ALBERT

Description: The blockade of Bcr-Abl signaling suppresses cellular growth and induces cell death in Bcr-Abl+ cells. While they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we assessed the regulatory mechanisms for cellular survival and death of Bcr-Abl+ leukemias more precisely, using a novel Bcr-Abl tyrosine kinase inhibitor, INNO-406 (formerly NS-187) which is more selective and 25-55-fold more active than imatinib (Kimura S, Blood 2005), in four CML-derived Bcr-Abl+ cell lines (K562, KT-1, BV173 and MYL), Ba/F3 harboring wild type bcr-abl (Ba/F3/wt bcr-abl), and in vivo CML mouse model. INNO-406 induces apoptosis in all lines examined, as were demonstrated by typical apoptotic morphology, loss of mitochondrial outer membrane potential (reduction of DiOC6 uptake), increase of cells in subG1 fraction by propidium iodide (PI) staining, DNA fragmentation and caspase-3 activation. However, when we inhibit caspase activity by zVAD-fmk (zVAD), a pan-caspase inhibitor, two modes of cell death execution were observed. In K562, KT-1 and BV173 cells treated with INNO-406, zVAD almost completely prevented apoptosis (i.e. showing atypical feature for apoptosis, no DNA fragmentation and no accumulation of subG1 fraction), with cell death resulting from morphologically non-apoptotic, so-called caspase-independent necrosis-like cell death (CIND). While, in MYL and Ba/F3/wt bcr-abl cells, despite the sufficient inhibition of caspases’ activity, the inhibition of the cell death by zVAD was only partial and these cell lines still underwent apoptosis (i.e. showing DNA fragmentation and the accumulation of subG1 population), suggesting the presence of alter cell death pathway which is caspase-independent apoptosis (CIA) in MYL and Ba/F3/wt bcr-abl. The propensity towards CIND or CIA in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity, that is CIND with a high apoptosome activity potential while CIA with low. Freshly isolated leukemic cell samples from Bcr-Abl+ leukemia patients also had either low or high apoptosome activity potential. Moreover, cells undergoing CIND exhibited hallmarks of autophagy (i.e. the autophagosome formation, punctate formations of LC3 and the accumulation of LC3-II isoform), suggested the participation of autophagy in response to Bcr-Abl blockade. Inhibition of autophagy with chloroquine enhanced INNO-406-induced cell death, which indicates that the autophagic response of the tumor cells is protective. While, in vivo CML model, INNO-406 treatment increased apoptotic cells regardless of the caspase-3 activation, further implicating the involvement of caspase-independent cell death regulatory pathway in vivo in primary Bcr-Abl+ leukemic cells. These findings suggest new insights into the biology and therapy of Bcr-Abl+ leukemias.

Description: ß-catenin is the downstream effector of the Wnt signaling pathway and has cyclin D1 and c-myc as its target genes. Activation of Wnt signaling is greatly involved in the process of carcinogenesis, and its overexpression has reported to be positively correlated with prognosis in many types of malignant tumors, as colorectal cancer and hepatocellular carcinoma, or hematological malignancy, as acute myeloid leukemia and chronic myeloid leukemia. Multiple myeloma (MM) is a relatively common hematological malignancy and remains incurable with conventional treatments. Here, we assessed the expression of ß-catenin in MM and growth inhibitory effect of ß-catenin siRNA on MM. We first examined the expression of ß-catenin on human MM cell lines: AMO-1, EJM, IM-9, KMS-12-BM, LP-1, NCIH929, OPM-2, RPMI8226, and U266. All cell lines examined expressed a significantly higher level of ß-catenin than normal human mononuclear cells. Moreover, ß-catenin was also overexpressed in myeloma cells from patients. We then assessed the in vivo growth inhibitory effects of ß-catenin siRNA in mouse model. Six -week-old male Balb/c nu/nu mice were subcutaneously inoculated in the right flank with 5 × 106 RPMI8226 myeloma cells in 100μl PBS. Three to four weeks later, when palpable tumors (100 mm3 in diameter) developed, mice (n = 5 for all groups) were treated with either subcutaneous (around tumors) injections of group A: ß-catenin siRNA (2.5μM) / atelocollagen complex (final atelocollagen concentration 0.5%), group B: control siRNA (2.5μM) /atelocollagen complex, group C: ß-catenin siRNA (2.5μM)/PBS, group D: PBS/atelocollagen twice a week for a total of eight injections. Tumor size was measured in two dimensions using a caliper, and tumor volume (mm3) was calculated as a2 × b /2 mm3 (a;minor axis,b;major axis). The mean tumor volume of each group after two weeks treatment was 412.2 mm3 in groupA, 1317.9 mm3 in group B, 2075.9 mm3 in group C, and 1802.3 mm3 in group D, respectively, and the treatment with ß-catenin siRNA (2.5μM) / atelocollagen complex significantly reduced tumor burdens and retarded tumor growth, measured as tumor volumes (p

Description: Background Primary central nervous system lymphoma (PCNSL) is a rare type of non-Hodgkin lymphoma. High-dose (HD) methotrexate (MTX) plus HD cytarabine (AraC) followed by whole-brain radiation therapy (WBRT) is currently considered the standard therapy for PCNSL. However, at present, the prognosis of patients is not always favorable, because neurotoxicity, particularly dementia, occurs in long-surviving patients at a high rate, and affects their quality of life. In recent years, therapy without WBRT has been attempted in patients, particularly the elderly. In this study, we investigated whether the prognosis of PCNSL patients was improved by the use of different therapeutic regimens. Patients and Methods Japanese patients with newly diagnosed PCNSL who had been admitted to our insutitute between January 2002 and March 2013 were retrospectively analyzed. Their medical records were reviewed regarding the histopathological diagnosis, performance status (PS), prognostic factors used by the International Extranodal Lymphoma Study Group (IELSG), and therapeutic regimens. Data were tabulated, stratified, and analyzed in terms of response rates and overall and progression-free survival. The response to treatment was evaluated by brain MRI. Overall and progression-free survival were estimated using the Kaplan-Meier method. The statistical analysis of observed differences was assessed using the log-rank test. Results Thirty-two patients with newly diagnosed PCNSL were admitted. Their median age at the start of treatment was 71 years (range, 42-86 years), with a male-to-female ratio of 21: 11. All patients were HIV-antibody-negative. Whole-body CT or FDG-PET excluded secondary CNS lymphoma in all patients. All of them underwent biopsy, and 28, 1, 1, and 2 were histopathologically diagnosed with diffuse large B-cell lymphoma, MALT lymphoma, ALK-negative anaplastic large-cell lymphoma, and unclassified type, respectively. In 25 patients, cerebrospinal fluid examination was performed, enabling the calculation of the IELSG prognostic score. Based on the prognostic score, 8, 14, and 3 patients were classified as high-, intermediate- and low-risk groups, respectively. Twenty-two patients received 6 courses of 3-3.5 g/m2 of HD-MTX (adjusted based on creatinine clearance) plus 2 courses of 4-6 g/m2of HD-AraC for 2 days with or without WBRT (30-36 Gy), while 4 underwent HD-MTX plus rituximab (R)-CHOP (6 courses) with or without WBRT. Four and 1 received WBRT and R-CHOP alone, respectively. The median follow-up period was 13 months (range, 1-123 months), and the overall response rate was 81.2% (complete and partial responses in 19 and 7 patients, respectively). To date, 14 patients have died, and 9 patients have survived. In all patients, the overall survival (OS) and progression-free survival (PFS) were 30 and 25 months, respectively. The median OS were 5 and 29 months in the high- and intermediate-risk groups, respectively, and the median OS was not reached in the low-risk group (p=0.03). On the other hand, the median PFS were 5, 20, and 77 months, respectively (p=0.07). No significant difference in the OS or PFS was observed between the HD-MTX plus HD-AraC and HD-MTX plus R-CHOP groups. However, univariate analysis showed that the OS and PFS were significantly improved in both groups treated with regimens including WBRT (p=0.005 and 0.008, respectively). Analysis by age revealed that the overall survival rate was significantly poorer in patients older than 65 years (p=0.03). Stratification based on PS and the use/non-use of rituximab showed no significant differences in the treatment results. Conclusions The treatment results were comparable in the HD-MTX plus HD-AraC and HD-MTX plus R-CHOP groups. On the other hand, the results of regimens with were better than those without WBRT. Thus, HD chemotherapy alone is insufficient for the management of PSCNL, and this study reinforced the importance of WBRT. We consider that HD chemotherapy regimens including HD-MTX and WBRT remain the standard therapy for PCNSL. The results also suggest that R-CHOP can be substituted for HD-AraC, and that it is necessary to reconsider the treatment strategy for PCNSL in the era of rituximab. Disclosures: Taniwaki: celgene: Research Funding.

Description: [Introduction] Multiple myeloma (MM) remains mostly incurable despite major advances in treatment strategies. The complex interplay among various cell-intrinsic and -extrinsic molecular mechanisms confers inter-patients diversity and intraclonal heterogeneity in MM. However, as the universal and relevant therapeutic target molecule against MM, we have recently identified that 3-phosphoinositide-dependent protein kinase 1 (PDPK1) and its major downstream substrate RSK2 were universally active in MM (Shimura Y, Mol Caner Ther 2012; Chinen Y, Cancer Res 2014), while we also identified that the repression of miR-375 was found to be the universal underlying mechanism for the overexpression/activation of PDPK1 in MM (Tatekawa S, ASH meeting 2015). In this study, we further extended our study to assess the clinical relevance of miR-375 repression and the molecular mechanisms for the miR-375 repression in MM for the future clinical translation of miR-375/PDPK1/RSK2 signaling axis in the diagnosis and treatment development for MM. [Methods] The miR-375 expression level was analyzed by the quantitative RT-PCR in 11 HMCLs and 113 patient-derived myeloma cells isolated by CD138-positive cell sorting (normal plasma cells; N=10, MGUS; N=30, newly diagnosed MM (NDMM); N=34, relapsed/refractory (RRMM); N=39). The level of miR-375 expression was calculated with 2-ΔΔCt methods. Human U6 snRNA was examined as the reference and cDNA of RPMI8226 was used as a calibrator sample. The methylation status of miR-375 upstream regions including promoter site were analyzed by methylation-specific PCR (MSP) and bisulfite sequence. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) was performed to examine the histone modification status in miR-375 upstream regions. The circulating plasma RNA samples were converted to cDNA libraries followed by sequencing using the multiplex small RNA library primer set. This study was conducted in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Boards. Patient-derived samples were obtained with informed consent. [Result] When compared to normal plasma cells, the miR-375 expression was significantly decreased in CD138 positive plasma cells from MGUS (p

Description: Abstract 1338 Poster Board I-360 Galectins are a family of soluble animal lectins that differ in their affinity for b-galactosides. Fifteen members of the human galectin family have been described to date. Galectin-9 (Gal-9) is a tandem-repeat-type galectin that was recently found to serve as a ligand for T cell immunoglobulin and mucin domain-3 (Tim-3), which is a Th1-specific type 1 membrane protein. Gal-9 modulates immune reactions, such as induction of apoptosis in Th1 cells. We herein investigated the effects of Gal-9 treatment on acute graft-versus-host disease (aGVHD) in a murine model. First, we assessed whether recombinant human (rh) Gal-9 can inhibit the mixed lymphocyte reaction (MLR) by culturing irradiated (30 Gy) splenic cells from 7- to 8-week-old female BDF1 mice with splenic cells from 7- to 8-week-old female C57BL/6J mice in the presence of various concentrations of Gal-9 for 10 days. rhGal-9 inhibited MLR in a dose-dependent manner. We then studied whether this effect was mediated by rhGal-9-induced apoptosis by culturing splenic cells from BDF1 mice with plate-bound anti-CD3 monoclonal antibody and Gal-9. Flow cytometric analysis revealed that rhGal-9 increased the number of Annexin-V+ cells in a dose-dependent manner (Figure. 1A). Thus, rhGal-9 inhibited MLR by inducing splenic cell apoptosis. This suppressive effect of Gal-9 on MLR was inhibited by lactose but not by sucrose (Figure. 1B). Taken together, Gal-9 induces T cell apoptosis through Ca2+ influx induced by binding to b-galactoside, resulting in the suppression of MLR. We then assessed whether rhGal-9 treatment altered aGVHD. Recipient B6D2F1 mice received a myeloablative dose (9 Gy) of total body irradiation from an X-ray irradiator. Six to eight hours later, each recipient mouse was injected i.v. with 4 × 106 TCD-BM cells and 5 × 106 mononuclear splenocytes. aGVHD clinical scores were evaluated once or twice a week. After aGVHD developed, recipient mice were treated with rhGal-9 (30 mg/mouse) or vehicle by i.p. injection for 14 consecutive days. The administration of rhGal-9 significantly attenuated aGVHD as compared to vehicle-treated mice (Figure. 2). Histological analyses also confirmed that aGVHD was declined by rhGal-9 treatment. Additionally, we investigated the effects of Gal-9 treatment on different T ell subsets. To analyze the effect of Gal-9 on donor lymphocytes, splenic mononuclear cells from GFP Tg mice were used for the induction of aGVHD. The gating parameter was first set to isolate the lymphocyte population of peripheral blood leukocytes, and then set for GFP+ cells. Gal-9 treatment decreased the frequency of CD4+/Tim-3+ cells and CD8+/intracellular IFN-g+ cells, whereas CD4+/CD25+ and CD25+/Foxp3+ Treg cells were increased by rhGal-9 treatment. These results suggest that Gal-9 regulates aGVHD by increasing regulatory T cells. In conclusion, Gal-9 ameliorates aGVHD by inducing T-cell apoptosis and also by increasing regulatory T cells. Disclosures No relevant conflicts of interest to declare.

Description: Central nervous system (CNS) relapse accompanying the prolonged administration of imatinib mesylate has recently become apparent as an impediment to the therapy of Philadelphia chromosome–positive (Ph+) leukemia. CNS relapse may be explained by limited penetration of imatinib mesylate into the cerebrospinal fluid because of the presence of P-glycoprotein at the blood-brain barrier. To overcome imatinib mesylate–resistance mechanisms such as bcr-abl amplification, mutations within the ABL kinase domain, and activation of Lyn, we developed a dual BCR-ABL/Lyn inhibitor, INNO-406 (formerly NS-187), which is 25 to 55 times more potent than imatinib mesylate in vitro and at least 10 times more potent in vivo. The aim of this study was to investigate the efficacy of INNO-406 in treating CNS Ph+ leukemia. We found that INNO-406, like imatinib mesylate, is a substrate for P-glycoprotein. The concentrations of INNO-406 in the CNS were about 10% of those in the plasma. However, this residual concentration was enough to inhibit the growth of Ph+ leukemic cells which expressed not only wild-type but also mutated BCR-ABL in the murine CNS. Furthermore, cyclosporine A, a P-glycoprotein inhibitor, augmented the in vivo activity of INNO-406 against CNS Ph+ leukemia. These findings indicate that INNO-406 is a promising agent for the treatment of CNS Ph+ leukemia.

Description: Background: A common referral to hematology is for patients with suspected myelodysplastic syndrome (MDS) or cytopenias of undetermined origin. We recently implemented a Next Generation Sequencing (NGS) panel which covers a wide spectrum of genes related to the major myeloid disorders, including DNA based gene sequencing and RNA based gene fusion analysis, as a tier one clinical diagnostic test (Levy et al. Eur. J. Haematol 2019). We compared the diagnostic and prognostic information derived from conventional cytogenetics and NGS testing as well as the clinical impact on management in this patient group. Methods: We identified all new cases with suspected MDS or cytopenias of undetermined origin referred between January 2018 to February 2019 that had both NGS and cytogenetic testing at London Health Sciences Centre, a tertiary care centre servicing a population of approximately 2.5 million in Southwestern Ontario, Canada. From the retrospective review of electronic medical record, patient demographics, diagnosis and management were ascertained. Diagnosis was based on the 2016 WHO classification and the ICD-10.Previously reported definitions for ICUS, IDUS, CHIP, and CCUSwere also adopted (Bejar et al. Leukemia 2017). Patients not meeting these criteria were defined as either cytopenia not yet determined (NYD) or secondary to other systemic disease. The impact of NGS and cytogenetics results on diagnosis, prognosis, and management of each disease were assessed by referring to the latest National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology. Either a bone marrow (BM) or peripheral blood (PB) sample from each patient was assessed by the Oncomine Myeloid NGS panel (Thermo-Fisher, MA, USA), which examines DNA sequence variants in 40 genes (17 full genes and 23 hotspot genes) along with an RNA-based panel of 29 fusion driver genes and their over 600 fusion partners. Patients' BM samples were also tested by the conventional cytogenetic G-banding method. Results: Of the 1100 samples assessed by NGS, 178 met the study inclusion criteria. Overall, 120 (67.4%) patients had both cytogenetics and NGS performed on their BM samples. Of those 120 patients, 41 (34.2%) had DNA mutations, 17 (14.2%) had cytogenetic abnormalities, 22 (18.3%) had both molecular and cytogenetic findings, and 40 (33.3%) had neither abnormality identified. NGS Information contributed in diagnosing 13 (10.8%) patients, while cytogenetics in 6 (5%) patients. In addition, of 38 patients who were diagnosed as MDS with good/intermediate risk cytogenetics, 24 (63.2%) harbored poor prognostic risk mutations as detected by NGS (Table), influencing the management of disease. Additionally, 22 (12.4%) of all patients had NGS testing using PB and cytogenetics using BM samples, and of these 13 (52.2%) patients had NGS abnormalities. Finally, 36 (20.2%) of all patients had only NGS testing by PB samples. Of those, 12 (33.3%) were found to harbor at least one gene mutation including ASXL1, TP53, ZRSR2, and STAG1 suggesting poor prognostic significance for diagnoses of MDS, or JAK2, SF3B1 which could support a disease specific diagnosis. Conclusion: NGS had enhanced diagnostic capabilities including classification of newly described entities such as ICUS or CCUS and more importantly yielded additional prognostic information compared to cytogenetics alone for this patient population. Cytogenetic findings were mainly aneuploidy or deletions, either clinically evident constitutional abnormalities such as Trisomy 21 and +X in Klinefelter syndrome, or loss of Y chromosome in a small proportion of cells and thus of questionable clinical significance. Based on this information consideration should be given to using the NGS panel as the primary molecular diagnostic and prognostic tool with karyotyping being reserved for subsets of patients being assessed for suspected MDS or cytopenias of undetermined origin. Disclosures Hsia: Amgen: Honoraria; Jansen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Description: Background The treatment outcome of diffuse large B cell lymphoma (DLBCL) has been greatly improved by rituximab (R) incorporating R-CHOP-based immunochemotherapy. The purpose of this study was to design a new prognostic model which can accurately predict the treatment outcome of DLBCL by R-CHOP (-like) immunochemotherapy, especially for discriminating very high risk patients with rapid disease progression and a short survival period from other large proportion of patients with favourable treatment outcome. Patients and Methods We retrospectively analysed the clinical records of patients who were histologically diagnosed as DLBCL and treated with either R-CHOP or R-CHOP-like therapy at the Kyoto Prefectural University of Medicine and Japanese Red Cross Kyoto Daiichi Hospital from January 2006 to December 2013 and at the Japanese Red Cross Kyoto Daini Hospital from January 2006 to April 2014. Patients were randomly divided into two groups for each institution; 70% for the training sample to construct a new prognostic model and 30% for validation of predictive performance. To evaluate the qualities of discrimination and prediction of risk groups by individual indices, we examined the c-index and the relative Brier score reduction (RBSR) in the validation cohort. The revised-International Prognostic Index (R-IPI) and the NCCN-IPI were also evaluated as the references. Results With a median follow-up time of 32.2 months, the 3-year overall survival (OS) and progression-free survival (PFS) of all patients were 78.5% and 67.4%, respectively. In the study cohort of 323 randomly selected patients, multivariate analyses revealed that the serum LDH level, ECOG performance status ≥2, serum albumin level

Description: Abstract 2590 Poster Board II-566 Chronic myelogenous leukemia (CML) is caused by a consistent genetic abnormality, termed the Philadelphia chromosome (Ph). It results in the production of BCR-ABL fusion protein, a constitutively active tyrosine kinase. Imatinib mesylate (IM, Gleevec®), the first generation tyrosine kinase inhibitor (TKI), has revolutionized therapy for CML patients. However, resistance for IM develops in a significant proportion of cases, and is predominantly mediated by single point mutations within the BCR-ABL kinase domain. Second generation TKIs such as dasatinib (Sprycel®) and nilotinib (Tasigna®) represent viable alternatives for IM-resistant or intolerant CML patients. Each mutated BCR-ABL has different sensitivity to those TKIs. Thus, it is significantly important to detect early the existence of BCR-ABL mutations and their specificities in treating Ph+ leukemias. We have developed a novel automated method that has high sensitivity to detect a few copies of mutation sequences that are mixed in many copies of normal sequences. This method consists of PCR amplification step and Tm (melting temperature) analysis step that uses a quenching probe. And we have already shown that this system has clinical efficacy in JAK2V617F mutation that is one of the genetic hallmarks of chronic myeloproliferative diseases. (Tanaka R, et al. Leuk Res, 2008). When a whole blood sample or a purified DNA sample reacts with reagents, PCR and Tm analysis automatically processed in the same tube, and whole procedure finishes in approximately 1 hour. The detection of mutation is extremely accurate because the quenching probe is designed perfectly matched for mutated sequence. As Tm value of mutation sequence is higher than that of normal one, it is easy to detect the existence of mutation from the Tm analysis data. We have constructed the probes for 14 mutations concerned for IM-resistance (M244V, G250E, Q252H, Y253F, Y253H, E255V, E255K, T315I, T315A, F317L, M351T, E355G, F359V, and H396R). Considering the clinical significance of T315I mutation, which renders resistance to all currently available TKIs, we refined this method to higher sensitivity for detecting T315I mutation. First, we analyzed the sensitivity of this system on BCR-ABL. In dilution assays using wt and mutated plasmid, the system reliably quantified the mutation in a population containing as few as 3.0% mutant. Moreover, for T315I setting, we successfully detected as few as 0.3% (30 copies from 10,000 copies) mutations by a higher-sensitive assay. Next, we examined the clinical samples. Each sample was also examined by direct sequencing in comparison to our method. Kinase domain mutations were identified in 24 of the 50 (48%) patients. Our automated analysis was enabled to detect mutations in 19 patients, including p-loop mutations (G250E: n=3; E255K: n= 5), IM-binding domain mutations (T315I: n=10), and an activation-loop mutation (H396R: n=1). And all the positive cases (19 of 19) showed a concordance with the result of direct sequencing. On the other hand, 5 cases were detected just by direct sequencing, but all that cases were out of our setting mutations (Q252E, V379I, S417F, E459K). Impressively, in one case, only higher-sensitivity assay could reveal T315I mutation, although it was detected as a wild type both by direct sequence and our usual method. It suggests that the higher-sensitive system could detect low amount of T315I mutation in the earlier stage of disease. In conclusion, sensitivity of our system (3%) is significantly greater than that of direct sequencing (15 – 25%), and results can be obtained within one hour. By the serial monitoring, it is demonstrated the availability of the higher-sensitive analysis (0.3%) to detect T315I mutation. This rapid and accurate detection of clinically significant mutations enables us to contribute to better clinical practice in treating Ph+ leukemia patients, such as in selecting alternative strategies of IM dose escalation, second generation TKIs, or allogeneic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

Description: [Introduction] Multiple myeloma (MM) is a cytogenetically/molecularly heterogeneous hematologic malignancy that remains mostly incurable, and the identification of a universal and relevant therapeutic target molecule is essential for the further development of therapeutic strategy. We have recently identified that 3-phosphoinositide-dependent protein kinase 1 (PDPK1), a serine threonine kinase, and its major downstream substrate RSK2, a member of the 90 kDa ribosomal S6 kinase family of serine threonine kinases, were universally active in eleven human MM-derived cell lines (HMCLs) examined regardless of the type of cytogenetic abnormality, the mutation state of RAS, RAF and FGFR3 genes and myeloma cells of approximately 90% of symptomatic patients at diagnosis. Our study also disclosed that PDPK1/RSK2 signaling axis played pivotal roles in myeloma pathophysiology by regulating series of downstream molecules, such as c-MYC, IRF4, D-type cyclins, or PLK1, while the inactivation of either PDPK1 or N-terminal domain of RSK2 resulted in the induction of apoptosis in myeloma cells which was accompanied by the activation of BH3-only proteins BIM and BAD (Shimura Y, Mol Caner Ther 2012; Chinen Y, Cancer Res 2014). Here we assessed the underlying mechanism for PDPK1 overexpression in MM. [Methods] The miR-375 expression level was analyzed by the quantitative RT-PCR in 11 HMCLs and 92 patient-derived myeloma cells isolated by CD138-positive cell sorting (normal plasma cells (N=10), MGUS (N=21), newly diagnosed MM (NDMM) (N=27), relapsed/refractory (RRMM) (N=34). The pre-miR-375 precursor molecule (miR-375 mimics), the siRNA targeted against PDPK1, or a negative control RNA-oligonucleotides was transfected into 8 cell lines by utilizing Hemagglutinating Virus of Japan (HVJ)-envelope vector. The copy number abnormality of PDPK1 gene was assessed by double-color FISH for PDPK1 gene and the centromere of chromosome 16. The methylation status of miR-375 promoter site was analyzed by methylation-specific PCR (MSP). This study was conducted in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Boards. Patient-derived samples were obtained with informed consent, and normal bone marrow plasma cells were obtained from volunteers who were not affected by hematologic disease. [Result] The level of miR-375 expression was calculated with 2-ΔCt methods. Human U6 snRNA was examined as the reference. The median log102-ΔCt ± SD of normal plasma cells, MGUS, NDMM, RRMM and HMCLs were -2.46 ± 0.67, -3.64 ± 0.68, -4.23 ± 0.95, -3.92 ± 1.24 and -3.69 ± 0.29 respectively. When compared to normal plasma cells, the miR-375 expression was significantly decreased in NDMM and RRMM (p