Illegitimate V(D)J Recombination Involving Notch1 and Bcl11b in Precursor T-Cell Lymphoblastic Leukemia/Lymphoma

Abstract 1323

V(D)J recombination of antigen receptor loci confers diversity to the mammalian immune system. However, off-target, illegitimate V(D)J recombination between non-antigen receptor loci, leading to site-specific interstitial deletion has been reported in humans and mice with lymphoid malignancies. These recurrent, site-specific interstitial deletions are thought to be oncogenic, leading to activation of proto-oncogenes or deletion of tumor suppressor genes. Notch1 and Bcl11b are known targets for illegitimate V(D)J recombination in murine precursor T-cell leukemia/lymphoma (pre-T-LBL). The Notch1 5'-deletions remove exon1 and 2; as a consequence Notch1 transcription initiates from a cryptic promoter immediately 5' of exon 25. These alternative transcripts are translated from exon 28, and lack the N-term ligand binding domain, resulting in a Notch1 protein that is independent of ligand binding. The known Bcl11b deletions excise exon2 and 3, and result in loss of function of the Bcl11b tumor suppressor gene. The frequency with which Notch1 and Bcl11b deletions occur in concert is unknown. Here we examined both Notch1 and Bcl11b deletion mutations, as well as Notch1 mutations involving the PEST, HD, and TM domains in primary murine pre-T LBL samples (N=44), and murine pre-T LBL cell lines (N=21), that arose in specific genetically engineered mouse strains (Scl-Lmo1, NUP98-PHF23, HoxA9, Lin28B, Survivin-TCR). Analysis of 44 primary tumor samples identified 17 Notch1 5'-interstitial deletions (38.6%) and 8 Bcl11b deletions (18.1%), while analysis of 21 cell lines revealed 10 Notch1 5' deletions (47.6%) and 5 Bcl11b deletions (24%). The percent of Notch1 PEST, HD, and TM mutations were 84.1%, 13.6%, and 4.5%, respectively, in the primary tumors and 95%, 5%, and 0% in the cell lines.

Two primary tumor samples showed several different 5' deletion mutations, representing multiple independent leukemic clones. Of the 13 primary tumor/cell line pairs we analyzed, 5 pairs showed identical Notch1 and Bcl11b mutations. However, for 7 pairs, the primary tumor was oligoclonal with respect to Notch1 and Bcl11b mutations, with one clone being biologically selected for growth in vitro. In 3 cases, Bcl11b deletion was not detected in primary tumor but was detected in the cell line, suggesting that the Bcl11b deletion was a secondary event in these cell lines. Notch1 PEST mutations were present in almost all cell lines, and were often accompanied by an additional Notch1 mutation (5'deletion, HD, or TM). Conversely, 5' deletion, HD, and TM mutations were mutually exclusive. These findings support the hypothesis that the 5'deletion, HD, and TM mutations lead to ligand independent activation, while the PEST domain mutations prevent proteasomal degradation leading to persistence of the transcriptionally active intracellular Notch1. In several cases, samples had an identical PEST mutation, but distinct 5' deletion or HD mutations, indicating the PEST mutation preceded the 5' deletion or HD mutation. Three of 21 cell lines had both a 5' deletion and Bcl11b deletion, however there was no statistically significant correlation between these events.

One 5'-recombination recognition sequence (RSS) and 2 3'-RSSs have been reported for Notch1 5' deletions. H3K4me3 ChIP-sequence data clearly demonstrated alternative transcript initiation from exon 25 in cell lines with a 5' Notch1 deletion. Curiously, one cell line (106A) which was negative for the 5' deletion showed a marked increase of H3K4Me3 at Notch1 exon25, suggesting transcription initiation at this alternative site, and leading to the hypothesis that this cell line may have a variant form of Notch1 5' deletion which was undetectable by conventional assays. We were able to identify an alternative RSS for this cell line, leading to a novel 5' Notch1 deletion. We then reanalyzed our samples and found an additional example of this alternate 5' deletion, and a third sample which displayed yet another form of Notch1 5' deletion. Of note, this deletion did not have a cryptic RSS, and had developed in a RAG1 deficient mouse. In summary, we have identified three novel forms of Notch1 5' deletions, shown that Notch1 mutations are very common in murine pre-T LBL, shown that PEST mutations generally precede 5'deletion mutations, and shown that 5' Notch1 deletions are not consistently associated with other illegitimate VDJ recombinase-mediated events (such as Bcl11b deletions).