NOTCH1 mutations occur in approximately 10% of patients with chronic lymphocytic leukemia (CLL). However, the relationship between the genetic aberrations and tumor cell drug resistance or disease progression remains unclear. Frameshift deletions were detected by gene sequencing in the NOTCH1 PEST domain in three naive CLL patients. These mutations were associated with chromosomal abnormalities including trisomy 12 or 13q deletion. Of note, one of the patients developed Richter’s transformation during FCR treatment. Immunofluorescent and western blot analyses revealed a markedly higher intracellular domain of NOTCH (ICN) expression in the mutated cells compared with their unmutated counterparts and normal CD19+ B lymphocytes (P<0.01 and P<0.001, respectively). In addition, strong DNA-κB binding activities were observed in the mutant cells by gel shift assays. RT-PCR analysis revealed elevated RelA mRNA expression in the mutant cells, while RelB levels were variable. Reduced levels of RelA and RelB mRNA were observed in unmutated CLL and normal B cells. Compared to unmutated CLL and normal B cells, increased apoptosis occurred in the mutant cells in the presence of GSI (ICN inhibitor) and PDTC (NF-κB inhibitor), particularly under the synergistic effects of the two drugs (P=0.03). Moreover, IKKα and IKKβ, the active components in the NF-κB pathway, were markedly inhibited following prolonged treatment with GSI and PDTC. These results suggested that NOTCH1 mutations constitutively activate the NF-κB signaling pathway in CLL, which is likely related to ICN overexpression, indicating NOTCH1 and NF-κB as potential therapeutic targets in the treatment of CLL.

Abstract.The majority of patients with chronic lymphocytic leukemia (CLL) are not cured by traditional chemotherapy. One possible explanation for this is that the microenvironment protects CLL cells from both spontaneous-and cytotoxic-mediated apoptosis. The present study was designed to investigate the mechanisms accounting for these effects, since this information is crucial to understanding CLL physiopathology and identifying potential treatment targets. The CLL cell line L1210 and primary CLL cells were cultured under different conditions: With serum, cyclophosphamide (CTX), or with monolayers and conditioned medium (CM) from the stromal cell line HESS-5. Apoptosis, Hes family BHLH transcription factor 1 (HES-1) gene and protein expression, and histone H3K27me3 DNA demethylation were determined. Co-culture of L1210 cells with HESS-5 cells significantly inhibited serum deprivation-and CTX-induced apoptosis of leukemia cells, and resulted in a significant increase in short-term proliferation. Soluble factors in the CM from HESS-5 cells had a negligible effect. The HESS-5 cell-mediated inhibition of apoptosis of CLL cells was associated with increased HES-1 expression and hypomethylation of the H3K27me3 gene in the leukemia cells. These results indicate that stromal cells enhance the survival of CLL cells by regulating the HES-1 gene and protein expression, as well as H3K27me3 DNA demethylation, and suggest that specific interactions between stromal and leukemia cells may enhance the resistance of leukemia cells to chemotherapy.