Acute Myeloid Leukemia (AML) Cells Alter the Bone Marrow Microenvironment By Inducing Osteogenic and Suppressing Adipogenic Differentiation of MSCs through BMP-RUNX2-CTGF Mediated Mechanisms

Osteoprogenitor cells are a critical component of the bone marrow microenvironment (BME), support AML cell growth (Raaijmakers et al., Nature, 2010); however the mechanism has not been fully elucidated. Here, we hypothesize that AML cells induce osteogenic differentiation in mesenchymal stem/stromal cells to gain growth advantage. We have isolated age matched (between 40-70 years) bone marrow derived MSCs from AML patients (AML-MSC, n=20) and normal (N-MSC, n=10) individuals and analyzed their cell growth, cell surface phenotype and multi-lineage differentiation. AML-MSCs are phenotypically different with larger cell surface area and lower cell growth compared to N-MSCs. The average doubling time of AML-MSCs is 52±8hrs compared to 34±6hours for N-MSCs (p<0.01). Phenotypic and enzyme activity studies revealed that tissue non-specific alkaline phosphatase (TNAP), a protein that is highly expressed in naïve MSCs and osteoprogenitor cells and osteoblasts, is expressed 10-14 fold higher in AML- compared to N-MSCs (p<0.01). As TNAP is associated with osteogenic lineage, we next assessed the expression of osteogenic markers including RUNX-2, osteopontin, TNAP and osterix.  Interestingly, these genes were up-regulated by 5-10-fold in AML-MSCs compared to N-MSCs. To validate these observations, N-MSCs were cultured with OCI-AML3 cell derived conditioned medium (CM) for 3-5 days and then induced to osteogenic or adipogenic differentiation. As expected, alkaline phosphatase enzyme activity and Alizarine Red S staining was twice as high in MSCs cultured with AML-CM compared to MSCs cultured in regular cell culture medium. In addition, we found expression of osteogenic genes including RUNX-2, osteopontin, TNAP and osterix  3-4 fold upregulated in MSCs cultured with AML-CM compared to control MSCs. These data indicate that AML cells prime MSCs to undergo osteogenic differentiation. Adipocyte differentiation was assessed by Oil-Red O staining for lipid droplets and revealed a >95% reduction (p<0.001) in the number of mature adipocytes in AML-MSCs compared to N-MSCs. Gene expression analysis by qRT-PCR revealed that adipogenic markers including aP2, lipoprotein lipase and PPARγ were down-regulated 10-20 fold in AML-MSCs compared to N-MSCs in a time-dependent manner suggesting that AML-MSCs lack the ability to differentiate into adipocytes. To evaluate these findings in-vivo, we developed a Human Bone Marrow Implant (HBMI) mouse model by subcutaneous implantation of human femur derived bone pieces into NSG mice. In this model, mice bearing HBMI with leukemia expressed higher osteogenic related proteins compared to mice without leukemia. Importantly, multispectral image analysis revealed 5-10 fold higher osteogenic activity in AML patient BM-biopsies compared to normal bones. Bone morphogenic proteins (BMP) are the most essential factors during osteogenic differentiation and new bone formation in humans. We therefore treated MSCs with AML cell-derived conditioned medium, and observed a 6-8 fold increase in pSmad1/5 levels in N-MSCs in a time dependent manner. In addition, AML induced pSmad1/5 up-regulation was inhibited when MSCs were treated with BMP-type1 receptor specific inhibitor LDN-212854, in a dose dependent manner. In addition, inhibition of BMP signaling also inhibited AML-induced osteogenic differentiation in MSCs. Mechanistic studies revealed that bone morphogenetic proteins (BMPs) derived from AML cells induce connective tissue growth factor (CTGF) expression in MSCs. When transplanted in Col1a2-CTGF transgenic mice, AML cells engrafted faster compared to wild type mice suggesting that CTGF enhances AML cell growth. Our data suggest that AML cells induce osteogenic differentiation in MSCs and receive growth advantage through BMP-CTGF signaling. Inhibition of these pathways may reduce leukemia propagation and improve AML patient survival.