Dysregulated Nucleotide Excision Repair (NER) Is a New Target in Multiple Myeloma

Abstract Nucleotide excision repair (NER) is involved in the removal of bulky adducts and DNA crosslinks induced by various genotoxins including alkylating agents. In cancer, somatic mutations, genes' up-regulation, and epigenetic silencing of NER may lead to abnormal DNA damage responses. Little is known about the role of NER in biology of multiple myeloma (MM), a heterogeneous disease characterized by genomic instability which is often treated by alkylating agents. In our genome sequencing data we did not observe recurrent mutation in NER although non silent mutations affected 13 genes involved in NER in 20out of 272 distinct patients. Using a functional assay based on the purified DNA-Damage Binding protein 2 (DDB2) proteo-probe complex that allows monitoring of photoproducts removal after UV irradiation, we observed NER variability in MM cell lines (MMCL) allowing to separate the cell lines in 2 groups according to their ability to repair: namely rapid (repair>90% after 2 hours) versus slow (repair < 85% after 2 hours). The separation in two NER phenotypes did not correlate with P53 loss of function or any cytogenetic event with the exception of t (4;14) translocation which was associated with the group showing a repair rate > 90%. Interestingly, the LR5 melphalan resistant cell line harbored a rapid repair phenotype as compared to the parental RPMI-8226 cell line which exhibited a slow repair. We evaluated melphalan sensitivity in relationship to NER activity in rapid and slow MMCL and observed that MMCL with slow NER were more sensitive to melphalan. We next evaluated whether inhibiting NER modified drug sensitivity. We utilized spironolactone which is known to inhibit NER. With the DDB2 proteo-probe assay, we confirmed the ability of spironolactone to inhibit NER in MMCL by degrading the Xeroderma Pigmentosum group B (XPB) protein. Combination of melphalan with NER inhibition in the 20 MMCL induced a significant increase in melphalan sensitivity in MMCL (increase from 15 to 79%). We have also confirmed similar NER activity in CD138 positive patient plasma cells. We then developed a gene expression signature using rapid and slow NER cell lines. In a multivariate analysis, the rapid NER signature was significantly associated with low survival in the IFM 2005-01 trial dataset that included 170 patients treated with high-dose melphalan. In conclusion, we have observed that NER affects MM cells and plays a significant role in drug sensitivity; we have also shown that NER can be targeted to increase melphalan sensitivity and potentially improve patient outcome. Disclosures Avet-Loiseau: onyx: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Consultancy; Millennium: Consultancy; BMS: Consultancy; Gilead: Consultancy; Oncopep: Equity Ownership; Acetylon: Equity Ownership.

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

Blood ◽

10.1182/blood.v126.23.917.917 ◽

2015 ◽

Vol 126 (23) ◽

pp. 917-917 ◽

Cited By ~ 1

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.

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SL-401, a Novel IL-3Rα/CD123-Directed Agent Targets Stem-like Cells in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.4463.4463 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4463-4463 ◽

Cited By ~ 1

Author(s):

Arghya Ray ◽

Yan Song ◽

Deepika Sharma Das ◽

Vincent Macri ◽

Janice Chen ◽

...

Keyword(s):

Multiple Myeloma ◽

Stem Cell ◽

Board Of Directors ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Drug Resistant ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership ◽

Rpmi 8226

Abstract Introduction Multiple myeloma (MM) remains incurable despite novel therapies, highlighting the need for further identification of factors mediating disease progression and resistance. One such factor is the development of a drug-resistant stem cell-like subpopulation. Previous studies have shown that MM side population cells (MM-SPs) exhibit stem-cell like features and contribute to relapse of MM. Recent research efforts, which have focused the biology of MM stem-like cells in order to derive specific therapy, have shown that stem-cell transcription factor Oct-4 is linked to stemness and chemoresistance. Here we examined the effect of enforced expression of Oct4 in MM cells and MM-SPs on the development of stem cell-like characteristics and drug-resistance in MM. These studies allow us to establish stable MM cell lines with characteristic stem-cell like features, which in turn facilitate screening of novel agents that effectively target this cell population in MM. Methods MM-SPs were isolated from RPMI-8226 cells by flow-cytometry based Hoechst 33342 staining. RPMI-8226 and RPMI-8226-SP cells were transfected with a phOct4-GFP construct (Gerrard et al., Stem Cell 2005, 23:124-133), and selected with G418 (0.5 mg/ml) to derive stable RPMI-8226-Oct4 and RPMI-8226-SP-Oct4 cell lines. Oct-4 expression was confirmed using FACS. Cell viability was analyzed by WST assays. SL-401 is a targeted therapy directed to IL-3Rα/CD123, comprised of recombinant human IL-3 fused to truncated diphtheria toxin. Drug and reagent source: SL-401 was obtained from Stemline Therapeutics; Bortezomib and flow antibodies were purchased from Selleck Chemicals and BD Biosciences, respectively. Statistical significance was derived using GraphPad Prism. Results 1) RPMI-8226 and RPMI-8226-Oct4 cells were analyzed for the expression of surface markers associated with stem cells (CD123/IL-3Rα, CD133 and CD27) by multicolor flow analysis. Oct-4 transfection does not affect the overall CD123 expression in RPMI-8226 cells, as the % MFI-CD123hi in RPMI-8226 versus RPMI-8226-Oct4 remains unchanged. However, the stable selection makes RPMI-8226-Oct4 more clonal in nature (%CD123hi : RPMI8226; 14.9% vs RPMI-8226-Oct4; 60%). 2) A significant increase in the frequency of CD133+ve cells was observed in RPMI-8226-SP-Oct4-tranfected cells versus either RPMI-8226-SP cells or RPMI-8226-Oct-4 cells [RPMI-8226-SP: 6.3%; RPMI-8226-Oct4: 27.8%; RPMI-8226-SP-Oct4: 40%; p< 0.05]. 3) Analysis of CD27 surface marker showed highest expression in RPMI-8226-SP-Oct4 cells compared to RPMI-8226-Oct4, RPMI-8226-SP, or RPMI-8226 cells (% MFI: RPMI-8226-SP-Oct4 > RPMI-8226-Oct4 > RPMI-8226-SP > RPMI-8226 cells). 4) Treatment of RPMI-8226 and RPMI-8226-Oct4 cells with proteasome inhibitor bortezomib decreased the viability of RPMI-8226 cells; in contrast, bortezomib did not significantly alter the viablity of RPMI-8226-Oct4 cells [% Viability after bortezomib: RPMI-8226; <50% versus RPMI-8226-Oct4; 95%]. Finally, 5) SL-401 significantly decreased the viability of RPMI-8226-Oct4 cells [IC50: RPMI-8226-Oct4 cells: 75 pM; RPMI-8226-SP cells: 350 pM; RPMI-8226 cells: 1367 pM]. We have previously shown anti-MM activity of SL-401 by an additional mechanism of targeting IL-3Rα-expressing plasmacytoid dendritic cells (pDCs) localized in the tumor microenvironment and blocking pDC-induced MM cell growth. Conclusions Our data show that stem-like cells in MM are relatively resistant to proteasome inhibitor therapy. Importantly, a novel agent SL-401 effectively targets these cells. Oct4-driven stable RPMI-8226 MM cell line serves as a novel tool to screen and develop newer agents targeting stem-like cells in MM. Overall, we show the ability of SL-401 to target a drug-resistant stem-like cell population in MM, and provide an additional rationale for clinical evaluation of SL-401 to improve patient outcome. A clinical trial of SL-401 in MM is currently ongoing (NCT02661022). Disclosures Macri: Stemline Therapeutics, Inc.: Employment. Chen:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Chauhan:Oncopeptide AB: Consultancy; Epicent Rx: Consultancy; C4 Therapeutics: Equity Ownership; Stemline Therapeutics, Inc.: Consultancy. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Sonofi Aventis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Gilead: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.

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Arginine Methylation Mediated By PRMT5 Has Significant Biological and Clinical Impact in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.1140.1140 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1140-1140

Author(s):

Annamaria Gulla ◽

Teru Hideshima ◽

Giada Bianchi ◽

Mariateresa Fulciniti ◽

Mehmet K. Samur ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Board Of Directors ◽

Cell Lines ◽

Prognostic Significance ◽

Arginine Methylation ◽

Gene Set Enrichment Analysis ◽

Advisory Committees ◽

Protein Levels ◽

Equity Ownership

Abstract Arginine-specific methyltransferases critically regulate cellular homeostasis by dictating the biological outcome of target proteins. Among them, Protein Arginine Methyltransferase 5 (PRMT5) has attracted growing interest due to its role as an enzyme mediating epigenetic regulation of anti-cancer target genes, as well as in methylation of non-histone proteins involved in growth-regulating and survival pathways including p53. However, little is known about its biologic function in multiple myeloma (MM). To first evaluate the clinical significance of PRMT5 in MM pathogenesis, we analyzed RNA-seq data from newly-diagnosed MM patients and identified highly upregulated PRMT5 in 320 patients' CD138+ cells compared to 16 samples of normal bone marrow (BM) plasma cells. Additional analysis of PRMT5 expression in two independent datasets also showed further PRMT5 mRNA upregulation during progression of MM. Immunohistochemical staining also confirmed elevated expression of PRMT5 in BM biopsies from MM patients as compared to healthy individuals and monoclonal gammopathy of undetermined significance (MGUS). Moreover, analysis of the prognostic significance of PRMT5 expression in MM patients enrolled on IFM/DFCI 2009 clinical study showed that high PRMT5 expression was associated with poor prognosis in terms of both event free (p= 0.016) and overall survival (p=0.018). Consistently, we also found upregulated PRMT5 expression at both mRNA and protein levels in MM cell lines (N=11) and patients CD138+ MM cells (N=3) as compared to PBMCs from healthy volunteers, associated with a parallel increase of cellular symmetric arginine di-methylation (SDMA) substrates. Interestingly, genetic depletion of PRMT5 in H929 (p53wt) and KMS11 (p53null) MM cell lines by shRNA decreased SDMA levels, associated with cell growth inhibition in a p53-independent manner. Likewise, pharmacological inhibition of PRMT5 with the small molecule inhibitor EPZ015666 triggered decreased SDMA levels, cell growth, survival, and clonogenicity, as well as induction of caspase-dependent apoptosis in MM cell lines. Moreover, although PRMT5 and SDMA levels were increased in MM cells cultured in the presence of BM stromal cell supernatant, cytotoxic activity of EPZ015666 was maintained. Notably, drug treatment significantly impaired cell proliferation of patient MM cells (n=2) even in the presence of BM mononuclear or stromal cells, without toxicity on normal PBMCs. At the level of gene expression modulation, PRMT5 inhibition was associated with downregulation of NF-kB-dependent transcription, evidenced by both gene set enrichment analysis (GSEA) and Ingenuity Upstream Regulator Analysis. Moreover, analysis of protein levels confirmed reduction of both canonical and non-canonical NF-kB pathways, evidenced by significantly decreased NF-kB DNA binding activity by ELISA. Importantly, Mass Spectrometry analysis identified TRIM21 as a new PRMT5 interactor; and EPZ015666-treated cells showed that PRMT5 methylates TRIM21 evidenced by WB analysis. Since TRIM21 mediates monoubiquitination of IKKbeta, thereby triggering its selective autophagy-mediated degradation, we next analyzed EPZ015666 effects on IKKbeta. Treatment increased both monoubiquitination of IKKbeta and the formation of IKKbeta-TRIM21-pBECLIN1-pULK1 autophagic complexes. Conversely, inhibition of autophagosome formation by 3-methyladenine abrogated the anti-MM activity of EPZ015666 and IKKbeta degradation, indicating that selective autophagic degradation of IKKbeta and inhibition of NF-kB signaling mediates EPZ015666-triggered anti-MM activity. Consistent with this view, confocal microscopy analysis also confirmed co-localization of IKKbeta in the autophagosome after EPZ015666 treatment. Finally, stable silencing of TRIM21 in MM cell lines significantly abrogated the anti-proliferative effect of EPZ015666. Collectively, these data delineate arginine methylation as a new control mechanism of MM cell growth, and demonstrate that inhibiting PRMT5 decreases tumor cell survival via blockade of NF-kB signaling, even in the context of the BM milieu. These data demonstrate the biologic and prognostic significance of PRMT5 in MM pathogenesis, and provide the rationale for novel therapies targeting PRMT5 to improve patient outcome in MM. Disclosures Hideshima: Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Munshi:Takeda: Consultancy; Celgene Corporation: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Consultancy, Equity Ownership. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

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p21-Associated Kinase 4 (PAK4) Addiction in Multiple Myeloma: A Potential Therapeutic Application with Enhanced Activity in FGFR3 Expressing Myeloma

Blood ◽

10.1182/blood.v128.22.3302.3302 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3302-3302

Author(s):

Mariateresa Fulciniti ◽

Joaquin Martinez Lopez ◽

William Senapedis ◽

Stefania Oliva ◽

Raphaël Szalat ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Cell Lines ◽

Chromosome Region ◽

Adaptor Protein ◽

Tyrosine Kinase Receptor ◽

Early Event ◽

Advisory Committees ◽

Equity Ownership

Abstract Dysregulated oncogenic serine/threonine kinases (STKs) play a pathological role in diverse forms of malignancies, including multiple myeloma (MM), and thus represent potential therapeutic targets. Here, we evaluated the biological and functional role of p21-activated kinase 4 (PAK4), and its potential as a new therapeutic target in MM. The PAK4 gene lies within chromosome region 19q13.2 commonly amplified in 30% of MM patients. We confirmed a correlation between copy number amplification and increased expression of PAK4 in two large myeloma patient datasets. High expression of total and phosphorylated PAK4 was also observed in the premalignant MGUS stage, suggesting that its overexpression may be an early event in the pathogenesis of myeloma. Myeloma cells with high PAK4 expression display sensitivity to conditional PAK4 knockdown, implying that an oncogene-addicted state exists in such cells. Moreover, we show that PAK4 promotes myeloma cell proliferation in vitro as well as in vivo in murine models of human myeloma through activation of the MEK/ERK pathway. This is further highlighted by the correlation between the basal levels of ERK activity and sensitivity to a novel small molecule inhibitor of PAK4, KPT-9274, a lead clinical agent. KPT-9274 effectively inhibits MM cell growth and survival via ERK inhibition in a large panel of MM cell lines and primary MM cells, also in the presence of bone marrow microenvironment, with no significant effect on normal PBMCs suggesting a potentially favorable therapeutic index. Inhibition of ERK by KPT-9274 also correlates with decreased DNA binding activity of ERK-dependent transcription factors AP1, ETS, CREB and EGR and decreased expression of ERK target genes such as CCND2, CCR1 and MYC. Finally, using a protein binding array, we have identified FGFR3, a commonly disrupted tyrosine kinase receptor, and Grb2, an adaptor protein involved in RAS activation, as novel PAK4 binding partners in myeloma and report disruption of this binding by KPT-9274. As a results, t(4;14)-positive MM cells expressing FGFR3 show greater sensitivity to PAK4 inhibition compared to MM cell lines without FGFR3 aberrations (e.g. U266 and ANBL6). FGFR3 may therefore serve as a predictive marker of KPT-9274 sensitivity in MM patients, with the potential for broader application to other malignancies associated with dysregulation of FGFR3 such as human bladder and cervical carcinomas. In conclusions, our study sheds light on the oncogenic role of the serine/threonine kinase PAK4 as survival and anti-apoptotic factor in myeloma and its inhibition with a new allosteric modulator, KPT-9274, as a potential novel therapeutic intervention in MM especially in high risk t(4:14)-positive patients. Disclosures Senapedis: Karyopharm Therapeutics Inc: Employment. Oliva:Celgene: Honoraria; Takeda: Honoraria; Amgen: Honoraria. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

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Targeting IRE1α-XBP1 Pathway Is a Novel Therapeutic Strategy In Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.4074.4074 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4074-4074 ◽

Cited By ~ 1

Author(s):

Naoya Mimura ◽

Teru Hideshima ◽

Gullu Gorgun ◽

Diana Cirstea ◽

Loredana Santo ◽

...

Keyword(s):

Multiple Myeloma ◽

Er Stress ◽

Cell Survival ◽

Board Of Directors ◽

Cell Lines ◽

Therapeutic Option ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership ◽

Novel Therapeutic

Abstract Abstract 4074 Aberrant protein folding results in the accumulation of misfolded/unfolded proteins in the endoplasmic reticulum (ER), which in turn triggers ER stress followed by unfolded protein response (UPR), an adaptive response against ER stress. Since multiple myeloma (MM) cells have high protein synthesis, they are sensitive to ER stress and require strict ER quality control for cell survival. Upon UPR, IRE1α is activated by auto-phosphorylation resulting in activation of its endoribonuclease domain to splice XBP1 mRNA from XBP1 unspliced form (XBP1u: inactive) to XBP1 spliced form (XBP1s: active). Since XBP1 is a transcription factor regulating genes which are responsible for protein folding and ER associated degradation (ERAD), IRE1α-XBP1 pathway acts as a pro-survival signaling pathway under the UPR condition. In this study, we examined whether IRE1α-XBP1 pathway is a potential novel therapeutic option in MM. We first examined IRE1α expression and confirmed its expression in all MM cell lines. In contrast, XBP1s was not detected by RT-PCR in most cell lines except in for RPMI8226 cells. To assess biologic significance of IRE1α in MM cell, we knock-downed its expression using shRNA and found that downregulation of IRE1α inhibited MM cell growth, indicating that IRE1α has a crucial role in MM cell survival. We next examined the impact of inhibition of XBP1 splicing by a small molecule IRE1α endoribonuclease inhibitor MKC-3946 (Mannkind, Valencia CA) in MM cells in vitro. As expected, MKC-3946 significantly inhibited tunicamycin-induced XBP1s without affecting phosphorylation of IRE1α. MKC-3946 induced only modest cytotoxicity in MM cell lines without toxicity in normal mononuclear cells from healthy donors; however, it significantly enhanced cytotoxicity in combination with bortezomib or 17-AAG. Both bortezomib and 17-AAG induced ER stress evidenced by induction of XBP1s; conversely, MKC-3946 blocked XBP1s triggered by these agents. Furthermore, apoptosis induced by these agents was enhanced with MKC-3946 associated with increased CHOP, which is a known pro-apoptotic protein induced in uncompensated ER stress condition. Importantly, MKC-3946 enhanced the cytotoxicity of bortezomib or 17-AAG in INA6 cells, even in the presence of increased IL-6 or bone marrow stromal cells. Finally, MKC-3946 was active inhibiting XBP1 splicing in a model of ER stress and significantly inhibited growth of RPMI8226 plasmacytoma in a xenograft murine model when used in combination with a low dose of bortezomib. Taken together, our results demonstrate that inhibition of XBP1 splicing by blockade of IRE1α is a promising therapeutic option in MM. Disclosures: Blumenthal: Mannkind Corporation: Employment, Equity Ownership. Tam:Mannkind Corporation: Employment, Equity Ownership. Kertesz:Mannkind Corporation: Employment, Equity Ownership. Zeng:Mannkind Corporation: Employment, Equity Ownership. Patterson:Mannkind Corporation: Employment, Equity Ownership. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

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Overcoming CD28-Mediated Multi-Drug Resistance With a Novel PIM2 Inhibitor

Blood ◽

10.1182/blood.v122.21.1931.1931 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1931-1931

Author(s):

Carmen M. Baldino ◽

Jayakumar R. Nair ◽

Justin Caserta ◽

Megan Murray ◽

Stephane Dumas ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Board Of Directors ◽

Cell Lines ◽

Tumor Xenograft ◽

Multi Drug Resistance ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Abstract Drug resistance in multiple myeloma (MM) is the major cause of treatment failure and is significantly mediated by pro-survival interactions with bone marrow microenvironment. A key myeloma receptor involved in this interaction is CD28, which has been largely characterized as the prototypic T cell costimulatory receptor. However, CD28 expression on myeloma cells is significantly correlated with disease progression, worse prognosis, and is significantly higher in the poor prognosis t(14;16) MAF subgroup. We now report that CD28 signaling mediates significant drug resistance and protects MM against death from multiple chemotherapeutics with different mechanisms of action – including dexamethasone, arsenic trioxide, melphalan or bortezomib. Inhibition of specific signaling (PI3K or Akt) or targets (Foxo3A or Bim) downstream of CD28 activation abrogates this protection. Unexpectedly, we found evidence that the PIM2 kinase (which is largely uncharacterized in MM but is also significantly overexpressed in the MAF subgroup) may be a previously unreported component of the CD28 pro-MM survival pathway. The novel small molecule PIM2 inhibitor JP_11646 (IC50 0.5 nM) abrogates CD28-mediated protection against apoptosis in MM cell lines in vitro, which we have not previously seen for any chemotherapeutic tested. In addition, blockade of CD28 activation sensitized MM cells significantly to JP_11646-induced death. Altogether, these data suggested that PIM2 inhibitors can overcome a major mechanism of multi drug resistance in MM. Jasco’s novel and selective pan-PIM inhibitor (JP_11646) has demonstrated biochemical IC50s of 24, 0.5 and 1 nM for PIM1, PIM2 and PIM3 respectively. The PIM mechanism of action has been confirmed through cell based transphosphorylation assays, where JP_11646 decreased PIM dependent phoshphorylation of the proapoptotic protein BAD at nM levels. JP_11646 increases apoptosis and decreases cell viability in multiple myeloma cell lines with the MAF translocations (<100 nM). JP_11646 is orally bioavailable and has demonstrated in vivo efficacy, inhibiting tumor growth by >80% in a MM1.S tumor xenograft study. These data provide solid rationale for further development of JP_11646 as a targeted therapy in MM, and specifically for patients exhibiting the MAF translocation. Disclosures: Baldino: Jasco pharmaceuticals: Employment, Equity Ownership, Founder and President Other, Membership on an entity’s Board of Directors or advisory committees. Caserta:Jasco Pharmaceuticals: Co-Founder Other, Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Dumas:Jasco Pharmaceuticals: Employment. Flanders:Jasco Pharmaceuticals: Employment. Lee:Jasco Pharmaceuticals: Research Funding.

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Deubiquitylating Enzyme Rpn11/POH1/PSMD14 As Therapeutic Target in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.4469.4469 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4469-4469 ◽

Cited By ~ 3

Author(s):

Yan Song ◽

Arghya Ray ◽

Deepika Sharma Das ◽

Mehmet K. Samur ◽

Ruben D. Carrasco ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Cell Viability ◽

Board Of Directors ◽

Cell Lines ◽

20S Proteasome ◽

Advisory Committees ◽

Healthy Donors ◽

Equity Ownership ◽

Rpmi 8226

Abstract Introduction The ubiquitin proteasome pathway is a validated therapeutic target in multiple myeloma (MM), evidenced by the FDA approval of proteasome inhibitors bortezomib, carfilzomib, and ixazomib. However, these agents are associated with possible off-target toxicities and the eventual development of drug-resistance. Therapeutic strategies directed against deubiquitylating (DUB) enzymes upstream of the 20S proteasome may allow for more specific targeting of the UPS, with fewer off-target activities and toxicities. Rpn11 is a 19S-proteasome-associated DUB enzyme that facilitates protein degradation by the 20S proteasome core particle. Here we examined the role of Rpn11 in MM using both biochemical and RNA interference strategies. Materials and Methods Drug sensitivity, cell viability, and apoptosis assays were performed using WST, MTT, Annexin V staining, respectively. MM.1S MM cells were transiently transfected with control short interfering RNA (siRNA), RPN11 ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. In the xenograft mouse model, CB-17 SCID-mice were subcutaneously inoculated with MM.1S cells as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419). Signal transduction pathways were evaluated using immunoblotting. Isobologram analysisand CalcuSyn software program were utilized to assesssynergistic/additive anti-MM activity. Statistical significance of observed differences were determined using a Student's t test. O-phenanthroline (OPA) was purchased from EMD Millipore, USA; and dex, lenalidomide, and pomalidomide were purchased from Selleck chemicals, USA. Results We found a statistically significant inverse correlation between Rpn11 levels and overall patient survival (p =0.022). Gene expression (GEP) analysisof Rpn11 showed a significantly higher level in patient MM cells versus normal plasma cells or PBMCs (p = 0.002 or p = 0.001 respectively). Immunohistochemical analysis of bone marrow biopsies from MM patients and normal healthy donors showed higher Rpn11 expression in MM cells than normal cells. Similarly, western blot analysis showed higher Rpn11 levels in MM cell lines and patient cells versus normal PBMCs.Rpn11 knockdown in MM cells significantly decreased cell viability (p < 0.001; n=3). To validate our siRNA data, we utilized Rpn11 inhibitor O-phenanthroline (OPA) (Verma et al., Science 2002, 298:611-5). Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, ARP-1, Dox40, LR5, INA6, ANBL6.WT, and ANBL6.BR) and patient MM cells with OPA significantly decreased cell viability (IC50 range 8µM to 60µM; p < 0.001 for all cell lines; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting selective anti-MM activity and a favorable therapeutic index for OPA. Importantly, the anti-MM activity of OPA was observed against tumor cells obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. In concert with these data, the cytotoxicity of OPA was observed in MM cell lines sensitive and resistant to conventional and novel therapies. Furthermore, OPA inhibits proliferation of MM cells even in the presence of BM stromal cells or plasmacytoid dendritic cells (pDCs). OPA inhibits Rpn11 DUB activity without blocking 20S proteasome activities. Mechanistic studies show that OPA-triggered MM cell apoptosis is associated with 1) activation of caspases; 2) accumulation of polyubiquitinated proteins; 3); induction of ER stress; and 4) induction of autophagy. OPA-induced apoptosis occurs in a p53-independent manner, since OPA triggered apoptosis in both p53-null (ARP-1) and p53-mutant (RPMI-8226) MM cells. OPA inhibits MM cell growth in vivo and prolongs survival in a MM xenograft mouse model. Finally, combining OPA with lenalidomide, pomalidomide, or dex induces synergistic/additive anti-MM activity, and overcomes drug resistance. Conclusion Our preclinical data showing efficacy of OPA in MM models both validates targeting 19S proteasome-associated DUB Rpn11, and provides the framework for clinical evaluation of Rpn11 inhibitors to overcome proteasome inhibitor resistance and improve patient outcome in MM. Disclosures Munshi: Celgene Corporation: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Consultancy, Equity Ownership; Takeda: Consultancy. Chauhan:C4 Therapeutics: Equity Ownership; Epicent Rx: Consultancy; Oncopeptide AB: Consultancy; Stemline Therapeutics, Inc.: Consultancy. Anderson:Sonofi Aventis: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: Scientific Founder; Acetylon: Other: Scientific Founder; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

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Results of a Phase I Study of Pnk-007, Allogeneic, Off the Shelf NK Cell, Post Autologous Transplant in Multiple Myeloma (NCT02955550)

Blood ◽

10.1182/blood-2019-128467 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4451-4451

Author(s):

Sarah A. Holstein ◽

Sarah Cooley ◽

Parameswaran Hari ◽

Sundar Jagannath ◽

Catherine R Balint ◽

...

Keyword(s):

Multiple Myeloma ◽

Maintenance Therapy ◽

Board Of Directors ◽

Research Funding ◽

Phase I Study ◽

Nk Cell ◽

Single Infusion ◽

Advisory Committees ◽

Cd34 Cells ◽

Equity Ownership

Background: PNK-007 is an allogeneic, off the shelf cell therapy product enriched for CD56+/CD3- NK cells expanded from placental CD34+ cells. PNK-007 cells exhibit cytotoxicity against various cancer cell types, including multiple myeloma (MM), and secrete cytokines during co-culture with cancer cells. This is a Phase I study of single infusion PNK-007 after autologous stem cell transplant (ASCT) in MM. Methods: Placental CD34+ cells were cultivated in the presence of cytokines for 35 days to generate PNK-007 under cGMP standards followed by release testing. HLA matching and KIR mismatching were not used. Four treatment arms were evaluated on patients (pts) following ASCT: 10 million (M) cells/kg Day (D) 14 with or without recombinant human IL-2 (rhIL-2), 30M cells/kg D14 with rhIL-2, or 30M cells/kg D7 with rhIL-2. rhIL-2 was administered subcutaneously at 6M units every other day for up to 6 doses to facilitate PNK-007 expansion. Pts received variable pre-ASCT induction therapy. Maintenance therapy was permitted after the Day 90-100 visit (D90). Subjects were followed for up to 1-year. Results: 15 pts who received PNK-007 (12 of whom received rhIL-2) were followed on this study. Pts aged 44-69 yrs included 12 newly diagnosed (ND)MM and 3 relapsed/refractory (RR)MM. The 3 RRMM pts had received 1, 2 or 5 prior lines of therapy, with 2 pts having previous ASCT. All pts had been exposed to immunomodulatory drug (IMiDs) and proteasome inhibitors (PIs). No serious adverse events (AEs) were attributable to PNK-007 and no dose-limiting toxicity, GvHD, graft failure or graft rejection were observed. 12/15 pts started maintenance therapy following the transplant while participating in this study, at the physician's discretion. Based on physician assessed responses by International Myeloma Working Group pre-ASCT, of the NDMM pts 10/12 achieved VGPR or better (1 CR and 9 VGPR), 1/12 achieved PR and 1/12 was not assessed during pre-ASCT induction. By D90 10/12 pts achieved VGPR or better (5 CR or sCR and 5 VGPR), 1/12 maintained PR and 1/12 stable disease. At 1-year 9/11 achieved VGPR or better (4 CR or sCR and 5 VGPR), 2/11 were not assessed and 1 was removed from the study prior to 1 year due to failure to respond to ASCT. Of the RRMM pts 2/3 achieved PR and 1/3 was not assessed during pre-ASCT induction, by D90 2/3 achieved VGPR and the pt that had not been assessed pre-ASCT achieved PR. At 1-year, 1 pt maintained VGPR, 1 pt was not assessed and 1 pt did not continue to the 1-year visit. Using a validated Euro-flow minimal residual disease (MRD) assay of bone marrow aspirate (BMA) samples, of the NDMM pts 4/12 were MRD negative (MRD-) pre-ASCT; by D90 9/12 were MRD-. At 1-year 6/12 were MRD-, 2/12 had insufficient BMA to perform testing, 2/12 refused BMA procedure, 1/12 did not convert to MRD-, and 1 was removed from the study prior to 1-year due to failure to respond to ASCT. Of the RRMM pts 0/3 were MRD- pre-ASCT with 1/3 having insufficient BMA to perform testing; by D90 1/3 were MRD-. At 1-year 1/3 was MRD-, 1/3 did not convert to MRD- and 1 pt did not continue to the 1-year visit. PNK-007 infusion did not interfere with immune reconstitution kinetics. Platelet, neutrophil, and absolute lymphocyte counts recovered by day 28 post-ASCT in 12/15 patients. All pts' sera tested negative for the presence of anti-HLA antibodies at all timepoints indicating the absence of humoral immunity and alloantibodies to PNK-007. Conclusion: PNK-007 is the first fully allogeneic, off the shelf CD34+ derived NK cell product in MM clinical trials. A single infusion of PNK-007 up to 30M cells/kg with and without rhIL-2 was well tolerated in the post-ASCT setting. We established the feasibility of infusing PNK-007 as early as 7 days post-ASCT without negative impact on blood count recovery or successful engraftment. BMA MRD- status was observed in 7/9 MRD evaluable pts at 1-year post ASCT. These clinical data are encouraging and warrant further evaluation. Disclosures Holstein: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Takeda: Membership on an entity's Board of Directors or advisory committees; Sorrento: Consultancy; GSK: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees. Cooley:Fate Therapeutics, Inc: Employment, Equity Ownership. Hari:Cell Vault: Equity Ownership; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Research Funding; Janssen: Consultancy, Honoraria; Kite: Consultancy, Honoraria; Amgen: Research Funding; Spectrum: Consultancy, Research Funding; Sanofi: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria. Jagannath:BMS: Consultancy; Merck: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Medicom: Speakers Bureau; Multiple Myeloma Research Foundation: Speakers Bureau. Balint:Celgene: Equity Ownership; Celularity, Inc: Employment. Van Der Touw:Celularity, Inc: Employment. Zhang:Celularity Inc: Employment. Hariri:Celularity Inc: Employment. Vij:Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria; Janssen: Honoraria; Karyopharm: Honoraria; Sanofi: Honoraria; Takeda: Honoraria, Research Funding.

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Phase II Trial of the Combination of Ixazomib, Lenalidomide, and Dexamethasone in High-Risk Smoldering Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-117871 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 804-804 ◽

Cited By ~ 7

Author(s):

Mark Bustoros ◽

Chia-jen Liu ◽

Kaitlen Reyes ◽

Kalvis Hornburg ◽

Kathleen Guimond ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

High Risk ◽

Disease Progression ◽

Board Of Directors ◽

Rna Sequencing ◽

Research Funding ◽

Response Rate ◽

Advisory Committees ◽

Equity Ownership

Abstract Background. This study aimed to determine the progression-free survival and response rate using early therapeutic intervention in patients with high-risk smoldering multiple myeloma (SMM) using the combination of ixazomib, lenalidomide, and dexamethasone. Methods. Patients enrolled on study met eligibility for high-risk SMM based on the newly defined criteria proposed by Rajkumar et al., Blood 2014. The treatment plan was designed to be administered on an outpatient basis where patients receive 9 cycles of induction therapy of ixazomib (4mg) at days 1, 8, and 15, in combination with lenalidomide (25mg) at days 1-21 and Dexamethasone at days 1, 8, 15, and 22. This induction phase is followed by ixazomib (4mg) and lenalidomide (15mg) maintenance for another 15 cycles. A treatment cycle is defined as 28 consecutive days, and therapy is administered for a total of 24 cycles total. Bone marrow samples from all patients were obtained before starting therapy for baseline assessment, whole exome sequencing (WES), and RNA sequencing of plasma and bone marrow microenvironment cells. Moreover, blood samples were obtained at screening and before each cycle to isolate cell-free DNA (cfDNA) and circulating tumor cells (CTCs). Stem cell collection is planned for all eligible patients. Results. In total, 26 of the planned 56 patients were enrolled in this study from February 2017 to April 2018. The median age of the patients enrolled was 63 years (range, 41 to 73) with 12 males (46.2%). Interphase fluorescence in situ hybridization (iFISH) was successful in 18 patients. High-risk cytogenetics (defined as the presence of t(4;14), 17p deletion, and 1q gain) were found in 11 patients (61.1%). The median number of cycles completed was 8 cycles (3-15). The most common toxicities were fatigue (69.6%), followed by rash (56.5%), and neutropenia (56.5%). The most common grade 3 adverse events were hypophosphatemia (13%), leukopenia (13%), and neutropenia (8.7%). One patient had grade 4 neutropenia during treatment. Additionally, grade 4 hyperglycemia occurred in another patient. As of this abstract date, the overall response rate (partial response or better) in participants who had at least 3 cycles of treatment was 89% (23/26), with 5 Complete Responses (CR, 19.2%), 9 very good partial responses (VGPR, 34.6%), 9 partial responses (34.6%), and 3 Minimal Responses (MR, 11.5%). None of the patients have shown progression to overt MM to date. Correlative studies including WES of plasma cells and single-cell RNA sequencing of the bone microenvironment cells are ongoing to identify the genomic and transcriptomic predictors for the differential response to therapy as well as for disease evolution. Furthermore, we are analyzing the cfDNA and CTCs of the patients at different time points to investigate their use in monitoring minimal residual disease and disease progression. Conclusion. The combination of ixazomib, lenalidomide, and dexamethasone is an effective and well-tolerated intervention in high-risk smoldering myeloma. The high response rate, convenient schedule with minimal toxicity observed to date are promising in this patient population at high risk of progression to symptomatic disease. Further studies and longer follow up for disease progression are warranted. Disclosures Bustoros: Dava Oncology: Honoraria. Munshi:OncoPep: Other: Board of director. Anderson:C4 Therapeutics: Equity Ownership; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Takeda Millennium: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ghobrial:Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; BMS: Consultancy.

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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 ◽

10.1182/blood.v114.22.1729.1729 ◽

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.

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