The phosphatidylinositol-3-kinase inhibitor NVP-BKM120 overcomes resistance signals derived from microenvironment by regulating the Akt/FoxO3a/Bim axis in chronic lymphocytic leukemia cells

Abstract

Phosphatidylinositol-3-kinase pathway is constitutively activated in chronic lymphocytic leukemia mainly due to microenvironment signals, including stromal cell interaction and CXCR4 and B-cell receptor activation. Because of the importance of phosphatidylinositol-3-kinase signaling in chronic lymphocytic leukemia, we investigated the activity of the NVP-BKM120, an orally available pan class I phosphatidylinositol-3-kinase inhibitor. Sensitivity to NVP-BKM120 was analyzed in chronic lymphocytic leukemia primary samples in the context of B-cell receptor and microenvironment stimulation. NVP-BKM120 promoted mitochondrial apoptosis in most primary cells independently of common prognostic markers. NVP-BKM120 activity induced the blockage of phosphatidylinositol-3-kinase signaling, decreased Akt and FoxO3a phosphorylation leading to concomitant Mcl-1 downregulation and Bim induction. Accordingly, selective knockdown of BIM rescued cells from NVP-BKM120-induced apoptosis, while the kinase inhibitor synergistically enhanced the apoptosis induced by the BH3-mimetic ABT-263. We also found NVP-BKM120 to inhibit B-cell receptor- and stroma-dependent Akt pathway activation, thus sensitizing chronic lymphocytic leukemia cells to bendamustine and fludarabine. Furthermore, NVP-BKM120 down-regulated secretion of chemokines after B-cell receptor stimulation and inhibited cell chemotaxis and actin polymerization upon CXCR4 triggering by CXCL12. Our findings establish that NVP-BKM120 effectively inhibits the phosphatidylinositol-3-kinase signaling pathway and disturbs the protective effect of the tumor microenvironment with the subsequent apoptosis induction through the Akt/FoxO3a/Bim axis. We provide here a strong rationale for undertaking clinical trials of NVP-BKM120 in chronic lymphocytic leukemia patients alone or in combination therapies.

Figure 1.

NVP-BKM120 cytotoxicity in primary CLL cells. (A) CLL cells (n=6) were treated with increasing doses of NVP-BKM120 (range 1–10 μM) for 48 h and cytotoxicity was measured by Annexin V. Mean ± SEM of all the samples analyzed. (B) Primary CLL cells (n=37) and PBMCs from healthy donors (n=4) were incubated with 2 3M NVP-BKM120 for 48 h before cytotoxicity was assessed by Annexin V labeling. **, P<0.01. (C) CLL cells were treated with NVP-BKM120 (1 and 2 μM) for 48 h and apoptosis hallmarks were determined by flow cytometry. A representative case was shown (CLL n. 5). Percentages inside each chart refer to the population in black.

Figure 2.

Modulation of PI3K/Akt/FoxO3a pathway and Bcl-2 anti-apoptotic family in CLL cells exposed to NVP-BKM120. (A) Primary CLL cells (n=3) were treated with 2 μM NVP-BKM120 for 30 min and PI3K activity was assessed. Data represent the mean ± SEM of the 3 cases analyzed. *, P<0.05. (B) CLL cells were incubated with NVP-BKM120 (1 and 2 μM) for 6 h before Western blot analysis was performed. Two representative cases out of 9 were showed.

Figure 3.

Role of Bim and Mcl-1 in NVP-BKM120-induced apoptosis in CLL cells. (A) Analysis of mRNA expression by quantitative RT-PCR in primary CLL cells (n=8) incubated with 2 μM NVP-BKM120 for 6 h. Mean ± SEM of the cases analyzed. **, P<0.01; ns=not significant. (B) Western blot analysis of several kinases implicated in the translational machinery in CLL primary cells exposed to NVP-BKM120 (1 and 2 μM) for 6 hours. A representative case was showed (CLL n. 32). (C) Primary CLL cells were transfected by electroporation with BIM siRNA and non-silencing siRNA in 3 independent experiments. Transfected cells were then incubated with 2 μM NVP-BKM120 for 24 h. Viability was assessed by flow cytometry labeling of AnnexinV and knockdown of Bim protein was quantified by RT-PCR. Mean ± SEM of the cases analyzed. *, P<0.05, **, P<0.01, ***, P<0.001; ns=not significant. (D) Primary CLL cells (n=5) were simultaneously exposed to NVP-BKM120 (1 μM) and ABT-263 (2.5 and 5 nM) for 48 h. Bars represent the mean ± SEM of cell viability referred to untreated control cells. ***, P<0.001.

Figure 4.

NVP-BKM120 abrogates BCR-derived signals. (A) Primary CLL cells (n=8) were incubated simultaneously with 2 μM NVP-BKM120 and anti-IgM and cell viability was assessed at 24 h by Annexin V/PI flow cytometry. Horizontal lines represent the mean. **, P<0.01, ***, P<0.001. (B) Western blot analysis after stimulation of CLL cells for 30 minutes with anti-IgM in the presence of 2 μM NVP-BKM120. A representative case was showed (CLL n. 4). (C) Primary CLL cells (n=6) were incubated with 2 μM NVP-BKM120 in presence or absence of anti-IgM for 6 h. Analysis of BIM was then determined by RT-PCR and Western blot analysis. Mean ± SEM of the cases analyzed. *, P<0.05. A representative case was shown (CLL n. 4). (D) Analysis of mRNA expression by RT-PCR of CCL3 and CCL4 chemokines in 4 CLL cases incubated simultaneously with NVP-BKM120 and anti-IgM for 6 h. Mean ± SEM of the cases analyzed. *, P<0.05, **, P<0.01.

Figure 5.

NVP-BKM120 induces cytotoxicity in the presence of microenvironment survival signals on CLL cells. (A) Primary CLL cells (n=8) were co-cultured with or without HS-5 and incubated with 2 μM NVP-BKM120. Cell viability was assessed at 24 h by Annexin V/PI flow cytometry. Horizontal lines represent the mean. **, P<0.01. (B) Western blot analysis after co-culture of CLL cells with HS-5 in the presence of 2 μM NVP-BKM120 for 6 h. A representative case was showed (CLL n. 4). (C) Primary CLL cells (n=8) co-cultured with or without HS-5 were incubated with 2 μM NVP-BKM120 for 6 h. Analysis of Bim was then determined by RT-PCR and Western blot analysis. A representative case was shown (CLL n.4). Mean ± SEM of the cases analyzed. **, P<0.01. (D) CLL cells (n=6) were simultaneously incubated with 1 μM NVP-BKM120 and flu-darabine (0.5 μg/mL) or bendamustine (10 μM) with or without stroma. Viability at 48 h was calculated relative to the respective untreated control, with or without stroma. Mean ± SEM of the cases analyzed. ***, P<0.001.

Figure 6.

NVP-BKM120 inhibits CXCL12-induced CLL migration and actin polymerization. (A) Primary CLL cells (n=6) were pre-incubated with 2 μM NVP-BKM120 for 1 h before CXCL12 addition. Cell viability was assessed by Annexin V/PI flow cytometry at 24 h. Horizontal lines represent the mean. ***, P<0.001. (B–D) Primary CLL cells derived from peripheral blood (PB, n=9), bone marrow (BM, n=5) and lymph node (LN, n=4) were assayed for migration in the presence of CXCL12 after NVP-BKM120 treatment as above. Total number of migrating cells is represented. Bars correspond to the mean ± SEM. *, P<0.05. (E) CLL cells (n=9) were exposed to 2 μM NVP-BKM120 for 1 h and F-actin content was determined at the indicated time points after CXCL12 addition. Results are displayed relative to samples before chemokine stimulation (100%). *, P<0.05, **, P<0.01.