Methylation of HAI-2/SPINT2 in Bone Marrow Mesenchymal Stromal Cells of MDS and AML Patients Affects Hematopoietic Stem Cell Survival and Adhesion

In recent years, the role of tumor microenvironment in neoplasm initiation and malignant evolution has been increasingly recognized. However, the bone marrow mesenchymal stem/stromal cell (BMMSC) contribution to disease progression remains poorly explored. We had previously performed a microarray analysis of myelodysplastic syndrome (MDS) patient-derived BMMSC (MDS-BMMSC) and found an underexpression of HAI-2/SPINT2, an endogenous inhibitor of the hepatocyte growth factor (HGF) activator. This gene has been described as methylated in various cancer types and has been associated with disease progression. Despite of being related to the pathogenesis of several neoplasms, the role of HAI-2/SPINT2 has not yet been fully elucidated in hematological diseases, such as MDS and acute myeloid leukemia (AML). Thus, the aim of this study was to evaluate HAI-2/SPINT2 expression in derived BMMSC and total bone marrow (BM) of healthy donors (HD), MDS and AML patients as well as in BMMSC treated with 5-Azacitidine (Aza), a DNA methyltransferase (DNMT) inhibitor. To achieve this, we collected BM hematopoietic cells and plastic-adherent BMMSC from aspirates of HD, MDS and AML patients. BMMSC were expanded to passage 4 and defined as CD73+/CD90+/CD105+/CD45–/CD34–/CD31–/HLA-DR–. A total of 29 HD and 121 patients at diagnosis (MDS=72 [low-risk=46, high-risk=26], AML with myelodysplastic related changes (AML-MRC)=17 and de novo AML=32) were included. HAI-2/SPINT2 mRNA was significantly decreased in MDS- (0.34[0.01-2.06];P<.01) compared to HD-BMMSC (0.89[0.46-1.59]). When patients were stratified according to WHO classification, HAI-2/SPINT2 expression was lower in both low-risk (0.31[0.01-1.33]) and high-risk (0.43[0.01-2.06]) MDS-BMMSC. Similar results were found in total BM: HAI-2/SPINT2 transcripts were significantly decreased in MDS (0.41[0.01-2.53];P<.01), AML-MRC (0.38[0.014-0.84];P<.01) and AML patients (0.33[0.01-2.07];P<.001) compared to HD (0.91[0.19-4.79]). To investigate whether this loss of expression was due to HAI-2/SPINT2 methylation, BMMSC were treated with Aza (1µM or IC₅₀ value) for 48h. In MDS- and AML-BMMSC, Aza treatment resulted in a pronounced upregulation of HAI-2/SPINT2 mRNA and protein levels. Moreover, Aza treatment of HD-BMMSC did not improve the HAI-2/SPINT2 mRNA and protein levels as much as the observed in MDS- and AML-BMMSC. To better understand the role of HAI-2/SPINT2 downregulation in BMMSC physiology, its expression was inhibited in a BM stromal cell line (HS5). As previously reported, HAI-2/SPINT2 silencing resulted in an increased secretion of HGF, known to be overexpressed in plasma of MDS patients and considered a prognostic factor in MDS and AML patients (Matsuda et al., Leukemia, 2004). Moreover, after co-culture, HAI-2/SPINT2 knockdown improved survival of blasts isolated from AML-MRC and AML patients. We also observed an increased adhesion of CD34⁺ hematopoietic stem cells (HSC) to HAI-2/SPINT2 silenced HS5 cells. This prompted us to analyze the expression of cell adhesion molecules in MDS- and AML-BMMSC. We observed a significant augment in the expression of CD49b and CD49d integrins in MDS- and AML- compared to HD-BMMSC. Taken together, SPINT2 inhibition improves HGF secretion, consequently with alteration in molecule receptor adhesion, resulting in an increased expression of integrins (CD49b and CD49d) responsible for cell-to-cell adhesion. Thus, reactivation of HAI-2/SPINT2 levels after Aza treatment indicates that this gene is probably epigenetically silenced by methylation in MDS and AML, and is possibly a tumor suppressor gene. Interestingly, nowadays, epigenetic therapy by Aza is the first-line treatment for MDS patients, and induces prolonged survival and delayed AML evolution. Likewise, our results suggest that HAI-2/SPINT2 may play a role in deregulation of HGF cytokine secretion with consequently alteration in HSC adhesion and growth/survival. Tumor microenvironment niche is currently known to play a critical role in cancer initiation and progression, thus HAI-2/SPINT2 may contributes to functional and morphological abnormalities of microenvironment niche and with the stem/progenitor cancer cell progression. Hence, downregulation in HAI-2/SPINT2 gene expression, due to methylation in MDS- and AML-BMMSC, provides novel insights into the pathogenic role of the leukemic bone marrow microenvironment.