MAGE-A3 Inhibits p53 and Promotes Proliferation and Survival in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1795-1795
Author(s):  
Hearn J. Cho ◽  
Anna Mei ◽  
Tricia Nardiello ◽  
Maurizio DiLiberto ◽  
Xiangao Huang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Research Funding ◽  
Myeloma Cell ◽  
Type I ◽  
Newly Diagnosed ◽  
Apoptosis Pathway ◽  
Myeloma Cells ◽  
Intrinsic Apoptosis Pathway

Abstract Abstract 1795 Poster Board I-821 The type I Melanoma Antigen GEne (MAGE) proteins MAGE-A3 and CT7 (MAGE-C1) were commonly detected in primary tumor cells from multiple myeloma patients and their expression was correlated with advanced disease and proliferation. They belong to the Cancer-Testis antigen (CTAg) family of tumor-associated proteins. In gene expression analyses of primary myeloma cells, CTAg were associated with proliferative gene signatures and poor clinical outcome. These findings suggest that type I MAGE may play a pathogenic role in proliferation or survival in multiple myeloma cells. To test this hypothesis, we examined MAGE expression, proliferation, and apoptosis in primary myeloma specimens and human myeloma cell lines (HMCL). First, we examined CTAg expression and proliferation in vivo at two critical clinical milestones, in newly diagnosed, untreated patients and patients who relapsed after chemotherapy. MAGE-A3 was detected in a higher percentage of tumor specimens from relapsed patients (77%) compared to those from newly diagnosed patients (36%, p=0.0003), whereas CT7 was detected in about 75% of both patient populations. The percentage of proliferating myeloma cells, as measured by staining for the proliferation marker Ki-67, was significantly higher in relapsed specimens (19.0 ± 3.5%) compared to newly diagnosed (6.9 ± 1.3%, p=0.0002), demonstrating an association between MAGE-A3, progression of disease and proliferation. Second, we investigated the functional role of MAGE-A3 by silencing this gene in HMCL by shRNA interference. Targeted lentiviral shRNA transduction efficiently knocked down MAGE-A3 mRNA (≥90% compared to controls) and protein in MM.1r and Arp-1 HMCL by 48 hours and this effect was maintained up to 96 hours. Pulse labeling of HMCL with bromodeoxyuridine for 30 minutes revealed that silencing of MAGE-A3 led to cell cycle arrest, as evidenced by the complete loss of cells in S phase and accumulation of cells in both G1 and G2. This was accompanied by increased expression of the tumor suppressor p53 and the endogenous cyclin-dependent kinase (CDK) inhibitor p21Cip1, a p53 target that inhibits CDKs in both late G1 and G2. However, CDK4/6-specific phosphorylation of the retinoblastoma gene product (Rb) was unimpaired, indicating that control of the mid-G1 cell cycle checkpoints by Rb remained intact and suggesting that MAGE-A3 acted in part to promote G1-S progression. Within 24 hours of cell cycle arrest, 70-80% of MAGE-A3-silenced cells underwent apoptosis as measured by Annexin V staining, compared to '20% in cells transduced with a non-target control lentivirus or untreated. Furthermore, this apoptosis was caspase-dependent, as it was completely prevented by the pan-caspase inhibitor Quinoline-Val-Asp-CH2-OPh, and was triggered by the loss of mitochondrial outer membrane potential in the activation of the intrinsic apoptosis pathway. Taken together, the in vivo and in vitro results suggest that MAGE-A3 promoted myeloma cell proliferation by inhibiting p53-dependent expression of p21, and loss of this activity leads to growth arrest and cell cycle-coupled apoptosis via activation of the intrinsic apoptosis pathway. Understanding the biochemical mechanism of MAGE-A3 in cell cycle regulation and survival may identify novel therapeutic strategies for multiple myeloma. Proof of principle in this disease may lead to broader application of these strategies in other cancers that express MAGE-A3. Disclosures Niesvizky: Proteolix: Research Funding, data monitoring committee; Seattle Genetics, Inc: Research Funding; Celgene: Research Funding, Speakers Bureau; Millenium: Research Funding, Speakers Bureau.

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.

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.

Differential Expression of Type I MAGE in New and Relapsed Multiple Myeloma: Evidence for Association with Proliferation and Progression of Disease.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3397-3397
Author(s):  
Hearn J. Cho ◽  
Scott Ely ◽  
Wayne R. Austin ◽  
Ruben Niesvizky ◽  
Roger Pearse ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Myeloma Cell ◽  
Proliferation Index ◽  
Type I ◽  
Newly Diagnosed ◽  
Tumor Vaccines ◽  
Proliferating Cells ◽  
Myeloma Cells ◽  
Incorporated Brdu

Abstract The type I Melanoma Antigen GEne (MAGE) proteins belong to the Cancer-Testis family of tumor-associated antigens and are found in a broad range of solid and hematologic malignancies. We previously showed that the type I MAGE proteins CT7 (MAGE-C1) and MAGE-A3 were commonly detected in primary myeloma by both RT-PCR and immunohistochemistry (IHC). Higher levels of MAGE protein expression had a positive correlation with abnormally elevated proliferation as measured by the Plasma Cell Proliferation Index (PCPI, percentage of Ki-67+ cells in the CD138+ myeloma cell compartment). These findings suggest that MAGE may play a role in abnormal cell cycle regulation in myeloma. We explored this hypothesis by examining type I MAGE gene expression and proliferation by IHC in 46 newly-diagnosed, untreated and 35 relapsed myeloma patients, based on the clinical observation that relapsed patients exhibit lower response rates to therapy and shorter time to progression, indicative of more aggressive disease. PCPI was significantly higher in relapsed patients (19.0 ± 3.5%) compared to newly diagnosed (6.9 ± 1.3%, p<0.0002). Expression of CT7 and CT10 (MAGE-C2), a type I MAGE not previously associated with myeloma, was stable between newly-diagnosed and relapsed patients (76.0% of new samples vs. 77.1% of relapsed for CT7, 48.5% vs. 50.0% for CT10). In contrast, MAGE-A3 was detected in a significantly greater percentage of relapsed patients (77.1%) compared to newly diagnosed (35.6%, p=0.0003). The link between MAGE expression and unrestricted proliferation was further supported by in vitro studies with human myeloma cell lines. Proliferating myeloma cells were metabolically labeled with the nucleotide analog bromodeoxyuridine (BrdU) followed by intracellular staining and flow cytometry. This assay demonstrated that proliferating myeloma cells that incorporated BrdU into their genomic DNA expressed higher levels of type I MAGE protein compared to non-proliferating cells. Arresting cells at the G1-S interface by double thymidine blockade lead to the accumulation of cells expressing high levels of MAGE, which rapidly entered S phase and incorporated BrdU upon release from block. These results strongly suggest that expression of CT7 and CT10 are relatively early and stable events in the pathogenesis of myeloma, whereas activation of MAGE-A3 expression is associated with disease progression. Furthermore, MAGE expression is correlated with abnormal proliferation in vitro and in vivo, suggesting a potential functional role in the dysregulation of the cell cycle that is a hallmark of this disease. These results support the further exploration of the role of type I MAGE expression in myeloma and the development of therapeutic agents targeting them, especially tumor vaccines.

scholarly journals Inhibition of sphingosine kinase 2 downregulates the expression of c-Myc and Mcl-1 and induces apoptosis in multiple myeloma

Blood ◽  
2014 ◽  
Vol 124 (12) ◽  
pp. 1915-1925 ◽  
Author(s):  
Jagadish Kummetha Venkata ◽  
Ningfei An ◽  
Robert Stuart ◽  
Luciano J. Costa ◽  
Houjian Cai ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Survival ◽  
Specific Inhibitor ◽  
Sphingosine Kinase ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Sphingosine Kinase 2 ◽  
Key Points

Key Points SK2 is overexpressed in myeloma cells and contributes to myeloma cell survival and proliferation. SK2-specific inhibitor promotes proteasome degradation of Mcl-1 and c-Myc and inhibits myeloma growth in vitro and in vivo.

scholarly journals Interleukin-6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 11-13 ◽  
Author(s):  
XG Zhang ◽  
B Klein ◽  
R Bataille
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
S Phase ◽  
Myeloma Cells ◽  
Severity Of The Disease

Abstract It has recently been demonstrated that interleukin-6 (IL-6) is a potent myeloma-cell growth factor in the majority of patients with multiple myeloma (MM). Using an anti-bromodeoxyuridine monoclonal antibody (MoAb) to specifically count myeloma cells in the S-phase (ie, labeling index, LI), we demonstrate that the IL-6 responsiveness of myeloma cells in vitro is directly correlated with their LI in vivo. Myeloma cells from all 13 patients with high LIs in vivo (greater than or equal to 1%) responded in vitro to IL-6, the strongest response occurring in cells from five patients with plasma-cell leukemia. In contrast, the cells of only two of eight patients with low myeloma-cell LIs in vivo (less than 1%) responded to IL-6 in vitro. After seven days of culturing with 1,000 U/mL recombinant IL-6 (rIL-6), the median LI value in the first group of patients (in vivo LI greater than or equal to 1%) was 11%, ie 11 times higher (P less than .01) than the median LI value (1%) in the second group of patients (in vivo LI less than 1%). Thus, the in vitro IL-6 responsiveness of myeloma cells is directly related to their in vivo proliferative status, and hence to the severity of the disease.

CD38-Targeted Immunochemotherapy Of Multiple Myeloma: Preclinical Evidence For Its Combinatorial Use In Lenalidomide and Bortezomib Refractory/Intolerant MM Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 277-277 ◽  
Author(s):  
Inger S. Nijhof ◽  
Willy A. Noort ◽  
Jeroen Lammerts van Bueren ◽  
Berris van Kessel ◽  
Joost M. Bakker ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Research Funding ◽  
Plasma Cells ◽  
Cell Lysis ◽  
In Vitro Assays ◽  
Human Antibody ◽  
Myeloma Cells ◽  
Clinical Phase

Abstract Multiple myeloma (MM) remains an incurable malignancy of clonal plasma cells. Although the new generation of immunomodulatory agents, such as lenalidomide (LEN), and the potent proteasome inhibitor bortezomib (BORT) have significantly improved the overall survival of MM patients, all chemotherapy strategies are eventually hampered by the development of drug-resistance. The outcome of patients who are refractory to thalidomide, lenalidomide (LEN) and bortezomib (BORT) is very poor. Set out with the idea that targeted immunotherapy with human antibodies may offer new perspectives for MM patients, we have recently developed daratumumab (DARA), a CD38 human antibody with broad-spectrum killing activity, mainly via ADCC (antibody dependent cellular cytotoxicity) and CDC (complement dependent cytotoxicity). In our previous preclinical studies and in current clinical phase I/II trials, DARA induces marked anti-MM activity. Based on these encouraging results, we now explored the potential activity of DARA for patients who are refractory to LEN- and/or BORT. In a recently developed human-mouse hybrid model that allows the in vivo engraftment and outgrowth of patient-derived primary myeloma cells in immune deficient Rag2-/-gc-/- mice, single dose DARA treatment appeared to effectively inhibit the malignant expansion of primary MM cells derived from a LEN- and BORT-refractory patient, indicating the potential efficacy of DARA even in LEN/BORT refractory patients. To substantiate the conclusions of these in vivo data, we conducted in vitro assays, in which full BM-MNCs from LEN (n=11) and LEN/BORT (n=8) refractory patients were treated with DARA alone or the combination of DARA with LEN or BORT to induce MM cell lysis. As expected, LEN alone induced no or little lysis of MM cells in the LEN-refractory patients and also BORT was not able to induce any lysis in the BORT-refractory patients. On the contrary, DARA induced substantial levels of MM cell lysis in all LEN and LEN/BORT-refractory patients. This lysis was significantly enhanced by combination with LEN or BORT. The combination of DARA and BORT improved MM lysis by additive mechanisms. However, LEN improved DARA-mediated lysis of MM cells in a synergistic manner through the activation of effector cells involved in DARA-mediated ADCC. In conclusion, our results demonstrate that DARA is also effective against multiple myeloma cells derived from LEN- and BORT-refractory patients. Especially LEN seems to improve responses in a synergistic manner. Our results provide a rationale for clinical evaluation of DARA in combination with LEN to achieve more effective results in LEN- and BORT-refractory patients. Disclosures: Lammerts van Bueren: Genmab: Employment. Bakker:Genmab: Employment. Parren:Genmab: Employment. van de Donk:Celgene: Research Funding. Lokhorst:Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria.

Constitutive and Immunoproteasome Inhibitors Increase IκB and Decrease NF-κB Expression In Dendritic Cell

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3493-3493
Author(s):  
Ahmad-Samer Samer Al-Homsi ◽  
Zhongbin Lai ◽  
Tara Sabrina Roy ◽  
Niholas Kouttab
Keyword(s):  
Multiple Myeloma ◽  
T Cell ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Peripheral Blood ◽  
Research Funding ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell

Abstract Introduction Constitutive and immunoproteasome inhibitors (C&IPI) were thought to suppress nuclear factor-κB (NF-κB) pathway by preventing IκB degradation, which prevents NF-κB translocation into the nucleus. This mechanism of action has since been questioned by a number of studies. First, bortezomib promoted constitutive NF-κB activity in endothelial cell carcinoma. Second, NF-κB constitutive activity was resistant to bortezomib in multiple myeloma cell lines. Third, bortezomib increased IκB mRNA but post-transcriptionally downregulated IκB in normal cells and in multiple myeloma cell lines resulting in induced canonical NF-κB activation. Lastly, bortezomib increased nuclear levels of IκB as opposed to lowering cytoplasmic levels in cutaneous T cell lymphoma cell line suggesting that nuclear translocation of IκB was possibly responsible for NF-κB inhibition. The inhibitory activity of C&IPI on dendritic cells (DC) is of interest in the prevention of graft versus host disease (GvHD). It has been shown that different C&IPI impede DC maturation and T cell priming both in vitro and in vivo. Herein we sought to understand the mechanism of action of proteasome and immunoproteasome inhibitors on DC and to test their effect on IκB and NF-IκB expression. Materials and Methods We first performed RT PCR on lysates of DC obtained from the peripheral blood of 7 patients who received post-transplant cyclophosphamide and bortezomib as prevention of GvHD on a phase I clinical trial. Patients received allogeneic transplantation from matched-related or unrelated donors. Patients received no other immunosuppressive therapy except for rabbit anti-thymocyte globulin for those receiving graft from unrelated donor. Steroids were not allowed on the study. Samples were obtained on days +1, +4, and +7. The results were analyzed in comparison to samples obtained on day 0 before stem cell infusion. We then performed the same experiment on lysates of DC obtained from the peripheral blood of healthy volunteer donors. DC were untreated or incubated with bortezomib (10 nM for 4 h), carfilzomib (30 nM for 1 h), oprozomib (100 nM and 300 nM for 4 h), ONX 0914 (200 nM for 1 h), PR-825 (125 nM for 1 h), or PR-924 (1000 nM for 1 h). The drug concentration and duration of exposure were chosen based on the IC50 on proteasome activity and to reproduce in vivo conditions. We also performed IκB western blot on DC isolated from peripheral blood of healthy volunteers, untreated or incubated with bortezomib (10 nM for 4 h) or oprozomib (300 nM for 4 h). Each experiment was performed at least in triplicate. Results We found that the combination of cyclophosphamide and bortezomib significantly and progressively increased IκB mRNA while decreasing NF-κB mRNA in DC studied ex vivo. We also found that all studied C&IPI increased IκB mRNA to a variable degree while only oprozomib (300 nM) decreased NF-κB mRNA in DC in vitro. Finally, both bortezomib and oprozomib increased IκB protein level in DC in vitro (figure). Conclusion Our data suggest that C&IPI increase IκB expression in DC. As opposed to the previously reported data in other cell types, the effect is not associated with post-transcriptional downregulation. Cyclophosphamide and bortezomib also decrease NF-κB expression in DC in vivo while only oprozomib had the same effect in vitro. The effect of C&IPI on IκB and NF-κB expression may represent a new mechanism of action and suggests their effect may be cell-type dependent. Disclosures: Al-Homsi: Millennium Pharmaceuticals: Research Funding. Off Label Use: The use of cyclophosphamide and bortezomib for GvHD prevention. Lai:Millennium Pharmaceuticals: Research Funding.

Interfering with Arginine Metabolism As a New Treatment Strategy for Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3005-3005
Author(s):  
Bjoern Jacobi ◽  
Lea Stroeher ◽  
Nadine Leuchtner ◽  
Hakim Echchannaoui ◽  
Alexander Desuki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Caspase 3 ◽  
Xenograft Model ◽  
Myeloma Cell ◽  
Intracellular Protein ◽  
Myeloma Cells ◽  
Induction Of Apoptosis

Abstract Introduction Starvation of tumor cells from the amino acid arginine has recently gained particular interest because of the downregulation of the rate-limiting enzyme argininosuccinate synthethase 1 (ASS1) in various cancer entities. ASS1-deficient cells cannot resynthesize arginine from citrulline and are therefore considered arginine auxotrophic. The arginine depleting enzyme arginine deiminase (ADI-PEG20, Polaris Pharmaceuticals) is currently tested in phase I-III clinical trials for different arginine auxotrophic cancers. The natural arginine analogue canavanine can compete with arginine for arginyl-tRNA-binding sites and consequently be incorporated into nascent proteins instead of arginine. Canavanine could therefore potentially further disturb intracellular protein homeostasis, especially under arginine deprivation. The sensitivity of myeloma cells towards arginine depletion strategies has not been analyzed so far. Methods Human myeloma cell lines and CD138-sorted primary human myeloma cells from patient bone marrow were screened for ASS1 expression by western blotting (WB). The cells were cultured in arginine free medium and assessed for proliferation and metabolic activity (CFSE/MTT assays), apoptosis (caspase-3 cleavage) and cell death (annexinV/propidium iodide). Canavanine was supplied in both arginine-sufficient and -deficient conditions. The level of intracellular protein stress was determined by WB and/or flow cytometry analysis for ubiquitinated proteins, phosphorylated eukaryotic initiation factor 2α (peIF2α) and the spliced isoform of the X-Box binding protein 1 (Xbp1s). Repetitive ADI-PEG20 ± canavanine application i.p. were tested in vivo in an U266 myeloma xenograft model in NOD/SCID/IL2Rcg-/- (NSG) mice. Arginine and canavanine levels in plasma were determined by HPLC. Tumor growth was measured, mice were assessed for survival, weight and side effects. Tumor tissues were analyzed for caspase-3 cleavage and Ki67 expression by immunohistochemistry. Results 5 of 6 myeloma cell lines were negative for ASS1. Also, ASS1 was either not or only weakly expressed in the majority of primary CD138+ myeloma patient samples. Arginine starvation induced an arrest of cell proliferation and/or metabolic activity of primary myeloma cells and myeloma cell lines after 18-24 h. Addition of citrulline could only rescue ASS1 positive myeloma cells due to the intracellular resynthesis of arginine. Arginine starvation alone led to delayed induction of apoptosis (e.g. 35% cell death of NCI-H929 cells after 72 h of treatment). Addition of 100 mM canavanine strongly increased cell death specifically in the context of arginine deficiency (e.g. cell death in NCI-H929 cells: 87% after 24 h, 100 % after 48h) while it was non-toxic and had no effect on cell viability under physiological arginine conditions. Co-application of canavanine induced ubiquitination of cellular proteins and led to the prolongation of a fatal unfolded protein response (UPR) as measured by markedly elevated Xbp1s levels. Prolonged UPR ultimately led to the induction of apoptosis as reflected by annexin V binding and caspase-3 cleavage. In an U266 myeloma NSG xenograft model, systemic arginine depletion by ADI-PEG20 suppressed tumor growth in vivo and significantly prolonged median survival of mice when compared with the control group (22±3 vs. 15±3 days). Canavanine treatment alone had no influence on viability (13±0 days). However, the combination of ADI-PEG20 and canavanine demonstrated the longest median survival (27±7 days). Histological examination of explanted tumors showed the highest rates of caspase-3 cleavage in the ADI-PEG20/canavanine group. Conclusion Myeloma cells are mostly arginine auxotrophic and can be selectively targeted by arginine starvation. Combination of arginine depletion with the arginine analogue canavanine leads to a highly efficient and specific tumor cell eradication and should be further optimized in multiple myeloma preclinical models. Disclosures Bomalaski: Polaris Pharmaceuticals Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Targeting EZH2 in Multiple Myeloma Could be Promising for a Subgroup of MM Patients in Combination with IMiDs

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 311-311 ◽  
Author(s):  
Laurie Herviou ◽  
Alboukadel Kassambara ◽  
Stephanie Boireau ◽  
Nicolas Robert ◽  
Guilhem Requirand ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Newly Diagnosed ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Ezh2 Expression ◽  
Bone Marrow Plasma

Abstract Multiple Myeloma is a B cell neoplasia characterized by the accumulation of clonal plasma cells within the bone marrow.Epigenetics is characterized by a wide range of changes that are reversible and orchestrate gene expression. Recent studies have shown that epigenetic modifications play a role in multiple myeloma (MM) by silencing various cancer-related genes. We investigated the epigenetic genes differentially expressed between normal bone marrow plasma cells (BMPC ; N=5) and MM plasma cells from patients (N=206). Using SAM (Significance Analysis of Microarrays) analysis, only 12 genes significantly differentially expressed between BMPC and MM cells (ratio > 2 and FDR (false discovery rate) < 5%) were identified, including the EZH2 histone methyltransferase. EZH2, the enzymatic subunit of Polycomb Repressive Complex 2, is a histone methyltransferases able to repress gene expression by catalyzing H3K27me3 histone mark. EZH2 overexpression has been associated with numerous hematological malignancies, including MM. We thus studied EZH2 role in MM physiopathology and drug resistance. EZH2 expression was analyzed in normal bone marrow plasma cells (BMPCs; N=5), primary myeloma cells from newly diagnosed patients (MMCs; N=206) and human myeloma cell lines (HMCLs; N=40) using Affymetrix microarrays. EZH2 gene is significantly overexpressed in MMCs of patients (median 574, range 105 - 4562) compared to normal BMPCs (median = 432; range: 314 - 563) (P < 0.01). The expression is even higher in HMCLs (median 4481, range 581 - 8455) compared to primary MMCs or BMPCs (P < 0.001). High EZH2 expression is associated with a poor prognosis in 3 independent cohorts of newly diagnosed patients (Heidelberg-Montpellier cohort - N=206, UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N =158). Furthermore, GSEA analysis of patients with high EZH2 expression highlighted a significant enrichment of genes involved in cell cycle, downregulated in mature plasma cells vs plasmablasts, and EZH2 targets. Specific EZH2 inhibition by EPZ-6438 EZH2 inhibitor induced a significant decrease of global H3K27me3 in all the HMCLs tested (P < 0.01) and inhibited MM cell growth in 5 out of the 6 HMCLs tested. The inhibitory effect of EZH2 inhibitor on MM cell growth appeared at day 6 suggesting that it is mediated by epigenetic reprogramming. To confirm that EZH2 is also required for the survival of primary MMCs from patients, primary MM cells (n = 17 patients) co-cultured with their bone marrow microenvironment and recombinant IL-6 were treated with EPZ-6438. As identified in HMCLs, EZH2 inhibition significantly reduced the median number of viable myeloma cells by 35% (P = 0.004) from a subset of patients (n=9) while the other group (n=8) was resistant. Of interest, EPZ-6438 induced a significant global H3K27me3 decrease in both groups of patient. RNA sequencing of 6 HMCLs treated with EPZ-6438 combined with H3K27me3 ChIP analyses allowed us to create an EZ GEP-based score able to predict HMCLs and primary MM cells sensitivity to EZH2 inhibitors. We also observed a synergy between EPZ-6438 and Lenalidomide, a conventional drug used for MM treatment. More interestingly, pretreatment of myeloma cells with EPZ-6438 significantly re-sensitize drug-resistant MM cells to Lenalidomide. Investigating the effect of EPZ-6438/Lenalidomide combination in MMC, we identified that IKZF1, IRF4 and MYC protein levels were significantly more inhibited by the combination treatment (65.5%, 63.9% and 14.8% respectively) compared with Lenalidomide (51.5%, 43% and 2.2%) or EPZ-6438 (45.2%, 38.7% and 6.2%) alone. Clinical trials are ongoing with EZH2 inhibitors in lymphoma and could be promising for a subgroup of MM patients in combination with IMiDs. Furthermore, the EZ score enables identification of MM patients with an adverse prognosis and who could benefit from treatment with EZH2 inhibitors. Disclosures Goldschmidt: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Hose:EngMab: Research Funding; Takeda: Other: Travel grant; Sanofi: Research Funding.

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