Oncogenic Transcriptional Programs Controlled by TLX1/HOX11 and TLX3/HOX11L2 in T-ALL.

Abstract 676

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with the activation of transcription factor oncogenes. TLX1/HOX11 was originally isolated from the recurrent t(10;14)(q24;q11) translocation and is aberrantly expressed in 5% to 10% of pediatric and up to 30% of adult T-ALL. The TLX3/HOX11L2 oncogene is closely related to TLX1 and is activated by the cryptic t(5;14)(q35;q32) chromosomal translocation in 25% of pediatric T-ALLs. TLX1 and TLX3 positive T-ALLs have a distinct gene expression profile resembling that of thymocytes blocked at the early double-positive stage of development, suggesting that aberrant expression of TLX1 and TLX3 interferes with critical transcriptional regulatory networks that control cell proliferation, differentiation and survival during T-cell development. However, the identity of such oncogenic pathways and the mechanisms through which they operate are still largely unknown. Here we used an integrated systems biology approach to elucidate the transcriptional regulatory networks controlled by aberrant expression of TLX1 and TLX3 in T-ALL. Towards this goal, we performed gene expression profiling studies and ChIP-on-chip analysis of TLX1 and TLX3 direct target genes in leukemic lymphoblasts. ChIP-on-chip analysis of TLX1 direct target genes identified 5,550 promoters bound by TLX1 in ALL-SIL T-ALL cells with a significance cutoff of P <0.0001. Similarly, ChIP-on-chip analysis of TLX3 direct target genes identified 7,183 promoters bound by this transcription factor oncogene in HPB-ALL cells at the P <0.0001 significance level. Most notably, TLX1 direct targets showed >75% overlap with the TLX3 ChIP-on-chip target genes (P<10E-16), suggesting a common mechanism of action for TLX1 and TLX3 in T-cell transformation. Notably, inhibition of TLX1 expression by shRNA in the ALL-SIL cell line resulted in global transcriptional upregulation of TLX1 direct target genes, establishing a direct connection between TLX1 binding and promoter regulation and defining TLX1 as a transcriptional repressor in T-ALL. Following on these results, we used Affymetrix arrays to analyze the pattern of gene expression associated with TLX1 and TLX3 in a series of 82 primary T-ALLs. This analysis identified a gene expression signature dominated by genes downregulated in TLX1 and TLX3 positive cases, which was markedly and significantly enriched in TLX1 and TLX3 direct targets (P <0.0001). These results define the core transcriptional program controlled by TLX1 and TLX3 in T-cell transformation and open two possible scenarios. First, most, if not all, TLX1 and TLX3 target genes may play an equivalent role downstream of these transcription factor oncogenes to modulate multiple functional pathways some of which will contribute to T-cell transformation. Alternatively, TLX1 and TLX3 target genes may be organized in hierarchical transcriptional regulatory modules. This second model would be characterized by the presence of master regulator genes with a high level of network connectivity and the capacity to redundantly control multiple genes in the core transcriptional program controlled by TLX1 and TLX3. In order to annotate the functional relevance of TLX1 and TLX3 direct target genes we used ARACNe, an algorithm for the reverse engineering of transcriptional networks, in a series of 228 primary T-ALL samples analyzed using Affymetrix U133 plus 2.0 expression arrays. This analysis established that the transcriptional network controlled by TLX1 and TLX3 has a hierarchical scale free structure in which a limited number of master regulator genes show a high level of connectivity. Notably, this structure defines RUNX1, a master regulator of hematopoietic and T-cell development, as the most prominent direct target gene controlled by TLX1 and TLX3 in T-ALL. Consistent with these results, integrative analysis of expression signatures in human (n=82) and mouse (n=55) T-ALLs, identified Runx1 downregulation as a prominent feature associated with aberrant expression of TLX1. Overall, these results identify the transcriptional regulatory program controlled by TLX1 and TLX3 in T-ALL, establish the role of TLX1 and TLX3 as transcriptional repressors in T-cell transformation and point to a prominent role of RUNX1 downregulation in the pathogenesis of T- ALL.