The Impact of ATR Kinase Inhibitors on CD8+ T Cell Expansion and Memory Development

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

Investigators

Linked publications & trials

Abstract

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.