Not So Fast: Tumor Cells Resisting Death Drive CAR T-Cell Dysfunction Michael R

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IN THE SPOTLIGHT Not So FASt: Tumor Cells Resisting Death Drive CAR T-cell Dysfunction Michael R. Green 1 ,2 and Sattva S. Neelapu 1 Summary: In this issue, Singh and colleagues describe a novel tumor-intrinsic mechanism of resistance to chimeric antigen receptor (CAR) T-cell therapy targeting CD19 in B-cell malignancies. They show that reduced expression of death receptor genes in the tumors mediates resistance to killing by CAR T cells, leads to progressive CAR T-cell dysfunction, and is associated with unfavorable clinical outcome in patients.

See related article by Singh et al., p. 552 (7 ).

Chimeric antigen receptor (CAR) T-cell therapy targeting may be infl uenced by host conditioning prior to infusion CD19 is highly effective in refractory B-cell malignancies, ( 5 ). The CAR T cells could also become dysfunctional after inducing response rates in up to 80% to 90% of patients with they traffi c to the tumor site due to immunosuppressive acute lymphoblastic leukemia (ALL) and various non-Hodgkin mechanisms in the tumor microenvironment mediated by lymphoma subtypes ( 1, 2 ). However, long-term durability regulatory T cells, myeloid-derived suppressor cells, immu- of these responses is observed in only approximately 40% to nosuppressive cytokines, and/or other immune checkpoints. 50% of these patients, highlighting the need to understand Immune escape due to CD19 loss has been reported in a the mechanisms of primary as well as acquired resistance signifi cant proportion in patients with ALL and lymphomas following CAR T-cell therapy. Resistance mechanisms can treated with anti-CD19 CAR T-cell therapy ( 1, 2 ). Loss of be categorized into those that are related to the fi tness and CD19 may be mediated by acquired mutations in CD19 and/ functional state of the T cells in the apheresis and/or infu- or alternative splicing of CD19 with deletion of exon 2 result- sion products, and tumor-intrinsic mechanisms of resistance, ing in sequestration of the CD19 protein in the endoplasmic with multiple levels of cross-talk likely existing between the reticulum or deletion of exons 5–6 that encode the trans- biology of the tumor cells and the functionality of a patient’s membrane domain ( 6 ). Relapse of the tumor with CD19 loss T cells (Fig. 1). would suggest that the CAR T-cell product used was of good The fi tness of the CAR T cells may be affected because of quality, as it likely eliminated all CD19-expressing tumor dysfunctional T cells in the leukapheresis product resulting cells. In contrast, if the relapsed tumor is CD19 positive, it from prior lines of therapy, excessive stimulation or other cul- raises the possibility that the resistance is likely because of ture conditions during manufacturing, or insuffi cient host impaired T-cell fi tness or alternative mechanisms of tumor- conditioning preinfusion, or resulting from the effects of intrinsic resistance to CAR T-cell killing. the tumor microenvironment on T cells prior to apheresis or In this issue of Cancer Discovery, Singh and colleagues after infusion. Indeed, in a cohort of patients with chronic describe a novel tumor-intrinsic mechanism that confers lymphocytic leukemia who were treated with anti-CD19 CAR resistance to cytotoxicity by anti-CD19 CAR T (CART19) T-cell therapy, Fraietta and colleagues found that the func- cells and also drives progressive CART19 dysfunction ( 7 ). tional phenotype of the T cells in the leukapheresis product To understand the mechanism of primary resistance to affected the quality of the CAR T-cell product that could be CART19 therapy, they performed an unbiased CRISPR-based generated, which was in turn associated with clinical effi cacy genome-wide loss-of-function screen in an ALL cell line, (3 ). Rossi and colleagues reported an association between Nalm6, and found that guides targeting genes associated the polyfunctionality of the preinfusion CAR T cells and with proapoptotic death receptor signaling pathway includ- clinical outcome in patients with lymphoma ( 4 ). Turtle and ing FADD, BID, CASP8, and TNFRSF10B were signifi cantly colleagues showed that the function of CAR T cells in vivo, as enriched for CART19 resistance. Conversely, guides targeting suggested by their expansion and persistence postinfusion, antiapoptotic molecules such as CFLAR, TRAF2 , and BIRC2 were depleted. They validated these fi ndings by generating CRISPR-edited Nalm6 cells lacking FADD, a critical adaptor molecule for signaling from all proapoptotic death receptors, 1Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, Texas. 2 Department of Genomic Medi- or BID, a cell death regulator that is activated downstream of cine, The University of Texas MD Anderson Cancer Center, Houston, Texas. FADD and induces release of cytochrome c from mitochon- Corresponding Author: Sattva S. Neelapu, The University of Texas MD Ander- dria, leading to apoptosis. Long-term cultures of a mixture of son Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: wild-type (WT) and knockout (KO) Nalm6 cells with CART19 713-792-2860; Fax: 713-563-3469; E-mail: [email protected] resulted in progressive enrichment of KO cells. Additional Cancer Discov 2020;10:492–4 studies further corroborated the role of death receptor sign- doi: 10.1158/2159-8290.CD-20-0037 aling in cytotoxic killing by CART19 cells. Coculture of WT ©2020 American Association for Cancer Research. Nalm6 cells with CART19 in the presence of an inhibitor

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Mechanisms of CAR T resistance

CAR T-cell intrinsic Tumor intrinsic Apheresis product Antigen escape Exhaustion Functional state Alternative Epitope masking Lineage switch splicing/mutation PD-1+ LAG3+ T CD8 T SCM EM

T cell T cell AML B cell

Infusion product Immunosuppressive tumor microenvironment Exhaustion Functional state B cell T CD8 T PD-1+ LAG3+ SCM EM Cross- talk Myeloid Treg CAR T CAR TCAR T

Polyfunctionality

Insufficient conditioning Impaired death receptor signaling Reduced expansion Reduced FAS/TRAIL B cell CAR T T-cell signaling dysfunction DR CAR T cells/mL (t)

Figure 1. Mechanisms of resistance to CAR T-cell therapy. Resistance mechanisms to CAR T-cell therapy may be T cell–intrinsic because of exhausted or dysfunctional T cells in the apheresis or infusion product and inadequate numbers of effector, stem cell memory, or polyfunctional T cells. It may also be due to insufficient conditioning of the host, which results in reduced expansion of CAR T cells. Alternatively, resistance may be mediated by tumor- intrinsic mechanisms such as antigen escape due to alternate splicing or mutation with loss of CAR-binding epitope, epitope masking due to inadvertent transduction of the CAR molecule into the malignant tumor cell, or lineage switch from B-cell ALL to acute myeloid leukemia (AML). Immunosuppressive mechanisms in the tumor microenvironment may also inhibit the function of CAR T cells once they traffic to the tumor site. In this issue, Singh and colleagues showed that impaired death receptor (DR) signaling in tumor cells leads to persistence of the tumor, resulting in chronic antigenic exposure and CAR T-cell dysfunction. of BIRC2, birinapant, resulted in enhanced killing, whereas induction of CAR T-cell dysfunction may have been mediated KO of FasL or TRAIL in CART19 decreased their cytotoxic by prolonged antigen exposure analogous to classic T-cell function. Similar effects were observed with additional ALL exhaustion rather than impaired death receptor signaling. models, a diffuse large B-cell lymphoma model, CAR T cells Transcriptional profiling of CART19 cells exposed long-term targeting CD22, and CAR T cells with alternative costimula- to BIDKO Nalm6 cells was also consistent with a dysfunctional tory domains. Importantly, the resistance to CART19 ther- T-cell signature compared with CART19 cells exposed to apy was also demonstrated in vivo in NOD/SCID/γc−/− mice WT Nalm6. Moreover, evaluation by ATAC-seq revealed an engrafted with FADDKO or BIDKO Nalm6 cells. epigenetic signature indicative of exhaustion, although not Because T cells may mediate killing of target cells by acti- completely identical to changes observed with classic T-cell vating the extrinsic apoptotic pathway via the surface death exhaustion. Integrative analysis of transcriptomic and epi- receptors or by activating the intrinsic apoptotic pathway genomic data showed upregulation of immunosuppressive through secretion of cytotoxic molecules such as granzymes transcription factors (TOX2, IRF8, and PRDM1) along with and perforin, the resistance observed with only disrup- increased promoter accessibility of these loci. Taken together, tion of the death receptors was unexpected. To explain this these results suggested that disruption of the death receptor finding, the authors reasoned that impairing the extrinsic signaling pathway leads to decreased tumor killing and per- apoptotic pathway likely led to CAR T-cell dysfunction. Con- sistent antigen exposure that in turn results in global tran- sistent with this notion, they found that long-term culture scriptomic and epigenetic reprogramming of CAR T cells, of CART19 with BIDKO Nalm6 cells caused reduced pro- adversely affecting their function (Fig. 1). liferation, cytokine secretion, and production of perforin To determine whether these observations are relevant clini- and granzymes. Similar effects were observed with continuous cally, the authors then assessed the baseline tumor samples exposure of CART19 to WT Nalm6 cells, suggesting that the from patients with ALL treated on two independent clinical

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Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. VIEWS trials with tisagenlecleucel, an anti-CD19 CAR T-cell product Disclosure of Potential Conflicts of Interest with 4-1BB and CD3ζ signaling domains (1). They found that a M.R. Green is a consultant at VeraStem Oncology and has owner- lower death receptor gene signature score representative of the ship interest in KDAc Therapeutics. S.S. Neelapu is a consultant at extrinsic apoptotic pathway, but not a gene signature represent- Kite/Gilead, Celgene, Calibr, Legend Biotech, Novartis, Unum Thera- ative of the intrinsic apoptotic pathway, was associated with peutics, Pfizer, Merck, Precision Biosciences, Cell Medica, Incyte, primary resistance with persistent CD19-expressing tumors and Allogene; reports receiving commercial research grants from following tisagenlecleucel. In contrast, a higher death receptor Kite/Gilead, Cellectis, Poseida, Merck, Acerta, Karus, Bristol-Myers gene signature score was associated with durable remission and Squibb, Unum Therapeutics, Allogene, and Precision Biosciences, better overall survival in these patients. Moreover, consistent and has patents related to cell therapy. No other potential conflicts of interest were disclosed. with preclinical studies, patients with a lower death receptor gene signature score had decreased expansion and persistence Acknowledgments of CAR T cells in vivo. Finally, single-cell transcriptomic analysis This work was supported by the MD Anderson Cancer Center Sup- of CAR T cells obtained from patients 10 days after infusion port Grant P30 CA016672 from the NIH. We thank Dave Aten for showed a higher level of exhaustion-associated genes in a non- assistance with the graphics illustration. responder compared with a patient achieving durable response. Collectively, this study has important implications for Published online April 1, 2020. both basic and translational research in the rapidly growing field of cellular therapy. From a basic biology perspective, it suggests that, like T cells with native T-cell receptors, REFERENCES CAR T cells may mediate cytotoxicity by activating the . 1 Maude SL, Laetsch TW, Buechner J, Rives S, Boyer M, Bittencourt intrinsic or extrinsic apoptosis pathway and that they can H, et al. Tisagenlecleucel in children and young adults with B-cell acquire exhaustion-like features with chronic antigen expo- lymphoblastic leukemia. N Engl J Med 2018;378:439–48. 2. Neelapu SS, Locke FL, Bartlett NL, Lekakis LJ, Miklos DB, Jacobson sure. From a translational standpoint, it sheds light on CA, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory a novel mechanism of resistance to CAR T cells that is large B-cell lymphoma. 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PTPN2 mechanism further could lead to development of novel strat- phosphatase deletion in T cells promotes anti-tumour immunity and egies to improve the efficacy of CAR T-cell therapy. CAR T-cell efficacy in solid tumours. EMBO J 2020;39:e103637.

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Michael R. Green and Sattva S. Neelapu

Cancer Discov 2020;10:492-494.

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