Activation of the LMO2 Oncogene in T-ALL through a Somatically Acquired Neomorphic Promoter

LMO2is a crucial regulator of normal hematopoiesis but is progressively silenced from the early T-cell progenitor stage of thymic development. Aberrant LMO2 expression leads to a block in differentiation, increased self-renewal and aggressive T-cell acute lymphoblastic leukemia (T-ALL). Although ≈50% of T-ALL patients overexpress LMO2, this can be attributed to a cytogenetic lesion in only ≈10% of patients, leaving a significant portion mechanistically unaccounted for. We recently discovered somatic mutations that create an oncogenic super-enhancer driving TAL1 expression (Mansour et al., 2014, Science). Therefore, we investigated whether comparable mutations may be causing dysregulated LMO2 expression.

Aberrant H3K27ac marks indicative of active chromatin were identified prior to and encompassing the non-coding exon 2 of the LMO2 gene by ChIP-seq in DU.528 and PF-382 T-ALL cell lines, both of which exhibit upregulated LMO2 expression but lack chromosomal lesions at this locus. Sequencing across this peak revealed a heterozygous 20bp duplication in PF-382 cells and a heterozygous 1bp deletion in DU.528 cells, located close to a region recently described as an intermediate promoter. Both indels generated a de novo MYB consensus motif. MYB ChIP-seq showed that ≥96% of reads aligned to the mutant rather than the wild-type allele, suggesting that MYB was preferentially recruited to the mutant allele. Heterozygous mutations were also detected in diagnostic samples from 3% (5/159) of pediatric and 6% (10/164) of adult T-ALL patients. Absence of the mutations in 7 available patient-matched remission samples confirmed that they were somatic. The mutations were densely distributed around highly conserved native ETS1, MYB and GATA motifs; 5 patients had an additional MYB site, 3 both a MYB and an ETS1 site, 1 an ETS1 site alone, and 4 had new RUNX1 binding sites. This suggests that this region may have a regulatory role that is exploited by the mutations to constitutively activate LMO2.

All 6 mutant-positive patients with available RNA showed LMO2 overexpression as determined by qRT-PCR, consistent with the hypothesis that these mutations activate gene expression. Furthermore, monoallelic LMO2 expression could be demonstrated in DU.528 cells and 3 of 4 informative T-ALL samples using a heterozygous germline SNP. Expression of the mutations in luciferase reporter assays also indicated that they all markedly activated luciferase activity to between 2x and 57x compared to the wild-type sequence.

To assess causality between the mutations and LMO2 dysregulation, we used CRISPR/Cas9 genome-editing with a guide RNA designed to target the mutant MYB site in PF-382 cells. Reduction of LMO2 expression to ≤10% of PF-382 mutant activity was observed in a clone where the mutant allele had been reverted to wild-type and in another clone with a single T>C substitution disrupting the mutant MYB binding site. Interestingly, 2 clones that increased the distance between the native and the mutant MYB sites also resulted in a reduction in LMO2 expression to 19% and 25% of PF-382 activity, suggesting that there are functionally limiting spatial constraints on the mutant MYB site in relation to other neighboring transcription factor binding sites. This was further validated by the lack of reduction in LMO2 expression in a clone where the sequence between the two MYB sites was altered but the spacing distance was unchanged.

In conclusion, we have identified and functionally validated a novel recurrent mutation hotspot occurring in a non-coding site whereby introduction of additional binding sites for a number of different transcription factors drives monoallelic LMO2 overexpression from a neomorphic promoter in a substantial proportion of both adult and pediatric T-ALL patients. This mechanism of oncogene activation may be relevant to a wide variety of human cancers.