Proteomic, Gene Expression, and Micro-RNA Analysis Of Bone Marrow Mesenchymal Stromal Cells In Acute Myeloid Leukemia Identifies Pro-Inflammatory, Pro-Survival Signatures In Vitro and In Vivo
Authors: Andreeff Michael , Wang Rui-yu , Davis Richard E. , Jacamo Rodrigo , Ruvolo Peter P. , McQueen Teresa , Huang Xielin , Battula Venkata L , Chen Ye , Majumdar Shonali , Benito Juliana M , Zeng Zhihong , Benton Christopher B. , Carter Bing Z , Mu Hong , Ma Wencai , Ruvolo Vivian R , Hodgson Lauren , Konoplev Sergej , Pan Rongqing , Somanchi Anitha , Strunck Dirk , Zhang Qiguo , Kornblau Steven M. , Kantarjian Hagop M , Marcucci Guido , Garzon Ramiro , Zuber Johannes , Lowe Scott W. , Croce Carlo M. , Konopleva Marina 
Published: October 2019

Abstract
The bone marrow microenvironment (BME) in acute myeloid leukemia (AML) generates resistance signals that protect AML cells/stem cells from chemotherapy. The mechanisms how the BME might support leukemia cell survival are unclear but elucidation of this process could prove useful for therapy design. Here we report new insights specific to stroma functionality in AML. A series of novel experimental approaches were developed including : 1) nanostring micro-RNA and proteomic analysis using reverse-phase protein arrays (RPPAs) of MSC derived from AML patients and normal donors; 2) genome-wide RNA analysis of FACS-sorted MSCs using Illumina arrays of genetically-defined human and murine AML cell lines/primary AML samples after co-culture with normal MSC in vitro; 3) in vivo interaction between genetically-defined murine AML and stromal cells in syngenic C57BL/6J mice, followed by harvesting and FACS-isolation of specific MSC after leukemia engraftment; 4) use of genetically-modified human MSC in vivo in our ectopic humanized bone marrow model in NSG mice (Blood 2012 : 119,4971), followed by bioluminescence growth and homing analysis of human leukemia cells. This model allows the study of in vivo effects of altered MSC on human AML development.
1) Proteomic and transcriptomic analysis of primary MSC from AML patients (n = 106) and normal MSC (n = 71) by RPPA using validated mAbs to 150 proteins and phospho-proteins demonstrated major differences by hierarchical clustering analysis: GSKA, STAT1, PDK1, PP2A, CDKN1A, CDK4, and STAT5AB were significantly over-expressed in AML- vs. normal MSC, while STMN1, SIRT1, SMAD1, SMAD4, HSP90 and EIF2S1 were under-expressed. Differences were observed between MSC from newly diagnosed vs. relapsed AML-MSC. Nanostring analysis of 38 AML-MSC and 24 normal MSC identified differential expression of numerous miRs, a select group of which has been validated so far by qRT-PCR. AML MSC express reduced levels of let-7g, let-7c, miR 21 and miR93, and elevated levels of miR410 compared to normal MSC. Pathways were identified in MSC that might contribute to leukemia survival.
2) Analysis OCI-AML3 cells co-cultured with normal -MSC revealed upregulation of a variety of genes in MSC encoding cytokines and chemokines and gene set enrichment analysis (GSEA) identified activation of NFkB in MSC as a potential cause of these changes. When the ectopic humanized bone marrow model system in NSG mice was used, suppression of NFkB in MSC resulted in a ∼ 50% reduction of AML burden. When murine MSC cultured with wt p53 MLL/ENL-Luc-FLT3-ITD cells were compared to isogenic cells with deleted p53, striking differences were seen in the MSC transcriptome: 429 differentially expressed genes were identified that distinguished co-cultures with p53wt and p53-/- cells, suggesting that AML cells may communicate signals to their microenvironment in a p53-dependent manner. GSEA identified NFkB and HIF-1a as targets, data were confirmed independently, and HIF-1a knockout MSC were found to be inhospitable for AML in the ectopic in vivo model.
3) These syngenic cells were introduced into B57BL/6J mice and MSC were isolated after leukemia engraftment: 147 genes were consistently upregulated and 236 genes downregulated in MSC by their interactions with AML in vivo. Upregulated genes included CTGF, CXCL12, genes related to complement (C4A, C4B, Serpin G1), and IGFBP5, an inhibitor of osteoblast differentiation. Identification of CXCL12 was intriguing as Link's group recently reported the critical role of CXCL12 produced by early MSC in normal hematopoiesis (Nature 2013 : 495,227). Both AML-ETO and MLL-ENL leukemias caused upregulation of CTGF, metalloproteinases, adhesion molecules, and NFkB-related genes in vivo. IPA analysis showed responses in BM-MSC associated with inflammation, cellular movement, cell-cell signaling, cellular growth and proliferation and immune cell trafficking.

Conclusion
AML cells induce changes in MSC, in short term co-cultures in vitro, or in syngenic systems in vivo, that are consistent with pro-survival, anti-apoptotic, and growth-stimulatory signals that mimic inflammatory responses. Large-scale analysis of primary AML-derived MSC confirms and extends this data. Results facilitate the development of therapeutic strategies to render the BM microenvironment inhospitable to leukemia cells but supportive of normal hematopoiesis.

Disclosures:
Lowe: Blueprint Medicines: Consultancy; Constellation Pharmaceuticals: Consultancy; Mirimus Inc.: Consultancy.