiation><Clinical><Clinical Trials><Clonal Expansion><Communicable Diseases><Cyclin-Dependent Kinases><Cyclin-Dependent Protein Kinases><DNA><DNA Damage><DNA Damage Repair><DNA Helicases><DNA Injury><DNA Integration><DNA Repair><DNA Repair Pathway><DNA Replication><DNA Sequence><DNA Synthesis><DNA Unwinding Proteins><DNA biosynthesis><DNA replication fork><DNA unwinding enzyme><Data><Deoxyribonucleic Acid><Deoxyuridine><Development><Drug Therapy><Drug usage><ETAA1><Effector Cell><Enzyme Gene><Enzymes><Ewing's Tumor associated antigen 1><Ewing's tumor associated antigen><Excision><Exclusion><Extirpation><First Gap Phase><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><G1 Phase><G1 period><Gap Phase 1><Gene Transcription><Genetic Alteration><Genetic Change><Genetic Transcription><Genetic defect><Genome><Genome Stability><Genomic Stability><Genotype><Germ-Line Mutation><Germline Mutation><Goals><Harvest><Hereditary Mutation><IFN><IFN Alpha><IFN α><IFN-α><IFNa><IFNα><Immune><Immune Modulation Therapy><Immune mediated therapy><Immune memory><Immune response><Immunes><Immunoblotting><Immunologic Memory><Immunological Memory><Immunological response><Immunologically Directed Therapy><Immunotherapy><In Vitro><Infection><Infectious Disease Pathway><Infectious Diseases><Infectious Disorder><Injections><Interferon Alfa-n3><Interferon alpha><Interferon-α><Interferons><Intracellular Communication and Signaling><KO mice><Kinases><Knock-out><Knock-out Mice><Knockout><Knockout Mice><LCM Viruses><LCMV><Lab Findings><Laboratories><Laboratory Finding><Leukocyte Interferon><Licensing><Lymphoblast Interferon><Lymphoblastoid Interferon><Lymphocytic choriomeningitis virus><Malignant Cell><Malignant Neoplasms><Malignant Tumor><Mediating><Memory><Memory Deficit><Memory impairment><Mice><Mice Mammals><Modeling><Molecular Interaction><Murine><Mus><Mutation><Non-Polyadenylated RNA><Nucleosides><Null Mouse><Nutrient><OKT3 antigen><Operative Procedures><Operative Surgical Procedures><Parents><Pathway interactions><Pharmacotherapy><Phenotype><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Play><Procedures><Proliferating><Protein Phosphorylation><Protocol><Protocols documentation><Publishing><R-Series Research Projects><R01 Mechanism><R01 Program><RNA><RNA Expression><RNA Gene Products><Radiation therapy><Radiotherapeutics><Radiotherapy><Removal><Replication Unit><Replicon><Research><Research Design><Research Grants><Research Project Grants><Research Projects><Research Proposals><Ribonucleic Acid><Ribonucleosides><Role><S Period><S phase><Schedule><Signal Transduction><Signal Transduction Systems><Signaling><Spleen><Spleen Reticuloendothelial System><Study Type><Surgical><Surgical Interventions><Surgical Procedure><Surgical Removal><Synthesis Period><Synthesis Phase><System><T cell based therapeutics><T cell based therapy><T cell directed therapies><T cell response><T cell targeted therapeutics><T cell therapy><T memory cell><T-Cell Activation><T-Cell Development><T-Cell Ontogeny><T-Cells><T-Lymphocyte><T-Lymphocyte Development><T-cell therapeutics><T-cell transfer therapy><T3 Antigens><T3 Complex><T3 molecule><T44><T8 Cells><T8 Lymphocytes><Teff cell><Testing><Thymidin><Thymidine><Time><Training><Transcription><Transphosphorylases><Transplantation><Tumor Cell><Tumor-infiltrating immune cells><Unscheduled DNA Synthesis><Update><Viral Diseases><Virus Diseases><Western Blotting><Western Immunoblotting><activate T cells><adoptive T cell transfer><adoptive T-cell therapy><anamnestic reaction><anti-tumor immune response><autoimmune condition><autoimmune disorder><autoimmunity disease><biological signal transduction><biosynthesis><cancer cell><cancer microenvironment><cdk Proteins><cell cycle check point><cell growth><cell killing><check point blockade><checkpoint blockade><chemo/radiation therapy><chemotherapy><chemotherapy and radiotherapy><compare to control><comparison control><conditional knock-out><conditional knockout><developmental><drug treatment><drug use><effector T cell><exhaust><experiment><experimental research><experimental study><experiments><flow cytophotometry><genome mutation><germ-line defect><germline variant><helicase><host response><immune cell infiltration of tumors><immune cells infiltrating the tumor><immune cells that infiltrate the tumor><immune check point blockade><immune checkpoint blockade><immune modulating therapies><immune modulatory therapies><immune system response><immune therapeutic approach><immune therapeutic interventions><immune therapeutic regimens><immune therapeutic strategy><immune therapy><immune-based therapies><immune-based treatments><immune-modulation treatment><immuno therapy><immunogen><immunomodulation therapy><immunomodulation treatment><immunomodulator therapies><immunomodulator treatment><immunomodulator-based therapies><immunomodulatory therapies><immunomodulatory therapy><immunomodulatory treatment><immunoresponse><in vivo><in vivo Model><infiltration of tumors by immune cells><inhibitor><innovate><innovation><innovative><intratumoral immune cell><intratumoral immune infiltrate><irradiation response><kinase inhibitor><malignancy><memory T lymphocyte><memory dysfunction><mouse model><murine model><necrocytosis><neoplasm/cancer><neoplastic cell><new approaches><novel approaches><novel strategies><novel strategy><parent><parent grant><pathway><pharmacologic><protein blotting><radiation or chemotherapy><radiation response><radiation treatment><repair><repaired><replication fork><resection><response><response to radiation><secondary immune response><social role><study design><surgery><therapeutic T-cell platform><thymus derived lymphocyte><tissue culture><transplant><transplant model><treatment with radiation><tumor><tumor immune cell><tumor immune infiltrate><tumor infiltration of immune cells><tumor microenvironment><viral infection><virus infection><virus-induced disease>