A second NOTCH1 chromosome rearrangement: t(9;14)(q34.3;q11.2) in T-cell neoplasia

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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.

Figure 1
figure1

Cytogenetic and molecular breakpoint analysis of t(9;14)(q34;q11). (a) Breakpoint data, clone maps and the genomic structures of NOTCH1, including protein domain schema, and TRAD Note the intra-heterodimerization domain (HD) breakpoints in HD-MAR/HT-1 downstream of the S2 metalloprotease cleavage site, and the cis-HD breakpoint in SUP-T1. (b) Chromosome painting of t(9;14), and (c and d) FISH analyses of NOTCH1 and TRAD, respectively, in HD-MAR4 and HT-1.5 TRAD/NOTCH1 breakpoint junction sequences determined by long-distance inverse (LDI)-PCR are shown in (e) and (f), respectively. Nucleotides are shown in red, green and black to indicate TRAD, filler DNA and NOTCH1, respectively, with breakpoints shown as black dots. LDI-PCR was performed as described elsewhere.6 Primer sequences are given in the Supplementary data.

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.

Figure 2
figure2

NOTCH1 protein expression in T-cell acute lymphocytic leukemia (T-ALL) cell lines with t(9;14). (a) Western analysis of T-ALL cell lines: NOTCH1-ANK domain antibody (Ab) to mN1A (top), bTAN20 Ab to transcriptional activation domains (TADs) (middle) and Ab8925 (activated NOTCH1) to TM/RAM (regulator of amino acid metabolism) (bottom). Ab8925 epitopes include a non-specific band (star) and require prior γ-secretase exposure (manufacturer's data). Note the weak TM/RAM epitopes in SUP-T1 NTM, which are attributable to impaired γ-secretase cleavage. (b) Western analysis of full-length NOTCH1 at longer exposure: note the faint (HD-MAR/HT-1) or absent (SUP-T1) full-length NOTCH1 bands, implying translocation-driven expression. Western blots were performed after lysis with 50 μl of RIPA buffer (1 μl aprotinin (1 mg/ml), 5 μl PMSF (20 ng/ml) and 50 μl 2 × SDS). Antibodies used: NOTCH1 (mN1A; BD Biosciences, San Diego, CA, USA, bTAN-20; Hybridoma Bank, Iowa City, IA, USA and Ab8925; Abcam, Cambridge, UK). Loading was checked using Ponceau dye and anti-human-MSH6 Ab (Santa Cruz Biotechnology, Heidelberg, Germany).

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.

Figure 3
figure3

Responses of t(9;14) cells to γ-secretase inhibitors (GSi) treatment .T-ALL cell lines were cultured for 96 h with the indicated concentrations of DAPT or mock-treated with dimethylsulfoxide (DMSO). Cellular proliferation and DNA content were, respectively, measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (a) and by flow cytometry (b) after staining with propidium iodide. Note GSi hypersensitivities of HD-MAR and HT-1 t(9;14) cells at both endpoints. To analyze the effects of GSi on NOTCH1 signaling, t(9;14) and t(7;9) cells were treated for 16 h with 2 μM DAPT and subsequently analyzed by RQ-PCR to measure expression of HES1 (c) and MYC (d), both shown in black. The DMSO-control was set at 100% (gray). Bars show standard deviations. Again note the greatest HES1/MYC dependence on NOTCH1 signaling in t(9;14) cells. For reverse transcription quantitative (RQ)-PCR, cDNA was synthesized from 5 μg total RNA extracted from 2 × 106 cells with TRIzol (Invitrogen, Karlsruhe, Germany) by random priming in 20 μl using Superscript II (Invitrogen). RQ-PCR used the 7500 Real-Time System (Applied Biosystems, Darmstadt/Germany). Expression of HES1, MYC and the control gene TBP was analyzed using commercial primer sets (Applied Biosystems). To analyse the effects of GSi on NOTCH1 polypeptides, western analysis was performed on HD-MAR and HT-1 cells treated with 2 μM DAPT for the times indicated. (e) NOTCH1 polypeptides were immunoprecipitated from whole cell extracts with antibodies recognizing either the intracellular domain of NOTCH1 (mN1A) or the GS-cleaved form of NOTCH1 (ab8925). Brackets show four accumulating ankyrin-positive (ANK+) species (taken as NTM/NTM*I) and concomitant loss of TM/RAM+ (regulator of amino acid metabolism-positive) species (taken as ICN1) following GSi treatment. A fifth truncated 100 kDa NTM/NTM* accumulated in HT-1 cells (arrow). The starred band is unspecific. (f) Effects of GSi on nuclear localization of NOTCH1 polypeptides in HD-MAR and SUP-T1. Cells treated for 24 h with DMSO (left) or 2 μM DAPT (right) were stained with the intracellular domain of NOTCH1 (mN1A) Ab. Reticular staining after GSi treatment reflects the redistribution of NOTCH1 polypeptide to the cell membrane.

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.

Table 1 Summary of t(9;14) cases

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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Acknowledgements

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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Correspondence to R A F MacLeod.

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Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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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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