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Multiple myeloma (MM) remains an incurable haematological malignancy despite substantial advances in therapy. Hypoxic bone marrow induces metabolic rewiring in MM cells contributing to survival and drug resistance. Therefore, targeting metabolic pathways may offer an alternative treatment option. In this study, we repurpose two FDA-approved drugs, syrosingopine and metformin. Syrosingopine was used as a dual inhibitor of monocarboxylate transporter 1 and 4 (MCT1/4) and metformin as an inhibitor for oxidative phosphorylation (OXPHOS). Anti-tumour effects were evaluated for single agents and in combination therapy. Survival and expression data for MCT1/MCT4 were obtained from the Total Therapy 2, Mulligan, and Multiple Myeloma Research Foundation cohorts. Cell death, viability, and proliferation were measured using Annexin V/7-AAD, CellTiterGlo, and BrdU, respectively. Metabolic effects were assessed using Seahorse Glycolytic Rate assays and LactateGlo assays. Differential protein expression was determined using western blotting, and the SUnSET method was implemented to quantify protein synthesis. Finally, the syngeneic 5T33MMvv model was used for in vivo analysis. High-level expression of MCT1 and MCT4 both correlated with a significantly lower overall survival of patients. Lactate production as well as MCT1/MCT4 expression were significantly upregulated in hypoxia, confirming the Warburg effect in MM. Dual inhibition of MCT1/4 with syrosingopine resulted in intracellular lactate accumulation and reduced cell viability and proliferation. However, only at higher doses (>10 μm) was syrosingopine able to induce cell death. By contrast, combination treatment of syrosingopine with metformin was highly cytotoxic for MM cell lines and primary patient samples and resulted in a suppression of both glycolysis and OXPHOS. Moreover, pathway analysis revealed an upregulation of the energy sensor p-AMPKα and more downstream a reduction in protein synthesis. Finally, the combination treatment resulted in a significant reduction in tumour burden in vivo. This study proposes an alternative combination treatment for MM and provides insight into intracellular effects.

While multi-drug combinations and continuous treatment have become standard for multiple myeloma, the disease remains incurable. Repurposing drugs that are currently used for other indications could provide a novel approach to improve the therapeutic efficacy of standard multiple myeloma treatments. Here, we assessed the anti-tumor effects of cardiac drugs called β-blockers as a single agent and in combination with commonly used anti-myeloma therapies. Expression of the β 2-adrenergic receptor correlated with poor survival outcomes in patients with multiple myeloma. Targeting the β 2-adrenergic receptor (β 2AR) using either selective or non-selective β-blockers reduced multiple myeloma cell viability, and induced apoptosis and autophagy. Blockade of the β 2AR modulated cancer cell metabolism by reducing the mitochondrial respiration as well as the glycolytic activity. These effects were not observed by blockade of β 1-adrenergic receptors. Combining β 2AR blockade with the chemotherapy drug melphalan or the proteasome inhibitor bortezomib significantly increased apoptosis in multiple myeloma cells. These data identify the therapeutic potential of β 2AR-blockers as a complementary or additive approach in multiple myeloma treatment and support the future clinical evaluation of non-selective β-blockers in a randomized controlled trial.

Supplemental Figure 1 shows the prognostic value of the classical RASSF proteins RASSF1, RASSF3, RASSF5 and RASSF6 in MM patients. Supplemental Figure S2 shows the cellular localization of RASSF4. Supplemental Figure S3 demonstrates the inducible doxycycline-dependent overexpression of human RASSF4 and provides additional evidence for the anti-myeloma activity of RASSF4 overexpression in human cell lines. Supplemental Figure S4 provides a comparison of the RASSF4 expression levels of the primary MM cells (n=10) and a selected panel of HMCL (n=9) obtained through microarray analysis. Supplemental Figure S5 shows additional data on the effect of RASSF4 overexpression on tumor development and the confirmation of RASSF4 overexpression on protein level. Supplemental Figure S6 provides an overview of the top 20 differentially expressed genes between all control and RASSF4 overexpressing human multiple myeloma cell lines and subsequent pathway enrichment analysis of the genes downregulated after RASSF4 overexpression. Supplemental Figure S7 provides additional evidence for the anti-myeloma activity of trametinib in combination with the HDACi quisinostat or panobinostat and shows the effect of the HDACi panobinostat and/or the MEK1/2 inhibitor trametinib on RASSF4 mRNA expression.

RAS mutations occur frequently in multiple myeloma (MM), but apart from driving progression, they can also stimulate antitumor effects by activating tumor-suppressive RASSF proteins. Although this family of death effector molecules are often silenced in cancers, functional data about RASSF proteins in MM are lacking. Here, we report that RASSF4 is downregulated during MM progression and correlates with a poor prognosis. Promoter methylation analysis in human cell lines revealed an inverse correlation between RASSF4 mRNA levels and methylation status. Epigenetic modulating agents restored RASSF4 expression. Enforced expression of RASSF4 induced G2-phase cell-cycle arrest and apoptosis in human cell lines, reduced primary MM cell viability, and blocked MM growth in vivo. Mechanistic investigations showed that RASSF4 linked RAS to several pro-death pathways, including those regulated by the kinases MST1, JNK, and p38. By activating MST1 and the JNK/c-Jun pathway, RASSF4 sensitized MM cells to bortezomib. Genetic or pharmacological elevation of RASSF4 levels increased the anti-MM effects of the clinical relevant MEK1/2 inhibitor trametinib. Kinome analysis revealed that this effect was mediated by concomitant activation of the JNK/c-Jun pathway along with inactivation of the MEK/ERK and PI3K/mTOR/Akt pathways. Overall, our findings establish RASSF4 as a tumor-suppressive hub in MM and provide a mechanistic rationale for combining trametinib with HDAC inhibitors or bortezomib to treat patients with tumors exhibiting low RASSF4 expression.Significance: These findings provide a mechanistic rationale for combining trametinib with HDAC inhibitors or bortezomib in patients with multiple myeloma whose tumors exhibit low RASSF4 expression. Cancer Res; 78(5); 1155–68. ©2017 AACR.

Information about material and methods, that further support the date described in this article. Additional references and legends to the supplmental figures are also provided.

Supplemental Table S1 summarizes the patient characteristics. Supplemental Table S4 provides an overview of all kinases differentially phosphorylated in RASSF4 overexpressing cells treated with trametinib compared to parental cells treated with trametinib and/or RASSF4 overexpressing cells. Supplemental Table S5 summarizes significance and combination index (CI) values calculated for the different drug concentrations used in the combination experiments.

List of differentially expressed genes between all control and RASSF4 overexpressing human multiple myeloma cell lines

BackgroundImmunotherapy emerged as a promising treatment option for multiple myeloma (MM) patients. However, therapeutic efficacy can be hampered by the presence of an immunosuppressive bone marrow microenvironment including myeloid cells. S100A9 was previously identified as a key regulator of myeloid cell accumulation and suppressive activity. Tasquinimod, a small molecule inhibitor of S100A9, is currently in a phase Ib/IIa clinical trial in MM patients (NCT04405167). We aimed to gain more insights into its mechanisms of action both on the myeloma cells and the immune microenvironment.MethodsWe analyzed the effects of tasquinimod on MM cell viability, cell proliferation and downstream signaling pathways in vitro using RNA sequencing, real-time PCR, western blot analysis and multiparameter flow cytometry. Myeloid cells and T cells were cocultured at different ratios to assess tasquinimod-mediated immunomodulatory effects. The in vivo impact on immune cells (myeloid cell subsets, macrophages, dendritic cells), tumor load, survival and bone disease were elucidated using immunocompetent 5TMM models.ResultsTasquinimod treatment significantly decreased myeloma cell proliferation and colony formation in vitro, associated with an inhibition of c-MYC and increased p27 expression. Tasquinimod-mediated targeting of the myeloid cell population resulted in increased T cell proliferation and functionality in vitro. Notably, short-term tasquinimod therapy of 5TMM mice significantly increased the total CD11b+cells and shifted this population toward a more immunostimulatory state, which resulted in less myeloid-mediated immunosuppression and increased T cell activation ex vivo. Tasquinimod significantly reduced the tumor load and increased the trabecular bone volume, which resulted in prolonged overall survival of MM-bearing mice in vivo.ConclusionOur study provides novel insights in the dual therapeutic effects of the immunomodulator tasquinimod and fosters its evaluation in combination therapy trials for MM patients.

Multiple myeloma (MM) cells derive proliferative signals from the bone marrow (BM) microenvironment via exosomal crosstalk. Therapeutic strategies targeting this crosstalk are still lacking. Bortezomib resistance in MM cells is linked to elevated expression of xCT (the subunit of system Xc

International audience; Background: The aggressive B-cell non-Hodgkin lymphomas diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are characterised by a high proliferation rate. The anaphase-promoting complex/cyclosome (APC/C) and its co-activators Cdc20 and Cdh1 represent an important checkpoint in mitosis. Here, the role of the APC/C and its co-activators is examined in DLBCL and MCL. Methods: The expression and prognostic value of Cdc20 and Cdh1 was investigated using GEP data and immunohistochemistry. Moreover, the therapeutic potential of APC/C targeting was evaluated using the small-molecule inhibitor proTAME and the underlying mechanisms of action were investigated by western blot. Results: We demonstrated that Cdc20 is highly expressed in DLBCL and aggressive MCL, correlating with a poor prognosis in DLBCL. ProTAME induced a prolonged metaphase, resulting in accumulation of the APC/C-Cdc20 substrate cyclin B1, inactivation/degradation of Bcl-2 and Bcl-xL and caspase-dependent apoptosis. In addition, proTAME strongly enhanced the anti-lymphoma effect of the clinically relevant agents doxorubicin and venetoclax. Conclusion: We identified for the first time APC/C as a new, promising target in DLBCL and MCL. Moreover, we provide evidence that Cdc20 might be a novel, independent prognostic factor in DLBCL and MCL.

Multiple myeloma (MM) cells derive proliferative signals from the bone marrow (BM) microenvironment via exosomal crosstalk. Therapeutic strategies targeting this crosstalk are still lacking. Bortezomib resistance in MM cells is linked to elevated expression of xCT (the subunit of system Xc(-) ). Extracellular glutamate released by system Xc(-) can bind to glutamate metabotropic receptor (GRM) 3, thereby upregulating Rab27-dependent vesicular trafficking. Since Rab27 is also involved in exosome biogenesis, we aimed to investigate the role of system Xc(-) in exosomal communication between BM stromal cells (BMSCs) and MM cells. We observed that expression of xCT and GRMs was increased after bortezomib treatment in both BMSCs and MM cells. Secretion of glutamate and exosomes was simultaneously enhanced which could be countered by inhibition of system Xc(-) or GRMs. Moreover, glutamate supplementation increased exosome secretion by increasing expression of Alix, TSG101, Rab27a/b and VAMP7. Importantly, the system Xc? inhibitor sulfasalazine reduced BMSC-induced resistance to bortezomib in MM cells in vitro and enhanced its anti-MM effects in vivo. These findings suggest that system Xc? plays an important role within the BM and could be a potential target in MM.

International audience; Multiple myeloma (MM) account for approximately 10% of hematological malignancies and is the second most common hematological disorder. Kinases inhibitors are widely used and their efficiency for the treatment of cancers has been demonstrated. Here, in order to identify kinases of potential therapeutic interest for the treatment of MM, we investigated the prognostic impact of the kinome expression profile in large cohorts of patients. We identified 36 kinome-related genes significantly linked with a prognostic value to MM, and built a kinome index based on their expression. The Kinome Index (KI) is linked to prognosis, proliferation, differentiation, and relapse in MM. We then tested inhibitors targeting seven of the identified protein kinas-es (PBK, SRPK1, CDC7-DBF4, MELK, CHK1, PLK4, MPS1/TTK) in human myeloma cell lines. All tested inhibitors significantly reduced the viability of myeloma cell lines, and we confirmed the potential clinical interest of three of them on primary myeloma cells from patients. In addition, we demonstrated their ability to potentialize the toxicity of conventional treatments, including Melphalan and Lenalidomide. This highlights their potential beneficial effect in myeloma therapy. Three kinases inhibitors (CHK1i, MELKi and PBKi) overcome resistance to Lenalidomide, while CHK1, PBK and DBF4 inhibitors re-sensitize Melphalan resistant cell line to this conventional therapeutic agent. Altogether, we demonstrate that kinase inhibitors could be of therapeutic interest especially in high-risk myeloma patients defined by the KI. CHEK1, MELK, PLK4, SRPK1, CDC7-DBF4, MPS1/TTK and PBK inhibitors could represent new treatment options either alone or in combination with Melphalan or IMiD for refractory/relapsing myeloma patients.

Diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are among the most aggressive B cell non-Hodgkin lymphomas. Maternal embryonic leucine zipper kinase (MELK) plays a role in cancer cell cycle progression and is associated with poor prognosis in several cancer cell types. In this study, the role of MELK in DLBCL and MCL and the therapeutic potential of MELK targeting is evaluated. MELK is highly expressed in DLBCL and MCL patient samples, correlating with a worse clinical outcome in DLBCL. Targeting MELK, using the small molecule OTSSP167, impaired cell growth and survival and induced caspase-mediated apoptosis in the lymphoma cells. Western blot analysis revealed that MELK targeting decreased the phosphorylation of FOXM1 and the protein levels of EZH2 and several mitotic regulators, such as Cdc25B, cyclin B1, Plk-1, and Aurora kinases. In addition, OTSSP167 also sensitized the lymphoma cells to the clinically relevant Bcl-2 inhibitor venetoclax by strongly reducing Mcl1 levels. Finally, OTSSP167 treatment of A20-inoculated mice resulted in a significant prolonged survival. In conclusion, targeting MELK with OTSSP167 induced strong anti-lymphoma activity both in vitro and in vivo. These findings suggest that MELK could be a potential new target in these aggressive B cell malignancies.

Introduction Multiple myeloma (MM) remains an incurable cancer despite advances in therapy. Therefore, the search for new targets is still essential to uncover potential treatment strategies. Metabolic changes, induced by the hypoxic bone marrow, contribute to both cancer cell survival and drug resistance. In this study, we aimed to identify which metabolic changes and downstream pathways are involved in myeloma cell growth and persistence. Methods Correlation of pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) with overall survival was investigated in the gene-expression data of MM patients (MMRF CoMMpass trial). To perform a tracer study, RPMI-8226 cells were supplemented with 13C-glutamine for 48h in both normoxia and hypoxia ( Results Pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) are 2 mitochondrial enzymes that facilitate the last step in the enzymatic conversion of glutamine to proline. High expression of both enzymes correlated with a lower overall survival in the CoMMpass trial. Moreover, MM cells from relapse/refractory patients expressed significant higher levels of PYCR1. We performed a tracer study with RPMI-8226 cells, revealing an increased conversion of 13C-glutamine to proline in hypoxia compared to normoxia. We confirmed these results by increased proline production after 48h of hypoxic culture. SiRNA-mediated knockdown of PYCR1 or both PYCR1/2 combined with bortezomib increased apoptotic cell death in OPM-2 and RPMI-8226, which we confirmed by detecting upregulation of cleaved PARP and cleaved CASPASE 3 levels. In contrast, PYCR2 knockdown combined with bortezomib did not significantly alter apoptosis. Further investigation revealed that PYCR1 knockdown reduced proliferation, and led to a decrease in p-AKT, p-p42/44 MAPK and c-MYC levels. Mechanistically, we found that PYCR1 silencing affected protein synthesis, as shown by a downregulation of p-PRAS40, p-MTOR, p-p70, p-S6, p-4EBP1 and p-EIF4e levels. Next, we evaluated whether the clinically relevant anti-hypertensive agent and PYCR1 inhibitor, pargyline, was capable of inducing myeloma cell death. In vitro, pargyline reduced proline production, MM viability and increased apoptotic cell death. Pargyline was also capable of reducing viability in CD138+ cells of primary patient samples . Finally, in vivo combination of pargyline with bortezomib significantly reduced tumor burden in the 5TGM1 model. On protein level, we also observed a significant decrease in p-4EBP1 and p-EIF4e in the freshly isolated 5TGM1 cells for the combination therapy. Conclusion Hypoxia increased glutamine-to-proline conversion in myeloma cells by stimulating PYCR activity. Knockdown of PYCR1 and PYCR1/2 increased bortezomib efficacy and inhibited proliferation. Mechanistically, PYCR1 interference reduced PRAS40-mediated protein synthesis. Pargyline, a PYCR1 inhibitor, also reduced MM viability and increased apoptosis. In vivo, pargyline combined with bortezomib significantly reduced tumor burden in the 5TGM1 model compared to both single agents. In conclusion, this study identifies PYCR1 as a novel target in MM therapy. Disclosures De Veirman: Active Biotech AB: Research Funding. OffLabel Disclosure: Pargyline is a antihypertensive agent and irreversible MAO B inhibitor that also inhibits PYCR1. Pargyline is not approved by the FDA as a PYCR1 inhibitor.

International audience; Multiple Myeloma (MM) is an incurable malignancy of terminally differentiated plasma cells, which are predominantly localized in the bone marrow. Myeloid-derived suppressor cells (MDSC) are described to promote MM progression by immunosuppression and induction of angiogenesis. However, their direct role in drug resistance and tumor survival is still unknown. In this study, we performed co-culture experiments of myeloma cells with 5TMM derived MDSC in vitro, leading to increased survival and proliferation of MM cells. Co-culture experiments resulted in MDSC-induced AMPK phosphorylation in MM cells, which was associated with an increase in the anti-apoptotic factors MCL-1 and BCL-2, and the autophagy-marker LC3II. In addition, 5TMM cells inoculated in mice showed a clear upregulation of AMPK phosphorylation in vivo. Targeting the AMPK pathway by Compound C resulted in apoptosis of human myeloma cell lines, primary MM cells and 5TMM cells. Importantly, we observed that the tumor-promoting effect of MDSC was partially mediated by AMPK activation. In conclusion, our data clearly demonstrate that MDSC directly increase the survival of MM cells, partially through AMPK activation, identifying this pathway as a new target in the treatment of MM patients.

RAS mutations occur frequently in multiple myeloma (MM), but apart from driving progression, they can also stimulate antitumor effects by activating tumor-suppressive RASSF proteins. Although this family of death effector molecules are often silenced in cancers, functional data about RASSF proteins in MM are lacking. Here, we report that RASSF4 is downregulated during MM progression and correlates with a poor prognosis. Promoter methylation analysis in human cell lines revealed an inverse correlation between RASSF4 mRNA levels and methylation status. Epigenetic modulating agents restored RASSF4 expression. Enforced expression of RASSF4 induced G2-phase cell-cycle arrest and apoptosis in human cell lines, reduced primary MM cell viability, and blocked MM growth in vivo. Mechanistic investigations showed that RASSF4 linked RAS to several pro-death pathways, including those regulated by the kinases MST1, JNK, and p38. By activating MST1 and the JNK/c-Jun pathway, RASSF4 sensitized MM cells to bortezomib. Genetic or pharmacological elevation of RASSF4 levels increased the anti-MM effects of the clinical relevant MEK1/2 inhibitor trametinib. Kinome analysis revealed that this effect was mediated by concomitant activation of the JNK/c-Jun pathway along with inactivation of the MEK/ERK and PI3K/mTOR/Akt pathways. Overall, our findings establish RASSF4 as a tumor-suppressive hub in MM and provide a mechanistic rationale for combining trametinib with HDAC inhibitors or bortezomib to treat patients with tumors exhibiting low RASSF4 expression. Significance: These findings provide a mechanistic rationale for combining trametinib with HDAC inhibitors or bortezomib in patients with multiple myeloma whose tumors exhibit low RASSF4 expression. Cancer Res; 78(5); 1155–68. ©2017 AACR.

Proper cell cycle progression through the interphase and mitosis is regulated by coordinated activation of important cell cycle proteins (including cyclin-dependent kinases and mitotic kinases) and several checkpoint pathways. Aberrant activity of these cell cycle proteins and checkpoint pathways results in deregulation of cell cycle progression, which is one of the key hallmarks of cancer. Consequently, intensive research on targeting these cell cycle regulatory proteins identified several candidate small molecule inhibitors that are able to induce cell cycle arrest and even apoptosis in cancer cells. Importantly, several of these cell cycle regulatory proteins have also been proposed as therapeutic targets in the plasma cell malignancy multiple myeloma (MM). Despite the enormous progress in the treatment of MM the past 5 years, MM still remains most often incurable due to the development of drug resistance. Deregulated expression of the cyclins D is observed in virtually all myeloma patients, emphasizing the potential therapeutic interest of cyclin-dependent kinase inhibitors in MM. Furthermore, other targets have also been identified in MM, such as microtubules, kinesin motor proteins, aurora kinases, polo-like kinases and the anaphase promoting complex/cyclosome. This review will provide an overview of the cell cycle proteins and checkpoint pathways deregulated in MM and discuss the therapeutic potential of targeting proteins or protein complexes involved in cell cycle control in MM.

The anaphase promoting complex/cyclosome (APC/C) is an ubiquitin ligase involved in cell cycle. During the metaphase-anaphase transition the APC/C is activated by Cdc20. The aim of this study is to elucidate the importance and therapeutic potential of APC/C and its co-activator Cdc20 in multiple myeloma (MM). Gene expression analysis revealed that Cdc20 was expressed at higher levels in gene expression-based high-risk MM patients. Moreover, high Cdc20 expression correlated with poor prognosis. Treatment of human myeloma cell lines with proTAME, an APC/C inhibitor, resulted in an accumulation of APC/CCdc20 substrate cyclin B1 and an accumulation of cells in metaphase. Moreover we observed a significant dose-dependent decrease in viability and increase in apoptosis in MM cells upon proTAME treatment. The induction of apoptosis was accompanied with caspase 3, 8, 9 and PARP cleavage. A similar metaphase arrest and induction of apoptosis were obtained with specific knockdown of Cdc20. In addition, we demonstrated the accumulation of Bim was partially responsible for the observed cell death. Combining proTAME with another APC/C inhibitor apcin or the alkylating agent melphalan resulted in enhanced anti-MM activity. This study suggests that the APC/C and its co-activator Cdc20 could be a new and promising target especially in high-risk MM patients.

Multiple myeloma (MM) is a genetic highly heterogeneous plasma cell malignancy. In MM, the RAS/MAPK pathway is the most frequently mutated pathway, leading to aberrant MEK/ERK and PI3K/Akt signaling and thus stimulating MM cell growth and survival. Increasing evidence indicates that next to their classical oncogenic effects, RAS proteins can also induce growth inhibitory effects and apoptosis. These tumor suppressive effects are mainly mediated through the Ras-Association Domain Family (RASSF), a group of 10 RAS effector proteins (RASSF1-10) and numerous isoforms. Based on the localization of the Ras-association (RA) domain this family is further subdivided into two groups. RASSF1 to 6 harbor a C-terminal RA domain and are termed the classical group, while RASSF7-10 contain a N-terminal RA domain and are referred to as non-classical RASSF proteins. The classical RASSF members also carry a SARAH domain on the C-terminus, allowing the RASSF proteins to bind each other and the mammalian sterile 20-like kinases MST and MST-2. Currently, no data about the biological role of RASSF proteins in the pathogenesis of MM is available. Here, we first investigated the expression of the classical RASSF proteins RASSF1-6 in MM using publically available gene expression profiling data of 2 independent newly diagnosed patient cohorts (namely the Heidelberg-Montpellier and the UAMS-TT2 cohort) and 1 cohort of relapsed patients (Mulligan-cohort). We report that RASSF4 was the only classical RASSF protein that was consistently found downregulated in MM cells compared to normal bone marrow plasma cells, correlating with a bad prognosis in all cohorts. Treatment with epigenetic modulating agents including histone deacetylase inhibitors (HDACi) and DNA methyltransferases inhibitors significantly increased RASSF4 expression both in vitro and in vivo, indicating that RASSF4 downregulation in MM is due to epigenetic silencing. Forced RASSF4 expression induced a strong G2-phase arrest and caspase-3 mediated apoptosis in human MM cell lines and strongly reduced the viability of primary CD138+ MM cells. Moreover, RASSF overexpression significantly reduced in vivotumor growth. In addition, we showed RASSF4-MST1 binding and the activation of the downstream signaling pathways JNK/Jun, p38 and p53. RNA sequencing and gene set enrichment analysis following forced RASSF4 expression furthermore revealed downregulation of genes involved in protein metabolism, the unfolded protein response and translation. RASSF4 overexpression also sensitized MM cells to the proteasome inhibitor bortezomib, the specific MEK1/2 inhibitor trametinib and the ROS inducer Prima-1Met. Consequently, combining trametinib with HDACi, e.g. panobinostat, resulted in very strong synergistic anti-MM effects. In conclusion, we identified RASSF4 as a new potent tumor suppressor in MM that is epigenetically silenced in malignant plasma cells and provide a rationale for testing novel therapeutic strategies enhancing RASSF4 expression (using HDACi or gene therapy) in combination with MEK/ERK inhibitors and bortezomib. Disclosures Hose: Takeda: Other: Travel grant; EngMab: Research Funding; Sanofi: Research Funding.

Perfluorooctanoic acid and perfluorooctane sulfonate were determined in the sediments from Winam Gulf, which is in the Kenyan side of Lake Victoria and in its source rivers. The sources of perfluorinated compounds within the Gulf of Lake Victoria have been identified and their levels determined for the first time, in this study, using SPE and HPLC-MS-MS analytical methodology. Variability in the concentrations of perfluorooctanoic acid and perfluorooctane sulfonate ranged from 1.4–99.1 and

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are manmade, stable perfluorosurfactants. The properties of perfluoroalkylated compounds that cause them to persist in the environment are also the properties that made them attractive compounds for industrial usage for over 50 years. Due to the unique properties of the carbon-fluorine bond and the polarity of perfluoroalkyl groups, potential substitutes to replace perfluorinated surfactants in most cases continue to be perfluoroalkyl based. Thus, issues of persistence in the environment remain. There is a need to test emerging new substitute surfactants for biodegradability. This study involved degradability measurements of emerging perfluorinated surfactant substitutes. The stability of the substitutes of perfluorinated surfactants was tested by employing advanced oxidation processes, which were based on degradation by ultraviolet lamp, hydrogen peroxide, or both, followed by conventional tests, among them an automated method based on the manometric respirometry test (OECD 301 F; OxiTop), closed-bottle test (OECD 301 D), and standardized fixed-bed bioreactor on perfluorobutane sulfonate, fluorosurfactant Zonyl, two fluoraliphatic esters (NOVEC FC-4430 and NOVEC FC-4432), and 10-(trifluoromethoxy) decane 1 sulfonate. Most of these new surfactants are well established in the marketplace and have been used in several applications as alternatives to PFOS- and PFOA-based surfactants. Ready biodegradation tests for fluoroaliphatic esters, the fluorosurfactant Zonyl, perfluorobutane sulfonate, and 10-(trifluoromethoxy) decane-1-sulfonate using the manometric respirometry test (OxiTop) did not meet the ready biodegradability test criteria. However, 10-(trifluoromethoxy) decane-1-sulfonate was observed to be degradable when a standardized fixed-bed bioreactor test was applied.

Unknown bisethylisooctanollactone isomers (BIOL isomers) which are chemical by-products of butyraldehyde synthesis, were isolated from industrial waste water applying various purification methods with subsequent semi-preparative high-performance liquid chromatography. Through interpretation of mass spectra after gas chromatographic separation the individual BIOL isomers were identified as stereoisomers of 2,4-diethyl-3-n-propyl-δ-valerolactone. Thus, it was possible for the first time to quantify the BIOL isomers in the river Rhine, Germany, with a mean sum concentration of 1.6 μg l−1. A regular analysis performed over a period of almost two years of the river Rhine always gave the same ratio among the individual isomers. Drinking water production out of such water was studied, revealing that activated carbon filtration led to a 95% reduction of the BIOL concentration. Additional subsoil passage and a subsequent slowsand filtration led to a total elimination due to microbial degradation. Even if the BIOL isomers proved not to be relevant to drinking water, their behavior in the aquatic environment needs to be more thoroughly investigated since these compounds have been discharged for many years in high amounts into the river Rhine.

Polar organic contaminants in surface water for drinking water purposes are relevant if they are poorly degradable, and analyses of such compounds are challenging because they are extremely water soluble. The problem is enhanced with more polar and unknown metabolites such as Sarkosin-N-(phenylsulfonyl) (SPS) which was detected in the Rhine river constantly in concentrations between 0.5 and 2 μg/L. The ubiquitous presence of SPS in the water was initially hypothesized to be due to the microbial degradation in sewage plants of 6-[methyl(phenylsulfonyl)amino]hexanoic acid (HPS) which is extensively used in Germany as a corrosion inhibiting agent. Soil suspension as well as closed-bottle degradation experiments with sewage water confirmed this hypothesis. Immobilized cell systems simulated the behavior in underground passage. HPS was totally degraded within hours, while SPS and other metabolites such as 4-[methyl(phenylsulfonyl)amino]butanoic acid (BPS) and methyl(phenylsulfonyl)amide (MPS) were identified. ...

The coupling of ion chromatography (IC) with electrospray mass spectrometry (ES-MS) opens new ways for the determination of polar organic micropollutants in water samples. The technique of conductivity suppression has been found to reduce the background signal in the range of about two-orders of magnitude leading to a significant increase in sensitivity. In addition, the formation of salt adducts has been avoided. The usefulness of this method was proven on several polar and environmentally relevant micropollutants such as the herbicide glyphosate and its metabolite aminomethylphosphonic acid (AMPA), the chelating agent ethylenediamine tetraacetate (EDTA) and diacetonketogulonic acid (DAG). This present study has shown that IC–ES-MS is a simple, sensitive and quick method for the determination of these polar organic traces in water samples after separation on an anion-exchange column without any derivatization. In this work, several possibilities of applications of IC–ES-MS (with varying conditions) are presented. Analysis of glyphosate, AMPA, DAG and EDTA in ground and surface waters has been achieved by IC–ES-MS without additional sample preparation at a concentration level of 1 μg/l.

The discovery of novel agents such as the proteasome inhibitor bortezomib has significantly increased the survival of multiple myeloma (MM) patients. However MM remains an incurable disease mainly due to relapse, associated with significant resistance to therapy including bortezomib. Therefore further investigation to elucidate the disease and the mechanisms leading to drug resistance is necessary. The success of bortezomib highlights the importance of the ubiquitin-proteasomal system (UPS) in MM. The UPS regulates protein turnover and plays a key role is several cellular processes such as apoptosis, cell cycle progression, cell proliferation and DNA replication. The Anaphase Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase protein complex involved in controlling cell cycle progression. The regulation of APC/C is dependent on 2 co-activators: Cdc20 and Cdh1. The APCCdc20 complex is controlling the metaphase to anaphase transition in mitosis, while APCCdh1 controls mitotic exit and early G1 phase. During metaphase, the activity of APCCdc20 is inhibited by the spindle assembly checkpoint. When all kinetochores are properly attached, the spindle assembly checkpoint is silenced and APCCdc20 becomes activated. When APCCdc20is active, cell cycle proteins are targeted for degradation by the proteasome such as securin and cyclin A and B leading to mitotic exit. Recent studies described that spindle assembly checkpoint is defective in MM cells and that patient samples after chemotherapy and at relapse displayed an increased chromosomal instability signature including Cdc20. The aim of our study is to elucidate the importance and therapeutic potential of APC/C and its co-activators Cdc20 and Cdh1 in MM. Analysis of gene expression in the data of Zhan et al. (Blood 108, 2020-8, 2006) revealed that the co-activator Cdc20 was higher expressed in certain MM sub-groups (PR, MS, CD1, MF) compared to healthy bone marrow plasma cells. Moreover, high Cdc20 expression is correlated with poor prognosis. Cdh1 on the other hand was significantly lower expressed in all MM sub-groups compared to healthy bone marrow plasma cells. Interestingly, lower Cdh1 expression is correlated with poor prognosis. Next, we analyzed whether blocking APC/C would affect MM cells. For this study the pro-drug of TAME (tosyl-L-arginine methyl ester) that has been described as an inhibitor of the APC/C, was used. When the human myeloma cell lines LP-1 and RPMI-8226 were treated with proTAME, an accumulation of the APCCdc20 substrate cyclin B1 was seen already after 6 hours. However the levels of Skp2, an APCCdh1 substrate, were not affected by proTAME treatment. This suggests that proTAME inhibits the APCCdc20 complex but not the APCCdh1complex. We morphologically assessed the effect on number of metaphases on May-Grünwald Giemsa stained cytospins. ProTAME clearly induced an accumulation of LP-1 and RPMI-8226 cells in metaphase. Since a metaphase arrest can lead to cell death, we investigated the effect of proTAME on the viability and apoptosis. A significant dose-dependent decrease in viability and increase in apoptosis was observed after treatment with proTAME of human myeloma cell lines and primary MM cells purified from human and 5T33MM diseased mice. In contrast, other cells from the bone marrow microenvironment were not affected upon proTAME treatment. The induction of apoptosis was accompanied with caspase 3, 8, 9 and PARP cleavage. Western Blot analysis also showed phosphorylation of H2AX suggesting DNA damage upon proTAME treatment. Previous studies showed that MM is a heterogeneous disease consisting of a bulk CD138+ population and a minor CD138- population which is less sensitivity to drugs such as bortezomib. Interestingly, treatment of CD138+/- 5T33MM cells with proTAME demonstrated an equal targeting of both populations. From these results we can conclude that overexpression of Cdc20 by MM cells is correlated with a bad prognosis. Inhibition of APCCdc20 results in a metaphase arrest in MM cells which is associated with reduced viability and induction of apoptosis. Moreover, APC/C inhibition equally targets CD138+ and the more resistant CD138- 5T33MM cells. This study suggests that APC/C and its co-activator Cdc20 could be a new and promising target in MM. Disclosures No relevant conflicts of interest to declare.