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1389 Decitabine-Venetoclax Treatment Reactivates Endogenous Retrovirus RNA Signaling in AML Cells and Induces T Cell Activation

Program: Oral and Poster Abstracts
Session: 604. Molecular Pharmacology and Drug Resistance: Myeloid Neoplasms: Poster I
Hematology Disease Topics & Pathways:
Research, Acute Myeloid Malignancies, AML, Translational Research, Diseases, Myeloid Malignancies, Biological Processes
Saturday, December 7, 2024, 5:30 PM-7:30 PM

Kotoko Yamatani, MD1*, Kaori Saito1*, Abhishek Maiti, MBBS2, Abdullah Khasawneh1*, Faith Jessica Paran, PhD3*, Tastuya Suzuki, PhD4*, Toru Okamoto, PhD4*, Kala Hayes, PhD5*, Zhihong Zeng, MD5, Courtney D. DiNardo, MD, MSc5, Michael Andreeff, MD, PhD6, Marina Konopleva7 and Yoko Tabe, MD, PhD1,5*

1Department of Clinical Laboratory Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan
2Department of Leukemia, The University of Texas Health Science Center At Houston, Houston, TX
3Intractable Disease Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan
4Department of Microbiology, Juntendo University Graduate School of Medicine, Tokyo, Japan
5Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX
6Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX
7Montefiore Einstein Comprehensive Cancer Center, Albert Einstein College of Medicine, Bronx, NY

The combination of hypomethylating agents such as decitabine (DEC) with venetoclax (VEN) has become the standard of care for AML patients who are not suitable for intensive chemotherapy. In this study, we investigated the reactivation of endogenous retroviruses (ERVs) in AML patients treated with DEC-VEN, activation of interferon (IFN) responses and cytotoxic T cells in the bone marrow (BM) microenvironment. Bulk and single-cell (sc) RNA-seq analyses of primary samples from AML patients participating in the prospective DEC-VEN clinical trial (NCT03404193) were performed to assess the effect of DEC-VEN treatment on immune-related gene expression in AML cells and T cells. In addition, the mechanisms of ERV double-stranded RNA (dsRNA)-induced INF immune signaling in AML cell lines were investigated.

The primary AML samples included BM and peripheral blood samples from newly diagnosed or relapsed/refractory (RR) AML patients (n= 13 and 13, respectively). Patients were classified as responders if they achieved complete remission (CR, n = 9), or non-responders if they failed to achieve CR (n= 17). First, bulk RNA-seq analysis was performed on 21 pre-treatment samples (9 responders and 12 non-responders) and 5 post-treatment samples (all non-responders) containing > 58% blasts. Gene ontology enrichment analysis showed that in the RR cohort, transcriptome signatures of immune system-related genes in pre-treatment samples were reduced in non-responders. In turn, comparisons of pre- and post-treatment gene expression in the RR cohort showed that IFN-stimulated genes (ISGs), reflecting IFN activation, were upregulated after treatment (e.g. post- vs. pre-treatment fold change: IFNGR2 2.4, CCL3 4.5, IRF8 3.6, p < 0.05).

To determine whether DEC-VEN induces ERV dsRNA known to stimulate ISGs, we analyzed the expression of 45 well-annotated ERVs in pre- and post-treatment samples from patients with RR AML. Differential expression analysis revealed significant upregulation of ERVFRD-2, ERVK3-1 and ERVK-27 following DEC-VEN treatment. We confirmed that mRNA levels of three ERVs (ERVFAD-1, ERVW-1, ERVK-7) were upregulated after DEC-VEN treatment in vitro in AML cell lines HL60 and MV4;11.

Next, to determine if ERV-derived dsRNA induces ISGs in a TANK-binding kinase 1 (TBK1)-dependent manner, we examined IFNB mRNA expression by HRVW-1 dsRNA in TBK1-deficient mouse embryonic fibroblasts (MEFs). ERV-dsRNA containing the 3' UTR, 5' UTR or ORF regions enhanced IFNB in wild-type MEFs but not in TBK1-deficient MEFs. These results were confirmed in AML cell lines; ERVFRD-1 dsRNA of ORF increased IFNB, 3.0- and 220-fold, and IFI27, 4.5- and 1,143-fold in HL-60 and MV4;11, respectively. TBK1 inhibitor GSK8612 diminished DEC-Ven-induced IFNB and IFI27 mRNA expression in HL-60 and MV4;11.

Finally, to investigate changes in T cell signatures after DEC-VEC treatment in the AML microenvironment, we utilized scRNA seq data of 12 BM samples (39,607 cells in total) from 8 patients with RR AML (4 responders, 4 non-responders, and 4 pre- and post-treatment pairs). In AML cell clusters, MX1 and IFITM, genes involved in cellular antiviral response, were significantly up-regulated after DEC-VEN treatment in all analyzed samples. Focusing on the responders, IFI27, an INF alpha inducible gene, was significantly increased. Sub-classification of T cells from scRNA-seq profiling showed that CD8+ T and CD8 CTL cells were significantly increased after treatment, especially in responders. Furthermore, significant increases in gene expression of cytotoxicity-related genes such as PRF1 and NKG7, and CD2, an activator of T cell differentiation and IFNγ production, were observed in CD8+ T cells in responders.

Taken together, these results indicated that DEC-VEN treatment reactivates ERV dsRNA, TBK1 signaling with upregulation of ISGs, translating into CD8+ T-cell activation which may contribute to therapeutic efficacy. These findings provide insights into the immunomodulatory effects of DEC-VEN therapy. To address anti-leukemia mechanisms of IFN response by ERV reactivation, we are currently studying the impact of TBK1 inhibitors on patient-derived AML cells co-cultured with T cells under DEC-VEN treatment using cytometry by time of flight (CyTof) analysis, and these results will be reported.

Disclosures: Maiti: Lin Biosciences: Research Funding; Indapta Therapeutics: Research Funding; Inspirna: Research Funding; Chimeric Therapeutics: Research Funding; Hibercell Inc.: Research Funding; CytoMed Therapeutics: Research Funding. DiNardo: Foghorn: Research Funding; Cleave: Research Funding; Schrodinger: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Loxo: Research Funding; Rigel: Research Funding; Astellas: Consultancy, Honoraria; Gilead: Consultancy; GenMab: Consultancy, Honoraria, Other: data safety board; Amgen: Consultancy; ImmuneOnc: Research Funding; GSK: Consultancy, Honoraria; Notable Labs: Honoraria; Astex: Research Funding; Servier: Consultancy, Honoraria, Other: meetingsupport, Research Funding; Immunogen: Honoraria; Riegel: Honoraria; Genetech: Honoraria; BMS: Consultancy, Honoraria, Research Funding; AstraZeneca: Honoraria; Abbvie: Consultancy, Honoraria, Research Funding; Stemline: Consultancy. Andreeff: SentiBio: Current holder of stock options in a privately-held company, Honoraria, Research Funding; Roivant: Honoraria; Chimerix: Current holder of stock options in a privately-held company; Syndax: Honoraria, Research Funding; Ona: Honoraria; Ellipses: Research Funding; Kintor Pharmaceutical: Research Funding; Sellas: Honoraria, Research Funding; Glycomimetics: Honoraria; Eterna: Current holder of stock options in a privately-held company, Honoraria, Research Funding; Paraza: Honoraria; Aptose: Honoraria; Oxford Biomedical: Research Funding; Boehringer-Ingelheim: Honoraria; Daiichi-Sankyo: Research Funding; Oncolyze: Current holder of stock options in a privately-held company. Konopleva: Intellisphere: Speakers Bureau; Vincerx: Consultancy; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: clinical trials, Research Funding; Syndax: Membership on an entity's Board of Directors or advisory committees; Auxenion GmbH: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Other: clinical trials; Novartis: Membership on an entity's Board of Directors or advisory committees; Menarini Group: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Consultancy; MEI Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Klondike Biopharma: Research Funding; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Servier: Speakers Bureau; Curis: Consultancy; Dark Blue Therapeutics: Membership on an entity's Board of Directors or advisory committees; Adaptive: Consultancy.

*signifies non-member of ASH