Tissue-Nonspecific Alkaline Phosphatase Is Required for MC3T3 Osteoblast-Mediated Protection of Acute Myeloid Leukemia Cells from Apoptosis

Abstract

The bone marrow microenvironment harbors and protects leukemic cells from apoptosis-inducing agents via mechanisms that are incompletely understood. We previously showed SDF-1 (CXCL-12), a chemokine readily abundant within the bone marrow microenvironment, induces apoptosis in acute myeloid leukemia (AML) cells that express high levels of the SDF-1 receptor CXCR4. However, differentiating osteoblasts found within this niche protect cocultured AML cells from apoptosis. Additionally, this protection was abrogated upon treatment of the differentiating osteoblasts with histone deacetylase inhibitors (HDACi). In this study, we begin to characterize and target the molecular mechanisms that mediate this osteoblast protection. Quantitative RT-PCR revealed that HDACi treatment of differentiating osteoblasts (mouse MC3T3 osteoblast cell line) reduced expression of multiple genes required for osteoblast differentiation, including genes important for producing mineralized bone matrix. Interestingly, pretreating differentiating osteoblasts with cyclosporine A, a drug known to inhibit osteoblast differentiation, similarly impaired osteoblast-mediated protection of cocultured AML cells (KG1a and U937 human AML cell lines). Both HDACi and cyclosporine A reduced osteoblast expression of the key mineralization enzyme tissue-nonspecific alkaline phosphatase (TNAP; encoded by Alpl). Moreover, specifically reducing TNAP expression or activity in differentiating osteoblasts significantly impaired the ability of the osteoblasts to protect cocultured AML cells. Together, our results indicate that inhibiting osteoblast matrix mineralization by specifically targeting TNAP is sufficient to significantly impair osteoblast-mediated protection of AML cells. Therefore, designing combination therapies that additionally target the osteoblast-produced mineralized bone matrix may improve treatment of AML by reducing the protection of leukemic cells within the bone marrow microenvironment.

FIGURE 1. HDACi inhibit expression of osteogenic genes in MC3T3 cells.

(A) Depiction of the timeline describing the MC3T3 osteoblast cell line culture for qRT-PCR. On Day 0, MC3T3 osteoblasts were treated with osteogenic differentiation medium. On Day 1, the MC3T3 cells were treated with HDACi (10 μM SAHA or 1 μM LBH-589) or vehicle control (0.1% DMSO) and harvested on Day 2 for qRT-PCR. (B) qRT-PCR results were normalized to Gapdh, which was set to 100. Each bar represents mean+standard deviation for three independent experiments with * representing statistical significance (p<0.05) from DMSO treated cells.

FIGURE 2. CSA reduces MC3T3 osteoblast-mediated protection of KG1a-CXCR4 and U937-CXCR4 AML cells from SDF-1-induced apoptosis.

(A) Depiction of MC3T3 osteoblast and KG1a-CXCR4 AML cell line co-culture model. On Day 0, MC3T3 osteoblasts were treated with osteogenic differentiation media and either 0.1% DMSO or 0.025 mg/mL CSA. On Day 2, the osteoblasts were washed to remove the DMSO or CSA pretreatment and were then co-cultured with KG1a-CXCR4 AML cells. The indicated wells were treated with 30 μM AMD3100 1 hr prior to 1.3 × 10⁻⁸ M SDF-1 challenge. On Day 3, the KG1a-CXCR4 AML cells were harvested and assayed for apoptosis utilizing annexin-V staining and flow cytometry. (B) A representative experiment as performed in (A) shows the percentage of annexin-V positive KG1a-CXCR4 cells from each culture within the YFP gate indicated (gating on cells with high levels of CXCR4 expression as the CXCR4 is YFP-tagged). (C) Summary of multiple experiments performed as in (A). Bars depict mean results + S.E.M., n=3; *, indicates p<0.05. (D) Summary of multiple experiments performed as in (A) except with U937-CXCR4 AML cells instead of KG1a-CXCR4 AML cells. Bars depict mean results + S.E.M., n=3; *, indicates p<0.05.

FIGURE 3. CSA pretreatment of early differentiating MC3T3 osteoblasts reduces TNAP expression.

(A) MC3T3 cells were treated as in Fig. 2A except whole cell lysates were harvested on Day 2 (without the addition of KG1a-CXCR4 AML cells, AMD3100, or SDF-1). Immunoblot depicting the effect of CSA on TNAP expression in MC3T3 cells. The same membrane was stripped and re-probed for total ERK2 as a control, n=3. (B) MC3T3 cells were treated as in Fig. 2a except live/dead staining and imaging occurred on Day 2 (without the addition of KG1a-CXCR4 AML cells, AMD3100, or SDF-1). Live/dead staining and confocal imaging of live (green) and dead (red) cells were used to ensure MC3T3 cell viability in the presence of CSA. Images were acquired on three separate days for a total of 15 images analyzed for each condition. (C) Statistical summary of (B). Bars depict mean results + S.E.M., n=3.

FIGURE 4. Depletion of TNAP via siRNA reduces MC3T3 osteoblast-mediated protection of KG1a-CXCR4 and U937-CXCR4 AML cells from SDF-1-induced apoptosis.

(A) Depiction of MC3T3 osteoblast and KG1a-CXCR4 AML cell line co-culture model. On Day −1, MC3T3 osteoblasts were electroporated with 0.8 nanomoles of control or Alpl (TNAP) siRNA. The maintenance medium was changed 6 hours later. On Day 0, MC3T3 osteoblasts were treated with osteogenic differentiation media. On Day 2, the osteoblasts were washed and then co-cultured with KG1a-CXCR4 AML cells. The indicated samples were challenged with 1.3 × 10⁻⁸ M SDF-1. On Day 3, the KG1a-CXCR4 AML cells were harvested and assayed for apoptosis utilizing annexin-V staining and flow cytometry. (B) MC3T3 cells were treated as in (A) except whole cell lysates were harvested on Day 2 (without the addition of KG1a-CXCR4 AML cells or SDF-1). Immunoblot of TNAP expression in control siRNA- and TNAP-siRNA transfected MC3T3 cells. The same membrane was stripped and re-probed for total ERK2 as a control, n=3. (C) A representative experiment as performed in (A) shows the percentage of annexin-V positive KG1a-CXCR4 cells from each culture within the YFP gate indicated (gating on cells with high levels of CXCR4 expression as the CXCR4 is YFP tagged). (D) Summary of multiple experiments performed as in (A). Bars depict mean results + S.E.M., n=3; *, indicates p<0.05. (E) Summary of multiple experiments performed as in (A) except with U937-CXCR4 AML cells instead of KG1a-CXCR4 AML cells. Bars depict mean results + S.E.M., n=4; *, indicates p<0.05. (F) MC3T3 cells were treated as in Fig. 4a except live/dead staining and imaging occurred on Day 2 (without the addition of KG1a-CXCR4 AML cells, AMD3100, or SDF-1). Live/dead staining and confocal imaging of live (green) and dead (red) cells were used to ensure MC3T3 cell viability in the presence of Alpl (TNAP) siRNA. Images were acquired on three separate days for a total of 15 images analyzed for each condition. (G) Statistical summary of (F). Bars depict mean results + S.E.M., n=3.

FIGURE 5. TNAP inhibition reduces MC3T3 osteoblast-mediated protection of KG1a-CXCR4 and U937-CXCR4 AML cells from SDF-1-induced apoptosis.

(A) Depiction of MC3T3 osteoblast and KG1a-CXCR4 AML cell line co-culture model. On Day 0, MC3T3 osteoblasts were treated with osteogenic differentiation media and either 0.1% DMSO or 10μM TNAP inhibitor MLS-0038949. On Day 2, the osteoblasts were washed to remove the DMSO or TNAP inhibitor pretreatment and were then co-cultured with KG1a-CXCR4 AML cells. The indicated wells were challenged with 1.3 × 10⁻⁸ M SDF-1. On Day 3, the KG1a-CXCR4 AML cells were harvested and assayed for apoptosis utilizing annexin-V staining and flow cytometry. (B) A representative experiment as performed in (A) shows the percentage of annexin-V positive KG1a-CXCR4 cells from each culture within the YFP gate indicated (gating on cells with high levels of CXCR4 expression as the CXCR4 is YFP tagged). (C) Summary of multiple experiments performed as in (A). Bars depict mean results + S.E.M., n=3; *, indicates p<0.05. (D) Summary of multiple experiments performed as in (A) except with U937-CXCR4 AML cells instead of KG1a-CXCR4 AML cells. Bars depict mean results + S.E.M., n=3; *, indicates p<0.05. (E) MC3T3 cells were treated as in Fig. 4a except live/dead staining and imaging occurred on Day 2 (without the addition of KG1a-CXCR4 AML cells, AMD3100, or SDF-1). Live/dead staining and confocal imaging of live (green) and dead (red) cells were used to ensure MC3T3 cell viability in the presence of TNAP inhibitor. Images were acquired on three separate days for a total of 15 images analyzed for each condition. (F) Statistical summary of (F). Bars depict mean results + S.E.M., n=3.

FIGURE 6. Reduction of TNAP expression or activity in early differentiating osteoblasts inhibits protection of AML cells.

Differentiating osteoblasts protect AML cells from SDF-1 induced apoptosis. HDACi and CSA inhibit osteoblast differentiation and TNAP expression as well as osteoblast-mediated protection of AML cells. Reducing TNAP expression or activity reduces osteoblast-mediated protection of AML cells.