NOX4 Inhibition Potentiates Immunotherapy by Overcoming Cancer-Associated Fibroblast-Mediated CD8 T-cell Exclusion from Tumors

Abstract

Determining mechanisms of resistance to aPD-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, no CAF-specific inhibitors are clinically available. Here we generated CAF-rich murine tumor models (TC1, MC38, and 4T1) to investigate how CAFs influence the immune microenvironment and affect response to different immunotherapy modalities [anticancer vaccination, TC1 (HPV E7 DNA vaccine), aPD-1, and MC38] and found that CAFs broadly suppressed response by specifically excluding CD8 þ T cells from tumors (not CD4 þ T cells or macrophages); CD8 þ T-cell exclusion was similarly present in CAF-rich human tumors. RNA sequencing of CD8 þ 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 nondepleting antibody overcame the CD8 þ T-cell exclusion effect without affecting Tregs. 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 with TGFb1 inhibition, a key regulator of the CAF phenotype. siRNA knockdown or pharmacologic inhibition [GKT137831 (Setanaxib)] of NOX4 "normalized" CAF to a quiescent phenotype and promoted intratumoral CD8 þ T-cell infiltration, overcoming the exclusion effect; TGFb1 inhibition could prevent, but not reverse, CAF differentiation. Finally, NOX4 inhibition restored immunotherapy response in CAFrich 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. Significance: NOX4 is critical for maintaining the immunesuppressive CAF phenotype in tumors. Pharmacologic inhibition of NOX4 potentiates immunotherapy by overcoming CAFmediated CD8 þ T-cell exclusion.

Spatially discrete signalling niches regulate fibroblast heterogeneity in human lung cancer

“…Our analysis suggests TGF-β1 triggers myofibroblast differentiation, but additional stimuli are required to complete the process. This is consistent with previous studies showing that TGF-β1 is not required to maintain myofibroblast activation 5 . The ligands we identified as associated with myofibroblast activation are likely to represent these additional stimuli (Fig.…”

“…Fibroblasts are abundant but poorly characterised cells that play pivotal roles in wound healing 1 , extracellular matrix (ECM) remodelling 2 and inflammation 3 . In cancer, fibroblasts are the most common type of stromal cell and promote multiple hallmarks of malignancy [4][5][6] . These cells have been a putative therapeutic target in cancer for over a decade, but clinically effective strategies are yet to be identified.…”

Cancer-Associated Fibroblasts: Understanding Their Heterogeneity

“…TGF-β, CXCL1 and IL-10 secreted by CAFs inhibit the function of NK cells, CD8+ T lymphocytes, Th1 lymphocytes and DCs [ 121 , 137 , 138 ]. These cytokines upregulate the expression of transcription factors such as T-bet and NOX4 in tumor cells, contributing to immune escape [ 118 , 121 , 122 , 123 , 124 ]. CAFs also suppress immune cells through direct contact by expressing βig-h3, a TGFβ-induced RGD-containing surface protein that binds to the integrin β3 (CD61) present on CD8+ T lymphocytes, inhibiting their cytotoxic function [ 125 ].…”

Cancer-Associated Fibroblast Mediated Inhibition of CD8+ Cytotoxic T Cell Accumulation in Tumours: Mechanisms and Therapeutic Opportunities

“…Therefore, selective blocking of CAF derived IL-6 in particular may represent a clinically relevant therapeutic approach, with the potential to ameliorate both immune checkpoint inhibition and adoptive T cell therapy through enhanced CD8+ T cell recruitment. Similarly, a recent study revealed that the pharmacological blockade of NOX4, a ROS-producing enzyme with established roles in CAF activation [ 99 ], sensitised murine models of lung, breast and colorectal cancer to both anticancer vaccination and anti-PD-1 checkpoint inhibition through enhanced CD8+ T cell infiltration [ 100 ]. Importantly, Setanaxib, the selective small molecule inhibitor of NOX-4 has been subject to clinical evaluation through phase II in the context of liver fibrosis (NCT03226067), displaying a favourable safety profile; this compound could therefore be feasibly repurposed for treatment of CAF-rich, immune-excluded tumours.…”