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.

Proteolytic Targeting Chimeric Molecules (PROTACs) Specific for Bromodomain-Containing Protein (BRD) 4 Are Active Against Pre-Clinical Models of Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 917-917 ◽  
Author(s):  
Xiaohui Zhang ◽  
Jing Lu ◽  
Yimin Qian ◽  
Robert Z. Orlowski
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Myeloma Cell ◽  
Employment Equity ◽  
Advisory Committees ◽  
Clinical Models ◽  
Equity Ownership ◽  
Rpmi 8226

Abstract Background: BRD4, a bromodomain and extraterminal domain (BET) family member, has an important role in modulating the expression of essential oncogenes such as c-MYC, and is emerged as a promising therapeutic target in diverse cancer types. Pharmacologic BET inhibitors in development such as JQ1 and OTX015 display preclinical anti-myeloma activity, and induce preferential loss of BRD4 bound to super-enhancers leading to transcriptional repression of c-MYC. Another approach to target this pathway is through the use of bi-functional molecules, which incorporate a small molecule BRD4 binding moiety with an E3 ubiquitin ligase recognition motif, such as ARV-825 and dBET1 (Lu et al. Chem Biol. 22:755, 2015, Winter et al. Science 348:1376, 2015). These agents induce Cereblon (CRBN)-dependent BRD4 ubiquitination and then proteasome-mediated degradation, thereby also reducing downstream c-MYC protein levels. Methods: We performed pre-clinical studies in myeloma cell lines and primary samples using ARV-825 and ARV-763, which are PROTACs that target BRD4 to either the CRBN or the Von Hippel-Lindau (VHL) E3 ligases, respectively. Downstream effects were studied using viability and apoptosis assays, cell cycle profiling, and Western blotting, among others. Results: Tetrazolium assays showed that both PROTACs were able to reduce the viability of a panel of myeloma cell lines, including MM1.S, U266, RPMI 8226, ANBL-6, KAS-6/1, and OPM-2 cells, and this occurred with greater potency than was the case for the BRD4 inhibitors JQ1 or OTX015. Median inhibitory concentrations were 5.66-91.98 nM for ARV-825, and 13.22-1522 nM for ARV-763, respectively. This reduction in viability was both time- and concentration-dependent, and was associated with a reduction of cells in the S phase, and an increase in G0/G1 cells, as well as cells with sub-G0/G1 DNA content, suggesting the onset of apoptosis. Programmed cell death was indeed found to be induced based on the appearance of an increase in Annexin V-positive cells by flow cytometry, and in cleaved caspase 8, caspase 9, caspase 3, and poly-ADP-ribose polymerase by Western blotting. The latter was associated with a specific reduction in the expression levels of both BRD4 and c-MYC that did not influence the abundance of other cellular proteins that were not BRD4 targets, and in a reduction in BRD4 and c-MYC mRNA. In contrast, JQ1 and OTX015 exposure resulted in a slight increase in BRD4 protein expression and a lesser decrease of c-MYC protein. Studies of drug combinations showed that, as expected, lenalidomide and pomalidomide were antagonistic to the effects of the CRBN-targeted ARV-825 PROTAC, but these immunomodulatory drugs showed additive or synergistic effects in combination with the VHL-targeted agent ARV-763. Also as expected, bortezomib and carfilzomib reduced the ability of both ARV-825 and ARV-763 to induce BRD4 degradation, but enhanced anti-proliferative and pro-apoptotic effects were seen in a manner that was influenced by the sequence of drug addition. In studies of drug-resistant cell lines, both PROTACs were able to overcome dexamethasone, melphalan, lenalidomide, and bortezomib resistance, but cross-resistance was seen in RPMI 8226/Dox40 cells, suggesting that these compounds are substrates for P-glycoprotein, which is over-expressed in these cells. Finally, we tested BRD4 PROTACs in primary cells isolated from patients with multiple myeloma, and observed rapid loss of viability of these plasma cells. Conclusions: Taken together, our data demonstrate that BRD4 degraders have promising activity against pre-clinical models of multiple myeloma, and support their translation to the clinic for patients with relapsed/refractory disease. Additional combination and mechanistic studies, as well as data from ongoing in vivo studies, will be presented at the meeting. Disclosures Lu: Arvinas, LLC: Employment, Equity Ownership. Qian:Arvinas, LLC: Employment, Equity Ownership. Orlowski:Acetylon: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Research Funding; Forma Therapeutics: Consultancy; Celgene: Consultancy, Research Funding; Millennium Pharmaceuticals: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; BioTheryX, Inc.: Membership on an entity's Board of Directors or advisory committees; Spectrum Pharmaceuticals: Research Funding.

Identification Of Tight Junction Protein (TJP)-1 As a Modulator and Biomarker Of Proteasome Inhibitor Sensitivity In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 123-123 ◽  
Author(s):  
Xing-Ding Zhang ◽  
Veerabhadran Baladandayuthapani ◽  
Heather Yan Lin ◽  
Bart Barlogie ◽  
Saad Z Usmani ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Rna Interference ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Myeloma Cell ◽  
Advisory Committees ◽  
Junction Protein ◽  
Rpmi 8226

Abstract Background Inhibition of the ubiquitin-proteasome pathway through the use of proteasome inhibitors (PIs) has been validated by our group and others as a successful strategy against multiple myeloma that has improved patient outcomes. However, these agents are currently used without patient selection, as no biomarkers have been validated that identify patients most or least likely to benefit. Also, drug resistance emerges in the vast majority through largely undefined mechanisms, and limits the activity of further therapy based on PIs. There is therefore an urgent need to identify such biomarkers, especially if they could also represent novel therapeutic targets to achieve resensitization. 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. In addition, using the Lentiviral GeneNet™ small hairpin (sh) RNA Library, we performed genome-wide RNA interference (RNAi) to identify genes whose knockdown conferred resistance. Genes of interest were subjected to further validation using myeloma cell lines,primary samples, murine models, and using clinically annotated GEP databases. These studies were supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results Bortezomib resistance was associated with decreased expression of TJP1 by GEP studies of isogenic bortezomib-resistant and -sensitive cell lines. TJP1 was also identified as a chemoresistance factor by RNA interference designed to detect genes that conferred a survival advantage after drug treatment. Suppression of TJP1 using shRNAs in RPMI 8226 and U266 myeloma cell lines with high TJP1 expression reduced sensitivity to both bortezomib and carfilzomib. Conversely, its over-expression in MOLP-8 cells, which had low TJP1 levels, conferred enhanced sensitivity to both PIs. Also, forced expression of TJP1 in bortezomib-resistant RPMI 8226 cells that had lost endogenous TJP1 levels restored drug sensitivity. In these resistant cells, TJP1 promoter hypermethylation was found, and treatment with decitabine restored both TJP1 expression, and sensitivity to bortezomib or carfilzomib. GEP studies showed TJP1 suppression was associated with enhanced expression of MHC class II region genes, including PSMB8 and PSMB9. This was mirrored at the protein level by enhanced PSMB8 and PSMB9 protein by Western blotting. As a result, TJP1 suppression was associated with increased activity of the chymotrypsin-like activity of the proteasome, while TJP1 overexpression reduced proteasome activity. A link between TJP1 and PSMB8 and 9 was supported by studies showing that TJP1 influenced activity of EGFR and STAT3 and indeed, EGFR inhibition with erlotinib enhanced PI sensitivity. Consistent with a role for TJP1 in vivo, treatment of mice with bortezomib showed a greater reduction of myeloma growth in tumors with high TJP1 expression compared with isogenic lines with low TJP1 expression. Finally, analysis of the Millennium Pharmaceuticals database of bortezomib studies in the relapsed and relapsed/refractory settings showed high TJP1 expression was associated with a greater likelihood of responding to bortezomib (p<0.0002), and a longer median overall survival (OS)(p=0.008). In addition, in the Total Therapy (TT) databases, higher TJP1 expression was associated with a better progression-free and OS in both TT3a (p=0.004 and <0.0001, respectively), and TT3b (p=0.001 and <0.0001). Conclusions Taken together, these data support the hypothesis that TJP1 modulates PI sensitivity in myeloma through effects on the proteasome’s protein turnover capacity, thereby tilting the load versus capacity balance in favor of cell death. Also, they indicate that strategies targeting TJP1 signaling should be studied as approaches to overcome both primary and secondary resistance. Finally, they support the possibility that TJP1 could be a useful biomarker to identify patients who are most likely to benefit from PI-based therapies, and prospective studies to validate this further are currently underway. Disclosures: Usmani: Celgene: Consultancy, Research Funding, Speakers Bureau; Onyx: Research Funding, Speakers Bureau. Orlowski:Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Resverlogix: Research Funding; Onyx: 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; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity’s Board of Directors or advisory committees.

Mucin 20 (MUC20) Modulates Proteasome Assembly Chaperones through the c-MET Pathway and Is a Biomarker of Proteasome Inhibitor Sensitivity in Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 274-274 ◽  
Author(s):  
Huihan Wang ◽  
Veerabhadran Baladandayuthapani ◽  
Heather Yan Lin ◽  
Xiaobin Wang ◽  
Bingzong Li ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Research Funding ◽  
Progression Free Survival ◽  
Myeloma Cell ◽  
Mobility Shift ◽  
Advisory Committees ◽  
Arq 197 ◽  
Drug Naïve

Abstract Background: Modulation of the activity of the ubiquitin-proteasome pathway with the proteasome inhibitor (PI) bortezomib is an established component of therapy for plasma cell disorders. More recently, the second-generation, irreversible proteasome inhibitor carfilzomib has been validated both pre-clinically and clinically, which can overcome bortezomib resistance in some patients. However, resistance emerges to carfilzomib as well, and the emergence of PI resistance in general is incompletely understood. Therefore, there is an urgent need to identify these mechanisms and develop biomarkers that could help guide therapy, and provide novel targets to overcome resistance. Methods: We generated carfilzomib-resistant (CR) myeloma cell lines by exposing drug-naive ANBL-6, KAS-6/1, U266, and OPM-2 cells to increasing concentrations of carfilzomib. These were then subjected to gene expression profiling (GEP) to identify prominent changes compared to their vehicle-treated counterparts. Molecular and pharmacologic approaches were then pursued to probe the significance of the observed changes to PI resistance in myeloma cell lines, murine models, and clinically annotated GEP databases. Results: Profiling and pathway analysis of CR myeloma cell lines identified suppressed expression of MUC20 as the most conserved and significant change compared to drug-naïve cells. Decreased levels of MUC20 in CR cells were confirmed by quantitative PCR and Western blotting, and also identified in bortezomib-resistant (BR) cell lines. Moreover, suppression of MUC20 with shRNAs was itself sufficient to confer carfilzomib resistance, which was associated with an increase in the proteasome chymotrypsin-like (ChT-L) activity. Reduced expression of MUC20 led to increased phosphorylation and activation of c-MET, STAT3, and ERK-1/2. In addition, exposure of drug-naïve cells to the c-MET ligand HGF induced activation of this pathway and of the proteasome ChT-L activity, but reduced carfilzomib sensitivity. Conversely, inhibition of c-MET with INCB28060, ARQ-197, or XL-184 restored carfilzomib sensitivity to the CR cell lines in culture, and ARQ-197 overcame resistance in a murine myeloma model. To explore the mechanisms behind the increase in ChT-L activity, we evaluated expression of proteasome components, and while 20S subunits were unchanged, Proteasome maturation protein (POMP), a key proteasome assembly chaperone, was more abundant. The POMP promoter has a consensus ETS-Like Gene 1 (ELK1) binding site, and ELK1 is a target for ERK-1/2. Therefore, we performed chromatin immunoprecipitation and gel mobility shift studies, which documented ELK1 binding, while mutation of this site reduced POMP expression in a reporter assay. Finally, analysis of the Millennium Pharmaceuticals GEP database of patients treated with bortezomib in the relapsed and relapsed/refractory settings showed patients who had higher expression of MUC20 had superior overall and progression-free survival compared to those who had lower expression. Conclusions: Taken together, our data support a role for signaling through the c-MET/ERK-1/2/ELK1/POMP axis in enhancing proteasome assembly and capacity, thereby reducing sensitivity to proteasome inhibitors like carfilzomib or bortezomib in myeloma. Also, they validate use of drugs suppressing c-MET as a potentially attractive strategy to overcome resistance to carfilzomib in the clinic. Finally, they indicate that MUC20 expression may be a viable biomarker to differentiate patients who have proteasome inhibitor-sensitive or relatively –resistant disease. Disclosures Orlowski: Onyx Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

High Expression of the Thalidomide-Binding Protein Cereblon (CRBN) Is Associated with Improved Clinical Response in Patients with Multiple Myeloma Treated with Lenalidomide and Dexamethasone

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2879-2879 ◽  
Author(s):  
Daniel Heintel ◽  
Arnold Bolomsky ◽  
Martin Schreder ◽  
Sabine Pfeifer ◽  
Niklas Zojer ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Univariate Analysis ◽  
Myeloma Cell ◽  
Full Data ◽  
Advisory Committees ◽  
Starting Dose

Abstract Abstract 2879 Background: Immunomodulatory drugs (IMiDs) are highly active in the treatment of multiple myeloma (MM), but the mechanisms of action are still not completely understood. Recently, cereblon (CRBN) has been identified as the primary target of thalidomide teratogenicity (Ito K et al, 2010) and, moreover as an essential requirement for IMiD therapy (Zhu YX et al, 2011). We wanted to investigate, if expression levels of CRBN could serve as a predictor of response. Patients and Methods: We measured CRBN mRNA expression in bone marrow samples of 44 well characterized MM patients treated with lenalidomide containing regimens, myeloma cell lines, and normal bone marrow (BM), using real time PCR. The median age of patients was 65 years (range: 37–85 years). Nine patients had ISS-stage I, 9 stage II, and 26 had stage III. All patients, except 12, were newly diagnosed. None of the patients had been exposed to lenalidomide before study entry. Full data documentation for response evaluation (> 2 cycles) was available in 37 patients (84%). Of these, lenalidomide was given in combination with dexamethasone in 27 patients with a starting dose of 25 mg per day on days 1–21 in a 28 days cycle, in combination with melphalan and prednisone (MPR, starting dose of lenalidomide 10 mg per day on days 1–21) in 9 patients, and in combination with bendamustine in 1 patient. Results: Normal BM was used as a reference with an expression level of one. All multiple myeloma cell lines tested (U266, KMS-12-BM, OPM-2, NCL-H929, MM.1S, SK-MM-1, and RPMI8226), had a higher CRBN expression than normal BM. CRBN was detected in all 44 MM samples distributed over a range covering 3 orders of magnitude (0.31 to 462.08-fold relative to normal BM; median: 3.61). Lenalidomide-based therapy resulted in CR in 3 (8%), nCR in 2 (5%), VGPR in 4 (11%), PR in 17 (46%), and in MR in 4 patients (11%), respectively. Three patients (8%) had SD, and 4 (11%) had PD. Median CRBN expression was three times higher in responding (≥MR) patients compared to non-responders (3.65 vs. 0.99, p<0.01). In addition, a significant correlation between quality of response and CRBN expression (r=0.34) was observed. This correlation remained statistically significant after exclusion of previously treated patients (r=0.37, p=0.02). Interestingly, among 9 available patients who had been pretreated, the lowest level of CRBN expression (0.90) was noted in the patient who progressed during lenalidomide treatment, indicating a predictive potential of CRBN expression also in pre-treated patients. When the analysis was restricted to the 27 patients who had uniformly been treated with lenalidomide and dexamethasone, an even more pronounced association between myeloma response and CRBN expression was noted (r=0.45; p=0.008). In patients with SD or PD, median CRBN expression was lower than in normal BM, while a higher CRBN expression was found in all patients with CR, nCR, VGPR, PR or MR (Table 1), suggesting that CRBN was required for anti-myeloma activity in these patients. This applied also to patients with marked myeloma response (CR, nCR, VGPR) and patients with PD (r=0.82; p= 0.003) (Figure 1). Univariate analysis between established prognostic factors such as beta-2-microglobulin, albumin, ISS stage, Hb, FISH defined cytogenetic aberrations, and response revealed no significant correlation in this patient cohort. A highly significant correlation between expression of CRBN and IRF4, an important transcription factor required for myeloma survival (p=0.00007), and XBP1 (p=0.00004) was observed. For PAX5 and BLIMP no such association were noted. When primary myeloma cells of 5 patients and cell lines (U 266, KMS-12-M) were exposed to lenalidomide, a significant down regulation of IRF4, but not of CRBN was found. Conclusion: Our studies show a significant association between CRBN expression and myeloma response in patients treated with lenalidomide containing regimens, especially in those receiving lenalidomide and dexamethasone therapy. These findings should be confirmed in larger, prospective clinical trials. Disclosures: Jäger: Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; AMGEN: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Research Funding.

Bim-Targeting Therapy Circumvents Adaptive Bortezomib-Resistance In Myeloma Through a Novel Cross-Link Between Autophagy and Apoptosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 601-601 ◽  
Author(s):  
Shuang Chen ◽  
Yun Dai ◽  
Yu Zhang ◽  
Liang Zhou ◽  
Hui Lin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Expression Profiles ◽  
Advisory Committees ◽  
Bh3 Mimetics ◽  
Targeting Therapy ◽  
Drug Naïve ◽  
Resistant Cells

Abstract In diverse human cancers, oncogene-driven pathways antagonize the lethal effects of Bim, a major apoptosis executioner implicated in the activity of various anti-cancer agents including bortezomib (btz). As loss of Bim plays an important role in tumor development and growth as well as in acquisition of drug resistance, Bim represents an attractive therapeutic target, particularly for efforts to circumvent resistance to standard chemotherapy and diverse novel therapies. In the present study, the role of Bim in acquired resistance of multiple myeloma (MM) cells to btz was investigated. Bcl-2 family member expression profiles revealed high basal levels of Bim in most MM cell lines (Bimhi) and in primary CD138+ MM samples. However, Bimlow cells (e.g., H929) were highly susceptible to btz, arguing against the possibility that endogenous Bim levels correlate with btz sensitivity. In sharp contrast, shRNA Bim knockdown in Bimhi cells conferred pronounced resistance to btz. Interestingly, btz-resistant cells (PS-R) generated by exposure of U266 cells (Bimhi) to progressively increasing btz concentrations (to 20 nM) displayed markedly diminished Bim expression, a phenomenon validated in btz-resistant cells derived from other Bimhi cell lines (e.g., OPM2, RPMI8226). Notably, Bim up-regulation by histone deacetylase inhibitors (HDACIs e.g., SBHA), when combined with BH3-mimetics (e.g., ABT-737) that release Bim from binding to Bcl-2/Bcl-xL, effectively killed btz-resistant MM cells. Moreover, this strategy was highly active against primary CD138+ cells isolated from relapsed MM patients. In view of recent evidence that Bim negatively regulates autophagy (Luo et al., Mol Cell 47:359-70, 2012), the possibility that autophagy might also play a role in Bim-targeting strategies was investigated. Whereas drug-naïve and btz-resistant cells exhibited little difference in expression of autophagy-inducing proteins or autophagy initiation (e.g., by thapsigargin or tunicamycin), resistant cells displayed deficiencies in autophagosome maturation, reflected by delayed removal of GFP-LC3 puncta. Interestingly, SBHA co-administration largely abrogated ABT-737-induced autophagy and markedly increased the association between Beclin-1 and Bcl-2, accompanied by diminished binding of Bim to Bcl-2. Significantly, shRNA Bim knockdown strikingly increased autophagy in Bimhi cells. Moreover, SBHA was unable to inhibit autophagy in such cells due to failure of Bim up-regulation, resulting in a pronounced attenuation of SBHA/ABT-737-induced apoptosis. In Bimlow H929 cells, SBHA also failed to up-regulate Bim and potentiate ABT-737 lethality. However, disruption of autophagy by chloroquine (CQ) dramatically restored the sensitivity of these cells to this Bim-targeting regimen, an event associated with a striking increase in Bim expression. In contrast, CQ only modestly increased lethality in Bimhi cells in which autophagy was already disabled by SBHA-mediated Bim up-regulation. Moreover, compared to parental drug-naïve cells (Bimhi), btz-resistant cells displaying acquired loss of Bim were significantly more susceptible to potentiation of SBHA/ABT-737 lethality by CQ (e.g., 50% vs 22% increase in apoptosis; P < 0.01). Finally, in bim-/- MEFs derived from gene knockout mice, SBHA/ABT-737 co-treatment, with or without the addition of CQ, was unable to induce apoptosis, arguing that the presence of the bim gene is required for restoration of drug sensitivity by Bim-targeting therapy. Together, these findings argue that Bim deficiency represents a novel mechanism of adaptive (rather than intrinsic) btz-resistance in MM cells, and that simultaneous up-regulation of Bim (e.g., by HDACIs) and unleashing of Bim from binding to anti-apoptotic Bcl-2 family members (e.g., by BH3-mimetics such as ABT-737) can circumvent this form of resistance. They also raise the possibility that in addition to direct induction of apoptosis, Bim-mediated suppression of autophagy contributes to the lethality of Bim-targeting strategies, and that disruption of the latter process (e.g., by CQ) may be particularly beneficial in cells in which Bim up-regulation is disabled. Collectively, these findings suggest that Bim-targeting therapy may represent a novel and effective strategy in overcoming btz resistance in MM. This study was supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. 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.

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.

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.

Proteasome Maturation Protein (pomp) Is Associated With Proteasome Inhibitor Resistance In Myeloma, and Its Suppression Enhances The Activity Of Bortezomib and Carfilzomib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 280-280 ◽  
Author(s):  
Bingzong Li ◽  
Hua Wang ◽  
Robert Z. Orlowski
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Xenograft Model ◽  
Proteasome Inhibition ◽  
Over Expression ◽  
Murine Xenograft Model ◽  
Drug Naïve ◽  
Inhibitor Resistance ◽  
Rpmi 8226

Abstract Background Proteasome inhibition with bortezomib has revolutionized the treatment of multiple myeloma, but the vast majority of patients eventually develop clinical bortezomib resistance through poorly understood mechanisms. One of the most conserved cellular responses to proteasome inhibition is to up-regulate proteasome subunit expression, presumably with the goal of enhancing proteasome activity and restoring intracellular protein homeostasis. We therefore hypothesized that proteasome inhibitor resistance could be associated with enhanced proteasome assembly, and that suppression of this assembly process could help restore drug sensitivity. The current studies focused on POMP, which is involved in addition of catalytically active b subunits to the hemiproteasome ring initially formed by structural a subunits. Methods We studied ANBL-6, KAS-6/1, OPM-2, and RPMI 8226 multiple myeloma cell lines which had acquired bortezomib resistance through prolonged exposure to increasing drug concentrations, and compared them to their drug-naïve, vehicle-treated counterparts. In addition, we evaluated primary cells derived from patients with myeloma, and examined an in vivo murine xenograft model of human myeloma. Results Bortezomib-resistant (V10R) ANBL-6, KAS-6/1, OPM-2, and RPMI 8226 cell lines showed enhanced levels of POMP mRNA by quantitative (q) PCR compared to their drug-sensitive counterparts, which was associated with higher POMP protein levels seen by immunoblotting. Exogenous over-expression of POMP in drug-naïve OPM-2 and KAS-6/1 cells was sufficient by itself to induce resistance to both bortezomib and carfilzomib. Conversely, suppression of POMP with one of two different Lentiviral small hairpin (sh) RNAs restored sensitivity in OPM-2 and KAS-6/1 V10R cells to bortezomib and carfilzomib. Since no known pharmaceuticals directly target POMP, we examined its promoter region, and found a consensus binding site for nuclear factor (erythroid-derived 2)-like (NRF) 2. Consistent with a role of NRF2 in POMP expression, NRF2 mRNA and protein were increased in V10R myeloma cells, and in drug-naïve cells treated with bortezomib. Moreover, transfection of cells with NRF2 cDNA activated a POMP promoter reporter, while chromatin immunoprecipitation with anti-NRF2 antibodies preferentially precipitated sequences near the POMP promoter. Also, NRF2 over-expression induced POMP and enhanced proteasome chymotrypsin-like activity, while its suppression had the opposite effects. All trans-retinoic acid (ATRA) blocked nuclear accumulation of NRF2 in OPM-2 and KAS-6/1 V10R cells, and reduced expression of POMP. Combinations of bortezomib with ATRA showed enhanced activity against these drug-resistant cell lines in association with greater proteasome inhibition, and were synergistic in drug-naïve cells. In primary samples, ATRA with bortezomib induced a greater reduction in viability than did either treatment alone. Finally, in a murine xenograft model with OPM-2 V10R cells, neither ATRA nor bortezomib showed substantial activity, while the combination regimen, by comparison, retained efficacy. Conclusions Taken together, our data support the hypotheses that NRF-2-influenced POMP over-expression contributes to proteasome inhibitor resistance in multiple myeloma, while approaches targeting POMP hold promise in overcoming resistance. Moreover, they provide a framework for translation of proteasome inhibitors with ATRA to the clinic to enhance activity, and to overcome resistance to this important class of anti-myeloma agents. Disclosures: No relevant conflicts of interest to declare.

RNA Polymerase I Inhibition with CX-5461 As a Novel Strategy to Target MYC in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3010-3010 ◽  
Author(s):  
Hans Lee ◽  
Hua Wang ◽  
Heather Lin ◽  
Veera Baladandayuthapani ◽  
Jin He ◽  
...  
Keyword(s):  
Western Blot ◽  
Board Of Directors ◽  
Cell Lines ◽  
Ribosomal Proteins ◽  
Research Funding ◽  
Myeloma Cell ◽  
Ribosomal Biogenesis ◽  
Advisory Committees ◽  
Pol I

Abstract Background: The role of dysregulation of the proto-oncogene MYC in both early and late myeloma progression events is well established. Among key MYC -downstream targets is upregulation of ribosomal biogenesis, resulting in increased protein translational capacity and biomass accumulation that is characteristic of neoplastic cells. Thus, given the relationship between myeloma pathobiology, MYC dysregulation, and ribosomal biogenesis, we hypothesized that selective targeting of ribosomal RNA (rRNA) transcription with the small molecule RNA polymerase (pol) I inhibitor CX-5461 (Senhwa Biosciences) may represent a novel therapeutic strategy in myeloma. Methods: Studies with CX-5461 were performed in human myeloma cell lines, isogenic p53 wild-type (wt) and knock-out (KO) p53 cells generated using sequence-specific zinc-finger nucleases, drug-resistant cell lines, primary patient samples, and myeloma murine xenograft models using NOD-SCID IL2Rgnull mice. Results: CX-5461 treatment of p53 wt (MM1.S, MOLP-8) and p53 mutant (U266, RPMI-8226) myeloma cell lines demonstrated a time- and dose-dependent decrease in cell proliferation with a median inhibitory concentration (IC50) at nM levels after 72 hours. A corresponding increase in cleaved-PARP, cleaved caspase-9, and cleaved caspase-3 expression was seen on Western blot as well as increased Annexin V staining on flow cytometry analysis, although this was more pronounced in p53 wt versus mutant cell lines. CX-5461 also retained activity in a panel of cell lines resistant to standard myeloma therapeutic agents (bortezomib, carfilzomib, lenalidomide, and doxorubicin) and in primary patient samples, including a heavily pretreated relapsed/refractory patient and a de novo plasma cell leukemia patient with del 17p. In vivo studies using a systemic isogenic MM1.S p53 wt and KO myeloma murine xenograft model demonstrated significant improvement in median overall survival in the CX-5461-treated p53 wt cohort (41 days vs. not reached, P .05), although outcomes were more modest in the p53 KO cohort with only a trend towards improved survival (P.1) in the drug-treated mice. To probe the p53-independent effects of CX-5461, gene expression profiling and gene set enrichment analysis was performed on isogenic MM1.S and MOLP-8 p53 wt and KO myeloma cell lines treated with CX-5461 or vehicle. These results suggested downregulation of MYC downstream targets as one p53-independent effect of RNA pol I inhibition. qPCR and Western blot studies revealed rapid downregulation of MYC at the transcript level within 1-hour of CX-5461 treatment followed by decreases in MYC protein levels. Previous studies have suggested ribosomal biogenesis is tightly controlled by an auto-regulatory feedback mechanism in which ribosomal proteins such as RPL5 and RPL11 can bind to the 3'UTR of MYC mRNA and facilitate its degradation through the RNA-induced silencing complex (RISC). Because RNA pol I inhibition is known to induce a nucleolar stress response and increase the availability of free ribosomal proteins, RISC-mediated degradation of MYC mRNA was explored as one possible mechanism of CX-5461-mediated MYC downregulation. Indeed, treatment with CX-5461 led to increased pull-down of RPL5 when immunoprecipitated with the RISC subunit TAR (HIV-1) RNA Binding Protein 2 (TARBP2) compared to vehicle-treated controls, and RNA immunoprecipitation assays with the catalytic RISC subunit, Argonaute 2 (AGO2), demonstrated enrichment of MYC mRNA with CX-5461 treatment. These results suggest that CX-5461 may induce degradation of MYC through the cooperative binding of ribosomal proteins, RISC subunits, and MYC mRNA. Finally, to evaluate the role of MYC expression and ribosomal biogenesis in relation to CX-5461 sensitivity, MYC was overexpressed in the H1112 myeloma cell line, which at baseline does not harbor a MYC translocation. MYC overexpression in H1112pCDH-myc cells led to increased basal pre-rRNA transcript levels compared to H1112pCDH cells, and furthermore, led to enhanced sensitivity to CX-5461. Conclusion: RNA pol I inhibition by CX-5461 is a promising target in myeloma therapy, with downregulation of MYC representing one mechanism of action. Moreover, increased MYC expression enhances sensitivity to CX-5461, providing rationale for the clinical translation of CX-5461 for the treatment of myeloma and other MYC-driven cancers. Disclosures O'Brien: Senhwa Biosciences, Inc.: Employment. Keats:Translational Genomic Research Institute: Employment. Orlowski:Bristol-Myers Squibb: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Spectrum Pharmaceuticals: Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Research Funding; Millennium Pharmaceuticals: Consultancy, Research Funding; Forma Therapeutics: Consultancy; Genentech: Consultancy; BioTheryX, Inc.: Membership on an entity's Board of Directors or advisory committees; Array BioPharma: Consultancy, Research Funding.

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