MLN4924, a Novel Investigational NEDD8 Activating Enzyme Inhibitor, Exhibits Preclinical Activity In Multiple Myeloma and Waldenström's Macroglobulinemia through Mechanism Distinct From Existing Proteasome Inhibitors

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2988-2988
Author(s):  
Douglas W. McMillin ◽  
Zachary Hunter ◽  
Jake Delmore ◽  
Val Monrose ◽  
Peter G Smith ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Normal Donor ◽  
Advisory Committees ◽  
Safety Studies ◽  
Ubiquitin Proteasome ◽  
Efficacy Studies

Abstract Abstract 2988 Background: Multiple myeloma (MM) and Waldenström Macroglobulinemia (WM) have both shown clinical responses to Bortezomib therapy which blocks the elimination of ubiquitin tagged regulatory proteins by the proteasome. The NEDD8 activating enzyme (NAE)-inhibitor MLN4924 is a novel agent which demonstrates selective inhibition of the proteins for degradation in the ubiquitin pathway and may offer benefits to MM and WM patients through the more targeted approach. Methods: A panel of human MM and WM cell lines were tested for their in vitro response to MLN4924 using MTT colorimetric survival assays. MM and WM cell lines tested exhibited dose and time dependent decrease of their viability upon exposure to MLN4924 (IC50=25-150 nM). In addition, miRNA and gene expression studies in response to MLN4924 were compared to treatment of the same cells with bortezomib. In vivo safety studies were performed in mice and animal efficacy studies are ongoing in both MM and WM engrafted mice. Results: A panel of MM and WM cells were treated with MLN4924 for 72hrs and compared to the colon carcinoma line HCT116 and normal cell lines HS-5 (stroma) and THLE-3 (hepatocytes). In addition, a longitudinal assessment of viability of MM1S (MM) and BCWM1 (WM) cells during a 72hr incubation with MLN4924 (500nM) showed commitment to death <48hrs. This result, coupled with the observation that normal donor peripheral blood mononuclear cells (PBMCs) and HS-5 stromal cells were less sensitive (IC50 >1000 nM) than the MM or WM cell lines tested, suggest that this compound exhibits a rapid, tumor-selective effect at clinically relevant conditions. We also evaluated primary MM (CD138+) and WM (CD19+) patient bone marrow cells and observed sub-μ M activity by MLN4924. In addition, we tested a series of combinations of MLN4924 with dexamethasone, doxorubicin and bortezomib in both MM1S and BCWM1 cells lines and observed additive activity or greater with MLN4924. Gene expression profiling revealed distinct signatures, in MM1S and BCWM1 lines, as well as distinct patterns of gene expression changes which were induced by MLN4924 vs. bortezomib. For instance, while bortezomib potently induces a compensatory upregulation of transcripts for ubiquitin/proteasome and heat shock protein genes which, in MM1S or BCWM1 cells, were not observed in response to MLN4924 treatment. Additional studies with the proteasome inhibitor MLN9708 revealed similar patterns of expression as bortezomib. These results indicate that MLN4924 does not induce pronounced proteotoxic stress in MM or WM cells, highlighting the distinct effect of MLN4924 on the ubiquitin/proteasome pathway compared to inhibitors which target the 20S proteasome subunit. Longitudinal miRNA profiling revealed a distinct pattern of miRNA expression in MLN4924-treated vs. bortezomib-treated MM and WM cells. Lastly, animal safety studies showed that MLN4924 was tolerated at doses up to 60mg/kg 2x daily for 1 week. Efficacy studies in MM and WM are ongoing. Conclusions: MLN4924 induces cell killing at sub-μ M concentrations for both MM and WM cells with higher sensitivity of tumor cells compared to normal tissues, exhibits selective gene expression and miRNA regulation and can be safely administered to mice. These studies provide the framework for the clinical investigation of MLN4924 in MM and WM. Disclosures: McMillin: Axios Biosciences: Equity Ownership. Smith:Millennium: Employment. Birner:Millennium: Employment. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Treon:Millennium Pharmaceuticals, Genentech BiOncology, Biogen IDEC, Celgene, Novartis, Cephalon: Consultancy, Honoraria, Research Funding; Celgene Corporation: Research Funding; Novartis Corporation: Research Funding; Genentech: Consultancy, Research Funding. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

The Interaction of Bortezomib with P-Gp, MRP-1 and BCRP Drug Transporters: Implications for Therapeutic Applications of Bortezomib in Advanced Multiple Myeloma and Other Neoplasias.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1729-1729
Author(s):  
Melissa G Ooi ◽  
Robert O'Connor ◽  
Jana Jakubikova ◽  
Justine Meiller ◽  
Steffen Klippel ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Multiple Myeloma ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Significant Activity ◽  
Cancer Resistance ◽  
Advisory Committees ◽  
Bortezomib Treatment

Abstract Abstract 1729 Poster Board I-755 Background Multidrug transporters are energy-dependent transmembrane proteins which can efflux a broad range of anticancer drugs and thereby play a role in resistance to the actions of substrate agents. Classically, three transporters, p-glycoprotein (Pgp; MDR-1; ABCB1), multidrug resistant protein-1 (MRP-1; ABCC1) and breast cancer resistance protein (BCRP; MXR; ABCG2), have been found to have the broadest substrate specificity and a strong correlation with drug resistance in vitro and in vivo in many models and forms of cancer. We have sought to characterize the interaction of bortezomib with these transporters and thereby explore the potential for these agents to play a role in resistance. Bortezomib is a novel proteosome inhibitor with significant activity in multiple myeloma, although subsets of patients remain refractory to the activity of the drug. Hence, better characterization of the interactions of this drug with classical resistance mechanisms may identify improved treatment applications. Methods and Results We investigated the role of these transporters by using isogenic cell line models which are resistant due to overexpression of a particular transporter: DLKP lung cancer cell line that overexpresses MRP-1; DLKP-A which overexpresses Pgp; and DLKP-SQ-Mitox which overexpresses BCRP. DLKP-A cells exhibited a 4.6-fold decrease in responsiveness to bortezomib compared to parental DLKP cells. In DLKP-SQ-Mitox, bortezomib-induced cytotoxicity was comparable to DLKP. When bortezomib was combined with elacridar, a Pgp and BCRP inhibitor, significant synergy was evident in DLKP-A (100% viable cells with single agent treatment versus 11% with the combination), but not DLKP-SQ-Mitox. Sulindac, an MRP-1 inhibitor, combined with bortezomib failed to produce any synergy in MRP-1 positive DLKP cells. Conversely, combination assays of Pgp substrate cytotoxics such as doxorubicin with Bortezomib were largely additive in nature. This indicates that bortezomib has little, if any, direct Pgp inhibitory activity, as combinations of a traditional Pgp inhibitor (such as elacridar) and doxorubicin would show marked synergy rather than just an additive effect in Pgp positive cells. To further characterize the extent of this interaction with Pgp, we conducted cytotoxicity assays in cell lines with varying levels of Pgp overexpression. NCI/Adr-res (ovarian cancer, high Pgp overexpression), RPMI-Dox40 (multiple myeloma, moderate Pgp overexpression) and A549-taxol (lung cancer, low Pgp overexpression). The combination of bortezomib and elacridar that produced the most synergy was in cell lines expressing moderate to high levels of Pgp expression. Cell lines with lower Pgp expression produced an additive cytotoxicity. We next examined whether bortezomib had any direct effect on Pgp expression. In RPMI-Dox40 cells, Pgp expression is reduced in a time-dependent manner with bortezomib treatment. Conclusions Our studies therefore show that bortezomib is a substrate for Pgp but not the other drug efflux pumps. In tumor cells expressing high levels of Pgp, the efficacy of bortezomib is synergistically enhanced by combinations with a Pgp inhibitor, while bortezomib treatment itself can reduce the expression of Pgp. This study suggests that in the subset of patients with advanced multiple myeloma or solid tumors which express high levels of Pgp, inhibition of its function could contribute to enhanced responsiveness to bortezomib. Disclosures Richardson: millenium: Membership on an entity's Board of Directors or advisory committees, Research Funding; celgene: Membership on an entity's Board of Directors or advisory committees, speakers bureau up to 7/1/09; MLNM: speakers bureau up to 7/1/09. Mitsiades:Millennium Pharmaceuticals : Consultancy, Honoraria; Novartis Pharmaceuticals : Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: licensing royalties ; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono : Research Funding; Sunesis Pharmaceuticals: Research Funding. Anderson:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Biotest AG: Consultancy, Research Funding.

Analysis of Serial Patient Samples Reveals a Variety of Clonal Dynamics In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1923-1923
Author(s):  
Jonathan J Keats ◽  
Esteban Braggio ◽  
Scott Van Wier ◽  
Patrick Blackburn ◽  
Angela Baker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Leukemia ◽  
Advisory Committees ◽  
Tumor Mass ◽  
Time Points ◽  
Tumor Genome ◽  
Time Point

Abstract Abstract 1923 Our understanding of the genetic abnormalities associated with the development of multiple myeloma has increased significantly in the last decade. However, very little is known about how, or if, myeloma tumor genomes change with time and if therapeutic interventions influence these events. To address these issues we studied a cohort of 29 patients for whom at least two serial samples (1-65 months, median 19 months) were available for analysis. Each serial pair was analyzed by both array-based comparative genomic hybridization (aCGH) and microarray gene expression profiling (GEP) to identify DNA copy number abnormalities (CNA) at a 25kb resolution and gene expression differences present in the bulk of the tumor mass. Though this does not address the intra-clonal heterogeneity that may exist at a given time point, it does answer if the bulk of the tumor mass is changing with time. This study has unearthed several surprising and clinically relevant findings. First, myeloma tumor genomes are not as unstable as previous cytogenetic analyses suggest. In 40% of patients we observed no detectable CNA changes (1-37 months, median 12 months). In 24% of patients we observed the exclusive acquisition of new CNA (1-12, median 3.5) (3-22 months, median 18 months). In 36% of patients we observed both the loss (1-20, median 3) and gain (1-33, median 21) of CNA (5-43 months, median 20 months). Because time was not a significant influence on the detection of stable or unstable genomes we compared CNA changes with TC class and found patients with the high-risk 4p16 and maf IgH translocations were over-represented in the latter subset of patients. These observations raise the question of what happens between multiple rounds of therapy and if different regimens influence these phenotypes differently. For two patients with no CNA changes between the first two time points there was an additional sample that extended the follow-up by 52 and 12 months. Again no CNA changes were seen between diagnosis and these final samples taken 63 and 50 months later. For one patient with CNA changes (5 shared, 29 lost, and 32 gained) we have a detailed time course of 5 samples from diagnosis through to end-stage plasma cell leukemia. This patient received continuous lenalidomide-dexamethasone (Rd) for 20 months and progessed with a clone containing a BIRC2/3 deletion, which activates the NFKB pathway. The patient received single agent PR-171 and a bortezomib containing regimen and unexpectedly, the tumor genome observed in the third sample was almost identical (32 shared, 2 lost, and 4 gained CNA) to the first time point, including two copies of BIRC2/3. Subsequently, the patient received melphalan-prednisone-bortezomib (MPV) and the tumor genome observed in the fourth and fifth samples, which were identical, were similar to that seen in the second sample (24 shared, 13 lost, and 39 gained CNA). To understand these observations better we performed FISH to ascertain if the observed clones were detectable earlier, albeit at a low frequency. These experiments proved that the two dominant subclones observed at time points 1 and 3 versus 2, 4, 5 were mutually exclusive at the single cell level. Moreover, both of these clones were detectable at diagnosis with 12% of the tumor mass being the second subclone that eventually evolved into plasma cell leukemia. Interestingly, we assayed 5 of the 39 unique CNA observed in the final two samples and only one, the 17p13 deletion, was detectable earlier. This suggests the MPV regimen effectively eliminated a clone that was previously sensitive to Rd and selected for a dramatically evolved subclone that was previously sensitive to two different proteasome inhibitors. Although it is clear that the high-risk patients are enriched in the subset with the most changes, it is not clear if the specific drugs used (Melphalan vs IMID vs proteasome inhibitor) or intervention strategy (Cycled vs continuous/maintenance) and perhaps the response achieved (PR vs CR) influences these events. These observations do highlight two important clinical concepts that need to be considered in the future. First, the meaning of a partial response needs further investigation as this may reflect effective elimination of one subclone but not another. Second, because some patients are not changing or can revert back to a previous subclone we need to consider re-chanllenging patients with previously effective regimens when patients progress. Disclosures: Fonseca: Genzyme: Consultancy; Medtronic: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Otsuka: Consultancy; Celgene: Consultancy, Research Funding; Intellikine: Consultancy; Cylene: Research Funding; Onyx: Research Funding; FISH probes prognostication in myeloma: Patents & Royalties. Stewart:Millennium: Consultancy; Celgene: Honoraria. Bergsagel:Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Millennium: Speakers Bureau; Novartis: Speakers Bureau.

CYC065, a Potent Derivative of Seliciclib Is Active In Multiple Myeloma In Preclinical Studies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2999-2999 ◽  
Author(s):  
Samantha Pozzi ◽  
Diana Cirstea ◽  
Loredana Santo ◽  
Doris M Nabikejje ◽  
Kishan Patel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Preliminary Data ◽  
Research Funding ◽  
Preclinical Studies ◽  
Dependent Manner ◽  
Advisory Committees

Abstract Abstract 2999 Multiple myeloma (MM) is a treatable but incurable hematological malignancy and novel targeted therapies are under investigation. MM is characterized by dysregulation of the cell cycle, consequent to the overexpression of cyclins and their related kinases, the cyclins dependent kinases (CDK), a group of Ser/Thr proteine kinases. CDKs represent a promising therapeutic target, and inhibitors have been developed for anticancer treatment. We have previously studied seliciclib in the context of MM. CYC065, a second generation CDK inhibitor is the more potent derivative of seliciclib. It is mainly active on CDK 2, 5 and 9, involved in progression of the cell cycle and protein transcription. It has already shown promising results in preclinical studies in breast cancer and acute leukemia. We tested CYC065 in in vitro experiments in MM. Our preliminary data in 7 MM cell lines showed cytotoxicity of CYC065, both in MM cell lines sensitive as well as resistant to conventional chemotherapy, with an IC50 ranging between 0.06 and 2μ M, at 24 and 48h. Tritiated thymidine uptake assay confirmed the antiproliferative effects of CYC065 in MM, and its ability to overcome the growth advantage conferred by co-culture with bone marrow stromal cells derived from MM patients, and cytokines like interleukin 6 (10ng/ml) and insulin like growth factor-1 (50ng/ml). The anti-proliferative effect was evident both at 24 and 48h, starting at concentrations as low as 0.015μ M. The AnnexinV/PI assay in the MM1.s cell line confirmed CYC065's ability to induce apoptosis in a time dependent manner starting at 9 hours of treatment, at a concentration of 0.125 μ M, inducing 82% of apoptosis after 48h of exposure. Cell cycle analysis in the same MM1.s cell line showed an increase of subG1 phase, starting at 9 hours of treatment, at 0.125 μ M of CYC065. Preliminary results of western blot analysis confirmed the apoptotic effect of CYC065 in the MM1s cell line, highlighted by the cleavage of caspase 3, 8, 9 and PARP. The compound was tested in primary CD138+ cells isolated from three refractory MM patients, confirming its efficacy at 0.125 μ M, both at 24 and 48h. Comparative analysis in PBMCs from normal donors, for the evaluation of the drug toxicity is ongoing and will be presented. In conclusion our preliminary data confirm the efficacy of CYC065 in MM cell lines and primary MM cells, at nanomolar concentrations. Ongoing mechanistic and in vivo studies will delineate its role in the now increasing spectrum of CDK inhibitors in MM and better define its potential for clinical development in MM. Disclosures: Green: Cyclacel: Employment. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Acetylon: Research Funding.

Lin28B/Let-7 Axis Regulates Multiple Myeloma Proliferation By Enhancing c-Myc and Ras Survival Pathways

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 273-273
Author(s):  
Salomon Manier ◽  
John T Powers ◽  
Antonio Sacco ◽  
Michaela R Reagan ◽  
Michele Moschetta ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Board ◽  
Loss Of Function ◽  
Advisory Committees ◽  
Expression Levels ◽  
Gain And Loss

Abstract Background MicroRNAs (miRNAs) play a pivotal role in tumorigenesis, due to their ability to target mRNAs involved in the regulation of cell proliferation, survival and differentiation. Lin28B is an RNA binding protein that regulates Let-7 miRNA maturation. Lin28B and Let-7 have been described to act as oncogenes or tumor suppressor genes, respectively, as demonstrated both in solid cancer and hematologic malignancies. However, the role of the Lin28B/Let-7 axis in Multiple Myeloma (MM) has not been studied. Method Lin28B level expression in MM patients was studied using previously published gene expression profiling (GEP) datasets. Let-7 expression levels were assessed in CD138+ primary MM cells and bone marrow stromal cells (BMSCs) by using PCR, as well as in circulating exosomes using miRNA array (Nanostring® Technology). Exosomes were collected from both normal and MM peripheral blood, using ultracentrifugation; and further studied by using electron microscopy and immunogold labeling for the detection of CD63 and CD81. The knockdown of Lin28B was performed on MM cell lines (U266, MM.1S, MOLP-8) by using a lentiviral Lin28B shRNA. Gain- and loss-of function studies for Let-7 were performed using Let-7 mimic and anti-Let-7 transfection in MM cell lines (MM1S, U266) and primary BMSCs. Cell proliferation has been evaluated by using thymidine assays. Effects of Let-7 and Lin28B on signaling cascades have been evaluated by western blot. Results Two independent GEP datasets (GSE16558; GSE2658) were analyzed for Lin28B expression, showing a significantly higher level in MM patients compared to healthy controls. In addition, high Lin28B levels correlated with a shorter overall survival (p = 0.0226). We next found that the Let-7 family members are significantly down-regulated in MM primary cells, particularly Let-7a and b (5 fold change, p < 0.05), as demonstrated by using qRT-PCR. Similarly, miRNA arrays showed a lower expression of Let-7-related miRNAs in circulating exosomes obtained from MM patients compared to healthy individuals. We further dissected the functional relevance of Lin28B in MM cells, by performing Lin28 knockdown (KD) in MM cell lines (U266, MOLP-8). This led to a significant decrease in MM cell proliferation associated with G1 phase cell cycle arrest. This was supported by up-regulation of Let-7 and down-regulation of c-Myc, Ras and Cyclin D1 in Lin28 KD MM cells. To further prove that Lin28B-dependent effects on MM cells are mediated by Let7, we next showed that let-7 gain- and loss-of-function studies regulate MM cell proliferation and Myc expression. Lin28B regulation in MM cells is dependent on Let-7, as demonstrated by an increase of both cell proliferation and c-Myc expression after anti-Let-7 transfection in the Lin28B KD cells. We therefore studied the regulation of Let-7 in MM cells through the interaction with BMSCs. Let-7 expression levels were significantly lower in BMSCs obtained from MM patients compared to healthy donors. Interestingly, the Let-7 expression level in MM cells was increased after co-culture with Let-7 over-expressing BMSCs, associated with a decrease of both cell proliferation and c-Myc expression. This suggests a potential transfer of Let-7 from BMSCs to MM cells. Conclusion This work describes a new signaling pathway involving Lin28B, Let-7, Myc and Ras in MM. Let-7 expression in MM cells is also regulated through the interaction of MM cells with BMSCs, leading to cell proliferation and Myc regulation in MM. Interference with this pathway might offer therapeutic perspectives. Disclosures: Leleu: CELGENE: Honoraria; JANSSEN: Honoraria. Daley:Johnson and Johnson: Consultancy, Membership on an entity’s Board of Directors or advisory committees; MPM Capital: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Verastem: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Epizyme: Consultancy, Membership on an entity’s Board of Directors or advisory committees; iPierian: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Solasia, KK: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

Expression Of Truncated Protein Tyrosine Phosphatase, Receptor Type, O (PTPROt) Influences Multiple Myeloma Sensitivity To Bortezomib Through Akt Signaling, and Is a Favorable Clinical Prognostic Factor

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2520-2520
Author(s):  
Hua Wang ◽  
Veerabhadran Baladandayuthapani ◽  
Zhiqiang Wang ◽  
Jiexin Zhang ◽  
Heather Yan Lin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Myeloma Cell ◽  
Advisory Committees ◽  
Over Expression ◽  
Resistant Disease ◽  
Drug Naïve ◽  
Rpmi 8226

Abstract Background Proteasome inhibitors such as bortezomib and carfilzomib are an important part of our current chemotherapeutic armamentarium against multiple myeloma, and have improved outcomes in the up-front, relapsed, and relapsed/refractory settings. Their efficacy has been demonstrated both as single agents, and as part of rationally designed combination regimens, but they are at this time used empirically, since biomarkers to identify patients who would most or least benefit from their application have not been clinically validated. Moreover, the vast majority of patients eventually develop drug-resistant disease which precludes further proteasome inhibitor use through mechanisms that have not been fully elucidated. Methods We compared gene expression profiles (GEPs) of a panel of bortezomib-resistant myeloma cell lines and their vehicle-treated, drug-naïve counterparts to identify significant changes associated with drug resistance. The list of genes whose expression was changed by at least 2-fold was compared with independent RNA interference studies whose goal was to identify genes whose suppression conferred drug resistance. Further validation of genes of interest was pursued in a panel of myeloma cell lines, and in clinically annotated GEP databases. Results Suppression of PTPROt expression was noted in bortezomib-resistant RPMI 8226 and ANBL-6 myeloma cells compared to isogenic, drug-naïve controls, and this was confirmed by quantitative PCR. Overexpression of PTRPOt in RPMI 8226, ANBL-6 and other myeloma cell lines was by itself sufficient to increase the level of apoptotic, sub-G0/G1 cells compared to vector controls, or cells expressing a phosphatase-dead PTPROt mutant. Moreover, PTPROt enhanced the ability of bortezomib to reduce myeloma cell viability, in association with increased activation of caspases 8 and 9. Exogenous over-expression of PTPROt was found to reduce the activation status of Akt, a known anti-apoptotic pathway that reduces bortezomib activity, based on Western blotting with antibodies to phospho-Akt (Ser473), and Akt kinase activity assays. Notably, we also found that exogenous over-expression of PTPROt resulted in increased expression levels of p27Kip1. Interestingly, array CGH data from studies of myeloma cell lines and primary cells showed that the PTPROt gene was located in a genomic region with a high propensity for loss. Analysis of the Total Therapy databases of GEP and patient outcomes available on the Multiple Myeloma Genomics Portal showed that higher than median expression of PTPROt was associated with better long-term survival (P=0.0175). Finally, analysis of the Millennium Pharmaceuticals database of studies of bortezomib in the relapsed and relapsed/refractory setting showed high PTRPOt expression was more frequently seen in patients who achieved complete remission (P<0.01), and was associated with a better median overall survival (P=0.0003). Conclusions Taken together, the data support the possibility that high expression of PTPROt is a good prognostic factor for response to bortezomib-containing therapies, and that this may occur through modulation by PTPROt of the Akt pathway. Moreover, they suggest that strategies to enhance the expression of PTPROt should be investigated to restore bortezomib sensitivity in patients with proteasome inhibitor-resistant disease. Disclosures: Orlowski: Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, 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, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees.

scholarly journals Dysregulation of Splicing in Multiple Myeloma: The Splicing Factor SRSF1 Supports MM Cell Proliferation Via Splicing Control

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4500-4500
Author(s):  
Mariateresa Fulciniti ◽  
Michael A Lopez ◽  
Anil Aktas Samur ◽  
Eugenio Morelli ◽  
Hervé Avet-Loiseau ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Alternative Splicing ◽  
Board Of Directors ◽  
Research Funding ◽  
Splicing Factor ◽  
Splicing Factors ◽  
Rna Seq ◽  
Advisory Committees ◽  
Disease Biology

Abstract Gene expression profile has provided interesting insights into the disease biology, helped develop new risk stratification, and identify novel druggable targets in multiple myeloma (MM). However, there is significant impact of alternative pre-mRNA splicing (AS) as one of the key transcriptome modifier. These spliced variants increases the transcriptomic complexity and its misregulation affect disease behavior impacting therapeutic consideration in various disease processes including cancer. Our large well annotated deep RNA sequencing data from purified MM cells data from 420 newly-diagnosed patients treated homogeneously have identified 1534 genes with one or more splicing events observed in at least 10% or more patients. Median alternative splicing event per patient was 595 (range 223 - 2735). These observed global alternative splicing events in MM involves aberrant splicing of critical growth and survival genes affects the disease biology as well as overall survival. Moreover, the decrease of cell viability observed in a large panel of MM cell lines after inhibition of splicing at the pre-mRNA complex and stalling at the A complex confirmed that MM cells are exquisitely sensitive to pharmacological inhibition of splicing. Based on these data, we further focused on understanding the molecular mechanisms driving aberrant alternative splicing in MM. An increasing body of evidence indicates that altered expression of regulatory splicing factors (SF) can have oncogenic properties by impacting AS of cancer-associated genes. We used our large RNA-seq dataset to create a genome wide global alterations map of SF and identified several splicing factors significantly dysregulated in MM compared to normal plasma cells with impact on clinical outcome. The splicing factor Serine and Arginine Rich Splicing Factor 1 (SRSF1), regulating initiation of spliceosome assembly, was selected for further evaluation, as its impact on clinical outcome was confirmed in two additional independent myeloma datasets. In gain-of (GOF) studies enforced expression of SRSF1 in MM cells significantly increased proliferation, especially in the presence of bone marrow stromal cells; and conversely, in loss-of function (LOF) studies, downregulation of SRSF1, using stable or doxy-inducible shRNA systems significantly inhibited MM cell proliferation and survival over time. We utilized SRSF1 mutants to dissect the mechanisms involved in the SRSF1-mediated MM growth induction, and observed that the growth promoting effect of SRSF1 in MM cells was mainly due to its splicing activity. We next investigated the impact of SRSF1 on allelic isoforms of specific gene targets by RNA-seq in LOF and confirmed in GOF studies. Splicing profiles showed widespread changes in AS induced by SRSF1 knock down. The most recurrent splicing events were skipped exon (SE) and alternative first (AF) exon splicing as compared to control cells. SE splice events were primarily upregulated and AF splice events were evenly upregulated and downregulated. Genes in which splicing events in these categories occurred mostly did not show significant difference in overall gene expression level when compared to control, following SRSF1 depletion. When analyzing cellular functions of SRSF1-regulated splicing events, we found that SRSF1 knock down affects genes in the RNA processing pathway as well as genes involved in cancer-related functions such as mTOR and MYC-related pathways. Splicing analysis was corroborated with immunoprecipitation (IP) followed by mass spectrometry (MS) analysis of T7-tagged SRSF1 MM cells. We have observed increased levels of SRSF phosphorylation, which regulates it's subcellular localization and activity, in MM cell lines and primary patient MM cells compared to normal donor PBMCs. Moreover, we evaluated the chemical compound TG003, an inhibitor of Cdc2-like kinase (CLK) 1 and 4 that regulate splicing by fine-tuning the phosphorylation of SR proteins. Treatment with TG003 decreased SRSF1 phosphorylation preventing the spliceosome assembly and inducing a dose dependent inhibition of MM cell viability. In conclusions, here we provide mechanistic insights into myeloma-related splicing dysregulation and establish SRSF1 as a tumor promoting gene with therapeutic potential. Disclosures Avet-Loiseau: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, 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, Research Funding. Munshi:OncoPep: Other: Board of director.

scholarly journals High Levels of APOBEC3B Gene Expression Contribute to Poor Prognosis in Multiple Myeloma Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3897-3897
Author(s):  
Valeriy V Lyzogubov ◽  
Pingping Qu ◽  
Cody Ashby ◽  
Adam Rosenthal ◽  
Antje Hoering ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Board Of Directors ◽  
Research Funding ◽  
Poor Prognosis ◽  
Target Genes ◽  
Plasma Cells ◽  
Nuclear Fraction ◽  
Advisory Committees ◽  
Significant Difference

Abstract Introduction: Poor prognosis and drug resistance in multiple myeloma (MM) is associated with increased mutational load. APOBEC3B is a major contributor to mutagenesis, especially in myeloma patients with t(14;16) MAF subgroup. It was shown recently that presence of the APOBEC signature at diagnosis is an independent prognostic factor for progression free survival (PFS) and overall survival (OS). We hypothesized that high levels of APOBEC3B gene expression at diagnosis may also have a prognostic impact in myeloma. To consider APOBEC3B as a potential target for therapy more studies are necessary to understand how APOBEC3B expression is regulated and how APOBEC3B generates mutations. Methods: Gene expression profiling (GEP, U133 Plus 2.0) of MM patients was performed. APOBEC3B gene expression levels were investigated in plasma cells of healthy donors (HD; n=34), MGUS (n=154), smoldering myeloma (SMM; n=219), MM low risk (LR; n=739), MM high risk (HR; n=129), relapsed MM (RMM; n=74), and primary plasma cell leukemia (pPCL; n=19) samples. The samples from relapse were taken on or after the progression/relapse date but within 30 days after progression/relapse from Total Therapy trials 3, 4, 5 & 6. GEP70 score was used to separate samples into LR and HR groups. We also investigated APOBEC3B expression in different MM molecular subgroups and used logrank statistics with covariate frequency distribution to determine an optimal cut off APOBEC3B expression value. Gene expression was compared in cases with low expression of APOBEC3B (log2<7.5) and high expression of APOBEC3B (log2>10), and an optimal cut-point in APOBEC3B expression was identified with respect to PFS. To explore the role of MAF and the non-canonical NF-ĸB pathway we performed functional studies using a cellular model of MAF downregulation. TRIPZ lentiviral shRNA MAF knockdown in the RPMI8226 cell lines was used to explore MAF-dependent genes. NF-ĸB proteins, p52 and RelB, were investigated in the nuclear fraction by immunoblot analysis. Results: Expression of APOBEC3B in HD control samples (log2=10.9) was surprisingly higher than in MGUS (log2=9.51), SMM (log2=9.09), and LR (log2=9.40) and was comparable to HR (log2=10.4) and RMM (log2=10.6) groups. Expression levels of APOBEC3B were gradually increased as disease progressed from SMM to pPCL. The high expression of APOBEC3B in HD places plasma cells at risk of APOBEC induced mutagenesis where the regulation of APOBEC3B function is compromised. The correlation between APOBEC3B expression and GEP70 score in MM was 0.37, and there was a significant difference in APOBEC3B expression between GEP70 high and low risk groups (p=0.0003). An optimal cut-point in APOBEC3B expression of log2=10.2 resulted in a significant difference in PFS (median 5.7 yr vs.7.4 yr; p=0.0086) and OS (median 9.1 yr vs. not reached; p<0.0001), between high and low expression. The highest APOBEC3B expression was detected in cases with a t(14;16). We analyzed t(14;16) cases with the APOBEC mutational signature and compared them to t(14;16) cases without the APOBEC signature and found elevated MAF (2-fold) and APOBEC3B (2.7-fold) gene expression in samples with the APOBEC signature. No APOBEC signature was detected in SMM cases, including those with a t(14;16). High APOBEC3B levels in myeloma patients was associated with overexpression of genes related to response to DNA damage and cell cycle control. Significant (p<0.05) increases of NF-κB target genes was seen in high APOBEC3B cases: TNFAIP3 (4.4-fold), NFKB2 (1.7-fold), NFKBIE (1.9-fold), RELB (1.4-fold), NFKBIA (2.0-fold), PLEK (2.5-fold), MALT1 (2.5-fold), WNT10A (2.4-fold). However, in t(14;16) cases there was no significant increase of NF-κB target genes except BIRC3 (2.5-fold) and MALT1 (2.0-fold). MAF downregulation in RPMI8226 cells did not lead to changes in NF-κB target gene expression but MAF-dependent genes were identified, including ETS1, SPP1, RUNX2, HGF, IGFBP2 and IGFBP3. Analysis of nuclear fraction of NF-ĸB proteins did not show significant changes in expression of p52 and RelB in RPMI8226 cells after MAF downregulation. Conclusions: Increased expression of APOBEC3B is a negative prognostic factor in multiple myeloma. MAF is a major factor regulating expression of APOBEC3B in the t(14;16) subgroup. NF-ĸB pathway activation is most likely involved in upregulation of APOBEC3B in non-t(14;16) subgroups. Disclosures Davies: TRM Oncology: Honoraria; MMRF: Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy. Morgan:Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Takeda: Consultancy, Honoraria.

scholarly journals Baseline and on-Treatment Bone Marrow Microenvironments Predict Myeloma Patient Outcomes and Inform Potential Intervention Strategies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1882-1882 ◽  
Author(s):  
Samuel A Danziger ◽  
Mark McConnell ◽  
Jake Gockley ◽  
Mary Young ◽  
Adam Rosenthal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Expression Profiles ◽  
Cell Types ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Introduction The multiple myeloma (MM) tumor microenvironment (TME) strongly influences patient outcomes as evidenced by the success of immunomodulatory therapies. To develop precision immunotherapeutic approaches, it is essential to identify and enumerate TME cell types and understand their dynamics. Methods We estimated the population of immune and other non-tumor cell types during the course of MM treatment at a single institution using gene expression of paired CD138-selected bone marrow aspirates and whole bone marrow (WBM) core biopsies from 867 samples of 436 newly diagnosed MM patients collected at 5 time points: pre-treatment (N=354), post-induction (N=245), post-transplant (N=83), post-consolidation (N=51), and post-maintenance (N=134). Expression profiles from the aspirates were used to infer the transcriptome contribution of immune and stromal cells in the WBM array data. Unsupervised clustering of these non-tumor gene expression profiles across all time points was performed using the R package ConsensusClusterPlus with Bayesian Information Criterion (BIC) to select the number of clusters. Individual cell types in these TMEs were estimated using the DCQ algorithm and a gene expression signature matrix based on the published LM22 leukocyte matrix (Newman et al., 2015) augmented with 5 bone marrow- and myeloma-specific cell types. Results Our deconvolution approach accurately estimated percent tumor cells in the paired samples compared to estimates from microscopy and flow cytometry (PCC = 0.63, RMSE = 9.99%). TME clusters built on gene expression data from all 867 samples resulted in 5 unsupervised clusters covering 91% of samples. While the fraction of patients in each cluster changed during treatment, no new TME clusters emerged as treatment progressed. These clusters were associated with progression free survival (PFS) (p-Val = 0.020) and overall survival (OS) (p-Val = 0.067) when measured in pre-transplant samples. The most striking outcomes were represented by Cluster 5 (N = 106) characterized by a low innate to adaptive cell ratio and shortened patient survival (Figure 1, 2). This cluster had worse outcomes than others (estimated mean PFS = 58 months compared to 71+ months for other clusters, p-Val = 0.002; estimate mean OS = 105 months compared with 113+ months for other clusters, p-Val = 0.040). Compared to other immune clusters, the adaptive-skewed TME of Cluster 5 is characterized by low granulocyte populations and high antigen-presenting, CD8 T, and B cell populations. As might be expected, this cluster was also significantly enriched for ISS3 and GEP70 high risk patients, as well as Del1p, Del1q, t12;14, and t14:16. Importantly, this TME persisted even when the induction therapy significantly reduced the tumor load (Table 1). At post-induction, outcomes for the 69 / 245 patients in Cluster 5 remain significantly worse (estimate mean PFS = 56 months compared to 71+ months for other clusters, p-Val = 0.004; estimate mean OS = 100 months compared to 121+ months for other clusters, p-Val = 0.002). The analysis of on-treatment samples showed that the number of patients in Cluster 5 decreases from 30% before treatment to 12% after transplant, and of the 63 patients for whom we have both pre-treatment and post-transplant samples, 18/20 of the Cluster 5 patients moved into other immune clusters; 13 into Cluster 4. The non-5 clusters (with better PFS and OS overall) had higher amounts of granulocytes and lower amounts of CD8 T cells. Some clusters (1 and 4) had increased natural killer (NK) cells and decreased dendritic cells, while other clusters (2 and 3) had increased adipocytes and increases in M2 macrophages (Cluster 2) or NK cells (Cluster 3). Taken together, the gain of granulocytes and adipocytes was associated with improved outcome, while increases in the adaptive immune compartment was associated with poorer outcome. Conclusions We identified distinct clusters of patient TMEs from bulk transcriptome profiles by computationally estimating the CD138- fraction of TMEs. Our findings identified differential immune and stromal compositions in patient clusters with opposing clinical outcomes and tracked membership in those clusters during treatment. Adding this layer of TME to the analysis of myeloma patient baseline and on-treatment samples enables us to formulate biological hypotheses and may eventually guide therapeutic interventions to improve outcomes for patients. Disclosures Danziger: Celgene Corporation: Employment, Equity Ownership. McConnell:Celgene Corporation: Employment. Gockley:Celgene Corporation: Employment. Young:Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment, Equity Ownership. Reiss:Celgene Corporation: Employment, Equity Ownership. Davies:MMRF: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; TRM Oncology: Honoraria; Abbvie: Consultancy; ASH: Honoraria; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria. Copeland:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Fitch:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership. Barlogie:Celgene: Consultancy, Research Funding; Dana Farber Cancer Institute: Other: travel stipend; Multiple Myeloma Research Foundation: Other: travel stipend; International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; Millenium: Consultancy, Research Funding; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend; Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC. Trotter:Celgene Research SL (Spain), part of Celgene Corporation: Employment, Equity Ownership. Hershberg:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Dervan:Celgene Corporation: Employment, Equity Ownership. Ratushny:Celgene Corporation: Employment, Equity Ownership. Morgan:Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding.

scholarly journals Hypersialylation Protects Multiple Myeloma Cells from NK Cell-Mediated Immunosurveillance and This Can be Overcome By Targeted Desialylation Using a Sialyltransferase Inhibitor

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 138-138
Author(s):  
John Daly ◽  
Subhashis Sarkar ◽  
Alessandro Natoni ◽  
Robert Henderson ◽  
Dawn Swan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Sialic Acid ◽  
Nk Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Immune Evasion ◽  
Research Funding ◽  
Nk Cell ◽  
Advisory Committees

Introduction: Evading Natural Killer (NK) cell-mediated immunosurveillance is key to the development of Multiple Myeloma (MM). Recent attention has focused on the role of hypersialylation in facilitating immune-evasion of NK cells. Abnormal cell surface sialylation is considered a hallmark of cancer and we have implicated hypersialylation in MM disease progression. Certain sialylated glycans can act as ligands for the sialic acid-binding immunoglobulin-like lectin (Siglec) receptors expressed by NK cells (Siglec-7 and Siglec-9). These ITIM motif-containing inhibitory receptors transmit an inhibitory signal upon sialic acid engagement. We hypothesized that desialylation of MM cells or targeted interruption of Siglec expression could lead to enhanced NK cell mediated cytotoxicity of MM cells. Methodology: MM cells were treated with the sialidase neuraminidase prior to co-culture with primary NK (PNK) cells. MM cells were treated with 300µM 3Fax-Neu5Ac (sialyltransferase inhibitor) for 3 days prior to co-cultures with PNK cells. PNK cells were expanded, IL-2 activated (500U/ml) overnight, or naïve (resting). Primary MM samples/MM cell lines were screened with Siglec-7/9 chimeras (10µg/ml). PNK (IL-2 activated) cells were stained with anti-Siglec-7 and anti-Siglec-9 antibodies. Siglec-7 was targeted for knockout (KO) using the CRISPR/Cas9 system, a pre-designed guideRNA and the MaxCyteGT transfection system. MM cells were treated with 10µg/ml of Daratumumab prior to co-culture with expanded PNK cells. Results: Using recombinant Siglec-7/9 chimeras a panel of MM cell lines (MM1S, RPMI-8226, H929, JJN3 and U266) were shown to express ligands for Siglec-7 and Siglec-9 (&gt;85%, n=3). Primary MM cells isolated from BM of newly diagnosed (n=3) and relapsed patients (n=2) were also shown to express Siglec-7 ligands (72.5±17.5%, 36.5% respectively). PNK cells express Siglec-7 and Siglec-9 (94.3±3.3% and 61±8.8% respectively, n=6). Desialylation of the MM cell lines JJN3 and H929 using neuraminidase significantly enhanced killing of MM cells by healthy donor (HD) derived PNK cells (expanded, IL-2 activated and naïve, n=7) at multiple effector:target (E:T) cell ratios. Furthermore, de-sialylation of JJN3 and H929 using neuraminidase resulted in increased NK cell degranulation (CD107α expression), compared to a glycobuffer control (n=7). De-sialylation, using 300µM 3Fax-Neu5Ac, resulted in strongly enhanced killing of MM1S by expanded HD-derived PNK cells at multiple E:T ratios (n=5, p&lt;0.01 at 0.5:1, p&lt;0.001 at 1:1, p&lt;0.01 at 2.5:1). Furthermore, CD38 expression on H929 MM cells significantly increased after treatment with 300µM 3Fax-Neu5Ac for 3 days (p&lt;0.01, n=3). In a cytotoxicity assay, expanded PNK cell-mediated antibody dependent cellular cytotoxicity (ADCC) of H929 MM cells pre-treated with Daratumumab (anti-CD38 moAb) and 3Fax-Neu5Ac was significantly higher than H929 cells pre-treated with Dara (p&lt;0.05 at 0.5:1, p&lt;0.01 at 1:1) or 3Fax-Neu5Ac (p&lt;0.01 at 0.5:1, p&lt;0.01 at 1:1) alone (n=5). Using CRISPR/Cas9, over 50% complete KO of Siglec-7 was observed on expanded PNK cells, yet did not result in enhanced NK cell-mediated cytotoxicity against either H929 or JJN3 (n=7). Siglec-9 KO using CRISPR/Cas9 is ongoing. Discussion: Hypersialylation of MM cells facilitates immune evasion and targeted removal of sialic acid strongly enhances the cytotoxicity of NK cells against MM. However, to date the role of Siglecs remains inconclusive. Nevertheless, our data suggest that targeted desialylation is a novel therapeutic strategy worth exploring in MM. In particular, upregulation of CD38 provides a strong rationale for combinatory strategies employing targeted desialylation with CD38 moAbs such as Daratumumab, with the goal of maximizing ADCC. Disclosures Sarkar: Onkimmune: Research Funding. O'Dwyer:Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; GlycoMimetics Inc: Research Funding; AbbVie: Consultancy.

scholarly journals Activating KRAS, NRAS, and BRAF Mutants Enhance Proteasome Capacity and Reduce Endoplasmic Reticulum Stress in Multiple Myeloma, Thereby Promoting Plasma Cell Survival and Proteasome Inhibitor Resistance

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 406-406
Author(s):  
Fazal Shirazi ◽  
Richard J. Jones ◽  
Isere Kuiatse ◽  
Zuzana Berkova ◽  
Hua Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Transcription Factor ◽  
Board Of Directors ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Research Funding ◽  
Mek Inhibitor ◽  
Mapk Signaling ◽  
Dominant Negative ◽  
Advisory Committees

Abstract Introduction: Multiple myeloma, a malignant proliferation of differentiated plasma cells, is the second most commonly diagnosed hematologic malignancy, and the number of cases may grow by almost 60% between 2010 and 2030. Recent therapeutic advances, including the use of proteasome inhibitors (PIs), have contributed to a doubling of the median overall survival in myeloma patients. This has been paralleled by an increased understanding of the mutational spectrum in this disease, which was first noted almost three decades ago to harbor KRAS and NRAS mutations. KRAS, NRAS, and BRAF mutations which induce p44/42 Mitogen-activated protein kinase (MAPK) signaling are found in about half of myeloma patients, and seem to contribute to proteasome inhibitor (PI) resistance, but the underlying mechanisms still remains elusive. Methods: ANBL-6 and U266 human-derived myeloma cell lines have endogenous wild-type (WT) KRAS, NRAS, and BRAF, and were used in this study. All cell lines were validated through The MD Anderson Cancer Center Characterized Cell Line Core Facility. We established lines stably expressing WT, constitutively active (CA)(G12V/G13D/Q61H), or dominant negative (DN)(S17N) KRAS and NRAS mutants, or V600E or DN BRAF. Cell viability was evaluated using the WST-1 tetrazolium reagent, while the chymotrypsin-, trypsin- and caspase-like activities were determined using fluorogenic substrates. Results: CA KRAS, NRAS, and BRAF mutants reduced the sensitivity of ANBL-6 and U266 cells to bortezomib and carfilzomib, while their DN variants sensitized cells to both PIs. This was associated with an induction by these CA mutants of the proteasome chymotrypsin-, trypsin- and caspase-like activities, while the DN variants reduced proteasome activity. These activity changes occurred in parallel with increased expression at both the mRNA and protein levels of catalytically active Proteasome subunit beta (PSMB)-8, PSMB9, and PSMB10, and of the proteasome assembly chaperone Proteasome maturation protein (POMP). Mechanistic studies showed that MAPK induction by the CA mutants caused activation of the ETS transcription factor (ELK1), which was found to have consensus binding sites in the promoters of PSMB8, PSMB9, PSMB10, and POMP. Notably, ELK1 suppression reduced PSMB8, PSMB9, PSMB10, and POMP expression, directly linking RAS/RAF/MAPK signaling to proteasome biology, and this suppression enhanced PI sensitivity. Inhibition of MAPK signaling with either the MAPK kinase (MEK) inhibitor selumetinib or the pan-RAF inhibitor TAK-632 showed synergistic activity with either bortezomib or carfilzomib that was more consistent in cell lines harboring CA mutants as opposed to the DN or WT constructs. Combination regimens of selumetinib or TAK-632 with either bortezomib or carfilzomib induced greater inhibition of the proteasome chymotrypsin-, trypsin- and caspase-like activities than the PIs as single agents. Finally, CA KRAS, NRAS, and BRAF mutants reduced expression levels of genes and proteins involved in the unfolded protein response (UPR), including Activating transcription factor (ATF)-4, -5, and -6, as well as C/EBP homologous protein transcription factor (CHOP) and the spliced variant of X-box binding protein 1 (XBP1s). In contrast, their dominant negative counterparts enhanced expression of the UPR effectors, consistent with an increase in endoplasmic reticulum (ER) stress. Conclusion: Taken together, the data support the hypothesis that activating MAPK pathway mutations enhance PI resistance by increasing proteasome capacity, and provide a rationale for targeting such patients with PI/RAF or PI/MEK inhibitor combinations. Moreover, they argue that these mutations promote plasma cell survival by reducing cellular stress, thereby distancing myeloma cells from the apoptotic threshold, potentially explaining their high frequency in myeloma. Disclosures Lee: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies Corporation: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Chugai Biopharmaceuticals: Consultancy; Takeda Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees. Dick:Takeda Oncology: Employment, Equity Ownership. Chattopadhyay:Takeda Oncology: Employment. Orlowski:Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; BioTheryX, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Poseida: Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Sign in / Sign up

Export Citation Format

Share Document