ALBERT

Description: Introduction: Daratumumab (DARA) is a first-in-class human CD38 IgG1κ monoclonal antibody that has demonstrated activity as monotherapy and in combination with standard of care regimens for multiple myeloma (MM). Population pharmacokinetics (PPK) analyses were conducted to describe the PK characteristics of DARA following its administration in combination therapies, to evaluate the influence of covariates on its disposition in patients with MM who had received ≥1 prior therapy, and to compare its PK in combination therapies with that of monotherapy. Exposure-efficacy/safety analyses were performed to investigate the relationship between DARA exposure and selected efficacy and safety endpoints. Methods: The PPK analysis primarily included data from two phase 3 studies in which DARA was combined with background regimens: MMY3003 (POLLUX; lenalidomide [R]/dexamethasone [d]) and MMY3004 (CASTOR; bortezomib [V]/d). Data from two phase 1/2 studies (GEN503 [Rd] and MMY1001 [pomalidomide/d; Vd; V-thalidomide-d; V-melphalan-d]) were also used. Most patients included in the analysis (684 of 694) received 16 mg/kg DARA intravenously. A PPK model based on previous monotherapy studies was used to fit the concentration-time data from combination studies. Subgroup analyses were conducted to evaluate the influence of patient and disease characteristics on exposure to DARA. Based on data from MMY3003 and MMY3004, the exposure-efficacy analyses investigated the relationship between maximal trough concentrations (Cpre-infusion,max) and progression-free survival (PFS), duration of response (DOR), and overall response rate (ORR), while the exposure-safety relationship was explored for infusion-related reactions (IRRs), thrombocytopenia, anemia, neutropenia, lymphopenia, and infections. Results: Exposure to DARA was similar between the monotherapy and combination therapies. Based on combination therapy data, the effects of the intrinsic and extrinsic factors (age, sex, race, renal and hepatic impairment, baseline albumin, type of MM, region, type of combination therapy, ECOG, refractory status, and number of prior lines of therapy) were similar to or smaller than those in the monotherapy studies. Consistent with the monotherapy studies, none of the investigated intrinsic and extrinsic factors had clinically important effects on the exposure to DARA as all the covariate effects were within 25%. Although the clearance and volume of distribution of DARA increased with increasing body weight, the exposure to DARA was relatively consistent across the range of body weights after administration on a mg/kg-basis. Despite the decreasing concentration of DARA over time due to less frequent dosing, the current dosing schedule was adequate to produce concentration levels that maintained target saturation during the Q4W dosing period in the dosing schedules for MMY3003 (QW for 8 weeks, Q2W for 16 weeks, and Q4W, thereafter) and MMY3004 (QW for 9 weeks, Q3W for 15 weeks, and Q4W, thereafter). The exposure-efficacy analyses on the data from combination therapies suggest that maximum clinical benefit to PFS, DOR, and ORR was attained for the majority of the patients with an acceptable safety profile at the recommended dose of 16 mg/kg. No apparent relationships between drug exposure and IRRs, thrombocytopenia, anemia, neutropenia, and lymphopenia were identified within the studied concentration range. Although the overall rate of infection (any grade) appeared to increase with drug exposure, this trend was not observed for grade ≥3 infections. These findings were consistent with results from the monotherapy studies. Conclusion: The PK of DARA was similar between monotherapy and combination studies. No clinically relevant demographic or clinical characteristics were identified. Therefore, no dose adjustment based on these factors is recommended. Population PK and exposure-response analyses for combination therapies support the recommended body weight-based 16 mg/kg dose and the dosing schedules for the MMY3003 and MMY3004 studies. Disclosures Xu: Janssen: Employment, Equity Ownership. Liao:Pharmax: Employment; Janssen Research & Development: Consultancy; Johnson & Johnson: Equity Ownership. Dimopoulos:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Sonneveld:Amgen, Celgene, Janssen, Karyopharm, Takeda: Consultancy, Honoraria; Amgen, Celgene, Janssen, Karyopharm: Research Funding. Ho:Janssen, Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Belch:Amgen, Celgene, Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Capra:Janssen: Speakers Bureau. Gomez:Janssen, Bristol Myers Squibb, Celgene, Amgen: Consultancy. Medvedova:Oregon Health & Science University: Employment. Iida:Janssen Pharmaceuticals, Takeda Pharmaceuticals Co, Celgene, Bristol-Myers Squibb, Ono Pharmaceuticals Co: Honoraria; Janssen Pharmaceuticals, Takeda Pharmaceuticals Co, Celgene, Bristol-Myers Squibb, Chugai Pharmaceuticals, Kyowa Hakko Kirin Co, Eli Lilli Japan, Novartis Pharma, Sanofi, Bayer Yakuhin, Toyama Chemical Co, Teijin Pharma, Astellas Pharma: Research Funding. Qi:Janssen: Employment. Schecter:Janssen: Employment, Equity Ownership. Khokhar:Janssen: Employment. Yan:Janssen: Employment; Johnson & Johnson: Equity Ownership. Zhang:Janssen: Employment, Equity Ownership. Clemens:Johnson & Johnson: Equity Ownership; Janssen: Employment.

Description: Nowadays, patients with multiple myeloma (MM) have multiple choices of therapy including monoclonal antibodies, proteasome inhibitors, and immunomodulatory drugs (IMiDs), whereas some patients still develop resistance to these drugs and require novel therapeutic modalities. Here, we focused on inhibition of HDAC and AKT to overcome drug resistance. Lenalidomide (Len) selectively binds to cereblon (CRBN), which mediates recruitment of specific substrates like IKZF1 to E3 ubiquitin ligase and subsequent degradation, resulting in downregulation of IRF-4 and c-Myc. Then, we developed Len-resistant myeloma cells by RNAi-mediated downregulation of CRBN. Treatment of these cells with HDAC inhibitors reduced IKZF1 mRNA, suggesting potential efficacy of HDAC inhibitors against CRBN-low expressing or mutated MM. According to the integrated database for expression profile and disease prognosis (GenomicScape, http://www.genomicscape.com), higher expression of MICA was significantly associated with better overall survival in MM. MICA is an NK cell-activating ligand and plays an important role in ADCC. We observed that ADCC activity of both daratumumab and elotuzumab against MM cells was enhanced in the presence of HDAC inhibitors, which was compatible with our previous data that HDAC inhibitors upregulated MICA mRNA expression via inhibition of IKZF1 (ASH2018 abstract #4435). We also observed that HDAC inhibitors upregulated MICA mRNA in CRBN-deficient cells, suggesting promise of the combination of HDAC inhibitors and monoclonal antibodies against Len-resistant MM. Len-resistance is also affected by phosphorylation status of GSK-3. PI3K/AKT pathway is frequently activated in MM cells, and AKT inactivates GSK-3 by direct phosphorylation, resulting in c-Myc stabilization. Enhanced phosphorylation of GSK-3 was observed in CRBN-deficient H929 cells after long-term culture with Len, and such a phosphorylation status of GSK-3 was correlated with less CRBN amount and higher Len concentration (Figure 1). Afuresertib, an AKT inhibitor, suppressed GSK-3 phosphorylation (p-GSK-3) with or without ACY-1215, an HDAC inhibitor, leading to a substantial decrease of c-Myc (Figure 2). On the other hand, CHIR 99021, a GSK-3 inhibitor, partially counteracted to cytotoxic effect of afuresertib on H929 cells (Figure 3). These results suggest that increased p-GSK-3 is involved in acquired Len-resistance, and that combined inhibition of HDAC and AKT can overcome Len-resistance through decreased p-GSK-3. Furthermore, we examined the efficacy of CUDC-907, a dual HDAC and PI3K inhibitor. CUDC-907 had a cytotoxic effect on the MM cell lines including those had low CRBN expression. Bortezomib, doxorubicin, and dexamethasone resistant MM cell lines were also sensitive to CUDC-907. CUDC-907 upregulated MICA mRNA expression, but downregulated IKZF1 mRNA expression. Treatment of RPMI-8226 cells with CUDC-907 enhanced the ADCC activity of daratumumab (Figure 4). Furthermore, CUDC-907 was effective on primary MM cells which were resistant to bortezomib and Len (Figure 5). Thus, dual inhibition of HDAC and AKT with or without monoclonal antibodies is a promising therapeutic approach to multi-drug resistant MM. Disclosures Imai: Celgene: Honoraria, Research Funding; Janssen Parmaceutical K.K.: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Futami:Torii Pharmaceutical: Research Funding. Ri:Janssen Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Daiichi Sankyo: Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Kyowa Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Sanofi: Honoraria, Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; MSD: Research Funding; Novartis Pharma: Research Funding; Gilead Sciences: Research Funding; Astellas Pharma: Research Funding; Teijin Pharma: Research Funding. Yasui:TokioTHERA Holdings, Inc.: Equity Ownership. Iida:Teijin Pharma: Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Astellas: Research Funding; Gilead: Research Funding; Sanofi: Research Funding; MSD: Research Funding; Abbvie: Research Funding; Kyowa Kirin: Research Funding; Chugai: Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Daichi Sankyo: Honoraria, Research Funding. Tojo:Torii Pharmaceutical: Research Funding; AMED: Research Funding.

Description: Introduction: Daratumumab (DARA) is a human IgGκ mAb targeting CD38 with both direct on-tumor and immunomodulatory mechanisms of action, and has been approved as monotherapy for RRMM and in combination with standard of care (SOC) regimens for RRMM and newly diagnosed MM (NDMM). Across three phase 3 DARA studies in RRMM and NDMM, DARA plus SOC reduced the risk of progression or death by ≥50%, enabled a doubling of CR rates, and elicited a ≥3-fold increase in MRD-negative rates. Among MRD-negative RRMM pts, pts treated with D-Rd or D-Vd rapidly achieved MRD negativity and demonstrated prolonged progression-free survival (PFS) vs MRD-positive pts (Avet-Loiseau H, et al. ASH 2016. Abstract 246). MRD assessment is being investigated as a potential surrogate for established endpoints such as overall survival (OS). When measured sequentially, sustained MRD-negativity provides an index of deep clinical responses that may provide a more robust assessment of disease control (Kumar S, et al. Lancet Oncol 2016. 17[8]:e328-e346). Here, we evaluate sustained MRD negativity with DARA plus SOC regimens and its association with PFS/OS outcomes in RRMM. Methods: Eligible pts in POLLUX and CASTOR received ≥1 prior line of therapy and were randomized (1:1) to receive SOC treatment regimens ± DARA. Pts in the POLLUX study were given lenalidomide (25 mg PO) on Days 1-21 and dexamethasone (40 mg) once per week in each 28-day cycle ± DARA (16 mg/kg IV) given weekly for Cycles 1-2, Q2W for Cycles 3-6, and Q4W thereafter. CASTOR pts received 8 cycles (21 d/cycle) of bortezomib (1.3 mg/m2 SC) on Days 1, 4, 8, and 11 and dexamethasone (20 mg) on Days 1, 2, 4, 5, 8, 9, 11, and 12 ± DARA (16 mg/kg IV) given weekly for Cycles 1-3, Q3W for Cycles 4-8, and Q4W thereafter. MRD was assessed at the time of suspected CR and at 3 and 6 months following confirmed CR in POLLUX, and at time of suspected CR and 6 and 12 months following the first treatment dose in CASTOR. Additional MRD evaluation was required in both studies every 12 months post-CR. MRD was assessed via next generation sequencing using the clonoSEQ® assay V2.0 (Adaptive Biotechnologies, Seattle, WA). Sustained MRD negativity was defined as the maintenance of MRD negativity in the bone marrow confirmed ≥6 or ≥12 months apart and was evaluated in the intent-to-treat (ITT) population. Sustained MRD negativity was also evaluated among ≥CR pts to account for different sustained MRD negativity rates between treatment arms. Results: A total of 569 (D-Rd, n = 286; Rd, n = 283) pts in POLLUX and 498 pts (D-Vd, n = 251; Vd, n = 247) in CASTOR were randomized; median (range) number of prior lines received was 1 (1-11) and 2 (1-10), respectively. Median duration of follow up was 39.5 months in POLLUX and 31.3 months in CASTOR for this analysis. Using the ≥6-month sustained MRD cutoff, a significantly higher proportion of pts achieved sustained MRD negativity for ≥6 months when treated with D-Rd vs Rd (16% vs 0.7%; P

Description: Background Although the proteasome inhibitor bortezomib (Btz) shows excellent efficacy in multiple myeloma (MM), some patients exhibit sub-optimal or no response to this agent. In addition, Btz-induced toxicity, such as peripheral neuropathy (PN) and skin disorders, limits its use in some patients. A comprehensive analysis of metabolites (metabolomics) in biofluids can be a potential novel strategy to predict the efficacy or adverse events of Btz treatment. With respect to the metabolic pathways in MM cells, a few studies have applied metabolomics using serum/plasma samples to elucidate MM pathogenesis or the mechanisms underlying the malignant transformation of MM cells. In addition, no metabolomic profile has been examined in terms of efficacy or toxicity of the specific MM treatment. In this study, we performed lipid metabolomics analysis using plasma samples from patients with newly diagnosed MM (NDMM) prior to the initial Btz therapy, and have attempted to identify the association between the level of specific biomarkers in plasma lipid metabolites and the efficacy or severity of Btz-related toxicity. Materials & Methods Fifty-four plasma samples were analyzed from transplant-ineligible patients with NDMM enrolled in a randomized phase II study comparing two less intensive regimens of melphalan, prednisolone, and Btz (MPB) (JCOG1105; UMIN000011180). Informed consent to participate in the JCOG-BioBank, Japan Biorepository project, was obtained from the patients prior to sample acquisition. Frozen plasma samples obtained prior to MPB therapy were subjected to lipid metabolomics analysis, and the levels of phospholipids, sphingolipids, neutral lipids, and fatty acids (FAs) were measured using Liquid Chromatography/Mass spectrometry. The levels of lipid metabolites were relatively quantified as the ion peak ratio of each metabolite to the internal standard. We then evaluated whether the level of each lipid metabolite associated with the depth of response to MPB therapy and the grade of Btz-induced toxicity, such as peripheral neuropathy (PN) and skin disorders. Statistical analysis was performed by a multivariate permutation test using the Welch's t-statistic for multiple comparisons of the metabolite levels between the two groups categorized based on their response to MPB therapy or the grade of toxicity, ie. responder vs non-responder to the therapy, and grade 0-1 vs grade 2 or higher in the toxicities. Results and Discussion Lipid metabolomics analysis detected approximately 485 lipid metabolites in 54 plasma samples. We then evaluated the association between the level of each lipid metabolite and the grade of Btz-induced PN (BiPN) or skin disorders. As shown in Figure 1, high levels of seven phospholipids, including four lysophosphatidylcholine (LPC) and three phosphatidylcholine (PC), were associated with BiPN of grade 2 or higher (n=11). LPC species were reported to facilitate neuropathic pain and nerve demyelination of the dorsal root ganglion and cuneate nucleus in the experimental model of PN. Therefore, high level of plasma LPC might worsen BiPN. Moreover, low levels of three fatty acids-FA (18:2), FA (18:1), and FA (22:6)-were observed in patients who developed severe skin disorders of grade 2 or higher (n=10). Low level of FA (18:2), also called linoleic acid, has been reported to impair the anti-inflammatory response and repair skin barrier during the healing process. Therefore, low level of plasma FA (18:2) might associate with the aggravation of Btz-induced skin disorders. No metabolite was significantly associated with tumor response, such as CR (n=9), in 53 samples evaluated for the best response to MPB therapy. Conclusion To our knowledge, this is the first plasma lipid metabolomics study to demonstrate that plasma lipid metabolite levels are associated with the severity of BiPN and skin disorders in patients with MM. These metabolites may serve as candidate biomarkers to predict Btz-induced toxicity in patients with MM before initiating Btz therapy. Our exploratory results need to be confirmed in further validation studies. Disclosures Ri: Janssen Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Daiichi Sankyo: Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Kyowa Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Sanofi: Honoraria, Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; MSD: Research Funding; Novartis Pharma: Research Funding; Gilead Sciences: Research Funding; Astellas Pharma: Research Funding; Teijin Pharma: Research Funding. Iida:Celgene: Honoraria, Research Funding; Chugai: Research Funding; Kyowa Kirin: Research Funding; Abbvie: Research Funding; MSD: Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Teijin Pharma: Research Funding; Janssen: Honoraria, Research Funding; Daichi Sankyo: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Astellas: Research Funding; Gilead: Research Funding; Sanofi: Research Funding. Maruyama:Eisai: Honoraria, Research Funding. Tohkin:Towa Pharmaceutical: Research Funding. Tobinai:Kyowa Kirin: Honoraria, Research Funding; Meiji Seika: Honoraria; Zenyaku Kogyo: Consultancy, Honoraria; Takeda Pharmaceutical: Consultancy, Honoraria, Research Funding; Verastem: Honoraria; Ono Pharmaceutical: Consultancy, Honoraria, Research Funding; HUYA Bioscience: Consultancy, Honoraria; Janssen Pharmaceutical: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Solasia: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria; Eisai: Honoraria, Research Funding; Yakult: Honoraria; Mundi Pharma: Consultancy, Honoraria, Research Funding; AbbVie: Research Funding. Fukuhara:Chugai Pharmaceutical Co., Ltd.: Honoraria; Zenyaku: Honoraria; Eisai: Honoraria, Research Funding; Janssen Pharma: Honoraria; Mochida: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Mundi: Honoraria; Kyowa-Hakko Kirin: Honoraria; AbbVie: Research Funding; Celgene Corporation: Honoraria, Research Funding; Nippon Shinkyaku: Honoraria; Bayer: Research Funding; Gilead: Research Funding; Solasia Pharma: Research Funding. Miyazaki:Ono Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Eisai: Honoraria; SymBio Pharmaceuticals: Honoraria; Takeda: Honoraria; Nippon Shinyaku: Honoraria; Celgene: Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; Chugai: Honoraria; Janssen Pharmaceutical: Honoraria. Tsujimura:Chugai Pharmaceutical: Honoraria; Eisai: Honoraria; Kyowa Kirin: Honoraria; Takeda Pharmaceutical: Honoraria. Yoshimitsu:Novartis: Speakers Bureau; Bristol-Myer-Squibb,: Speakers Bureau; Shire: Speakers Bureau; Takeda: Speakers Bureau; Chugai: Speakers Bureau; Sanofi: Speakers Bureau. Tsukasaki:Byer: Research Funding; Kyowa Kirin: Honoraria; Mundi Pharma: Honoraria; Ono Pharmaceutical: Consultancy; Daiichi Sankyo: Consultancy; Eisai: Research Funding; Celgene: Honoraria, Research Funding; Huya: Consultancy, Honoraria, Research Funding; Chugai Pharmaceutical: Honoraria, Research Funding. Nagai:SymBio Pharmaceuticals Limited: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; HUYA Bioscience International: Research Funding; Solasia Pharma K.K.: Research Funding; Zenyaku Kogyo: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria, Research Funding; AbbVie: Research Funding; Bayer Pharma: Honoraria, Research Funding; Mundi Pharma: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen Pharmaceutical: Honoraria, Research Funding; Chugai Pharmaceutical: Honoraria, Research Funding; MSD: Honoraria; IQVIA: Research Funding; Otsuka Pharmaceutical: Research Funding; Sanofi: Honoraria; Novartis Pharma: Honoraria.

Description: Abstract 2945 Background: Despite recent advances of therapeutic strategies, multiple myeloma (MM) still remains mostly incurable due to its drug resistance by both various cell intrinsic molecular abnormalities and the supports of myeloma microenvironment factors. The search for new therapeutic target molecule upon which various MM-promoting signalings converge is essential for the development of more effective therapy for MM. RSK2 is a member of the 90 kDa ribosomal S6 kinases (RSKs) family of serine (Ser) /threonine (Thr) kinases and lies downstream of RAS/ERK-1/2 signaling cascade. In MM, it has been shown that RSK2 mediates signaling initiated by FGFR3 activation for cell survival, but the functional role and the value as the therapeutic target of RSK2 have not been clearly defined. In this study, we precisely studied the association with RSK2 activation and chromosomal status, especially focusing on RSK2Ser227, which is responsible for downstream signaling phosphorylation, and the value of RSK2 as the therapeutic target in MM. Methods: Twelve human myeloma-derived cell lines and primary MM cells were utilized in this study. The use of human samples was approved by the ethical review board of our institute. Cell killing assay was performed using flow cytometry. Growth inhibitory assay was performed with modified MTT assay. Protein levels were examined by Western blotting. BI-D1870, a RSK2 inhibitor, was purchased from Symansis Limited (Auckland, New Zealand). RNA interference for RSK2 was performed by transfection of small interfering RNA (siRNA) into NCI-H929 cells by means of CLB-Transfection Kit (Lonza, Swiss) with protocol 9. Drug combination assays were evaluated with CalcuSyn software (Biosoft, Cambridge, UK). Results: RSK2Ser227 was shown to be phosphorylated in all 12 MM cell lines and 6 of 9 primary MM cells. t(4;14) involving IgH/FGFR3 was detected only in 7 cell lines and high FGFR3 expression was identified only in 6 cell lines. RSK2Ser227 was phosphorylated even in the absence of ERK1/2 or RSK2Tyr529 phosphorylation in several MM cell lines, indicating that their activation was not mandatory to RSK2Ser227 phosphorylation. Our study also did not show any positive relationship between RSK2Ser227 phosphorylation and other so-called “high-risk” cytogenetic abnormalities, such as 13q-, t(11;14), t(8;14), t(14;16), 1q21+, or 17p-. Treatment with BI-D1870 (RSK2 inhibitor) resulted in complete dephosphorylation of RSK2Ser227, but not RSK2Tyr529. The phosphorylation status of ERK1/2, or related signaling kinases, such as AKT, p38 MAPK, or JNK was also not affected. BI-D1870 showed dose-dependent growth inhibition effect in 6 MM-derived cell lines by inducing apoptosis. At molecular level, Mcl-1, p21WAF1/CIP1, cyclin D2 and c-Myc was significantly down-regulated and Bim was up-regulated by BI-D1870 intervention in NCI-H929 cell. BI-D1870 didn't modulate expression level of IRF-4. These change were also observed in other MM cell lines (KMS-34, AMO-1). To exclude the possibility that above BI-D1870 induced changes in protein expression and cell death are due to its off-target effects, we performed gene knockdown experiments of RSK2. Transient gene knockdown of RSK2 by means of RNAi caused downregulation of cyclin D2, p21WAF1/CIP1, c-Myc, while it induced cell death in NCI-H929 cells. In contrast, Bim expression was not altered by RSK2 knockdown. Lenalidomide (LEN) is the most powerful currently available IMiD for MM treatment. While cell death induction by LEN (100μM) was partly prevented by IL-6 (50ng/ml) in NCI-H929 cells, co-administration of BI-D1870 (0.2 μM, at the degree of low concentration not to affect cell viability when used solely) overcame the resistance to LEN induced by IL-6. KMS-11/BTZ were highly resistant to bortezomib (BTZ)-induced cell death, by contrast, it showed the similar sensitivities to BI-D1870-induced cell death when compared with their parental cells. And more, BI-D1870 showed synergistic or additive effects with upcoming new anti-MM agents, such as RAD001, MS-275 and ABT-263 in MM cell lines. Conclusions: RSK2, especially RSK2Ser227, may be a universal therapeutic target for MMs with diverse molecular signatures. Disclosures: Iida: Janssen Pharmaceutical K.K.: Honoraria.

Description: Despite recent progress in treatment for multiple myeloma (MM), a complete cure remains elusive. To further improve the therapeutic outcome of patients with MM, elucidation of the pathology of refractory cases is important. Hyperamylasemia, which is associated with ectopic amylase (AMY) production by MM cells, is a rare condition, and it has been reported to present with poor prognosis showing rapid tumor growth, extramedullary tumor mass formation, and refractoriness of the condition. However, to date, there have been no biological analyses of MM cells ectopically producing AMY. In this study we generated transfectants that stably expressed AMY with human MM cells, and investigated the impact that ectopic AMY production has on tumor proliferation and changes in drug susceptibility in vitro and in vivo. Two human MM cell lines (RPMI8226 and KMS11) and the cDNA encoding AMY1 were used to establish transfectants with ViraPower™ Lentiviral Gateway Expression Kit (Invitrogen), because the increased AMY isotype was salivary type, which is coded in AMY1, in all MM patients previously reported. The constitutive expression and production of AMY1 were confirmed in the AMY-transfectants (8226/AMY and KMS11/AMY), while they were not in the mock controls. These transfectants were assayed for proliferation and apoptosis after exposure to dexamethasone (Dex), bortezomib (Bz) and lenalidomide (Len) in vitro. The anti-myeloma activity of Bz was also tested in vivo in a xenograft model generated by injecting 8226/AMY or the mock cells into NOD-SCID mice. 8226/AMY had no growth advantage in vitro but grew rapidly when subcutaneously transplanted in mice compared with the mock control (2,177±878 vs 970±131 mm3, p = 0.044). 8226/AMY showed a higher cell proliferation rate than the mock control in vitro when treated with Dex (40uM), Bz (2nM), and Len (1mM). The number of apoptotic 8226/AMY cells decreased after exposure to Bz and Len, but the number after exposure to Dex was equivalent compared with the mock control by the Annexin / Propidium Iodide assay. Therefore, 8226/AMY became less sensitive to Bz and Len partly through the inhibition of apoptosis induced by these drugs. 8226/AMY grew rapidly subcutaneously in mice compared with the mock control when treated with Bz (0.3mg/kg, twice weekly) (p = 0.017). As for KMS11/AMY, the AMY-transfectant showed a higher proliferation rate than the mock control in vitro. KMS11/AMY showed reduced susceptibility to Dex, no change in the susceptibility to Bz, and an enhanced susceptibility to Len unexpectedly in comparison with the mock control. The reason for a difference in the effect of ectopic AMY expression on the susceptibility to anti-MM drugs between 8226/AMY and KMS11/AMY is unclear; however, it might be due to the nature of their parental cells. No significant difference was observed in the gene expression profiling between both AMY-transfectants and each of the respective mock controls, except for AMY1, suggesting that ectopic AMY expression did not affect the expression level of the specific gene in MM. In conclusion, we found that 8226/AMY had reduced susceptibility to Dex, Bz, and Len in vitro and also rapid tumor growth with a weakened anti-tumor effect of Bz in vivo. All of these were consistent with the clinical course of previously reported patients with ectopic AMY-producing MM. On the other hand, KMS11/AMY showed an enhanced susceptibility to Len compared with the mock control, indicating that Len might be effective for some patients with AMY-producing MM. Our data provided beneficial clues for elucidating the molecular pathology and developing a treatment strategy for this clinical setting. Disclosures No relevant conflicts of interest to declare.

Description: Key Points BAY 1143572, a novel and selective P-TEFb/CDK9 inhibitor, possessed significant antitumor activity against primary ATL cells in vitro. BAY 1143572 possessed significant antitumor activity in an ATL mouse model based on tumor cells from a patient.

Description: Abstract 3772 Poster Board III-708 [Purpose] Bortezomib (BTZ), a proteasome inhibitor, has been introduced into the treatment of multiple myeloma (MM). It shows remarkable response against both relapsed/refractory and newly diagnosed MM. However, it is often encountered that BTZ treatment achieves very short duration of response and permits early drug resistance. Therefore, understanding the mechanisms underlying this drug resistance is necessary to develop novel treatments to overcome this problem. [Materials & Methods] We established two stable BTZ-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, whose IC50 values were respectively 24.7- and 16.6-fold higher than their parental cell lines, under continuous exposure to BTZ. Using these resistant cells, we investigated on their proteasome activity, the alteration of proteasome β5 subunit (PSMB5) gene, misfolded protein accumulation, endoplasmic reticulum (ER) stress, and apoptosis signals including BH3 only proteins in comparison with their parental cells at clinically achievable concentration of BTZ treatment. [Results & Discussion] No activation of caspase -3,-8, and -9 and BH3 only protein, Noxa, which were initially up-regulated in BTZ-treated cells, were noted in BTZ-resistant cells even in the presence of BTZ. These results indicate avoidance of fatal intracellular stress may block transcriptional activation of Noxa in resistant cells at an early phase after BTZ exposure. In gel shift assay detecting NF-kB-DNA complexes, BTZ-resistant cells maintained constitutive NF-kB activation, whereas their parental cells lost its activity in the presence of BTZ. In addition, cellular proteasome activities including chymotrypsin-like and caspase-like activity were markedly inhibited by BTZ treatment in parental cells, and moderately also in BTZ-resistant cells, when detected by fluorogenic substrates specific for each proteasome activity. While time-dependent accumulation of ubiquitinated proteins was prevented only in BTZ-resistant cells, but not in their parental cells after BTZ exposure. Resistance was partly explained by the presence of a unique point mutation, G322A, in the gene encoding PSMB5 in both BTZ-resistant cell lines, which substituted Thr for Ala at the codon 49 in amino acid level. This constitution has been reported to gives rise to the conformational change of BTZ-binding pocket in β5 subunit, which results in partial disruption of the contact between BTZ and chymotrypsin-like active site. Furthermore, BTZ-resistant and parental MM cells had nearly equal expression of cytoplasmic and ER chaperons, however, only BTZ-resistant cells could prevent misfolded protein accumulation and therefore avoid fatal ER stress represented as activation of CHOP and of caspase-4, -12 after BTZ treatment. [Conclusion] Two kinds of stable BTZ-resistant MM cell lines were established, which acquired the unique point mutation (G322A) in BTZ-binding pocket of PSMB5, prevented the accumulation of misfolded proteins probably via reduced affinity of 26S proteasome to BTZ and avoided the development of catastrophic ER stress unlike their parental cells. These cell lines will provide better understanding of the underlying mechanisms of the BTZ-resistance, and will lead to the development of novel treatment strategies for overcoming BTZ-resistance in the patients with MM. Disclosures: Iida: JANSSEN PHARMACEUTICAL: Honoraria; KYOWA KIRIN: Research Funding. Nakashima:KYOWA KIRIN: Employment. Miyazaki:KYOWA KIRIN: Employment. Shiotsu:KYOWA KIRIN: Employment.