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PI3K pan-inhibition impairs more efficiently proliferation and survival of T-cell acute lymphoblastic leukemia cell lines when compared to isoform-selective PI3K inhibitors.

Lonetti A, Cappellini A, Spartà AM, Chiarini F, Buontempo F, Evangelisti C, Evangelisti C, Orsini E, McCubrey JA, Martelli AM - Oncotarget (2015)

Bottom Line: Intriguingly, the dual p110γ/δ inhibitor IPI-145 was effective in Loucy cells, which are representative of early T-precursor (ETP)-ALL, a T-ALL subtype associated with a poor outcome.PTEN gene deletion did not confer a peculiar reliance of T-ALL cells on PI3K activity for their proliferation/survival, as PTEN was inactivated in PTEN non deleted cells, due to posttranslational mechanisms.PI3K pan-inhibition suppressed Akt activation and induced caspase-independent apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy.

ABSTRACT
Class I phosphatidylinositol 3-kinases (PI3Ks) are frequently activated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to the loss of PTEN function. Therefore, targeting PI3Ks is a promising innovative approach for T-ALL treatment, however at present no definitive evidence indicated which is the better therapeutic strategy between pan or selective isoform inhibition, as all the four catalytic subunits might participate in leukemogenesis. Here, we demonstrated that in both PTEN deleted and PTEN non deleted T-ALL cell lines, PI3K pan-inhibition exerted the highest cytotoxic effects when compared to both selective isoform inhibition or dual p110γ/δ inhibition. Intriguingly, the dual p110γ/δ inhibitor IPI-145 was effective in Loucy cells, which are representative of early T-precursor (ETP)-ALL, a T-ALL subtype associated with a poor outcome. PTEN gene deletion did not confer a peculiar reliance of T-ALL cells on PI3K activity for their proliferation/survival, as PTEN was inactivated in PTEN non deleted cells, due to posttranslational mechanisms. PI3K pan-inhibition suppressed Akt activation and induced caspase-independent apoptosis. We further demonstrated that in some T-ALL cell lines, autophagy could exert a protective role against PI3K inhibition. Our findings strongly support clinical application of class I PI3K pan-inhibitors in T-ALL treatment, with the possible exception of ETP-ALL cases.

No MeSH data available.


Related in: MedlinePlus

PI3K pan-inhibition induces autophagy which plays a protective role(A) Western blotting demonstrated autophagy activation in Loucy, ALL-SIL, and DND-41 cell lines in response to PI3K inhibition. Thirty μg of protein was blotted to each lane. Antibody to β-Actin served as a loading control. Molecular weights are indicated at right. (B) T-ALL cells were treated with 5 μM ZSTK-474 for 48 h with or without the autophagy inhibitor 3-MA (200 μM) and cell death fraction was assessed by Annexin V-FITC/PI staining. Autophagy inhibition significantly increased cell death in Loucy, ALL-SIL, and DND-41 cell lines, whereas it did not affect Jurkat cells. Results are the mean of three different experiments ± SD. Asterisks indicate statistically significant differences with respect to untreated cells (*p < 0.05; **p < 0.01; ***p < 0.001). (C) Real-time PCR expression profiling of 82 autophagy-related genes in T-ALL cell lines untreated (Ctr) or treated for 24 h with 5 μM ZSTK-474 were visualized using an unsupervised heat map. Data are presented as 2−ΔCt (ΔCt = Ct target gene – Ct RLP0). (D) Histograms represent the relative gene expression of several autophagy-related genes in T-ALL cells treated with ZSTK-474 and compared to untreated paired sample. Data are presented as 2−ΔΔCt (ΔΔCt = ΔCt treated sample – ΔCt Ctr sample). When fold change values are = 1, the regulation in treated samples is equal to the paired control sample. When fold change values are > 1 or < 1, the autophagy-related genes are up- or down-regulated, respectively, compared to untreated samples.
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Figure 6: PI3K pan-inhibition induces autophagy which plays a protective role(A) Western blotting demonstrated autophagy activation in Loucy, ALL-SIL, and DND-41 cell lines in response to PI3K inhibition. Thirty μg of protein was blotted to each lane. Antibody to β-Actin served as a loading control. Molecular weights are indicated at right. (B) T-ALL cells were treated with 5 μM ZSTK-474 for 48 h with or without the autophagy inhibitor 3-MA (200 μM) and cell death fraction was assessed by Annexin V-FITC/PI staining. Autophagy inhibition significantly increased cell death in Loucy, ALL-SIL, and DND-41 cell lines, whereas it did not affect Jurkat cells. Results are the mean of three different experiments ± SD. Asterisks indicate statistically significant differences with respect to untreated cells (*p < 0.05; **p < 0.01; ***p < 0.001). (C) Real-time PCR expression profiling of 82 autophagy-related genes in T-ALL cell lines untreated (Ctr) or treated for 24 h with 5 μM ZSTK-474 were visualized using an unsupervised heat map. Data are presented as 2−ΔCt (ΔCt = Ct target gene – Ct RLP0). (D) Histograms represent the relative gene expression of several autophagy-related genes in T-ALL cells treated with ZSTK-474 and compared to untreated paired sample. Data are presented as 2−ΔΔCt (ΔΔCt = ΔCt treated sample – ΔCt Ctr sample). When fold change values are = 1, the regulation in treated samples is equal to the paired control sample. When fold change values are > 1 or < 1, the autophagy-related genes are up- or down-regulated, respectively, compared to untreated samples.

Mentions: Autophagy is a homeostatic cellular process which regulates protein and organelle turnover through their lysosomal destruction [35]. However, autophagy also executes cell death, and autophagic cell death is one of the better recognized caspase-independent programmed cell death mechanisms [36]. To analyze possible autophagy induction, we investigated the expression of LC3B I/II, a recognized autophagy marker [37]. Western blot analysis demonstrated a marked increase in LC3B II, the lipidated form of the protein which is bound to the autophagosome membranes, in Loucy, ALL-SIL and DND-41 cells, especially following 24 h ZSTK-474 treatment, whereas no changes were observed in Jurkat cells (Fig. 6A). Because PI3K pan-inhibition with ZTK-474 treatment induced a considerable percentage of cell death in Jurkat cells compared to the other cell lines, we supposed a protective role of autophagy in this context. To test our hypothesis, we inhibited autophagy with the early-stage autophagy inhibitor 3-methyladenine (3-MA) and subsequently evaluated cell death induced by treatment with ZSTK-474. The results demonstrated that 3-MA increased the cytotoxic effect of pan PI3K inhibition, as the percentage of Annexin V/PI positive cells was significantly higher in Loucy, ALL-SIL, and DND-41 cells compared to that of samples treated with ZSTK-474 alone (Fig. 6B). On the contrary, in Jurkat cells, where pan-inhibition did not induce LC3B lipidation, inhibition of autophagy did not increase cytotoxicity (Fig. 6B). To ascertain whether the different behavior between Jurkat cells and the other cell lines was related to a different modulation of autophagy-related genes, a screening for gene expression was performed using a quantitative real-time PCR assay which interrogates 82 genes related to the autophagic pathway (Tab. 1 and Fig. 6C). Unsupervised hierarchical clustering showed similarities in autophagy gene expression in each paired cell line (untreated and treated samples) (Fig. 6C), although untreated Loucy cells showed a basal higher expression of these genes compared to the other cell lines. Moreover, no differentially clustered transcripts were observed in Jurkat cells, despite the fact that this cell line did not activate the autophagy process following PI3K pan-inhibition. Nevertheless, we further investigated the modulation of autophagy in more detail, by comparing for each cell line untreated and treated samples and assessing for each gene the fold change, expressed as 2−ΔΔCt. A 24 h treatment with ZSTK-474 had limited effects on autophagy at a transcriptional level, as the majority of genes resulted expressed equally to the control (2−ΔΔCt = 1) or slightly reduced (2−ΔΔCt < 1), especially in Loucy cells (Fig. 6C). Nevertheless, in some instances we observed a > 2 fold increase both in components of the autophagic machinery and in genes involved in autophagy regulation (Tab. 2 and Fig. 6D). In particular, in Loucy, ALL-SIL, and DND-41 cells, PI3K pan-inhibition increased the expression of DRAM1, GABARAPL1, GABARAPL2, WIPI1, MAP1LC3B, and ATG16L2, all involved in autophagic vesicle nucleation and expansion, as well as increased expression of genes involved in autophagy induction and regulation (INS, PIK3C) or prosurvival genes (BCL2, EIF2AK3). In contrast, in Jurkat cells, pan-inhibition had limited effects on autophagy-related gene induction, as the only upregulated gene was ULK1. However, we observed a > 2 fold change in expression of the tumor suppressor gene CDKN1B, as well as in CTSS (cathepsin S) gene. Interestingly, low levels of cathepsin S or its pharmacological inhibition have been related to the induction of autophagy in cancer cells [38, 39].


PI3K pan-inhibition impairs more efficiently proliferation and survival of T-cell acute lymphoblastic leukemia cell lines when compared to isoform-selective PI3K inhibitors.

Lonetti A, Cappellini A, Spartà AM, Chiarini F, Buontempo F, Evangelisti C, Evangelisti C, Orsini E, McCubrey JA, Martelli AM - Oncotarget (2015)

PI3K pan-inhibition induces autophagy which plays a protective role(A) Western blotting demonstrated autophagy activation in Loucy, ALL-SIL, and DND-41 cell lines in response to PI3K inhibition. Thirty μg of protein was blotted to each lane. Antibody to β-Actin served as a loading control. Molecular weights are indicated at right. (B) T-ALL cells were treated with 5 μM ZSTK-474 for 48 h with or without the autophagy inhibitor 3-MA (200 μM) and cell death fraction was assessed by Annexin V-FITC/PI staining. Autophagy inhibition significantly increased cell death in Loucy, ALL-SIL, and DND-41 cell lines, whereas it did not affect Jurkat cells. Results are the mean of three different experiments ± SD. Asterisks indicate statistically significant differences with respect to untreated cells (*p < 0.05; **p < 0.01; ***p < 0.001). (C) Real-time PCR expression profiling of 82 autophagy-related genes in T-ALL cell lines untreated (Ctr) or treated for 24 h with 5 μM ZSTK-474 were visualized using an unsupervised heat map. Data are presented as 2−ΔCt (ΔCt = Ct target gene – Ct RLP0). (D) Histograms represent the relative gene expression of several autophagy-related genes in T-ALL cells treated with ZSTK-474 and compared to untreated paired sample. Data are presented as 2−ΔΔCt (ΔΔCt = ΔCt treated sample – ΔCt Ctr sample). When fold change values are = 1, the regulation in treated samples is equal to the paired control sample. When fold change values are > 1 or < 1, the autophagy-related genes are up- or down-regulated, respectively, compared to untreated samples.
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Figure 6: PI3K pan-inhibition induces autophagy which plays a protective role(A) Western blotting demonstrated autophagy activation in Loucy, ALL-SIL, and DND-41 cell lines in response to PI3K inhibition. Thirty μg of protein was blotted to each lane. Antibody to β-Actin served as a loading control. Molecular weights are indicated at right. (B) T-ALL cells were treated with 5 μM ZSTK-474 for 48 h with or without the autophagy inhibitor 3-MA (200 μM) and cell death fraction was assessed by Annexin V-FITC/PI staining. Autophagy inhibition significantly increased cell death in Loucy, ALL-SIL, and DND-41 cell lines, whereas it did not affect Jurkat cells. Results are the mean of three different experiments ± SD. Asterisks indicate statistically significant differences with respect to untreated cells (*p < 0.05; **p < 0.01; ***p < 0.001). (C) Real-time PCR expression profiling of 82 autophagy-related genes in T-ALL cell lines untreated (Ctr) or treated for 24 h with 5 μM ZSTK-474 were visualized using an unsupervised heat map. Data are presented as 2−ΔCt (ΔCt = Ct target gene – Ct RLP0). (D) Histograms represent the relative gene expression of several autophagy-related genes in T-ALL cells treated with ZSTK-474 and compared to untreated paired sample. Data are presented as 2−ΔΔCt (ΔΔCt = ΔCt treated sample – ΔCt Ctr sample). When fold change values are = 1, the regulation in treated samples is equal to the paired control sample. When fold change values are > 1 or < 1, the autophagy-related genes are up- or down-regulated, respectively, compared to untreated samples.
Mentions: Autophagy is a homeostatic cellular process which regulates protein and organelle turnover through their lysosomal destruction [35]. However, autophagy also executes cell death, and autophagic cell death is one of the better recognized caspase-independent programmed cell death mechanisms [36]. To analyze possible autophagy induction, we investigated the expression of LC3B I/II, a recognized autophagy marker [37]. Western blot analysis demonstrated a marked increase in LC3B II, the lipidated form of the protein which is bound to the autophagosome membranes, in Loucy, ALL-SIL and DND-41 cells, especially following 24 h ZSTK-474 treatment, whereas no changes were observed in Jurkat cells (Fig. 6A). Because PI3K pan-inhibition with ZTK-474 treatment induced a considerable percentage of cell death in Jurkat cells compared to the other cell lines, we supposed a protective role of autophagy in this context. To test our hypothesis, we inhibited autophagy with the early-stage autophagy inhibitor 3-methyladenine (3-MA) and subsequently evaluated cell death induced by treatment with ZSTK-474. The results demonstrated that 3-MA increased the cytotoxic effect of pan PI3K inhibition, as the percentage of Annexin V/PI positive cells was significantly higher in Loucy, ALL-SIL, and DND-41 cells compared to that of samples treated with ZSTK-474 alone (Fig. 6B). On the contrary, in Jurkat cells, where pan-inhibition did not induce LC3B lipidation, inhibition of autophagy did not increase cytotoxicity (Fig. 6B). To ascertain whether the different behavior between Jurkat cells and the other cell lines was related to a different modulation of autophagy-related genes, a screening for gene expression was performed using a quantitative real-time PCR assay which interrogates 82 genes related to the autophagic pathway (Tab. 1 and Fig. 6C). Unsupervised hierarchical clustering showed similarities in autophagy gene expression in each paired cell line (untreated and treated samples) (Fig. 6C), although untreated Loucy cells showed a basal higher expression of these genes compared to the other cell lines. Moreover, no differentially clustered transcripts were observed in Jurkat cells, despite the fact that this cell line did not activate the autophagy process following PI3K pan-inhibition. Nevertheless, we further investigated the modulation of autophagy in more detail, by comparing for each cell line untreated and treated samples and assessing for each gene the fold change, expressed as 2−ΔΔCt. A 24 h treatment with ZSTK-474 had limited effects on autophagy at a transcriptional level, as the majority of genes resulted expressed equally to the control (2−ΔΔCt = 1) or slightly reduced (2−ΔΔCt < 1), especially in Loucy cells (Fig. 6C). Nevertheless, in some instances we observed a > 2 fold increase both in components of the autophagic machinery and in genes involved in autophagy regulation (Tab. 2 and Fig. 6D). In particular, in Loucy, ALL-SIL, and DND-41 cells, PI3K pan-inhibition increased the expression of DRAM1, GABARAPL1, GABARAPL2, WIPI1, MAP1LC3B, and ATG16L2, all involved in autophagic vesicle nucleation and expansion, as well as increased expression of genes involved in autophagy induction and regulation (INS, PIK3C) or prosurvival genes (BCL2, EIF2AK3). In contrast, in Jurkat cells, pan-inhibition had limited effects on autophagy-related gene induction, as the only upregulated gene was ULK1. However, we observed a > 2 fold change in expression of the tumor suppressor gene CDKN1B, as well as in CTSS (cathepsin S) gene. Interestingly, low levels of cathepsin S or its pharmacological inhibition have been related to the induction of autophagy in cancer cells [38, 39].

Bottom Line: Intriguingly, the dual p110γ/δ inhibitor IPI-145 was effective in Loucy cells, which are representative of early T-precursor (ETP)-ALL, a T-ALL subtype associated with a poor outcome.PTEN gene deletion did not confer a peculiar reliance of T-ALL cells on PI3K activity for their proliferation/survival, as PTEN was inactivated in PTEN non deleted cells, due to posttranslational mechanisms.PI3K pan-inhibition suppressed Akt activation and induced caspase-independent apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy.

ABSTRACT
Class I phosphatidylinositol 3-kinases (PI3Ks) are frequently activated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to the loss of PTEN function. Therefore, targeting PI3Ks is a promising innovative approach for T-ALL treatment, however at present no definitive evidence indicated which is the better therapeutic strategy between pan or selective isoform inhibition, as all the four catalytic subunits might participate in leukemogenesis. Here, we demonstrated that in both PTEN deleted and PTEN non deleted T-ALL cell lines, PI3K pan-inhibition exerted the highest cytotoxic effects when compared to both selective isoform inhibition or dual p110γ/δ inhibition. Intriguingly, the dual p110γ/δ inhibitor IPI-145 was effective in Loucy cells, which are representative of early T-precursor (ETP)-ALL, a T-ALL subtype associated with a poor outcome. PTEN gene deletion did not confer a peculiar reliance of T-ALL cells on PI3K activity for their proliferation/survival, as PTEN was inactivated in PTEN non deleted cells, due to posttranslational mechanisms. PI3K pan-inhibition suppressed Akt activation and induced caspase-independent apoptosis. We further demonstrated that in some T-ALL cell lines, autophagy could exert a protective role against PI3K inhibition. Our findings strongly support clinical application of class I PI3K pan-inhibitors in T-ALL treatment, with the possible exception of ETP-ALL cases.

No MeSH data available.


Related in: MedlinePlus