Abstract
Activating mutations and deletions affecting specific NOTCH1 protein domains have been recently shown to occur widely in T-cell neoplasia, e.g. in T-acute lymphoblastic leukemia (T-ALL). However, knowledge of NOTCH1 chromosomal alterations is largely based on a single cell line model (SUP-T1) with t(7;9)(q35;q34) in which NOTCH1 truncated at exon 24 is juxtaposed with TCRB. We describe the characterization of a novel rearrangement, t(9;14)(q34.3;q11) in two T-cell lymphoma cell lines, HD-MAR and HT-1. FISH analysis using fosmid clones and sequencing of fragments identified by long distance inverse PCR showed that in both cases t(9;14) effected tail-to-tail juxtaposition of intron 27 of NOTCH1 with TCRA genes, namely 5′-TRAV40 in HD-MAR, and intron 2 of TRAV5 in HT-1. Thus, in both cell lines t(9;14) places NOTCH1, truncated immediately 3′ of the HD-domain, under transcriptional control of TCRA. The 14q11.2 breakpoints in HD-MAR and HT-1 lie, respectively, near the proximal E-delta enhancer and amid a cryptic enhancer region represented by a cluster of T-cell specific DNase-I hypersensitive sites. Western blotting revealed prominent expression of truncated activated NOTCH1 polypeptides, ranging in size from 100 to 115 kDa in both cell lines. Antibodies recognizing ANK and TAD domains, believed essential for inducing T-ALL, detected the aberrant polypeptides. Moreover, treatment with gamma-secretase inhibitor (GSI) altered expression patterns of NOTCH1 polypeptides and induced growth inhibition due to G0/G1 cell cycle arrest in both t(9;14) cell lines, in stark contrast to GSI-resistant SUP-T1 cells wherein truncation occurs before the heterodimerization (HD) domain. (Another recently described t(7;9) cell line (CUTLL1) which is GSI-sensitive also carries a NOTCH1 breakpoint at intron 27.) The same protein species were not detectable by antibodies recognizing the transmembrane domain of NOTCH1 which requires GS for exposure suggesting nuclear access requires GS-cleavage. Immunostaining confirmed extranuclear blocking of NOTCH1 in response to GSI in HD-MAR/HT-1 but not in SUP-T1. In contrast, repression of HES1 occurred in response to GSI irrespective of NOTCH1 breakpoint location, suggesting its non-involvement in growth signaling. In addition to providing cell line models for a new NOTCH1 disease translocation, these data suggest that the sensitivities of T-cell neoplasias bearing NOTCH1 translocations may critically depend on whether 9q34 breakpoints lie upstream or downstream of the HD domain.
Molecular characterization of subcutaneous panniculitis-like T-cell lymphoma reveals upregulation of immunosuppression- and autoimmunity-associated genes
