The Characteristics of AML1-ETO Fusion Gene Induced Pre-Leukemia Immune Microenvironment

Objective: The immune-privileged microenvironment plays a critical role in immune escape of acute myeloid leukemia (AML), however the characteristics of the immune micro-environment in pre-leukemia states have not yet been clarified. In this study, the Aml1^Eto/+; Mx1-Cre mouse model was employed to explore the remodeling of the immune microenvironment by leukemic precursor cells, and to elucidate the characteristics of the immune microenvironment in the pre-leukemic state.

Methods: Four weeks after PIPC induction (5mg/kg), bone marrow-derived immune cells such as T cells, B cells and macrophages(Mφ) that do not express the AML1-ETO (AE) fusion gene were sorted from Aml1^Eto/+; Mx1-Cre mice (experimental group) and Aml1^Eto/+;w/o Mx1-Cre mice (control group), respectively. The immune cell subsets were analyzed using CIBERSORT after bulk RNA-sequence, and TCR and BCR information was reconstructed by TRUST4.

Results: After PIPC induction, the proportion of the AE fusion gene expressed hematopoietic precursors cells expanded significantly in the experiment group. The absolute counts of T cells and dendritic cells that did not express the fusion gene showed a considerable increment (p<0.0001; p=0.0067), and the monocyte-macrophage cells decreased (p<0.0001; p=0.0009). In addition, the expressions of Gzma and other cytotoxicity-related genes were significantly up-regulated in the T cells of experiment group. As for reconstructed TCR, the clonal diversity of TCR expanded. Although the proportion and absolute count of T cell increased when AE fusion protein induced, there didn't appear some hyperexpanded clones. The clonotype relative abundance and gene usage preference of TCR & BCR are similar between experiment and control group. There were 11 pre-leukemia-specific TCR amino acid sequences appeared, though no dominant clone was formed, suggesting that T cells produced relevant but not strong enough immune responses to pre-leukemia precursor cells. Further analysis found that the T cells in experiment group were composed of more Treg cells and expressed higher exhaustion-related genes such as PD-1, indicated that T cells began to accumulate negative immune regulation pathways in the bone marrow microenvironment of pre-leukemic state.

For the antigen presentation, the expression of MHC-I molecules on AE fusion gene expressed LT-HSC and GMP in the experimental group was up-regulated, while the expressions of MHC-I and MHC-II molecules in dendritic cells were not significantly different from those in the control group. Meanwhile, CIBERSORT and flow cytometry analysis showed more classically activated M1-Mφ in the experiment group, in which the expressions of INF-γ, IL-1, and other genes were up-regulated. In terms of B cells, the production of immunoglobulins related genes and antigen processing and presenting genes were up-regulated. The BCR clonal diversity expanded, and 73 pre-leukemia-specific BCR amino acid sequences appeared, but no dominant clones were formed. Among the heavy chain sequences, the clonal diversity of IgD and IgM in the experimental group increased, the relative abundance of IgM increased, and the relative abundance of IgA decreased.

Conclusion: We performed a comprehensive cell-type based dissection of the AML1-ETO fusion gene induced pre-leukemia immune microenvironment. The results demonstrates that in pre-leukemia stage, activated immune microenvironment is involved in the fight with AML1-ETO expressed cells. And there is also an accumulation of negative immune regulation mechanisms which may potentially contribute to weaken "the power" of immune surveillance. Although further validation is required, restoring dysfunctional immune cells or enhance the AML1-ETO specific immune response might be a potential early therapeutic approach for patients.

Wang:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy.