ONC201 Overcomes Chemotherapy Resistance By Upregulation of Bim in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4476-4476 ◽  
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.

scholarly journals An inhibitor of the EGF receptor family blocks myeloma cell growth factor activity of HB-EGF and potentiates dexamethasone or anti–IL-6 antibody-induced apoptosis

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1829-1837 ◽  
Author(s):  
Karène Mahtouk ◽  
Michel Jourdan ◽  
John De Vos ◽  
Catherine Hertogh ◽  
Geneviève Fiol ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Cell Lines ◽  
Stromal Cells ◽  
Egf Receptor ◽  
Myeloma Cell ◽  
Factor Activity ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor–like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells, we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma, particularly by monocytes and stromal cells, but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration, HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines, through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti–IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether, these data identify ErbB receptors as putative therapeutic targets in multiple myeloma.

Expression of functional interleukin-15 receptor and autocrine production of interleukin-15 as mechanisms of tumor propagation in multiple myeloma

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 610-618 ◽  
Author(s):  
Inge Tinhofer ◽  
Ingrid Marschitz ◽  
Traudl Henn ◽  
Alexander Egle ◽  
Richard Greil
Keyword(s):  
Multiple Myeloma ◽  
Cell Survival ◽  
Cell Lines ◽  
Plasma Cells ◽  
Constitutive Expression ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Cytotoxic Treatment ◽  
Interleukin 15 ◽  
Induced Apoptosis

Interleukin-15 (IL-15) induces proliferation and promotes cell survival of human T and B lymphocytes, natural killer cells, and neutrophils. Here we report the constitutive expression of a functional IL-15 receptor (IL-15R) in 6 of 6 myeloma cell lines and in CD38high/CD45low plasma cells belonging to 14 of 14 patients with multiple myeloma. Furthermore, we detected IL-15 transcripts in all 6 myeloma cell lines, and IL-15 protein in 4/6 cell lines and also in the primary plasma cells of 8/14 multiple myeloma patients. Our observations confirm the existence of an autocrine IL-15 loop and point to the potential paracrine stimulation of myeloma cells by IL-15 released from the cellular microenvironment. Blocking autocrine IL-15 in cell lines increased the rate of spontaneous apoptosis, and the degree of this effect was comparable to the pro-apoptotic effect of depleting autocrine IL-6 by antibody targeting. IL-15 was also capable of substituting for autocrine IL-6 in order to promote cell survival and vice versa. In short-term cultures of primary myeloma cells, the addition of IL-15 reduced the percentage of tumor cells spontaneously undergoing apoptosis. Furthermore, IL-15 lowered the responsiveness to Fas-induced apoptosis and to cytotoxic treatment with vincristine and doxorubicin but not with dexamethasone. These data add IL-15 to the list of important factors promoting survival of multiple myeloma cells and demonstrate that it can be produced and be functionally active in an autocrine manner.

Identification of FAM46C As a Multiple Myeloma Repressor

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 836-836 ◽  
Author(s):  
Yuan Xiao Zhu ◽  
Chang-Xin Shi ◽  
Patrick Jedlowski ◽  
Klaus Martin Kortum ◽  
Laura Ann Bruins ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Growth Inhibition ◽  
Cell Viability ◽  
Cell Survival ◽  
Pathway Analysis ◽  
Myeloma Cell ◽  
Newly Diagnosed ◽  
Wild Type ◽  
Myeloma Cells

Abstract Introduction: Partial loss of chromosome arm 1p frequently occurs in multiple myeloma (MM), and is associated with a poor prognosis. Several minimally altered regions on 1p have been identified, including 1p32.3, 1p31.3, 1p22.1-1p21.3, and 1p12. Cytoband 1p12 was deleted in 19% of cases, and this deletion was associated with shorter overall survival (OS) in univariate analysis. The target of homozygous deletion 1p12 was FAM46C. In addition, mutations of FAM46C were identified in 3.4% to 13% of primary MM tumors and 25% of 16 human myeloma cell lines (HMCLs), implying its potential pathogenic relevance. In other work we have suggested that FAM46C mutation is a progression event and have shown that it is rarely seen in newly diagnosed del17 patients, inferring some overlap in function. However, there is no published functional annotation of FAM46C and its role in MM remains unknown. In the present study, we aimed to identify the biological role of FAM46C in myeloma cells. Materials/Method: The expression of FAM46C in HMCLs was analyzed by western blot. Lentiviral constructs expressing wild type and mutated FAM46C were generated and transduced into HMCLs, followed by cell viability assay and cell cycle analysis. Cells were harvested and processed to measure gene expression and cell signaling changes after introduction of FAM46C by mRNAseq, pathway analysis and immunoblotting assay. Results: The expression of FAM46C protein is generally low in most HMCLs. Introduction of wild type FAM46C in HMCLs induced a substantial cell growth inhibition and apoptosis, especially in two HMCLs including MM1.S and KMS11. Cell viability of KMS11 and MM1.S was reduced by 50% to 80% at day 6 after introduction of FAM46C, compared to 0-30% growth retardation detected in HMCLs and non-myeloma cell lines that do not carry FAM46C deletion. We identified 88 genes whose mRNA expression was significantly altered after enforced expression of FAM46C in MM1.S cells. Pathway analysis revealed that FAM46C-regulated genes are enriched in the canonical pathways associated with unfolded protein response, cell cycle control and DNA damage repair. Critical MM genes that are downregulated by FAM46C expression include IRF4 and MYC, which are also downstream targets of immunomodulatory drugs (IMiDs). Consistently, some HMCLs such as KMS11 and OPM2 show an enhanced sensitivity to lenalidomide after introduction of FAM46C. Next, lentiviral constructs expressing various FAM46C mutants were generated in order to understand the consequence of FAM46C mutation. The mutant constructs mimic mutations identified in MM patients or HMCLs. Those mutants and wild type FAM46C were transduced and tested together in MM1.S cells. We found that three published misssense mutations, one frame-shift mutation and deletion of the sequence between aa172 and aa186 of FAM46C (which has been found in previous studies as a hot spot of mutation) all abolished FAM46C-mediated anti-myeloma activity, thus would be expected to confer a MM cell survival advantage. Conclusion: Our data demonstrated that enforced FAM46C expression in myeloma cells induced myeloma growth inhibition and apoptosis. Mutations in FAM46C and TP53 in newly diagnosed patients seem mutually exclusive but not in relapsed patients from our patients sequencing studies, suggesting it may associate with disease progression. Together, these studies suggest that FAM46C may function as a tumor suppressor in myeloma. We also found that published mutations of FAM46C confer a survival advantage to MM cells, and that FAM46C overexpression downregulates IRF4 and MYC and is thus associated with loss of myeloma cell survival. Disclosures Stewart: Novartis: Consultancy; Oncospire Inc.: Equity Ownership; Celgene: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees.

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

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4337-4337
Author(s):  
Chang-Xin Shi ◽  
Yuan Xiao Zhu ◽  
Laura Ann Bruins ◽  
Cecilia Bonolo De Campos ◽  
William Stewart ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Viability ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Resistance Function ◽  
Proteasome Subunits ◽  
Underlying Mechanisms

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.

Triggering of Toll-Like Receptor-4 In Human Multiple Myeloma Cells Promotes Proliferation and Alters Cell Responses to Immune and Chemotherapy Drug Attack

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1905-1905
Author(s):  
Zhen Cai ◽  
Hanying Bao ◽  
Peilin Lu ◽  
Lijuan Wang ◽  
Donghua He ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Response To Chemotherapy ◽  
Tlr4 Ligand ◽  
Induced Apoptosis

Abstract Abstract 1905 Multiple myeloma (MM) is a fatal plasma cell malignancy mainly localized in the bone marrow. The clonal expansion of tumor cells is associated with the disappearance of normal plasma cells and with a marked depression in the production of normal immunoglobulin (Ig). This makes MM patients highly vulnerable to bacterial, fungal and viral infections and recurrent infections remain to be a major cause of death in MM patients. It has been shown that most primary myeloma cells and cell lines express multiple Toll-like receptors (TLRs). Among them, TLR4 is most frequently expressed. To investigate TLR-initiated responses in MM cells including proliferation, anti-apoptosis and immune escape, we first screened four commonly used human myeloma cell line (HMCL) for the expression of major TLRs by RT-PCR. Surprisingly, all the HMCL expressed multiple TLRs. We also examined primary myeloma cells from 4 patients with MM and our results showed that TLR4 was expressed by all the tumor cells. We incubated myeloma cells with LPS, the natural ligand for TLR4, and found that cell proliferation increased significantly. Targeting TLRs on malignant B cells can induce resistance to chemotherapeutic agents but can also be exploited for combined therapeutic approaches. As mechanisms involved in the resistance to apoptosis play a major role in MM escape to therapies, we sought to determine the capacity of TLR4 ligand to promote the survival of HMCL cells. Myeloma cells were pretreated for four hours with LPS before being induced apoptosis by adriamycin. Results showed that LPS pretreatment partially protected the cells from adriamycin-induced apoptosis. The TLR signaling pathway activates several signaling elements, including NF-kB and ERK/JNK/p38 MAPKs, which regulate many immunologically relevant proteins. Time-dependent MAPK phosphorylation was measured to assess the activation of these kinases upon treatment with LPS in cell lines. ERK1/2, p38, and JNK phosphorylation and NF-kB were significantly up-regulated following LPS treatment. Moreover, our findings demonstrated that LPS-induced cell proliferation was dependent on JNK, ERK and p38 signaling. IL-18, a recently described member of the IL-1 cytokine superfamily, is now recognized as an important regulator of innate and acquired immune responses. In this study, we found that LPS induced IL-18 secretion and activated MAPK and NF-kB signaling simultaneously. Therefore, our results suggest that activation of the MAPK signaling and secretion of IL-18 are interconnected. Tumors evade immune surveillance by multiple mechanisms, including the production of factors such as TGF-β and VEGF, which inhibit and impair tumor-specific T cell immunity. Our study also showed that T cell proliferation induced by allostimulatory cells decreased when the HMCL were pre-treated with LPS. Moreover, immunoregulatory molecules on HMCL, such as B7-H1, B7-H2 and CD40, were upregulated after treatment with LPS, suggesting that TLR4 ligand LPS facilitates tumor cell evasion of the immune system. Our results show that TLRs are functional on myeloma tumor cells, and the ligands to these TLRs have a functional role in affecting myeloma cell proliferation, survival, and response to chemotherapy and immune attacks. Disclosures: No relevant conflicts of interest to declare.

SDX-101 Is Cytotoxic and Overcomes Drug Resistance in Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3466-3466
Author(s):  
Makoto Hamasaki ◽  
Teru Hideshima ◽  
Kenji Ishitsuka ◽  
Hiroshi Yasui ◽  
Norihiko Shiraishi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Antineoplastic Agent ◽  
Parp Cleavage ◽  
Family Protein ◽  
Dose Dependent Fashion ◽  
Peripheral Mononuclear Cells ◽  
Induced Apoptosis

Abstract SDX-101 is an oral antineoplastic agent, has been evaluated in a phase I/II study in B-cell malignancies. It induces cytotoxicity at least in part, by significantly inhibiting expression of Mcl-1, an anti-apoptotic Bcl-2 family protein which is highly expressed in CLL cells. Here, we examined the cytotoxicity of SDX-101 against multiple myeloma (MM) cell lines. SDX-101 significantly inhibited growth in MM.1S, U266, RPMI8226 MM cell lines using MTT assays in a time- and dose-dependent fashion, with IC50s of 0.6mM, 1.0mM, and 0.4mM, respectively. In contrast, SDX-101 did not induce cytotoxicity in normal peripheral mononuclear cells (PBMCs) at these concentrations. Importantly, SDX-101 induced cytotoxicity even in dexamethasone (MM.1R)-, doxorubicin (RPMI-Dox40)-, and melphalan (LR5)- resistant MM cell lines. SDX-101 (0.3–1.25mM) triggered apoptosis associated with pro-caspase-3, pro-caspase-8, and PARP cleavage, as confirmed by immunoblotting. Although, interleukin-6 (IL-6) and insulin-like growth factor (IGF)-1 completely abrogates Dex-induced MM cell apoptosis, neither protects against SDX-101-induced apoptosis in MM.1S and RPMI8226 cells. Moreover, dexamethasone, Melphalan, and AS2O3 augment apoptosis induced by SDX-101. Importantly, SDX-101 downregulated both b-catenin and cyclin D1 expression in RPMI8226 cells. Finally, our recent studies demonstrate that the bone marrow (BM) microenvironment promotes MM cell growth, survival, and drug resistance while SDX-101 inhibits viability even if MM cells adherent to BM stromal cells; an environment in which cytotoxic potency is lost with other anti-neoplastic agents. Our data therefore demonstrate that SDX-101 induces apoptosis in MM cells via a mechanism different from conventional MM drugs, and support clinical trials of this agent, alone or in combination with conventional and/or novel agents to improve therapeutic outcome in MM.

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

Blood ◽  
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.

Role Of RECQ1 Helicase In Multiple Myeloma Biology and Drug Resistance

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1889-1889
Author(s):  
Elena Viziteu ◽  
Bernard Klein ◽  
Angelique Bruyer ◽  
Dirk Hose ◽  
Hartmut Goldschmidt ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Alkylating Agents ◽  
Myeloma Cell ◽  
Mitotic Catastrophe ◽  
Myeloma Cells ◽  
Dnmt Inhibitor ◽  
Promising Therapeutic Approach

Abstract Multiple Myeloma (MM) is a still lethal disease in 2013 characterized by the accumulation in the bone marrow of a clone of malignant plasma cells. Recent studies have shown that epigenetic modifications play a role by silencing various cancer-related genes in MM. We initiated a microarray-based genome-wide screen for genes responding to DNMT inhibition in MM cells and built a “DNA methylation gene score” that makes it possible identification of myeloma patients that will be sensitive to DNMT inhibitors. Among the genes regulated by DNMT inhibitor and associated with the worst prognostic value in patients, RECQ1 was identified. RECQ helicase are DNA unwinding enzymes involved in the maintenance of chromosome stability. RECQ1 is highly expressed in various types of solid tumors. RECQ1 silencing in cancer cells results in mitotic catastrophe and prevents tumor growth in murine models. In glioblastoma cells, depletion of RECQ1 induces reduction in cellular proliferation, spontaneous γ-H2AX foci formation and hypersensitivity to drugs. Furthermore, it was described that RECQ1 protein could interact with MSH proteins, RAD51 and PARP1 involved in DNA repair pathways. RECQ1 protein is expressed in human myeloma cell lines (HMCLs) and primary myeloma cells of patients. In four HMCLs (XG2, XG7, XG19 and LP1), RECQ1 was downregulated by conditional shRNA expression through lentiviral delivery. RECQ1 knock down inhibits growth of myeloma cells, induces 53BP1 foci formation and apoptosis. RECQ1 depletion sensitizes myeloma cells to DNA alkylating agent (melphalan) but not to corticosteroid (dexamethasone) or proteasome inhibitor (bortezomib). Using immunoprecipitation of myeloma cell nuclear proteins with anti-RECQ1 antibody, RECQ1 was shown to interact with PARP1 but not RAD51 or MSH2. An increased association of the two proteins was found upon DNA damages induced by melphalan. In agreement, RECQ1 depletion sensitizes myeloma cell lines to the PJ34 hydrochloride hydrate PARP inhibitor. In conclusion, RECQ1 could represent a biomarker of drug resistance in MM, which is targeted by DNMT inhibitor. This suggests association of alkylating agents and/or PARP inhibitors with DNMT inhibitor may represent a promising therapeutic approach. Disclosures: Goldschmidt: Celgene and Janssen: Membership on an entity’s Board of Directors or advisory committees.

scholarly journals Hexokinase-2 Regulates Hypoxia-Inducible Autophagy, Leading to Enhance Anti-Apoptotic Capability of Refractory Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1787-1787
Author(s):  
Sho Ikeda ◽  
Fumito Abe ◽  
Matsuda Yuka ◽  
Akihiro Kitadate ◽  
Takahiro Kobayashi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Research Funding ◽  
Pharmaceutical Research ◽  
Proteasome Inhibitors ◽  
Myeloma Cell ◽  
Hypoxic Stress ◽  
Myeloma Cells ◽  
Viable Cells

(background) The drug resistance of multiple myeloma (MM) cells is thought to be induced by various factors of the bone marrow microenvironment. Of these factors, hypoxic stress may be associated with drug resistance in various hematologic malignancies, including MM. Hypoxic stress lead MM cells to induce distinct gene expressions. It has been reported that oncogenic transcription factors such as IRF4 and Myc are suppressed under hypoxia. Instead, accumulation of another transcription factor, HIF-1α upregulates anti-apoptotic proteins, increases glycolysis, and enhances neovascularization leading MM cells to represent anti-apoptotic phenotype. Autophagy is an intracellular process that encapsulates cytoplasmic components, which are directed to the lysosome for degradation. Autophagy and proteasomal degradation prevent apoptosis caused by endoplasmic reticulum (ER) stress. Although proteasome inhibitor such as bortezomib, is a key drug for MM, it may induce treatment resistance. This might be because autophagy is induced in hypoxic microenvironment. Autophagy associated molecules might be therapeutic target in MM cells adapted to hypoxia. (Aim and methods) To clarify the association of hypoxia inducible genes and autophagy, and to obtain rational basis for a new therapeutic strategy against MM, we performed following experiments in vitro using myeloma cell lines (MM.1S, KMS-12-PE, KMS-11, and H929) and primary samples (n=6) that were subjected to hypoxia (1% O2). (Results) First, we examined volcano plot analysis on our cDNA microarray data (GSE80545) of patient samples incubated in normoxia or hypoxia for 48 hours. 546 probes were significantly elevated in hypoxia (fold change > 2.0, p < 0.05). Gene ontology analysis revealed that "Glycolytic Process" contained 13 genes such as PFKFB4, ENO2, ALDOC, PFKFB3, HK2, PFKP, GPI, PGK1, LDHA, ALDOA, ENO1, PKM, and GAPDH. We focused on hexokinase-2 (HK2) because it has been reported that HK2 activates autophagy under stress conditions. Western blot analysis for patient samples revealed that HK2 expression was remarkably upregulated under hypoxia. Apoptosis assay showed that viable cells of HK2 knockdowned cell lines were significantly lower than that of control cells under hypoxia, but not under normoxia. Also, in hypoxia, we found that number of 3-bromopyruvate (3-BrPA, a HK2 inhibitor) subjected viable cells were significantly lower than that of normoxia. This suggested that HK2 contributes to anti-apoptotic phenotype of MM cells under hypoxia. Next, we examined the role of HK2 in autophagy under hypoxia. Because degradation of p62 and increase of LC3-II/LC3-I ratio is considered to be useful for autophagy detection, we examined these factors by Western blot analysis. We found that hypoxic stress decreased expression of p62 and increased the ratio of LC3-II/LC3-I in myeloma cell lines, indicating that hypoxia activates autophagy. However, under hypoxia, these changes were canceled by HK2 knockdown. We confirmed that the number of autophagosome were significantly decreased in HK2-knockdowned myeloma cells by electron microscopy analysis. These data suggested that HK2 is required for hypoxia-inducible autophagy in MM. Finally, we examined the effect of combined inhibition of HK2 and proteasome. In hypoxia, apoptosis by bortezomib was significantly increased in HK2-knockdowned myeloma cells when compared with control. Moreover, we found that the combination of 3-BrPA and bortezomib increased apoptotic cells in both normoxia and hypoxia. These results suggested that HK2-inhibition can induce apoptosis against MM cells with enhancement of sensitivity to proteasome inhibitors. (Conclusion) These results suggest that hypoxia induced HK2 promotes autophagy and inhibits apoptosis. Thus, the combination of proteasome inhibitors and HK2 inhibition may bring about a deep response against treatment resistant MM. Disclosures Ikeda: Nippon Shinyaku Research Grant: Research Funding. Takahashi:Bristol-Myers Squibb: Speakers Bureau; Eisai Pharmaceuticals: Research Funding; Pfizer: Research Funding, Speakers Bureau; Otsuka Pharmaceutical: Research Funding, Speakers Bureau; Kyowa Hakko Kirin: Research Funding; Chug Pharmaceuticals: Research Funding; Ono Pharmaceutical: Research Funding; Novartis Pharmaceuticals: Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; Asahi Kasei Pharma: Research Funding.

Targeting NF-κB and Induction of Apoptosis by Dehydroxymethylepoxyquinomicin (DHMEQ) in Multiple Myeloma Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3393-3393
Author(s):  
Yoshitaka Miyakawa ◽  
Kanoko Kohmura ◽  
Kaori Saito ◽  
Hiroshi Yoshida ◽  
Asako Ikejima ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Caspase 3 ◽  
Regulatory Protein ◽  
Active Form ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract We previously designed and synthesized a new NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) (J Biol Chem, 2002). DHMEQ is a derivative of the weak antibiotics epoxyquinomicin C, which was isolated from the culture broth of Amycolaptosis sp. NF-κB is a critical regulatory protein that activates the transcription of a number of genes, including growth factors, angiogenesis modifiers, cell adhesion molecules and anti-apoptotic factors. As NF-κB has been shown as a good target for the new therapies such as bortezomib, we studied the effects of the new specific NFκB inhibitor, DHMEQ, to myeloma cells. In the present study, we demonstrated that DHMEQ inhibited the proliferation of human myeloma cell lines, RPMI8226 and U266 in dose- and time-dependent manners. Apoptosis was detected using fluorescein-conjugated Annexin-V by FACS. Around 45.3%of RPMI8226 and 45.2% of U266 were in apoptosis 12 hours after treatment with 10 μg/ml DHMEQ. Formation of apoptotic bodies were observed 24 hour-treatment with DHMEQ in both cell lines by Giemsa staining. In contrast, no obvious cell cycle arrest was observed with DHMEQ, indicating DHMEQ directly induces apoptosis without cell cycle arrests in these myeloma cell lines. The activation of caspase-3 in RPMI8226 and U266 cells were detected with the specific antibody against the active form of caspase-3 by FACS. When the myeloma cells were pretreated with 20 μM pan-caspase inhibitor, z-VAD-FMK, DHMEQ-induced apoptosis was inhibited by 62.1% in RPMI8226 and 71.9% in U266 cells, indicating DHMEQ-induced apoptosis was caspase-dependent. The binding activities of nuclear NF-κB protein to the oligonucleotides including NF-κB binding sites was suppressed by 81.9% in RPMI8226 and 69.0% in U266 1 hour after treatment with DHMEQ. NF-κB protein seemed more accumulated in cytoplasm of myeloma cells after treatment with DHMEQ under the confocal microscope, indicating DHMEQ prevents the translocation of NF-κB protein into the nucleus. Bcl-XL is the anti-apoptotic factor and its transcription is regulated by NF-κB. However, the expression level of Bcl-XL protein was not altered 24 hours after treatment with DHMEQ in RPMI8226 and U266. We also studied the effects of DHMEQ to the patient materials. We found that DHMEQ induced apoptosis in CD138-positive plasma cells from the myeloma patients (n=3), demonstrating that DHMEQ is also effective for primary cells. We previsously developed the model of human multiple myeloma by simply injecting U266 cells into the tail vein of the immunodeficient NOG mice. This myeloma model demostrated the massive osteolytic lesions and hind leg paralysis around 7 weeks after transplantation. We did not observe any invasion of U266 cells into other organs except bone marrow. As NF-κB regulates the proliferation of myeloma cells and osteoclasts, we expect DHMEQ will inhibit the tumor growth and prevent pathological fractures by inducing apoptosis in both myeloma cells and osteoclasts in vivo. We are currently evaluating the in vivo efficacies of DHMEQ using this experimental animal model of multiple myeloma. In conclusion, we demonstrated that DHMEQ targets NF-κB and induces apoptosis in myeloma cells through caspase-dependent pathways.

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