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

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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TAS-117, a Novel Selective Akt Inhibitor Demonstrates Significant Growth Inhibition in Multiple Myeloma Cells in Vitro and in Vivo

Blood ◽

10.1182/blood.v120.21.942.942 ◽

2012 ◽

Vol 120 (21) ◽

pp. 942-942 ◽

Cited By ~ 1

Author(s):

Naoya Mimura ◽

Hiroto Ohguchi ◽

Diana Cirstea ◽

Francesca Cottini ◽

Gullu Topal Gorgun ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Cell Growth ◽

Board Of Directors ◽

Cell Lines ◽

Akt Inhibitor ◽

Advisory Committees ◽

Significant Growth Inhibition

Abstract Abstract 942 The PI3K/Akt pathway mediates multiple myeloma (MM) cell growth and drug resistance, and targeting this molecule is a promising therapeutic option. In this study, we examined anti-MM activities of TAS-117 (TAIHO PHARMACEUTICAL CO., LTD., JAPAN), a selective potent Akt inhibitor in MM cell lines including MM.1S, MM.1R, OPM1 and H929 cells with high level of baseline Akt phosphorylation. TAS-117 induced significant growth inhibition in these cell lines, associated with downregulation of phosphorylation (Ser473 and Thr308) of Akt and downstream molecule FKHR/FKHRL1, without cytotoxicity in normal peripheral blood mononuclear cells. TAS-117 triggered G0/G1 arrest followed by apoptosis, evidenced by increased annexin V-positive cells, in both MM.1S and H929 cell lines. Apoptosis was further confirmed by cleavage of caspase-8, -3 and PARP. Interestingly, TAS-117 also induced: autophagy, evidenced by increased LC3-II; as well as endoplasmic reticulum (ER) stress, confirmed by induction of phospho-eIF2α, phospho-IRE1α and a molecular chaperone BiP/GRP78. Since the bone marrow (BM) microenvironment plays a crucial role in MM cell pathogenesis including drug resistance, we further examined the effect of TAS-117 in the presence of BM stromal cells (BMSCs). TAS-117 induced significant cytotoxicity in MM cells even in the presence of BMSCs, associated with downregulation of phospho-Akt. Importantly, TAS-117 inhibited secretion of IL-6 from BMSCs, and exogenous IL-6 and IGF-1 did not block cytotoxicity induced by this agent. We have previously shown the bortezomib activates Akt, and that Akt inhibition with bortezomib triggers synergistic MM cell cytotoxicity. TAS-117 enhanced bortezomib-induced cytotoxicity in MM.1S cells, associated with increased CHOP followed by PARP cleavage, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. TAS-117 also enhanced cytotoxicity induced by other therapeutic agents (ie, rapamycin, dexamethasone, 17-AAG) in MM.1S cells. Finally, we examined anti-MM activities of TAS-117 in a xenograft murine model. Oral administration of TAS-117 for 14 days significantly inhibited growth of H929 plasmacytoma and was well tolerated. Taken together, the novel and selective Akt inhibitor TAS-117 blocks MM cell growth in vitro and in vivo, providing the preclinical framework for clinical evaluation of this agent to improve patient outcome in MM. Disclosures: Shimomura: TAIHO PHARMACEUTICAL CO., LTD.: Employment. Utsugi:TAIHO PHARMACEUTICAL CO., LTD.: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder, Scientific Founder Other.

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Inhibition Of Nuclear Transport Modulator Xpo1/CRM1 For The Treatment Of Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v122.21.237.237 ◽

2013 ◽

Vol 122 (21) ◽

pp. 237-237 ◽

Cited By ~ 2

Author(s):

Michael P. Rettig ◽

Matthew Holt ◽

Julie Prior ◽

Sharon Shacham ◽

Michael Kauffman ◽

...

Keyword(s):

Flow Cytometry ◽

Board Of Directors ◽

Cell Lines ◽

Nuclear Export ◽

Employment Equity ◽

Advisory Committees ◽

Ic50 Values ◽

Equity Ownership

Abstract Background Exportin 1 (XPO1) also called CRM1, is a widely expressed nuclear export protein, transporting a variety of molecules including tumor suppressor proteins and cell cycle regulators. Targeted inhibition of XPO1 is a new strategy to restore multiple cell death pathways in various malignant diseases. SINEs are novel, orally available, small molecule Selective Inhibitors of Nuclear Export (SINE) that specifically bind to XPO1 and inhibit its function. Methods We used WST-1 cell proliferation assays, flow cytometry, and bioluminescence imaging to evaluate the efficacy of multiple SINEs to induce apoptosis alone and in combination with cytarabine (AraC) or doxorubicin in vitro in chemotherapy sensitive and resistant murine acute promyelocytic leukemia (APL) cells. This murine model of APL was previously generated by knocking in the human PML-RARa cDNA into the 5’ regulatory sequence of the cathepsin G locus (Westervelt et al. Blood, 2003). The abnormal co-expression of the myeloid surface antigen Gr1 and the early hematopoietic markers CD34 and CD117 identify leukemic blasts. These Gr1+CD34+CD117+ APL cells partially retain the ability to terminally differentiate toward mature granulocytes (mimicking more traditional AML models) and can be adoptively transferred to secondary recipients, which develop a rapidly fatal leukemia within 3 weeks after tumor inoculation. To assess the safety and efficacy of SINEs in vivo, we injected cryopreserved APL cells intravenously via the tail vein into unconditioned genetically compatible C57BL/6 recipients and treated leukemic and non-leukemic mice (n=15/cohort) with 15 mg/kg of the oral clinical staged SINE KPT-330 (currently in Phase 1 studies in patients with solid tumors and hematological malignancies) alone or in combination with 200 mg/kg cytarabine every other day for a total of 2 weeks. Peripheral blood was obtained weekly from mice for complete blood counts and flow cytometry to screen for development of APL. Results The first generation SINE, KPT214, inhibited the proliferation of murine APL cell lines in a dose and time dependent manner with IC50 values ranging from of 95 nM to 750 nM. IC50 values decreased 2.4-fold (KPT-185) and 3.5-fold (KPT-249) with subsequent generations of the SINEs. Consistent with the WST-1 results, Annexin V/7-aminoactinomycin D flow cytometry showed a significant increase of APL apoptosis within 6 hours of KPT-249 application. Minimal toxicity against normal murine lymphocytes was observed with SINEs even up to doses of 500 nM. Additional WST-1 assays using AraC-resistant and doxorubicin-resistant APL cell lines demonstrated cell death of both chemotherapy-resistant cell lines at levels comparable to the parental chemosensitive APL cell lines. Combination therapy with low dose KPT-330 and AraC showed additive effects on inhibition of cell proliferation in vitro. This additive effect of KPT-330 and chemotherapy on APL killing was maintained in vivo. As shown in Figure 1, treatment with AraC or KPT-330 alone significantly prolonged the survival of leukemic mice from a median survival of 24 days (APL + vehicle) to 33 days or 39 days, respectively (P < 0.0001). Encouragingly, combination therapy with AraC + KPT-330 further prolonged survival compared to monotherapy (P < 0.0001), with some mice being cured of the disease. Similar in vivo studies with the AraC-resistant and doxorubicin-resistant APL cells are just being initiated. Conclusions Our data suggests that the addition of a CRM1 inhibitor to a chemotherapy regimen offers a promising avenue for treatment of AML. Disclosures: Shacham: Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. McCauley:Karyopharm Therapeutics, Inc: Employment, 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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Concomitant Suppression of IKZF1, IRF4 and MYC Contribute to the Anti-Tumor Activity of the BET Inhibitor, Cpi-0610, in Disease Models of Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.3320.3320 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3320-3320 ◽

Cited By ~ 1

Author(s):

Ka Tat Siu ◽

Janani Ramachandran ◽

Andrew J. Yee ◽

Homare Eda ◽

Loredana Santo ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Research Funding ◽

Employment Equity ◽

Advisory Committees ◽

Bet Inhibitor ◽

Bet Inhibitors ◽

Equity Ownership ◽

Anti Tumor Activity

Abstract Inhibition of the bromodomain and extra-terminal (BET) proteins is a promising therapeutic strategy for various hematologic malignancies. Previous studies suggest that BET inhibitors constrain tumor cell proliferation and survival mainly through suppression of MYC transcription and activity. However, suppression of the transcription of additional genes also contributes to the anti-tumor activity of BET inhibitors but is less well understood. Here we investigated the therapeutic potential of CPI-0610, a novel BET inhibitor that is currently in a phase I clinical trial in relapsed multiple myeloma (MM) (ClinicalTrials.gov Identifier: NCT02157636). CPI-0610 displays potent in vitro cytotoxicity against MM cell lines and patient-derived MM cells by inducing G1 cell cycle arrest and caspase-dependent apoptosis. Furthermore, CPI-0610-mediated BET inhibition overcomes the protective effects conferred by cytokines and bone marrow stromal cells. We also confirmed the in vivo efficacy of CPI-0610 in a MM xenograft mouse model. CPI-0610 significantly delayed tumor growth and increased the survival of MM-bearing SCID mice. Our study found IKZF1 and IRF4 to be among the primary targets of CPI-0610, along with MYC. These findings indicate that BET inhibition not only results in a robust reduction of MYC transcription and activity but also suppresses the expression of IKZF1 and IRF4 in MM. Given that immunomodulatory drugs stabilize cereblon and facilitate Ikaros degradation in MM cells, we combined it with CPI-0610. Combination studies of CPI-0610 with lenalidomide or pomalidomide show in vitro synergism, in part due to concomitant suppression of IKZF1, IRF4, and MYC, providing a rationale for clinical testing of this drug combination in MM patients. Disclosures Mertz: Constellation Pharmaceuticals, Inc.: Employment, Equity Ownership. Sims:Constellation Pharmaceuticals, Inc.: Employment, Equity Ownership. Cooper:Constellation Pharmaceuticals, Inc.: Employment, Equity Ownership. Raje:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Eli Lilly: Research Funding.

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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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A Phase 1/2 Trial of TH-302 and Dexamethasone without or with Bortezomib (TBorD) in Patients with Relapsed/Refractory Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.2142.2142 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2142-2142

Author(s):

Jacob P. Laubach ◽

Noopur S. Raje ◽

Andrew J. Yee ◽

Philippe Armand ◽

Robert L. Schlossman ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Research Funding ◽

Progressive Disease ◽

Response Rate ◽

Phase 1 ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Abstract Background: TH-302 is a 2-nitroimidazole prodrug of the DNA alkylator bromo-isophosphoramide mustard that is selectively activated under hypoxic conditions. TH-302 has demonstrated anti-myeloma activity in preclinical models both in vitro and in vivo, as well as synergistic cytotoxic activity with bortezomib (Bor) in vitro. An ongoing Phase 1/2 study (NCT01522872) investigates TH-302 with dexamethasone (dex) without Bor or with Bor (TBorD) in patients (pts) with relapsed/refractory multiple myeloma (RR MM). The maximum-tolerated dose (MTD) of TH-302 plus dex was previously established at 340 mg/m2. We report preliminary results from pts treated at the MTD of TH-302 plus dex; enrollment in the TBorD arm is ongoing. Methods: The Phase 1/2 open-label multicenter study investigates IV TH-302 (240-480 mg/m²) plus PO dex (40 mg) with or without Bor (1.3 mg/m2) on Days 1, 4, 8 and 11 of a 21-day cycle. At the MTDs, Simon two-stage designs are implemented to pursue a regimen of TH-302 plus dex if ≥25% response rate or discontinue if ≤5% (90% power, 10% alpha), and pursue TBorD if ≥50% response rate or discontinue if ≤25% (85% power, 10% alpha). Treatment at the MTD of TH-302 plus dex, and establishment of the MTD of TH-302 in TBorD, is ongoing. Results: 24 pts (19 male, 5 female) with median age 65 years (range: 53 – 86) were enrolled at the 340 mg/m2 MTD of the TH-302 plus dex biweekly regimen. Ten pts had 18 severe adverse events (SAEs), of which 9 were related to TH-302, including 3 pts with cellulitis and 2 pts with pneumonia. Of 17 pts assessable for response at the time of abstract submission, 3 pts achieved a partial response (PR) and 2 pts achieved a minimal response (MR) for an overall response rate of 18% (PR) and a clinical benefit rate of 29% (PR+MR). Nine pts achieved stable disease and 3 pts had progressive disease. Eight pts are undergoing treatment; 16 pts discontinued: progressive disease (10), subject decision (4), AE (1) and alternative therapy (1). The initial dose escalation with TBorD has been completed at 240 mg/m2 TH-302, with enrollment ongoing at 340 mg/m2 TH-302. Conclusions: TH-302 can be administered at 340 mg/m2 biweekly with dex. Preliminary clinical activity has been noted in pts with heavily pre-treated RR MM. Data from the complete cohort of pts treated with dex and initial patients treated with TBorD will be updated and presented at the meeting. Disclosures Laubach: Onyx: Research Funding; Novartis: Research Funding; Millenium: Research Funding; Celgene: Research Funding. Raje:Acetylon: Research Funding; Eli Lilly: Research Funding; Millenium: Consultancy; Celgene: Consultancy; Onyx: Consultancy; Novartis: Consultancy; Amgen: Consultancy. Schlossman:Millennium: Consultancy. Matous:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx: Speakers Bureau; Millenium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Seattle Genetics: Speakers Bureau. Reynolds:Threshold: Honoraria. Shain:Onyx/Amgen: Research Funding; Celgene: Research Funding; Envision/Celgene: Speakers Bureau; L&M Healthcare/Onyx/Amgen: Speakers Bureau. Zackon:Millenium: Speakers Bureau. Mar:Threshold: Employment. Handisides:Threshold: Employment, Equity Ownership. Kroll:Threshold: Employment, Equity Ownership. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership. Richardson:Millenium: Membership on an entity's Board of Directors or advisory committees; JNJ: Membership on an entity's Board of Directors or advisory committees. Ghobrial:Millennium/Takeda: 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, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees.

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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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Carfilzomib, Lenalidomide, and Dexamethasone In Newly Diagnosed Multiple Myeloma: Initial Results of Phase I/II MMRC Trial

Blood ◽

10.1182/blood.v116.21.862.862 ◽

2010 ◽

Vol 116 (21) ◽

pp. 862-862 ◽

Cited By ~ 5

Author(s):

Andrzej J Jakubowiak ◽

Dominik Dytfeld ◽

Sundar Jagannath ◽

David H. Vesole ◽

Tara B. Anderson ◽

...

Keyword(s):

Multiple Myeloma ◽

Phase I ◽

Board Of Directors ◽

Newly Diagnosed ◽

Employment Equity ◽

Advisory Committees ◽

Level 3 ◽

Equity Ownership ◽

Level 1 ◽

Level 2

Abstract Abstract 862 Background: Carfilzomib (Cfz) is a novel, irreversible proteasome inhibitor that has demonstrated promising single-agent activity and favorable toxicity profile, including very low rates of peripheral neuropathy and neutropenia in relapsed/refractory multiple myeloma (MM). Combining Cfz with Lenalidomide (Revlimid®, Len), and Dexamethasone (Dex) into CRd shows an additive anti-MM effect in preclinical studies and lack of overlapping toxicity allowing for the use of these agents at full doses and for extended duration of time in relapsed/refractory MM (Niesvizky et al, ASH, 2009). This Phase I/II study was designed to determine the maximum tolerated dose (MTD) of CRd and to assess safety and evaluate efficacy of this combination in newly diagnosed MM. Methods: In Phase I, dose escalation follows the TITE-CRM algorithm, with Cfz as the only escalating agent starting at 20 mg/m2 (level 1), maximal planned dose 27 mg/m2 (level 2), and 15 mg/m2, if needed (level -1), given IV on days 1, 2, 8, 9, 15, 16 in 28-day cycles. Len is used at 25 mg PO (days 1–21), and Dex at 40/20 mg PO weekly (cycles 1–4/5-8) for all dose levels. Based on toxicity assessment, the study was amended to add dose level 3 with Cfz at 36 mg/m2 and the number of pts in the Phase I was increased to 35. A total of 36 pts are planned to be treated at the MTD in Phase I/II. Pts who achieve ≥ PR can proceed to stem cell collection (SCC) and autologous stem cell transplant (ASCT) after ≥ 4 cycles, although per protocol design, ASCT candidates are offered to continue CRd treatment after SCC. After completion of 8 cycles, pts receive 28-day maintenance cycles with Cfz (days 1, 2 15, 16), Len days 1–21, and Dex weekly at the doses tolerated at the end of 8 cycles. Responses are assessed by IMWG criteria with the addition of nCR. Results: The study has enrolled 24 pts to date, 4 pts at level 1 (Cfz 20), 14 at level 2 (Cfz 27) and at 6 at level 3 (Cfz 36). Toxicity data are available for 21 pts, of which 19 have completed at least the first cycle required for DLT assessment; 2 pts were removed during the first cycle for events unrelated to study therapy (1 at level 1 and 1 at level 2), and 3 are currently within their first cycle of treatment. There was a single DLT event at dose level 2 (non-febrile neutropenia requiring dose reduction of Len per protocol) and the MTD has not been reached. Hematologic toxicities were reversible and included Grade (G) 3/4 neutropenia in 3 pts, G3/4 thrombocytopenia in 3, and G3 anemia in 2. There have been additional G3 non-hematologic AEs including 1 case of DVT while on ASA prophylaxis, 1 fatigue, 1 mood alteration, and 5 glucose elevations; the last 2 AEs were related to Dex. There was no emergence of peripheral neuropathy (PN), even after prolonged treatment, except in 2 pts who developed G1 sensory PN. Twenty-three pts continue on treatment, most (20 pts) without need for any dose modifications. After a median of 4 (range 1–8) months of treatment, preliminary response rates by IMWG in 19 evaluable pts who completed at least 1 cycle are: 100% ≥ PR, 63% ≥ VGPR, 37% CR/nCR, including 3 pts with sCR. Responses were rapid with 17 of 19 pts achieving PR after 1 cycle and improving responses with continuing therapy in all pts. To date, 7 pts proceeded to SCC using growth factors only, with a median 6.3 × 106 CD34+ cells/kg collected (range 4.1–8.2), after a median of 4 cycles of CRd (range 4–8); all resumed CRd treatment after SCC. After a median of 4 months of follow-up, none of evaluable pts progressed and all are alive. Conclusion: CRd is well tolerated and highly active in newly diagnosed MM with ≥ PR of 100%, including 63% ≥VGPR and 37% CR/nCR. Accrual is ongoing, with updated toxicity and efficacy data to be presented at the meeting. The results of this study represent the first report of treatment of frontline myeloma with Cfz to date, and provide additional support to recently initiated Phase 3 trial of CRd vs. Rd in relapsed MM. Disclosures: Jakubowiak: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Exelixis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Centocor OrthoBiotech: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Off Label Use: Lenalidomide for newly diagnosed multiple myeloma. Jagannath:Millennium: Honoraria; OrthoBiotech (Canada): Honoraria; Celgene: Honoraria; Merck: Honoraria; Onyx Pharmaceuticals: Honoraria; Proteolix, Inc: Honoraria; Imedex: Speakers Bureau; Medicom World Wide: Speakers Bureau; Optum Health Education: Speakers Bureau; PER Group: Speakers Bureau. Vesole:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millennium: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau. Stockerl-Goldstein:Celgene: Speakers Bureau; Millennium: Speakers Bureau. Barrickman:Celgene: Employment, Equity Ownership. Kauffman:Onyx Pharmaceuticals: Employment, Equity Ownership. Vij:Proteolix: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Onyx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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A Novel Acanthoic Acid Analog NPI-1342 Blocks IκB Kinase-α and Trigger In Vitro and In Vivo cytotoxicity in Multiple Myeloma Cells

Blood ◽

10.1182/blood.v118.21.1841.1841 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1841-1841

Author(s):

Dharminder Chauhan ◽

Ajita V. Singh ◽

Arghya Ray ◽

Teru Hideshima ◽

Paul G. Richardson ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Board Of Directors ◽

Tumor Cells ◽

Cell Lines ◽

Inhibitory Effect ◽

Advisory Committees ◽

Acid Analog

Abstract Abstract 1841 Introduction: The dimeric Nuclear Factor-kappa B (NF-κB) transcription factor plays a key role during multiple myeloma (MM) cell adhesion-induced cytokine secretion in bone marrow stromal cells, which in turn triggers MM cell growth in a paracrine manner. NF-κB signaling pathway is mediated via canonical (IKK-α/IKK-β/NEMO-P50/65 or NF-κB1) and non-canonical (IKK-α/IKK-α/NIK-p52/RelB or NF-κB2) components. Prior studies have also linked constitutive activation of non-canonical NF-κB pathway to genetic abnormalities/mutation, allowing for an autocrine growth of MM cells. Other recent studies showed that constitutive NF-κB activity in tumor cells from MM patients renders these cells refractory to inhibition by bortezomib; and in fact, that bortezomib induces canonical NF-κB activity. These reports provided the impetus for the development of an agent with ability to modulate canonical and/or non-canonical NF-κB axis, allowing for a more robust and specific inhibition of NF-κB. Recent research and development efforts at Nereus Pharmaceuticals, Inc., have identified a novel small molecule acanthoic acid analog NPI-1342 as a potent NF-κB inhibitor. Here, we examined the effects of NPI-1342 on canonical versus non-canonical NF-κB signaling pathways, as well as its anti-tumor activity against MM cells using both in vitro and in vivo model systems. Methods: We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12PE, NCI-H929, OCI-MY5, LR5, Dox-40, OPM1, and OPM2 human MM cell lines, as well as purified tumor cells from patients with MM. Cell viability assays were performed using MTT and Trypan blue exclusion assays. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Animal model studies were performed using the SCID-hu model, which recapitulates the human BM milieu in vivo. Results: We first examined the effects of NPI-1342 on lipopolysaccharides (LPS)-induced NF-κB activity. Results showed that NPI-1342 inhibits LPS-stimulated NF-κB activity in vitro, as measured by phosphorylation of IkBa. To determine whether NPI-1342 triggers a differential inhibitory effect on IKKβ versus IKKα, MM.1S MM cells were treated with NPI-1342 for 48 hours, and protein lysates were subjected to kinase activity assays. NPI-1342 blocked IKKα, but not IKKβ or IKKγ phosphorylation. We next assessed whether the inhibitory effect of NPI-1342 on NF-κB activity is associated with cytotoxicity in MM cells. We utilized a panel of MM cell lines: at least five of these have mutations of TRAF3 (MM.1S, MM.1R, DOX40 and U266); one has no known NF-κB mutations (OPM2), and one has amplification of NF-κB1 (OCI-MY5). Treatment of MM cell lines and primary patient (CD138 positive) MM cells for 48 hours significantly decreased their viability (IC50 range 15–20 μM) (P < 0.001; n=3) without affecting the viability of normal peripheral blood mononuclear cells, suggesting selective anti-MM activity and a favorable therapeutic index for NPI-1342. NPI-1342-induced a marked increase in Annexin V+ and PI- apoptotic cell population (P < 0.001, n=3). Mechanistic studies showed that NPI-1342-triggered apoptosis in MM cells is associated with activation of caspase-8, caspase-9, caspase-3, and PARP cleavage. We next examined the in vivo effects of NPI-1342 in human MM xenograft models. For these studies, we utilized the SCID-hu MM model, which recapitulates the human BM milieu in vivo. In this model, MM cells are injected directly into human bone chips implanted subcutaneously in SCID mice, and MM cell growth is assessed by serial measurements of circulating levels of soluble human IL-6R in mouse serum. Treatment of tumor-bearing mice with NPI-1342 (20 mg/kg intraperitoneally, QD1-5 for 2 weeks), but not vehicle alone, significantly inhibits MM tumor growth in these mice (10 mice each group; P = 0.004). The doses of NPI-1342 were well tolerated by the mice, without significant weight loss. Finally, immunostaining of implanted human bone showed robust apoptosis and blockade of NF-κB in mice treated with NPI-1342 versus vehicle alone. Conclusions: We demonstrate the efficacy of a novel small molecule inhibitor of NF-κB NPI-1342 in MM using both in vitro and in vivo models. NPI-1342 blocks NF-κB activity with a preferential inhibitory activity against IKK-α component of NF-κB signaling. Our preclinical studies support evaluation of NPI-1342 as a potential MM therapy. Disclosures: Hideshima: Acetylon: Consultancy. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: 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. Palladino:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Anderson:Celgene: Consultancy; Millennium: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acetylon:; Nereus Pharmaceuticals, Inc: Consultancy.

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