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

NOX4 inhibition potentiates immunotherapy by overcoming cancer-associated fibroblast-mediated CD8 T-cell exclusion from tumors
NOX4 inhibition potentiates immunotherapy by overcoming cancer-associated fibroblast-mediated CD8 T-cell exclusion from tumors
Determining mechanisms of resistance to PD-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];PD-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 compared this to TGF-062;1 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-062;1 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, K.
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Hanley, C.J.
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Mellone, M.
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Szyndralewiez, C.
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Heitz, F.
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Wiesel, P.
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Wood, O.
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Machado, M.
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Lopez, M.-A.
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Ganesan, A.-P.
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Wang, C.
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Chakravarthy, A.
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Fenton, T.R.
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King, E.V.
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Vijayanand, P.
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Ottensmeier, C.H.
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Al-Shamkhani, A.
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Savelyeva, N.
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Thomas, G.J.
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Ford, K.
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Hanley, C.J.
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Mellone, M.
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Szyndralewiez, C.
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Heitz, F.
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Wiesel, P.
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Wood, O.
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Machado, M.
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Lopez, M.-A.
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Ganesan, A.-P.
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Wang, C.
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Chakravarthy, A.
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Fenton, T.R.
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King, E.V.
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Vijayanand, P.
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Ottensmeier, C.H.
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Al-Shamkhani, A.
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Savelyeva, N.
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Thomas, G.J.
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Ford, K., Hanley, C.J., Mellone, M., Szyndralewiez, C., Heitz, F., Wiesel, P., Wood, O., Machado, M., Lopez, M.-A., Ganesan, A.-P., Wang, C., Chakravarthy, A., Fenton, T.R., King, E.V., Vijayanand, P., Ottensmeier, C.H., Al-Shamkhani, A., Savelyeva, N. and Thomas, G.J. (2020) NOX4 inhibition potentiates immunotherapy by overcoming cancer-associated fibroblast-mediated CD8 T-cell exclusion from tumors. Cancer Research, 80 (9), 1846-1860. (doi:10.1158/0008-5472.CAN-19-3158).

Record type: Article

Abstract

Determining mechanisms of resistance to PD-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];PD-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 compared this to TGF-062;1 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-062;1 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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Ford et al., 2020_ online version - Accepted Manuscript
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Accepted/In Press date: 4 February 2020
e-pub ahead of print date: 2 March 2020
Published date: 4 May 2020
Additional Information: Copyright: 2020 American Association for Cancer Research.

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 M. Mellone: ORCID iD orcid.org/0000-0002-4964-9340
ORCID for T.R. Fenton: ORCID iD orcid.org/0000-0002-4737-8233
ORCID for P. Vijayanand: ORCID iD orcid.org/0000-0001-7067-9723
ORCID for A. Al-Shamkhani: ORCID iD orcid.org/0000-0003-0727-4189

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Date deposited: 10 Mar 2020 17:30
Last modified: 17 Mar 2024 05:23

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Contributors

Author: K. Ford
Author: C.J. Hanley
Author: M. Mellone ORCID iD
Author: C. Szyndralewiez
Author: F. Heitz
Author: P. Wiesel
Author: O. Wood
Author: M. Machado
Author: M.-A. Lopez
Author: A.-P. Ganesan
Author: C. Wang
Author: A. Chakravarthy
Author: T.R. Fenton ORCID iD
Author: E.V. King
Author: P. Vijayanand ORCID iD
Author: A. Al-Shamkhani ORCID iD
Author: N. Savelyeva
Author: G.J. Thomas

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