ALBERT

Description: Multiple myeloma (MM) is a neoplasm of plasma cells that often remains fatal despite the use of high-dose chemotherapy with hematopoietic stem cell transplantation. In the clinical setting, the introduction of novel agents, such as proteasome inhibitors and immunomodulatory drugs, has improved the clinical outcomes of both patients with newly diagnosed MM and patients with advanced MM. However, most patients eventually relapse and develop drug resistance. T-LAK cell-originated protein kinase (TOPK), also known as PDZ-binding kinase (PBK), is a mitogen-activated protein kinase kinase (MAPKK)-like serine/threonine kinase that plays a critical role in many cellular functions, such as cell proliferation, apoptotic cell death, and inflammation, in normal tissues. Because the expression of TOPK is up-regulated during mitosis and is activated by the Cdk1/cyclin B1 complex, TOPK is thought to have a role in cytokinesis. While the expression of TOPK is very low in most normal human tissues except for testis and placenta, it is overexpressed in various malignant neoplasms, indicating its crucial role in tumorigenesis. Phosphorylation of TOPK leads to the activation of the MAPK signaling pathway including p38 and Ras extracellular signal-regulated kinase (ERK). Moreover, TOPK interacts with p53 tumor suppressor protein and inhibits its function. Ribosomal protein S6 kinase (RSK2) is a downstream target of the ERK/MAPK signaling cascade and it has a pivotal role in cell survival and proliferation. Recent studies suggest that RSK2 inhibition induces apoptotic cell death and sensitizes MM cells to lenalidomide. Suppression of p53 function is also involved in MM progression. Taken together, these data suggest that TOPK might be an attractive target for new therapeutic agents against this incurable hematological malignancy. HI-TOPK-032, which is a potent and specific inhibitor of TOPK, occupies the ATP-binding site of TOPK and thereby suppresses TOPK kinase activity. In the present study, we investigated the role of TOPK/PBK in MM as a potential therapeutic target by using HI-TOPK-032. MTSand trypan blue dye exclusion assays showed that HI-TOPK-032 inhibited the proliferation of various MM cell lines, including U266, RPMI8226, MM1.S, OPM-2, and KMS-11, in a dose- (0 to 10 mM) and time- (0 to 72 h) dependent manner. To examine the mechanisms behind the growth inhibition effect induced by HI-TOPK-032, assays for apoptotic cell death were performed; these assays demonstrated that HI-TOPK-032 induced both early and late apoptosis in MM cells. To investigate the molecular mechanisms of HI-TOPK-032-induced cell death in MM cells, the expression of various cell death-associated proteins and down-stream molecules of TOPK was examined. Western blotting analysis showed that HI-TOPK-032 arrested cell growth and induced apoptotic cell death in MM cells in a dose-dependent manner by reducing t he phosphorylation of ERK and RSK2, thereby reducing the expression of the target molecules of RSK2, i.e., MCL1 and c-Myc. Moreover, HI-TOPK-032 induced p53 expression in a dose-dependent manner. We next examined the effects of HI-TOPK-032 on bortezomib (BTZ)-resistant MM cells, which represent an urgent issue in clinics and for which a therapeutic solution is important. Interestingly, HI-TOPK-032 inhibited the proliferation of both BTZ-sensitive wild-type KMS cells and BTZ-resistant KMS cells, suggesting that BTZ resistance can be overcome by targeting TOPK. Because our results showed that HI-TOPK-032 reduced the phosphorylation of RSK2, and previous studies have suggested that RSK2 inhibition sensitized MM cells to lenalidomide, we next studied the effects of HI-TOPK-032 in combination with lenalidomide on MM cell growth. HI-TOPK-032 and lenalidomide synergisticallyinduced growth arrest in not only lenalidomide-sensitive MM cells, but also in lenalidomide-resistant cells. To determine whether HI-TOPK-032 can re-sensitize BTZ-resistant cells to the anti-MM activity of BTZ, the effects of the combination of HI-TOPK-032 and BTZ were tested using an MTS assay. Interestingly, HI-TOPK-032 was able to re-sensitize BTZ-resistant MM cells to BTZ. These results indicate that the inhibition of TOPK may serve as an attractive therapeutic option for both patients with BTZ- or lenalidomide-resistant MM. In conclusion, these data suggest that TOPK/PBK can be a promising molecular target for the treatment of MM. Disclosures Kizaki: Nippon Shinyaku Co., Ltd.: Research Funding; Ono Phranacutical Co., Ltd.: Consultancy; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Chugai Phrarmaceutical Co., Ltd.: Research Funding.

Description: Background and Aim: Therapy-related chronic myeloid leukemia (TR-CML), which is defined as CML that developed after exposure to cytotoxic chemotherapy and/or radiotherapy, rarely exists in clinical practice, although its incidence rates are relatively lower than those of acute myeloid leukemia or myelodysplastic syndromes, accounting for 1.2-30.4% of secondary leukemias. The clinical behavior of TR-CML, including patient outcome, is reportedly not different from that of de novo CML before the era of imatinib treatment. While the recent advancement of CML treatment by the introduction of tyrosine kinase inhibitors (TKIs) has dramatically improved treatment outcomes in patients with CML, little is known about the treatment response and outcomes in patients with TR-CML treated with TKI. In this regard, we investigated the clinical entity of TR-CML in the era of TKIs, including treatment response to TKI and prognosis, in patients enrolled to the CML Cooperative Study Group. Patients and Methods: We retrospectively reviewed the data of patients enrolled in the CML Cooperative Study to identify patients diagnosed with TR-CML. This study included patients aged 〉15 years who were diagnosed with CML in the chronic phase between April 2001 and January 2016, and treated with any TKIs as initial therapy and followed up for at least 3 months. The study was approved by the research ethics board of each institution and conducted in accordance with the Declaration of Helsinki. A major molecular response (MMR) was defined as ≤0.1% on the International Scale (IS) or 100 copies of the BCR-ABL1 transcript/μg RNA in a transcription-mediated amplification and hybridization protection assay. A deep molecular response (DMR) was defined ≤0.0032% in the IS. Event-free survival (EFS) was defined as the period from the date of initial treatment with TKI to the date of onset of the first adverse event (loss of treatment efficacy, progression to the accelerated or blastic phase, or any cause of death) or the last follow-up. Statistical analyses were performed by using EZR. Results and Discussion: We identified 308 patients with newly diagnosed CML in the chronic phase, including 11 (3.6%) with TR-CML and 297 with de novo CML. Regarding the primary cancer, 2 of the 11 patients had breast cancer and the remaining 9 had prostate cancer, pharyngeal cancer, mesothelioma, lung cancer, colon cancer, ureter cancer, acute leukemia, gastric cancer, or bladder cancer, respectively. Eight cases were treated with chemotherapy, 2 were treated with radiotherapy, and the remaining case was treated with both chemotherapy and radiotherapy. The results of a cytogenetic analysis by G-banding were exclusive t(9;22)(q34;q11) in all the patients. The median time to diagnosis of CML from the initiation of chemotherapy and/or radiotherapy was 7 years (range, 1.2-33 years). No significant differences in patient age, sex, white blood cell count, hemoglobin level, platelet count, or European Treatment and Outcome Study risk were observed between the TR-CML and de novo CML groups. Among the patients whose cytogenetic and/or molecular responses were assessable, all had excellent treatment response to TKI. Seven patients unexpectedly reached MMR within 6 months after TKI initiation. Finally, 8 patients attained DMR or undetectable leukemia in the bone marrow and the remaining 3 attained MMR. The 5-year EFS of the patients in the de novo CML group was 90%. None of the patients in the TR-CML group experienced any adverse event. In conclusion, in the present study, we revealed that patients with TR-CML could attain a good clinical course with TKI therapy. Detailed investigations of TR-CML may provide new insights into the CML biology. Disclosures Iriyama: Novartis: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau. Takaku:Bristol: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Nakazato:Mundipharma KK: Research Funding. Fujita:Chugai Pharmaceutical Co.,LTD: Honoraria. Tokuhira:Bristol Myers Squibb Co., Ltd: Honoraria; Pfizer Co., Ltd: Honoraria; Eizai Co., Ltd: Honoraria. Kawaguchi:Novartis: Honoraria.

Description: Background: Substantial efficacy has been demonstrated with bortezomib (Bor)-containing regimens for the treatment of refractory/relapsed (RR) patients with transplant-ineligible (TI) multiple myeloma (MM). However, the dosing schedules for induction therapy and the significance of maintenance therapy using Bor in clinical settings have yet to be standardized. Discontinuations and dose reduction of Bor owing to severe adverse events (AEs) such as peripheral neuropathy (PN) and infection are frequent causes of adjustments to the standard 9 cycles of Bor/dexamethasone (BD) therapy used for induction in clinical practice. We have previously demonstrated that a change in Bor administration to a schedule of weekly or longer intervals had similar or greater efficacy for patients with RR-MM including transplant-eligible (Tokuhira M, et al. Leuk Res. 35:591-7, 2011). To investigate the efficacy of this modified Bor dosing schedule in the treatment focusing of TI-RR-MM, we retrospectively analyzed 22 patients receiving weekly-BD induction followed by a modified schedule of biweekly or longer intervals using Bor as maintenance therapy in a single institution. Methods: Data on the 22 TI-RR-MM patients treated with BD therapy in our institution were retrospectively analyzed. The induction therapy consisted of intravenous injections of Bor (1.3 mg/m2) on days 1, 8, 15, and 22 in combination with dexamethasone (10–20 mg) on days 1 and 2 after Bor infusion, every 5 weeks. After BD induction, Bor maintenance was initiated with a schedule of biweekly or longer intervals. The dose of BD could be reduced based on AEs or other social aspects at the discretion of the treating physician. Overall survival (OS) was defined as the period between BD initiation of treatment to the last follow-up. Results: The median age of patients at the time of BD induction was 72.6 years (range, 49–84 years), and the subtypes of MM were the IgG type in 14 patients, IgA type in 6 patients, and BJP in 2 patients. Twelve patients were men and 10 were women. On the basis of the International Staging System classification, the clinical stage was I in 3 patients, II in 16 patients, and III in 3 patients. The median number of prior treatment regimens was 2.0 (range, 1–6). The median follow-up duration was 4.3 years (inter-quartile range, 3.0–7.8 years). Ten patients were alive, and 12 patients had died at the time of reporting. The median duration from the start of the first line treatment to BD initiation was 1.8 years (range, 0.1–14.8 years). The median number of BD induction cycles was 2.0 (range, 1–6), and 8 patients (36%) achieved a partial response (PR), although the other 14 patients (64%) showed stable disease or only a minimal response. Although AEs such as PN and gastrointestinal tract symptoms developed in 10 of 22 patients during BD induction, all 22 patients could move on to the maintenance phase. The median duration of Bor maintenance was 14.5 months (range, 1.8–76.5 months). Three patients were still receiving maintenance therapy at the time of reporting. Six patients (27.2%) received maintenance therapy for over 2 years, and 3 (13.6%) received it for over 4 years. The median OS was 3.6 years (range, 0.4–6.5 years), and the duration of Bor therapy from induction to the last administration was 2.5 years. The median progression free survival was 1.6 years (range, 0.3–6.5 years). During maintenance therapy, 5 patients achieved a PR. The reasons for cessation of Bor maintenance therapy were AEs in 8 patients and progressive disease in 11 patients. No patient died during Bor administration in this study. Discussion: We retrospectively analyzed 22 TI-RR-MM patients receiving modified BD therapy. Pantani L et al. reported the results of using BD therapy for 85 RR-MM patients, and demonstrated that twice-weekly Bor administration resulted in a clinical response rate of 19% and a median OS of 22 months (Ann Hematol, 2014). Although the clinical response rate in our study was inferior to that of Pantani L et al., the median OS in our study was superior (3.6 years) than those of previous reports. Because the majority of patients in this study were frail or elderly, the early decision to change to maintenance therapy before they developed severe AEs resulted in a good clinical outcome and a longer OS. Continuing BD therapy without a satisfactory molecular response might be an attractive therapeutic approach for TI-RR-MM patients. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2939 Multiple myeloma (MM) remains incurable despite the use of conventional and high-dose chemotherapy with stem cell transplantation. However, novel agents such as thalidomide, lenalidomide, and bortezomib have already achieved promising clinical results. In particular, bortezomib, a proteasome inhibitor, is widely used in the treatment of MM and has remarkable clinical effects in both refractory/relapsed and newly diagnosed patients. However, acquired drug resistance and serious toxicities such as peripheral neuropathy are often triggered by bortezomib in the clinical settings. Therefore, novel targeted agents with less toxicity are necessary to overcome bortezomib-resistance and improve clinical outcomes in MM. We have previously reported that 1'-acetoxychavicol acetate (ACA), a component of a traditional Asian condiment obtained from the rhizomes of the commonly used ethno-medicinal plant Languas galanga, has an inhibitory effect on NF-κB, and induces cell death in MM cells both in vitro and in vivo (Cancer Res, 2005; 65: 4417). With the aim of discovering more potent NF-κB inhibitors, we subsequently developed several ACA analogs based on quantitative structure-activity relationship (QSAR) analysis. This showed that a novel benzhydrol-type analog of ACA (TM-233) exhibited greater inhibition of NF-κB activity in MM cells when compared with ACA. In the present study, we examined the effects of TM-233 on various MM cells, including those resistant to bortezomib and we investigated the molecular mechanism of TM-233-induced cell death in these cells. TM-233 inhibited cell proliferation and induced cell death in various MM cell lines (U266, RPMI8226, and MM-1S cells) in a time (0–48 hours)- and dose (0–5 μM)- dependent manner. Similarly potent activity was observed against primary MM cells. Exposure of bone marrow mononuclear cells from 3 patients with MM to TM-233 revealed selective activity under the same conditions as for cell lines. Moreover, TM-233 did not inhibit colony formation in CD34-positive cells from human umbilical cord blood; therefore, TM-233 did not influence the normal blood cells. We next investigated the effects of TM-233 on the various signaling pathways including JAK/STAT and NF-κB in MM cells. TM-233 activated apoptosis-related caspase-3, -8, -9, and PARP, suggesting that cell death related to TM-233 is mediated by both mitochondrial- and Fas-dependent pathways. Furthermore, treatment with TM-233 inhibited constitutive activation of JAK2 and STAT3, and then downregulated the expression of anti-apoptotic Mcl-1 protein, but not that of Bcl-2 and Bcl-xL proteins. TM-233 directly bound and activated the transcription of the Mcl-1 gene promoter, suggesting that TM-233-induced cell death in MM cells down-regulated Mcl-1 via modulation of the JAK/STAT pathway. In addition, the DNA-binding activity of NF-κB in MM cells was inhibited by the treatment of TM-233. Further, TM-233 rapidly decreased the nuclear expression of NF-κB, but increased the accumulation of cytosol NF-κB, suggesting that TM-233 inhibits the translocation of NF-κB from the cytosol to the nucleus. These results indicate that TM-233 induced cell death in MM cells occurs via both JAK/STAT and NF-κB pathways. We finally examined the effects of TM-233 on bortezomib-resistant MM cells. We have recently established the bortezomib-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ (Leukemia 2010; 24: 1506). We found that these cells have a unique point mutation, G322A, in the gene encoding the proteasome b5 subunit, resulting in bortezomib-resistance mediated through prevention of the accumulation of unfolded proteins and fatal ER stress. TM-233, but not bortezomib, inhibited cellular proliferation and induced cell death in KMS-11/BTZ and OPM-2/BTZ cells in a time- and dose-dependent manner. Interestingly, the combination of TM-233 and bortezomib significantly induced cell death in these bortezomib-resistant MM cells via inhibition of NF-κB activity. These results indicate that TM-233 could overcome bortezomib resistance in MM cells by acting according to a different mechanism from that of bortezomib, possibly inhibition of the JAK/STAT pathway. In conclusion, TM-233, a novel analog of ACA, might be a more potent NF-κB inhibitor than ACA and could overcome bortezomib-resistance in MM cells. Further studies investigating clinical applications are necessary and may yield interesting results. Disclosures: Iida: Janssen Pharmaceutical K.K.: Honoraria. Kizaki:Janssen Pharmaceutical K.K.: Honoraria.

Description: Background and Aim: Treatment with a tyrosine kinase inhibitor (TKI) is the standard of care for patients with chronic myeloid leukemia (CML). Based on the results of the ENESTnd and DASISION studies, the European LeukemiaNet 2013 guidelines now recommend the use of TKIs, imatinib (400 mg once daily), nilotinib (300 mg twice daily), and dasatinib (100 mg once daily) as first-line treatment for patients with CML in the chronic phase (CML-CP). Compared to imatinib, the new generation TKIs, nilotinib and dasatinib, were found to have deeper and faster treatment response rates with acceptable toxicities. However, a direct comparison between nilotinib and dasatinib has never been reported previously. Our study aims to compare the outcomes and molecular responses achieved following the first-line use of these two agents in patients with CML-CP. Patients and Methods: The database of the CML Cooperative Study Group, which includes patients who were initially diagnosed with CML after the introduction of imatinib (April 2001), was reviewed, and patients who were given nilotinib or dasatinib as first-line therapy were identified. The event-free survival (EFS, defined as the loss of treatment efficacy, disease progression, or any cause of death), and rates of cumulative major molecular response (MMR), and deep molecular response (DMR, defined as the depth of MR 4.5) were compared between the nilotinib and dasatinib groups. Further, the predictive ability of the Sokal, Hasford, and European Treatment and Outcome Study (EUTOS) scoring systems for the achievement of molecular responses was also evaluated. For the analysis of molecular responses, patients who switched from their initial treatment agent to another TKI before achievement of MMR or DMR were considered to have no MMR or DMR. Results and Discussion: Out of 361 patients with CML-CP enrolled in our database, 58 and 63 were treated with conventional doses of nilotinib (300 mg twice daily) and dasatinib (100 mg once daily), respectively, as first-line therapy. Patients who had been started on a low dose TKI therapy were excluded from this analysis. The patient demographics, including age, sex, observation periods, and the Sokal, Hasford, and EUTOS scores did not show significant differences between the groups. In total, there were five events during the observation period (1 in the nilotinib group and 4 in the dasatinib group), and all events were deaths unrelated to CML, except for one in a patient in the dasatinib group who showed loss of complete cytogenetic response. The disease did not progress to the accelerated or blastic phase in any of the cases. The EFS did not differ between these two groups (p = 0.214). The MMR rates by 6, 12, 18, and 24 months were 59%, 72%, 74%, and 81%, respectively, in the nilotinib group and 52%, 73%, 81%, and 86%, respectively, in the dasatinib group. The DMR rates by 6, 12, 18, and 24 months was 7%, 17%, 24%, and 28%, respectively, in the nilotinib group and 3%, 16%, 25%, and 29%, respectively, in the dasatinib group. During the first 24 months of treatment, 4 (7%) patients in the nilotinib group and 11 (17%) in the dasatinib group had been switched to other TKIs (p = 0.0983). Among the three scoring systems, only the Hasford score could predict the achievement of DMR, and all of them failed to predict the achievement of MMR in the entire cohort. Our data suggest that both nilotinib and dasatinib have comparable efficacies, with high molecular response rates and promising outcomes. The validity of our findings should be tested in a randomized study, which is currently underway in Japan. Disclosures Takaku: Bristol-Myers Squibb: Honoraria, Speakers Bureau; Novartis Pharma K.K.: Honoraria, Speakers Bureau. Tokuhira:Mitsubishi Tanabe Pharma Corporation: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; AYUMI Pharmaceutical Corporation: Speakers Bureau; Chugai: Speakers Bureau. Asou:Eisai Co., Ltd.: Research Funding; SRL Inc.: Consultancy; Yakult Honsha Co., Ltd.: Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Asahi Kasei Pharma Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Kizaki:Nippon Shinyaku,: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis: Speakers Bureau. Kawaguchi:Novartis Pharma K.K.: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Alexion: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau.

Description: Multiple myeloma (MM) is a hematological malignancy characterized by abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novel agents such as proteasome inhibitors, immunomodulatory drugs, and antibodies that have significantly improved clinical outcomes of the patients of MM, most patients eventually relapse and develop drug resistance. In particular, the prognosis of patients harboring either chromosome TP53 deletion or mutations remains very poor, suggesting the prevalence of TP53 abnormalities increases with disease progression. Therefore, novel therapeutic strategies to overcome this unfavorable feature are urgently needed in clinical settings. WEE1 is a cell-cycle checkpoint kinase and a key regulator of DNA damage surveillance pathways. In response to extrinsically induced DNA damage, WEE1 kinase induces cell cycle arrest, allowing damaged DNA to be repaired before the cell undergoes DNA replication in S phase, and preventing cells harboring unrepaired, damaged DNA from mitotic lethality. Furthermore, WEE1 overexpression has been observed in many types of cancers. In addition, our previous studies revealed that monotherapy with AZD1775, a potent and highly selective inhibitor of WEE1, inhibited the proliferation of various MM cell lines irrespective of TP53 status. (Blood 2016; 128: 3256). On the other hand, one of the defining features of MM cells is the production of large amounts of protein, such as immunoglobulin, that must be processed within the endoplasmic reticulum (ER). Due to the accumulation of abundant immunoglobulin in ER, MM cells are constitutively under conditions of ER stress. The unfolded protein response (UPR)-signaling pathway is a cytoprotective mechanism against ER stress, and is therefore activated in MM cells to survive these conditions. Activation of the UPR has been observed in many types of cancers, and loss of TP53 has shown to enhance the UPR. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is one of three ER transmembrane protein kinases implicated as primary effectors of the UPR. Recent studies have suggested that PERK inhibition resulted in dose-dependent inhibition of tumor growth both in vitro and in vivo. In addition, more recent studies have proposed that PERK induces resistance to cell death elicited by chemotherapy. The combination of WEE1 and PERK inhibitors might thus offer an attractive therapeutic option against this incurable hematological malignancy. Here, we investigated the therapeutic utility of AZD1775 and GSK2606414, a highly selective inhibitor of PERK kinase, alone and in combination in various MM cells including TP53 wild-type (MM1.S) as well as TP53-deficient (KMS-11) and TP53-mutated (U266, RPMI8226, OPM-2) cell lines. AZD1775 and GSK2606414 alone induced dose-dependent cell growth inhibition in all investigated MM cells irrespective of TP53 status. Interestingly, GSK2606414 in combination with AZD1775 inhibited proliferation of all MM cells more effectively than either single agent. Assays for apoptotic cell death demonstrated that AZD1775 in combination with GSK2606414 induced significant and marked apoptotic cell death in MM cells used in this study compared to monotherapy alone. Next, western blotting analysis was performed to address the mechanisms of apoptotic cell death by the treatment of WEE1 and PERK inhibitors in MM cells. GSK2606414 inhibited PERK activation and decreased its downstream substrates (phospho-eIF2a, ATF4, and CHOP). Combination treatment with WEE1 inhibitor and various doses of PERK inhibitor significantly increased PARP and caspase 3 cleavage, CDK1 phosphorylation, and histone H2AX expression. Taken together, these data suggest that combining AZD1775 and GSK2606414 synergistically induced DNA damage and promoted premature mitotic entry, resulting in apoptotic cell death of TP53-deleted or -mutated MM cells. In conclusion, dual targeting of WEE1 and PERK might be a promising therapeutic approach for MM irrespective of TP53 status. Disclosures Tokuhira: Bristol-Myers Squibb: Speakers Bureau; AYUMI Pharmaceutical Corporation: Speakers Bureau; Mitsubishi Tanabe Pharma Corporation: Speakers Bureau; Chugai: Speakers Bureau. Kizaki:Novartis: Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Nippon Shinyaku,: Research Funding, Speakers Bureau.

Description: Abstract 3837 Poster Board III-773 Multiple myeloma (MM) is a plasma cell malignancy that remains incurable despite the use of conventional chemotherapy. Recent advances in the field of cell biology could lead to new therapeutic agents based upon molecular targeting, such as thalidomide and proteasome inhibitor bortezomib. These agents have remarkable activity against both newly diagnosed and refractory patients with MM, but prolonged exposure to them may result in the development of de novo drug resistance. Thus, it is necessary to identify and validate novel agents with less toxicity to overcome drug resistance and to improve the clinical outcome of MM. Progression and chemoresistance are thought to involve IL-6, whose expression is induced by NF-κB, through its regulation of the growth and survival of MM cells. IL-6 leads to constitutive activation of STAT3, which in turn results in the expression of high levels of Mcl-1. Thus, the constitutive activation of both NF-κB and STAT3 plays an important role in MM cell proliferation, and both molecules are important targets for the treatment of MM. Auranofin (AF: RIDAURA®; GSK) is a coordinated gold compound that has been widely used for the treatment of rheumatoid arthritis based on its anti-inflammatory properties through the inhibition of NF-κB activation. Therefore, assuming that AF has the potency to induce apoptosis in MM cells by interfering with NF-κB and STAT pathways, it may become a candidate for a novel therapeutic agent. To address our hypothesis, the effects of AF on inducing apoptosis of various MM cells were examined. Further, the molecular mechanism of AF-induced apoptosis in MM cells was investigated. AF inhibited the growth of U266 cells in a time- and dose-dependent manner with IC50 of 50 nM at 24 h. AF significantly induced cell cycle arrest at the G1 phase and subsequent apoptosis of U266 cells. AF-induced apoptosis in various human MM cell lines and CD138-positive plasma cells from patients with MM involved the activation of caspases-3, -8, and -9. Treatment with AF inhibited the constitutive and IL-6-induced activation of STAT3, and then downregulated the expression of Mcl-1 but not that of Bcl-2 or Bcl-xL proteins. To clarify the biological significance of Mcl-1 in AF-induced apoptosis of MM cells, Mcl-1 expression vector (pEGFP-hmcl-1) and control vector were introduced into U266 cells (designated as U266/mcl and U266/neo cells). Induction of apoptosis by AF was abrogated in U266/mcl, but not in U266/neo cells. We next examined the effect of AF on the DNA binding activity of STAT3. Electrophoretic mobility gel shift assay (EMSA) using U266 nuclear extracts demonstrated that IL-6-induced STAT3 binding activity was inhibited by the presence of AF. These results suggest that AF inhibits the IL-6-induced JAK/STAT pathway selectively and induces apoptosis in MM cells via the downregulation of Mcl-1. In addition, AF downregulated the activation of NF-κB in an IκB-independent manner, and also inhibited DNA binding activity in U266 cells. Although NF-κB inhibitory peptide (SN-50) did not directly induce the expression of Mcl-1, the combination of SN-50 with AF reduced the levels of Mcl-1, suggesting that inhibition of NF-κB potentiates the apoptotic effect of AF. Finally, our in vitro data prompted us to examine whether or not the effects of AF are equally valid in a clinical settings. The protocol for assessing the clinical benefit of oral administration of AF (RIDAURA®) in patients with relapsed or refractory MM was approved by the Ethics Committee of Saitama Medical University. Since 2008, 7 patients with refractory MM were treated with oral RIDAURA® 6 mg daily. The median follow-up for patients was 7.5 months. All 7 patients responded to AF, including one PR (partial response) and 6 SD (stable disease) with no progression. No adverse events have been observed to date. Details of the clinical outcome will be presented. In conclusion, gold compound AF inhibited constitutive activation of both STAT3 and NF-κB, resulting in the downregulation of anti-apoptotic Mcl-1 protein in MM cells with clinical relevance. A low pharmacological concentration (50 nM) of AF is widely employed for the treatment of rheumatoid arthritis without any side effects; therefore, it may be used to treat MM without the risk of severe toxicity. We propose that AF (RIDAURA®) may have potential use as a new molecular-targeted agent for the treatment of MM. Disclosures: Off Label Use: Auranofin in the treatment of multiple myeloma.

Description: Background: According to VISTA study, the prognosis of elderly multiple myeloma (MM) patients has been improved with VMP therapy. However, intensive chemotherapy has induced severe adverse events (AEs), resulting in highly discontinued rate, especially often seen in unfit or frail patients. Several studies have demonstrated the potent of weekly bortezomib administration in induction therapy (GIMEMA, GEM2005MAS65), and maintenance therapy with bortezomib. Based on these fact, we designed phase 2 clinical study, BoRtezomib-based Optimized therapy Aiming Disease control in Japan (BROAD-J study), based on a weekly VMP as induction therapy followed by maintenance therapy with bortezomib (Bor-MT) with every two weeks administration for newly-diagnosed (ND) symptomatic non-transplant eligible MM patients. (UMIN 00007335) Method: From August 2011 to June 2016, according to the International Myeloma Working Group (IMWG) criteria, 87 patients older than 65 years with symptomatic MM were eligible for this trial (Table 1). The primary objectives were included maximum response and time to progression duration. Secondary objective was the continued treatment duration and adverse event rate. The induction phase consisted of VMP treatment: Melpharan: on days 1-4 (6 mg/m2), every 35-day cycle; prednisolone: on days 1-4 (40mg/d, every 35-day cycle; Bortezomib: 1.3mg/m2, d1, 8, 15, 22 every 35-day cycle. After 9 cycles of VMP therapy, the maintenance therapy starts. The maintenance phase was consisted of twice a month bortezomib administration (1.3 mg/m2) without dexamethasone until progressive disease. Dose reductions of bortezomib were allowed according to instructorfs recommendation. Patients could receive supportive therapy including bisphosphonates and transfusions as necessary. AEs were graded according to NCI-CTCAE v4.0. Response was assessed prior to every treatment cycle. Response categories were based on the International Myeloma Working Group uniform response criteria. Results: Mean age was 75 years (66-88), sex ratio was 44:43 (M: F), and ISS stage was I 12%, II 43%, III 43%. High risk cytogenetics abnormalities (i.e. del17p, t(14;16)) were observed in 13 patients (15%). Overall response rate was 87% and complete response was obtained in 22 patients (25%) (Table 1). Median progression free survival (PFS) was 36 months (Figure 1a) and median overall survival was 57 months (Figure 1b). Median PFS (Kaplan-Meier estimate) was significantly higher with patients who obtained CR or VGPR than PR or SD (P

Description: Multiple myeloma (MM) is a hematological malignancy that derives from the proliferation of unregulated plasma cells. Dramatic improvement in the clinical outcomes of both newly diagnosed and relapsed/refractory patients with MM has been achieved using many clinical approaches, including use of high-dose chemotherapy followed by hematopoietic stem cell transplantation, and new drugs, such as proteasome inhibitors, immunomodulatory drugs, and histone deacetylase inhibitors. However, most patients eventually relapse and develop drug resistance. Moreover, the prognosis of patients with bortezomib (BTZ) and/or lenalidomide (LEN)-resistant MM (key drugs in the treatment of MM) is very poor. Therefore, novel therapeutic approaches to overcome BTZ and LEN resistance are urgently needed in clinical settings. WEE1 is a cell-cycle checkpoint kinase and a key regulator of DNA damage surveillance pathways. In response to extrinsically induced DNA damage, WEE1 catalyzes inhibitory phosphorylation of both cyclin-dependent kinase1 and 2 (CDK1 and CDK2), leading to CDK1- and CDK2-induced cell cycle arrest at the G1, S, or G2-M phases. This cell-cycle arrest, in turn, allows for the damaged DNA to be repaired before the cell undergoes DNA replication, and prevents cells harboring unrepaired damaged DNA from mitotic lethality. Furthermore, recent research has shown that knockdown of WEE1 leads to DNA double-strand breaks specifically in S-phase cells undergoing DNA replication, and that WEE1 is most active in the S-phase, suggesting that WEE1 is involved in DNA synthesis. Overexpression of WEE1 has been observed in many types of cancers, including hepatic cancer, breast cancer, glioblastoma and gastric cancer, and high expression of WEE1 has been shown to correlate with poor prognosis. In addition, research has shown that inhibition of checkpoint kinase 1 (Chk1), a critical transducer of the DNA damage response, potentiates the cytotoxicity of chemotherapy on p53-deficient MM cells, which are regarded as chemotherapy-resistant, suggesting that inhibition of cell-cycle checkpoint kinase is involved in re-sensitization of refractory MM cells to anticancer drugs. These data suggest that WEE1 might be an attractive target for novel therapeutic agents against this incurable hematological malignancy. MK-1775 is a potent and highly-selective small-molecule inhibitor of WEE1. In the present study, we investigated the role of WEE1 in MM as a potential therapeutic target using MK-1775. MTSassays showed that single agent MK-1775 inhibited the proliferation of various MM cell lines, including the intrinsically LEN-resistant cell line, RPMI-8226, in a dose- (0 to 10 mM) and time- (0 to 72 h) dependent manner. Furthermore, the growth inhibition effect is irrespective of p53 status. To examine the mechanisms behind the growth inhibition effect induced by MK-1775, assays for apoptotic cell death were performed. These assays demonstrated that MK-1775 induces both early and late apoptosis in MM cells. To investigate the molecular mechanisms of MK-1775-induced cell death in MM cells, the expression of various cell death-associated proteins and downstream molecules of WEE1 were examined. Western blotting analysis showed that MK-1775 arrested cell growth and induced apoptotic cell death in MM cells in a dose-dependent manner by inhibiting both, the expression of the target molecules of Bcl-2 and MCL1, and the cleavage of PARP and Caspase 3. Similarly, there was a substantial inhibition of CDK1 phosphorylation downstream of WEE1. Moreover, an increased expression of histone H2AX was observed following administration of MK-1775, suggesting that MK-1775 results in cytotoxicity by direct DNA damage. Next, we examined the effects of MK-1775 on BTZ-resistant MM cells. Interestingly, MK-1775 inhibited the proliferation of both BTZ-sensitive wild-type MM cells and BTZ-resistant MM cells, suggesting that BTZ resistance can be overcome by targeting WEE1. Furthermore, in combination with BTZ, MK-1775 was able to re-sensitize BTZ-resistant MM cells to BTZ. These results indicate that inhibition of WEE1 might serve as an attractive therapeutic option for patients with both BTZ-resistant and LEN-resistant MM. In conclusion, our data suggest that WEE1 might be a promising molecular target for the treatment of MM. Disclosures Tokuhira: Bristol Myers Squibb Co., Ltd: Honoraria; Pfizer Co., Ltd: Honoraria; Eizai Co., Ltd: Honoraria.