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H. Larose et al.
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Figure 3. NOTCH1 T349P and T311P mutants confer a growth advantage to cells. (A) The most frequent variants (of the amino acids involved in the NOTCH1-JAG1 interface) of NOTCH1 reported on COSMIC, and the frequency at which they have been reported. (B) Quantitative polymerase chain reaction (qPCR) showing the fold- increase in transcript levels of NOTCH1 compared to that in cells transduced with an empty vector (n=3). (C) Western blot for full-length and cleaved intracellular NOTCH1 (ICN) and α-tubulin in HEK293FT cells 48 h after transfection to express wild-type (WT) or mutant (T349P or T311P) NOTCH1, or an empty vector (EV). Only the relevant sections of the whole blot are shown and the contrast of the whole image was modified in order to improve legibility. Data are representative of three biological repeats. (D) Proliferation of HEK293FT cells expressing the indicated proteins or empty vector control as measured by the Real Time Glo assay at 24, 48 and 72 h after transfection (***P<0.001; n=3). (E) Fold-change in expression of DLL1 over the WT NOTCH1 control as assessed by qPCR in HEK293FT cells express- ing the indicated proteins and co-cultured with either WT or DLL1-expressing OP9 cells (*P<0.05; **P<0.01; n=3). All bar plots display the mean of biological repli- cates, and error bars represent the standard deviation.
HEK293FT cells expressing NOTCH1 mutants compared to those transfected with either an empty vector or WT NOTCH1 (Online Supplementary Figure S3F and S3G). These data are suggestive of increased NOTCH1 activity as a consequence of the NOTCH1 T349P mutation com- pared to WT NOTCH1.
To determine why the T349P NOTCH1 mutant led to enhanced cell proliferation, in silico modeling was per- formed to predict the effects of the mutation on protein conformation using a published crystal structure of NOTCH1 bound to one of its ligands (Jagged1; PDBID 5UK5) (Online Supplementary Figure S3C and S3D).36 The model demonstrated that NOTCH1 residues 349 and 311 mediate binding to NOTCH1 ligands. Therefore, to determine whether increased cell proliferation (Figure 3D) was dependent on NOTCH1 ligand, NOTCH1 WT or mutant-expressing HEK293FT cells were co-cultured with OP9 cells expressing or not the NOTCH1 ligand DLL1. There was no discernible difference in prolifera- tion between cells co-cultured with wild-type OP9, or OP9-DLL1 cells (Online Supplementary Figure S3E). However, increased transcription of endogenous DLL1 was observed on expression of the mutant forms of NOTCH1 compared to WT protein, suggesting that the mutant proteins might themselves lead to transcription of ligand in an autonomous fashion (Figure 3E). In an
attempt to validate this, we silenced DLL1 using a specific siRNA (Online Supplementary Figure S3H), which reduced the proliferation advantage induced by the NOTCH1 T349P mutant (Online Supplementary Figure S3I).
NPM-ALK induces expression of NOTCH1 via STAT3 transcriptional activity in anaplastic large cell lymphoma
Given that ALCL cell lines express high levels of WT NOTCH1,8 the mechanism of NOTCH1 expression was investigated. NPM-ALK activity was inhibited by incuba- tion with the ALK/ROS/MET inhibitor crizotinib (Figure 4A) or expression silenced with a specific shRNA (Figure 4D) in three and two cell lines, respectively. In both cases, a significant decrease in transcripts for NOTCH1 (Figure 4B and E) and its transcriptional target HES1 (Figure 4C and F), was observed, suggesting that NOTCH1 transcrip- tion and NOTCH1 activity are dependent on NPM-ALK. Given that STAT3 is a key nodal downstream target of NPM-ALK, STAT3 expression was inhibited by employ- ing specific shRNA in ALCL cell lines (Figure 4G). As pre- dicted, transcript levels of NOTCH1 (Figure 4H), HES1 (Figure 4I) and HEY1 (Figure 4J) were all significantly downregulated as a result of STAT3 silencing in both ALK+ and ALK– ALCL cell lines (Figure 4H-J, Online Supplementary Figure S4A). Analysis of published chro-
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