Antitumor Activity of a Novel Proteasome Inhibitor BSc2118 Against Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5725-5725
Author(s):  
Meirong Zang ◽  
Lanting Liu ◽  
Xin Li ◽  
Wei Li ◽  
Ulrike Kuckelkorn ◽  
...  
Keyword(s):  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Molecular Mechanisms ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Proteasome Activity ◽  
Autophagy Flux ◽  
Ubiquitinated Proteins ◽  
Rpmi 8226

Abstract Background: Although the first generation of proteasome inhibitor bortezomib is very effective, the development of resistance limits it long-term utility. In this study, we explored the efficiency and molecular mechanisms of the novel, irreversible proteasome inhibitor BSc2118, particularly, the reversal of bortezomib resistance. Materials and Methods: Human MM cell lines (MM.1S, MM.1R, RPMI-8226, U266, NCI-H929) were treated with BSc2118 at various concentrations for 48h, and assessment for cell viability by CCK-8 assay. MM.1S and MM.1R cells were treated with BSc2118 for 24 hours, and cell cycle and apoptosis analysis were conducted by flow cytometry. Associated molecules were detected by qRT-PCR and western blot. Chymotrypsin-like proteasome activity assay was performed by using the 20S proteasome assay kit. Ubiquitinated proteins were isolated and determined with ubiquitin enrichment kit. Results: Our results revealed that treatment of MM cell lines with BSc2118 inhibits the chymotrypsin-like proteasome activity and induces accumulation of ubiquitinated proteins. BSc2118 inhibits MM cell growth and induces MM apoptosis via induction of G2/M phase arrest, activation of cleaved caspase-3, caspase-8 and caspase-9 and PARP, increasing p53, p21 and E2F1, and inhibition of autophagy in MM.1S, MM.1R and RPMI-8226 cell lines. In addition, BSc2118 dramatically inhibits cytokines mRNA, such as IL-6, VEGF and bFGF in both myeloma cells line and primary bone marrow stromal cells from myeloma patients. More importantly, BSc2118 could overcome bortezomib resistance in vitro by using primary CD138 positive plasma cells from bortezomib-resistant myeloma patients and bortezomib resistance cell line ANBL-6 (ANBL-6.BR), most likely as the consequence of inhibition of autophagy flux which is responsible for bortezomib resistance. Conclusion: Our study revealed BSc2118, a novel irreversible proteasome inhibitor, exerts anti-MM effect, mainly through activation of caspase pathway and inhibition of basal autophagy. It is of great importance that BSc2118 could overcome bortezomib resistance via inhibition of autophagy flux. A head to head of BSc2118 versus Bortezomib is performing in human plasmacytoma xenograft tumor model to evaluate drug safety, anti-tumor efficiency, in particular, reversal of bortezomib resistance. Our preclinical study supports clinical evaluation of BSc2118, particularly, overcomes bortezomib resistance, as a potential MM therapy. Disclosures No relevant conflicts of interest to declare.

ABT-737, an Inhibitor of Bcl-2/Bcl-XL Proteins, Is Cytotoxic in Multiple Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1589-1589
Author(s):  
Michael Kline ◽  
Terry Kimlinger ◽  
Michael Timm ◽  
Jessica Haug ◽  
John A. Lust ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Tumor Microenvironment ◽  
Cell Lines ◽  
Plasma Cells ◽  
Annexin V ◽  
Significant Activity ◽  
Rpmi 8226 ◽  
Dose Dependent

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that is incurable with the currently available therapeutics. New therapies based on better understanding of the disease biology are urgently needed. MM is characterized by accumulation of malignant plasma cells predominantly in the bone marrow. These plasma cells exhibit a relatively low proliferative rate as well as a low rate of apoptosis. Elevated expression of the anti-apoptotic Bcl-2 family members has been reported in MM cell lines as well as in primary patient samples and may be correlated with disease stage as well as resistance to therapy. ABT-737 (Abbott Laboratories, Abbott Park, IL) is a small-molecule inhibitor designed to specifically inhibit anti-apoptotic proteins of the Bcl-2 family and binds with high affinity to Bcl-XL, Bcl-2, and Bcl-w. ABT-737 exhibits toxicity in human tumor cell lines, malignant primary cells, and mouse tumor models. We have examined the in vitro activity of this compound in the context of MM to develop a rationale for future clinical evaluation. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity of ABT-737 was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Flow cytometry was also used to measure BAX: Bcl-2 ratios after ABT-737 treatment and cell permeabilization with FIX & PERM (Caltag Laboratories, Burlingame, CA) Results: ABT-737 exhibited cytotoxicity in several MM cell lines including RPMI 8226, KAS-6/1, OPM-1, OPM-2, and U266 with an LC50 of 5-10μM. The drug also had significant activity against MM cell lines resistant to conventional agents such as melphalan (LR5) and dexamethasone (MM1.R) with similar LC50 (5-10 μM), as well as against doxorubicin resistant cells (Dox40), albeit at higher doses. Furthermore, ABT-737 retained activity in culture conditions reflective of the permissive tumor microenvironment, namely in the presence of VEGF, IL-6, or in co-culture with marrow-derived stromal cells. ABT-737 was also cytotoxic to freshly isolated primary patient MM cells. Time and dose dependent induction of apoptosis was confirmed using Annexin V/PI staining of the MM cell line RPMI 8226. Flow cytometry analysis of cells treated with ABT-737 demonstrated a time and dose dependent increase in pro-apoptotic BAX protein expression without significant change in the Bcl-XL or Bcl-2 expression. Ongoing studies are examining the parameters and mechanisms of ABT-737 cytotoxicity to MM cells in more detail. Conclusion: ABT-737 has significant activity against MM cell lines and patient derived primary MM cells in vitro. It is able to overcome resistance to conventional anti-myeloma agents suggesting a different mechanism of toxicity that may replace or supplement these therapies. Additionally, it appears to be able to overcome resistance offered by elements of the tumor microenvironment. The results of these studies will form the framework for future clinical evaluation of this agent in the clinical setting.

Proteasome Activity As a Biomarker in Multiple Myeloma (MM): In MM Cells Treated with Bortezomib (BTZ) or Epoxomicin (EPX) Caspase 3/7 Activation Is Inversely Related to Baseline β1 and β5 Activity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1431-1431
Author(s):  
Ping Zhou ◽  
Xun Ma ◽  
Raymond L. Comenzo
Keyword(s):  
Cell Lines ◽  
Dose Level ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
High Reliability ◽  
Proteasome Activity ◽  
Fluorescent Intensity ◽  
Apoptotic Signaling ◽  
The Mean ◽  
Rpmi 8226

Abstract Abstract 1431 An irony of the era of increased knowledge of molecular and genetic aspects of MM, and of improved MM patient survival with proteasome inhibitor therapy (PSI), is that the sensitivity of MM cells to PSI is not related to specific addictive oncogenic abnormalities but rather to generic plasma-cell dependence on the ubiquitin-proteasome system. MM patients develop resistance to PSI, however, and a better understanding of factors that modulate sensitivity and resistance to PSI is needed to improve therapies. We hypothesized that in MM cells the differences in baseline levels of proteasome activity inversely correlated with activation of apoptosis in response to PSI, as has been suggested by qualitative experimental evidence (Blood 2009;113: 3040). A corollary of this hypothesis is that proteasome activity in MM patient cells may provide a biomarker allowing customized therapy. We characterized the chymotrypsin-like (β5), trypsin-like (β2) and caspase-like (β1) activity of five MM cell lines (MM1S, MM1R, ALMC1, KMS12-BM and RPMI 8226) using the site-specific peptide substrates Suc-LLVY-aminoluciferin (AML) (β5), Z-LRR-AML (β2) and Z-nLPnLD -AML (β1) in buffers optimized for cell permeabilization and for proteasome and luciferase activity (Promega) following manufacturer's instructions with read-out in relative luminescent units (RLU) (minus control). We also characterized intracellular proteasome-directed and signaling-related ubiquitinated proteins in these cell lines by flow cytometry with anti-ubiquitin lysine48- and lysine63-specific monoclonal antibodies (Millipore). Read-out was mean fluorescent intensity minus isotype control (MFI). Cells were cultured for 24 hours with increasing doses of BTZ or EPX (0, 3.125, 6.25, 12.5 and 25nM) and evaluated for caspase 3/7 activation in a cell-based luminescence assay (Promega). Read-out was the mean RLU of each dose level minus the mean baseline RLU. We used 104 cells per well in all luminescence assays and all experiments were performed in triplicate for each situation on 3 different days. Statistical analyses were performed with GraphPad PRISM. The differences in baseline β1, β2 and β5 activity among these cell lines were significant by unpaired t test (P <0.05) in 52/60 comparisons and trended to significance in 4/60 (P ≤0.10). RPMI8226 cells had the highest and MM1S/R the lowest levels of overall activity while KMS12-BM and ALMC1 were intermediate. The coefficients of variation for each assay were 0.10+/−0.02 (β1), 0.2+/−0.05 (β2) and 0.17+/−0.04 (β5) (means+/−SE). The lys48 MFI was highest for MM1S/R and lowest for KMS12-BM and RPMI 8226 while lys63 MFI was highest for KMS12-BM and ALMC1 and lowest for MM1S/R. The caspase 3/7 dose-response curves with BTZ are shown in Figure 1 and were comparable for EPX. For BTZ and EPX the 12.5nM dose level was the point at which slopes were maximal. We used linear regression calculating caspase 3/7 activity with 12.5nM BTZ or EPX as a function of baseline β1, β2, β5 and both MFI, and obtained non-significant results for β2 and both MFI but significant results for β1 (r2 0.92, 0.82; P <0.01, 0.03) and β5 activity (r2 0.76, 0.64; P 0.05, 0.11). Figure 2 shows the typical inverse relationship with 95% CI. We used multivariate regression analysis evaluating baseline β1, β2, β5 and both MFI as predictors of caspase 3/7 activity at 12.5nM BTZ or EPX and identified baseline β1 activity as the only significant predictor of outcome. We conclude from these results that in MM baseline β1 and β5 activity affect the level of apoptotic signaling in response to PSI. Of note, these results highlight the importance of β1 activity in modulating apoptotic signaling, downplay the significance of proteasome load, and also, given the CV measures, identify a need for further refinement of these assays, perhaps with the inclusion of proteasome 20S controls, in order to apply them with high reliability to the assessment of CD138+ patient MM samples. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Heat Shock Protein 70 Inhibitor, Alone or in Combination with Bortezomib, Prevented Plasmacytoma Development in Immunodeficient Mice Transplanted with Myeloma Cell Lines

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5658-5658
Author(s):  
Mariana Bleker de Oliveira ◽  
Angela Isabel Eugenio ◽  
Veruska Lia Fook Alves ◽  
Daniela Zanatta ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Cell Line ◽  
Tumor Cells ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Immunodeficient Mice ◽  
Late Apoptosis

Abstract Introduction: HSP70 has an integrative role in protein degradation due to the interaction with many pathways, such as ubiquitin proteasome (UPS), unfolded protein response (UPR) and autophagy. In multiple myeloma (MM) HSP70 is overexpressed and helps to prevent proteotoxic stress and cell death caused by overload of unfolded/misfolded proteins produced by tumor cells. Aims: To explore the role of HSP70 inhibition, isolated or in association with proteasome inhibitor, as therapeutic strategy for MM through in vitro and in vivo analyses. Methods: RPMI8226-LUC-PURO and U266-LUC-PURO bioluminescent cell lines were treated with HSP70 inhibitor (VER155008- 50 μM or 80μM) and proteasome inhibitor (bortezomib 100nM) for evaluation of apoptosis induction by flow cytometry using annexin V and propidium iodide. NOD.Cg-rkdcscid Il2rgtm1Wjl/SzJ immunodeficient mice were used for plasmacytoma xenograft model and treated with intravenous VER155008 (40mg/kg) and bortezomib (1mg/kg), immediately after transplant of RPMI8226-LUC-PURO and U266-LUC-PURO bioluminescent cell lines (N=3 for each group, including controls, bortezomib, VER155008, and combination of bortezomib and VER155008). Bioluminescence was measured in IVIS Kinetic (Capiler Life Science) once a day for seven days. Results: Bortezomib used as single treatment was able to induce apoptosis in RPMI8226-LUC-PURO cell line: the best result for in vitro studies RPMI8226-LUC-PURO was 65% of late apoptosis after treatment with bortezomib. On the other hand, U266-LUC-PURO cell line presented higher percentage of apoptosis when treated with bortezomib and VER155008 combination: U266-LUC-PURO cell line presented more than 60% of late apoptosis after VER155008 (80μM) combined with bortezomib, showing that inhibition of HSP70 could overcome U266-LUC-PURO resistance to bortezomib alone. Mice treated with VER155008, alone or in combination with bortezomib, showed complete inhibition of tumor growth (absence of bioluminescence) for both cell lines when compared with control group after one week of treatment (p<0.001, Two-way ANOVA). Therefore, in vivo studies using mice treated with VER155008, alone or in combination with bortezomib, prevented tumor development after one week of treatment, independent of the cell line used in the xenotransplant. Conclusion: Our study shows that HSP70 and proteasome inhibitors combination induced apoptosis in tumor cells in vivo for both MM cell lines. Since HSP70 is overexpressed in MM and connects several signaling pathways that maintain cell survival, such as UPS, UPR and autophagy, it can represent a key role to establish a new approach for the treatment of MM. Financial support: FAPESP 2010/17668-6 and CNPq (155272/2013-6). UNIFESP Ethics Committee (0219/12). Disclosures No relevant conflicts of interest to declare.

RFWD2 Induces Cellular Proliferation and Proteasome Inhibitor Resistance By Mediating p27 Ubiqutinaiton in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3068-3068
Author(s):  
Ye Yang ◽  
Mengjie Guo ◽  
Chunyan Gu
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Drug Resistance ◽  
Proteasome Inhibitor ◽  
Cellular Proliferation ◽  
Conflicts Of Interest ◽  
Scid Mice ◽  
Rpmi 8226

Purpose: In recent years, with the emergence of targeted proteasome inhibitors (PIs), the treatment of multiple myeloma (MM) has made great progress and significantly improves the survival rate of patients. However, MM remains an incurable disease, mainly due to the recurrence of drug resistance. The constitutive photomorphogenic 1 (RFWD2, also known as COP1), is closely related to the occurrence and development of tumors, but its role in MM is largely unknown. This study was aimed to explore the mechanism of RFWD2 on cell proliferation and resistance to proteasome inhibitor in MM. Experimental Design: Using gene expression profiling (GEP) samples, we verified the relation of RFWD2 to MM patients' survival and drug-resistance. The effect of RFWD2 on cell proliferation was confirmed by MTT and cell cycle analysis in RFWD2-overexpressed and RFWD2-knockdown MM cells. MTT and apoptosis experiments were performed to evaluate whether RFWD2 influenced the sensitivity of MM cells to several chemotherapy drugs. MM xenografts were established in immunodeficient NOD/SCID mice by injecting wild-type or RFWD2 over-expression MM cells with drug intervention. The mechanism of drug resistance was elucidated by analyzing the association of RFWD2 with E3 ligase of p27. Bortezomib-resistant RPMI 8226 cells were used to construct RFWD2 knockdown cells, which were injected into NOD/SCID mice to assess the effect of RFWD2 on bortezomib resistance in vivo. Results: RFWD2 expression was closely related to poor outcome, relapse and bortezomib resistance in MM patients' GEP cohorts. Elevated RFWD2 induced cell proliferation, while decreased RFWD2 inhibited cell proliferation and induced apoptosis in MM cells. RFWD2-overexpression MM cells resulted in PIs resistance, however, no chemotherapy resistance to adriamycin and dexamethasone was observed in vitro. In addition, overexpressing RFWD2 in MM cells led to bortezomib resistance rather than adriamycin resistance in myeloma xenograft mouse model. RFWD2 regulated the ubiquitination degradation of P27 by interacting with RCHY1 ubiquitin ligase. The knockdown of RFWD2 in bortezomib-resistant RPMI 8226 cells overcame bortezomib resistance in vivo. Conclusions: Our data demonstrate that elevated RFWD2 induces MM cell proliferation and resistance to PIs, but not to adriamycin and dexamethasone both in vitro and in vivo through mediating the ubiquitination of p27. Collectively, RFWD2 is a novel promising therapeutic target in MM. Disclosures No relevant conflicts of interest to declare.

An Investigational Proteasome Inhibitor MLN9708 (MLN2238) Induces Apoptosis In Human Multiple Myeloma Cells In Vitro

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3066-3066
Author(s):  
Aisha Masood ◽  
Kasyapa Chitta ◽  
Kiersten M Miles ◽  
Nazmul H Khan ◽  
Remi Adelaiye ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Conflicts Of Interest ◽  
Active Form ◽  
Biologically Active ◽  
Mitochondrial Outer Membrane ◽  
Dependent Manner ◽  
In Vivo Models

Abstract Abstract 3066 Targeting the proteasome has proven to be one of the most effective therapeutic strategies in the treatment of multiple myeloma (MM), and the proteasome inhibitor bortezomib is approved for treatment of MM. However its clinical efficacy is compromised by the acquired resistance in patients, necessitating the development of new therapeutics. Several new proteasome inhibitors are under investigation for their therapeutic efficacy in MM. MLN9708 (Millennium Pharmaceuticals, Inc., Cambridge, MA) is a proteasome inhibitor which shows refined pharmacokinetic and pharmacodynamic properties in preclinical studies and is currently in Phase I clinical development. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to MLN2238, the biologically active form. MLN2238 was used for all of the studies reported here, in which we report the efficacy of MLN2238 on three established MM cell lines-KMS11, OPM2 and U266. MLN2238 was found to inhibit the chymotrypsin-like proteasomal activity of all MM cell lines in a dose dependent manner. Investigation of the IC50 of MLN2238 on these cell lines demonstrated that KMS11 is the most sensitive (IC50 of 15.9 nM) while U266 was found to be the least sensitive cell line (IC50 of 511 nM). OPM2 cells also showed intermediate sensitivity with an IC50 of 58.6 nM. MLN2238 induced apoptosis in KMS11 cells as evidenced by annexin V staining and PARP-1 cleavage. Cleavage of caspases 9 and 3 suggested activation of the intrinsic apoptotic pathway by MLN2238. Furthermore, MLN2238 treatment was shown to increase the mitochondrial outer membrane permeability (MOMP) and decrease BCL-2 levels. Evaluation of the expression of PSMB5, the preferred proteasomal subunit target for both bortezomib and MLN2238, revealed that it is expressed at approximately 3 fold more in KMS11 cells as compared to U266, suggesting a possible mechanism for higher sensitivity of KMS11 to the proteasomal inhibitor, MLN2238. This preclinical evaluation confirms the anti-myeloma effects of MLN2238, warranting further in-depth evaluation in both in vitro and in vivo models of MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Deptor Inhibitor Induces Its Degradation with Resulting Anti-Myeloma Cytotoxicity in Vitro and In Vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1821-1821
Author(s):  
Mario I Vega ◽  
Yijiang Shi ◽  
Patrick Frost ◽  
Sara Huerta-Yepez ◽  
Alan Lichtenstein
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Cytotoxic Effect ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Inhibition Of Proliferation ◽  
Induction Of Apoptosis

Multiple myeloma (MM) is a hematological disorder characterized by a proliferation of malignant monoclonal plasma cells in the bone marrow (BM) and / or in extramedullary sites. Despite recent progress in OS rates, MM remains an incurable disease and most patients will relapse and require treatment. Deptor is a component of mTOR complexes and a constitutive inhibitor of their activities. It is known that the inhibition of Deptor results in the inhibition of the proliferation and induction of apoptosis in MM cells. In addition, high levels of Deptor are predictive of a poor response to conventional therapies, indicating that Deptor expression are important as a prognostic marker for patients with myeloma and is a possible therapeutic target. Our group previously identified a drug which prevents mTOR-Deptor binding (NSC126405) and induces cellular cytotoxicity in MM (Shi Y, et al 2016). In this study, we developed a new related chemical inhibitor (43 M) capable of inducing the inhibition of the mTOR / Deptor interaction and results in the negative regulation of Deptor that leads to the inhibition of proliferation and induces apoptosis in several MM cell lines. The cytotoxic effect of 43 M is not dependent of caspase activation and induces the activation of p70 and AKT (T308). This leads to the induction of apoptosis in MM cell lines and tumor cells derived from MM patients. The degradation of Deptor induced by 43 M is dependent on the proteasome complex since it was prevented in the presence of MG132. In vivo, 43 M prevents the expression of Deptor in a xenograft tumor, and delayed tumor growth and interestingly, induces the eradication of tumors in 40% of mice in a murine model of MM, without significant toxic implications. Recent studies show that Deptor expression protects MM cells against Bortezomib treatment, suggesting that anti-Deptor drugs can synergize with proteasome inhibitors (PIs). However, the combination of 43 M + Bortezomib was not synergistic, and was antagonistic in vitro. These results are probably due to the prevention of the proteasomal degradation of Deptor, suggesting a possible use of the 43 M inhibitor in MM in the absence of the current PIs. This study describes for the first time the possible role of Deptor as a therapeutic target using a chemical inhibitor capable of degrading and inducing a cytotoxic effect in MM cell lines. In addition, Deptor is reported as an important therapeutic target in an in vivo MM model. Shi Y, Daniels-Wells TR, Frost P, Lee J, Finn RS, Bardeleben C, Penichet ML, Jung ME, Gera J, Lichtenstein A. Cytotoxic Properties of a DEPTOR-mTOR Inhibitor in Multiple Myeloma Cells. Cancer Res. 2016 Oct 1;76(19):5822-5831 Disclosures No relevant conflicts of interest to declare.

A Novel Role for Histone Deacetylase 11 (HDAC11) in B Cell Lymphopoiesis and Plasma Cell Survival in Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4715-4715
Author(s):  
Jason B. Brayer ◽  
Eva Sahakian ◽  
John Powers ◽  
Mark B Meads ◽  
Susan Deng ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Proteasome Inhibitors ◽  
Myeloma Cell ◽  
Resistant Cell ◽  
Rpmi 8226 ◽  
Human Myeloma Cell

Abstract While multiple myeloma (MM) remains incurable presently, expanded therapeutic options over the past decade have improved patient survival markedly. Proteasome inhibitors have redefined the treatment paradigm for myeloma, often serving as the backbone of front-line treatment. Histone deacetylase (HDAC) inhibitors (HDI), although only marginally active as single agent therapy in hematological malignancies, have demonstrated an ability to salvage bortezomib responsiveness in refractory patients, prompting heightened interest in this class of targeted therapeutics in myeloma. HDAC’s represent a family of enzymes, currently with 11 known members in the classical HDAC family, and subdivided into 4 sub-classes. HDAC11 is currently the only member of the sub-class IV and, as the newest member of the HDAC family, its impact on B cell lymphopoiesis and myeloma development is only starting to be unveiled. Intriguingly, we show that mice with germ-line silencing of HDAC11 (HDAC11KO mice) exhibit a 50% decrease in plasma cells in both the bone marrow and peripheral blood plasma cell compartments relative to wild-type mice. Consistent with this, Tg-HDAC11-eGFP mice, a transgenic strain engineered to express GFP under control of the HDAC11 promoter (Heinz, N Nat. Rev. Neuroscience 2001) reveals that HDAC11 expression is increased in the plasma cell population and to a lesser extent B1 B cells, as compared to earlier lineage stages. Similar observations based on measurements of HDAC11 mRNA were seen in normal human plasma cells. Significant increases in HDAC11 mRNA expression were observed in 7 of 11 primary human multiple myeloma samples and 11 of 12 human myeloma cell lines as compared to normal plasma cells, further emphasizing the potential relevance of HDAC11 to the underlying pathologic processes driving myeloma development and/or survival. Targeted silencing of HDAC11 in RPMI-8226 cells lines using siRNA results in a modest decrease in cell viability as measured by Annexin/PI staining and detection of activated caspase-3. Quisinostat, a second generation pan-HDI, has previously demonstrated activity against human myeloma cell lines in vitro (Stuhmer, Brit J Haematol, 2010), and suppressed bone destruction in an in vivo murine myeloma model (Deleu, Cancer Res, 2009). We similarly observe dose-dependent survival impairment in 10 human myeloma cell lines when cultured in the presence of quisinostat, with EC50’s consistently in the 1-10nM range. Importantly, quisinostat acts synergistically with proteasome inhibitiors (bortezomib and carfilzomib) in RPMI-8226 cells; more importantly, the degree of synergism is amplified in the RPMI-6226-B25 bortezomib-resistant cell line. Although a clear mechanism of action remains to be elucidated, preliminary data suggests that RPMI-8226 cells exposed to quisinostat appear to exhibit a decrease nuclear, but not cytosolic HDAC11. Collectively, these data illustrate a previously unknown role for HDAC11 in plasma cell differentiation and survival. Increased HDAC11 expression seen in myeloma patient specimens and primary myeloma cell lines highlights the potential of HDAC11 as a therapeutic target. Furthermore, we show that quisinostat, a pan-HDI with selectivity towards HDAC11 at lower dosing, acts synergistically with proteasome inhibitors in vitro in proteasome inhibitor sensitive and resistant cell lines. Future work will focus on further elucidating the role of HDAC11 in myeloma survival and drug response, with particular emphasis on proteasome inhibitors. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Mucolipin TRPML2 Channel Enhances the Sensitivity of Multiple Myeloma Cell Lines to Ibrutinib and/or Bortezomib Treatment

Biomolecules ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 107
Author(s):  
Giorgio Santoni ◽  
Consuelo Amantini ◽  
Federica Maggi ◽  
Oliviero Marinelli ◽  
Matteo Santoni ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Plasma Cells ◽  
Biological Effects ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell ◽  
Bortezomib Treatment

Multiple myeloma (MM) is a haematological B cell malignancy characterised by clonal proliferation of plasma cells and their accumulation in the bone marrow. The aim of the present study is the evaluation of biological effects of Ibrutinib in human MM cell lines alone or in combination with different doses of Bortezomib. In addition, the relationship between the expression of TRPML2 channels and chemosensitivity of different MM cell lines to Ibrutinib administered alone or in combination with Bortezomib has been evaluated. By RT-PCR and Western blot analysis, we found that the Ibrutinib-resistant U266 cells showed lower TRPML2 expression, whereas higher TRPML2 mRNA and protein levels were evidenced in RPMI cells. Moreover, TRPML2 gene silencing in RPMI cells markedly reverted the effects induced by Ibrutinib alone or in combination with Bortezomib suggesting that the sensitivity to Ibrutinib is TRPML2 mediated. In conclusion, this study suggests that the expression of TRPML2 in MM cells increases the sensitivity to Ibrutinib treatment, suggesting for a potential stratification of Ibrutinib sensitivity of MM patients on the basis of the TRPML2 expression. Furthermore, studies in vitro and in vivo should still be necessary to completely address the molecular mechanisms and the potential role of TRPML2 channels in therapy and prognosis of MM patients.

scholarly journals JAM-A as a Prognostic Factor and New Therapeutic Target in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 307-307 ◽  
Author(s):  
Antonio Solimando ◽  
Andreas Brandl ◽  
Mattenheimer Katharina ◽  
Carolin Graf ◽  
Miriram Ritz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Healthy Subjects ◽  
Plasma Cells ◽  
Cell Membrane Protein ◽  
And Migration ◽  
Rpmi 8226

Abstract Cell adhesion in the multiple myeloma (MM) microenvironment is a mechanism by which MM plasma cells escape the effects of therapy and survive. To improve clinical strategies and overcome drug resistance, approaches directed to both MMPCs and bone marrow microenvironment are under investigation. Here, we examined the cell membrane protein Junctional adhesion molecule-A (JAM-A) as a clinical biomarker and novel therapeutic target for MM. We evaluated JAM-A expression by real time PCR (RT-PCR), flow cytometry and immunofluorescence microscopy in 132 MM patients at different stages and various MM cell lines. Next, we measured the concentrations of soluble JAM-A from MM and healthy subjects sera by enzyme linked immune assay (ELISA). We investigated JAM-A functionally in vitro and in vivo by transient gene silencing (siRNA) and with blocking antibodies. Patient-derived plasma cells (MMPCs) expressed increased JAM-A expression levels when compared to control PC from healthy individuals. Elevated JAM-A expression correlated with poor prognosis (Figure 1A,B). Furthermore, soluble JAM-A was significantly increased in MM patient sera when compared to healthy subjects. Additionally, MM cell lines showed high expression of both membrane and cytoplasmic JAM-A. Consequently, inhibition of JAM-A using specific siRNA treatment resulted in diminished tumorigenic potential, including decreased colony formation, chemotaxis and migration. Importantly, treatment of luciferase+RPMI-8226 MM bearing NSG with a JAM-A blocking monoclonal antibody reduced significantly MM progression and dissemination in vivo when compared to MM bearing mice that received an non-specific isotype control antibody (Figure 1C). Conclusively, our data suggest that JAM-A can serve as a biomarker of malignancy in MM patients. Soluble plasma JAM-A could contribute to serum-based clinical stratification. Furthermore, therapeutic targeting of JAM-A appears attractive for clinical translation. Figure 1 Figure 1. Disclosures Einsele: Celgene: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria.

scholarly journals Repurposing Leflunomide for Multiple Myeloma Treatment

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5667-5667
Author(s):  
Ralf Buettner ◽  
Corey Morales ◽  
Natalie Perret ◽  
Joycelynne Palmer ◽  
Amrita Krishnan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Plasma Cells ◽  
Single Agent ◽  
Differentially Expressed ◽  
Dependent Manner ◽  
Rpmi 8226

Abstract Background: Despite recent advances in treatment that have improved the prognosis for patients with multiple myeloma (MM), the disease remains incurable. There is a need for MM treatments with new mechanisms of action. Leflunomide, a commercially available oral immunosuppressive agent that has been FDA-approved since 1998 for the treatment of rheumatoid arthritis (RA) was evaluated as a potential MM therapy. The primary mechanism of action is de novo inhibition of pyrimidine synthesis by targeting dihydroorotate dehydrogenase (DHODH), and thus achieving an anti-proliferative effect in B- and T-lymphocytes. A secondary mechanism of action is inhibition of cytokine and growth factor receptor-associated tyrosine kinase activity. Methods and Results: Pre-clinical studies of teriflunomide, the active metabolite of leflunomide, showed that it inhibited cell growth and induced apoptosis in MM cell lines (MM.1S, MM.1R, U266, H929, RPMI-8226) and primary MM patients' (CD138+) plasma cells at clinically achievable concentrations (50-200 uM) in a time- and dose-dependent manner. We also found that teriflunomide induces cell-cycle arrest in both, glucocorticoid-sensitive (MM.1S) and resistant (MM.1R) MM cell lines at <200 uM. In addition, teriflunomide and dexamethasone synergized in the in vitro growth inhibition of MM cell line MM.1S. To identify MM-associated mRNAs and miRNAs whose expression levels are frequently altered upon teriflunomide exposure, MM cell lines (RPMI-8226, U266, MM.1S, NCI-H929) and CD138-enriched primary plasma cells from two MM patient samples were treated with 200 µM teriflunomide or DMSO control for 24 h before extraction and purification of mRNA and microRNA. mRNA-seq and miRNA-seq analysis from teriflunomide-treated MM samples revealed that similar changes were present between patient samples and cell lines. A total of 382 genes were found to be differentially expressed (225 upregulated, 157 downregulated). Upregulated genes included those that participate in defense response and negative regulation of cell growth. Genes involved in mitosis, rRNA biogenesis/processing, and immune response were generally downregulated. Analysis of microRNA-seq data from these samples revealed five differentially expressed, mostly newly discovered miRNAs that have unknown function. Conclusions: Leflunomide and its analogues demonstrated anti MM effects in vitro as well as synergy with dexamethasone. Based on our promising pre-clinical results we have initiated a single-agent phase I/II clinical trial in patients with relapsed/refractory MM. Disclosures No relevant conflicts of interest to declare.

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