The Contribution of Proteasome Subunits to Myeloma Cell Viability and Proteasome Inhibitor Sensitivity

Background Bortezomib (BTZ) is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. The underlying mechanisms of such proteasome inhibitor resistance are still incompletely understood. Methods To further understand its resistant mechanism, we generated eight multiple myeloma (MM) cell lines resistant to bortezomib (BTZ) by exposure to increasing drug concentration: five of them acquired novel PSMB5 mutations. Given the rarity of similar mutations in over 1,500 analyzed MM patients, we explored in depth the role of the proteasome on MM cell viability and BTZ sensitivity by systematically deleting the major proteasome targets of BTZ by CRISPR. Results We demonstrated that MM cell lines without PSMB5 were surprisingly viable (mutation corresponding yeast gene pre2 is lethal). PSMB5 mutated, BTZ resistant, MM cell lines were re-sensitized to BTZ when PSMB5 was experimentally deleted, implying that this mutation is activating in its drug resistance function. In contrast PSMB6 knockout was lethal to MM cell lines, which were efficiently rescued by re-introduction of wild type PSMB6. Interestingly, reduction in PSMB6 levels also prevented the splicing of the major catalytic subunits PSMB5, PSMB7, PSMB8 and PSMB10. PSMB6 engineered with no splicing function or catalytic activity, also restored viability, inferring that the contribution of PSMB6 to proteasome structure is more important than functional activity. Supporting this observation, BTZ sensitivity was restored in resistant MM cells line by introducing low level expression of mutated PSMB6 lacking splicing function. As with PSMB6, PSMB7 knockout was lethal to MM cell lines. In contrast, loss of immunoproteasome subunits PSMB8 and PSMB9 was neither lethal nor restored sensitivity to BTZ. Our results demonstrate that expression of the three constitutive proteasome subunits PSMB5, PSMB6 and PSMB7 is highly co-dependent. This dependence is relying on the structure, but not the function, of PSMB5 and PSMB6. Conclusions In summary, PSMB5 and PSMB6, but not PSMB8 and PSMB9, are highly relevant for BTZ sensitivity in MM. Absence of PSMB6 or PSMB7, but not PSMB5, was lethal in MM cell lines. Expression of PSMB5, PSMB6 and PSMB7 was highly co-dependent. Together these findings suggest that the modulation of expression rather than function of PSMB5, PSMB6 or PSMB7 may be a new therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

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Rational Drug Design: Proteasome Inhibitor Mediated Down-Regulation of the FA/BRCA Pathway Is Synergistic with PARP Inhibition in Myeloma Cell Lines

Blood ◽

10.1182/blood.v118.21.2922.2922 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2922-2922

Author(s):

Liang Nong ◽

Linda Mathews ◽

Mark B Meads ◽

William Dalton ◽

Kenneth H. Shain

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Dna Repair ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Myeloma Cell ◽

Sequential Treatment ◽

Parp Inhibition ◽

Repair Pathway ◽

Myeloma Cells

Abstract Abstract 2922 The development of new and biologically-based therapeutic regimens is critical for the successful control, if not cure, of multiple myeloma. Incorporation of the novel agents, including the proteasome inhibitor bortezomib, harbored large strides in disease modification. However, even with the success of bortezomib containing regimens, drug resistance and disease relapse remain inevitable. As such, it is critical that we use preclinical models to not only develop drugs, but also to consider strategies for co-development of novel drug combinations capitalizing on complementary biological activities. Our investigations in drug resistance recently revealed that increased homologous recombination (HR) potential, via over-expression of the FA/BRCA DNA repair pathway (FA/BRCA pathway), contributed to acquired melphalan-resistance in myeloma cell lines.(Yarde et al 2009) Drug resistance was causally linked to a novel transcriptional regulation of the FA/BRCA by NF-κB. Further examination demonstrated that bortezomib attenuated this component of the HR repair pathway and reversed melphalan resistance. To this end, we anticipated that bortezomib treatment may sensitize cells to inhibitors of complementary DNA repair pathways in a manner similar to the synthetic lethality elicited in by PARP1/2 inhibition in BRCA1 or FANCD1/ BRCA2 mutant cancers.(Farmer 2005, Bryant 2005) Consistent with this rationale, treatment of myeloma cells with bortezomib and the PARP inhibitor AZD2281/olaparib demonstrated synergism in specific myeloma cell lines. Pre-treatment of RPMI8226 myeloma cells with bortezomib for 6 hours greatly enhanced myeloma cell sensitivity to PARP inhibition with AZD2281/olaparib. The inhibitory concentration(IC)-50 was decreased by 17.7-fold (n=3; IC50 AZD2281 alone: 62.7 microM (39.0–84.0) and pretreated with bortezomib 3.54 microM (2.4–4.6)). Combination Index (CI) demonstrated a mean of 0.41 in 8226 and 0.43 in U266 myeloma cells, consistent with a synergistic relationship. Further analysis confirmed that synergism correlated with decreased expression of FANCD2 mRNA and protein by 6 hours. In contrast to sequential treatment, concomitant treatment with these agents did not elicit the synergistic phenotype. Interestingly, sequential treatment of NCIH929 myeloma cells did not demonstrate the same synergistic response (CI :0.89, slight synergism). Consistent with this, treatment of NCIH929 cells with bortezomib did not negatively regulate FANCD2 mRNA or protein expression, suggesting that FA/BRCA pathway can be differentially regulated in myeloma cells. To more specifically determine if FANCD2 was a key factor regulated by bortezomib, we targeted FANCD2 with siRNA. Pretreatment of myeloma cells with FANCD2 siRNA also sensitized cells to AZD2281/olaparib relative to siRNA control (IC50: 19.0 microM vs 35.0 microM n=4; p<0.05). These results show that bortezomib (or other proteosome inhibitors) and AZD2281/olaparib (or other PARP inhibitors) may represent an exciting new combination therapy for myeloma. We are currently examining the applicability of these studies to other proteosome inhibitors and the clinical relevance with ex vivo studies with myeloma patient samples. We believe that data presented here are innovative as they introduce a novel biological rationale, the abrogation complementary pathways in DNA damage repair, for the preclinical development of novel targeted drug combinations in myeloma. Further, we anticipate that although this study has focused on multiple myeloma, the results of the proposed research will be applicable to a wide range of hematologic and solid tumors. Disclosures: No relevant conflicts of interest to declare.

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ONC201 Overcomes Chemotherapy Resistance By Upregulation of Bim in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.4476.4476 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4476-4476 ◽

Cited By ~ 2

Author(s):

Yong-sheng Tu ◽

Jin He ◽

Huan Liu ◽

Richard Eric Davis ◽

Robert Z. Orlowski ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Cell Viability ◽

Cell Lines ◽

Myeloma Cell ◽

Parp Cleavage ◽

Wild Type ◽

Myeloma Cells ◽

Induced Apoptosis ◽

Tumor Types

Abstract In multiple myeloma, disease relapse and drug resistance occurs in the majority of myeloma patients after standard treatment despite recent improvements offered by new therapies. Therefore, there is an urgent need for new drugs that can overcome drug resistance and prolong patient survival after failure of standard therapies. ONC201, the founding member of a novel class of anti-tumor agents called impridones, has selective preclinical efficacy against a variety of tumor types. It is currently in phase I and phase II clinical trials for patients with advanced solid tumors and hematological malignancies. Given the pronounced sensitivity of B-cell lymphomas to ONC201, we assessed the efficacy of ONC201 in preclinical models of multiple myeloma. We treated human myeloma cell lines and primary myeloma cells isolated from bone marrow aspirates of myeloma patients with ONC201 for 72 hours. CellTiter-Glo Luminescent and annexin-V binding assays for assessing myeloma cell viability and apoptosis were performed, along with immunoblotting for cleavage of caspases, phosphorylation of signaling kinases, and expression of pro- or anti-apoptotic proteins. ONC201 treatment decreased myeloma cell viability, with IC50 values that were 1 μM to 1.5 μM, even in high risk myeloma cell line RPMI8226. The status of TP53 did not appear to affect the efficacy of ONC201, as MM.1S or NCI-H929 cells with wild-type TP53 and OPM-2 or RPMI8226 with mutated TP53 had a similar sensitivity towards ONC201. These results agree with prior reports in other tumor types that have demonstrated that the efficacy of ONC201 is independent of TP53. Western blot analysis showed increased apoptosis, cleavage of caspase-9, caspase-3, and PARP. We also found that ONC201 induced expression of the pro-apoptotic protein Bim in myeloma cells, which can occur downstream of ERK inactivation. Knockdown of Bim expression in myeloma cells by shRNAs abrogated ONC201-induced apoptosis. Phosphorylation of Bim at Ser69 by Erk1/2 has been shown to promote proteasomal degradation of Bim. In accordance with this mechanism, we observed that ONC201 treatment reduced levels of phosphorylated Erk1/2, an indicator of Erk1/2 kinase activity, and Bim pSer69. In addition, ONC201 induced apoptosis in dexamethasome-, bortezomib-, and carfilzomib-resistant myeloma cell lines with the same efficacy as in wild-type cells. As a rational strategy to increase the efficacy of ONC201 by enhancing its inhibition of proteasome-mediated Bim degradation, we tested combinations of ONC201 with proteasome inhibitors bortezomib or carfilzomib. These combinations were synergistic in reducing cell viability and enhancing Bim expression and PARP cleavage in myeloma cells. Overall, these findings demonstrate that ONC201 inhibits the Erk1/2 signaling pathway and induces Bim expression to induce apoptosis in multiple myeloma regardless of resistance to standard-of-care therapies. Our studies suggest that ONC201 should be evaluated clinically in relapsed/refractory multiple myeloma. Disclosures Allen: Oncoceutics: Employment, Equity Ownership.

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RNAi Screen of the Druggable Genome Identifies Modulators of Proteasome Inhibitor Sensitivity in Myeloma Including CDK5.

Blood ◽

10.1182/blood.v114.22.602.602 ◽

2009 ◽

Vol 114 (22) ◽

pp. 602-602

Author(s):

Yuan Xiao Zhu ◽

Rodger E. Tiedemann ◽

Chang-Xin Shi ◽

Jessica Schmidt ◽

Laura Bruins ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Conflicts Of Interest ◽

Shrna Expression ◽

Primary Screening ◽

A Genome ◽

Proteasome Subunits ◽

Druggable Genome ◽

Sensitization Effect

Abstract Abstract 602 The molecular target(s) which co-operate with proteasome inhibition in inducing drug sensitivity or resistance in Multiple Myeloma (MM) remain unknown. We therefore conducted a genome scale small interfering RNA (siRNA) lethality study in KMS11 MM cells in the presence or absence of bortezomib without regard to pre-conceived mechanistic notions. Primary screening was performed in a single-siRNA-per-well format with the human druggable genome siRNA set V4 comprising 13,984 siRNA targeting 6,992 genes and comprising two RNAi per gene. siRNA were transfected at low concentration (13nM) to minimize off-target effects using conditions that resulted in transfection of >95% cells and <5% background cytotoxicity. Bortezomib was added (at the IC10, 25, 70 and 90) 24 hours post transfection. After 96 hours (72 hours after bortezomib), viability was measured by ATP-dependent luminescence. In primary screening 320 candidate bortezomib sensitizing genes were identified and rescreened with four siRNA oligos each gene. 57 of the top sensitizer hits were selected for which at least two distinct siRNA decreased the EC50 by 2 standard deviations from cells treated with control siRNAs. By cross referencing gene expression profile (GEP) data of KMS11, we furthered pared the list to 37 plausibly expressed targets (0.5% of those genes originally screened) as bortezomib sensitizers. After silencing, 50% of these 37 genes also sensitized the lung cancer cell line A549 to bortezomib. After further parsing of genes which also modulated the sensitivity of MM to Melphalan (non specific chemosensitizers), 34 genes remained: The strongest sensitizers to bortezomib were the proteasome subunits PSMA5, PSMB2, PSMB3, PSMB7 but included less obvious targets such as BAZ1B, CDK5, CDC42SE2, MDM4, NME7, TFE3, TNFAIP3, TNK1, TOP1, VAMP2 and YY1 were also identified. Of these, the most potent synergetic effects were observed with siRNAs against the proteasome subunits and against cyclin dependent kinase 5 (CDK5), which caused the greatest shift in EC50. CDK5 is of particular interest as a therapeutic target as it is expressed at high levels in MM and neural tissues but has low expression in other organs. Using viral shRNA expression, silencing CDK5 consistently increased the sensitivity of genetically variable MM cell lines (n=5) to all of the proteasome inhibitors tested: bortezomib, carfilzomib and PR047, and the effect could be at least partially rescued by overexpression of an RNAi resistant CDK5. To explore therapeutic relevance the small molecule CDK5 inhibitor, Roscovitin, was shown to be synergistic or additive with bortezomib in both MM cell lines and primary patient samples. Gene expression profiling was then performed to seek an explanation for the CDK5 sensitization effect and regulation of a proteasome subunit PSMB5 by CDK5 was identified as a probable route to sensitization. In summary inhibition of the existing proteasome either directly by suppression of proteasome subunits, or indirectly by suppression of modulators such as CDK5 appears to confer the greatest sensitization effect suggesting that combinations of bortezomib with other unique proteasome inhibitor drugs or combinations with inhibitors of CDK5 is a logical avenue for clinical exploration. Disclosures: No relevant conflicts of interest to declare.

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Serum/Glucocorticoid-Regulated Kinase 1 (SGK1) Is a Prominent Target Gene of the Transcriptional Response to Cytokines In Multiple Myeloma and Supports the Growth of Myeloma Cells

Blood ◽

10.1182/blood.v116.21.1915.1915 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1915-1915

Author(s):

Unn-Merete Fagerli ◽

Thorsten Stühmer ◽

Toril Holien ◽

Randi Utne Holt ◽

Ove Bruland ◽

...

Keyword(s):

Multiple Myeloma ◽

Growth Factors ◽

Cell Lines ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Transcriptional Response ◽

Myeloma Cell ◽

Myeloma Cells ◽

Growth And Survival ◽

Stat3 Phosphorylation

Abstract Abstract 1915 Multiple myeloma is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We hypothesized that the intracellular signals evoked by cytokines converge and regulate transcription of a set of genes that are common targets for several growth factors and therefore constitute pivotal mediators of the tumor-promoting effects of autocrine or paracrine stimuli. To identify such targets, we determined the changes in gene expression induced by IL-6, TNFalpha, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase SGK1, which is a down-stream effector of PI3-kinase and highly homologous to AKT. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the JAK/STAT pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, shRNA-mediated knock-down of STAT3 reduced basal and induced SGK1 levels, demonstrating the involvement of the JAK/STAT3 signaling pathway in SGK1 induction. Furthermore, down-regulation of SGK1 by shRNAs resulted in decreased proliferation and viability of myeloma cell lines. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their growth and survival and represents an attractive candidate for further evaluation as a therapeutic target. Disclosures: No relevant conflicts of interest to declare.

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Identification of Tight Junction Protein (TJP)-1 As a Modulator of Sensitivity to Doxorubicin and Cisplatin in Models of Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.3962.3962 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3962-3962

Author(s):

Xing-Ding Zhang ◽

Robert Z. Orlowski ◽

Lin Yang

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Hela Cells ◽

Conflicts Of Interest ◽

Myeloma Cell ◽

Cancer Center ◽

In Vivo Models ◽

Junction Protein ◽

Rpmi 8226

Abstract Abstract 3962 Background: Therapeutic advances in multiple myeloma have improved the outcomes of patients with this malignant plasma cell disorder, but the disease course is still strongly influenced by both innate, or primary, as well as acquired, or secondary mechanisms of drug resistance. Identification and validation of genes that may mediate these phenotypes is therefore of importance, since they could be useful prognostic markers, and also potential targets to overcome the emergence of resistance, or possibly preclude its emergence altogether. Methods: To identify non-redundant determinants of chemoresistance, we designed a robust, high-throughput RNA interference (RNAi) screen targeting 9610 human genes. The screen involved retroviral-mediated transduction first of HeLa cervical carcinoma cells with either the RNAi library, or with non-targeting retrovirus particles. After infection, cells were selected with puromycin, and treated with different concentrations of doxorubicin and cisplatin. Doxorubicin (Dox) treatment led to 33 surviving colonies from the cells transduced with the shRNA library, cisplatin (Cis) treatment led produced 22 surviving colonies, while non-targeting retrovirus-infected cells failed to form colonies after treatment. Screening was performed to identify the shRNA target gene(s) in each colony, and genes that were identified in both Dox- and Cis-treated HeLa cells, and that were expressed in myeloma cells, were selected for further study. These studies were supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results: TJP1 (zona occludens (ZO)-1) was identified as one gene whose knockdown promoted survival in Dox- and Cis-treated HeLa cells, and which was expressed in myeloma cell lines and in primary plasma cells. To further examine its potential role in myeloma chemosensitivity, we performed mRNA and protein expression profiling in a panel of 11 cell lines and observed that TJP1 expression was silenced in 3 cell lines (ARP-1, INA-6, and MOLP-8), while it was moderately to highly expressed in 7 cell lines (including RPMI 8226, MM1.S, and U266). Comparing TJP1-positive MM1.S cells to TJP1-null MOLP-8 cells, the latter displayed a significantly higher median inhibitory concentration to Dox and Cis. Knockdown of TJP1 in RPMI 8226 and U266 cells, which produced a >75% target suppression, was sufficient to reduce the proportion of apoptotic cells in the sub-G1 fraction after treatment with Dox or Cis compared to control cells. Conversely, MOLP-8 cells transfected with human TJP1 cDNA exhibited an increase in the sub-G1 population in response to Dox and Cis treatment compared to vector controls. Conclusion: Taken together, these studies support the hypothesis that TJP1 expression mediates myeloma cell resistance to the DNA damaging agents doxorubicin and cisplatin. Further studies are underway to determine the mechanism by which TJP1 influences chemosensitivity, and to validate its impact using in vivo models. Disclosures: No relevant conflicts of interest to declare.

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Anti-CD138-IFNα Fusion Proteins Are Effective in Treating Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.939.939 ◽

2012 ◽

Vol 120 (21) ◽

pp. 939-939

Author(s):

Esther Yoo ◽

Alex Vasuthasawat ◽

Danh Tran ◽

Alan Lichtenstein ◽

Sherie Morrison

Keyword(s):

Multiple Myeloma ◽

Fusion Protein ◽

Cell Lines ◽

Dna Content ◽

Fusion Proteins ◽

Conflicts Of Interest ◽

Myeloma Cell ◽

Inhibition Of Proliferation ◽

Discontinue Therapy

Abstract Abstract 939 Although IFNα has shown some efficacy in the treatment of multiple myeloma (MM), this efficacy has been limited in large part because systemic toxicity makes it difficult if not impossible to reach therapeutically effective doses at the site of the tumor. The short half-life of IFN also makes it difficult to sustain high levels during treatment, and because of the side effects, the patients often discontinue therapy. To address these issues, we have genetically fused IFNα2 to a chimeric IgG1 antibody specific for the antigen CD138 expressed on the surface of MM cells, yielding anti-CD138-IFNα. We have also produced a fusion protein (anti-CD138-mutIFNα) using a mutant IFNα that binds the IFN receptor (IFNAR) more tightly. The fusion proteins continued to bind CD138 and retained IFN activity and showed anti-proliferative activity against a broad panel of myeloma cell lines (HMCL) representing MM with different characteristic. To investigate the events responsible for the inhibition of proliferation, 8226/S, ANBL-6, MM1-144, H929, OCI-My5 and U266 cells were incubated with 500 pM anti-CD138-IFNα for 72 h and their DNA content analyzed by FLOW cytometry following permeabilization and staining with PI. The different cell lines exhibited different responses. All of the cell lines except OCI-My5 underwent apoptosis. For 8226/S, OCI-My5 and U266 there was little change in DNA content following treatment. ANBL-6 showed a slight increase in the number of cells in S. However, MM1-144 and H929 showed a marked accumulation in G2 with H929 also showing accumulation of cells with sub-G0content of DNA. Therefore, there is heterogeneity in the response of different HMCL to treatment with targeted IFNα2. For many but not all of the cell lines, anti-CD138-mutIFNα was more effective than anti-CD138-IFNα in inhibiting proliferation and causing DNA fragmentation. Anti-CD138-mutIFNα was more effective than anti-CD138-IFNα in inducing senescence-associated β-galactosidase and STAT1 activation in OCI-My5 cells. Treatment with anti-CD138-IFNα or anti-CD138-mutIFNα resulted in a decrease in the amount of IRF4 present in U266, suggesting that this may be responsible for the efficacy of the fusion proteins in this cell line. Treatment of the other cell lines did not alter the level of IRF4 present, but anti-CD138-IFNα and anti-CD138-mutIFNα treatment caused a decrease in the amount of ppRB present in 8226/S, OCI-My5 and MM1-144, and to a lesser extent in H929. To determine the in vivo efficacy of fusion protein treatment, SCID mice were injected subcutaneously with OCI-My5 cells and treated intravenously on days 14, 16 and 18 with 100 μg of the indicated proteins and monitored for tumor growth (Figure 1). Mice were sacrificed when tumors exceeded 1.5 cm in diameter. Treatment with anti-CD138-IFNα provided some protection (p ≤ 0.0001 compared to PBS). However, treatment with anti-CD138-mutIFNα was even more effective (p = 0.0004 compared to anti-CD138-IFNα). Anti-CD138-mutIFNα was also found to be more effective than anti-CD138-IFNα against primary MM cells. Patients with active myeloma were biopsied while off therapy and the marrow cells isolated by a negative antibody selection to >95% purity. After 72 h incubation with 25 nM of protein, anti-CD138 was found to have little effect. In contrast treatment with anti-CD138-IFNα caused a decrease in viability with anti-CD138-mutIFNα treatment leading to an even greater decrease in cell viability. Following 72 h of treatment, 25 nM of anti-CD138-mutIFNα was found to have more potent cytoreductive effects than 100 nM of anti-CD138-IFNα. Disclosures: No relevant conflicts of interest to declare.

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Combined Carfilzomib and Selective PI3Kδ Inhibition (TGR1202) Results In Enhanced Myeloma Cell Apoptosis

Blood ◽

10.1182/blood.v122.21.3224.3224 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3224-3224

Author(s):

Claire Torre ◽

Yanyan Gu ◽

Lawrence H. Boise ◽

Sagar Lonial

Keyword(s):

Multiple Myeloma ◽

Western Blot ◽

Signaling Pathway ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Feedback Loop ◽

Myeloma Cell ◽

Pi3k Signaling ◽

Mtor Signaling Pathway ◽

The Impact

Abstract Introduction The PI3K signaling pathway plays a vital role in regulating cell growth, proliferation and survival in multiple myeloma (MM) as well as in many other cancers. TGR-1202, an isoform-specific PI3Kδ inhibitor, with efficacy in preclinical models of hematologic malignancies, is currently in Phase I clinical development. In multiple myeloma, PI3K signaling appears to be very important for many extracellular signals, yet inhibition with -pan PI3K inhibitors have not exhibited significant activity. However, literature reports indicate that there are several MM cell lines that express PI3K -δ, and do appear to have differential sensitivity to specific isoform inhibition as opposed to pan PI3K inhibition. In this report, we sought to evaluate the effects of TGR-1202 alone and in combination with the proteasome inhibitor carfilzomib, with the intent of further understanding the mechanism of action and evaluating the impact of the combination. Methods Human myeloma cell lines (MM.1S, MY5, RPMI8226, U266, KMS11, ARH-77, OPM1, OPM2, LP1, JJN3 and L363) were treated with TGR-1202 alone, carfilzomib alone, or with the combination of TGR-1202 and carfilzomib. Annexin V/PI staining and Western blot were used to identify the cellular and molecular sequelae of the combination. Result 10 µM TGR-1202 alone did not cause significant cell death in the MM cell lines tested at 48 hours. When cells were treated with the combination of TGR-1202 and 3 nM carfilzomib, we observed enhanced apoptosis in all of the tested cell lines. In the U266 cell line, 3 nM carfilzomib and 10 µM TGR-1202 induced 16% and 14% cell apoptosis respectively. In the combination treatment, apoptosis increased to 75%. To explore the molecular mechanisms underlying the combination, we used a Western blot assay to evaluate the impact of the combination on the mTOR signaling pathway, a known reciprocal feedback loop when PI3K is blocked. TGR-1202 alone did not have an obvious effect on the mTOR signaling pathway, yet combining TGR-1202 with carfilzomib, significantly inhibited phospho-mTOR, suggesting total pathway blockade. Conclusion The combination of TGR-1202 with carfilzomib induces synergistic apoptosis in MM cell lines. Preliminary data suggests that this occurs through blockade of the entire reciprocal feedback loop of mTOR activation. Additional data from primary patient samples and underlying mechanisms will be presented at the meeting. These findings support the rationale for future clinical studies of TGR-1202, a selective PI3K-δ inhibitor in combination with the proteasome inhibitor carfilzomib. Disclosures: Boise: Onyx: Consultancy. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy.

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High Trip13 and Low P31 Comet Cause Drug Resistance and Poor Prognosis In Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.3820.3820 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3820-3820

Author(s):

Yi Tao ◽

Zhimin Gu ◽

Ye Yang ◽

Hongwei Xu ◽

Xiaojing Hu ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Cell Lines ◽

Poor Prognosis ◽

Caspase 3 ◽

Plasma Cells ◽

Myeloma Cell ◽

Newly Diagnosed ◽

Caspase 9 ◽

Over Expression

Abstract Background We have recently established that increased chromosomal instability (CIN) signature is linked to drug resistance and poor outcome in multiple myeloma (MM) and other cancers. Thyroid Hormone Receptor Interactor 13 (Trip13), one of the 56 drug-resistant genes, plays a key role in chromosomal recombination and structure development during meiosis and has been reported to be increased in some malignancies including lung cancer, prostate cancer and breast cancer. In this study, we investigated how important Trip13 is in myelomagenesis and progression. Materials and Methods Gene expression profiling (GEP) was analyzed on plasma cells from 22 healthy donors, 44 patients with monoclonal gammopathy of undetermined significance (MGUS), 351 patients with newly diagnosed multiple myeloma, and 9 human myeloma cell lines, as well as on 36 sequential samples at diagnosis, pre-1st, pre-2nd and post-2nd autologous stem cell transplantation (ASCT). Over-expression and knock-down experiments of Trip13 were performed on myeloma cell lines by lentivirus transfection. Cell viability was assessed by trypan exclusion assay. Western blots were used to detect the expression of Trip13, P31 comet, caspase-8, caspase-9, caspase-3 and PARP, and checkpoint related proteins MAD2 and CDC20 in Trip13 overexpressed or Trip13 shRNA-transfected myeloma cells. Results Sequential GEP samples showed that Trip13 expression increased in 8 of 9 patients after chemotherapy and ASCT compared to the samples at diagnosis strongly suggesting that increased Trip13 is associated with drug resistance. Trip13 was already significantly increased in MGUS patients, newly diagnosed MM patients and MM cell lines compared with normal plasma cells. Furthermore, Trip13 was significantly higher in high-risk MMs than in low-risk MMs and increased Trip13 was linked to an inferior event-free survival (EFS) (p<0.01) and overall survival (OS) (p<0.01) in 351 newly diagnosed MMs. In contrast, the Trip13-interacting gene P31 comet was down-regulated in high-risk MMs and high expression of P31 was associated with good outcome. Interestingly, patients with high Trip13 and low P31 comet have the worst outcome compared to patients with only one of these, suggesting the interaction of Trip 13 and p31 has a synergistic effect on MM progression. Transfection of Trip13 into ARP1 and OCI-My5 cells significantly increased cell proliferation, while knock-down Trip13 in OCI-My5, H929, RPMI8226 cells inhibited cell growth and induced MM cell apoptosis with increases of cleaved caspase-8, caspase-9, caspase-3 and PARP. Mechanistic studies showed that Trip13 over-expression decreased P31comet and MAD2 expression by western blotting, but increased CDC20. Conclusions The association of increased Trip13 and decreased p31 is a good biomarker for MM drug resistance and poor prognosis. Our results also show Trip13 and P31 comet could be potential targets to overcome drug resistance in MM. Disclosures: No relevant conflicts of interest to declare.

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Targeting MAGE-C1/CT7 Expression Increases Cell Sensitivity to the Proteasome Inhibitor Bortezomib in Multiple Myeloma Cell Lines

PLoS ONE ◽

10.1371/journal.pone.0027707 ◽

2011 ◽

Vol 6 (11) ◽

pp. e27707 ◽

Cited By ~ 16

Author(s):

Fabricio de Carvalho ◽

Erico T. Costa ◽

Anamaria A. Camargo ◽

Juliana C. Gregorio ◽

Cibele Masotti ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Myeloma Cell ◽

Cell Sensitivity ◽

Multiple Myeloma Cell

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Xenograft Models of Multiple Myeloma Reveal That PU.1 Serves As a Tumor Suppressor for Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.2047.2047 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2047-2047

Author(s):

Nao Nishimura ◽

Shinya Endo ◽

Niina Ueno ◽

Shikiko Ueno ◽

Hiromichi Yuki ◽

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Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Tumor Suppressor ◽

Cell Lines ◽

Conflicts Of Interest ◽

Myeloma Cell ◽

Suppressor Activity ◽

Enhancer Region ◽

Tumor Suppressor Activity

Abstract PU.1 is an essential transcription factor for hematopoiesis and important for differentiation of both myeloid and lymphoid lineages. In mice conditionally knocked-out of 3.4 kb length of the enhancer region located in14 kb 5’ upstream of the PU.1 gene (URE), PU.1 is down-regulated in myeloid cells and B cells by 20% of that of wild type, and such mice develop acute myeloid leukemia and CLL-like diseases. These data strongly suggest that PU.1 has tumor suppressor activity in hematopoietic cells. We previously reported that human PU.1 is down-regulated in the majority of myeloma cell lines through the methylation of the promoter and the 17 kb upstream enhancer region (URE) of the PU.1 gene that is homologous to that in 14 kb 5’ upstream of the murine PU.1 gene. Conditionally expressed PU.1 with tet-off system induced cell growth arrest and apoptosis in two myeloma cell lines, KMS12PE and U266, suggesting that the down-regulation of PU.1 is necessary for myeloma cell growth. We have also reported that PU.1 is expressed in normal plasma cells and in contrast, PU.1 is down-regulated in primary myeloma cells from a subset of myeloma patients, who appear to have poor prognosis. In the present study, to test whether PU.1 has tumor suppressor activity in vivo, we generated xenograft mouse models. 0.6 - 1 x 107 KMS12PE cells were subcutaneously injected in 16 immunodeficient mice (Rag2-/- Jak3-/- bulb/c). The mice were then administered doxycycline through drinking water. Half of the mice (N=8) stopped taking doxycycline when the tumor sizes reached 1 cm in diameter, whereas the other half (N=8) kept taking doxycycline. Although the tumors in the mice taking doxycycline continued to grow, the tumor growth in the mice not taking doxycycline significantly slowed down. Flow cytometry analysis of the tumors in the mice that stopped taking doxycycline revealed that the cells from the tumor had completely lost PU.1 expression. Moreover, when U266 cells conditionally expressing PU.1 were subcutaneously injected to another 10 mice and the same experiment was conducted, although the tumors in the mice taking doxycycline (N=5) kept growing, the tumors in the mice not taking doxycycline (N=5), did not grow any further. The present data suggest that PU.1 serves as a tumor a suppressor in the multiple myeloma cell lines as examined in vivo. Disclosures No relevant conflicts of interest to declare.

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