A critical evaluation of PI3K inhibition in Glioblastoma and Neuroblastoma therapy

Members of the PI3K/Akt/mTor signaling cascade are among the most frequently altered proteins in cancer, yet the therapeutic application of pharmacological inhibitors of this signaling network, either as monotherapy or in combination therapy (CT) has so far not been particularly successful. In this review we will focus on the role of PI3K/Akt/mTOR in two distinct tumors, Glioblastoma multiforme (GBM), an adult brain tumor which frequently exhibits PTEN inactivation, and Neuroblastoma (NB), a childhood malignancy that affects the central nervous system and does not harbor any classic alterations in PI3K/Akt signaling. We will argue that inhibitors of PI3K/Akt signaling can be components for potentially promising new CTs in both tumor entities, but further understanding of the signal cascade’s complexity is essential for successful implementation of these CTs. Importantly, failure to do this might lead to severe adverse effects, such as treatment failure and enhanced therapy resistance

Sequential dosing in chemosensitization : targeting the PI3K/Akt/mTOR pathway in neuroblastoma

Breaking resistance to chemotherapy is a major goal of combination therapy in many tumors, including advanced neuroblastoma. We recently demonstrated that increased activity of the PI3K/Akt network is associated with poor prognosis, thus providing an ideal target for chemosensitization. Here we show that targeted therapy using the PI3K/mTOR inhibitor NVP-BEZ235 significantly enhances doxorubicin-induced apoptosis in neuroblastoma cells. Importantly, this increase in apoptosis was dependent on scheduling: while pretreatment with the inhibitor reduced doxorubicin-induced apoptosis, the sensitizing effect in co-treatment could further be increased by delayed addition of the inhibitor post chemotherapy. Desensitization for doxorubicin-induced apoptosis seemed to be mediated by a combination of cell cycle-arrest and autophagy induction, whereas sensitization was found to occur at the level of mitochondria within one hour of NVP-BEZ235 posttreatment, leading to a rapid loss of mitochondrial membrane potential with subsequent cytochrome c release and caspase-3 activation. Within the relevant time span we observed marked alterations in a ~30 kDa protein associated with mitochondrial proteins and identified it as VDAC1/Porin protein, an integral part of the mitochondrial permeability transition pore complex. VDAC1 is negatively regulated by the PI3K/Akt pathway via GSK3β and inhibition of GSK3β, which is activated when Akt is blocked, ablated the sensitizing effect of NVP-BEZ235 posttreatment. Our findings show that cancer cells can be sensitized for chemotherapy induced cell death – at least in part – by NVP-BEZ235-mediated modulation of VDAC1. More generally, we show data that suggest that sequential dosing, in particular when multiple inhibitors of a single pathway are used in the optimal sequence, has important implications for the general design of combination therapies involving molecular targeted approaches towards the PI3K/Akt/mTOR signaling network

The effect of several inhibitors of PI3K/mTOR signaling on SHEP NB cell survival.

<p><b>A</b> SHEP NB cells were treated with doxorubicin for 24(control), or in the presence of the indicated pharmacological inhibitor, which was given 12 hrs prior to doxorubicin (Pre), or simultaneously with doxorubicin (Co), or 12 hrs after doxorubicin (Post). Apoptosis was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. <b>B</b> Comparison of different treatment strategies, either using the pharmacolgical inhibitors as single agents or in combination. The sensitization effect is depicted as X-fold increase in cell death (as determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei) over treatment with doxorubicin alone. <b>C</b> Cells were either left untreated or treated with either NVP-BEZ235 posttreatment, or the complex combination therapy shown to work best in B, in the presence or absence of doxorubicin. Cells were treated with Nu7026 for 24.5 hrs, with doxorubicin and/or rapamycin for 12.5 hrs, 0.5 hrs with NVP-BEZ235 and allowed to grow 10 days. In A and B mean+s.e.m. values of three independent experiments carried out in triplicate are shown, in C a representative result of two independent experiments is depicted. Statistical analysis was carried out by two-sided Student's <i>t</i>-test; * P-value <0.01; ** P-value <0.001; # P-value <0.0001.</p

Apoptosis sensitization occurs at the mitochondrial level.

<p><b>A</b> SHEP NB cells were either left untreated (control) or treated as indicated, followed by a Western blot analysis of the caspase-3 processing kinetic, with β-actin as loading control. <b>B</b> The loss of mitochondrial membrane potential (MMP) was analyzed after indicated treatment. Cells were incubated with TMRM dye prior to FACS analysis. <b>C</b> Cells were either left untreated or treated as indicated. The DNA damage was assayed by single cell gel electrophoresis (Comet) assay and expressed as Mean Olive Tail Moment. <b>D</b> Cells were treated for the indicated length of time with 0.6 µM NVP-BEZ235, 0.2 µg/ml doxorubicin, 20 nM Bafilomycin A1 (a inhibitor of the late stages of autophagy that blocks fusion between autophagosomes and lysosomes), or combinations thereof. The percentage of autophagic cells was then determined by counting cells with LC3 foci. In A representative results of two independent experiments are shown, in B mean+s.e.m. of three independent experiments carried out in triplicate are shown, while in C mean+s.d. of two independent experiments are depicted. In D mean+s.d. Of three independent experiments are depicted. Statistical analysis was carried out by two-sided Student's <i>t</i>-test; * P-value <0.01; ** P-value <0.001; # P-value <0.0001.</p

Sensitization for doxorubicin-induced apoptosis via posttreatment with NVP-BEZ235 is mediated via VDAC1.

<p><b>A</b> SHEP NB cells were treated for 12.5-BEZ235 for the last 0.5 hr. Either Bim, Bax or Bad was then immunoprecipitated and interaction partners that are phosphorylated on Serine or Threonine were visualized by Western blot analysis. A ∼30 kD protein, the presence of which appears to depend on NVP-BEZ235 addition, was identified as VDAC by VDAC1/Porin-specific antibody. IgG_H – heavy chain. <b>B</b> Cells were left untreated, treated for 12.5 hrs with Doxorubicin, or after 12 hrs for 0.5 hr with NVP-BEZ235, or a combination of both (first 12 hrs with Doxorubicin alone, followed by the addition of NVP-BEZ235 for 0.5 hr). VDAC was immunoprecipitated and its phosphorylation status was probed. IgG_L – light chain. <b>C</b> Cells were left untreated, treated for 12.5 hrs with doxorubicin, or after 12 hrs for 0.5 hr with NVP-BEZ235, or a combination of both (first 12 hrs with doxorubicin alone, followed by the addition of NVP-BEZ235 for 0.5 hr). Protein expression levels and phosphorylation status of GSK3β were analyzed by Western blotting, GAPDH served as loading control. <b>D</b> Cells were treated either for 12.5 hrs with doxorubicin, or a combination of doxorubicin and NVP-BEZ235, (first 12 hrs with doxorubicin alone, followed by the addition of NVP-BEZ235 for 0.5 hr). This was followed by immunoprecipitation of GSK3β and analysis of this protein's interaction with VDAC via immunoblotting. <b>E</b> Cells were again treated with a combination of doxorubicin and NVP-BEZ235, (first 12 hrs with doxorubicin alone, followed by the addition of NVP-BEZ235 for 0.5 hr), during the last hour in the absence or presence of the GSK3β-specific inhibitor SB415286. This was followed by immunoprecipitation of GSK3β and analysis of this protein's interaction with VDAC. <b>F</b> Apoptosis in cells treated for 24 hrs with doxorubicin, for 12.5 hrs with SB415286, for 12 hrs with NVP-BEZ235, or a combination of those substances was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei, and percentage of specific DNA fragmentation is shown. Shown in A to E are representative blots of at least two independent experiments, in F the mean+s.e.m. of three independent experiments performed in triplicate is depicted. Statistical analysis was carried out by two-sided Student's <i>t</i>-test; * P-value <0.01; ** P-value <0.001; # P-value <0.0001.</p

The superiority of posttreatment with NVP-BEZ235 is not restricted to one NB cell line and doxorubicin.

<p><b>A</b> SH-SY5Y NB cells were treated as indicated by the scheme and apoptosis was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. <b>B</b> Kelly NB cells were treated as indicated by the scheme and apoptosis was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. <b>C</b> SHEP NB cells were treated as indicated by the scheme, substituting doxorubicin with either 1.0 µg/ml Cisplatin (Cis), 0.03 µg/ml Topotecan (Topo) or 5.0 µg/ml Etoposide (VP16). Apoptosis was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. <b>D</b> D54 glioblastoma cells were treated as indicated by the scheme and apoptosis after doxorubicin (0.3 µg/ml doxorubicin) treatment was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. In A to D mean+s.e.m. values of three independent experiments carried out in triplicate are shown. Statistical analysis was carried out by two-sided Student's <i>t</i>-test; * P-value <0.01; ** P-value <0.001; # P-value <0.0001.</p

Altered timing affects the potency of NVP-BEZ235/doxorubicin combination therapy in SHEP NB cells.

<p>Three different treatment combinations were tested on SHEP NB cells, giving NVP-BEZ235 12 hrs prior to doxorubicin (Pre), giving both substances concurrently (Co), or giving NVP-BEZ235 12 hrs after the chemotherapeutic (Post). Importantly, the maximal incubation time with doxorubicin was kept constant at 24 hrs (earlier time points also shown in C and D). <b>A</b> SHEP NB cells were treated with NVP-BEZ235 and indicated concentrations of doxorubicin for 24 hrs, according to the scheme outlined above. Apoptosis was determined by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei, and percentage of specific DNA fragmentation is shown. <b>B</b> An alternative depiction of the data presented in A, highlighting the difference between the three NVP-BEZ235/doxorubicin combinations. For all following experiments 0.2 µg/ml doxorubicin was used. <b>C</b> Cells were either left untreated or treated as indicated and mitochondrial release of immunofluorescent-labeled cytochrome c was determined by FACS analysis. <b>D</b> Cells were either left untreated or treated as indicated. A Western blot analysis of caspase-3 processing served as surrogate read-out of caspase activation (appearance of the ∼12 kD cleavage fragment), β-actin was used as loading control. In A and B mean+s.e.m. values of three independent experiments carried out in triplicate, in C mean+s.d. of three independent experiments are shown, while in D a representative result of three independent experiments is depicted. Statistical analysis was carried out by two-sided Student's <i>t</i>-test; * P-value <0.01; ** P-value <0.001; # P-value <0.0001.</p

The effects of the PI3K/mTOR inhibitor NVP-BEZ235 on SHEP NB cells.

<p><b>A</b> Cells were either left untreated, treated for 24 µM PI-103, a well-characterized pan-PI3K inhibitor used as positive control, or the indicated concentrations of NVP-BEZ235. Protein expression levels and phosphorylation status of Akt and S6 ribosomal protein served as surrogate read-outs for PI3K and mTOR activity, respectively, and were analyzed by Western blotting, β-actin served as loading control. <b>B</b> Cells were either left untreated, treated for 24 hrs with either 0.6 µM PI-103 as positive control, or 0.6 µM NVP-BEZ235 for the indicated lengths of time. Protein expression levels and phosphorylation status of Akt and S6 ribosomal protein were analyzed by Western blotting, β-actin served as loading control. <b>C</b> Cells were either left untreated, treated for indicated length of time with 0.6 µM NVP-BEZ235, or treated for 24 hrs with 0.2 µg/ml doxorubicin as positive control. Protein expression levels and cleavage of caspase-.3 protein were analyzed by Western blotting, β-actin served as loading control. <b>D</b> Cells were cultured either in the presence or absence of 0.6 µM of NVP-BEZ235 for 24, 48 and 72 hrs, followed by FACS analysis of the DNA fragmentation of propidium iodide-stained nuclei. The percentage of absolute DNA fragmentation is shown as readout of apoptosis. <b>E</b> 24, 48 and 72 hrs after treatment with 0.6 µM NVP-BEZ235 total cell numbers of treated and untreated cells were counted. <b>F</b> Cell cycle distribution (untreated control and samples treated with 0.6 µM of NVP-BEZ235) was determined after indicated times by FACS analysis of propidium iodide-stained nuclei. <b>G</b> Either untreated controls, or cells treated for 12 and 24 hrs with 0.6 µM NVP-BEZ235 were stained for Ki67 protein expression and evaluated by immunofluorescent microscopy. In A–C and F a representative result of two independent experiments is depicted, while in D and E mean+s.e.m. values of at least three independent experiments carried out in triplicate are shown. Shown in F is the mean of three independent experiments carried out in triplicate, in G the mean+SD of three independent experiments. Statistical analysis was carried out by two-sided Student's <i>t</i>-test; * P-value <0.01; ** P-value <0.001; # P-value <0.0001.</p

MicroRNA-497/195 is tumor-suppressive and cooperates with CDKN2A/B in pediatric acute lymphoblastic leukemia

none21siWe previously identified an association of rapid engraftment of patient-derived leukemia cells transplanted into NOD/SCID mice with early relapse in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In search for the cellular and molecular profiles associated with this phenotype, we investigated the expression of microRNAs (miRNAs) in different engraftment phenotypes and patient outcomes and found high miR-497/195 expression in patient-derived xenograft samples with slow engraftment, derived from patients with favorable outcome. In contrast, epigenetic repression and low expression of these miRNAs was observed in rapidly engrafting samples associated with early relapse. Overexpression of miR-497/195 in patient-derived leukemia cells suppressed in vivo growth of leukemia and prolonged recipient survival. Conversely, inhibition of miR-497/195 led to increased leukemia cell growth. Key cell cycle regulators were downregulated upon miR-497/195 overexpression and we identified CDK4/CCND3-mediated control of G1/S transition as a principal mechanism for the suppression of BCP-ALL progression by miR-497/195. The critical role for miR-497/195-mediated cell cycle regulation was underscored by the finding in an additional independent series of patient samples, showing that high miR-497/195 expression together with a full sequence of CDKN2A/B was associated with excellent outcome, while deletion of CDKN2A/B together with low expression of miR-497/195 was associated with clearly inferior relapse-free survival. These findings point to the cooperative loss of cell cycle regulators as new prognostic factor indicating possible therapeutic targets for pediatric BCP-ALL.noneBoldrin, Elena; Gaffo, Enrico; Niedermayer, Alexandra; Boer, Judith M; Zimmermann, Martin; Weichenhan, Dieter; Claus, Rainer; Münch, Vera; Sun, Qian; Enzenmüller, Stefanie; Seyfried, Felix; Demir, Salih; Zinngrebe, Julia; Cario, Gunnar; Schrappe, Martin; Den Boer, Monique L; Plass, Christoph; Debatin, Klaus-Michael; te Kronnie, Geertruij; Bortoluzzi, Stefania; Meyer, Lüder HinrichBoldrin, Elena; Gaffo, Enrico; Niedermayer, Alexandra; Boer, Judith M; Zimmermann, Martin; Weichenhan, Dieter; Claus, Rainer; Münch, Vera; Sun, Qian; Enzenmüller, Stefanie; Seyfried, Felix; Demir, Salih; Zinngrebe, Julia; Cario, Gunnar; Schrappe, Martin; Den Boer, Monique L; Plass, Christoph; Debatin, Klaus-Michael; te Kronnie, Geertruij; Bortoluzzi, Stefania; Meyer, Lüder Hinric

Prediction of venetoclax activity in precursor B-ALL by functional assessment of apoptosis signaling

Deregulated cell death pathways contribute to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Intrinsic apoptosis signaling is regulated by different proapoptotic and antiapoptotic molecules: proapoptotic BCL-2 homology domain 3 (BH3) proteins activate prodeath molecules leading to cellular death, while antiapoptotic molecules including B-cell lymphoma 2 (BCL-2) prevent activation of prodeath proteins and counter-regulate apoptosis induction. Inhibition of these antiapoptotic regulators has become a promising strategy for anticancer treatment, but variable anticancer activities in different malignancies indicate the need for upfront identification of responsive patients. Here, we investigated the activity of the BCL-2 inhibitor venetoclax (VEN, ABT-199) in B-cell precursor acute lymphoblastic leukemia and found heterogeneous sensitivities in BCP-ALL cell lines and in a series of patient-derived primografts. To identify parameters of sensitivity and resistance, we evaluated genetic aberrations, gene-expression profiles, expression levels of apoptosis regulators, and functional apoptosis parameters analyzed by mitochondrial profiling using recombinant BH3-like peptides. Importantly, ex vivo VEN sensitivity was most accurately associated with functional BCL-2 dependence detected by BH3 profiling. Modeling clinical application of VEN in a preclinical trial in a set of individual ALL primografts, we identified that leukemia-free survival of VEN treated mice was precisely determined by functional BCL-2 dependence. Moreover, the predictive value of ex vivo measured functional BCL-2 dependence for preclinical in vivo VEN response was confirmed in an independent set of primograft ALL including T- and high risk-ALL. Thus, integrative analysis of the apoptosis signaling indicating mitochondrial addiction to BCL-2 accurately predicts antileukemia activity of VEN, robustly identifies VEN-responsive patients, and provides information for stratification and clinical guidance in future clinical applications of VEN in patients with ALL