SALL4-Dependent Signaling Networks Are Required for Survival of Leukemia Cells.

Abstract

Our group and others have recently shown that a murine transcriptional factor, SALL4 plays an essential role in maintaining the properties of embryonic stem cells and in governing the fate of the primitive inner cell mass through regulation of Oct4. Over-expression of SALL4 in mice exhibit myelodysplastic syndrome (MDS) which transforms to acute myeloid leukemia (AML) associated with expansion of leukemic stem cells. Aberrant expression of SALL4 in leukemic blasts is commonly seen in a variety of AML and MDS cases. However, the transcriptional programs downstream of the SALL4 stem cell gene in leukemia are unknown. We mapped its global gene targets using chromatin-immunoprecipitation followed by microarray hybridization (ChIP-on-ChIP). We identified over two thousands high-confidence SALL4 binding sites in the acute leukemic cell line, NB4. Analysis of the SALL4 binding sites revealed that genes involved in cell death, cancer, DNA replication/repair, and cell cycle were highly enriched (p<0.05) when functionally classified. This is consistent with promoter binding of over 280 apoptosis-related genes including important apoptosis and cell-cycle arrest induction genes (DAP3, CARD9, TP53, DAXX, CASP6, CASP9, CYCS, TNF, TAL1, and PTEN) and apoptosis inhibition genes (Bmi-1, BCL2, TEGT and XIAP). This indicates that SALL4 may play a role in leukemogenesis. Reduction of wild type SALL4 mRNA levels by approximately 50 percent resulted in a 21-fold increase in caspase-3 activity from 4.6 percent to 98.3 percent. In addition, suppression of SALL4 expression caused a significant decrease in NB4 cell growth rate. To further study the role of the SALL4 in cell growth, we monitored cell-cycle changes and cellular DNA synthesis in SALL4-suppressed and wild type NB4 cells through flow cytometry analysis and the BrdU incorporation assay. SALL4-reduced NB4 showed about a four-fold decrease in S phase cells and a significant increase in the G1 and G2 phases, which paralleled the drop in DNA synthesis as measurement from the level of BrdU incorporation. Similar results were observed in other cancer cell lines. In addition, SALL4-reduced NB4 cells showed significantly decreased tumorigenicity in immunodeficient mice. Our studies provide a foundation for developing a leukemia stem cell-specific therapy targeting SALL4.