NOTCH1 encodes a transmembrane signaling protein that plays key roles in development and neoplasia. Its leukemogenic involvement was first revealed by analysis of t(7;9)(q34;q34.3) in a T-cell acute lymphocytic leukemia (T-ALL) cell line (SUP-T1), which juxtaposes truncated NOTCH1 with TCRB, directing overexpression of N-terminally truncated polypeptides (reviewed in Grabher et al.1). Wild-type NOTCH1 is initially cleaved (S1 cleavage) into twin polypeptides: an extracellular N-terminal subunit (NEC) and a transmembrane C-terminal subunit (NTM). Binding of NOTCH ligands to the epidermal growth factor-like repeats region of NEC promotes metalloprotease cleavage of NTM (S2 cleavage) to create membrane-bound NTM*I monomers (Figure 1a). These are cleaved in turn (S3 cleavage) at multiple sites within the heterodimerization domain (HD) binding extracellular and transmembrane subunits by the γ-secretase (GS) protease complex to release the intracellular domain (ICN1), which forms a ternary complex stimulating effector transcription.1 ICN1 contains regulator of amino acid metabolism (RAM), ankyrin repeat and transcriptional activation domains, and a C-terminal polypeptide enriched with proline, glutamate, serine and threonine (PEST). Ankyrin and transcriptional activation domains are required for induction of T-ALL in mice.2 Whereas γ-secretase inhibitors (GSi) may prove effective against T-ALL subsets, most T-cell lines are resistant, including SUP-T1.3 We describe a second NOTCH1 rearrangement, t(9;14)(q34.3;q11.2), in T-ALL cell lines, HD-MAR4 and HT-1,5 both highly GSi-sensitive despite overexpressing truncated NOTCH1.
Cytogenetic analysis revealed t(9;14)(q34.3;q11.2) in both HD-MAR and HT-1 cells, with NOTCH1 breakpoints inside fosmid 80019F4 accompanied in HD-MAR cells by deletion of ∼80 kbp corresponding to NEC (Figures 1a–d). Fluorescence in situ hybridization (FISH) also showed 14q11.2 breakpoints in both HD-MAR and HT-1 cells within the TRAV locus. These observations define an hitherto unlisted rearrangement (http://atlasgeneticsoncology.org//index.html), t(9;14)(q34.3;q11.2) that juxtaposes the intragenic regions of both NOTCH1 and TRAV. Long-distance inverse (LDI)-PCR confirmed and extended these findings, revealing tail-to-tail fusions of intron-27 of NOTCH1 at 138 516 818 and 138 516 905 bp, with 5'-TRAV40 (21 852 526 bp) and intron-1 of TRAV5 (21 287 494 bp) in HD-MAR and HT-1, respectively (Figures 1e and f, Supplementary Figure S1 A/B). t(9;14) in HD-MAR and HT-1 cells places NOTCH1, truncated inside HD (predicted 5 amino acids trans- of the S2 cleavage site), under transcriptional control of TRAV (Figure 1a). The TRAV-40 breakpoint in HD-MAR lies close to the proximal Eδ enhancer, whereas that in HT-1 at TRAV-5 lies ∼600 kbp upstream near a cluster of DNase-I hypersensitive sites known to drive oncogene transcription in T-ALL. Thus, both t(9;14) cell lines carry breakpoints located inside HD, in contrast with that lying upstream (cis-) of HD in t(7;9) SUP-T1 cells.1
Protein (western blot) analyses of the minimal transforming regions, ankyrin and transcriptional activation domains, together with TM/RAM domains in T-ALL cells are shown in Figure 2a (top/middle). High protein expression of full-length NOTCH1 (∼300 kDa) and NTM (∼120 kDa) was detected in non-translocation cell lines excepting TALL-1 cells, which is monosomic for chromosome 9. Contrastingly, in NOTCH1 translocation cell lines the ∼300-kDa band was weak (HD-MAR, HT-1) or absent (SUP-T1), indicating translocation-driven expression therein (Figure 2b). N-truncated NTMs in both t(9;14) cells undercut 116 kDa wild-type polypeptide (Figure 2a top), in contrast with t(7;9) SUP-T1 cells, which also translated longer species implying impaired cleavage therein. Correspondingly, ∼110 TM/RAM+ forms in t(9;14) cells are taken to represent ICN1. Weak TM/RAM signals in cis-HD breakpoint SUP-T1 cells (Figure 2a bottom) also imply impaired GS-cleavage, as Ab8925 epitopes require prior GS exposure. These findings, linking peri-HD breakpoint location to aberrant protein expression, prompted further investigation into the effect of GSi treatments on NOTCH1 signaling and proliferation.
γ-Secretase inhibitor treatment effected dose-dependent reductions of cell growth and viability in HD-MAR and HT-1, while sparing SUP-T1 cells (Figure 3a). The GSi sensitivities of both t(9;14) cell lines significantly exceeded that of T-ALL1, which was described earlier as sensitive.3 Proliferation arrest in HD-MAR and HT-1 paralleled dose-dependent increases in G0/G1 arrest after GSi treatments, which evinced progressively weaker responses in TALL-1 and SUP-T1 cells (Figure 3b). To investigate these differential GSi sensitivities, downregulation of NOTCH1 transcriptional target genes, HES1 and MYC were respectively, measured in HD-MAR (92, 66%), HT-1 (94, 79%) and SUP-T1 (75, 39%) cells. Differential Gsi sensitivities of NOTCH1 effector gene expression thus paralleled both cell survival and cell cycle progression endpoints (Figures 3c and d), confirming the greater GS-dependence of NOTCH1 signaling in intra-HD breakpoint cells.
Inhibiting cleavage by GS-dependence (GSi) treatment effected the accumulation of four ANK+ species (105–116 kDa) taken as NTM/NTM*I (Figure 3e brackets), and dissipation of ∼110 kDa TM/RAM+ (ICN1), that is, a picture consistent with interconversion. This result underlines the dependence of TM/RAM expression on GS activity in t(9;14) cells. A fifth truncated ∼100 kDa NTM/NTM*I accumulated in HT-1 cells (arrow). Immunostaining revealed greater accumulation of perimembraneous NOTCH1 at the expense of intranuclear forms in HD-MAR than in SUP-T1 (Figure 3f). Taken together, GSi treatments revealed increased dependence of intra-HD breakpoint cells on GS activity for NOTCH1 expression, signaling and proliferation. The enhanced TM/RAM epitope exposure of both t(9;14) cell lines (Figure 2a bottom) suggests a protein structural basis underlying the reduced dependence of GS cleavage on prior S2 cleavage owing to their adjacent breakpoints. The correlation of GS sensitivity with intra-HD breakpoint placement is consistent with data from a second t(7;9) cell line CUTLL1 with an intra-HD breakpoint, also sensitive to GSi.7
In T-ALL mutations occur in the F-box protein FBXW7 (alias FBW7, hCdc4), which abrogate its binding to NOTCH1, affecting NOTCH1 longevity and GSi responses. However, analysis of HD-MAR, HT-1 and SUP-T1 cells showed normal sequences around Arg465, Arg479 and Arg505 hotspots, discounting a major contribution in modulating GSi sensitivity thereby, refocusing the spotlight on GS cleavage.
All the three molecularly karyotyped t(9;14)(q34;q11) cases report TRAD/NOTCH1 involvement (Gesk et al.,8 this report). All the five age-recorded t(9;14) cases were hitherto described in young adults (Table 1), whereas all four t(7;9) cases are pediatric (http://atlasgeneticsoncology.org/), raising the question whether different age groups might be targeted. However, only a few piecemeal cases have been described for either translocation and additional mapped examples are required to delineate breakpoints in both t(9;14) and t(7;9) T-ALL and help determine the clinical relationship of the cytogenetic entities.
Our findings highlight the role of intra-HD NOTCH1 breakpoint locations in promoting ligand-independent transcription and translation of GSi-sensitive polypeptides. Hightened GSi sensitivity bestowed by such intra-HD breakpoints may reflect increased molecular exposure near the HD region facilitating NOTCH1 cleavage. Independence from ligand-mediated cleavage may serve to promote the ‘non-oncogene addiction’ of intra-HD breakpoint cells on GS cleavage for NOTCH1 signaling.
In summary, we have characterized a new NOTCH1 alteration, t(9;14)(q34;q11), in T-ALL, which is only the second neoplastic NOTCH1 chromosome rearrangement described hitherto. Our data highlight breakpoints in the peri-HD region in determining GS activity and responses to inhibitor. Cell line models for the entity described here provide singular tools for investigating the leukemic role of NOTCH1, a topic of pressing clinical and scientific interest.
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We thank H Ben-Bassat and M Abe for HD-MAR and HT-1 cells, respectively, A Ferrando for reading the paper, and the José Carreras Leukemia Research Fund for support.
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Suzuki, S., Nagel, S., Schneider, B. et al. A second NOTCH1 chromosome rearrangement: t(9;14)(q34.3;q11.2) in T-cell neoplasia. Leukemia 23, 1003–1006 (2009) doi:10.1038/leu.2008.366
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