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, 9 (12), 1160

IL-4-dependent Jagged1 Expression/Processing Is Associated With Survival of Chronic Lymphocytic Leukemia Cells but Not With Notch Activation

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IL-4-dependent Jagged1 Expression/Processing Is Associated With Survival of Chronic Lymphocytic Leukemia Cells but Not With Notch Activation

Filomena De Falco et al. Cell Death Dis.

Abstract

As previously reported, chronic lymphocytic leukemia (CLL) cells show constitutive Notch1/2 activation and express the Notchligand Jagged1. Despite increasing knowledge of the impact of Notch alterations on CLL biology and pathogenesis, the role of Jagged1 expressed in CLL cells remains undefined. In other cell types, it has been shown that after Notch engagement, Jagged1 not only activates Notch in signal-receiving cell, but also undergoes proteolytic activation in signal-sending cell, triggering a signaling with biological effects. We investigated whether Jagged1 expressed in CLL cells undergoes proteolytic processing and/or is able to induce Notch activation through autocrine/paracrine loops, focusing on the effect that CLL prosurvival factor IL-4 could exert on the Notch-Jagged1 system in these cells. We found that Jagged1 was constitutively processed in CLL cells and generated an intracellular fragment that translocated into the nucleus, and an extracellular fragment released into the culture supernatant. IL-4 enhanced expression of Jagged1 and its intracellular fragments, as well as Notch1/2 activation. The IL-4-induced increase in Notch1/2 activation was independent of the concomitant upregulated Jagged1 levels. Indeed, blocking Notch-Jagged1 interactions among CLL cells with Jagged1 neutralizing antibodies did not affect the expression of the Notch target Hes1. Notably, anti-Jagged1 antibodies partially prevented the IL-4-induced increase in Jagged1 processing and cell viability, suggesting that Jagged1 processing is one of the events contributing to IL-4-induced CLL cell survival. Consistent with this, Jagged1 silencing by small interfering RNA partially counteracted the capacity of IL-4 to promote CLL cell survival. Investigating the pathways whereby IL-4 promoted Notch1/2 activation in CLL cells independent of Jagged1, we found that PI3Kδ/AKT and PKCδ were involved in upregulating Notch1 and Notch2 proteins, respectively. Overall, this study provides new insights into the Notch-ligand system in CLL cells and suggests that targeting this system may be exploited as a novel/additional therapy approach for CLL.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Jag1 is constitutively processed in CLL cells.
ac Western blot analysis of Jag1 was performed in 25 µg whole-cell lysates (WCL) from primary CLL cells (n = 21), MEC1 cell line, and PBL from healthy donors (n = 3), using three different C-terminal antibodies (Abs), as indicated on top of the blots. Data in panel a are obtained using the Jag1 C-20 Ab. Data in panels b and c are from representative CLL samples where the analysis of Jag1 was repeated using the Jag1 TS1.15H (b) and Jag1 E-12 (c) Abs. WCL isolated from IM9 cell line was used as a positive control for Jag1 expression. Protein loading was assessed reprobing the blots with an anti-GAPDH antibody. In each blot, separated panels are shown because different X-ray film exposures were necessary to detect Jag1-FL and Jag1 fragments. Positions of the molecular weight markers (kDa) are indicated on the right of the blots. In the blots with PBL, an empty lane has been placed between PBL2 and CLL11 to avoid cross-contamination. Vertical line inserted in the middle blot of panel b indicates a repositioned gel lane. Full images of all blots are shown in Supplementary Figure S1
Fig. 2
Fig. 2. Jag1 processing generates an extracellular fragment released into CLL culture medium and an intracellular fragment that translocates into the nucleus.
a Western blot analysis of sJag1-EC was performed, using the N-terminal Jag1 AF1277 antibody, in soluble proteins (40 µg) of conditioned medium (CM) collected from CLL cells (n = 10; CLL4-13, selected to include patients with different clinical and biological characteristics), and from MEC1 cell line cultured for the indicated times in serum-free conditions. Serum-free medium (indicated as medium) incubated in the same conditions of CLL and MEC1 cultures, but without leukemic cells, was used as negative control. WCL from each respective CLL sample and from MEC1 cell line were used as a control for the molecular weight of Jag1-FL. Ponceau red staining is shown to assess protein loading. Representative CLL cases are shown. Full images of the blots are shown in Supplementary Figure S2A. b Western blot analysis of Jag1 was performed, using the C-terminal Jag1 C-20 antibody, in 25 μg nuclear (NE) and cytoplasmic (Cyt) extracts from CLL (n = 10; CLL4-13) and MEC1 cells. Adequate fractionation and protein loading were assessed using anti-lamin B1 and anti-β-tubulin antibodies. WCL from each respective CLL sample and from MEC1 cells were used as a control for the molecular weight of Jag1-TM and Jag1-IC. An empty lane was placed between WCL, NE and Cyt lanes to avoid potential cross-contamination. Representative CLL cases are shown. Full images of the blots are shown in Supplementary Figure S2B. c Confocal microscopy images of subcellular localization of Jag1-IC in a representative CLL sample and in MEC1 cells. CLL cells (n = 5; CLL7-11) and MEC1 cells were stained with the C-terminal Jag1 HPA021555 antibody (green) and with DAPI for nuclei (blue), as described in “Materials and methods”, and then analyzed by confocal microscopy. Upper images show a representative field, lower images show one single slice from the z-stacks of a representative CLL or MEC1 cell in the field. Scale bars are indicated in each image
Fig. 3
Fig. 3. Analysis of the correlation between Jag1 expression levels and clinical status of CLL patients.
ad Comparison of Jag1-FL expression levels in CLL subgroup patients according to prognostic factors. The expression levels of Jag1-FL, calculated as a Jag1-FL/GAPDH ratio and expressed as densitometric units (U), were evaluated in the context of IGVH mutational status (a), ZAP70 expression (b), CD38 expression (c), and cytogenetic subgroups (d). Statistical analysis was performed using the Mann–Whitney test; ns: not significant. e Kaplan–Meier curves for overall survival (OS) in CLL patients according to Jag1-FL expression levels. CLL patients were divided into two subgroups: one subgroup (n = 11) defined as Jag1hi with a Jag1-FL/GAPDH ratio ≥ 0.53; the other subgroup (n = 10) defined as Jag1low with a Jag1-FL/GAPDH ratio < 0.53. Survival curves were compared using log-rank test. Differences were not significant
Fig. 4
Fig. 4. IL-4 enhances Jag1 expression in CLL cells.
af CLL cells were cultured for 24 h with or without 25 ng/ml IL-4 (n = 6; CLL7-12, selected to include patients with different clinical and biological characteristics). a, b Western blot analysis of Jag1 was performed, using the C-terminal Jag1 C-20 antibody, in 25 µg whole-cell lysates, and protein loading was assessed reprobing the blots with an anti-GAPDH antibody. a In each blot, separated panels are shown because different X-ray film exposures were necessary to detect Jag1-FL and Jag1 fragments. Representative cases are shown. Full images of the blots are shown in Supplementary Figure S4A. b The density of the bands corresponding to Jag1-FL, Jag1-TM, and Jag1-IC was evaluated by densitometric analysis, and densitometry units (U) were calculated relative to GAPDH. Data are the mean ± SD of six patients. **P < 0.01, ***P < 0.001 according to Student’s t test. c, d Western blot of Jag1-IC was performed, using the C-terminal Jag1 C-20 antibody, in 25 µg nuclear extracts. Protein loading and adequate fractionation were assessed using anti-lamin B1 and anti-β-tubulin antibodies. c Representative cases are shown. Full images of the cropped blots are shown in Supplementary Figure S4B. d Blots of each sample were subjected to densitometric analysis, and densitometry units (U) were calculated relative to lamin B1. Data are the mean ± SD of six patients. ***P < 0.001 according to Student’s t test. e mRNA levels of Jag1 were evaluated by quantitative real-time PCR, normalized to GAPDH and represented as fold change with respect to untreated cells. Results are the mean ± SD of six patients. *P < 0.05 according to Student’s t test. f Cell viability and apoptosis were evaluated by flow cytometric analysis of Annexin V/PI (An V/PI) staining. Results are presented as percentage of viable (An V/PI) cells and are the mean ± SD of six patients. ***P < 0.001 according to Student’s t test
Fig. 5
Fig. 5. Effects of IL-4 on Jag1 expression in CLL cells are mediated by the PI3Kδ/AKT signaling.
a Western blot analysis of AKT Ser473 phosphorylation (phospho-AKT) was performed in 25 µg whole-cell lysates from CLL cells cultured with or without 25 ng/ml IL-4 for the indicated times. Protein loading was assessed using anti-total AKT and anti-GAPDH antibodies. One representative case is shown. Full image of the blot is shown in Supplementary Figure S5A. be CLL cells, pretreated for 2 h with the PI3Kδ inhibitor Idelalisib (5 µM) or 0.01% DMSO as control, were incubated for further 24 h with or without 25 ng/ml IL-4 (n = 6; CLL10 and CLL12-16, selected to include patients with different clinical and biological characteristics). b Western blot analysis of Jag1 was performed, using the C-terminal Jag1 C-20 antibody, in 25 µg whole-cell lysates, and protein loading was assessed reprobing the blots with an anti-GAPDH antibody. Representative cases are shown. Full images of the blots are shown in Supplementary Figure S5B. c Blots of each sample were subjected to densitometric analysis, and densitometry units (U) were calculated relative to GAPDH. Data are the mean ± SD of six patients. ns: the difference between the two groups was not significant; **P < 0.01 according to Student’s t test. d, e Cell viability and apoptosis were evaluated by flow cytometric analysis of Annexin V/PI (An V/PI) staining. d Results are presented as the percentage of viable (An V/PI), early apoptotic (An V+/PI), late apoptotic (An V+/PI+), and necrotic (An V/PI+) cells. Representative cases are shown. e Results are presented as percentage of viable (An V/PI) cells and are the mean ± SD of six patients. *P < 0.05, ***P < 0.001 according to Student’s t test
Fig. 6
Fig. 6. IL-4 enhances Notch1 and Notch2 expression and activation in CLL cells.
ac CLL cells were cultured for 24 h with or without 25 ng/ml IL-4 (n = 6; CLL7-12, selected to include patients with different clinical and biological characteristics). a, b Western blot analysis of Notch1, Notch2, and Hes1 was performed in 25 µg whole-cell lysates, and protein loading was assessed using an anti-GAPDH antibody. a Representative cases are shown. Full images of the blots are shown in Supplementary Figure S6A–C. b The density of the bands corresponding to Notch1-TM, Notch1-IC, Notch2-TM, Notch2-IC, and Hes1 was evaluated by densitometric analysis, and densitometry units (U) were calculated relative to GAPDH. Data are the mean ± SD of six patients. ns: the difference between the two groups was not significant; *P < 0.05, **P < 0.01 according to Student’s t test. c Notch1 and Notch2 mRNA levels were evaluated by real-time PCR, normalized to GAPDH and represented as fold change with respect to untreated cells. Results are the mean ± SD of six patients. The effects of IL-4 are not significant
Fig. 7
Fig. 7. Blocking Notch–Jag1 interactions among CLL cells does not affect IL-4-induced Notch activation, but reduces IL-4-induced Jag1 processing and cell viability.
af CLL cells were cultured for 48 h with or without 25 ng/ml IL-4 in the presence of 60 µg/ml Jag1 neutralizing or goat IgG antibodies as isotype control (ctrl Ab) (n = 5; CLL7-8 and CLL10-12, selected to include patients with different clinical and biological characteristics). ad Western blot analysis of Hes1 and Jag1 was performed in 25 µg whole-cell lysates, and protein loading was assessed reprobing the blots with an anti-GAPDH antibody. a, c Representative cases are shown. a Full images of Hes1 blots are shown in Supplementary Figure S7A. c For Jag1 analysis, the C-20 antibody was used. In each blot, separated panels are shown because different X-ray film exposures were necessary to detect Jag1-FL and Jag1 fragments. Full images of Jag1 blots are shown in Supplementary Figure S7B. b, d The density of the bands corresponding to Hes1, Jag1-FL, Jag1-TM, and Jag1-IC was evaluated by densitometric analysis, and densitometry units (U) were calculated relative to GAPDH. Data are the mean ± SD of five patients. ns: the difference between the two groups was not significant; *P < 0.05, **P < 0.01 according to Student’s t test. e, f Cell viability and apoptosis were evaluated by flow cytometric analysis of Annexin V/PI (An V/PI) staining. e Results are presented as the percentage of viable (An V/PI), early apoptotic (An V+/PI), late apoptotic (An V+/PI+), and necrotic (An V/PI+) cells. Representative cases are shown. f Results are presented as percentage of viable (An V/PI) cells and are the mean ± SD of five patients. **P < 0.01, ***P < 0.001 according to Student’s t test
Fig. 8
Fig. 8. Jag1 silencing counteracts the IL4-dependent increase of CLL cell viability.
ad CLL cells were transfected with control siRNA (siCtrl) and Jag1 siRNA (siJag1), as described in “Materials and methods”, and then were cultured for 72 h in complete medium containing 25 ng/ml IL-4 (n = 5; CLL7-8 and CLL10-12). a Western blot analysis of Jag1 was performed, using the Jag1 C-20 antibody, in 25 µg whole-cell lysates, and protein loading was assessed reprobing the blots with an anti-GAPDH antibody. Representative cases are shown. In each blot, separated panels are shown because different X-ray film exposures were necessary to detect Jag1-FL and Jag1 fragments. Full images of the blots are shown in Supplementary Figure S8. b The density of the bands corresponding to Jag1-FL, Jag1-TM, and Jag1-IC was evaluated by densitometric analysis, and densitometry units (U) were calculated relative to GAPDH. Data are the mean ± SD of five patients. *P < 0.05 according to Student’s t test. c, d Cell viability and apoptosis were evaluated by flow cytometric analysis of Annexin V/PI (An V/PI) staining. c Results are presented as the percentage of viable (An V/PI), early apoptotic (An V+/PI), late apoptotic (An V+/PI+), and necrotic (An V/PI+) cells. Representative cases are shown. d Results are presented as percentage of viable (An V/PI) cells and are the mean ± SD of five patients. **P < 0.01, ***P < 0.001 according to Student’s t test
Fig. 9
Fig. 9. Signaling pathways involved in Notch1 and Notch2 upregulation induced by IL-4 in CLL cells.
CLL cells (n = 5; CLL7-8 and CLL10-12, selected to include patients with different clinical and biological characteristics) were preincubated for 2 h with 5 µM Idelalisib or 0.01% DMSO (a, b), or with 10 µM Rottlerin or 0.05% DMSO (cf), and then were cultured with or without 25 ng/ml IL-4 for further 24 h. a–d Western blot analysis of Notch1 and Notch2 was performed in 25 µg whole-cell lysates, and protein loading was assessed using an anti-GAPDH antibody. a, c Representative cases are shown. Full images of the blots are shown in Supplementary Figure S9A–C. b, d The density of the bands corresponding to Notch1-TM, Notch1-IC, Notch2-TM, and Notch2-IC was evaluated by densitometric analysis, and densitometry units (U) were calculated relative to GAPDH. Data are the mean ± SD of five patients. ns: the difference between the two groups was not significant; *P < 0.05, **P < 0.01, ***P < 0.001 according to Student’s t test. e, f Cell viability and apoptosis were evaluated by flow cytometric analysis of Annexin V/PI (An V/PI) staining. e Results are presented as the percentage of viable (An V/PI), early apoptotic (An V+/PI), late apoptotic (An V+/PI+), and necrotic (An V/PI+) cells. Representative cases are shown. f Results are presented as percentage of viable (An V/PI) cells and are the mean ± SD of five patients. *P < 0.05, **P < 0.01, ***P < 0.001 according to Student’s t test
Fig. 10
Fig. 10. Schematic representation of the signaling network of Jag1 and Notch1/Notch2 in CLL cells in the presence of the prosurvival factor IL-4.
CLL cells constitutively express both Notch1/Notch2 receptors and Jag1 ligand. IL-4 activates the PI3Kδ/AKT signaling to increase Jag1 expression and Notch1 activation, and the PKCδ signaling to increase Notch2 activation. Interactions between Jag1 and Notch1/Notch2 do not activate Notch signaling but induce Jag1 processing, generating an intracellular fragment (Jag1-IC) that migrates into the nucleus and contributes to CLL cell survival through mechanisms which remain to be defined (“?”). Even the mechanisms by which PI3Kδ/AKT and PKCδ activate Notch signaling in a ligand-independent manner are still not elucidated (“?”). The star symbols indicate potential druggable molecular events for targeting the Notch-ligand system in CLL

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