Complexity of NOTCH1 juxtamembrane insertion mutations in T-cell acute lymphoblastic leukemia

In T-cell acute lymphoblastic leukemia (T-ALL) constitutively active NOTCH1 signaling triggered by activating mutations in the NOTCH1 gene contributes to the malignant transformation of T-cell precursors and is a hallmark of this disease [1]. NOTCH1 mutations are found in more than 60% of T-ALL and affect critical protein domains responsible for preventing the spontaneous activation of the NOTCH1 receptor in the absence of ligand or for terminating NOTCH1 signaling in the nucleus [2]. The mutation hotspots within NOTCH1 are the heterodimerization domain (HD), the PEST, and the juxtamembrane domains (JMD). More specifically, NOTCH1 mutations cluster in exons 26–27 and 34, encoding the HD and PEST domains, respectively. While PEST domain mutations result in the loss of the domain and increased intracellular NOTCH1 levels due to its impaired proteasomal degradation, HD mutations induce ligand-independent activation of NOTCH1 [2]. Most HD mutations, comprising single amino acid substitutions and small in-frame deletions and insertions (class 1 mutations), destabilize the HD-LNR (LIN-12/ NOTCH1 repeats) interaction and reduce heterodimer stability [3]. In rare cases, insertions of extra amino acids between the distal part of the HD (HD-C) and the S2 cleavage site (class 2 mutations) displace the S2 away from the protective effects of the LNR-HD complex and expose it to cleavage by ADAM-type metalloproteases [2,3]. Moreover, another class of NOTCH1 mutations located in the vicinity of exon 28 encoding the extracellular juxtamembrane domain, termed juxtamembrane expansion mutations (JEMs), which distance the LNR-HD complex from the membrane and allow ligandindependent proteolytic processing of S2, has been identified [4]. Generally, both HD-C class 2 and JME mutations are generated by relatively long insertions resulting from, at least partial, internal tandem duplications (ITDs). However, in case of class 2 HD-C NOTCH1 mutations, aberrant S2 cleavage is induced by the insertion of extra amino acids immediately proximal to the S2 cleavage site, which is displaced closer to the membrane and out of reach of the protective effects of the LNR-HD complex [5]. In contrast, JME insertions are located distal to the S2 cleavage site and displace the LNR-HD complex and the S2 site away from the membrane without altering the primary structure of any of these elements [3,4,6]. While NOTCH1 HD and PEST domain mutations are found in about 40% and 20–25% of T-ALL, respectively, JMD mutations are rather rare and have been detected in roughly 3% of the cases [5,7]. However, since most studies have focused on identifying mutations in the HD and PEST domains and rarely included exon 28, the frequency of JMD mutations may have been underestimated and their complexity has not been explored in detail. Herein we describe five pediatric T-ALL patients with different types of JMD mutations and show, for the first time, their impact on HES1 expression in primary leukemia samples. Furthermore, we summarize all mutations within this region described so far and discuss how these mutations may affect NOTCH1 signaling. We screened 39 pediatric T-ALL patients registered in the Austrian ALL-BFM 90, 95 or 2000 clinical trials [8,9] for mutations in NOTCH1 exons 26–28 and 34 by RT-PCR and direct sequencing of the PCR products as previously described [10]. In three cases (3/39; 7.7%) we detected insertions in exon 28, which were analyzed in detail by sequence analysis of the cloned PCR products [Fig. 1A] and resulted in in-frame insertions of 7 (06-0195), 13 (96-0030) or 14 (04-0573) amino acids in the extracellular juxtamembrane region [Fig. 2; Supplementary Table SI (see http://dx.doi.org/10.3109/ 10428194.2015.1080366), available online]. Of note, in cases 96-0030 and 06-0195 the mutations led to amino acid substitutions at the insertion sites but only the