Heterodimerization Domain Mutations Research Articles

Mutational analysis of the Notch2 negative regulatory region identifies key structural elements for mechanical stability

The Notch signalling pathway is fundamental to cell differentiation in developing and self-renewing tissues. Notch is activated upon ligand-induced conformational change of the Notch negative regulatory region (NRR), unmasking a key proteolytic site (S2) and facilitating downstream events. The favoured model requires endocytosis of a tightly bound ligand to transmit force to the NRR region, sufficient to cause a structural change that exposes the S2 site. We have previously shown, using atomic force microscopy and molecular dynamics simulations, that application of force to the N-terminus of the Notch2 NRR facilitates metalloprotease cleavage at an early stage in the unfolding process. Here, mutations are made within the heterodimerization (HD) domain of the NRR that are known to cause constitutive activation of Notch1 whilst having no effect on the chemical stability of Notch2. Comparison of the mechanical stability and simulated forced unfolding of recombinant Notch2 NRR proteins demonstrates a reduced stability following mutation and identifies two critical structural elements of the NRR in its response to force – the linker region between Lin12-Notch repeats LNRA and LNRB and the α3 helix within the HD domain – both of which mask the S2 cleavage site prior to Notch activation. In two mutated proteins, the LNRC:HD domain interaction is also reduced in stability. The observed changes to mechanical stability following these HD domain mutations highlight key regions of the Notch2 NRR that are important for mechanical, but not chemical, stability. This research could also help determine the fundamental differences in the NRRs of Notch1 and Notch2.

NOTCH1 mutations are associated with favourable long‐term prognosis in paediatric T‐cell acute lymphoblastic leukaemia: a retrospective study of patients treated on BCH‐2003 and CCLG‐2008 protocol in China

Activating mutations of NOTCH1 are a common occurrence in T-cell acute lymphoblastic leukaemia (T-ALL), but its impact on T-ALL treatment is still controversial. In this study, the incidence, clinical features, and prognosis of 92 Chinese children with T-ALL treated using the Beijing Children's Hospital-2003 and Chinese Childhood Leukaemia Group-2008 protocols were analysed. NOTCH1 mutations were found in 42% of T-ALL patients and were not associated with clinical features, prednisone response, and minimal residual disease (MRD) at day 33 and 78. However, proline, glutamate, serine, threonine (PEST)/transactivation domain (TAD) mutations were associated with younger age (15/16 mutant vs. 48/76 wild-type, P=0·018) and more central nervous system involvement (4/16 mutant vs. 3/76 wild-type, P=0·016); while heterodimerization domain (HD) mutations were associated with KMT2A-MLLT1 (MLL-ENL; 4/30 mutant vs. 1/62 wild-type, P=0·037). Furthermore, prognosis was better in patients with NOTCH1 mutations than in those with wild-type NOTCH1 (5-year event-free survival [EFS] 92·0±4·5% vs. 64·0±7·1%; P=0·003). Long-term outcome was better in patients carrying HD mutations than in patients with wild-type HD (5-year EFS 89·7±5·6% vs. 69·3±6·2%; P=0·034). NOTCH1 mutations and MRD at day 78 were independent prognostic factors. These findings indicate that NOTCH1 mutation predicts a favourable outcome in Chinese paediatric patients with T-ALL on the BCH-2003 and CCLG-2008 protocols, and may be considered a prognostic stratification factor.

Unlike Paediatric T-ALL, Notch-1 and FBXW7 Mutations Do Not Seem to Predict a Better Outcome in Adult Patients: Data from the UKALLXII/ECOG2993 Protocol

A risk-adapted approach to the treatment of patients with acute lymphoblastic leukemia (ALL) has the potential of improving survival in high-risk patients and reducing therapy-related long-term sequelae in those at low-risk. The use of molecular markers that can further define risk are needed in this disease. Activating mutations of Notch-1 have been reported in over 50% of patients with T-ALL. Mutations in the heterodimerization domain (HD) lead to ligand-independent cleavage, whilst truncating mutations of the C-terminal PEST domain disrupt binding to the negative regulator FBXW7, leading to prolonged half-life of intracellular Notch (ICN). More recently, Notch-activating mutations in FBXW7 that prevent ICN degradation have been reported in 20% of patients. In pediatric patients treated on the ALL-BFM 2000 protocol, patients with Notch-1 mutations (MUT) had an excellent outcome with an event free survival (EFS) of 90% compared to 71% in wild type (WT) patients. The impact of FBXW7 mutations was not evaluated. We sought to address whether adults with Notch-1 and/or FBXW7 mutations also faired sufficiently well that they might avoid treatment intensification/transplantation. Notch-1 HD-N, HD-C, TAD and PEST domains, and FBXW7 WD40 domain were screened by denaturing high performance liquid chromatography and/or bidirectional sequencing in a cohort of 88 adult T-ALL patients treated on the MRC UKALLXII/ECOG2993 trial (54 UKALLXII, 34 ECOG2993). Overall, 51 patients (58%) had at least one Notch-1 mutation; 37 in the HD only, 8 in the PEST domain only and 6 in both HD and PEST domains. Sixteen patients (18%) had an FBXW7 mutation (7 R465C, 3 R505C, 2 R479Q, 2 R479L, 1 R465H, and 1 G423V) and, of note, 15 of these mutations altered conserved arginine residues in the WD40 domain thought to be responsible for binding to the Notch-1 PEST domain. Five FBXW7 MUT patients were Notch-1 WT. There was a positive association between having an FBXW7 mutation and a Notch-1 mutation in the HD only (Fishers exact test 2P.02). Furthermore, no patient had both an FBXW7 and a PEST mutation. This is consistent with the hypothesis that Notch-1 HD and FBXW7 mutations act in concert similar to dual HD and PEST mutations. Complete remission was achieved in 96% of patients. Median follow-up was 3.5 years. The Notch-1 WT and MUT groups received similar treatment (8 Vs 15 sibling allograft; 3 Vs 7 autograft; 3 Vs 3 matched unrelated donor allograft; 19 Vs 24 chemotherapy with maintenance alone; P.84). There was no significant association with white cell count (WCC) or age according to either Notch-1 or FBXW7 mutational status (Mantel Haenszel test for trend P>0.1 for each case). Outcome is shown in table 1. In contrast to the data in pediatric T-ALL, in this adult cohort Notch-1 mutational status was not associated with improved EFS or overall survival (OS); nor was there an association according to the type of Notch-1 mutation. Similarly, FBXW7 mutational status alone was not predictive of outcome. There was a suggestion that patients with either a Notch and/or an FBXW7 mutation had better EFS, although this did not reach statistical significance. Adjusting for treatment received in first remission did not affect the results. In conclusion, the outcome of adult patients remains poor irrespective of their Notch-1 and/or FBXW7 mutation status, and there is no evidence that these markers should influence therapy in this age group.5 yr EFS (WT v MUT)OR (CI)log rank 2pNotch37% v 47%0.79 (0.44–1.42)0.4FBXW741% v 53%0.85 (0.40–1.82)0.7NOTCH +/− FBXW731% v 49%0.70 (0.38–1.29)0.35 yr OS WT v MUT)OR (CI)log rank 2pNotch47% v 51%0.87 (0.47–1.62)0.7FBXW746% v 62%0.76 (0.35–1.66)0.5NOTCH +/− FBXW744% v 52%0.82 (0.43–1.56)0.5

CDC4 and NOTCH1 Mutations in Childhood T-Cell Acute Lymphoblastic Leukemia and T Cell Non-Hodgkin's Lymphoma; a Japan Association of Childhood Leukemia Study Group.

Risk-adapted therapy has resulted in a improvement of outcome in childhood acute lymphoblastic leukemia (ALL), however, T-cell ALL (T-ALL) have still relatively poor outcome compared to B-precursor ALL. Although activating mutations of the NOTCH1 gene have been reported in more than half of T-ALL, the prognostic significance of this finding has yet to be determined. CDC4 gene was isolated as a negative regulator of NOTCH1 and mutations were detected in T-ALL cell line and other cancers. We performed mutation analysis of the NOTCH1 and CDC4 gene in 14 T-ALL cell lines, 52 T-ALL and 17 T cell non-Hodgkin's lymphoma (T-NHL) fresh samples. All children with T-ALL and T-NHL were treated on Japan Association of Childhood Leukemia Study (JACLS) ALL-97 and NHL-98 protocol after obtaining informed consent. Mutation detection was performed via PCR based denaturing HPLC followed by direct sequence.Mutations of the NOTCH1 were identified in 10 (71.4%) of 14 T-ALL cell lines, 16 (30.8%) of 52 T-ALL and 6 (35.3%) of 17 T-NHL fresh samples. Twelve mutations in heterodimerization domain (HD) and 12 mutations in PEST domain (PD) were found in 69 fresh samples. The incidence of the NOTCH1 mutation is less frequent than that of previous reports. We found CDC4 mutations in 12 (35.3%) of 52 T-ALL and 2 (11.8%) of 17 T-NHL fresh samples. One insertion mutation in exon 3 and 11 missense mutations were detected in CDC4 gene. Both NOTCH1 and CDC4 mutations were found in 7 patients. Interestingly, the NOTCH1 mutations were only observed in T-ALL and T-NHL patients without relapse, suggesting to be associated with favorable prognosis. However, CDC4 gene was found not to be associated with prognosis.

Leukemia-Associated Mutations within the NOTCH1 Heterodimerization Domain Fall into at Least Two Distinct Mechanistic Classes

The NOTCH1 receptor is cleaved within its extracellular domain by furin during its maturation, yielding two subunits that are held together noncovalently by a juxtamembrane heterodimerization (HD) domain. Normal NOTCH1 signaling is initiated by the binding of ligand to the extracellular subunit, which renders the transmembrane subunit susceptible to two successive cleavages within and C terminal to the heterodimerization domain, catalyzed by metalloproteases and gamma-secretase, respectively. Because mutations in the heterodimerization domain of NOTCH1 occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL), we assessed the effect of 16 putative tumor-associated mutations on Notch1 signaling and HD domain stability. We show here that 15 of the 16 mutations activate canonical NOTCH1 signaling. Increases in signaling occur in a ligand-independent fashion, require gamma-secretase activity, and correlate with an increased susceptibility to cleavage by metalloproteases. The activating mutations cause soluble NOTCH1 heterodimers to dissociate more readily, either under native conditions (n = 3) or in the presence of urea (n = 11). One mutation, an insertion of 14 residues immediately N terminal to the metalloprotease cleavage site, increases metalloprotease sensitivity more than all others, despite a negligible effect on heterodimer stability by comparison, suggesting that the insertion may expose the S2 site by repositioning it relative to protective NOTCH1 ectodomain residues. Together, these studies show that leukemia-associated HD domain mutations render NOTCH1 sensitive to ligand-independent proteolytic activation through two distinct mechanisms.

Notch1 mutations are important for leukemic transformation in murine models of precursor-T leukemia/lymphoma

AbstractNOTCH1 is frequently mutated in human precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL). In the current study, we found that 13 of 19 cell lines and 29 of 49 primary tumors from SCL/LMO1, OLIG2/LMO1, OLIG2, LMO1, NUP98/HOXD13, and p27-/-/SMAD3+/- mice had Notch1 mutations in either the heterodimerization (HD) or the glutamic acid/serine/threonine (PEST) domain but not both. Thymocytes from clinically healthy SCL/LMO1 mice aged 5 weeks did not have Notch1 mutations, whereas thymocytes from clinically healthy SCL/LMO1 mice aged 8 to 12 weeks did have Notch1 mutations and formed tumors upon transplantation into nude mice. Remarkably, all of the HD domain mutations that we identified were single-base substitutions, whereas all of the PEST domain mutations were insertions or deletions, half of which mapped to 1 of 2 mutational “hot spots.” Taken together, these findings indicate that Notch1 mutations are very frequent events that are acquired relatively early in the process of leukemic transformation and are important for leukemic cell growth. (Blood. 2006;107: 2540-2543)

Functional Analysis of Leukemia-Associated Mutations Involving the Heterodimerization Domain of NOTCH1.

NOTCH1 is a type I transmembrane receptor that regulates T cell development via a signaling pathway that relies on regulated proteolysis. During its maturation, most NOTCH1 is cleaved at a position 70 amino acids external to the transmembrane domain by a furin-like protease, creating extracellular (NEC) and transmembrane (NTM) subunits that are held together non-covalently by a juxtamembranous heterodimerization (HD) domain. Ligand-binding to NEC promotes cleavage by i) metalloproteases at a site in the ectodomain of NTM, followed by ii) gamma-secretase within the transmembrane domain. This releases the NOTCH1 intracellular domain (ICN1), allowing it to translocate to the nucleus and activate target genes. Normally, proteolysis is constrained prior to ligand-binding by an extracellular negative regulatory region consisting of 3 iterated LNR repeats and the N- and C-terminal portions of the HD domain, which flank the furin cleavage site. Recent work has shown that human T-ALL is frequently associated with gain of function mutations that map to the HD domain of NOTCH1. These mutations are distributed in both parts of the HD domain and include point mutations, short insertions, and deletions, suggesting that there might be variation in their relative strength and the mechanisms by which they act. To investigate these issues, we introduced 16 of the HD domain mutations found in primary T-ALLs or T-ALL cell lines into a full-length NOTCH1 cDNA, and tested their ability to activate a NOTCH sensitive luciferase reporter gene. Except for the "mutation" R1609S, which was found in only one primary T-ALL sample, all of the mutations stimulated NOTCH1 signaling. These increases in signaling were abolished by a gamma-secretase inhibitor and were associated with increased rates of metalloprotease-mediated cleavage, indicating that activation proceeds through the normal series of proteolytic events. The mutations also caused gains in function when introduced into NOTCH1 polypeptides lacking the ligand-binding region of NEC, indicating that the HD domain mutations can cause ligand-independent receptor activation. Since NEC dissociation can lead to activation of NOTCH signaling (and is a proposed mechanism for normal ligand-mediated NOTCH activation), one simple way for HD domain mutations to act is through the destabilization of NOTCH1 heterodimers. To test this model, each mutation was introduced into soluble NOTCH1 mini-receptors bearing N-terminal FLAG and C-terminal HA tags. When expressed transiently, the normal NOTCH1 mini-receptor was secreted into conditioned media as a furin-processed heterodimer. Certain activating HD domain mutations, such as L1601P, resulted in complete dissociation of the furin-processed mini-receptor subunits under native conditions, and all other HD domain mutations save one were more sensitive to urea-induced dissociation than normal NOTCH1. The exception was an unusual insertional mutation (identified in the P12-Ichikawa cell line) that introduces a 14 amino acid direct repeat sequence at a position immediately N-terminal of the metalloprotease cleavage site. We hypothesize that this mutation, which was associated with the greatest increases in signaling in NOTCH1 reporter gene assays, displaces protective HD and LNR domain residues and thereby unveils the metalloprotease cleavage site. We conclude that most T-ALL-associated HD domain mutations confer ligand-independent gain-of-function on NOTCH1 receptors, but vary in strength and are likely to act through several distinct mechanisms.

Synergistic Induction of T Cell Acute Lymphoblastic Leukemia by Functionally Distinct Mutations in NOTCH1.

Recent observations that Notch1 mutations are present in more than 50% of human T-ALL patient samples indicate that Notch signals have a central role in the pathogenesis of T-ALL (Weng et al., 2004, Science, 306, 269–271). Notch1 mutations are of two types: i) mis-sense mutations in an extracellular heterodimerization (HD) domain; and ii) frameshift or stop codon mutations that result in deletion of an intracellular PEST destruction box. The likely effect(s) of these mutations is to i) cause heightened proteolytic activation of Notch1, an event that requires the enzyme gamma-secretase; and to ii) increase the duration of Notch1 signaling, respectively. To investigate the contribution of the HD and PEST domain mutations to the pathogenesis of human T-ALL, Notch1 alleles with common HD mutations were introduced retrovirally into hematopoietic progenitor cells, which were then transplanted into lethally irradiated mice. These mice developed transiently circulating abnormal CD4+CD8+ T cells, but failed to develop leukemia after 9 months of monitoring. Similar findings occurred when Notch1 alleles bearing only PEST mutations were introduced into mice, as previously noted by others (Feldman et al., 2000, Blood , 96, 1906–1913). However, when HD mutations were combined with a PEST mutation in cis, the mice developed persistent circulating CD4+CD8+ T cells, leukocytosis, and eventually T-ALL (median time, 18 weeks post-transplantation). These tumors infiltrated lymphoid organs, marrow, and viscera, were monoclonal or oligoclonal, and had intact proviral integrations. Cell lines derived from these tumors are susceptible to gamma-secretase inhibitor-mediated cell death, which indicates a continued reliance on Notch signaling for growth and survival. These results show the utility of the murine BMT assay to score Notch1 mutants found in human T-ALL with respect to leukemogenicity, and suggest that the level of signal that is necessary to induce and sustain Notch1-associated T-ALL is relatively high. It will be of interest to determine if HD domain mutations generally require PEST deletions in cis to induce T-ALL, or if weak and strong mutations of varying leukemogenic potential exist.

Gain-of-Function NOTCH1 Mutations Occur Frequently in Human T Cell Acute Lymphoblastic Leukemia.

NOTCH1 was discovered originally through its involvement in a rare (7;9) translocation found in human T cell acute lymphoblastic leukemia (T-ALL). Here, we report that >50% of human T-ALLs have activating NOTCH1 mutations, occurring as amino acid substitutions in an extracellular heterodimerization (HD) domain and/or as frameshift and stop codon mutations that result in the deletion of a C-terminal PEST destruction box. Normal pro-NOTCH1 is processed into a heterodimer consisting of an extracellular subunit and a transmembrane subunit, which associate non-covalently through the HD domain. NOTCH1 activation is triggered by binding of Serrate or Delta-like ligands to the extracellular subunit, which induces successive proteolytic cleavages in the transmembrane subunit that are dependent on i) metalloproteases and ii) gamma-secretase. The γ-secretase cleavage releases intracellular NOTCH1 (ICN1), which translocates to the nucleus and forms a transcriptional activation complex with the transcription factor CSL and co-activators of the Mastermind family. Normal turnover of ICN1 is regulated by the C-terminal PEST sequence. Data pointing to the existence of frequent abnormalities of NOTCH1 in T-ALL stemmed from a functional screen of 30 T-ALL cell lines. This identified five T-ALL cell lines that underwent growth arrest in response to i) treatment with an inhibitor γ-secretase, and ii) retroviral transduction of dominant negative Mastermind-like-1. Sequencing of of cDNAs from 4 of these 5 cell lines demonstrated both a missense mutation in the HD domain and a frameshift mutation in the PEST domain lying in cis in the same NOTCH1 allele. Subsequent sequencing of genomic DNA obtained from bone marrow lymphoblasts of 96 children and adolescents with T-ALL demonstrated identical or similar mutations in NOTCH1 in 53 samples (55.2%). Mutations in the HD domain alone were observed in 26 cases (27.1%), in the PEST domain alone in 11 cases (11.4%), and in both the HD and PEST domains in 16 cases (16.7%). Mutations were observed in tumors associated with expression of HOX11 (2/3), HOX11L2 (10/13; 77%), TAL1 (12/31; 39%), LYL1 (9/14; 64%), MLL-ENL (1/3) or CALM-AF10 (1/2), which span the major molecular T-ALL subtypes. In contrast, NOTCH1 mutations were not observed in genomic DNAs samples obtained from B-ALL lymphoblasts (N=89), or from T-ALL patients with NOTCH1-associated disease at the time of clinical remission (N=4). Reporter gene assays conducted with plasmids expressing normal and mutated forms of NOTCH1 showed that a PEST deletion or various HD mutations alone caused ~1.5-fold and 3–9-fold stimulations of reporter gene activity, respectively, whereas normal NOTCH1 lacked intrinsic signaling activity. More strikingly, the combination of various HD mutations and a PEST deletion in cis caused synergistic 20–40-fold stimulations of reporter gene activity that were completely abrogated by a γ-secretase inhibitor, indicating that signaling depends on proteolysis. These results suggest a model in which HD domain mutations promote ICN1 production, and PEST domain mutations enhance ICN1 stability. Our findings greatly expand the role of NOTCH1 in the pathogenesis of human T-ALL, and provide a rationale for targeted therapies that interfere with NOTCH signaling.