Inhibition of BMP-Smad Pathway Reduces Leukemic Stemness in Pediatric AML

Despite aggressive chemotherapy, relapse occurs in almost half of children with acute myeloid leukemia (AML), with very dismal survival. Novel and mechanism-driven therapies are desperately needed to conquer chemotherapy resistance and leukemic stemness in pediatric AML. Within the bone marrow niche, stromal cells protect leukemia cells from chemotherapy, maintain leukemic stemness, and eventually lead to disease recurrence. We developed an in vitro AML cell-stromal cell co-culture model to mimic bone marrow microenvironment. Stroma-leukemia cell interaction leads to activation of various signaling molecules in AML cells that allow them to evade apoptosis. One such example is extracellular signal-regulated kinases 1/2 (ERK1/2), important pro-survival proteins. ERK1/2 are activated by the Ras/Raf/ mitogen-activated protein kinase kinase (MAPK/ERK kinase or MEK) pathway downstream of signals from the stroma. We recently showed that stromal co-culture activates ERK1/2 in pediatric AML samples, contributing to chemotherapy resistance (Long, et al, 2017, Oncotarget, 8:90037).

To identify genes that are regulated in AML cells by ERK1/2 activation, 4 pediatric AML samples were cultured alone, or co-cultured with mOrange-expressing stroma for 24 hours, in the presence or absence of a selective MEK inhibitor, selumetinib (1 μM). Thereafter, cells were flow sorted to exclude mOrange+ stroma and CD45^high/SSC^low lymphocytes. Sorted AML cells underwent total RNA extraction for nCounter® PanCancer Pathways Panel (Nanostring Technologies) gene expression profiling study. We focused on the genes that were either up- or down-regulated by co-culture with stroma, and changed in the reverse direction by the addition of selumetinib. We chose a few genes among the list (BMP2, BNIP3, H2AFX, DUSP2, FZD3, BCL2L1, CHEK2) that are reported to be involved in oncogenesis, chemotherapy resistance, cell growth and survival. Using qRT-PCR, we confirmed bone morphogenic protein 2 (BMP2) to be upregulated in AML cells by stroma, and the effect of stroma was reversed by selumetinib. Further, we confirmed the same change of BMP2 at the protein level by FACS. Smad1, 5 and 8 are transcriptional factors immediately downstream from BMP receptors and play a central role in BMP signal transduction. Using FACS we discovered stroma-induced activation of Smad 1/8 in pediatric AML patient samples, which was partially alleviated by selumetinib and a selective BMP inhibitor, K02288 (10 μM).

BMPs are growth factors that belong to the transforming growth factor beta (TGF-beta) superfamily and are thought to be involved in stem cell properties such as self-renewal. To determine if the BMP-Smad pathway plays a role in chemotherapy resistance, 4 pediatric AML patient samples were cultured on or off stromal cells, and treated with cytarabine (10 μM) with or without K02288 (10 μM) for 24h. Cells were analyzed for cytarabine-induced apoptosis with Annexin V staining by FACS, excluding stromal cells and lymphocytes. K02288 treatment did not alter cytarabine-induced apoptosis. We next tested the potential role of BMP-Smad pathway in leukemic self-renewal in pediatric AML samples. Pediatric AML patient samples were plated at 50,000/ml or 100,000/ml in Methocult medium (H-4535) to quantify stem and progenitor cells. Samples were treated with vehicle or K02288 (10 μM). Colonies and viable cells were counted and normalized to control 7-14 days after plating. Harvested cells were stained for CD34, CD14 and CD11b to evaluate differentiation by FACS. Remaining cells were replated at the same density for 2 more rounds. We found that K02288 reduced colony counts (e.g., 100±0% in vehicle control, v. 25±10% in K02288, n=5, p<0.002, post 2^nd plating) and decreased the number of viable cells at 2^nd and 3^rd plating (Figure 1). The BMP inhibitor also promoted differentiation of leukemic stem cell in pediatric AML samples, as evidenced by increased CD14 and CD11b expression. In contrast, for normal pediatric bone marrow samples, K02288 did not change colony counts or the number of viable cells, and it did not promote differentiation. Those data suggested BMP-Smad is likely to contribute to leukemic stemness in pediatric AML without disrupting normal hematopoietic stem cells. Therefore, BMP-Smad pathway may be a promising therapeutic target to reduce leukemia burden and improve survival for children with AML.

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