Mesenchymal Stromal Cell Secretome in Acute Myeloid Leukemia Bone Marrow Niche

Bone marrow (BM) niche finely regulates hematopoiesis through a complex network of stromal factors, but it undergoes to drastic remodeling at Acute Myeloid Leukemia (AML) onset, impairing Hematopoietic Stem Cells (HSCs) functions and supporting leukemia cell development. To date, the BM niche various component interaction underlying mechanisms are not clear, but several lines of evidence suggested that Mesenchymal Stromal Cells (MSCs)-derived soluble factors might play a key role in suppressing HSCs activity contributing to the emergence of a leukemic niche, that supports tumor progression and patients’ mortality rate.

For this purpose, we aimed to characterize the secretome profile of primary AML-MSCs obtained from samples collected at diagnosis of AML as compared to MSCs derived from healthy donors (h-MSCs), and of h-MSCs after being in vitro co-cultured with AML blasts for 4 days (namely induced, iAML-MSCs). By transcriptional analysis of AML- and iAML-MSCs, we showed an enrichment of upregulated genes related to inflammation and tumor-associated soluble factors with respect to h-MSCs. We established an in vitro BM endosteal niche by using a biomimetic 3D structure, made up of engineered hydroxyapatite and collagen I, comprising AML-MSCs or h-MSCs, co-cultured with AML blasts or normal HSCs (CD34⁺ selected from cord blood) to study the mechanism responsible to the normal hematopoiesis impairment. After 7 days of 3D co-culture, we documented that AML-MSCs strongly support AML cell proliferation (increase of 46% by ATP and 28% by Ki67⁺ cells) but decrease CD34⁺ proliferation (reduction of 32%) with respect to h-MSCs. Conversely, CD34⁺ cells co-cultured with AML-MSCs increased their colony-forming unit capacity and maintained an immature-stem like cell phenotype. We observed same results when CD34⁺ cells were co-cultured with AML-MSCs but separated via a transwell-insert. These data suggest that AML-MSCs secretome influences HSCs differentiation and regulation. To identify key niche-derived factors, we explored AML-MSCs, h-MSCs and iAML-MSCs secretome in 3D long term cultures after 7, 14, 21 days with AML blasts, by performing Mass-Spectometry in combination with SILAC-labeling proteomic analysis. We identified that AML cells induced 15 differentially synthesized secreted factors involved in inflammation, angiogenesis and cancer pathways. By ELISA assay, we validated and determined the levels of each niche-derived factor in primary co-cultures in vitro and identified three novel top up-secreted factors in the AML-niche. We exogenously expressed them, alone or used in combination (namely the combo), in CD34⁺co-cultured with h-MSCs and showed that CD34⁺ cells recovered the features mediated by AML-MSCs in terms of proliferation (0.65-fold decrease of Ki67⁺ cells), and colonies number (1.85-fold increase), as well as the maintenance of the CD34⁺ expression (1.66-fold increase). Additionally, by using an in vitro AML blasts-HSCs competitive assay, we confirmed the potential of the combo favoring AML and reducing CD34⁺ proliferation. To support the role of the combo to impede hematopoiesis, we cultured iPSCs-derived healthy HSCs with the combo and monitored their differentiation toward the myeloid lineage. We observed a reduced CD34⁺proliferation, a delayed differentiation, and an increase in colony forming units.

In conclusion, secretome analysis in a 3D humanized AML niche model allowed the identification of three novel factors with strong commitment to disrupt HSCs homeostasis. Overall, these findings provide a potential route for novel secretome-targeted strategies aimed at supporting normal hematopoiesis, while disadvantaging AML cell growth.

Locatelli:AMGEN: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SANOFI: Membership on an entity's Board of Directors or advisory committees; NEOVII: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MEDAC: Speakers Bureau; JAZZ PHARMACEUTICALS: Speakers Bureau; TAKEDA: Speakers Bureau; BLUEBIRD BIO: Speakers Bureau; SOBI: Speakers Bureau; NOVARTIS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GILEAD: Speakers Bureau; MILTENYI: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Membership on an entity's Board of Directors or advisory committees. Pigazzi:Altheia Science: Consultancy.