Abstract. γ-Secretase inhibitors (GSI) suppress the growth of acute T-lymphoblastic leukemia (T-ALL) cells with NOTCH1 mutations. Recently, clinical trials of GSI for refractory T-ALL have commenced. In the present study, we examined the effects of three types of GSI; GSI-I, GSI-IX, and GSI-XII on the growth of four B-cell malignant lymphoma (B-ML) and four acute myeloid leukemia (AML) cell lines as well as four T-ALL cell lines. We found that GSI also suppressed the in vitro growth of some B-ML and AML cell lines in a dosedependent manner. Growth suppression occurred through induction of apoptosis. Expression of the HES1 gene, one of the targets of Notch signaling, was high in T-ALL cells with NOTCH1 mutations, but was low in GSI-sensitive B-ML and AML cells. GSI treatment decreased HES1 mRNA expression in T-ALL cells, while GSI increased HES1 mRNA in two GSI-sensitive B-ML and AML cell lines. In immunoblot analysis, the band for the intracellular fragment of Notch1, an active form of Notch1, was dense in T-ALL cells but was faint in GSI-sensitive B-ML and AML cells; attenuation of the band by GSI was not evident. These findings suggest that GSI may act on Notch 2, 3 or 4 protein, or some pathways other than Notch signaling in GSI-sensitive B-ML and AML cells. Namely, growth suppression by GSI may involve cell growth-related proteins, which are γ-secretase substrates. Taken together, we have shown that GSI may be useful for the treatment of hematological malignancies other than T-ALL. The mechanism behind the effects remains to be clarified. Our investigations lead to a novel molecular target therapy for chemotherapy-resistant leukemia and lymphomas.
Mutations in the NOTCH1 gene were investigated in 12 primary acute myeloid leukemia (AML) cell samples and eight AML cell lines. Mutations in the genomic DNA were screened using a nested PCR-SSCP analysis and confirmed by direct sequencing. A missense mutation, Pro2439Leu (7316C/T), was found in the PEST domain in one primary AML case. This mutation was different from those previously reported for T-cell acute lymphoblastic leukemia, in which more than half the cases had the mutations. This mutation was not detected in his sample in complete remission, which indicated that the mutation was not a single nucleotide polymorphism. The sample with the mutation expressed the intracellular Notch1 fragment by immunoblotting and HES1 mRNA by reverse transcription-polymerase chain reaction. This is the first paper to present an AML case with NOTCH1 mutation. The precise role of the mutation is to be determined.
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