Abstract 3026: Targeting GPX4 in tumor-associated stromal cells increases inflammatory-cell infiltration
Authors: Chattopadhyay Shrikanta , Huang Cherrie , Baryawno Ninib , Severe Nicolas , Viswanathan Vasanthi , Costa Carlotta , Scadden David , Schreiber Stuart 
Published: July 2017

Abstract
The lack of a T-cell inflamed microenvironment in tumors limits responsiveness to many immunotherapies. T-cell exclusion is often mediated by a dense infiltration of fibroblast-like stromal cells. Up to 55% of triple-negative breast cancers have ‘stroma-rich’ tumors with markedly lower T-cell inflammation. Here we report a therapeutic strategy that can potentially convert stroma-rich tumors into T-cell inflamed tumors by forcing stromal cells to secrete 5-lipoxygenase products which are powerful chemo-attractants for T cells.
We were initially interested in identifying selective inhibitors of stromal-cell function. To achieve this, we used phenotype-based small-molecule screening in which the phenotype of stroma-induced cancer cell migration in vitro was a surrogate for stroma-induced metastasis in vivo. We identified a compound, RSL3 that inhibited this migration. RSL3 was selectively cytotoxic to stromal cells over cancer cells, in comparisons of immortalized cell lines as well as comparisons of patient-derived primary breast cancer cells to cancer-associated fibroblasts (CAFs). We therefore undertook studies to identify its mechanism of action.
RSL3 was recently reported to target the redox enzyme glutathione peroxidase 4 (GPX4). GPX4 metabolizes lipid peroxides so we performed metabolomic profiling of RSL3-treated stroma-cancer co-cultures and found elevated arachidonic acid products of lipoxygenase enzymes. Stromal cells were found to contain &gt;10-fold higher levels of lipoxygenase products than carcinoma cells. Blocking either 5-lipoxygenase (5-LO) or 15-lipoxygenase (15-LO) with selective inhibitors abrogated RSL3’s cytotoxicity to stromal cells. Thus, high lipoxygenase activity in stromal cells increases their susceptibility to GPX4 inhibition.
Because 5-LO products like leukotriene B4 are powerful chemo-attractants for myeloid cells and T cells, we studied the impact of GPX4 knockdown in vivo using xenografts of cancer cells co-injected with stromal cells. GPX4 knockdown resulted in a large increase in myeloid-cell infiltration into tumors but, surprisingly, T-cell infiltration was suppressed. We reasoned that 15-LO products are immunosuppressive based on recent findings that 15-LO gene amplifications are inversely correlated with T-cell infiltration in breast cancers in The Cancer Genome Atlas. Consistent with this, GPX4 inhibition of stroma-cancer co-cultures suppressed T-cell chemotaxis but combined inhibition of GPX4 and 15-LO significantly enhanced T-cell chemotaxis over untreated controls in vitro. We are undertaking in vivo testing of this combination.
In summary, our unbiased chemical biology approach has revealed a therapeutic strategy to promote T-cell inflammation. We envision this to be a priming strategy for stroma-rich cancers to become responsive to a variety of different immunotherapies thereby unleashing their full curative potential.
Citation Format: Shrikanta Chattopadhyay, Cherrie Huang, Ninib Baryawno, Nicolas Severe, Vasanthi Viswanathan, Carlotta Costa, David Scadden, Stuart Schreiber. Targeting GPX4 in tumor-associated stromal cells increases inflammatory-cell infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3026. doi:10.1158/1538-7445.AM2017-3026