Paracaspase MALT1 regulates glioma cell survival by controlling endo‐lysosome homeostasis
2019; Springer Nature; Volume: 39; Issue: 1 Linguagem: Inglês
10.15252/embj.2019102030
ISSN1460-2075
AutoresKathryn Jacobs, Gwennan André‐Grégoire, Clément Maghe, An Thys, Ying Li, Elizabeth Harford‐Wright, Kilian Trillet, Tiphaine Douanne, Carolina Alves Nicolau, Jean‐Sébastien Frenel, Nicolas Bidère, Julie Gavard,
Tópico(s)Cell death mechanisms and regulation
ResumoArticle27 November 2019free access Source DataTransparent process Paracaspase MALT1 regulates glioma cell survival by controlling endo-lysosome homeostasis Kathryn A Jacobs Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Search for more papers by this author Gwennan André-Grégoire Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Integrated Center for Oncology, ICO, St. Herblain, France Search for more papers by this author Clément Maghe Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Search for more papers by this author An Thys orcid.org/0000-0002-6838-312X Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Search for more papers by this author Ying Li Tsinghua University-Peking University Joint Center for Life Sciences, Technology Center for Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Elizabeth Harford-Wright Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Search for more papers by this author Kilian Trillet Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Search for more papers by this author Tiphaine Douanne Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Search for more papers by this author Carolina Alves Nicolau Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Search for more papers by this author Jean-Sébastien Frénel Integrated Center for Oncology, ICO, St. Herblain, France Search for more papers by this author Nicolas Bidère Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Search for more papers by this author Julie Gavard Corresponding Author [email protected] orcid.org/0000-0002-7985-9007 Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Integrated Center for Oncology, ICO, St. Herblain, France Search for more papers by this author Kathryn A Jacobs Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Search for more papers by this author Gwennan André-Grégoire Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Integrated Center for Oncology, ICO, St. Herblain, France Search for more papers by this author Clément Maghe Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Search for more papers by this author An Thys orcid.org/0000-0002-6838-312X Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Search for more papers by this author Ying Li Tsinghua University-Peking University Joint Center for Life Sciences, Technology Center for Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Elizabeth Harford-Wright Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Search for more papers by this author Kilian Trillet Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Search for more papers by this author Tiphaine Douanne Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Search for more papers by this author Carolina Alves Nicolau Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Search for more papers by this author Jean-Sébastien Frénel Integrated Center for Oncology, ICO, St. Herblain, France Search for more papers by this author Nicolas Bidère Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Search for more papers by this author Julie Gavard Corresponding Author [email protected] orcid.org/0000-0002-7985-9007 Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France Integrated Center for Oncology, ICO, St. Herblain, France Search for more papers by this author Author Information Kathryn A Jacobs1, Gwennan André-Grégoire1,2, Clément Maghe1, An Thys1, Ying Li3, Elizabeth Harford-Wright1, Kilian Trillet1, Tiphaine Douanne1, Carolina Alves Nicolau1, Jean-Sébastien Frénel2, Nicolas Bidère1 and Julie Gavard *,1,2 1Team SOAP, CRCINA, Inserm, CNRS, Université de Nantes, Université d'Angers, Nantes, France 2Integrated Center for Oncology, ICO, St. Herblain, France 3Tsinghua University-Peking University Joint Center for Life Sciences, Technology Center for Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, China *Corresponding author. Tel: +33 2808 0327; E-mail: [email protected] EMBO J (2020)39:e102030https://doi.org/10.15252/embj.2019102030 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 Glioblastoma is one of the most lethal forms of adult cancer with a median survival of around 15 months. A potential treatment strategy involves targeting glioblastoma stem-like cells (GSC), which constitute a cell autonomous reservoir of aberrant cells able to initiate, maintain, and repopulate the tumor mass. Here, we report that the expression of the paracaspase mucosa-associated lymphoid tissue l (MALT1), a protease previously linked to antigen receptor-mediated NF-κB activation and B-cell lymphoma survival, inversely correlates with patient probability of survival. The knockdown of MALT1 largely impaired the expansion of patient-derived stem-like cells in vitro, and this could be recapitulated with pharmacological inhibitors, in vitro and in vivo. Blocking MALT1 protease activity increases the endo-lysosome abundance, impairs autophagic flux, and culminates in lysosomal-mediated cell death, concomitantly with mTOR inactivation and dispersion from endo-lysosomes. These findings place MALT1 as a new druggable target involved in glioblastoma and unveil ways to modulate the homeostasis of endo-lysosomes. Synopsis This study unveils that the paracaspase activity of MALT1, which was previously linked to antigen receptor-mediated NF-κB activation and lymphomas, is decisive for the expansion of glioblastoma stem-like cells (GSC), highlighting potential therapeutic strategies against brain cancers. Expression and catalytic activity of MALT1 are required for GSC expansion. Pharmacological targeting of MALT1 is lethal to GSCs and reduces the expansion of established tumors in mice. MALT1 depletion results in an increased endo-lysosomal compartment and decreased mTOR signaling. MALT1 expression negatively correlates to that of RNA-binding protein Quaking to control endo-lysosomal biogenesis. Introduction Glioblastoma multiforme (GBM) represents the most lethal adult primary brain tumors, with a median survival time of 15 months following diagnosis (Stupp et al, 2009, 2015). The current standard-of-care for the treatment of GBM includes a surgical resection of the tumor followed by treatment with alkylating agent temozolomide and radiation. While these standardized strategies have proved beneficial, they remain essentially palliative (Stupp et al, 2009; Chinot et al, 2014; Brown et al, 2016). Within these highly heterogeneous tumors exists a subpopulation of tumor cells named glioblastoma stem-like cells (GSCs). Although the molecular and functional definition of GSCs is still a matter of debate, there is compelling evidence that these cells can promote resistance to conventional therapies, invasion into normal brain, and angiogenesis (Singh et al, 2004; Bao et al, 2006; Chen et al, 2012; Yan et al, 2013; Lathia et al, 2015). As such, they are suspected to play a role in tumor initiation and progression, as well as recurrence and therapeutic resistance. Owing to their quiescent nature, GSCs resist to both chemotherapy and radiation, which target highly proliferative cancer cells (Bao et al, 2006; Chen et al, 2012). Hence, there is a clear need to identify novel therapeutic targets, designed to eradicate GSCs, in order to improve patient outcome. GSCs constantly integrate external maintenance cues from their microenvironment and as such represent the most adaptive and resilient proportion of cells within the tumor mass (Lathia et al, 2015). Niches provide exclusive habitat where stem cells propagate continuously in an undifferentiated state through self-renewal (Lathia et al, 2015). GSCs are dispersed within tumors and methodically enriched in perivascular and hypoxic zones (Calabrese et al, 2007; Jin et al, 2017; Man et al, 2017). GSCs essentially received positive signals from endothelial cells and pericytes, such as ligand/receptor triggers of stemness pathways and adhesion components of the extracellular matrix (Calabrese et al, 2007; Galan-Moya et al, 2011; Pietras et al, 2014; Harford-Wright et al, 2017; Jacobs et al, 2017). GSCs are also protected in rather unfavorable conditions where they resist hypoxic stress, acidification, and nutrient deprivation (Shingu et al, 2016; Jin et al, 2017; Man et al, 2017). Recently, it has been suggested that this latter capacity is linked to the function of the RNA-binding protein Quaking (QKI), in the down-regulation of endocytosis, receptor trafficking, and endo-lysosome-mediated degradation. GSCs therefore down-regulate lysosomes as one adaptive mechanism to cope with the hostile tumor environment (Shingu et al, 2016). Lysosomes operate as central hubs for macromolecule trafficking, degradation, and metabolism (Aits & Jaattela, 2013). Cancer cells usually show significant changes in lysosome morphology and composition, with reported enhancement in volume, protease activity, and membrane leakiness (Fennelly & Amaravadi, 2017). These modifications can paradoxically serve tumor progression and drug resistance, while providing an opportunity for cancer therapies. The destabilization of the integrity of these organelles might indeed ignite a less common form of cell death, known as lysosomal membrane permeabilization (LMP). LMP occurs when lysosomal proteases leak into the cytosol and induce features of necrosis or apoptosis, depending on the degree of permeabilization (Aits & Jaattela, 2013). Recent reports also highlighted that lysosomal homeostasis is essential in cancer stem cell survival (Shingu et al, 2016; Mai et al, 2017; Le Joncour et al, 2019). Additionally, it has been shown that targeting the autophagic machinery is an effective treatment against apoptosis-resistant GBM (Shchors et al, 2015; Zielke et al, 2018). The autophagic flux inhibitor chloroquine can decrease cell viability and acts as an adjuvant for TMZ treatment in GBM. However, this treatment might cause neural degeneration at the high doses required for GBM treatment (Weyerhäuser et al, 2018). Therefore, it is preferable to find alternative drugs that elicit anti-tumor responses without harmful effects on healthy brain cells. A growing body of literature supports the concept of non-oncogene addiction (NOA) in cancer. Although neither mutated nor involved in the initiation of tumorigenesis, NOA genes are essential for the propagation of the transformed phenotype (Luo et al, 2009). Because NOA gene products are pirated for the benefit of tumor cells’ own survival, their targeting therefore constitutes an Achilles’ heel. Among reported NOA genes and pathways (Staudt, 2010), the paracaspase mucosa-associated lymphoid tissue l (MALT1) might be of particular interest in GBM (please see Fig 1). This arginine-specific protease plays a key role in NF-κB signaling upon antigen receptor engagement in lymphocytes, via the assembly of the CARMA-BCL10-MALT1 (CBM) complex. In addition to this scaffold role in NF-κB activation, MALT1 regulates NF-κB activation, cell adhesion, mRNA stability, and mTOR signaling through its proteolytic activity (Rebeaud et al, 2008; Staal et al, 2011; Uehata et al, 2013; Hamilton et al, 2014; Jeltsch et al, 2014; Nakaya et al, 2014). MALT1 has been shown to be constitutively active in activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL), and its inhibition is lethal (Ngo et al, 2006; Hailfinger et al, 2009; Nagel et al, 2012). MALT1 was also recently reported to exert pro-metastatic effects in solid tumors (McAuley et al, 2019). However, the role of MALT1 in solid tumors has not been extensively investigated. Figure 1. MALT1 expression sustains glioblastoma cell growth A. STRING diagram representation of the network of proteins involved in NF-κB pathway. B. The Cancer Genome Atlas (TCGA RNAseq dataset) was used on the GlioVis platform (Bowman et al, 2007) to analyze the probability of survival (log-rank P-value) of 155 GBM patients, for each gene encoding for the mediators of the NF-κB pathway. C. Kaplan–Meier curve of the probability of survival for 155 GBM patients with low or high MALT1 RNA level, using median cutoff, based on the TCGA RNAseq dataset. D, E. Box and whisker plot of MALT1 mRNA expression in low-grade glioma (LGG, grades II and III) or in GBM (grade IV) (TCGA GBMLGG, RNAseq dataset) (D). Horizontal line marks the median, box limits are the upper and lower quartiles, and error bars show the highest and lowest values. Alternatively, MALT1 mRNA expression was plotted in non-tumor samples versus GBM samples (TCGA RNAseq dataset) (E). Each dot represents one clinical sample. F. Fraction of surviving cells over time in GSC#1 and GSC#9, transduced with control (shc) or bi-cistronic GFP plasmids using two different short hairpin RNA (shMALT1 sequences, seq #1 and #2). Data are plotted as the percentage of GFP-positive cells at the day of the analysis (Dx), normalized to the starting point (day 4 post-infection, D4). G. EdU incorporation (green, 2 h) was visualized by confocal imagery in GSC#1 or by FACS in GSC#9 transfected with sic or siMALT1. In GSC#1, the percentage of EdU-positive cells was quantified. Nuclei (DAPI) are shown in blue. n > 240 cells per replicate. Scale bar: 10 μm. Data are presented as the mean ± SEM on three independent experiments. H. FACS analysis of propidium iodide (PI) incorporation in GSC #1 and #9 transfected with non-silencing duplexes (sic) or MALT1 siRNA duplexes (siMALT1) and analyzed 72 h later. I. Linear regression plot of in vitro limiting dilution assay (LDA) for control (shc) or shMALT1 seq#1 and seq#2 transduced GSC#9. Data are representative of n = 2. Knockdown efficiency was verified at day 3 by Western blot using anti-MALT1 antibodies. GAPDH served as a loading control. J. Tumorspheres per field of view (fov) were manually counted in sic or siMALT1 transfected GSC#1, #4, and #9. Data are presented as the mean ± SEM on three independent experiments. Data information: All data are representative of n = 3, unless specified. Statistics were performed using pairwise comparisons (Tukey's honest significant difference (HSD) with a 95% confidence interval for panels C–E), and a two-tailed t-test with a 95% confidence interval for panels (G and J), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are available online for this figure. Source Data for Figure 1 [embj2019102030-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Here, we provide evidence of the role of MALT1 in disrupting GSC lysosomal homeostasis, which is associated with autophagic features. We found that targeting MALT1, notably through the phenothiazine family of drugs, including mepazine (MPZ), is lethal to GBM cells. We further established that MALT1 sequesters QKI and maintains low levels of lysosomes, while its inhibition unleashes QKI and hazardously increases endo-lysosomes, which subsequently impairs autophagic flux. This leads to cell death concomitant with mTOR inhibition and dispersion from lysosomes. Disrupting lysosomal homeostasis therefore represents an interesting therapeutic strategy against GSCs. Results MALT1 expression sustains glioblastoma cell growth Glioblastoma stem-like cells (GSCs) are suspected to be able to survive outside the protective vascular niche, in non-favorable environments, under limited access to growth factors and nutrients (Calabrese et al, 2007; Shingu et al, 2016; Jin et al, 2017). While many signaling pathways can influence this process, the transcription factor NF-κB has been demonstrated to be instrumental in many cancers as it centralizes the paracrine action of cytokines, in addition to playing a major role in cell proliferation and survival of tumor cells and surrounding cells (Bargou et al, 1996; Davis et al, 2001; Karin & Greten, 2005; Li et al, 2009; McAuley et al, 2019). Because of this dual influence on both tumor cells and their microenvironment, we revisited The Cancer Genome Atlas (TCGA) for known mediators of the NF-κB pathway (Fig 1A). We found that MALT1 expression was more significantly correlated with survival than other tested genes of the pathway (Fig 1B). This arginine-specific protease is crucial for antigen receptor-mediated NF-κB activation and B-cell lymphoma survival (Ngo et al, 2006). In addition, when GBM patients were grouped between low and high MALT1 expression levels, there was a significant survival advantage for patients with lower MALT1 expression (Fig 1C). Moreover, levels of MALT1 mRNA are elevated in GBM (Grade IV) when compared with lower grade brain tumors (grades II and III) or non-tumor samples (Fig 1D and E). Although this increased MALT1 expression may be due to tumor-infiltrating immune cells, we first explored whether MALT1 was engaged in patient-derived GSCs, as these cells recapitulated ex vivo features of the tumor of origin (Lathia et al, 2015). The functional impact of MALT1 knockdown was thus evaluated by their viability and expansion in vitro (Fig 1F–J). Two individual short hairpin RNA sequences targeting MALT1 (shMALT1) cloned in a lentiviral bi-cistronic GFP-expressing plasmid were delivered into GSC#1 (mesenchymal) and GSC#9 (classical) cells. We observed a reduced fraction of GFP-positive cells over time, while cells expressing non-silencing RNA plasmids (shc) maintained a steady proportion of GFP-positive cells, indicating that MALT1 silencing was detrimental to GSCs (Fig 1F). Likewise, cells transfected with siMALT1 had a lower percentage of EdU-positive cells as compared to non-silenced control cells (Fig 1G) and a higher incorporation of propidium iodide (PI) (Fig 1H). Additionally, GSCs either expressing shMALT1 or transfected with siMALT1 had less stem traits, as evaluated by limited dilution assay and tumorsphere formation (Fig 1I and J). Taken together, these results indicate that MALT1 expression may be important for glioblastoma cell ex vivo expansion. Pharmacological inhibition of MALT1 is lethal to glioblastoma cells Next, to evaluate the potential of targeting MALT1 pharmacologically, we treated GSC #1 (mesenchymal), #4 (mesenchymal), #9 (classical), and #12 (neural) with the MALT1 allosteric inhibitor mepazine (MPZ) at a dose of 20 μM, as initially described (Nagel et al, 2012). All four GSCs showed a significant reduction in stemness by both limited dilution and tumorsphere assays (Fig 2A–C). Additionally, the competitive inhibitor Z-VRPR-FMK induced similar decrease in tumorsphere formation (Fig 2C). This was accompanied by a marked reduction in the abundance of SOX2 and NESTIN stemness markers (Fig 2D). Alongside the in vitro self-renewal impairment, GSC viability was largely annihilated by MPZ treatment, including reduction in EdU staining and increase in PI incorporation (Fig 2E–G). In contrast, MPZ had no significant effect on viability of brain-originated human cells (endothelial cells, astrocytes, and neurons), ruling out a non-selectively toxic effect (Fig 2E). Differentiated sister GSCs (DGCs) also showed reduced viability in response to MPZ, indicating that targeting MALT1 may have a pervasive effect on differentiated GBM tumor cells (Fig 2H). Figure 2. MALT1 pharmacological inhibition is lethal to glioblastoma cells Linear regression plot of in vitro limiting dilution assay (LDA) for GSC#9 treated with MALT1 inhibitor, mepazine (MPZ, 20 μM, 14 days). DMSO vehicle was used as a control. Data are representative of n = 2. Stem cell frequency was calculated from LDA in GSCs #1, #4, and #12 treated with MPZ treatment (20 μM, 14 days). Data are presented as the mean ± SEM on two independent experiments. Tumorspheres per field of view (fov) were manually counted in GSCs #1, #4, #9, and #12 in response to MPZ (20 μM) and vehicle (DMSO), and in GSC#9 treated with Z-VRPR-FMK (75 μM) and vehicle (H2O) for 4 days. Data are presented as the mean ± SEM on 4 independent experiments for MPZ and three independent experiments for Z-VRPR-FMK. The expression of the stemness markers SOX2 and NESTIN was evaluated by Western blot and immunofluorescence (SOX2 in red NESTIN in green) in MPZ (+, 20 μM, 16 h) and vehicle (−, DMSO, 16 h) treated GSC#9. GAPDH served as a loading control. Scale bar: 10 μm. Cell viability was measured using Cell TiterGlo luminescent assay in GSCs #1, #4, #9, and #12, human brain endothelial cells (endo), human astrocytes (astro), and human neuron-like cells (neuron) treated for 48 h with DMSO or MPZ (20 μM). Data were normalized to their respective DMSO-treated controls and are presented as the mean ± SEM of three independent experiments in triplicate. FACS analysis of EdU staining was performed on GSC#1 treated overnight with MPZ (10 μM). Data are presented as the mean ± SEM on three independent experiments. FACS analysis of propidium iodide (PI) incorporation in GSC#9 treated for 48 h with vehicle (DMSO) or MPZ (20 μM). Cell viability was measured using Cell TiterGlo luminescent assay in differentiated GSC#1 #4, and #9 (DGCs) treated for 48 h with vehicle (DMSO) or MPZ (20 μM). Data were normalized to their respective DMSO-treated controls and are presented as the mean ± SEM of three independent experiments. Morphology of GSCs #1, #4, #9, and DGCs #1, #4, #9 was shown using brightfield images. Heatmap of cell viability of GSC#9 using increasing doses (0, 5, 10, 20, 40 μM) of phenothiazines: mepazine (MPZ), fluphenazine (FLU), cyamemazine (CYAM), chlorpromazine (CHLO), pipotiazine (PIPO), alimemazine (ALI), promethazine (PRO), and doxylamine (DOXY). Data were normalized to their respective DMSO-treated controls. Nude mice were implanted with GSC#9 (106 cells) in each flank, and randomized cages were treated with either vehicle (DMSO) or MPZ (8 mg/kg) daily i.p., for 14 consecutive days, once tumors were palpable. Tumor volume was measured from the start of treatment until 1 week after treatment was removed. Graph of tumor volume on day 21 post-treatment is presented. Data are presented as the mean ± SEM n = 10/group. Cryosections from GSC-xenografted tumors were stained for the endothelial marker PECAM1 (red) and tumor marker NESTIN (green). Nuclei (DAPI) are shown in blue. Scale bar: 20 μm. Data information: All data are representative of n = 3, unless specified. Statistics were performed using a two-tailed t-test with a 95% confidence interval for panels (B, C, E, F, H), a two-way ANOVA with Bonferroni post-test at 95% confidence interval for panel (J), a Wilcoxon–Mann–Whitney test for Expt #2 with P-values stated for panel (J). *P < 0.05 **P < 0.01, ***P < 0.001. Source data are available online for this figure. Source Data for Figure 2 [embj2019102030-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint MPZ is a drug, belonging to the phenothiazine family, and was formerly used in the treatment of schizophrenia (Lomas, 1957). Several anti-psychotic phenothiazines have been shown to potentially reduce glioma growth (Tan et al, 2018). We therefore evaluated whether clinically relevant phenothiazines could affect GSC viability (Fig EV1A–E). The effect on MALT1 inhibition was reflected in cell viability, with chlorpromazine (Oliva et al, 2017) and fluphenazine having robust effects on cell viability (Fig 2I). In addition to its effect on MALT1 protease activity (Fig EV1B and C) (Nagel et al, 2012; Schlauderer et al, 2013), MPZ may also exert off-target biological effects (Meloni et al, 2018). We took advantage of the well-characterized MPZ-resistant E397A MALT1 mutant (Schlauderer et al, 2013) to challenge the toxic action of phenothiazines in GSCs (Fig EV1F). E397A MALT1 expression in GSCs partially restored cell viability in phenothiazine-treated cells, suggesting that the main target of phenothiazine-mediated death involves MALT1 inhibition (Fig EV1F). Because MPZ has been shown to efficiently and safely obliterate MALT1 activity in experimental models (Nagel et al, 2012; McGuire et al, 2014; Kip et al, 2018; Di Pilato et al, 2019; Rosenbaum et al, 2019), ectopically implanted GSC#9 mice were challenged with MPZ. Daily MPZ administration reduced tumor volume in established xenografts, as well as NESTIN-positive staining (Fig 2J and K). This effect was prolonged for the week of measurement following treatment withdrawal (Fig 2J). Together, these data demonstrate that targeting MALT1 pharmacologically is toxic to GBM cells in vitro and in vivo. Click here to expand this figure. Figure EV1. Impact of phenothiazines on MALT1 protease activity and lysosomes Table summarizing eight phenothiazines used in clinics as either anti-psychotic or anti-histaminic, along with their generic and brand names (cap letters), and chemical structures. Western blot analysis of two MALT1 substrates, HOIL1 and CYLD, either full length (FL) or cleaved (c'd) in Jurkat T cells treated with vehicle (DMSO) or phenothiazines, as follows: 20 μM CYAM (cyamemazine), CHLO (chlorpromazine), PIPO (pipotiazine), DOXY (doxylamine), ALI (alimemazine), and PRO (promethazine), and 10 μM MPZ (mepazine) and FLU (fluphenazine) for 30 min and stimulated for 30 min more with PMA (20 ng/ml) and Ionomycin (Iono, 300 ng/ml). TUBULIN served as a loading control. Western blot analysis of CYLD processing in GSC#9 treated with vehicle (DMSO) or phenothiazines (20 μM CYAM, CHLO, PIPO, DOXY, ALI, and PRO, 10 μM MPZ and FLU) for 60 min. GAPDH served as a loading control. Western blot analysis of LAMP2 and LC3B in equal amount of total protein lysates from GSC#9 treated for 6 h with vehicle (DMSO) or 20 μM phenothiazines (MPZ, FLU, CYAM, CHLO, ALI, PRO). GAPDH served as a loading control. Cell viability of GSC#1 and GSC#9 using 20 μM of MPZ, FLU, CHLO, and CYAM, using Cell TiterGlo assays. Data were normalized to their respective DMSO-treated controls and are presented as the mean ± SEM of three independent experiments in triplicate. Schematic drawing of MALT1 structures highlighting the E397A substitution in the mepazine-resistant version. DD: death domain, C-like D: caspase-like domain, Ig: immunoglobulin domain. Western blot analysis of FLAG in equal amount of total protein lysates from HEK-293T cells transfected with empty vector (mock), MALT-WT, or MALT1-E397A. GAPDH serves as a loading control. GSC#9 were transduced with MALT-WT or MALT1-E397A and treated with phenothiazines (10 μM of MPZ, FLU, CYAM, CHLO) for 24 h. Cell Viability was analyzed using Cell TiterGlo assay. Data were normalized to their respective DMSO-treated controls and are presented as the mean ± SEM of three independent experiments in triplicate. Data information: All data were repeated in three independent experiments. Statistics were performed using a one-way ANOVA with a 95% confidence interval for all experiments with P-values stated. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure. Download figure Download PowerPoint GSCs maintain basal protease activity of MALT1 In addition to its scaffold function in the modulation of the NF-κB pathway, MALT1 also acts as a protease for a limited number of substrates (Juilland & Thome, 2018; Thys et al, 2018). No hallmarks of NF-κB activation such as phosphorylation and degradation of IκBα, or p65 and cREL nuclear translocation were observed, unless GSCs were treated with TNFα (Fig 3A and B). Nevertheless, the deubiquitinating enzyme CYLD (Staal et al, 2011) and the RNA-binding proteins ROQUIN 1 and 2 (Jeltsch et al, 2014), two known MALT1 substrates, were constitutively cleaved in GSCs (Fig 3C–F). This was, however, not the case of the MALT1 target HOIL1 (Douanne et al, 2016), suggesting that only a subset of MALT1 substrates is cleaved in GSCs (Fig 3C). Of note, CYLD proteolysis was not further increased upon stimulation with PMA plus ionomycin, in contrast to Jurkat lymphocytes, most likely due to a failure to co-opt this signaling route in GSCs (Fig 3C). However, CYLD processing was reduced in cells treated with MPZ or upon siRNA-mediated MALT1 knockdown (Fig 3D and E). The same was true when MALT1 competitive inhibitor Z-VRPR-FMK was used (Fig 3F). Further supporting a role for MALT1 enzyme in GSCs, the expression of a protease-dead version of MALT1 (C464A) weakened CYLD trimming (Fig 3G and H). Interestingly, we found that refreshing medium also reduced CYLD cleavage, suggesting that MALT1 basal activity may rely on outside-in signals rather than cell autonomous misactivation (Fig 3I). Figure 3. MALT1 is active in GSCs Total protein lysates from GSCs #1 and #9 challenged with TNFα (10 ng/ml, for the indicated times) were analyzed by Western blot for p-IκBα, IκBα, and p-JNK. Total JNK and GAPDH se
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