{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T13:54:30Z","timestamp":1781877270146,"version":"3.54.5"},"posted":{"date-parts":[[2026,6,17]]},"group-title":"Immunology","reference-count":77,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T00:00:00Z","timestamp":1781654400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NCI","award":["5R01CA262145"],"award-info":[{"award-number":["5R01CA262145"]}]},{"name":"NCI","award":["ARTNet 5U54CA224019"],"award-info":[{"award-number":["ARTNet 5U54CA224019"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,17]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  AML is an aggressive blood cancer associated with poor clinical outcomes. Chemotherapy remains the standard of treatment, but unfortunately relapse is very common, highlighting the need for alternative therapies. T cell dysfunction and exhaustion are prominent in AML and may represent a barrier to effective immunotherapy yet remains poorly studied in AML. DNA methylation is a major driver of T cell exhaustion and inhibition of\n                  <jats:italic>de novo<\/jats:italic>\n                  methylation can block exhaustion and restore T cell function in chronic viral infections and other cancers but is understudied in AML. Here, we investigated the impact of azacytidine (Aza), an FDA-approved hypomethylating agent, on T cell exhaustion in AML. Using a spontaneous AML mouse model and samples from patients with AML, we found that Aza treatment modulates T cell function.\n                  <jats:italic>In vivo<\/jats:italic>\n                  Aza-treatment of AML-bearing mice decreased tumor burden and reshaped CD8+ T cell states, with increases in frequencies of memory subsets and decreases in regulatory T cells (Tregs). Functionally, Aza treatment overcame the impaired proliferation displayed by both CD4 and CD8+ T cells in our model. DNA methylation sequencing of T cells after Aza treatment revealed hypomethylation and increased expression of stem-like precursor gene\n                  <jats:italic>TCF7<\/jats:italic>\n                  and\n                  <jats:italic>E2F2<\/jats:italic>\n                  , a regulator of cell cycle progression and proliferation. Similar changes in phenotypes were observed in cultures of AML patient samples treated with Aza. Collectively, we show that Aza remodels epigenetic and functional states in AML and has the potential to reverse T cell exhaustion, with enhanced memory and proliferation capacity. Our work generates a mechanistic framework that provides rationale of combining hypomethylating agents with T cell-based immunotherapies in this lethal disease.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Data Sharing Statement<\/jats:title>\n                  <jats:p>RRBS data is available in GEO under the accession number GSE328721. For original data please contact Dr. Evan F. Lind.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Key Points<\/jats:title>\n                  <jats:p>Azacytidine mediated epigenetic modulation can alleviate T cell exhaustion in AML<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Translational Relevance<\/jats:title>\n                  <jats:p>Immune therapy has shown limited efficacy in AML, despite increasing evidence of T cell dysfunction in this malignancy. Azacytidine (Aza) is an FDA approved drug for AML, but patients develop therapy resistance and relapse. Studies have mainly focused on Aza\u2019s tumor intrinsic effects. In this study, we investigated the impact of Aza on immune function, especially T cell exhaustion in AML, since exhaustion is a major mechanism of disease resistance. We demonstrated that Aza can modulate T cell phenotype and restore T cell proliferation. Mechanistically, Aza induces epigenetic reprogramming in T cells and increases the expression of a stem-like precursor marker, TCF7. By shifting the focus on T cell biology, our study provides a rationale for combining Aza with other immunotherapies that can enhance durable immune responses in this malignancy.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.06.14.732148","type":"posted-content","created":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T23:40:13Z","timestamp":1781739613000},"source":"Crossref","is-referenced-by-count":0,"title":["Azacytidine restores T cell function in AML by modulating DNA methylation"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6110-6608","authenticated-orcid":false,"given":"Ravina","family":"Pandita","sequence":"first","affiliation":[{"name":"Graduate Program in Biomedical Sciences, Oregon Health & Science University, Portland Oregon, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoko","family":"Kosaka","sequence":"additional","affiliation":[{"name":"Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland Oregon, USA"},{"name":"Knight Cancer Institute, Oregon Health & Science University, Portland Oregon, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jessica S.","family":"Mulkey","sequence":"additional","affiliation":[{"name":"Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland Oregon, USA"},{"name":"Knight Cancer Institute, Oregon Health & Science University, Portland Oregon, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cora E.","family":"Layman","sequence":"additional","affiliation":[{"name":"Knight Cardiovascular Institute, Oregon Health & Science University, Portland, OR, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7985-1921","authenticated-orcid":false,"given":"Brett A.","family":"Davis","sequence":"additional","affiliation":[{"name":"Knight Cardiovascular Institute, Oregon Health & Science University, Portland, OR, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lucia","family":"Carbone","sequence":"additional","affiliation":[{"name":"Knight Cancer Institute, Oregon Health & Science University, Portland Oregon, USA"},{"name":"Knight Cardiovascular Institute, Oregon Health & Science University, Portland, OR, USA"},{"name":"Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR, USA"},{"name":"Division of Genetics, Oregon National Primate Research Center, Beaverton, OR, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7026-2012","authenticated-orcid":false,"given":"Evan F.","family":"Lind","sequence":"additional","affiliation":[{"name":"Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland Oregon, USA"},{"name":"Knight Cancer Institute, Oregon Health & Science University, Portland Oregon, USA"},{"name":"Department of Cell, Development and Cancer Biology. Oregon Health & Science University, Portland, OR, USA"},{"name":"Department of Pediatrics, Oregon Health & Science University, Portland, OR, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026061906101523000_2026.06.14.732148v1.1","unstructured":"Acute Myeloid Leukemia - Cancer Stat Facts. SEER. Accessed 30 April 2026. https:\/\/seer.cancer.gov\/statfacts\/html\/amyl.html"},{"key":"2026061906101523000_2026.06.14.732148v1.2","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1808777"},{"key":"2026061906101523000_2026.06.14.732148v1.3","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-019-0400-z"},{"key":"2026061906101523000_2026.06.14.732148v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbmt.2006.02.005"},{"key":"2026061906101523000_2026.06.14.732148v1.5","doi-asserted-by":"publisher","DOI":"10.1002\/ajh.25750"},{"key":"2026061906101523000_2026.06.14.732148v1.6","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1916206117"},{"key":"2026061906101523000_2026.06.14.732148v1.7","doi-asserted-by":"publisher","DOI":"10.1182\/bloodadvances.2019000574"},{"key":"2026061906101523000_2026.06.14.732148v1.8","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2010-10-310425"},{"key":"2026061906101523000_2026.06.14.732148v1.9","doi-asserted-by":"publisher","DOI":"10.1158\/1078-0432.CCR-15-2626"},{"key":"2026061906101523000_2026.06.14.732148v1.10","doi-asserted-by":"publisher","DOI":"10.1172\/jci.insight.120974"},{"key":"2026061906101523000_2026.06.14.732148v1.11","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2023.1169144"},{"key":"2026061906101523000_2026.06.14.732148v1.12","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2023.1139517"},{"key":"2026061906101523000_2026.06.14.732148v1.13","doi-asserted-by":"publisher","DOI":"10.1186\/s40364-020-0185-8"},{"key":"2026061906101523000_2026.06.14.732148v1.14","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0801497105"},{"key":"2026061906101523000_2026.06.14.732148v1.15","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2019.09.013"},{"key":"2026061906101523000_2026.06.14.732148v1.16","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1903520116"},{"key":"2026061906101523000_2026.06.14.732148v1.17","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2018.12.021"},{"key":"2026061906101523000_2026.06.14.732148v1.18","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2020.04.014"},{"key":"2026061906101523000_2026.06.14.732148v1.19","doi-asserted-by":"publisher","DOI":"10.1038\/s41590-022-01224-z"},{"key":"2026061906101523000_2026.06.14.732148v1.20","doi-asserted-by":"publisher","DOI":"10.1126\/science.aae0491"},{"key":"2026061906101523000_2026.06.14.732148v1.21","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2017.06.007"},{"key":"2026061906101523000_2026.06.14.732148v1.22","doi-asserted-by":"publisher","DOI":"10.1126\/scitranslmed.abh0272"},{"key":"2026061906101523000_2026.06.14.732148v1.23","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-20696-x"},{"key":"2026061906101523000_2026.06.14.732148v1.24","doi-asserted-by":"publisher","DOI":"10.1016\/j.ccr.2010.11.015"},{"key":"2026061906101523000_2026.06.14.732148v1.25","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2011-02-339747"},{"key":"2026061906101523000_2026.06.14.732148v1.26","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa0810069"},{"key":"2026061906101523000_2026.06.14.732148v1.27","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1005143"},{"key":"2026061906101523000_2026.06.14.732148v1.28","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2015-01-621664"},{"key":"2026061906101523000_2026.06.14.732148v1.29","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0179501"},{"key":"2026061906101523000_2026.06.14.732148v1.30","doi-asserted-by":"publisher","DOI":"10.18632\/oncotarget.17395"},{"key":"2026061906101523000_2026.06.14.732148v1.31","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1712514114"},{"key":"2026061906101523000_2026.06.14.732148v1.32","doi-asserted-by":"publisher","DOI":"10.1038\/s41375-023-01808-0"},{"key":"2026061906101523000_2026.06.14.732148v1.33","doi-asserted-by":"publisher","DOI":"10.1016\/j.ccr.2007.08.031"},{"key":"2026061906101523000_2026.06.14.732148v1.34","doi-asserted-by":"publisher","DOI":"10.1016\/j.ccr.2011.06.001"},{"key":"2026061906101523000_2026.06.14.732148v1.35","doi-asserted-by":"publisher","DOI":"10.3324\/haematol.2023.283437"},{"key":"2026061906101523000_2026.06.14.732148v1.36","unstructured":"Andrews S , FastQC: A quality control tool for high throughput sequence data, Babraham Bioinformatics, http:\/\/www.bioinformatics.babraham.ac.uk\/projects\/fastqc\/"},{"key":"2026061906101523000_2026.06.14.732148v1.37","unstructured":"Krueger F , A wrapper tool around Cutadapt and FastQC to consistently apply quality and adapter trimming to FastQ files, with some extra functionality for MspI-digested RRBS-type (Reduced Representation Bisufite-Seq) libraries, Babraham Bioinformatics, https:\/\/www.bioinformatics.babraham.ac.uk\/projects\/trimgalore\/"},{"key":"2026061906101523000_2026.06.14.732148v1.38","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btr167"},{"key":"2026061906101523000_2026.06.14.732148v1.39","doi-asserted-by":"publisher","DOI":"10.1186\/gb-2012-13-10-r87"},{"key":"2026061906101523000_2026.06.14.732148v1.40","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btv145"},{"key":"2026061906101523000_2026.06.14.732148v1.41","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pcbi.1010378"},{"key":"2026061906101523000_2026.06.14.732148v1.42","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.1630"},{"key":"2026061906101523000_2026.06.14.732148v1.43","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1524490113"},{"key":"2026061906101523000_2026.06.14.732148v1.44","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2024.1437726"},{"key":"2026061906101523000_2026.06.14.732148v1.45","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2010.08.002"},{"key":"2026061906101523000_2026.06.14.732148v1.46","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0914127107"},{"key":"2026061906101523000_2026.06.14.732148v1.47","doi-asserted-by":"publisher","DOI":"10.1182\/bloodadvances.2018016279"},{"key":"2026061906101523000_2026.06.14.732148v1.48","doi-asserted-by":"publisher","DOI":"10.1016\/j.cellimm.2009.02.006"},{"key":"2026061906101523000_2026.06.14.732148v1.49","doi-asserted-by":"publisher","DOI":"10.1128\/MCB.21.24.8547-8564.2001"},{"key":"2026061906101523000_2026.06.14.732148v1.50","doi-asserted-by":"publisher","DOI":"10.3390\/ijms23010311"},{"key":"2026061906101523000_2026.06.14.732148v1.51","doi-asserted-by":"publisher","DOI":"10.1016\/j.ccell.2024.03.001"},{"key":"2026061906101523000_2026.06.14.732148v1.52","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.182.1.259"},{"key":"2026061906101523000_2026.06.14.732148v1.53","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2011-09-377044"},{"key":"2026061906101523000_2026.06.14.732148v1.54","doi-asserted-by":"publisher","DOI":"10.1080\/10428194.2024.2346755"},{"key":"2026061906101523000_2026.06.14.732148v1.55","doi-asserted-by":"publisher","DOI":"10.1111\/cts.13742"},{"key":"2026061906101523000_2026.06.14.732148v1.56","doi-asserted-by":"publisher","DOI":"10.1038\/s41375-021-01232-2"},{"key":"2026061906101523000_2026.06.14.732148v1.57","doi-asserted-by":"publisher","DOI":"10.1158\/2326-6066.CIR-18-0307"},{"key":"2026061906101523000_2026.06.14.732148v1.58","doi-asserted-by":"publisher","DOI":"10.3390\/cancers13164113"},{"key":"2026061906101523000_2026.06.14.732148v1.59","doi-asserted-by":"publisher","DOI":"10.1038\/bcj.2015.58"},{"key":"2026061906101523000_2026.06.14.732148v1.60","doi-asserted-by":"publisher","DOI":"10.3389\/fmed.2021.763773"},{"key":"2026061906101523000_2026.06.14.732148v1.61","doi-asserted-by":"publisher","DOI":"10.1358\/dot.2007.43.6.1062666"},{"key":"2026061906101523000_2026.06.14.732148v1.62","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.81.22.6993"},{"key":"2026061906101523000_2026.06.14.732148v1.63","doi-asserted-by":"publisher","DOI":"10.1038\/sj.onc.1208540"},{"key":"2026061906101523000_2026.06.14.732148v1.64","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(81)90248-8"},{"key":"2026061906101523000_2026.06.14.732148v1.65","doi-asserted-by":"publisher","DOI":"10.1002\/ajh.22141"},{"key":"2026061906101523000_2026.06.14.732148v1.66","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-05-3020"},{"key":"2026061906101523000_2026.06.14.732148v1.67","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2015.10.020"},{"key":"2026061906101523000_2026.06.14.732148v1.68","doi-asserted-by":"publisher","DOI":"10.1038\/s41375-024-02250-6"},{"key":"2026061906101523000_2026.06.14.732148v1.69","doi-asserted-by":"publisher","DOI":"10.1111\/bjh.18783"},{"key":"2026061906101523000_2026.06.14.732148v1.70","doi-asserted-by":"publisher","DOI":"10.1038\/leu.2013.355"},{"key":"2026061906101523000_2026.06.14.732148v1.71","doi-asserted-by":"publisher","DOI":"10.1038\/s41375-018-0070-8"},{"key":"2026061906101523000_2026.06.14.732148v1.72","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2024-210370"},{"key":"2026061906101523000_2026.06.14.732148v1.73","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2006-07-036863"},{"key":"2026061906101523000_2026.06.14.732148v1.74","doi-asserted-by":"publisher","DOI":"10.1186\/s12885-020-06961-8"},{"key":"2026061906101523000_2026.06.14.732148v1.75","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2018.01274"},{"key":"2026061906101523000_2026.06.14.732148v1.76","doi-asserted-by":"publisher","DOI":"10.3324\/haematol.2012.074823"},{"key":"2026061906101523000_2026.06.14.732148v1.77","doi-asserted-by":"publisher","DOI":"10.1016\/j.leukres.2012.03.026"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.06.14.732148","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T13:10:28Z","timestamp":1781874628000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.06.14.732148"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,17]]},"references-count":77,"URL":"https:\/\/doi.org\/10.64898\/2026.06.14.732148","relation":{},"subject":[],"published":{"date-parts":[[2026,6,17]]},"subtype":"preprint"}}