Knockdown of the Transcription Factor PU.1 Causes Characteristic Transcriptional Changes in Hematopoietic Stem Cells Prior to Leukemic Transformation.

Knockdown of the expression of the transcription factor PU.1 in mice results in an initial preleukemic expansion of myeloid progenitors and subsequent transformation to an immature acute myeloid leukemia (AML). Recent reports suggest that functional inactivation of PU.1 might also play a role in human AML. However, the molecular mechanisms underlying the malignant transformation are unknown. Examining the bone marrow of leukemic PU.1 knockdown mice we found an expansion of lin−, c-kit+, Sca1+ hematopoietic stem cells (HSC) as compared to wildtype animals. This finding prompted us to examine the transcriptome of HSC of PU.1 knockdown mice in the initial phase to look for early transcriptional changes underlying the malignant transformation. After lineage-depletion and FACS sorting of lin−, c-kit+, Sca1+ HSC we performed linear amplification of RNA which was then hybridized with Affymetrix Mouse Genome 430 2.0 arrays covering ~39,000 transcripts. Unsupervised hierarchical cluster analysis clearly distinguished PU.1 knockdown and wildtype HSC, demonstrating that reduced PU.1 expression levels lead to characteristic transcriptional changes in the stem cell compartment preceding the malignant transformation. 55 transcripts were upregulated, and 143 transcripts were downregulated in PU.1 knockdown HSC (>1.5-fold, p<0.05), reflecting the role of PU.1 as a transcriptional activator. We corroborated differential expression of 16 genes by quantitative real-time RT-PCR. Among the downregulated genes were c-fes, Bruton’s tyrosine kinase, transcription factor EC, and GM-CSF receptor α, demonstrating that those in-vitro targets of PU.1 are also affected in stem cells in vivo. BAFF-R and TACI, two members of the tumor necrosis factor receptor superfamily, receptor tyrosine kinase TEK and toll-like receptor 4 were downregulated, whereas PDGF receptor was upregulated, suggesting disturbed signaling in PU.1 knockdown HSC. We are currently investigating the causal contribution of these deregulated genes to the leukemic phenotype. In summary, our data delineate a distinct transcriptional pattern that precedes the leukemic transformation in PU.1 knockdown HSC, and provide novel insight into PU.1-mediated molecular pathways in vivo.