Natural killer (NK) cells are primary defenders against cancer precursors, but cancer cells can persist by evading immune surveillance. To investigate the genetic mechanisms underlying this evasion, we perform a genome-wide CRISPR screen using B lymphoblastoid cells. SPPL3, a peptidase that cleaves glycosyltransferases in the Golgi, emerges as a top hit facilitating evasion from NK cytotoxicity. SPPL3-deleted cells accumulate glycosyltransferases and complex N-glycans, disrupting not only binding of ligands to NK receptors but also binding of rituximab, a CD20 antibody approved for treating B cell cancers. Notably, inhibiting N-glycan maturation restores receptor binding and sensitivity to NK cells. A secondary CRISPR screen in SPPL3-deficient cells identifies B3GNT2, a transferase-mediating poly-LacNAc extension, as crucial for resistance. Mass spectrometry confirms enrichment of N-glycans bearing poly-LacNAc upon SPPL3 loss. Collectively, our study shows the essential role of SPPL3 and poly-LacNAc in cancer immune evasion, suggesting a promising target for cancer treatment.
Published by Elsevier Inc.

Natural killer (NK) cells derived or isolated from different sources have been gaining in importance for cancer therapies. In this study, we evaluate and compare key characteristics between NK cells derived or isolated from umbilical cord blood, umbilical cord blood hematopoietic stem/progenitor cells, peripheral blood, and induced pluripotent stem cells (iPSCs). Specifically, we find CD56+ NK cells isolated and expanded directly from umbilical cord blood (UCB56) and NK cells derived from CD34+ hematopoietic stem/progenitors in umbilical cord blood (UCB34) differ in their expression of markers associated with differentiation including CD16, CD2, and killer Ig-like receptors (KIRs). UCB56-NK cells also displayed a more potent cytotoxicity compared to UCB34-NK cells. NK cells derived from iPSCs (iPSC-NK cells) were found to have variable KIR expression, with certain iPSC-NK cell populations expressing high levels of KIRs and others not expressing KIRs. Notably, KIR expression on UCB56 and iPSC-NK cells had limited effect on cytotoxic activity when stimulated by tumor target cells that express high levels of cognate HLA class I, suggesting that in vitro differentiation and expansion may override the KIR-HLA class I mediated inhibition when used across HLA barriers. Together our results give a better understanding of the cell surface receptor, transcriptional, and functional differences between NK cells present in umbilical cord blood and hematopoietic progenitor-derived NK cells which may prove important in selecting the most active NK cell populations for treatment of cancer or other therapies.
Copyright © 2020 Goldenson, Zhu, Wang, Heragu, Bernareggi, Ruiz-Cisneros, Bahena, Ask, Hoel, Malmberg and Kaufman.

Cytokine-inducible SH2-containing protein (CIS; encoded by the gene CISH) is a key negative regulator of interleukin-15 (IL-15) signaling in natural killer (NK) cells. Here, we develop human CISH-knockout (CISH-/-) NK cells using an induced pluripotent stem cell-derived NK cell (iPSC-NK cell) platform. CISH-/- iPSC-NK cells demonstrate increased IL-15-mediated JAK-STAT signaling activity. Consequently, CISH-/- iPSC-NK cells exhibit improved expansion and increased cytotoxic activity against multiple tumor cell lines when maintained at low cytokine concentrations. CISH-/- iPSC-NK cells display significantly increased in vivo persistence and inhibition of tumor progression in a leukemia xenograft model. Mechanistically, CISH-/- iPSC-NK cells display improved metabolic fitness characterized by increased basal glycolysis, glycolytic capacity, maximal mitochondrial respiration, ATP-linked respiration, and spare respiration capacity mediated by mammalian target of rapamycin (mTOR) signaling that directly contributes to enhanced NK cell function. Together, these studies demonstrate that CIS plays a key role to regulate human NK cell metabolic activity and thereby modulate anti-tumor activity.
Copyright © 2020 Elsevier Inc. All rights reserved.

Advanced-stage cutaneous T-cell lymphoma (CTCL) is usually a fatal malignancy despite optimal use of currently available treatments. In this preclinical study of novel CTCL therapy, we performed in vitro and ex vivo experiments to determine the efficacy of combination treatment with a panel of BET bromodomain inhibitors (BETi) (JQ1, OTX015, CPI-0610, I-BET762) and HDAC inhibitors (HDACi) (SAHA/Vorinostat, Romidepsin). BETi/HDACi combinations were synergistic (combination index <1) against cell viability and induced G0/G1 cell cycle arrest. Apoptosis was uniformly enhanced. From a mechanistic standpoint, proliferative drivers c-Myc, Cyclin D1, NFkB, and IL-15Rα were reduced. Inhibitory CDKN1A was increased. CDKN1B, IL-7R, IL-17Rα, STAT3, and STAT5 alterations varied. There were significant increases in extrinsic apoptotic pathway death receptors and ligands (FasL, DR4, DR5, TRAIL, and TNFR1). At clinically tolerable levels of single agents, Romidepsin (1 nM) + OTX015 (125 nM) induced the greatest apoptosis (60%_80%) at 96 hours. Ex vivo studies of leukemic CTCL cells obtained from patients with Sezary syndrome also showed higher levels of apoptosis (about 60%-90%) in response to combination treatments relative to single agents. In contrast, combination treatment of normal CD4+ T cells induced only minimal apoptosis (<10%). Our findings show that the mechanism of action of BETi/HDACi therapy in CTCL involves induction of both cell cycle arrest and apoptosis with reduced proliferative drivers and enhanced expression of apoptotic extrinsic pathway death receptors and ligands. Relative to single agents, the superior anti-CTCL effects of BETi/HDACi combinations in vitro and ex vivo provide a rationale for clinical trials exploring their efficacy as therapy for CTCL.
Published by Elsevier Inc.

Chimeric antigen receptors (CARs) significantly enhance the anti-tumor activity of immune effector cells. Although most studies have evaluated CAR expression in T cells, here we evaluate different CAR constructs that improve natural killer (NK) cell-mediated killing. We identified a CAR containing the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3ζ signaling domain to mediate strong antigen-specific NK cell signaling. NK cells derived from human iPSCs that express this CAR (NK-CAR-iPSC-NK cells) have a typical NK cell phenotype and demonstrate improved anti-tumor activity compared with T-CAR-expressing iPSC-derived NK cells (T-CAR-iPSC-NK cells) and non-CAR-expressing cells. In an ovarian cancer xenograft model, NK-CAR-iPSC-NK cells significantly inhibited tumor growth and prolonged survival compared with PB-NK cells, iPSC-NK cells, or T-CAR-iPSC-NK cells. Additionally, NK-CAR-iPSC-NK cells demonstrate in vivo activity similar to that of T-CAR-expressing T cells, although with less toxicity. These NK-CAR-iPSC-NK cells now provide standardized, targeted "off-the-shelf" lymphocytes for anti-cancer immunotherapy.
Copyright © 2018 Elsevier Inc. All rights reserved.