Molecular Characterization of the Leukemic Niche in Chronic Myeloid Leukemia (CML) and Evaluation of a Leukemia / Niche Cross-Talk
Authors: Aggoune Djamel , Sorel Nathalie , Marsafy Sanaa El , Bonnet Marie Laure , Clay Denis , Meunier Marie Claude , Tarte Karin , Herault Olivier , Domenech Jorge , Guerci Agnès , Chomel Jean-Claude , Turhan Ali G 
Published: October 2019

Abstract
Abstract 908
There is growing evidence that the bone marrow microenvironment could participate to the progression of chronic myeloid leukemia (CML). Recent data show indeed that placental growth factor (PGF) expression is highly induced in stromal cells from CML patients although they are not part of the leukemic clone as they are Ph1-negative (Schmidt et al, Cancer Cell 2011). It is possible that leukemic cells instruct the niche components via extracellular or contact signals, transforming progressively the “normal niche” into a functionally “abnormal niche” by inducing aberrant gene expression in these cells, similar to the pattern that has been identified in cancer-associated fibroblasts (CAF). In an effort to identify the differential gene expression pattern in the CML niche, we have undertaken two strategies of gene expression profiling using a Taqman Low Density Arrays (TLDA) protocol designed for 93 genes involved in antioxidant pathways (GPX, PRDX, SOD families), stromal cell biology (Collagen, clusterin, FGF, DHH), stem cell self-renewal (Bmi1, MITF, Sox2) and hematopoietic malignancies (c-Kit, hTERT, Dicer, beta-catenin, FOXO3). The first strategy consisted in the analysis of mesenchymal stem cells (MSCs) isolated from the bone marrow of newly diagnosed CP-CML patients (n=11). As a control, we have used MSCs isolated from the bone marrow of age-matched donors (n=3). MSCs were isolated by culturing 6–8.106 bone marrow mononuclear cells in the presence of b-FGF (1 ng/ml). At 2–3 weeks, cells were characterized by the expression of cell surface markers (CD105+, CD90+) and by their potential of differentiation towards osteoblastic, chondrocytic and adipocytic lineages. The second strategy aimed to study the potential instructive influence of leukemic cells in the gene expression program of normal MSC after co-culture with either the UT7 cell line expressing BCR-ABL (3 days) or with CD34+ cells isolated from CP-CML at diagnosis (5 days) as compared to co-culture with cord blood CD34+ cells. After culture, CD45-negative MSC were cell-sorted and analyzed by TLDA. All results were analyzed using the StatMiner software.

Results:
TLDA analysis of gene expression pattern of MSC from CML patients (n=11) as compared to normal MSCs (n=3) identified 6 genes significantly over-expressed in CML-MSC: PDPN (10-Fold Increase), V-CAM and MITF (∼8 Fold increase), MET, FOXO3 and BMP-1 (∼ 5 Fold increase). To confirm these results we have performed Q-RT-PCR in a cohort of CML-MSC (n= 14, including the 11 patients as analyzed in TLDA) as compared to normal MSC. High levels of PDPN (Podoplanin, ∼8 fold increase), MITF (Microphtalmia Associated Transcription factor, 4-Fold) and VCAM (Vascular Cell Adhesion Protein, 2 fold increase) mRNA were again observed on CML MSCs. Our second strategy (co-culture of normal MSC with BCR-ABL-expressing UT7) revealed an increase of IL-8 and TNFR mRNA expression in co-cultured MSCs (∼5-fold ) whereas there was a major decrease in the expression of DHH (∼ 25-fold) upon contact with BCR-ABL-expressing cells. No modification of the expression of PDPN, MITF or VCAM was noted in normal MSC after this 3-day co-culture strategy using UT7-BCR-ABL cells. Current experiments are underway to determine if primary CD34+ cells from CML patients at diagnosis could induce a specific gene expression pattern in normal MSC after 5 days of co-culture. PDPN is a glycoprotein involved in cell migration and adhesion, acting downstream of SRC. It has been shown to promote tumor formation and progression in solid tumor models and is highly expressed in CAFs. MITF is a bHLH transcription factor involved in the survival of melanocyte stem cells and metastatic melanoma. Finally, high VCAM1 mRNA expression by MSCs from CML patients could be involved in increased angiogenesis known to be present on CML microenvironment. In conclusion, our results demonstrate an abnormal expression pattern of 3 important genes (PDPN, MITF and VCAM1) in MSC isolated in CP-CML patients at diagnosis. The mechanisms leading to an increased mRNA expression (instructive or not instructive by leukemic cells) and their relevance to CML biology are under evaluation. Our results, confirming previous data, suggest strongly the existence of a molecular cross-talk between leukemic cells and the leukemic niche. The elucidation of such aberrant pathways in the microenvironment could lead to the development of “niche-targeted” therapies in CML.

Disclosures:
Turhan: Novartis, Bristol Myers Squibb: Honoraria, Research Funding.