Notch signaling sustains the expression of Mcl-1 and the activity of eIF4E to promote cell survival in CLL

Abstract

In chronic lymphocytic leukemia (CLL), Notch1 and Notch2 signaling is constitutively activated and contributes to apoptosis resistance. We show that genetic inhibition of either Notch1 or Notch2, through small-interfering RNA, increases apoptosis of CLL cells and is associated with decreased levels of the anti-apoptotic protein Mcl-1. Thus, Notch signaling promotes CLL cell survival at least in part by sustaining Mcl-1 expression. In CLL cells, an enhanced Notch activation also contributes to the increase in Mcl-1 expression and cell survival induced by IL-4.Mcl-1 downregulation by Notch targeting is not due to reduced transcription or degradation by caspases, but in part, to increased degradation by the proteasome. Mcl-1 downregulation by Notch targeting is also accompanied by reduced phosphorylation of eukaryotic translation initiation factor 4E (eIF4E), suggesting that this protein is another target of Notch signaling in CLL cells.Overall, we show that Notch signaling sustains CLL cell survival by promoting Mcl-1 expression and eIF4E activity, and given the oncogenic role of these factors, we underscore the therapeutic potential of Notch inhibition in CLL.

Keywords: Mcl-1; Notch; cell survival; chronic lymphocytic leukemia; eIF4E.

Figure 1. Notch1 and Notch2 silencing decreases cell viability and expression of Mcl-1 protein in CLL cells

CLL cells were transfected with control siRNA (siCtrl), Notch1 siRNA (siNotch1), Notch2 siRNA (siNotch2) or combined siNotch1 and siNotch2 (siNotch1/2) as described in “siRNA transfection” and then cultured in complete medium for 72 hours. A., C. Western blot analysis of Notch1, Notch2, Mcl-1 and Bcl-2 expression was performed on 15 μg whole-cell lysates separated on a 7.5% SDS-PAGE. The antibodies used for Notch1 and Notch2 recognized the 120-kDa transmembrane/cytoplasmic subunit (TM) and the 100-kDa active intracellular domain (IC). Protein loading was assessed by reprobing the blots with an anti-GAPDH antibody. Vertical line inserted in Notch2 blot of CLL7 indicates a repositioned gel lane. Data of CLL7 and 18 are representative of six samples. D. The blots of Mcl-1 were subjected to densitometric analysis and densitometry units (U) were calculated relative to GAPDH. Data are the mean ± SD of six samples. *P < 0.05, **P < 0.01 (each siNotch transfection condition versus siCtrl) according to Student t test. B. Cell viability was evaluated by flow cytometric analysis of Annexin V/PI (An V/PI) staining. Viability (An V⁻/PI⁻) of siCtrl cells was set to 100%. Data are the mean ± SD of six samples. **P < 0.01 (each siNotch transfection condition versus siCtrl) according to Student t test.

Figure 2. Combined Notch1/2 silencing prevents the increase in Mcl-1 levels and cell viability induced by IL-4 in CLL cells

CLL cells, transfected with control siRNA (siCtrl) or combined Notch1 and Notch2 siRNA (siNotch1/2) as described in “siRNA transfection”, were cultured for 72 hours in complete medium with or without 25 ng/ml IL-4 (n = 6). A. Expression of Notch1, Notch2 and Mcl-1 was analyzed as described in Figure 1A,C. Vertical line inserted in Notch1 blot of CLL18 indicates a repositioned gel lane. The blots of Mcl-1 B. and those of Notch1 and Notch2 C. were subjected to densitometric analysis, and densitometry units (U) were calculated relative to GAPDH. A. Data of CLL7 and 18 are representative of six samples. B., C. Data are the mean ± SD of six samples. *P <0.05, **P < 0.01 calculated by Student t test. D. Cell viability was evaluated by flow cytometric analysis of Annexin V/PI (An V/PI) staining. Viability (An V⁻/PI⁻) of IL-4-untreated siCtrl cells was set to 100%. Data are the mean ± SD of six samples. **P < 0.01 calculated by Student t test.

Figure 3. Mcl-1 downregulation by Notch silencing partially depends on proteasome degradation

A. Mcl-1 downregulation by Notch silencing is independent of reduced transcription. CLL cells were transfected with control siRNA (siCtrl), Notch1 siRNA (siNotch1) or Notch2 siRNA (siNotch2) as described in “siRNA transfection” and then cultured in complete medium for 72 hours (n = 6). Mcl-1 mRNA levels were evaluated by real-time PCR, normalized to GAPDH and represented as fold change with respect to siCtrl cells. Data are the mean ± SD of six samples. Differences between each siNotch transfection and siCtrl were not significant. B., C. Mcl-1 downregulation by Notch silencing is independent of degradation by caspases. siCtrl, siNotch1 or siNotch2 transfected cells were cultured for 72 hours in complete medium with 50 μM pan-caspase inhibitor z-VAD-fmk or 0.005% DMSO as control (n = 6). B. PARP cleavage, indicator of caspase activity, and Mcl-1 expression were analyzed by western blot on 15 μg whole-cell lysates. Protein loading was assessed by reprobing the blots with an anti-GAPDH antibody. Data of CLL18 are representative of six samples. C. The blots of Mcl-1 were subjected to densitometric analysis, and densitometry units (U) were calculated relative to GAPDH. Data are the mean ± SD of six samples. ^*P < 0.05; ns, not significant (z-VAD-fmk-treated cells versus DMSO-treated cells in each transfection condition) according to Student t test. D., E. Mcl-1 downregulation by Notch silencing partially depends on degradation by proteasome. siCtrl, siNotch1 or siNotch2 transfected cells were cultured for 72 hours in complete medium additioned, during the last 4 hours, with 2.5 μM proteasome inhibitor MG132 or 0.001% DMSO as control (n = 6). D. Accumulation of polyubiquitinated proteins, indicator of proteasome inhibition, and Mcl-1 expression were analyzed by western blot on 15 μg whole-cell lysates. Protein loading was assessed by reprobing the blots with an anti-GAPDH antibody. Data of CLL18 are representative of six samples. E. The blots of Mcl-1 were subjected to densitometric analysis, and densitometry units (U) were calculated relative to GAPDH. Data are the mean ± SD of six samples. *P < 0.05, **P < 0.01 (MG132-treated cells versus DMSO-treated cells in each transfection condition) according to Student t test.

Figure 4. Mcl-1 downregulation by Notch silencing is associated with reduced phosphorylation of eIF4E

CLL cells were transfected with control siRNA (siCtrl), Notch1 siRNA (siNotch1), Notch2 siRNA (siNotch2) or combined siNotch1 and siNotch2 (siNotch1/2), as described in “siRNA transfection” and then cultured in complete medium for 72 hours (n = 6). A. Expression and phosphorylation of eIF4E (Ser209), 4E-BP1 (Thr37/46 and Ser65) and MNK1 (Thr197/202) were analyzed by western blot on 15 μg whole-cell lysates, using antibodies able to detect total and phosphorylated forms. Protein loading was assessed by reprobing the blots with an anti-GAPDH antibody. Data of CLL7 and 18 are representative of six samples. B. The blots of phosphorylated eIF4E (Ser209) were subjected to densitometric analysis, and densitometry units (U) were calculated relative to total eIF4E. Data are the mean ± SD of six samples. *P < 0.05, **P < 0.01 (each siNotch transfection condition versus siCtrl) according to Student t test.