Abstract

The NOTCH1 signaling pathway plays a critical role in the regulation of hematopoietic stem cell homeostasis and is required for lymphocytes to adopt a T-cell fate. Importantly, aberrant activation of NOTCH signaling due to activating mutations in the NOTCH1 gene is involved in the pathogenesis of over 50% human T-cell acute lymphoblastic leukemias (T-ALL) and the therapeutic efficacy of inhibition of NOTCH signaling via gamma-secretase inhibitors (GSI) is currently being tested in clinical trials. However, little is known about the transcriptional programs activated downstream of NOTCH1 activation that contribute to the transformation of T-cell progenitors. Here we used chromatin immunoprecipitation and promoter microarrays (ChIP-on-chip) to identify direct transcriptional targets of NOTCH1; and microarray gene expression analysis to decipher the oncogenic transcriptional network activated by the NOTCH1 oncoprotein in T-ALL cells. Using the Hu19K arrays that contain over 13,000 human promoter regions, we have identified 134 candidate direct targets (p<0.0001) of NOTCH1 in the HPB-ALL T-ALL cell line by ChIP-on-chip. NOTCH1 targets identified by ChIP -on-chip included known direct targets of NOTCH1 such as preTCRA, as well as genes involved in T-cell signaling (CD3D, ASE-1/CD3E associated protein), proliferation (CDK5, CDC25A, RBL1, BUB3 and ING3) and survival (Survivin).

Inhibition of NOTCH1 signaling with compound E, a strong GSI, induced a gene expression signature characterized by the downregulation of known NOTCH1 direct target genes such as DELTEX1, HES1 and HEY1. Analysis of different cell lines representative of different stages of maturation arrest and different oncogenic groups of T-ALL identified a core signature of transcriptional responses to NOTCH signaling inhibition, which included the downregulation of the MYC oncogene as well as numerous genes involved in nucleotide metabolism and protein synthesis. In addition, cell cycle inhibitors p27/KIP1 and p18/INK4D were upregulated. Importantly, 13 of the top direct target genes of NOTCH1 identified by ChIP-on-chip were consistently downregulated (p<0.005) upon NOTCH signaling inhibition in multiple cell lines. However, more restricted responses to NOTCH1 signaling inhibition, which included important developmental regulators of T-cell development such as the pre-TCRA, were present in specific groups of samples. These results demonstrate that NOTCH1 activation induces a complex transcriptional response in T-ALL cells, which is in part dependent on the stage of T-cell development and/or the interaction with other T-ALL transcription factor oncogenes. Common effector pathways downstream of NOTCH signaling may represent novel therapeutic targets for the treatment of T-ALL, while cell type specific responses may influence the cellular effects and the clinical efficacy of NOTCH signaling inhibitors currently under evaluation in clinical trials. Overall, our results indicate that NOTCH1 acts as a master transcriptional regulator at the top of a complex regulatory network that contributes to leukemogenesis by regulating multiple critical pathways involved in the regulation of T-cell differentiation, proliferation and survival.

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