NOTCH1 has been implicated in hematopoiesis, T-cell differentiation, and the pathogenesis of precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL). Although rare patients with pre-T LBL have chromosomal translocations that activate NOTCH1, it has recently been shown that over 50% of human pre-T LBL samples which did not have chromosomal aberrations involving NOTCH1 had activating mutations in the NOTCH1 heterodimerization (HD) and/or the PEST domain. We examined murine pre-T LBL cell lines as well as primary thymic tumors that arose in SCL/LMO1, OLIG2, OLIG2/LMO1, LMO1, NUP98/HOXD13 transgenic mice, and [p27(− /−) /SMAD3(−/+)] mice for evidence of Notch1 mutations. We also investigated the timing of Notch1 mutation in SCL/LMO1 transgenic mice.

We found that 13/19 cell lines and 29/49 primary thymic tumors had Notch1 mutations in either the HD or PEST domain, but not both. Of the thirteen cell lines with Notch1 mutations, twelve had mutations in the PEST domain. The cell lines with Notch1 mutations were sensitive to gamma-secretase inhibitor treatment, indicating that the mutations were important for the survival of these cells. Of twenty-nine tumors with Notch1 mutations, 23 were in the PEST domain and 6 in the HD. All HD mutations were single base substitutions, whereas all PEST domain mutations were insertion/deletion mutations. Intriguingly, half of the PEST domain mutations mapped to one of two mutational hot spots, suggesting that these regions may be prone to insertion/deletion mutations.

Thymocytes from clinically healthy SCL/LMO1 mice aged 5 weeks showed evidence of oligoclonal T-lymphocyte expansion, but did not have Notch1 mutations and did not form tumors when injected into nude mice (pre-malignant thymocytes), whereas thymocytes from SCL/LMO1 mice aged 8–12 weeks had Notch1 mutations and formed tumors upon transplantation into nude mice. Thus, Notch1 mutations are very frequent secondary events that can cooperate with a wide range of primary events as cells progress from a pre-malignant to a fully transformed state.

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