BCL11B Mutations In T-Cell Acute Lymphoblastic Leukemia

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<0.001).

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.