Abstract B11: Targeting mesenchymal cells in the tumor stroma by GPX4 inhibition
Authors: Chattopadhyay Shrikanta , Huang Cherrie , Baryawno Ninib , Severe Nicolas , Viswanathan Vasanthi , Boskovic Zarko , Mukherjee Siddhartha , Gentry Jeff , Wittner Ben , Ramaswamy Sridhar , Shamji Alykhan , Scadden David , Schreiber Stuart 
Published: October 2016

Abstract
Fibroblast-like mesenchymal cells are the most abundant component of tumor stroma and have been shown to promote tumor progression. However, there are no therapeutic agents that target these cells. Developing therapeutics against mesenchymal cells has been challenging because they lack clear “druggable” targets. Here we used a phenotypic screening approach to identify therapeutic targets in bone-marrow mesenchymal stem cells (MSCs), precursors of cancer-associated fibroblasts, that are reported to induce a metastatic phenotype in breast cancer cells (Karnoub et al, Nature 2007). Using the in vitro phenotype of enhanced migration of breast cancer cells (GFP-labeled MDA-MB-231; MDA) induced by MSCs (3-fold faster than MDA alone), we identified inflammatory signaling and glutathione peroxidase 4 as potential targets in MSCs.
To validate this screening model, we first confirmed that MSCs can enhance metastasis using orthotopic xenografts of luciferase-labeled MDA cells co-injected with primary human MSCs in NOD-SCID mice. Mice with MSC+MDA tumors had 5-fold greater thoracic bioluminescence (corresponding to lung metastasis) than mice with MDA tumors alone. We then performed gene-expression profiling of MSC+MDA co-cultures in vitro compared to cells grown alone. The top pathway upregulated in co-cultures was the interferon pathway, suggesting that an inflammatory response follows MSC-MDA interactions. Using publicly available gene-expression datasets, we prioritized transcripts that are expressed in patient stroma and correlate with poor survival in a meta-analysis of 20 whole-tumor datasets. To determine if these transcripts are necessary for MSC-induced metastatic behavior, we performed shRNA knockdown and measured effects on in vitro migration of MSC+MDA co-cultures compared to normal endothelial cells. Knockdown of 9 genes was specific for MSC+MDA migration but the effects were weak. This suggested functional redundancy and indicated that targeting individual upregulated genes is insufficient to block the phenotype.
We then performed a small-molecule screen on the MSC+MDA migration phenotype, counter-screening hits on endothelial cell migration. Only one compound, RSL3, showed a large and selective inhibition of MSC+MDA migration. RSL3 was selectively toxic to MSCs with no effect on MDA cells. The target of RSL3 was recently identified to be glutathione peroxidase 4 (GPX4) that metabolizes lipid peroxides. To identify lipid mediators of RSL3 toxicity, we profiled lipid levels in RSL3-treated MSC+MDA co-cultures and found that the top changes were lipoxygenase products of arachidonic acid. Pre-treatment with zileuton or PD146176, that inhibit 5- and 15-lipoxygenase respectively, abrogated RSL3's toxic effects on MSCs. We found that MSCs contain &gt;10-fold higher lipoxygenase products than MDA cells, highlighting an inflammatory role of MSCs. Fibroblasts from different tissues (lungs, spleen, breast, skin) were similarly sensitive to RSL3, identifying a previously unrecognized vulnerability in these mesenchymal cells.
Since RSL3 is a tool compound with poor plasma stability, we used GPX4 shRNA knockdown (KD) to determine in vivo effects of depleting MSCs in MSC+MDA xenografts. Unexpectedly, we found accelerated tumor growth in GPX4 KD tumors compared with controls. However, this is consistent with previous reports showing that mesenchymal cells restrain tumor growth and can have both pro- and anti-tumor effects. Histological examination revealed increased myeloid cell infiltration into GPX4 KD tumors reflecting increased inflammation mediated by lipoxygenase products like leukotriene B4.
In summary, phenotypic screening has identified GPX4 inhibition as a novel approach to target mesenchymal cells. This approach may be particularly effective at recruiting immune/ inflammatory cells into tumors with the exciting possibility of synergizing with cancer immunotherapy.
Citation Format: Shrikanta Chattopadhyay, Cherrie Huang, Ninib Baryawno, Nicolas Severe, Vasanthi Viswanathan, Zarko Boskovic, Siddhartha Mukherjee, Jeff Gentry, Ben Wittner, Sridhar Ramaswamy, Alykhan Shamji, David Scadden, Stuart Schreiber. Targeting mesenchymal cells in the tumor stroma by GPX4 inhibition. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B11.