A Novel Class of Activating Mutations in NOTCH1 in T-ALL.
Authors: Sulis Maria Luisa , Williams Odette , Tosello Valeria , Pallippukam Sasikala , Palomero Teresa , Barnes Kelly , Meijerink Jules , Ferrando Adolfo 
Published: October 2019

Aberrant activation of NOTCH1 signaling induces transformation of T-cell progenitors and plays a prominent role in the pathogenesis of over 50% of human T-cell acute lymphoblastic leukemias (T-ALL), which harbor activating mutations in the heterodimerization (HD) and PEST domains of NOTCH1. Here we report a new class of activating mutations in NOTCH1 in human T-ALL. These so called JuxtaMembrane Expansion (JME) mutants consist of internal tandem duplications of exon 28 and adjacent intronic sequences in the NOTCH1 gene, which result in expansions of the extracellular juxtamembrane region of the NOTCH1 receptor. Western blot analysis of T-ALL cell lines lacking known NOTCH1 mutations demonstrated high levels of activated NOTCH1 protein in Jurkat T-ALL cells, suggesting the presence of an as yet unidentified activating NOTCH1 mutation in this cell line. Sequence analysis of Jurkat NOTCH1 transcripts revealed an internal tandem duplication in exon 28 of NOTCH1, resulting in the insertion of 17 amino acids at position 1740 in the extracelullar juxtamembrane region of the receptor. Subsequent PCR amplification of NOTCH1 exon 28 sequences from 194 primary T-ALL lymphoblast samples identified seven additional in frame insertion mutations ranging from 11 to 36 amino acids in length, all of which were located in the vicinity of codon 1740 in the extracelullar juxtamembrane region of the NOTCH1 receptor. Luciferase assays showed that expression of the NOTCH1 Jurkat JME17 mutant allele induced over 200 fold activation of a NOTCH1 reporter construct compared to controls. Activation of NOTCH1 signaling requires proteolytic cleavage of the receptor, first by an ADAM metalloprotease (S2 clevage) and subsequently by the gamma-secretase complex. NOTCH1 signaling induced by the Jurkat JME17 mutant was completely abrogated by incubation with CompE, a highly active gamma-secretase inhibitor. Consistently, treatment of Jurkat T-ALL cells with CompE resulted in rapid clearance of activated NOTCH1 protein and in marked downregulation of NOTCH1 target genes such as HES1 and DELTEX1. Interestingly, and in contrast with previously described HD mutations, JME NOTCH1 alleles retain an intact HD domain and a protected canonical S2 metalloprotease cleavage site. Thus, we hypothesized that activation of NOTCH1 by JME mutations could be mediated by aberrant metalloprotease cleavage at ectopic S2 sites within the JME insertion sequence. However, mutation of the canonical S2 cleavage abrogated the function of the NOTCH1 Jurkat JME17 mutant allele. Furthermore, analysis of artificially generated JME insertions containing sequences unrelated to the leukemia-derived JME alleles, showed that activation of NOTCH1 by JME mutations depends primarily on the length of the inserted peptides and not on their specific amino acid sequences. Thus, shorter insertions of 5 to 13 amino acids in length induced moderate (5–10 fold) activation of the NOTCH1 receptor, while insertions of 14 amino acids or longer induced marked (>70 fold) increases in NOTCH1 signaling. Overall, these results provide further insight in the mechanisms that control the activation of the NOTCH1 receptor in T-ALL.