Noggin Overexpression Enhances Leukemic Progenitors Self-Renewal in AML by Abrogating the BMP Pathway Activation.

Noggin, encoded by Nog gene, is a secreted protein that inhibits the bone morphogenetic proteins (BMPs). BMPs regulate the proliferation and differentiation of many tissues, including bone, kidney and hematopoiesis, mainly through phosporylation of SMAD1, 5 or 8 . By cDNA-RDA, we found overexpression of Nog in OCI-AML2 and OCI-AML5 cell lines compared to normal bone marrow (NBM). To confirm this overexpression, we performed real-time RT-PCR on 6 NBM, 2 NBM CD34⁺ cells, 6 AML cell lines (OCI-AML1-5 and NB4) and 53 AML patients’ (pts) samples. NBM had very low Nog/GAPDH (Median: 1.63 X 10⁻⁵, range: 1.55 X 10⁻⁵-1.75 X 10⁻⁵). Similar levels were found in CD34⁺ cells. Five cells lines (OCI-AML-1, -2, -4, -5 and NB4) showed high Nog/GAPDH (range: 7.5 X 10⁻⁴ - 4.3 X 10⁻³), whereas OCI-AML3 expessed very low level (8.6 X 10⁻⁷). High Nog expression (>4-fold NBM level) was, also, found in 51/53 pts (96%), with a median 45.8-fold NBM level. Protein study revealed a positive correlation between Noggin and Nog RNA levels. In contrast, there was a negative correlation between Noggin and phosphorylated SMAD1, 5, 8, suggesting that Noggin plays a key role in the BMP pathway regulation in hematopoietic tissues. To analyze the effect of Noggin on leukemic behavior, we assessed the effect of Noggin on the clonogenicity of OCI-AML3. OCI/AML-3 cultured with Noggin showed enhanced plating efficiency (56±7 vs 33±6, p=0.005), increased colony size and more compact colonies. To determine whether this higher efficiency is due to enhanced leukemic progenitors self-renewal, cells were grown in suspension culture in the presence of Noggin, washed and plated in methylcellulose medium without Noggin on days 1–3. Cells treated with Noggin showed increasing colony-forming potential with time, suggesting an enhancement of leukemic progenitors self-renewal. To further characterize the effect of Noggin on OCI/AML-3 cells we performed cell-cycle and apoptosis assays. Noggin significantly increased the percentage of cells in S/G2/M phase as early as 3h after treatment with a maximum effect by 24h (38% vs 27%). Noggin, also, decreased the percentage of cells undergoing apoptosis (2.8% vs 9.1%), an effect that became noticeable within 24h. Moreover, Noggin protected the leukemic cells against serum-deprivation and daunorubicin-induced apoptosis. As Noggin is a negative regulator of the BMP pathway we assessed the effect of Noggin on the expression of genes known to be affected by BMP, including DKK1, Wnt1, Wnt3a, Wnt5b, P21, P27, Bcl2, Bax, MSX1, MSX2, Id1, Id2, Id3, DLX1 and DLX2. While complete inhibition of SMAD 1,5,8 phosphorylation could be detected within 1 h of the addition of Noggin, gene expression changes were noticed beginning after 4h. Significant decreased expression at the RNA level was noted for DKK1, Wnt1, Wnt3a, Wnt5b, MSX1, Id1, Id3 and DLX2. Although substantial downregulation of the Wnt-pathway-related genes was seen, there was no change in this pathway overall activity as assessed by B-catenin protein and Wnt-pathway target-genes RNA levels. In summary, AML cells express and respond to the BMP inhibitor Noggin. BMP pathway inhibition increases the leukemic progenitors growth and self- renewal. The reduced expression of MSX1 is of interesting importance as MSX1 promotes p53 apoptotic function. The loss of MSX1 may thus reduce the cells sensitivity to apoptotic stimuli, such as daunorubicin.