DNA Damage Signaling to Immune CheckpointsVendetti F (2015) Oncotarget ATR kinase inhibition...
Transcript of DNA Damage Signaling to Immune CheckpointsVendetti F (2015) Oncotarget ATR kinase inhibition...
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DNA Damage Signaling to Immune Checkpoints
Chris Bakkenist, PhD, Associate Professor
Department of Radiation Oncology,
UPMC Hillman Cancer Center
University of Pittsburgh
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The Bakkenist Lab has sponsored research agreements with AstraZeneca
Disclosure
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DNA Damage Signaling to Immune Checkpoints
1) ATR kinase inhibitors induce checkpoint blockade
2) ATR kinase inhibitors induce unscheduled origin firing
Frank Vendetti, PhD
Tatiana Moiseeva, PhD
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DNA Damage Signaling
>5,000 substrates
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• ATR kinase inhibitor AZD6738 – four clinical trials• VX-970 (VE-822) – nine clinical trials
• ATM kinase inhibitor AZ31• AZD0156 – one clinical trial• AZD1390
Inhibitors from Mark O’Connor, PhD
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Vendetti F (2015) Oncotarget
ATR kinase inhibition doesn’t kill mice
AZD6738 Pharmacokinetics t1/2 = 6 h
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* p=0.001
ATR kinase inhibitor with cisplatin delivers a
compete response in ATM-deficient xenograftscisplatin before ATR kinase inhibitor
ATM-deficient
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>40% of lung adenocarcinomas are
deficient for ATM
Lung SPORE TMA
>200 cases were included in the analysis. Inclusion criteria were +ve
staining in stroma and immune cell infiltrate, and at least 2 cores scored for
each patient.
Villaruz L (2016) Oncotarget
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Clinical and preclinical studies with ATR kinase inhibitors are justified
Summary
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Vendetti F (2017) Scientific Reports
ATM kinase inhibitor sensitizes
mice to total body irradiation ATR kinase inhibitor before radiation
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Vehicle ATMi
Vehicle
ATRi
ATMi
ATM kinase inhibitor with radiation kills
intestinal crypts
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CCSP-rtTAtetO-KrasG12D
Luc-tetO-Twist1
Tet-O LucTwist1
KrasG12D
Tet-O
Genetically engineered mouse model of NSCLC
Small Animal Radiation Research Platform (SARRP)
Frank Vendetti, PhD
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ATR kinase inhibitor with radiation delivers adelayed response
ATR kinase inhibitor before radiation
end of treatmentdelayed response
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ATM kinase inhibitor with radiationkills hair follicles
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Day 35
The impact of ATR kinase inhibitor is CD8+
T cell dependent
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Immunology
Adaptation by A Sharabi, original figure fromSharabi A et al. (2016) Oncology, (Williston Park) 29(5)
Adaptated fromPardoll DM. (2012) Nat Rev Cancer, 12(4)
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ATR kinase inhibitor potentiates radiation in
syngeneic CT26 colorectal carcinomasATR kinase inhibitor before radiation
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ATR kinase inhibitor attenuates radiation-induced
PD-L1 upregulation in vivo
Isotype
Vehicle
IR
ATRi + IR
PD-L1
Day 5 Day 5
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TIL IR (2 Gy x 2) TIL ATRi + IRSpleen Vehicle
ATR kinase inhibitor blocks T cell exhaustion after
radiation
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cytokine production
following PMA/ionomycin
ATR kinase inhibitor induces checkpoint
blockade after radiation
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Kinase inhibition does not phenocopy kinase knockout
>5,000 substrates
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Molecular Mechanism
•Cell intrinsic signaling - radiation
•cGAS-STING knockout in CT26
•Cell extrinsic signaling - interferon g
•Cancer cells - cell cycle checkpoint competent?
•Lung cancer patients will have been treated with platinum-based chemotherapy combinations
•Leonna Samson, PhD
•Trey Ideker, PhD and Mike Yaffe, MD, PhD
•Stromal cells
•Immune cells
•CD8+ T cells and Tregs
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ATR kinase inhibitor induces origin firing in undamaged cells
Couch et al. Cortez D. Genes Dev. 2013Kwok et al. Stankovic T. Blood 2016
Tatiana Moiseeva, PhD
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ATR kinase inhibitor induces an association of replisome components in chromatin
Moiseeva et al. (2017) Nature Communications
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ATR and Chk1 kinase inhibitors induce Cdc7-dependent MCM4 hyper-phosphorylation
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ATR kinase inhibitor induces MCM4 hyper-phosphorylation in S phase cells
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ATR kinase inhibitor induces Cdc7-dependent MCM4 hyper-phosphorylation
ATR kinase Cdc7 kinase
ATRi Cdc7 kinase
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DNA damage before ATR kinase inhibitor changes the signaling
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DNA damage before ATR kinase inhibitor changes the signaling
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ATR kinase Cdc7 kinase
ATRi Cdc7 kinase
ATR kinase ATRi Cdc7 kinase
DNA damage before ATR kinase inhibitor changes the signaling
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ATR kinase inhibitor induces origin firing and this leads to more DSBs when H2O2 is added
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Sequence is important
Summary
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ATR kinase inhibitor induces an association of replisome components in chromatin
Moiseeva et al. (2017) Nature Communications
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Ctf4/And-1 in yeastWHDH1/And-1 in humans
Simon…….Pellegrini
A Ctf4 trimer couples the CMG
helicase to DNA polymerase α in the
eukaryotic replisome.
Nature 510, 293-297 (2014)
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ATR and Chk1 kinase inhibitors induce the association of GINS, And-1 and DNA pol a
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ATR kinase inhibitor induces Cdc7-dependent phosphorylation on GINS4
Phosphorylated S12/S16 GINS associates with C terminus of And-1
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Inhibitors of DNA damage signaling are not inert
Summary
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PBMCs do not express ATR
Bakkenist CJ et al. (2015) Oncoscience
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no activationanti-CD3 + anti-CD28
+ IL-2 (100U/mL)
CFSE
CD
44
CD8+
103 104 105 106
106
105
104
12345
60
proliferation dye
activa
tio
n m
ark
er
dye is diluted
as T cells divide
T cell expansion ex vitro
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CFSE
CD
44
CD8+
103 104 105 106
ATRi
125 nM 250 nM 500 nM 1000 nM
ATR kinase inhibitors block
T cell expansion
ATR kinase inhibitor blocks T cell expansion ex vitro
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It is important to identify the structures generating
change within the complexity
Cancer Immune System
Search for ATR kinase inhibitors generates 2,500 hits on Pubmed
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Sequence is important
• If the cancer is cell cycle checkpoint deficient, genotoxic chemotherapy
before ATR kinase inhibitor
• If the cancer is DNA repair deficient, ATR kinase inhibitor and then
genotoxic chemotherapy
• The ATR kinase inhibitor must be removed to allow an anti-tumor immune
response
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Radiation Oncology
• Frank Vendetti
• Tatiana Moiseeva
• Pooja Karukonda
• Sandy Schamus
• Katie Lemon
• Andy Clump
Inova Schar Cancer Institute
• Tom Conrads
Pathology
• Brian Leibowitz
• Jian Yu
Immunology
• Greg Delgoffe
• Robert Ferris
• Saumen Sarkar
AstraZeneca
• Mark O’Connor
• Jennifer Barnes
• Elaine Barnes
• Andrew Pierce
Johns Hopkins University
• Phuoc Tran
Acknowledgements
Always have openings for new postdocs