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Introduction: Combined activation of specific oncogenes is a general feature of human cancer and suggests that co-occurrence of particular oncogenic factors provides a selective advantage during cellular transformation. However, the exact molecular mechanisms by which oncoproteins cooperate during malignant transformation often remains elusive. Here, we study the functional relationship between the cooperative oncogenes NOTCH1 and TLX1 in the context of T-cell acute lymphoblastic leukemia to better understand their cooperative mechanism of action during T cell transformation. Methods: In this study, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) for the TLX1 homeobox oncoprotein in the T-ALL cell line ALL-SIL and analyzed the transcriptional response before and after TLX1 modulation using microarray based methods. We performed integration of TLX1 ChIPseq data with publically available transcription factor binding profiles in T-ALL and evaluated the immunophenotypic and transcriptional effects of ectopic TLX1 expression in thymus-derived CD34+ T-cell progenitors. Results: Integration of TLX1 ChIP-seq data with gene expression profiles after TLX1 knockdown in the TLX1 positive T-ALL cell line ALL-SIL, confirmed the previously established role for TLX1 as transcriptional repressor in T-ALL biology. In line with previous reports (Della-Gatta et al., Nature Medicine, 2012), de novo TLX1 motif discovery identified RUNX1 and ETS1 as important mediators of the global TLX1 transcriptional network. Next, we used TLX1 ChIP-seq data to define TLX1 super-enhancers including several loci critically involved in T-cell biology (e.g. T-cell receptor loci, RAG2, MYB). Furthermore, Gene set Enrichment Analysis (GSEA) showed that TLX1-defined super-enhancers were significantly affected by JQ1 treatment in ALL-SIL. Integration of our TLX1 ChIP-seq data with publically available ChIP-seq data for ICN1, RUNX1 and ETS1 in T-ALL cells (Wang et al., PNAS, 2013) showed a remarkable genome-wide overlap between the binding sites of these four transcription factors. Integration of these binding patterns with transcriptional read-out revealed an unprecedented transcriptional antagonism between TLX1 and NOTCH1, in which TLX1 suppresses the oncogenic NOTCH1 transcriptional program including IL7R, NOTCH3 and c-MYC. In line with this observation, ectopic TLX1 expression in CD34+ human thymic precursor T-cells broadly interfered with the normal T-cell differentiation program causing differentiation arrest, massive apoptosis and a significant downregulation of NOTCH1 target genes including reduced IL7R-alpha surface expression. Conclusion: In conclusion, our study reveals unexpected transcriptional antagonism between the cooperative oncogenes TLX1 and NOTCH1 in the biology of T-ALL. TLX1 mediated suppression of NOTCH signaling might be critically involved in the pre-leukemic phenotype (reduced thymus size and decreased cellularity) that has been observed in Lck-TLX1 transgenic mice (De Keersmaecker et al., Nature Medicine, 2010). Moreover, our results suggests that full malignant transformation of TLX1-driven leukemias might only be possible through acquisition of secondary NOTCH1 mutations that can overcome the initial TLX1 mediated suppression of NOTCH1 signaling, which is in line with the high frequency of gain-of-function NOTCH1 mutations in TLX1 positive human T-ALL. Disclosures No relevant conflicts of interest to declare.

Abstract 471 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with the activation of oncogenic transcription factors. Thus, 5% to 10% of pediatric and up to 30% of adult T-ALL patients show aberrant expression of the TLX1 transcription factor oncogene. Aberrant expression of TLX1 in Lck-TLX1 transgenics induces transformation of T-cell progenitors and the development of clonal T-cell lymphoblastic tumors with over 80% penetrance and a latency of 29 weeks. The prolonged latency before T-ALL development in TLX1 transgenic mice and the clonal nature of these tumors suggested the presence of cooperating mutations involved in the pathogenesis of these leukemias. Array Comparative Genomic Hybridization (aCGH) analysis identified several recurring chromosomal alterations in these tumors including three heterozygous deletions on chromosome 12, with a common deleted region containing only the Bcl11b gene. Moreover, DNA sequence analysis of Bcl11b showed the presence of Bcl11b mutations in 4/15 (27%) mouse TLX1 induced T-ALLs, which together with the 3 Bcl11b deletions identified in our aCGH analysis brings the prevalence of Bcl11b alterations in mouse TLX1 induced tumors to 7/15 (47%). Notably, mutation analysis of Bcl11b in a panel of 21 non TLX1-transgenic mouse T-ALL tumors failed to detect any Bcl11b mutations (P Bcl11b encodes a zinc finger transcription factor with a critical role in the differentiation and survival of T-cell progenitors in the thymus. Given the similarities between our mouse model of TLX1-induced leukemia and human T-ALL, we hypothesized that BCL11B alterations could also be implicated in the pathogenesis of human T-ALL. Most notably, aCGH analysis of human T-ALL samples showed the presence of focal heterozygous deletions encompassing the BCL11B locus in 2/69 (3%) T-ALL cases. In addition, mutation analysis of BCL11B in human T-ALL demonstrated the presence of somatic truncating frameshift mutations and somatic missense point mutations in 9/71 (13%) cases analyzed. Interestingly, all 6 BCL11B missense mutations involved zinc finger domains. One sample showed compound heterozygous BCL11B mutations while the remaining cases harbored heterozygous BCL11B lesions. Finally, expression analysis of TLX1 or TLX3 in 8 T-ALLs harboring mutations or deletions in BCL11B with RNA available showed aberrant expression of these transcription factor oncogenes in 5/8 (63%) [TLX1 (4/8); TLX3 (1/8)] of these samples. The identification of a high prevalence of Bcl11b mutations and deletions in TLX1-induced tumors suggested a specific genetic interaction between these two genes in T-cell transformation. We contemplated two alternative scenarios. First, TLX1 could induce partial inactivation of BCL11B in T-cell transformation, an effect that would be reinforced by secondary genetic alterations in the BCL11B locus during tumor progression. Alternatively, BCL11B mutations and TLX1 overexpression could operate in parallel pathways that contribute to distinct but complementary aspects of tumor development. To test if BCL11B could be a direct transcriptional target of TLX1 in T-ALL, we analyzed the binding of TLX1 to the BCL11B promoter via chromatin immunoprecipitation analysis in ALL-SIL, a T-ALL cell line expressing high levels of TLX1 as result of the t(10;14)(q24;q11.2) translocation. This analysis revealed that indeed TLX1 binds to the BCL11B promoter. Moreover, siRNA knockdown of TLX1 in this cell line resulted in transcriptional upregulation of BCL11B, indicating that BCL11B is a direct transcriptional target downregulated by TLX1 in T-ALL. The model that emerges from these results is that the TLX1 oncogene directly downregulates the BCL11B tumor suppressor gene, and that the oncogenic effects of this transcriptional regulatory axis can be fixed and reinforced by secondary deletions and mutations in the BCL11B locus acquired during tumor progression. Altogether, these results highlight the power of integrated genomic analysis of human and mouse leukemias, identify a TLX1-BCL11B transcriptional regulatory axis in the pathogenesis of T-ALL and define BCL11B as a tumor suppressor gene recurrently deleted and mutated in 16% of human T-ALL patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 361 Chromosomal rearrangements involving the EVI1 gene are a recurrent finding in malignant myeloid disorders. These translocations or inversions contribute to ectopic expression or to the formation of fusion genes involving the EVI1 gene. EVI1 transcriptional activation has been reported in up to 10% of acute myeloid leukemia (AML) and is a prognostic marker of poor outcome. MicroRNA (miRNA) deregulation was recently identified as a major contributor to cancer initiation and progression. As miRNA genes were shown to be directly regulated by activated proto-oncogenes, we aimed to identify miRNAs under direct or indirect control of EVI1. To this purpose, we analyzed the expression of 366 miRNAs in 38 EVI1 rearranged/overexpressing patient samples, 6 normal bone marrow controls and 2 EVI1 knockdown model systems (siRNA mediated EVI1 knockdown in the EVI1 rearranged/overexpressing cell lines Kasumi-3 and UCSD-AML1). In total, 24 upregulated and 25 downregulated miRNAs (p Disclosures: No relevant conflicts of interest to declare.