Abstract 5085: Acute myeloid leukemia cells induce osteogenic differentiation in mesenchymal stem cells through bone morphogenetic protein- and RUNX-2- mediated signaling
Authors: Battula Venkata Lokesh , Le Phuong M. , Sun Jeff , Benton Christopher B. , Mc.Queen Teresa , Shpall Elizabeth J. , Bueso-Ramos Carlos E. , Konopleva Marina , Andreeff Michael 
Published: August 2015

Mesenchymal stem/stromal cells (MSCs) are a critical component of the bone marrow micro-environment (BME), induce cell survival signals in normal and acute myeloid leukemia (AML) cells and protect them from chemotherapy. The mechanisms underlying BME-induced cell growth and chemo-resistance however have not been fully elucidated. Here, we hypothesize that AML cells induce functional changes in BME to gain growth advantage. We have compared age matched (40-60) bone marrow derived MSCs from AML patients (AML-MSC, n = 10) 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. The average doubling time of AML-MSCs is 52±8hrs compared to 34±6hours for N-MSCs (p&lt;0.01). Cell surface phenotyping by flow cytometry revealed that tissue non-specific alkaline phosphatase (ALP/ALPL/TNAP), which is highly expressed in embryonic stem cells, naïve-MSCs and osteoblasts, is expressed 10-14 fold higher in AML- compared to N-MSCs. Since ALP is also an osteoblast specific marker, we compared the osteogenic differentiation potential of N- and AML-MSCs: an increase in baseline ALP activity (by BCIP/NBT substrate) was observed in AML- compared to N-MSCs. mRNA analysis by qRT-PCR revealed 5-10-fold up-regulation of osteoblast-specific genes including RUNX-2, osteopontin, TNAP and osterix in AML-MSCs compared to N-MSCs before osteogenic induction. These data indicate that AML-MSCs are primed to differentiate into osteoblasts. Adipocyte differentiation was assessed by Oil-Red O staining for lipid droplets and revealed a &gt;95% reduction (p&lt;0.001) in the number mature adipocytes in AML-MSCs compared to N-MSCs suggesting that AML-MSCs lack the ability to differentiate into adipocytes. To validate these observations, we co-cultured N-MSCs with OCI-AML3 cells for 3-5 days and FACS sorted the MSCs for gene expression and differentiation analysis. As expected, qRT-PCR analysis revealed that osteopontin, osterix and ALP were up-regulated 3-4 fold in N-MSCs co-cultured with OCI-AML3 cells. In addition, ALP activity was 2-3 fold higher in N-MSCs co-cultured with OCI-AMl3 cells compared to mono-cultured cells. Bone morphogenetic proteins (BMP) are the most essential factors during osteogenic differentiation and new bone formation in humans. We therefore treated MSCs with AML cell-derived condition medium, and observed a dramatic 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-193189, in a dose dependent manner. Our data suggest that AML cells induce osteogenic differentiation in BM-MSCs and inhibition of BMP signaling may suppress AML cell growth in the BM endosteal niche.

Citation Format: Venkata Lokesh Battula, Phuong M. Le, Jeff Sun, Christopher B. Benton, Teresa Mc.Queen, Elizabeth J. Shpall, Carlos E. Bueso-Ramos, Marina Konopleva, Michael Andreeff. Acute myeloid leukemia cells induce osteogenic differentiation in mesenchymal stem cells through bone morphogenetic protein- and RUNX-2- mediated signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5085. doi:10.1158/1538-7445.AM2015-5085