Published OnlineFirst December 17, 2020; DOI: 10.1158/2159-8290.CD-20-0756 RESEARCH BRIEF A Critical Role for Fas-Mediated Off-Target Tumor Killing in T-cell Immunotherapy Ranjan Upadhyay1,2,3, Jonathan A. Boiarsky1,2,3, Gvantsa Pantsulaia1,2,3, Judit Svensson-Arvelund1,2,3, Matthew J. Lin1,2,3, Aleksandra Wroblewska2,3,4, Sherry Bhalla3,4, Nathalie Scholler5, Adrian Bot5, John M. Rossi5, Norah Sadek1,2,3, Samir Parekh1,2,3, Alessandro Lagana4, Alessia Baccarini2,3,4, Miriam Merad2,3,6, Brian D. Brown2,3,4, and Joshua D. Brody1,2,3 ABSTRACT T cell–based therapies have induced cancer remissions, though most tumors ulti- mately progress, reflecting inherent or acquired resistance including antigen Bianca Dunn by Illustration escape. Better understanding of how T cells eliminate tumors will help decipher resistance mecha- nisms. We used a CRISPR/Cas9 screen and identified a necessary role for Fas–FasL in antigen-specific T-cell killing. We also found that Fas–FasL mediated off-target “bystander” killing of antigen-negative tumor cells. This localized bystander cytotoxicity enhanced clearance of antigen-heterogeneous tumors in vivo, a finding that has not been shown previously. Fas-mediated on-target and bystander killing was reproduced in chimeric antigen receptor (CAR-T) and bispecific antibody T-cell models and was augmented by inhibiting regulators of Fas signaling. Tumoral FAS expression alone predicted survival of CAR-T–treated patients in a large clinical trial (NCT02348216). These data suggest strate- gies to prevent immune escape by targeting both the antigen expression of most tumor cells and the geography of antigen-loss variants. SIGNIFIcancE: This study demonstrates the first report ofin vivo Fas-dependent bystander killing of antigen-negative tumors by T cells, a phenomenon that may be contributing to the high response rates of antigen-directed immunotherapies despite tumoral heterogeneity. Small molecules that target the Fas pathway may potentiate this mechanism to prevent cancer relapse. INTRODUCTION contributing to T-cell priming (4–6) and T cell–intrinsic fac- tors (7, 8) both influence antitumor immunity, but tumor T cell–based immunotherapies—including adoptive transfer cell–intrinsic factors have the most abundant clinical evidence of engineered T cells, bispecific antibodies, and checkpoint for contributing to treatment potency and failures. blockade—have revolutionized cancer treatment. However, The clearest such mechanism is target antigen (Ag) mod- even with the remarkably high response rates of chimeric anti- ulation—expression downregulation, lineage switching, or gen receptor (CAR)-T–treated patients, most either progress emergence of splice variants—which is the most common or relapse within one year (1–3). Microenvironmental factors cause of relapse following CAR-T therapy for B-cell acute 1Department of Medicine, Division of Hematology and Medical Oncology, Corresponding Author: Joshua D. Brody, Icahn School of Medicine at Icahn School of Medicine at Mount Sinai, New York, New York. 2Precision Mount Sinai, 1470 Madison Avenue, 5th Floor, Room 106, New York, NY Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, 10029. Phone: 212-241-6756; E-mail: [email protected] 3 New York. Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, Cancer Discov 2021;11:1–15 New York, New York. 4Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York. 5Kite Pharma, doi: 10.1158/2159-8290.CD-20-0756 Santa Monica, California. 6Department of Oncological Sciences, Icahn ©2020 American Association for Cancer Research. School of Medicine at Mount Sinai, New York, New York. Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). March 2021 CANCER DISCOVERY | OF1 Downloaded from cancerdiscovery.aacrjournals.org on September 30, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst December 17, 2020; DOI: 10.1158/2159-8290.CD-20-0756 RESEARCH BRIEF Upadhyay et al. lymphoblastic leukemia (B-ALL; ref. 9). Similar mechanisms an even better predictor of long-term CAR-T therapy out- of Ag escape have also been noted in CAR-T therapy for non- comes than expression of the target molecule, which may Hodgkin lymphoma (NHL; ref. 9), multiple myeloma (10), suggest that homogeneity of Ag expression is not needed to and glioblastoma multiforme (11). Although downregulation achieve full efficacy of Ag-directed therapies. Finally, we pre- of target Ag is a well-characterized occurrence, comparable sent evidence and rationale for combination strategies that rates of Ag+ relapses (12) that are unresponsive to CAR-T rein- augment both on-target and bystander cytotoxic pathways fusion (13–15) also indicate the presence of other immune in order to prevent both Ag+ and Ag− relapses after adoptive evasion mechanisms in tumor cells such as suppression of T-cell therapies. apoptotic pathways. The majority of preclinical and clinical data suggest that RESULTS the tumoricidal effects of murine and human T cells are perforin- and granzyme-mediated (16–18). Consequently, Fas Is a Critical T-Cell Effector Molecule expression of these and other effector molecules (e.g., IFNγ Using a reductionist 3-cell system comprising GFP-spe- and TNFα) is the primary focus of clinical biomarker studies cific (JEDI) CD8 T cells (29, 30), Ag+ A20-GFP cells, and across T cell–based therapies (19–22). However, equal efficacy Ag− A20-mCherry cells (Fig. 1A), we exerted strong iterative of adoptive cell therapy (23) and checkpoint blockade (24) in Ag-specific T-cell selection pressure on the GFP+ popula- perforin-deficient murine models, as well as recent evidence tion (Supplementary Fig. S1A) and sequenced surviving of death receptor signaling gene signatures correlating with cells to measure changes in the frequency of targeted genes CAR-T efficacy (25), supports the notion that other mecha- (Supplementary Fig. S1B). The 291 genes targeted in the nisms may have an underappreciated role in T-cell cytotoxic- screen (Supplementary Table S1) were manually curated ity and warrant further investigation. for high expression in our cancer model and enriched for Additionally, even with optimal Ag-specific tumor clear- immune-related functional annotations. Significant devia- ance, emergence of preexisting Ag-null variants is an tions from the expected frequency of targeted genes in expected mechanism of rapid relapse. Thus, the frequency of the GFP+ cells were internally controlled by the measured durable immunotherapy remissions despite tumoral Ag het- frequency in mCherry+ cells (Supplementary Fig. S1C). erogeneity is surprising. A majority of patients with B-ALL As expected, knockout clones of B2m and Tap1 (Supple- harbor >1% CD19− cells at diagnosis (26), though only ∼20% mentary Fig. S1D and S1E), genes crucial to Ag presen- of patients relapse with CD19− disease after CD19-directed tation, became highly enriched following selection (P < therapy (9). Similarly, response rates of CAR-T therapy for 1e−14; Fig. 1B; Supplementary Fig. S1C). Enrichment of NHL are comparable between subgroups with high and low/ Ag+ cells that had lost MHC class I expression was con- negative tumoral CD19 expression (27). Just as puzzling is firmed at the protein level (Supplementary Fig. S1F and that subclonal loss of β2m, effectively abolishing antigen S1G), in addition to the expected relative loss of PD-L1– presentation to CD8 T cells, in MSI-H colorectal tumors deleted cells (Supplementary Fig. S1F and S1H). Surpris- has no apparent effect on PD-1 blockade efficacy (28). These ingly, knockout clones of Fas became equally enriched (P < data are all highly suggestive of bystander cytotoxicity medi- 1e−14; Fig. 1B; Supplementary Fig. S1C and S1I), a finding ating clearance of Ag− cancer subclones. However, there are unique in our model system compared with other recent no published studies identifying tumor cell mechanisms T cell–based screens (31–34). For validation, we created of how this occurs in vivo despite its enormous therapeutic polyclonally derived stable Fas-knockout lines of A20 lym- potential. phoma as well as 4T1 breast cancer (Fig. 1C) that had no To dissect these mechanisms and address these impor- measurable surface Fas protein expression (Supplementary tant knowledge gaps, we performed a CRISPR/Cas9 screen Fig. S2A) and were resistant to Fas-induced cell death using a targeted library of genes highly expressed by the (Supplementary Fig. S2B). Although wild-type (WT) GFP+ A20 murine lymphoma cell line and identified the death cells were depleted by JEDI, both Fas-negative lymphoma receptor Fas as crucial to T-cell cytotoxicity. For the first (Fig. 1D; quantified in Supplementary Fig. S2C) and breast time, we demonstrate that Fas-mediated bystander killing cancer (Fig. 1E; quantified in Supplementary Fig. S2D) enhances in vivo control of heterogeneous tumors. We also demonstrated resistance to killing under the same con- demonstrate that pretreatment tumoral FAS expression is ditions, despite robust T-cell proliferation, granzyme B Figure 1. A pooled CRISPR/Cas9 screen for T cell cytolytic mechanisms identifiesFas as crucial to on-target anticancer immunity. A, Schema depict- ing screening strategy; A20-GFP cells (Ag+; green), A20-mCherry cells (Ag−; red), GFP-specific T cells (blue). NGS, next-generation sequencing. B, Summary of 291-gene screen across four pooled libraries after 0, 1, or 2 iterations