Mesenchymal niche contribution to normal and malignant hematopoiesis

Abstract

Normal hematopoiesis is tightly regulated by hematopoietic microenvironment/niche in bone marrow (BM) via direct hematopoietic cell-niche cell interaction and factors secreted by various types of cellular niches. The BM niche consists of cells of mesenchymal cell origin including mesenchymal stem cells (MSCs) and progenitor cells (MPCs). Accumulated evidence suggests the important role of BM mesenchymal cell niche for the maintenance of normal hematopoiesis and leukemogenesis. However, the exact role of different niche elements and the molecular mechanisms in leukemia development remain poorly defined. Knowledge is required for developing new therapeutic strategy to effectively treat the diseases. This thesis focuses on mesenchymal niche contribution to normal and malignant hematopoiesis, particularly, myeloproliferative neoplasms (MPN) and acute myeloid leukemia (AML). By using mouse models, multi-color flow cytometry, RNA-sequencing, transplantation and lineage tracing techniques, the thesis work has demonstrated the contribution of BM mesenchymal cell niche in the initiation of the myeloproliferative disease and progression of AML. The specific role of BM MSCs and laminin isoforms in AML progression and therapy response were studied. In addition, this thesis reports the features of native skin MSCs and their function in supporting normal hematopoietic and AML stem cells.

In paper I, the instructive role of BM niches in MDS/MPN initiation is studied by using signalinduced proliferation associated 1 (Sipa1) gene deleted mice (Sipa1-/-) that develop agedependent MPN. The loss of Sipa1 induces BM niche alterations prior to the disease onset, including biased differentiation of Sipa1-/- MSCs towards adipocytes and upregulated expression of pro-inflammatory genes (Il-6 and TGF-β). Concomitantly, hematopoiesis maintenance gene (Cxcl12, Angptl1, Kitl) expressions were reduced in Sipa1-/- BM MSCs and MPCs. Transplantation of Sipa1+/+ hematopoietic cells to young Sipa1-/- mice resulted in MDS/MPN development, supporting the causative role of Sipa1 deficient niche for the development of MDS/MPN.

The role of BM MSCs and MPCs during progression of AML is reported in paper II. By transplanting MLL-AF9+ AML cells to immunocompetent mice, we showed dynamic niche alterations induced by AML cells. During AML development, frequency of BM MSC & MPC were increased while hematopoiesis gene (Kitl, Cxcl12, Angptl1, Nov and Igf1) expression in BM MPCs were down regulated in correlation to AML engraftment in BM. Moreover, the expression of pro-inflammatory gene (Il-6) is elevated following the AML progression. Specifically, BM primitive subset of MSC (Ebf2+) is altered by AML cells to generate more progenies including Ebf2-MSC, MPC and CD44+ cells in the leukemic niche. The depletion of Ebf2+ cells accelerated AML development, demonstrating the suppressive role of Ebf2+ MSCs in AML progression possibly by maintaining normal hematopoiesis. In this study, upregulation of laminin 4 (Lama4) in both MSC and MPC was observed.

To further investigate the functional consequence of Lama4 during AML development, the Lama4 (Lama4-/-) deficient mice were employed in paper III. We firstly studied the role of Lama4 in hematopoiesis regeneration following irradiationinduced stress and observed impaired recovery of erythropoiesis and megakaryopoiesis in Lama4-/- mice. On the contrary, AML progression and relapse were accelerated post transplantation of MLL-AF9+ AML cells. Furthermore, the Lama4-/- MSCs promoted AML cell growth and confer AML stem cell chemoresistance to cytarabine (Ara-C) via providing more metabolic support to the AML stem cells (LSCs). Taken together, paper III shows critical role of Lama4 in hematopoiesis recovery following irradiation and during AML development.

Recent study has shown that AML LSCs infiltrate extramedullary organ. Meanwhile, skin has been reported to contain MSC-like population although the characteristics are not well defined. In paper IV, we employed Ebf2-gfp transgenic mice to prospectively characterize skin MSC phenotypically and functionally at bulk and single cell level. Skin Ebf2+ cells represent purified MSC while the Ebf2-fraction contained more differentiated MSCs that can be generated by the Ebf2+ cells, revealed by the in vivo lineage tracing of Ebf2+ MSCs. Both skin Ebf2+ cells and Ebf2-MSC displayed hematopoiesis supportive function, similar to their BM counterpart. Furthermore, co-culture of AML and AML CAFC on skin Ebf2+ and Ebf2-MSCs showed that skin MSCs also supported normal HSCs and provided chemoprotection for AML LSCs. In skin tissue of AML mice, infiltration of AML cells was observed and remained in skin tissue after Ara-C treatment, suggesting a possible contribution of skin MSCs to the persistence of AML cells. The skin Ebf2+ were found to be reduced in AML mice. However, the functional consequence of the skin MSCs remains to be investigated in the future. Altogether, paper IV reports skin harbors Ebf2+ and Ebf2-MSC with similar characteristics to BM MSC. Both skin Ebf2+ and Ebf2-MSCs support normal HSC and AML cells. Importantly, skin MSCs provide chemoprotection for AML LSC.

In conclusion, the work in this thesis shows the role of BM niche for the initiation and progression of the myeloid malignancies using several transgenic mouse models. The work also provides evidence for critical role of Lama4 in hematopoiesis recovery following irradiation and AML progression. Furthermore, the biological features of skin MSCs and their function in supporting normal hematopoietic and AML cells. During Ara-C treatment, skin MSCs also displayed protective role for AML LSCs, indicating skin MSC possible role as a reservoir of chemoresistant AML LSC.