EVI1 Promotes Immune-Evasive Microenvironment Via Cyclin D1 in Acute Myeloid Leukemia

Inappropriate expression of Ecotropic viral integration site 1 (EVI1) has been associated with dismal clinical outcomes in acute myeloid leukemia (AML), while EVI1^high (EVI1⁺) AML cells do not show clear resistance to cytotoxic agents in vitro. Since EVI1 is essential for normal hematopoietic stem cells (HSCs), we performed chromatin-immunoprecipitation (ChIP)- and RNA-sequencing of murine EVI1⁺ AML cells and normal hematopoietic cells to identify factors involved explicitly in AML, and thereby to elucidate molecular cues to refractory EVI1⁺ AML.

We firstly employed a murine EVI1-transformed AML (EVI1-AML) model, where murine hematopoietic cells exogenously expressing 3×FLAG-tagged EVI1 were transplanted into syngeneic mice. Using EVI1-AML cells, we next performed ChIP coupled to next-generation sequencing (ChIP-seq) by anti-FLAG tag antibody and transcriptome analysis by RNA-seq. We further generated another murine EVI1⁺ AML model by retrovirally transducing the KMT2A-MLLT1 fusion gene into Lineage- Sca-1+ c-kit+ cells from EVI1-GFP knock-in mice, where Evi1-IRES-GFP allele was inserted into the Evi1 locus to enable real-time monitoring of Evi1 expression by GFP, followed by transplantation into lethally irradiated syngeneic mice. Intriguingly, GFP^high L-GMPs from KMT2A-MLLT1 AML mice showed distinct expression profiles from GFP^low L-GMPs, including high NF-kB activity and low stem cell activity, along with the lower frequency of leukemia-initiating cells in GFP^high fraction. Consistently, an exogenous expression of EVI1 in KMT2A-MLLT1 AML cells without Evi1 expression decreased colony-forming and leukemia-initiating activity, suggesting a differential role of EVI1 in AML from normal HSCs, where EVI1 is essential in maintaining stem cell function.

Combined with functional screening by cell proliferation-related short hairpin RNAs (shRNAs), we identified that cyclin D1 (CCND1) was a downstream target, commonly implied in the above analyses, and a potential therapeutic vulnerability of EVI1-AML. EVI1 bound to the region 3.8 kb upstream of the transcription start site of Ccnd1, through which EVI1 demonstrated strong transcriptional activity in a luciferase reporter assay. Genetic silencing of Ccnd1 and pharmacologic inhibition of Cyclin D1 in EVI1- and EVI1⁺ KMT2A-MLLT1 AML cells negatively affected proliferation and colony-forming activity in vitro. Moreover, in vivo knockdown of Ccnd1 strikingly impaired the leukemogenic capacity of murine EVI1-AML models.

It was surprising that transcriptomic analysis of EVI1-AML cells with Ccnd1 silencing showed the downregulation of genes involved in chemokine production and response to interferon instead of cell cycle-related genes. The expression of Cd274 (PD-L1) as well as Stat1, the primary signaling mediator for type I and II IFN, was decreased in EVI1-AML cells with Ccnd1 knockdown. EVI1 directly bound to the promoter region of Stat1 and activated its transcription in line with a previous report, and indirectly up-regulated STAT1 expression via Cyclin D1. Accordingly, infiltrating CD4 and CD8 T cells from EVI1-shCcnd1 AML mice demonstrated decreased exhaustion markers, including LAG3, PDCD1, and TIGIT. Silencing of Stat1 and Ifngr, the receptor for IFN-γ, impaired the leukemogenic capacity of EVI1-AML in vivo, with a decrease in T and NK cells with exhaustion phenotypes, while Ifnar, the receptor for IFN-α, was not involved in this process. Genetic inhibition of the IFN-γ-STAT1 axis in non-EVI1⁺ AML models did not affect AML development, nor the frequency of immune cells expressing exhaustion-related molecules, suggesting this axis is specifically important in EVI1⁺ AML. By using human AML datasets, overexpression of EVI1 and CCND1 were associated with IFN-γ signature, including up-regulation of STAT1 and CD274, and elevated expression of chemokines, with increased exhaustion molecules in T cells.

These data collectively suggest that cyclin D1 is implicated in the development of EVI1-AML through the formation of IFN-γ signature and exhausted T cell phenotypes, which could be potentially targeted.

Masamoto:MSD K.K: Honoraria; Otsuka Pharmaceutical Co., Ltd: Honoraria; ONO PHARMACEUTICAL CO., LTD: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; SymBio Pharmaceuticals: Honoraria; AstraZeneca: Honoraria; Yamasa Corporation: Honoraria; Sanofi: Honoraria; Asahi Kasei Pharma: Honoraria; Bristol Myers Squibb: Honoraria; Janssen Pharmaceutical K.K: Honoraria; AbbVie GK: Honoraria; Nippon Shinyaku Co., Ltd: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Company: Honoraria. Kurokawa:AbbVie GK: Research Funding; MOCHIDA PHARMACEUTICAL CO.,LTD.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Sanofi K.K.: Honoraria; SymBio Pharmaceuticals Limited: Honoraria; Amgen Inc: Honoraria; AstraZeneca K.K.: Honoraria; Shionogi & Co., Ltd.: Research Funding; Asahi Kasei Pharma Corporation.: Research Funding; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Company: Honoraria, Research Funding; Takeda Pharmaceutical Company Limited: Honoraria, Research Funding; ONO PHARMACEUTICAL CO., LTD.: Honoraria, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc: Research Funding; SANWA KAGAKU KENKYUSHO CO., LTD.: Consultancy, Honoraria; Pfizer Japan Inc.: Honoraria, Research Funding; Daiichi Sankyo Company .: Honoraria, Research Funding; Nippon Shinyaku Co., Ltd.: Honoraria, Research Funding; Teijin Limited: Honoraria, Research Funding.