{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,28]],"date-time":"2026-10-28T18:22:37Z","timestamp":1793211757696,"version":"4.5.0"},"reference-count":41,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,3,7]],"date-time":"2025-03-07T00:00:00Z","timestamp":1741305600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000046","name":"National Research Council Canada","doi-asserted-by":"publisher","award":["CGT-501-2"],"award-info":[{"award-number":["CGT-501-2"]}],"id":[{"id":"10.13039\/501100000046","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009326","name":"Cancer Research Society","doi-asserted-by":"publisher","award":["#1057810"],"award-info":[{"award-number":["#1057810"]}],"id":[{"id":"10.13039\/100009326","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000024","name":"Canadian Institutes of Health Research","doi-asserted-by":"publisher","award":["PJT-518790, PJT-178197"],"award-info":[{"award-number":["PJT-518790, PJT-178197"]}],"id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:p>Allogeneic natural killer (NK) cell therapy has demonstrated significant potential in cancer immunotherapy by harnessing NK cells to target malignancies. CD138-targeting chimeric antigen receptor (CAR)-engineered NK cells offer a promising therapeutic option for multiple myeloma (MM). However, sustaining CAR expression on CAR-NK cells during <jats:italic>ex vivo<\/jats:italic> expansion poses a challenge to developing effective immunotherapies. In this study, primary NK cells were isolated, cryopreserved, and modified to express anti-CD138 CARs through retroviral transduction. Histone deacetylase inhibitors (HDACi), particularly entinostat (ENT), were applied to enhance CAR expression stability in CAR-NK cells. Our findings indicate that ENT treatment significantly improves and maintains CAR expression, thereby enhancing the cytotoxic activity of CAR-NK cells against CD138-positive multiple myeloma cells. ENT-treated CAR-NK cells exhibited prolonged persistence and more significant tumor reduction in an MM tumor-bearing mouse model, highlighting the therapeutic potential of HDACi-treated CAR-NK cells. This study provides the first evidence that HDAC inhibitors can sustain CAR expression in CAR-NK cells in a promoter-dependent manner, potentially enhancing anti-tumor efficacy in multiple myeloma and underscoring the possible need for further clinical evaluation.<\/jats:p>","DOI":"10.3389\/fimmu.2025.1533044","type":"journal-article","created":{"date-parts":[[2025,3,7]],"date-time":"2025-03-07T06:48:33Z","timestamp":1741330113000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":17,"title":["Entinostat, a histone deacetylase inhibitor, enhances CAR-NK cell anti-tumor activity by sustaining CAR expression"],"prefix":"10.3389","volume":"16","author":[{"given":"Dong-Hyeon","family":"Jo","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shelby","family":"Kaczmarek","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abrar Ul Haq","family":"Khan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jannat","family":"Pervin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Diana M.","family":"Clark","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suresh","family":"Gadde","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lisheng","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Scott","family":"McComb","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alissa","family":"Visram","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seung-Hwan","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,3,7]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1182\/blood.2022016200","article-title":"Allogeneic natural killer cell therapy","volume":"141","author":"Berrien-Elliott","year":"2023","journal-title":"Blood"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1038\/s41423-024-01145-x","article-title":"Development of NK cell-based cancer immunotherapies through receptor engineering","volume":"21","author":"Page","year":"2024","journal-title":"Cell Mol Immunol"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2023.1166038","article-title":"Advancements in CAR-NK therapy: lessons to be learned from CAR-T therapy","volume":"14","author":"Kilgour","year":"2023","journal-title":"Front Immunol"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1016\/j.omtm.2023.03.006","article-title":"Simultaneous engineering of natural killer cells for CAR transgenesis and CRISPR-Cas9 knockout using retroviral particles","volume":"29","author":"Jo","year":"2023","journal-title":"Mol Ther Methods Clin Dev"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-022-02003-x","article-title":"KIR-based inhibitory CARs overcome CAR-NK cell trogocytosis-mediated fratricide and tumor escape","volume":"28","author":"Li","year":"2022","journal-title":"Nat Med"},{"key":"B6","doi-asserted-by":"publisher","first-page":"102975","DOI":"10.1016\/j.ebiom.2020.102975","article-title":"CAR-NK cells: A promising cellular immunotherapy for cancer","volume":"59","author":"Xie","year":"2020","journal-title":"EBioMedicine"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-023-02785-8","article-title":"Safety, efficacy and determinants of response of allogeneic CD19-specific CAR-NK cells in CD19(+) B cell tumors: a phase 1\/2 trial","volume":"30","author":"Marin","year":"2024","journal-title":"Nat Med"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1080\/08830185.2021.1984449","article-title":"The immunologic aspects of\u00a0cytokine\u00a0release syndrome and graft versus host disease following CAR T\u00a0cell\u00a0therapy","volume":"41","author":"Mansouri","year":"2022","journal-title":"Int Rev Immunol"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2007-10-078022","article-title":"Multiple myeloma","volume":"111","author":"Kyle","year":"2008","journal-title":"Blood"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-023-02496-0","article-title":"Idecabtagene vicleucel for relapsed and refractory multiple myeloma: post hoc 18-month follow-up of a phase 1 trial","volume":"29","author":"Lin","year":"2023","journal-title":"Nat Med"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1002\/ajh.26590","article-title":"Multiple myeloma: 2022 update on diagnosis, risk stratification, and management","volume":"97","author":"Rajkumar","year":"2022","journal-title":"Am J Hematol"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1016\/j.clml.2019.02.006","article-title":"Indatuximab ravtansine (BT062) monotherapy in patients with relapsed and\/or refractory multiple myeloma","volume":"19","author":"Jagannath","year":"2019","journal-title":"Clin Lymphoma Myeloma Leuk"},{"key":"B13","doi-asserted-by":"publisher","first-page":"5562630","DOI":"10.1155\/2021\/5562630","article-title":"Selective cytotoxicity of single and dual anti-CD19 and anti-CD138 chimeric antigen receptor-natural killer cells against hematologic Malignancies","volume":"2021","author":"Luanpitpong","year":"2021","journal-title":"J Immunol Res"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.3389\/fonc.2024.1370854","article-title":"Targeted therapy for multiple myeloma: an overview on CD138-based strategies","volume":"14","author":"Riccardi","year":"2024","journal-title":"Front Oncol"},{"key":"B15","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1007\/978-1-0716-1441-9_1","article-title":"Generation of CAR-T cells by lentiviral transduction","volume":"2312","author":"Okuma","year":"2021","journal-title":"Methods Mol Biol"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1016\/j.cels.2022.11.005","article-title":"The sound of silence: Transgene silencing in mammalian cell engineering","volume":"13","author":"Cabrera","year":"2022","journal-title":"Cell Syst"},{"key":"B17","doi-asserted-by":"publisher","first-page":"556","DOI":"10.3390\/genes11050556","article-title":"Histone deacetylases (HDACs): evolution, specificity, role in transcriptional complexes, and pharmacological actionability","volume":"11","author":"Milazzo","year":"2020","journal-title":"Genes (Basel)"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.3389\/fonc.2023.1244035","article-title":"A review of the therapeutic potential of histone deacetylase inhibitors in rhabdomyosarcoma","volume":"13","author":"Selim","year":"2023","journal-title":"Front Oncol"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.12717\/DR.2013.17.4.379","article-title":"Enhancement of transgene expression by HDAC inhibitors in mouse embryonic stem cells","volume":"17","author":"Kim","year":"2013","journal-title":"Dev Reprod"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1002\/bit.v113.6","article-title":"The histone deacetylase inhibitor Entinostat enhances polymer-mediated transgene expression in cancer cell lines","volume":"113","author":"Elmer","year":"2016","journal-title":"Biotechnol Bioeng"},{"key":"B21","doi-asserted-by":"publisher","first-page":"23","DOI":"10.1016\/j.jviromet.2004.11.023","article-title":"Valproic acid enhances gene expression from viral gene transfer vectors","volume":"125","author":"Fan","year":"2005","journal-title":"J Virol Methods"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1016\/j.omto.2021.01.014","article-title":"HDAC inhibition prevents transgene expression downregulation and loss-of-function in T-cell-receptor-transduced T cells","volume":"20","author":"Moore","year":"2021","journal-title":"Mol Ther Oncolytics"},{"key":"B23","doi-asserted-by":"publisher","first-page":"98","DOI":"10.1038\/s41392-022-00915-1","article-title":"Identification of NOXA as a pivotal regulator of resistance to CAR T-cell therapy in B-cell Malignancies","volume":"7","author":"Yan","year":"2022","journal-title":"Signal Transduct Target Ther"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0014700","article-title":"The HDAC inhibitor FK228 enhances adenoviral transgene expression by a transduction-independent mechanism but does not increase adenovirus replication","volume":"6","author":"Danielsson","year":"2011","journal-title":"PloS One"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.3791\/2540-v","article-title":"Expansion, purification, and functional assessment of human peripheral blood NK cells","volume":"48","author":"Somanchi","year":"2011","journal-title":"J Vis Exp"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1016\/j.omtm.2020.01.012","article-title":"A high-throughput method for characterizing novel chimeric antigen receptors in jurkat cells","volume":"16","author":"Bloemberg","year":"2020","journal-title":"Mol Ther Methods Clin Dev"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2014-02-558163","article-title":"Baboon envelope pseudotyped LVs outperform VSV-G-LVs for gene transfer into early-cytokine-stimulated and resting HSCs","volume":"124","author":"Girard-Gagnepain","year":"2014","journal-title":"Blood"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.3343\/alm.2022.42.6.638","article-title":"Natural killer cell expansion and cytotoxicity differ depending on the culture medium used","volume":"42","author":"Koh","year":"2022","journal-title":"Ann Lab Med"},{"key":"B29","doi-asserted-by":"publisher","first-page":"1063","DOI":"10.1016\/j.cmet.2020.11.007","article-title":"SCENITH: A flow cytometry-based method to functionally profile energy metabolism with single-cell resolution","volume":"32","author":"Arguello","year":"2020","journal-title":"Cell Metab"},{"key":"B30","doi-asserted-by":"publisher","first-page":"39","DOI":"10.1016\/j.jim.2004.11.021","article-title":"Biophotonic cytotoxicity assay for high-throughput screening of cytolytic killing","volume":"297","author":"Brown","year":"2005","journal-title":"J\u00a0Immunol Methods"},{"key":"B31","doi-asserted-by":"publisher","first-page":"3208","DOI":"10.3390\/cells10113208","article-title":"Sensitivity and specificity of CD19.CAR-T cell detection by flow cytometry and PCR","volume":"10","author":"Schanda","year":"2021","journal-title":"Cells"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.2217\/fon-2016-0526","article-title":"Entinostat: a promising treatment option for patients with advanced breast cancer","volume":"13","author":"Connolly","year":"2017","journal-title":"Future Oncol"},{"key":"B33","doi-asserted-by":"publisher","first-page":"197","DOI":"10.3892\/ol.2024.14330","article-title":"Effect of valproic acid on histone deacetylase expression in oral cancer (Review)","volume":"27","author":"Al-Khafaji","year":"2024","journal-title":"Oncol Lett"},{"key":"B34","doi-asserted-by":"publisher","first-page":"58","DOI":"10.1016\/j.bcp.2016.03.010","article-title":"HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-kappaB p65 transcriptional activity","volume":"108","author":"Leus","year":"2016","journal-title":"Biochem Pharmacol"},{"key":"B35","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2022.864868","article-title":"Programmable attenuation of antigenic sensitivity for a nanobody-based EGFR chimeric antigen receptor through hinge domain truncation","volume":"13","author":"McComb","year":"2022","journal-title":"Front Immunol"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.1038\/cgt.2009.65","article-title":"An HDAC inhibitor enhances the antitumor activity of a CMV promoter-driven DNA vaccine","volume":"17","author":"Lai","year":"2010","journal-title":"Cancer Gene Ther"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.1038\/s41551-023-01013-5","article-title":"A lentiviral vector for the production of T cells with an inducible transgene and a constitutively expressed tumor-targeting receptor","volume":"7","author":"Reichenbach","year":"2023","journal-title":"Nat BioMed Eng"},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.1111\/imr.v320.1","article-title":"Coengineering specificity, safety, and function into T cells for cancer immunotherapy","volume":"320","author":"Giordano Attianese","year":"2023","journal-title":"Immunol Rev"},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.1016\/j.omtm.2020.06.014","article-title":"Superior expansion and cytotoxicity of human primary NK and CAR-NK cells from various sources via enriched metabolic pathways","volume":"18","author":"Yang","year":"2020","journal-title":"Mol Ther Methods Clin Dev"},{"key":"B40","doi-asserted-by":"publisher","first-page":"451","DOI":"10.3390\/cells13050451","article-title":"Building a better defense: expanding and improving natural killer cells for adoptive cell therapy","volume":"13","author":"Maia","year":"2024","journal-title":"Cells"},{"key":"B41","doi-asserted-by":"publisher","first-page":"100236","DOI":"10.1016\/j.crmeth.2022.100236","article-title":"Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV","volume":"2","author":"Naeimi Kararoudi","year":"2022","journal-title":"Cell Rep Methods"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1533044\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,3,7]],"date-time":"2025-03-07T06:48:36Z","timestamp":1741330116000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1533044\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,3,7]]},"references-count":41,"alternative-id":["10.3389\/fimmu.2025.1533044"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2025.1533044","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,3,7]]},"article-number":"1533044"}}