Anti‐tumor effects of PIM / PI 3K/ mTOR triple kinase inhibitor IBL ‐302 in neuroblastoma
2019; Springer Nature; Volume: 11; Issue: 8 Linguagem: Inglês
10.15252/emmm.201810058
ISSN1757-4684
AutoresSofie Mohlin, Karin Hansson, Katarzyna Radke, Sonia Martı́nez, Carmen Blanco‐Aparicio, Cristian Garcia‐Ruiz, Charlotte Welinder, Javanshir Esfandyari, Michael F. O’Neill, Joaquı́n Pastor, Kristoffer von Stedingk, Daniel Bexell,
Tópico(s)Signaling Pathways in Disease
ResumoArticle16 July 2019Open Access Source DataTransparent process Anti-tumor effects of PIM/PI3K/mTOR triple kinase inhibitor IBL-302 in neuroblastoma Sofie Mohlin Corresponding Author [email protected] orcid.org/0000-0002-2458-3963 Division of Pediatrics, Department of Clinical Sciences, Lund University, Lund, Sweden Search for more papers by this author Karin Hansson Department of Laboratory Medicine, Translational Cancer Research, Lund University Cancer Center, Lund University, Lund, Sweden Search for more papers by this author Katarzyna Radke Department of Laboratory Medicine, Translational Cancer Research, Lund University Cancer Center, Lund University, Lund, Sweden Search for more papers by this author Sonia Martinez orcid.org/0000-0003-2230-7794 Experimental Therapeutics Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Carmen Blanco-Apiricio Experimental Therapeutics Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Cristian Garcia-Ruiz orcid.org/0000-0003-2914-7914 Department of Laboratory Medicine, Translational Cancer Research, Lund University Cancer Center, Lund University, Lund, Sweden Search for more papers by this author Charlotte Welinder Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden Search for more papers by this author Javanshir Esfandyari Department of Laboratory Medicine, Translational Cancer Research, Lund University Cancer Center, Lund University, Lund, Sweden Search for more papers by this author Michael O'Neill Inflection Biosciences Ltd, Blackrock, Ireland Search for more papers by this author Joaquin Pastor Experimental Therapeutics Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Kristoffer von Stedingk Division of Pediatrics, Department of Clinical Sciences, Lund University, Lund, Sweden Department of Oncogenomics, University Medical Center, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Daniel Bexell Corresponding Author [email protected] orcid.org/0000-0001-9426-9550 Department of Laboratory Medicine, Translational Cancer Research, Lund University Cancer Center, Lund University, Lund, Sweden Search for more papers by this author Sofie Mohlin Corresponding Author [email protected] orcid.org/0000-0002-2458-3963 Division of Pediatrics, Department of Clinical Sciences, Lund University, Lund, Sweden Search for more papers by this author Karin Hansson Department of Laboratory Medicine, Translational Cancer Research, Lund University Cancer Center, Lund University, Lund, Sweden Search for more papers by this author Katarzyna Radke Department of Laboratory Medicine, Translational Cancer Research, Lund University Cancer Center, Lund University, Lund, Sweden Search for more papers by this author Sonia Martinez orcid.org/0000-0003-2230-7794 Experimental Therapeutics Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Carmen Blanco-Apiricio Experimental Therapeutics Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Cristian Garcia-Ruiz orcid.org/0000-0003-2914-7914 Department of Laboratory Medicine, Translational Cancer Research, Lund University Cancer Center, Lund University, Lund, Sweden Search for more papers by this author Charlotte Welinder Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden Search for more papers by this author Javanshir Esfandyari Department of Laboratory Medicine, Translational Cancer Research, Lund University Cancer Center, Lund University, Lund, Sweden Search for more papers by this author Michael O'Neill Inflection Biosciences Ltd, Blackrock, Ireland Search for more papers by this author Joaquin Pastor Experimental Therapeutics Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Kristoffer von Stedingk Division of Pediatrics, Department of Clinical Sciences, Lund University, Lund, Sweden Department of Oncogenomics, University Medical Center, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Daniel Bexell Corresponding Author [email protected] orcid.org/0000-0001-9426-9550 Department of Laboratory Medicine, Translational Cancer Research, Lund University Cancer Center, Lund University, Lund, Sweden Search for more papers by this author Author Information Sofie Mohlin *,1,‡, Karin Hansson2,‡, Katarzyna Radke2, Sonia Martinez3, Carmen Blanco-Apiricio3, Cristian Garcia-Ruiz2,†, Charlotte Welinder4, Javanshir Esfandyari2, Michael O'Neill5, Joaquin Pastor3, Kristoffer Stedingk1,6 and Daniel Bexell *,2 1Division of Pediatrics, Department of Clinical Sciences, Lund University, Lund, Sweden 2Department of Laboratory Medicine, Translational Cancer Research, Lund University Cancer Center, Lund University, Lund, Sweden 3Experimental Therapeutics Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain 4Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden 5Inflection Biosciences Ltd, Blackrock, Ireland 6Department of Oncogenomics, University Medical Center, University of Amsterdam, Amsterdam, The Netherlands †Present address: Hematology and Hemotherapy Research Group, IIS La Fe, Valencia, Spain ‡These authors contributed equally to this work *Corresponding author. Tel: +46 462226439; E-mail: [email protected] *Corresponding author. Tel: +46 462226423; E-mail: [email protected] EMBO Mol Med (2019)11:e10058https://doi.org/10.15252/emmm.201810058 Correction(s) for this article Anti-tumor effects of PIM/PI3K/mTOR triple kinase inhibitor IBL-302 in neuroblastoma09 January 2020 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The PI3K pathway is a major driver of cancer progression. However, clinical resistance to PI3K inhibition is common. IBL-302 is a novel highly specific triple PIM, PI3K, and mTOR inhibitor. Screening IBL-302 in over 700 cell lines representing 47 tumor types identified neuroblastoma as a strong candidate for PIM/PI3K/mTOR inhibition. IBL-302 was more effective than single PI3K inhibition in vitro, and IBL-302 treatment of neuroblastoma patient-derived xenograft (PDX) cells induced apoptosis, differentiated tumor cells, and decreased N-Myc protein levels. IBL-302 further enhanced the effect of the common cytotoxic chemotherapies cisplatin, doxorubicin, and etoposide. Global genome, proteome, and phospho-proteome analyses identified crucial biological processes, including cell motility and apoptosis, targeted by IBL-302 treatment. While IBL-302 treatment alone reduced tumor growth in vivo, combination therapy with low-dose cisplatin inhibited neuroblastoma PDX growth. Complementing conventional chemotherapy treatment with PIM/PI3K/mTOR inhibition has the potential to improve clinical outcomes and reduce severe late effects in children with high-risk neuroblastoma. Synopsis In this study, we report that the multikinase PIM/PI3K/mTOR inhibitor IBL-302 has therapeutic effects in multiple preclinical models of neuroblastoma. IBL-302 in combination with chemotherapy has the potential to improve therapy of high-risk neuroblastoma. The multikinase PIM/PI3K/mTOR inhibitor IBL-302 was developed. Among > 700 cell lines, neuroblastoma was the most sensitive to IBL-302 treatment. IBL-302 caused cell death and neuronal differentiation in MYCN-amplified neuroblastoma preclinical models. IBL-302 potentiated the effects of cisplatin in PDX models. Introduction Phosphoinositol-3-kinase (PI3K) activation is a driver in many cancers (Engelman, 2009). The mammalian target of rapamycin (mTOR), a downstream PI3K pathway effector, promotes the expression of proteins involved in cell growth and survival, and mTOR kinase inhibitors, either alone or in combination with PI3K inhibitors, have shown preclinical promise (Fruman et al, 2017). These encouraging data have led to clinical trials of small-molecule PI3K and PI3K/mTOR pathway inhibitors (Fruman et al, 2017). Neuroblastoma is a childhood malignancy of the sympathetic nervous system that accounts for 15% of all pediatric cancer fatalities. Children with high-risk disease have poor survival rates, with up to 40% 5-year mortality. Current treatment strategies include high-dose chemotherapy, surgery, radiotherapy, and anti-GD2 therapy. We and others have shown that targeting the PI3K/mTOR pathway could be a viable treatment for aggressive neuroblastoma (Chesler et al, 2006; Johnsen et al, 2008; Segerström et al, 2011; Chanthery et al, 2012; Mohlin et al, 2013, 2015; Cage et al, 2015; Stewart et al, 2015; Vaughan et al, 2016). Several studies reported anti-tumor effects and improved survival rates in preclinical neuroblastoma models using PI3K inhibitors LY294002 (Chesler et al, 2006), PI-103 (Segerström et al, 2011), dactolisib/NVP-BEZ235 (Chanthery et al, 2012; Stewart et al, 2015; Vaughan et al, 2016), ZSTK474 (Vaughan et al, 2016), PIK-75 (Cage et al, 2015), PW-12 (Cage et al, 2015), BKM120 (Stewart et al, 2015), and/or SF1126 (Erdreich-Epstein et al, 2016). In addition, AKT inhibitor perifosine (Li et al, 2010, 2011) and mTOR inhibitors rapamycin (Johnsen et al, 2008), CCI-779 (Johnsen et al, 2008), and Torin (Vaughan et al, 2016) were demonstrated to decrease proliferation in vitro and consequent tumor growth in vivo. One explanation to the promising preclinical results from inhibiting PI3K/AKT/mTOR could be the anti-angiogenic downstream effects (Johnsen et al, 2008; Chanthery et al, 2012; Mohlin et al, 2015). The majority of reports also showed that neuroblastoma cells with high expression of MYCN were more sensitive to PI3K inhibition and/or that treatment resulted in downregulated N-Myc protein levels in vitro and in vivo (Cage et al, 2015; Chanthery et al, 2012; Chesler et al, 2006, Erdreich-Epstein et al, 2016, Johnsen et al, 2008; Vaughan et al, 2016). Importantly, the PI3K inhibitor perifosine has recently been tested in Phase I/Ib clinical trials with promising therapeutic effects and negligible toxicity in 46 neuroblastoma patients (Kushner et al, 2017; Matsumoto et al, 2017). However, intrinsic resistance and acquired resistance to PI3K inhibitors are possible major obstacles to effective treatment with these agents. Resistance mechanisms include activation of downstream mTOR complexes and activation of other networked signaling pathways (Elkabets et al, 2013). The serine/threonine proviral insertion site in murine leukemia virus (PIM) kinases is overexpressed in many cancers and is associated with MYC overexpression and metastasis. Increased PIM1-3 expression has been linked to PI3K inhibitor resistance (Nawijn et al, 2011; Le et al, 2016). As a result, we synthesized the IBL-300 series multikinase inhibitors to specifically target PIM and PI3K to improve efficacy. Here, we show that PIM, PI3K, and mTOR inhibitor IBL-302 demonstrated robust target specificity. In 707 cell lines across 47 tumor types, neuroblastoma was most sensitive to IBL-302 treatment, and of 16 neuroblastoma cell lines, IBL-302 was generally more effective than PI3K inhibitors alone. We used neuroblastoma patient-derived xenografts (PDXs) and cell lines to investigate the benefit of combination therapies directed toward PIM, PI3K, and mTOR pathways. Nanomolar concentrations of IBL-302 induced apoptosis and tumor cell differentiation and reduced N-Myc protein levels. IBL-302 potentiated the effect of three clinically used chemotherapeutic agents in vitro and low-dose cisplatin in vivo. Finally, RNA sequencing and mass spectrometry analyses of IBL-302-treated PDX cells demonstrated that multitarget treatment induced apoptosis and cell death while diminishing cell growth and motility. Adding multikinase PIM/PI3K/mTOR inhibitors to current treatments to lower administered doses of highly toxic chemotherapy could decrease complications later in life and improve outcomes in these young patients. Results High PIM3 expression is associated with poor outcome We first investigated the potential roles of PIM isoforms in neuroblastoma. PIM1 and PIM3 were expressed in neuroblastoma cell lines and PDX-derived cell cultures (Appendix Fig S1A and B). High levels of PIM1 and PIM3 were also significantly associated with adverse neuroblastoma patient outcomes [Appendix Fig S1C (PIM1) and D (PIM3)]. Neuroblastoma is highly sensitive to triple PIM/PI3K/mTOR inhibition We then developed single compound multikinase inhibitors directed toward PIM, PI3K, and mTOR (covered under patent WO2012/156756). The precise synthetic structures of IBL-301 and IBL-302 are outlined in Fig 1A and B, respectively. Due to superior pharmaceutical profile over IBL-301 in vivo, the closely related analog IBL-302 was selected for further development. We also included the previously published dual PIM/PI3K inhibitor IBL-202 in our studies (Crassini et al, 2018). Target efficiency toward PI3Kα, mTOR, PIM1, PIM2, and PIM3 displayed as IC50 (nM) for all three inhibitors is shown in Fig 1C. Figure 1. Structures of multikinase inhibitors A, B. Chemical structures of IBL-301 (A) and IBL-302 (B). C. IC50 data of IBL-301, IBL-302, and IBL-202. IC50 values were calculated representing the percentage of inhibition against compound concentration and adjusting the experimental data to sigmoidal curve using the software Activity base from IDBS. Download figure Download PowerPoint In an initial screen for tumor type sensitivity to triple PIM/PI3K/mTOR inhibition by IBL-302, 707 cell lines derived from 47 different tumor types were tested by the Genomics of Drug Sensitivity in Cancer (GDSC) screening program (Yang et al, 2013). Selecting for tumor types represented by at least five cell lines, GI50 values for IBL-302 were compared for the remaining 35 tumor types. Neuroblastoma was the most sensitive cancer to IBL-302 treatment (Fig 2A). To establish whether neuroblastoma sensitivity to IBL-302 was due to triple target inhibition or whether the effects were conferred by PI3K pathway inhibition, GI50 values were compared between IBL-302 and the PI3K/mTOR inhibitors dactolisib, PI-103, and omipalisib, the mTOR inhibitor AZD8055, and the ribosomal S6 kinase (RSK)/PIM inhibitor SL0101 using GDSC data. Across a panel of 31 tumor types, neuroblastoma did not show particular sensitivity to any of the five PI3K-specific inhibitors (Appendix Fig S1E–I). Furthermore, analyzing GI50 values across a panel of 16 neuroblastoma cell lines showed that the majority were highly sensitive to IBL-302 compared with PI3K inhibitors alone (IBL-302 was among the top-two most sensitive compounds in 14 of the 16 cell lines; Fig 2B). Figure 2. Neuroblastoma is particularly sensitive to the multikinase inhibitor IBL-302 A. Screening of 707 cell lines from 47 tumor types for sensitivity against IBL-302. Tumor forms with n ≥ 5 cell lines screened are displayed. Comprehensive screening data are available in Appendix Supplementary Materials. Horizontal bands are the median line; the upper part of the box is the 1st quartile and the lower box is the 3rd quartile; the error bars are the maximum and minimum values excluding outliers; the X symbols are mean values. The diagram should show that the y-axis is cut at IC50 = 12, for visualization. Outliers range up to IC50 ~ 400. B. Screening of 16 neuroblastoma cell lines for sensitivity against IBL-302 vs. five PI3K only inhibitors. Download figure Download PowerPoint Neuroblastoma cells differentiate in response to PIM/PI3K/mTOR inhibition To assess the effects of IBL inhibitors, we treated neuroblastoma PDX cells (Appendix Table S1, and Braekeveldt et al, 2015; Persson et al, 2017) and conventional neuroblastoma cell lines with increasing drug concentrations surrounding their respective GI50 range (Appendix Table S2). We first verified that IBL-202 (PIM/PI3K) and IBL-301 (PIM/PI3K/mTOR) targeted the intended individual signaling pathways. Treatment with the IBL inhibitors downregulated the levels of phosphorylated Akt (Ser473 and Thr308), phosphorylated p70S6K and p85S6K (Thr412 and Thr389, respectively), and phosphorylated PRAS40 (Thr246; Figs 3A–C and EV1A–C). It has been suggested that tumors are more susceptible to chemotherapy when induced into a differentiated state due to the reduction in the stem cell pool (Pietras et al, 2010). Triple kinase inhibition with IBL-301 initiated profound neurite outgrowth, a morphological sign of differentiation, in PDX LU-NB-3 cells as well as SK-N-BE(2)c and SK-N-SH neuroblastoma cell lines (Figs 3D and EV1D). These results were confirmed by Tuj1 staining and neurite outgrowth quantification (Figs 3D and E, and EV1E). In addition, gene expression of the neuronal differentiation marker GAP43 was slightly upregulated (Fig EV2A), and Gap43 protein expression was induced in LU-NB-3 PDX and SK-N-BE(2)c cells (Fig 3F). Figure 3. PIM/PI3K/mTOR inhibition decreases N-Myc levels and increases cellular differentiationNeuroblastoma PDX and SK-N-BE(2)c cells treated with IBL inhibitors at indicated concentrations for 48 h. A, B. pAkt [at Ser473 (A) and Thr308 (B) sites] levels in LU-NB-3 and SK-N-BE(2)c cells determined by Western blotting. Total Akt levels were used as loading control. C. p-p70S6K and p-p85S6K levels in LU-NB-3 cells determined by Western blotting. Actin, p70S6K, and p85S6K levels were used as loading controls. D. Brightfield photomicrographs of LU-NB-3 and SK-N-BE(2)c cells treated with 0.36 μM IBL-202 or 0.05 μM IBL-301. Scale bars represent 100 μm (LU-NB-3) or 200 μm (SK-N-BE(2)c). Arrows indicate neurite outgrowths, and asterisks indicate where inserts are magnified. IBL-301-treated cells were stained for Tuj1. DAPI was used to visualize nuclei. E. Quantification of neurite outgrowth presented as number of neurites/cell in LU-NB-3 PDX and SK-N-BE(2) cells treated with IBL-301. For LU-NB-3 PDX cells, representative areas (n = 2) were used and n = 344 and n = 240 cells/condition for CTRL and IBL-301, respectively, were counted. For SK-N-BE(2)c cells, representative areas (n = 2 and n = 3 for CTRL and IBL-301, respectively) were used and n = 141 and n = 130 cells/condition for CTRL and IBL-301, respectively, were counted. Values are reported as mean ± SEM. Statistical significance was determined by two-sided Student's t-test. P = 0.003 for LU-NB-3 and P = 0.08 for SK-N-BE(2)c. F. Gap43 protein levels in LU-NB-3 and SK-N-BE(2)c cells determined by Western blotting. SDHA levels were used as loading control. G. N-Myc levels in LU-NB-2 and LU-NB-3 cells determined by Western blotting. SDHA levels were used as loading control. Source data are available online for this figure. Source Data for Figure 3 [emmm201810058-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Multikinase inhibitors target downstream signaling pathways A–C. Western blot analyses of downstream signaling protein pPRAS40 (A), pAkt(Ser473) (B), and pAkt(Thr308) (C) expression in LU-NB-3 and LU-NB-2 PDX cells after treatment with IBL-202 or IBL-301 at indicated concentrations for 48 h. PRAS40, total Akt, and SDHA levels were used as loading controls. D. Brightfield photomicrographs of SK-N-SH cells treated with 0.36 μM IBL-202 or 0.05 μM IBL-301 for 48 h. Scale bars represent 100 μm. E. Quantification of neurite outgrowth presented as number of neurites/cell in SK-N-SH cells treated with 0.05 μM IBL-301. Representative areas (n = 4 and n = 7 for CTRL and IBL-301, respectively) were used, and n = 249 and n = 245 cells/condition for CTRL and IBL-301, respectively, were counted. Values are reported as mean ± SEM. Statistical significance was determined by two-sided Student's t-test. P = 0.14. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. PIM3 is expressed at higher levels in MYCN-amplified tumors A. Relative mRNA expression levels of GAP43 in LU-NB-3 and SK-N-BE(2)c cells treated with IBL-202 or IBL-301 at indicated concentrations for 48 h as determined by qRT–PCR. Mean values from three biologically independent experiments. Error bars represent SEM. Statistical significance was determined by one-way ANOVA. *P = 0.013. B. PIM1 and PIM3 expression in non-MYCN-amplified vs. MYCN-amplified tumors in publicly available datasets SEQC498 (left panels) and Versteeg88 (right panels). Horizontal bands are the median line; the upper part of the box is the 1st quartile and the lower box is the 3rd quartile; the error bars are the maximum and minimum values excluding outliers; the X symbols are mean values. C. Determination of PIM1 and PIM3 MYCN dependence through multivariate cox regression analysis in publicly available dataset SEQC498. The text in the lowest row in this Table is random and not everything is included. D. Relative mRNA expression levels of MYCN in LU-NB-3 and SK-N-BE(2)c cells treated with IBL-202 or IBL-301 at indicated concentrations for 48 h as determined by qRT–PCR. Mean values from three biologically independent experiments. Error bars represent SEM. Statistical significance was determined by one-way ANOVA. No asterisk indicates no significance. Download figure Download PowerPoint N-Myc protein expression is downregulated following IBL inhibitor treatment Amplification of the MYCN oncogene correlates with aggressive neuroblastoma growth, and we thus investigated putative correlations between MYCN and PIM isoform expression levels. There were no differences in PIM1 expression in MYCN-amplified vs. non-amplified tumors, whereas PIM3 was expressed at significantly higher levels in MYCN-amplified tumors (Fig EV2B). We therefore investigated whether PIM1 and PIM3 had prognostic effects independent of MYCN through multivariate analyses. PIM3 expression did indeed fall out as significant independent prognostic variable in a multivariate cox regression analysis including MYCN status (P = 0.007), while PIM1 expression did not (P = 0.084; Fig EV2C). IBL-202/301 treatment of PDX cells and neuroblastoma cell lines resulted in unchanged MYCN mRNA levels (Fig EV2D) but pronounced decreases in N-Myc protein levels (Fig 3G). Multikinase PIM/PI3K/mTOR inhibition induces neuroblastoma cell death Treatment with IBL-202 and IBL-301 reduced cell viability in two PDX lines and two conventional neuroblastoma cell lines, and the triple PIM/PI3K/mTOR inhibitor IBL-301 had distinctly lower GI50 (Fig 4A and Appendix Table S2). To determine whether the decrease in viable cells following IBL treatment was due to cell death and not solely a result of decreased proliferation, we analyzed the cell cycle distribution of neuroblastoma LU-NB-3 PDX cells. The fraction of cells in sub-G1 phase (i.e., non-viable cells) increased after treatment, with the most profound induction by the triple inhibitor IBL-301 (Fig 4B and C). We further showed that the increase in cell death was mediated via apoptosis as assessed by increased cleaved caspase-3 levels (Fig 4D) as well as an increased fraction of Annexin V- and propidium iodide (PI)-positive cells in PDXs and conventional neuroblastoma cell lines (Fig 4E and F). Figure 4. Treatment with multikinase inhibitors results in cell death A. Viability of IBL-202- or IBL-301-treated cells determined by CellTiter-Glo. Values are reported as mean ± SEM. n = 2 replicates for LU-NB-2 and LU-NB-3, n = 3 replicates for SK-N-BE(2)c and SK-N-SH. B, C. Cell cycle distribution of LU-NB-3 cells determined by flow cytometry. Bars show mean values from two independent experiments. D. Cleaved caspase-3 (Cl Casp3) levels in LU-NB-3 cells determined by Western blotting. SDHA levels were used as loading control. E. Flow cytometry analyses of Annexin V and PI stainings following treatment with 360 nM IBL-202 or 50 nM IBL-301. F. Quantification of live and dead cells from the Annexin V/PI stainings. Dead cells = PI positive, live cells = PI negative. Dot plots from two independent experiments and error bars represent SEM. Source data are available online for this figure. Source Data for Figure 4 [emmm201810058-sup-0005-SDataFig4.pdf] Download figure Download PowerPoint IBL-302 reduces neuroblastoma cell viability in vitro and tumor growth in vivo IBL-302, a compound with comparable structure to IBL-301, but with increased bioavailability, was chosen for further testing. We first reproduced in vitro data and confirmed that IBL-302 inhibited downstream target activity (Fig 5A and B, and Appendix Fig S2A and B). IBL-302 induced neuronal differentiation in PDX as well as two neuroblastoma cell lines as assessed morphologically and by quantification of neurite outgrowth (Fig 5C and D, and Appendix Fig S2C). There was a concentration-dependent reduction in N-Myc protein expression (Fig 5E). In similarity to previous data, we observed reduced neuroblastoma cell viability at nanomolar concentrations (Fig 5F). We could confirm that these changes were due to induced cell death by the increased proportions of Annexin V- and PI-positive cells (Fig 5G). Figure 5. IBL-302 reduces neuroblastoma growth in vivoNeuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 h (pAkt and N-Myc expression, photomicrographs, and flow cytometry) or 72 h (cell viability, (F)). A. Expression of pAkt(Ser473) in LU-NB-3 and SK-N-BE(2)c cells. Total Akt and actin were used as loading controls. B. Expression of pAkt(Thr308) in LU-NB-3 and SK-N-BE(2)c cells. Total Akt and actin were used as loading controls. C. Brightfield photomicrographs of LU-NB-3 and SK-N-BE(2)c cells treated with 50 nM IBL-302. Scale bars represent 100 μm (LU-NB-3) or 200 μm (SK-N-BE(2)c). D. Quantification of neurite outgrowth presented as number of neurites/cell in LU-NB-3 PDX and SK-N-BE(2)c cells treated with IBL-302. For LU-NB-3 PDX cells, representative areas (n = 4) were used and n = 460 and n = 216 cells/condition for CTRL and IBL-302, respectively, were counted. For SK-N-BE(2)c cells, representative areas (n = 5 and n = 7 for CTRL and IBL-302, respectively) were used and n = 124 and n = 260 cells/condition for CTRL and IBL-301, respectively, were counted. Values are reported as mean ± SEM. Statistical significance was determined by two-sided Student's t-test. P = 0.009 for LU-NB-3 and P = 0.0003 for SK-N-BE(2)c. E. N-Myc protein expression determined by Western blotting. SDHA was used as loading control. F. Cell viability determined by CellTiter-Glo. Values are reported as mean ± SEM. n = 3. G. Flow cytometry analyses of Annexin V and PI stainings following treatment with 50 nM IBL-302. H. Quantification of live and dead cells. Dead cells = PI positive, live cells = PI negative. Dot plots from two independent experiments and error bars represent SEM. I. Neuroblastoma SK-N-BE(2)c carrying mice (n = 5 in each group) were treated with vehicle (CTRL) or 40 mg/kg IBL-302 for up to 35 days, and tumor growth was followed over time. Asterisks indicate each occasion a mouse within that particular group was sacrificed. Values are reported as mean ± SEM. J. Kaplan–Meier survival curves comparing mice treated with vehicle (CTRL) or IBL-302. Log-rank test was used to determine statistical significance. P = 0.0577. n = 5 in each group. Source data are available online for this figure. Source Data for Figure 5 [emmm201810058-sup-0006-SDataFig5.pdf] Download figure Download PowerPoint To assess how these results translated in vivo, we xeno-transplanted neuroblastoma cells into the flanks of immunodeficient Nu/Nu mice. Mice were randomly allocated to either control or IBL-302 groups (n = 5 in each group) and treated with IBL-302 per orally (p.o.) 5 days a week. IBL-302-treated mice did not display any signs of compound toxicity, and all maintained their body weight throughout treatment (Appendix Fig S2D). Within a few days of starting treatment, there were observable differences in tumor growth, which increased throughout the experiment (Fig 5I). IBL-302 markedly slowed tumor growth (Fig 5I and Appendix Fig S2E) and prolonged survival (Fig 5J). Anti-tumor effects from multikinase targeting To investigate putative superior effects that triple kinase inhibition holds over single-target inhibition, we set out to analyze the effect of adding PIM and mTOR inhibitors to single inhibition of PI3K. PI3K inhibitor PI-103, mTORC1/2 inhibitor PP242, and PIM inhibitor AZD1208 were used alone or in combination. First, we established EC50 values for all three single-target inhibitors in neuroblastoma PDX cells (Fig EV3A–C), and these EC50 values were used for subsequent combination analysis. There was a trend toward increased neuroblastoma cell death following the combination of PI3K, mTORC1/2, and PIM inhibition as compared to single-target inhibition (Fig EV3D and E). We expanded our analysis on the effects of multikinase inhibition by comparing IBL-302 to PI3K inhibitors alone in neuroblastoma cell lines SK-N-AS and SK-N-FI. Both PI3K inhibitors PI-103 and dactolisib induced a dose-dependent reductio
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