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Cystine–Glutamate Antiporter Xct Deficiency Suppresses Tumor Growth While Preserving Antitumor Immunity

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Cystine–Glutamate Antiporter Xct Deficiency Suppresses Tumor Growth While Preserving Antitumor Immunity Cystine–glutamate antiporter xCT deficiency suppresses tumor growth while preserving antitumor immunity Michael D. Arensmana, Xiaoran S. Yanga, Danielle M. Leahya, Lourdes Toral-Barzaa, Mary Mileskia, Edward C. Rosfjorda, Fang Wanga, Shibing Dengb, Jeremy S. Myersa, Robert T. Abrahamb, and Christina H. Enga,1 aOncology Research & Development, Pfizer, Pearl River, NY 10965; and bOncology Research & Development, Pfizer, San Diego, CA 92121 Edited by William G. Kaelin Jr., Dana-Farber Cancer Institute and Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, and approved April 2, 2019 (received for review September 1, 2018) T cell-invigorating cancer immunotherapies have near-curative Thus, tumor cells may rely on xCT to fulfill the majority of their potential. However, their clinical benefit is currently limited, as cysteine and GSH needs by importing cystine. only a fraction of patients respond, suggesting that these regimens Inhibition of xCT has been investigated as a therapeutic may benefit from combination with tumor-targeting treatments. As strategy for cancer based on observations that elevated xCT ex- oncogenic progression is accompanied by alterations in metabolic pression on tumor cells correlates with poor prognosis (10–12) pathways, tumors often become heavily reliant on antioxidant and that inhibition of xCT in preclinical studies suppresses tumor machinery and may be susceptible to increases in oxidative stress. growth (10, 12–14). However, these studies relied heavily on the The cystine–glutamate antiporter xCT is frequently overexpressed in use of sulfasalazine, a clinical compound used for the treatment cancer and fuels the production of the antioxidant glutathione; thus, of rheumatoid arthritis, ulcerative colitis, and Crohn’s disease. tumors prone to redox stress may be selectively vulnerable to xCT While sulfasalazine inhibits xCT-mediated uptake of cystine disruption. However, systemic inhibition of xCT may compromise (15), sulfasalazine also inhibits NF-κB (16), sepiapterin reductase antitumor immunity, as xCT is implicated in supporting antigen- (17), and reduced folate carrier (18). Thus, the interpretation of induced T cell proliferation. Therefore, we utilized immune-competent reports attributing sulfasalazine-mediated reductions in tumor murine tumor models to investigate whether cancer cell expres- growth to xCT inhibition is hampered by the lack of specificity of MEDICAL SCIENCES sion of xCT was required for tumor growth in vivo and if deletion this drug. Furthermore, immunocompromised mice bearing human of host xCT impacted antitumor immune responses. Deletion of tumor xenografts were used to investigate the effects of xCT in- xCT in tumor cells led to defective cystine uptake, accumulation hibition in vivo (10, 12–14). While these models provide evidence of reactive oxygen species, and impaired tumor growth, supporting a that tumors rely on xCT for proliferation in vivo, they do not ac- cancer cell-autonomous role for xCT. In contrast, we observed that, count for the possibility that whole-body xCT inhibition may have although T cell proliferation in culture was exquisitely dependent on deleterious effects on immune responses, potentially undermining xCT expression, xCT was dispensable for T cell proliferation in vivo the effects of xCT deficiency in tumor cells. and for the generation of primary and memory immune responses to Antigen-specific T cells play critical roles in immuno- tumors. These findings prompted the combination of tumor cell xCT surveillance and are effectors of tumor cell killing during cancer – deletion with the immunotherapeutic agent anti CTLA-4, which dra- immunotherapy. Naïve T cells proliferate, differentiate, and ac- matically increased the frequency and durability of antitumor re- quire effector functions in response to antigenic stimulation of sponses. Together, these results identify a metabolic vulnerability the T cell receptor (TCR) together with costimulatory signals. specific to tumors and demonstrate that xCT disruption can expand Upon stimulation, activated human T cells express xCT (19, 20), the efficacy of anticancer immunotherapies. and their expansion is dependent on adequate concentrations of xCT | cystine | cancer | T cells | immunotherapy Significance he development of T cell-stimulating immunotherapies, such xCT, the cystine–glutamate antiporter, has been implicated in Tas immune checkpoint blockade (ICB), has led to un- supporting both tumor growth and T cell proliferation; thus, precedented durable responses. However, not all tumor types antitumor effects of systemic xCT inhibition may be blunted by succumb to treatment, and the response rate even in sensitive compromised antitumor immunity. This report details the un- – tumor types is less than 30% (1 3). Thus, the clinical benefit of expected finding that xCT is dispensable for T cell proliferation anticancer immunotherapies may be expanded if used in combina- in vivo and for antitumor immune responses. Consequently, tion with therapeutics that target tumor cell-specific vulnerabilities. tumor cell xCT loss acts synergistically with the immunothera- Cancer cells are distinguished from normal cells by their peutic agent anti–CTLA-4, laying the foundation for utilizing oncogene-driven alteration of metabolic pathways (4). A conse- specific xCT inhibitors clinically to expand the efficacy of quence of this metabolic reprogramming is increased production existing anticancer immunotherapeutics. of reactive oxygen species (ROS), which must be countered by endogenous antioxidants to avoid loss of cell viability (5). A key Author contributions: M.D.A., E.C.R., F.W., J.S.M., and C.H.E. designed research; M.D.A., X.S.Y., D.M.L., L.T.-B., M.M., E.C.R., F.W., and C.H.E. performed research; M.D.A., F.W., participant in cellular redox homeostasis is xCT (encoded by the S.D., R.T.A., and C.H.E. analyzed data; and M.D.A., R.T.A., and C.H.E. wrote the paper. Slc7a11 Slc3a2 − gene). xCT together with CD98/ form system xc , Conflict of interest statement: All authors are employees and shareholders of Pfizer. which transports cystine (but not cysteine) into the cell in ex- This article is a PNAS Direct Submission. change for glutamate export (6, 7). Intracellularly, cystine is re- This open access article is distributed under Creative Commons Attribution-NonCommercial- duced to its monomeric form, cysteine, which is important for NoDerivatives License 4.0 (CC BY-NC-ND). several cellular processes, including the production of glutathi- 1To whom correspondence should be addressed. Email: [email protected]. one (GSH), a key intracellular antioxidant. Due to the oxidizing This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. extracellular environment, cystine is more abundant in plasma 1073/pnas.1814932116/-/DCSupplemental. (and in tissue culture medium) compared with cysteine (8, 9). Published online April 24, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1814932116 PNAS | May 7, 2019 | vol. 116 | no. 19 | 9533–9542 Downloaded by guest on October 2, 2021 cystine (19, 21) and the ability to produce GSH (22, 23). Taken resulted in a remarkable increase in durable responses, suggesting together, these findings indirectly support the concept that T cells that systemic inhibition of xCT is a viable strategy to expand the require xCT in a cell-autonomous fashion for proliferation. If this efficacy of anticancer immunotherapies. model is correct, then systemic inhibition of xCT may negatively impact T cell function. Although xCT has been implicated in pro- Results moting the pathophysiology of experimental autoimmune enceph- Loss of xCT Inhibits Tumor Growth. To examine the role of xCT in alomyelitis (EAE), a T cell-driven form of autoimmunity (24, 25), tumor growth, we generated xCT knockout cell lines by the requirement for xCT in supporting T cell proliferation or an- CRISPR-Cas9–mediated targeting of the Slc7a11 gene followed titumor immunity has not been evaluated in vivo. by expansion of single-cell clones. Murine MC38 colon cancer The effects of xCT loss on tumor cells in immunocompetent and Pan02 pancreatic cancer cell lines were selected for gene models and on antitumor immunity were evaluated by genetically editing based on their ability to grow in immunocompetent mice. deleting xCT in both murine tumor lines and immunocompetent Two MC38 clones (2-1 and 2-6) and two Pan02 clones (1-5 and hosts. Loss of xCT in cancer cells led to ROS accumulation, 1-11) that lacked expression of xCT compared with parental xCT resulting in decreased tumor growthinvitroandinvivo.Surpris- WT cells (Fig. 1A) were chosen for additional analysis. The ingly, T cell proliferation and antitumor immunity were not im- functional loss of xCT was assessed by measuring uptake of ra- − − paired in xCT knockout mice, leading us to evaluate the possibility dioactively labeled cystine. All xCT / clones were deficient in of combining systemic xCT loss with the immunotherapeutic agent cystine uptake (Fig. 1 B and C), demonstrating that xCT is the anti–CTLA-4. The combination of xCT deletion with anti–CTLA-4 predominant cystine transporter in these cell types. To confirm A MC38 Pan02 -11 2-6 2-1 WT 1 WT 1-5 xCT HI MC38 Proliferation Pan02 Proliferation 100 100 Vinculin 80 80 e e c MC38 Cystine Uptake c BCPan02 Cystine Uptake n **** en 60 60 u 40 150 **** * ue **** **** *** *** PM 40 40 30 onfl 100 C **** d C ed CPM % Confl 20 % 20 20 ze 50 10 0 0 rmali 0 o 0 0 244872 02448 72 N Normaliz -/- -/- -/- -/-
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