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NOX4 inhibition potentiates immunotherapy by overcoming cancer-associated fibroblast-mediated CD8 T-cell exclusion from tumors: CAF-targeting by NOX4 inhibition potentiates immunotherapy

NOX4 inhibition potentiates immunotherapy by overcoming cancer-associated fibroblast-mediated CD8 T-cell exclusion from tumors: CAF-targeting by NOX4 inhibition potentiates immunotherapy
NOX4 inhibition potentiates immunotherapy by overcoming cancer-associated fibroblast-mediated CD8 T-cell exclusion from tumors: CAF-targeting by NOX4 inhibition potentiates immunotherapy
Determining mechanisms of resistance to antiPD-1/PD-L1 immune checkpoint immunotherapy is key to developing new treatment strategies. Cancer-associated fibroblasts (CAF) have many tumor-promoting functions and promote immune evasion through multiple mechanisms, but as yet, there are no CAF-specific inhibitors clinically available. Here we generated CAF-rich murine tumor models (TC1, MC38, 4T1) to investigate how CAF influence the immune microenvironment and affect response to different immunotherapy modalities (anti-cancer vaccination; TC1, [HPV E7 DNA vaccine]; antiPD-1, MC38) and found that CAFs broadly suppressed response by specifically excluding CD8+ T-cells from tumors (not CD4+ T-cells or macrophages); CD8+ T-cell exclusion was similarly present in CAF-rich human tumors. RNA sequencing of CD8+ T-cells from CAF-rich murine tumors and immunochemistry analysis of human tumors identified significant upregulation of CTLA-4 in the absence of other exhaustion markers; inhibiting CTLA-4 with a non-depleting antibody overcame the CD8+ T-cell exclusion effect without affecting T-regs. We then examined the potential for CAF targeting, focusing on the ROS-producing enzyme NOX4, which is upregulated by CAF in many human cancers, and comparing this to TGF-beta1 inhibition, a key regulator of the CAF phenotype. siRNA knockdown or pharmacological inhibition (GKT137831 [Setanaxib]) of NOX4 ‘normalized’ CAF to a quiescent phenotype and promoted intratumoral CD8+ T-cell infiltration, overcoming the exclusion effect; TGF-beta1 inhibition could prevent, but not reverse, CAF differentiation. Finally, NOX4 inhibition restored immunotherapy response in CAF-rich tumors. These findings demonstrate that CAF-mediated immunotherapy resistance can be effectively overcome through NOX4 inhibition, and could improve outcome in a broad range of cancers.
0008-5472
1846-1860
Ford, Kirsty
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Hanley, Christopher
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Mellone, Massimiliano
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Szyndralewiez, Cedric
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Heitz, Freddy
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Wiesel, Philippe
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Wood, Oliver
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Machado, Maria J.C.
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Lopez, Maria-Antoinette
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Ganesan, Anusha-Preethi
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Wang, Chuan
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Chakravarthy, Ankur
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Fenton, Tim R.
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King, Emma
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Vijayanand, Pandurangan
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Ottensmeier, Christian
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Al-Shamkhani, Aymen
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Savelyeva, Natalia
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Thomas, Gareth J.
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Ford, Kirsty
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Hanley, Christopher
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Mellone, Massimiliano
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Szyndralewiez, Cedric
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Heitz, Freddy
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Wiesel, Philippe
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Wood, Oliver
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Machado, Maria J.C.
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Lopez, Maria-Antoinette
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Ganesan, Anusha-Preethi
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Wang, Chuan
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Chakravarthy, Ankur
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Fenton, Tim R.
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King, Emma
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Vijayanand, Pandurangan
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Ottensmeier, Christian
42b8a398-baac-4843-a3d6-056225675797
Al-Shamkhani, Aymen
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Savelyeva, Natalia
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Thomas, Gareth J.
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Ford, Kirsty, Hanley, Christopher, Mellone, Massimiliano, Szyndralewiez, Cedric, Heitz, Freddy, Wiesel, Philippe, Wood, Oliver, Machado, Maria J.C., Lopez, Maria-Antoinette, Ganesan, Anusha-Preethi, Wang, Chuan, Chakravarthy, Ankur, Fenton, Tim R., King, Emma, Vijayanand, Pandurangan, Ottensmeier, Christian, Al-Shamkhani, Aymen, Savelyeva, Natalia and Thomas, Gareth J. (2020) NOX4 inhibition potentiates immunotherapy by overcoming cancer-associated fibroblast-mediated CD8 T-cell exclusion from tumors: CAF-targeting by NOX4 inhibition potentiates immunotherapy. Cancer Research, 80 (9), 1846-1860. (doi:10.1158/0008-5472.CAN-19-3158).

Record type: Article

Abstract

Determining mechanisms of resistance to antiPD-1/PD-L1 immune checkpoint immunotherapy is key to developing new treatment strategies. Cancer-associated fibroblasts (CAF) have many tumor-promoting functions and promote immune evasion through multiple mechanisms, but as yet, there are no CAF-specific inhibitors clinically available. Here we generated CAF-rich murine tumor models (TC1, MC38, 4T1) to investigate how CAF influence the immune microenvironment and affect response to different immunotherapy modalities (anti-cancer vaccination; TC1, [HPV E7 DNA vaccine]; antiPD-1, MC38) and found that CAFs broadly suppressed response by specifically excluding CD8+ T-cells from tumors (not CD4+ T-cells or macrophages); CD8+ T-cell exclusion was similarly present in CAF-rich human tumors. RNA sequencing of CD8+ T-cells from CAF-rich murine tumors and immunochemistry analysis of human tumors identified significant upregulation of CTLA-4 in the absence of other exhaustion markers; inhibiting CTLA-4 with a non-depleting antibody overcame the CD8+ T-cell exclusion effect without affecting T-regs. We then examined the potential for CAF targeting, focusing on the ROS-producing enzyme NOX4, which is upregulated by CAF in many human cancers, and comparing this to TGF-beta1 inhibition, a key regulator of the CAF phenotype. siRNA knockdown or pharmacological inhibition (GKT137831 [Setanaxib]) of NOX4 ‘normalized’ CAF to a quiescent phenotype and promoted intratumoral CD8+ T-cell infiltration, overcoming the exclusion effect; TGF-beta1 inhibition could prevent, but not reverse, CAF differentiation. Finally, NOX4 inhibition restored immunotherapy response in CAF-rich tumors. These findings demonstrate that CAF-mediated immunotherapy resistance can be effectively overcome through NOX4 inhibition, and could improve outcome in a broad range of cancers.

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Accepted/In Press date: 4 February 2020
e-pub ahead of print date: 2 March 2020
Published date: 1 May 2020

Identifiers

Local EPrints ID: 438433
URI: http://eprints.soton.ac.uk/id/eprint/438433
ISSN: 0008-5472
PURE UUID: fbb26e80-f8e1-4473-b1a8-58ed5d0d278c
ORCID for Kirsty Ford: ORCID iD orcid.org/0000-0002-6997-428X
ORCID for Christopher Hanley: ORCID iD orcid.org/0000-0003-3816-7220
ORCID for Massimiliano Mellone: ORCID iD orcid.org/0000-0002-4964-9340
ORCID for Aymen Al-Shamkhani: ORCID iD orcid.org/0000-0003-0727-4189

Catalogue record

Date deposited: 10 Mar 2020 17:30
Last modified: 17 Mar 2021 05:03

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Contributors

Author: Kirsty Ford ORCID iD
Author: Massimiliano Mellone ORCID iD
Author: Cedric Szyndralewiez
Author: Freddy Heitz
Author: Philippe Wiesel
Author: Oliver Wood
Author: Maria J.C. Machado
Author: Maria-Antoinette Lopez
Author: Anusha-Preethi Ganesan
Author: Chuan Wang
Author: Ankur Chakravarthy
Author: Tim R. Fenton
Author: Emma King
Author: Pandurangan Vijayanand

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