Role of CD40 and Zeb1 in Shaping Normal and Leukemic Bone Marrow Niche

Bassani, Barbara (2021). Role of CD40 and Zeb1 in Shaping Normal and Leukemic Bone Marrow Niche. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0001325c

Abstract

Acute myeloid leukaemia (AML) is an aggressive disease impairing normal haematopoiesis. Beyond the genomic landscape and the mutational profile of AML cells, increasing evidence demonstrated that bone marrow (BM) microenvironment contributes to leukemia development and, in particular, the immunologic landscape is emerging as a crucial component of leukemic microenvironment correlating with clinical parameters. Leukemic cells express different antigens that are able to activate T-cells triggering an anti-leukemia immune response. Thus, we hypothesized that immunogenic leukemia cells could set up mechanisms for immune escape including the establishment of immunosuppression. To escape from immune recognition leukemic cells could instruct neighbouring mesenchymal (MSCs) cells to trigger inhibitory T cell pathways through the up-regulation of CD40 or autonomously gain immunosuppressive properties activating epithelial to mesenchymal transition-related programs (i.e. ZEB-1). Hence, the aim of this study is to evaluate the impact of ZEB-1 expression on AML cell ability to mold an immunosuppressive microenvironment. Moreover, we also evaluated the role of CD40 on BM-MSCs in shaping the BM tolerogenic niche during leukemia development and in bone marrow transplantation.

To this aim we firstly analysed the gene expression profile of 61 AML cases that were divided in “ZEB1low” and “ZEB1high”. The two groups showed difference between the karyotypic features and expressed different levels of immunomodulatory markers. Analysis performed using public datasets, showed that ZEB1high patients displayed a worse overall survival compared with ZEB1low patients. Immunohistochemistry analysis performed on archival BM biopsy showed that ZEB1high cases also showed an expansion of CD3+IL-17A+ cells. To unveil the real contribution of ZEB-1 in shaping a BM microenvironment, we moved from humans to mouse models. Zeb-1 expressing or silenced C1498 murine AML cells were injected intra-bone in immunocompetent mice. Mice injected with Zeb-1+ cells showed a higher engraftment compared with mice injected with silenced cells. This phenotype correlated with an expansion of Th17 and an increase of TIM3+PD1+CD8+ cells within the BM of Zeb-1+ cell injected mice. Molecular studies highlighted that Zeb-1 silencing in C1498 induced a significant reduction of Il-6, Tgfβ and Il-23, cytokines involved in Th17 differentiation and maintenance. In turn, IL-17A promoted AML cell proliferation and aggressiveness in-vitro. Moreover, Zeb-1 silenced cells showed a reduced expression of Arg1 that has been reported to negatively affect the activation of CD8+ T cells leading to the increase of cytotoxic CD8+ cells in mice injected with silenced C1498. In addition, Zeb-1 was able to up-regulate the expression of Cd40 and Ido-1 on BM-MSCs. Given the importance of stromal Cd40 expression in sustaining the progression of other haematological diseases, we hypothesized that its expression can be able to affect AML development. To this end, we injected C1498 cells in Cd40-KO mice and we showed that Cd40 deficiency was associated with graft failure. To evaluate the immune-regulatory role of stromal Cd40, we performed BM transplantation experiments. We observed that Cd40 deficiency leads to the persistent T-cell activation with the expansion of CD4+ INF-γ/TNF-α effector T cells and with the parallel depletion of regulatory T cells due to an increase production of Ox40l.

Our results suggest that ZEB1 expression could characterize a group of particularly aggressive and chemotherapy resistant AML whose poor outcome is the result of both intrinsic clone aggressiveness and the immunosuppressive microenvironment initiated by ZEB1.

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