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Background In the phase 2 KarMMa trial, patients with relapsed/refractory multiple myeloma (RRMM) achieved deep and durable responses with idecabtagene vicleucel (ide-cel), a B-cell maturation antigen-directed chimeric antigen receptor (CAR) T cell therapy. Here we report a sub-analysis of the Japanese cohort of KarMMa. Methods Adult patients with RRMM who had received ≥ 3 prior treatment regimens, including a proteasome inhibitor, an immunomodulatory agent, and an anti-CD38 antibody, and had disease refractory to last treatment received ide-cel at a target dose of 450 × 106 CAR positive T cells. Results Nine patients were treated with ide-cel. The overall response rate was 89% (median follow-up, 12.9 months). The best overall response was stringent complete response in 5 patients (56%), very good partial response in 3 (33%), and stable disease in 1. Median duration of response was not reached. All patients experienced grade ≤ 2 cytokine release syndrome and one patient experienced grade 2 neurotoxicity, but all resolved. Two patients died, one each from plasma cell myeloma and general health deterioration. Conclusion Ide-cel yielded deep, durable responses with a tolerable and predictable safety profile in Japanese patients with RRMM. These results are similar to those of the non-Japanese population in KarMMa.

Lenalidomide is a synthetic analog of thalidomide formed by the removal of one keto group (plus the addition of an amino group); it has anti-tumor activities beneficial for the treatment of hematologic malignancies. However, lenalidomide distribution to brain in animal models is reportedly low compared with that of thalidomide. The aim of this study was to evaluate plasma and cerebrospinal fluid concentrations of lenalidomide in three patients with malignant hematologic malignancies. Lenalidomide was detected in plasma from the three Japanese patients 1.5 h following oral administration of 20 mg lenalidomide using liquid chromatography/mass spectrometry, despite the in vitro gastrointestinal permeability of lenalidomide being low. Clinically observed cerebrospinal fluid-to-plasma ratios of lenalidomide were low (1.3-2.4%). Observed influx permeability values for lenalidomide in monkey blood-brain barrier model and human placental cell systems were one order of magnitude lower than those of thalidomide and another second-generation drug, pomalidomide along with a positive permeability control, caffeine. Because of the low cell-barrier permeability of lenalidomide demonstrated in in vitro assays, clinically relevant pharmacokinetic profiles of lenalidomide resulted in low penetrability from plasma into cerebrospinal fluid in patients with hematologic malignancies. Lenalidomide is conclusively suggested to expert its favorable immunomodulatory effects via systemic exposures in the patients.

RAS/RAF/MEK/ERK pathway inhibitors exhibit significant anti-tumor effects against various tumor types, including multiple myeloma (MM), and they are predicted to play a pivotal role in precision medicine. The XPO1 inhibitor KPT-330 has also exhibited promising efficacy in combination with other novel drugs in treating relapsed/refractory MM (RRMM). In this study, we explored the anti-tumor effects of a combination of the pan-RAF inhibitor TAK-580 and KPT-330. Importantly, TAK-580 enhanced KPT-330-induced cytotoxicity and apoptosis in human myeloma cell lines and primary myeloma cells from RRMM patients. Moreover, TAK-580 and KPT-330 synergistically inhibited nuclear phospho-FOXO3a and enhanced cytoplasmic phospho-FOXO3a in MM cells, leading to cytoplasmic enhanced Bim expression and finally apoptosis. This indicates that TAK-580 enhances KPT-330-induced cytotoxicity and apoptosis primarily via the FOXO3a-Bim axis. In addition, TAK-580 enhanced the cytotoxicity of KPT-330 against MM cells even in the presence of IGF-1. Taken together, our results demonstrate that a combination of pan-RAF inhibitor and XPO1 inhibitor is a potential therapeutic option in treating MM.

The prognosis of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL) has improved dramatically. Although measurable residual disease (MRD) kinetics during pretransplant treatment has been recently reported to correlate with patient outcomes, it is unclear whether prognosis is better if the MRD falls below the detection sensitivity soon after induction therapy. We retrospectively analyzed data of 37 Ph + ALL patients who were treated with autologous or allogeneic stem cell transplantation (auto-SCT, allo-SCT) at our institute from 2003 to 2019. Based on MRD kinetics, patients were divided into three groups: early responders (MRD became negative after induction therapy [n = 10, 27.0%]); late responders (MRD remained positive after induction therapy and became negative just before SCT [n = 12, 32.4%]); and poor responders (MRD was positive until just before SCT [n = 15, 40.5%]). The 5-year disease-free survival (DFS) rates for the three groups were 80.0%, 60.0%, and 29.9%, respectively (P = 0.037). The 5-year overall survival rates were not significantly different. The 5-year relapse rates were 0.0%, 31.7%, and 49.5%, respectively (P = 0.045). Non-relapse mortality (NRM) rates were similar among the three groups. Subgroup analysis for the cases that received posttransplantation tyrosine kinase inhibitor maintenance therapy revealed that DFS was similarly dependent on MRD kinetics (P = 0.022). This study clarified that MRD kinetics was a significant prognosticator for DFS and relapse rate in Ph + ALL.

Multiple myeloma (MM) is still extremely difficult to cure, and new therapeutic drugs are needed. We recently found that integrin β7 is constitutively activated in MM cells, and chimeric antigen receptor (CAR) T cells targeting activated integrin β7 have a significant anti-MM effect. In this study, we performed flow cytometry analysis of the expression of activated integrin β7 in bone marrow cells from 137 symptomatic MM patients. In 60/137 (44%) MM patients, activated integrin β7 was detected in most MM cells (> 80% of MM cells were in the positive gate). Activated integrin β7 was highly expressed in MM cells even in heavily treated patients. It also showed high expression in many CD38lo/−CD138−CD19+B cells, which reportedly include clonotypic B cells, in the bone marrow of MM patients. Taken together, these results suggest that CAR T-cell therapy targeting activated integrin β7 has the potential to benefit many patients with relapsed or refractory MM.

The Janus kinase/signal transducers and activators of transcription signaling pathway induces programmed death ligand-1 (PD-L1) expression. JAK2 mutation at position 617 (JAK2V617) is a frequent driver of myeloproliferative neoplasms (MPN) through PD-L1 expression. Although PD-1 inhibitors should be effective against MPN with JAK2V617F mutation, this has not yet been reported in humans. Thus, we assessed the efficacy of a PD-1 inhibitor in a lung cancer patient with JAK2V617F-positive essential thrombocythemia (ET). A 71-year-old man was diagnosed with ET, and with lung carcinoma 3 years later. After right lobectomy and postoperative chemotherapy, pembrolizumab [a PD-1 inhibitor (200 mg, every 3 weeks)] was initiated for refractory lung carcinoma. Lung cancer progression did not occur for 1.5 years under treatment. Most megakaryocytes were PD-L1-positive, and after pembrolizumab initiation, platelet count remained below 45 × 104/μL without the need for other cytoreductive therapies for ET. The JAK2V617F allele burden gradually decreased from 11.5% at diagnosis to 2.9% after 17 months of pembrolizumab treatment. Other peripheral blood lineages did not decrease, and pembrolizumab treatment was continued without any adverse events. This is the first report demonstrating the effectiveness of pembrolizumab in an MPN patient with JAK2V617F mutation.

Over the past 25 years, the application of immunomodulating and immunosuppressive agents in the treatment of multiple sclerosis (MS), an autoimmune demyelinating and neurodegenerative disease, has ...

A 67-year-old female was referred to our hospital with a sternal fracture in March 2008. She received a diagnosis of multiple myeloma (MM) BJP-κ type (ISS stage III). G-banding karyotype revealed 46, XX, t(11;22)(q23.3;q11.2) (Hubacek, Gene 592:193-9, 2016), which was later confirmed to be congenital. After repeated rounds of chemotherapy with bortezomib and lenalidomide, she obtained a very good partial response in August 2014, and she was followed up with no treatment. However, she relapsed in February 2016. At that time, fluorescence in situ hybridization identified del(13q) and t(4;14)(p16;q32), which are associated with a poor prognosis. Furthermore, PCR analysis showed that the chromosome 11 breakpoint was at the APOA5/APOA4 locus at 11q23.3, which is associated with malignancy, and that the chromosome 22 breakpoint was at the SEPT5 intron 1 locus, which also plays a role in leukemogenesis through formation of a fusion gene with MLL. Although she was treated with three further lines of therapy, she died from disease progression in August 2017. Synergism between t(11;22) and t(4;14) may have induced the double-refractory phenotype to proteasome inhibitor and lenalidomide, at least during the chemorefractory phase. We present a biological analysis of this case and a review of the literature.

The risk factors for bleomycin-induced lung injury (BLI), a fatal complication of cancer chemotherapy, are not well-established. The renin-angiotensin-aldosterone system (RAAS) has recently been suggested to play a role in the development of lung injury. This study clarified the impact of hypertension (HTN) and the administration of RAAS inhibitors on BLI occurrence in patients treated with bleomycin-containing regimens.We retrospectively analyzed the data of 190 patients treated with a bleomycin-containing regimen for Hodgkin lymphoma or germ cell tumors at our institutions from 2004 to 2018.Overall, 190 patients received bleomycin, and symptomatic BLI occurred in 21 (11.1%) cases. In the multivariate analysis, age ≥ 65 years (odd ratio, 10.90; 95% confidence interval, 3.72-32.20; P .001) and history of HTN (odds ratio, 3.32; 95% confidence interval, 1.07-10.30; P = .04) were found to be significant risk factors for BLI onset. BLI occurred in 3.6% (n = 5) of patients with no risk, 11.8% (n = 2) of those whose only risk factor was HTN, 31.6% (n = 6) of those whose only risk factor was age ≥ 65 years, and 57.1% (n = 8) of those with both risk factors (P .001). BLI-induced mortality rates in each group were 0.0% (n = 0), 5.9% (n = 1), 10.5% (n = 2), and 42.9% (n = 6) (P .001), respectively. Among 31 patients with HTN, BLI incidence was 12.5% in patients who were administered RAAS inhibitors and 53.3% in those who were not (P = .02).Older age and history of HTN were independent risk factors for the development of BLI, and the administration of RAAS inhibitors might reduce the onset of BLI.

Significant advancements have been achieved with regard to the outcomes of acute promyelocytic leukemia (APL) patients through the introduction of all-trans retinoic acid; however, early hemorrhagic death and differentiation syndrome remain the major causes of remission induction failure in patients with APL. To investigate early death, serious hemorrhage, and differentiation syndrome during remission induction therapy in terms of incidence, risk factors, influence on outcomes, and prophylactic effects of several new anticoagulants, the results of 344 patients enrolled in the Acute Promyelocytic Leukemia 204 study conducted by the Japan Adult Leukemia Study Group were analyzed. Early death was observed in 16 patients (4.7%), of whom 14 had serious hemorrhage and 2 had differentiation syndrome. Serious hemorrhage and differentiation syndrome of grade 2 or higher were observed in 21 and 54 patients, respectively. Patients who achieved complete remission had a 7-year disease-free survival of 84.8% if they did not experience serious hemorrhage and 40.0% if they experienced serious hemorrhage during remission induction therapy (P = 0.001). Risk factor analyses showed that higher white blood cell count was associated with early death, higher white blood cell count and lower platelet count with serious hemorrhage, and leukocytosis during induction therapy and higher body surface area with differentiation syndrome. In conclusion, these results indicate that patients with such high-risk features may benefit from more intensive supportive care. The hemorrhagic risk was not relieved by the introduction of new anticoagulants. Further studies are required to establish the predictive impact of body surface area on differentiation syndrome. This trial is registered with UMIN-CTR as C000000154 on September 13, 2005.

Between April 2004 and December 2010, we conducted a prospective randomized controlled study comparing tamibarotene with all-trans retinoic acid (ATRA) in the maintenance therapy of newly diagnosed acute promyelocytic leukemia (APL), and here report the final results of this study with a median follow-up of 7.3 years. Of 344 eligible patients who had received ATRA and chemotherapy, 319 (93%) achieved complete remission (CR). After completion of three courses of consolidation chemotherapy, 269 patients in molecular remission underwent maintenance randomization, 135 to ATRA (45 mg/m2 daily), and 134 to tamibarotene (6 mg/m2 daily) for 14 days every 3 months for 2 years. The primary endpoint was relapse-free survival (RFS). The 7-year RFS was 84% in the ATRA arm and 93% in the tamibarotene arm (p = 0.027, HR = 0.44, 95% CI, 0.21 to 0.93). The difference was prominent in high-risk patients with initial leukocytes ≥ 10.0 × 109/L (62% vs. 89%; p = 0.034). Tamibarotene was significantly superior to ATRA by decreasing relapse in high-risk patients. Overall survival after randomization did not differ (96% vs. 97%; p = 0.520). Secondary hematopoietic disorders developed in nine patients, secondary malignancies in 11, and grade 3 or more late cardiac comorbidities in three. These late complications did not differ between the two arms.

We analyzed the function of a SEPT9-ABL1 fusion identified in a case of T-prolymphocytic leukemia with tyrosine kinase inhibitor (TKI) resistance. Five isoforms with different N-termini, including SEPT9a-ABL1, SEPT9b-ABL1, SEPT9d-ABL1, SEPT9e-ABL1 and SEPT9f-ABL1, were detected in the leukemic cells. All isoforms except SEPT9d-ABL1 are localized in the cytoplasm, undergo autophosphorylation and phosphorylate the downstream targets, STAT-5 and Crkl, and provided IL-3-independence and in vivo invasiveness to 32D cells. Additionally, these SEPT9-ABL1 isoforms were resistant to TKIs in vitro and in vivo, in comparison to BCR-ABL1. These findings demonstrated that SEPT9-ABL1 had oncogenic activity and conferred resistance to TKIs.

Introduction Fingolimod is a sphingosine-1-phosphate (S1P) receptor modulator that prevents B and T lymphocyte egress from the lymph nodes, and reduces peripheral lymphocyte counts and entry of lymphocytes into the central nervous system. In 2010, the Food and Drug Administration (FDA) approved it for the treatment of relapsing-remitting multiple sclerosis (MS). It has been reported that Fingolimod treatment causes cutaneous side-effects such as opportunistic skin infections and cutaneous malignancies. We herein report the first case of peripheral T cell lymphoma, not otherwise specified (PTCL-NOS) induced by Fingolimod. Case report A 63-year-old man was diagnosed with MS at 33 years of age. Due to repeated MS relapses, the administration of Fingolimod was started at 61 years of age. After 2 years, skin rash appeared in the abdominal and right femoral regions. Computed tomography showed swelling of the left cervical, mesenteric and bilateral inguinal lymph nodes and cutaneous tumors in the abdominal and right femoral regions. He was diagnosed with PTCL-NOS based on biopsies of the skin in right femoral region and right inguinal lymph nodes. Immunohistochemistry was positive for CD3, CD4, CD5, CD7, and negative for CD8, CD25 and CD30. He was treated with methylprednisolone (500 mg/day) for 3 days followed by prednisolone. His skin tumors and lymph node swelling did not progress. Discussion Among 10 reported cases of malignant lymphoma induced by fingolimod, 7 had a T-cell lineage and 8 cases occurred in the cutaneous region. Fingolimod should be stopped if lymphomatous lesions are found. However, the treatment of the MS patients with lymphoma is very troublesome. There are few alternatives to fingolimod for the treatment of relapsing MS. Additionally, the performance status of the patients with relapsing MS is not usually good enough to undergo standard therapies. Further analyses are required to reveal the lymphomagenesis in patients undergoing fingolimod treatment.

Small-molecule multi-targeted cyclin-dependent kinase (CDK) inhibitors (CDKIs) are of particular interest due to their potent antitumor activity independent of p53 gene alterations. P53 deletion is associated with a very poor prognosis in multiple myeloma (MM). In this regard, we tested the anti-MM activity of RGB-286638, an indenopyrazole-derived CDKI with Ki-nanomolar activity against transcriptional CDKs. We examined RGB-286638's mode-of-action in MM cell lines with wild-type (wt)-p53 and those expressing mutant p53. RGB-286638 treatment resulted in MM cytotoxicity in vitro associated with inhibition of MM tumor growth and prolonged survival in vivo. RGB-286638 displayed caspase-dependent apoptosis in both wt-p53 and mutant-p53 cells that was closely associated with the downregulation of RNA polymerase II phosphorylation and inhibition of transcription. RGB-286638 triggered p53 accumulation via nucleolar stress and loss of Mdm2, accompanied by induction of p53 DNA-binding activity. In addition, RGB-286638 mediated p53-independent activity, which was confirmed by cytotoxicity in p53-knockdown and p53-mutant cells. We also demonstrated downregulation of oncogenic miR-19, miR-92a-1 and miR-21. Our data provide the rationale for the development of transcriptional CDKIs as therapeutic agents, which activate p53 in competent cells, while circumventing p53 deficiency through alternative p53-independent cell death mechanisms in p53-mutant/deleted cells.

Background: CD56 expression is reported to be associated with adverse prognosis in patients with acute promyelocytic leukemia (APL) treated with all-trans retinoic acid (ATRA) and chemotherapy (Murray et al, 1999, Ferrara et al, 2000, Montesinos et al, 2011, Ono T et al, 2014). However, the prognostic significance of CD56 has not been elucidated, particularly when more potent agents are used. We recently reported long term analysis of the Japan Adult Leukemia Study Group (JALSG) APL204 study and concluded that maintenance therapy with tamibarotene was more effective than ATRA by reducing relapse in APL patients (Takeshita et al, 2018). In this study, the clinical significance of CD56 was evaluated with other surface markers on APL cells. Patients and Methods: Newly diagnosed APL patients with documented cytogenetic and/or molecular evidence of t(15;17)/PML-RARA were registered to the APL204 study from April 2004 to December 2010. The eligibility criteria included age between 15 and 70 years, ECOG performance status between 0 and 3, and sufficient function of organs. Induction therapy was composed of ATRA and chemotherapy whose dose and duration were based on initial white blood cell (WBC) count. Patients who achieved molecular remission after three courses of consolidation therapy were randomly assigned to maintenance therapy with tamibarotene 6 mg/day for 14 days or ATRA 45 mg/day for 14 days, which was repeated every 3 months for 2 years. The primary endpoint was hematological or molecular relapse-free survival (RFS). Surface markers, including CD56, were defined as positive if more than 10% of the CD45-gated cells expressed a specific antigen. Clinical characteristics were compared by the chi-square test or the Fisher's exact test for categorical data and the Wilcoxon rank-sum test for continuous data. RFS, overall survival (OS) and event-free survival (EFS) were estimated by the Kaplan-Meier method, and compared using the log-rank test. Cumulative incidence of relapse (CIR) was compared by Gray's test. Multivariate analyses were also performed by the Cox-proportional-hazards-model. Clinical outcomes were renewed between January 2016 and June 2017 and the median follow-up period was 7.3 years. This study is registered at the University Hospital Medical Information Network Clinical Trials Registry as C000000154. Results: Of the 344 eligible patients, 319 (93%) achieved CR. After completing consolidation chemotherapy, 269 patients underwent maintenance random assignment; 135 to ATRA, and 134 to tamibarotene. Among 344 eligible patients, 325 were assessable for CD-phenotypes, and 45 (14%) were CD56-positive (CD56+). Among 269 patients who underwent the maintenance assignment, 34 (13%) were CD56+. CD56 expression was significantly associated with obvious bleeding (p Conclusions: CD56 expression is an independent unfavorable prognostic factor for RFS in APL patients treated with ATRA and chemotherapy followed by ATRA or tamibarotene maintenance therapy, especially in patients whose initial WBC count was more than 3.0 x 109/L. The present study supports the prognostic significance of CD56 in the treatment of APL using more potent agents. Figure. Figure. Disclosures Takeshita: Chugai Pharmaceutical Co. Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Astellas Pharma Inc.: Research Funding; Takeda Pharmaceutical Co. Ltd.: Research Funding; Bristol-Myers Squibb Co.: Research Funding; Kyowa Hakko Kirin Co. Ltd.: Research Funding. Asou:Asahi Kasei Pharma Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; SRL Inc.: Consultancy; Yakult Honsha Co., Ltd.: Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Astellas Pharma Inc.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Sawa:Celgene Corporation: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis International AG: Honoraria; CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria; Mundipharma K.K.: Honoraria. Dobashi:Celgene Co.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co. Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Sysmex Co.: Research Funding. Kobayashi:Pfizer: Research Funding; Ohtuka: Research Funding; Astellas: Research Funding. Kiyoi:Kyowa Hakko Kirin Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria; FUJIFILM Corporation: Research Funding; Celgene Corporation: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sanofi K.K.: Research Funding; Astellas Pharma Inc.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding.

Mutations in the fms-like tyrosine kinase 3 (FLT3) gene containing an internal tandem duplication (FLT3/ITD) or mutations in the nucleophosmin 1 gene (NPM1) are thought to be prognostic indicators in acute myeloid leukemia (AML). Previous studies suggested that FLT3/ITD mutation indicates a poor prognosis and that NPM1 mutation indicates a more favorable one, but these studies were often performed with selected patient populations. We investigated the clinical significance of these mutations at our institution with an unselected group of patients with newly diagnosed AML. This group included patientsor =60 years old and those with a poor performance status. Using polymerase chain reaction and sequencing analyses, we detected FLT3/ITD mutations in 12 patients (20.0%) and NPM1 mutations in 7 patients (11.7%) among a group of 60 patients. There was a nonsignificant trend for FLT3/ITD mutation to be associated with a poorer predicted overall survival (OS) probability in this population. In contrast, OS was significantly higher in patients with wild-type NPM1 than in patients with NPM1 mutation, both for all AML patients and for AML patients with a normal karyotype. In this general and unselected AML patient population, NPM1 mutation was not a prognostic indicator of a favorable outcome.

The Dickkopf-1 (DKK1) gene product is an extracellular Wnt inhibitor. Hypermethylation of the DKK1 promoter results in transcriptional silencing and may play an important role in cancer development. Here, we investigated hypermethylation of the DKK1 promoter in patients with acute myeloid leukaemia (AML), especially core-binding factor (CBF) leukaemia. The methylation status of DKK1 was analysed using methylation-specific polymerase chain reaction in 47 patients with AML. DKK1 methylation was found in 14 (29.8%) patients, and more frequently in those with CBF leukaemia (6 of 12 patients), than in those with acute promyelocytic leukaemia (APL) (0 of 6 patients) (P = 0.03). In contrast, Wnt inhibitory factor-1 methylation was found in APL (4 of 6 patients) but not in CBF leukaemia (0 of 12 patients) (P = 0.001). Multivariate analyses suggested that DKK1 methylation was a risk factor for poorer overall survival. Sequential analysis using four paired samples obtained at diagnosis and relapse suggested that DKK1 methylation was involved in the progression of leukaemia. Therefore, DKK1 methylation may be involved in leukaemogenesis, especially in CBF leukaemia, and may be a useful prognostic marker in AML.

Histone deacetylase (HDAC) inhibitors have been extensively investigated as therapeutic agents in cancer. However, the biological role of class IIa HDACs (HDAC4, 5, 7 and 9) in cancer cells, including multiple myeloma (MM), remains unclear. Recent studies show HDAC4 interacts with activating transcription factor 4 (ATF4) and inhibits activation of endoplasmic reticulum (ER) stress-associated proapoptotic transcription factor C/EBP homologous protein (CHOP). In this study, we hypothesized that HDAC4 knockdown and/or inhibition could enhance apoptosis in MM cells under ER stress condition by upregulating ATF4, followed by CHOP. HDAC4 knockdown showed modest cell growth inhibition; however, it markedly enhanced cytotoxicity induced by either tunicamycin or carfilzomib (CFZ), associated with upregulating ATF4 and CHOP. For pharmacological inhibition of HDAC4, we employed a novel and selective class IIa HDAC inhibitor TMP269, alone and in combination with CFZ. As with HDAC4 knockdown, TMP269 significantly enhanced cytotoxicity induced by CFZ in MM cell lines, upregulating ATF4 and CHOP and inducing apoptosis. Conversely, enhanced cytotoxicity was abrogated by ATF4 knockdown, confirming that ATF4 has a pivotal role mediating cytotoxicity in this setting. These results provide the rationale for novel treatment strategies combining class IIa HDAC inhibitors with ER stressors, including proteasome inhibitors, to improve patient outcome in MM.

Anti-tumor activities of selective HSP90α/β inhibitor, TAS-116, in combination with bortezomib in multiple myeloma

FLT3 is a member of receptor tyrosine kinases expressed in leukemia cells, as well as in hematopoietic stem cells. Recently, a somatic alteration of the FLT3 gene was found in acute myeloid leukemia, as an internal tandem duplication (FLT3/ITD) which caused elongation of the juxtamembrane (JM) domain of FLT3. Here we characterized the FLT3/ITD and investigated its clinical significance in acute promyelocytic leukemia (APL). Seventy-four newly diagnosed patients with APL, who were treated with the same protocol in a multi-institutional study, were studied for the FLT3/ITD. Genomic and message sequences of the FLT3 gene were amplified by means of polymerase chain reaction (PCR), and elongated PCR products were sequenced. Fifteen patients (20.3%) had FLT3/ITD, all of which were transcribed in frame. Location of the duplicated fragments (six to 30 amino acids) varied from patient to patient. However, they always contained either Y591 or Y599, but the tyrosine kinase domain was not significantly affected. This finding implied that signal transduction of FLT3 is amplified by the duplication. Clinically, the presence of FLT3/ITD was related to high peripheral white blood cell counts as well as peripheral leukemia cell counts (P < 0.0001), high LDH level (P = 0.04), and low fibrinogen concentration (P = 0.04). These data suggest that FLT3/ITD plays a significant role in progression of APL.

Histone deacetylases (HDACs) represent novel molecular targets for the treatment of various types of cancers, including multiple myeloma (MM). Many HDAC inhibitors have already shown remarkable antitumor activities in the preclinical setting; however, their clinical utility is limited because of unfavorable toxicities associated with their broad range HDAC inhibitory effects. Isoform-selective HDAC inhibition may allow for MM cytotoxicity without attendant side effects. In this study, we demonstrated that HDAC3 knockdown and a small-molecule HDAC3 inhibitor BG45 trigger significant MM cell growth inhibition via apoptosis, evidenced by caspase and poly (ADP-ribose) polymerase cleavage. Importantly, HDAC3 inhibition downregulates phosphorylation (tyrosine 705 and serine 727) of signal transducers and activators of transcription 3 (STAT3). Neither interleukin-6 nor bone marrow stromal cells overcome this inhibitory effect of HDAC3 inhibition on phospho-STAT3 and MM cell growth. Moreover, HDAC3 inhibition also triggers hyperacetylation of STAT3, suggesting crosstalk signaling between phosphorylation and acetylation of STAT3. Importantly, inhibition of HDAC3, but not HDAC1 or 2, significantly enhances bortezomib-induced cytotoxicity. Finally, we confirm that BG45 alone and in combination with bortezomib trigger significant tumor growth inhibition in vivo in a murine xenograft model of human MM. Our results indicate that HDAC3 represents a promising therapeutic target, and validate a prototype novel HDAC3 inhibitor BG45 in MM.

Histone methylations are tightly regulated by a balance between methyltransferases and demethylases that mediate the addition and removal of these modifications. Importantly, dysregulation of histone methylation is implicated in pathogenesis of cancers, including multiple myeloma (MM). For example, the t(4;14) (p16;q32) is present in 15 - 20% of MM patients and results in overexpression of WHSC1, a histone H3 lysine 36 (H3K36) methyltransferase. On the other hand, approximately 10% of MM patients without the t(4;14) have inactivating mutations in KDM6A, a H3K27 demethylase. KDM3A is a Jumonji C-domain-containing histone demethylase which catalyzes removal of H3K9 mono- and dimethylation (H3K9me1 and H3K9me2). KDM3A is implicated in pathogenesis of different types of cancers. Here we investigated the biological impact of KDM3A in MM. KDM3A expression was significantly elevated in MM patient samples compared to normal plasma cells in publicly available dataset (GSE5900, GSE6691). To evaluate the functional role of KDM3A, shRNAs targeting KDM3A were transduced into MM cell lines: knockdown of KDM3A significantly inhibited MM cell growth (RPMI8226, MM.1S, U266, H929) in vitro and in xenograft model (MM.1S). Apo2.7 staining showed that apoptotic cells were significantly increased after knockdown of KDM3A. We next examined gene expression profiles after knockdown of KDM3A in RPMI8226 cells. With a cutoff of > 1.5-fold downregulation, a total of 305 probe sets were downregulated in KDM3A-knockdown cells relative to control cells. Among putative KDM3A targets, a gene of particular interest is KLF2 which plays a key role in maintenance of B cell and plasma cell phenotype, and function. Another intriguing gene is IRF4, given its known crucial role in MM cell survival. We confirmed that expression of KLF2 and IRF4 was downregulated after knockdown of KDM3A by quantitative realtime PCR and immunoblots in RPMI82226, MM.1S, and U266 cells. KDM3A binding to KLF2 and IRF4 core promoters was demonstrated by chromatin immunoprecipitation (ChIP) assay in RPMI8226 cells. Moreover, knockdown of KDM3A increased H3K9me1 and me2 levels at both promoter regions, indicating that KDM3A directly regulates KLF2 and IRF4 expression by removing H3K9 methylation marks at their promoters in MM cells. shRNAs targeting KLF2 were next transduced into MM cell lines: silencing of KLF2 significantly reduced cell growth of MM cell lines, associated with decreased IRF4. Promoter reporter assays using human IRF4 promoter showed that KLF2 significantly increased luciferase expression in a dose-dependent manner. Moreover, ChIP assay showed that KLF2 bound to IRF4 promoter in RPMI8226 cells. Since transcription factors could form an autoregulatory feedback loop, we hypothesized that IRF4 might regulate KLF2 expression. As expected, knockdown of IRF4 downregulated KLF2 expression at both the mRNA and protein levels in 3 MM cell lines. In addition, ChIP assays demonstrated that IRF4 bound to KLF2 second intron that contains tandem IRF4 motifs in RPMI8226 cells. Collectively, these results suggest that KLF2 activates IRF4 expression and vice versa, forming an autoregulatory loop in MM cells. KLF2 has been reported to control homing of plasma cells to the bone marrow; we therefore hypothesized that KDM3A-KLF2-IRF4 axis might regulate adhesion and homing of MM cells to the bone marrow. Importantly, knockdown of KDM3A, KLF2, or IRF4 decreased adhesion of 3 MM cell lines to bone marrow stromal cells. Furthermore, bone marrow homing of MM.1S cells was significantly reduced after knockdown of KDM3A, KLF2, or IRF4 in a murine xenograft MM model, indicating that KDM3A-KLF2-IRF4 axis regulates, at least in part, MM cell adhesion and homing to the bone marrow. In conclusion, our study demonstrated that KDM3A is a crucial epigenetic regulator of MM cell survival, and that inhibition of KDM3A represents a novel therapeutic strategy in MM. Disclosures Raje: Amgen: Consultancy; Takeda: Consultancy; Novartis: Consultancy; Celgene Corporation: Consultancy; BMS: Consultancy; Acetylon: Research Funding; Eli Lilly: Research Funding; Onyx: Consultancy; AstraZeneca: Research Funding; Millenium: Consultancy. Richardson:Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Harigae:Chugai Pharmaceutical Co., Ltd.: Research Funding. Anderson:Oncopep: Equity Ownership; Gilead: Consultancy; BMS: Consultancy; Millennium: Consultancy; Celgene: Consultancy; Acetylon: Equity Ownership.

Chronic graft-versus-host disease (GVHD) remains the most common late complication of allogeneic haematopoietic stem cell transplantation (HSCT). Although improvements have been made in the prevention of acute GVHD, these advances have not resulted in a concomitant decrease in the incidence of chronic GVHD. Chronic GVHD is associated with decreased quality of life, impaired functional status, the need for extended immune-suppressive treatments, and impaired sur- vival. It occurs in approximately 40% of patients undergoing human leucocyte antigen (HLA)-identical sibling HSCT, 50% Summary Chronic graft-versus-host disease (GVHD) is the most common cause of poor outcomes after haematopoietic stem cell transplantation (HSCT), while the pathophysiology of chronic GVHD remains poorly understood. As both chronic GVHD and autoimmune disease share clinical features, we speculated that autoimmune disease-related genes might be candidate chronic GVHD-related genes. Recent large-scale cohort studies showed that Fc receptor-like 3 gene (FCRL3) single nucleotide polymorphism (SNP) and peptidylarginine deiminases citrullinating enzymes 4 gene (PADI4) haplotype were associated with autoimmune disease. The present study investigated the association between polymorphisms of these two genes and the incidence of chronic GVHD. We analysed 123 cases of Japanese human leucocyte antigen- matched sibling recipients and their donors who underwent HSCT. Although PADI4, which is the rheumatoid arthritis-specific related gene, was not associated with the occurrence of chronic GVHD, the recipient FCRL3-169C/ C genotype was significantly less frequent in chronic GVHD patients than in those without chronic GVHD (P ¼ 0AE0086). There was no relationship between FCRL3 polymorphism and acute GVHD. As FCRL3 is expressed by B cells and might have an important role in immunoregulation, this significant protective genetic effect raises the question of whether FCRL3 might also be involved in the pathogenesis of chronic GVHD.

Background: Multiple myeloma (MM) cells show a variable combination of chromosomal translocations, copy-number variations, somatic mutations and clonal heterogeneity, which makes every patient unique. We have recently shown that MM cells have signs of ongoing DNA-damage, which activates an ATM/ABL1-dependent DNA damage response (DDR) without overt apoptosis (Cottini et al., Nat Med, 2014). Here we further characterize the mechanisms of DNA damage and replicative stress in MM, which provide the basis for a novel synthetic lethality treatment approach. Results: MM cell lines with active DNA damage have enrichment in pathways of DNA replication and cell cycle. These same MM cell lines also present 53BP1, RPA and RAD51 foci with activated ATR and CHK1. Of note, 53BP1, RPA and RAD51 foci are markers of replicative stress, associated with DNA hyper-replication and stalled replication forks. Importantly, replicative stress markers are also present in primary MM cells. We also demonstrated a gene expression signature specific for increased chromosomal instability and DNA damage in a cohort of MM patients versus normal plasma cells. Remarkably, a subset (20 percent) of patients with myeloma overexpress genes belonging to the instability signature; this group also shows an unfavorable prognosis due to a more aggressive disease. These findings suggest that some patients present a similar phenotype to the cell lines, characterized by extensive replicative stress and activation of hyper-replicative pathways. We therefore hypothesized that MM cells might be sensitive to replicative stress overload, which occurs when cells fail to endure the presence of an excess of unrepaired DNA. To evaluate this hypothesis, we used shRNAs to silence ATR, a protein involved in the control of stalled replication origins, in two myeloma cell lines, one with normal TP53 (H929) and another with mutant TP53 (OPM-2). Inhibition of ATR caused a reduction in cell growth and induction of apoptosis, both more evident in MM cell lines with mutant TP53. A similar phenotype was observed when MM cell lines were incubated with VE-821, a specific ATR inhibitor. The strongest response occurred in TP53 mutant cell lines, which are representative of a model of aggressive MM, consistent with the concept of replicative stress overload. Indeed, p53 is normally phosphorylated and active in MM, while TP53 loss in the context of hyper-replication may prevent activation of salvage checkpoint, thereby favoring cell death in the absence of ATR. ATR inhibition also induces an increase in DNA double strand breaks, as evidenced by the higher number of γ-H2A.X foci. Reactive oxygen species (ROS) can also mediate DNA damage, and treatment with an antioxidant reagent N-Acetylcysteine (NAC), which helps scavenging ROS by replenishing glutathione stores, was indeed capable of reducing DNA double strand breaks and replicative stress markers. Since cancer cells are particularly sensitive to oxidative stress, we then evaluated the anti-MM activity of piperlongumine (PL), a drug that induces ROS accumulation. MM cell lines were sensitive to PL treatment, while PBMCs were minimally affected. As expected, the apoptotic effects of PL were abrogated upon co-incubation with NAC, indicating the specific activity of PL on ROS. We next exploited the possibility of combining replicative and oxidative stress in myeloma cells, hoping to overcome the threshold of tolerance to unrepaired DNA. H929 and OPM-2 cells were transfected with ATR shRNAs or treated with VE-821 and incubated with DMSO or 1-2.5 μM PL; synergic effects by the combination treatment were evident in both myeloma cell lines and also in patient MM cells. Conclusion: Replicative stress is present in a group of MM patients, who have aggressive disease, myeloma cell hyperproliferation and poor prognosis. Strategies aimed to shift the balance towards high DNA damage by ROS production and reduced DNA repair can decrease MM growth and may benefit patients with otherwise unfavorable outcomes. Disclosures No relevant conflicts of interest to declare.

Introduction: Recent studies have elucidated the importance of using 3-dimensional rather than 2-dimensional models in order to create an experimental system recapitulating the specialized properties of the bone marrow microenvironment. Since the neoplastic bone marrow (BM) milieu plays important roles in multiple myeloma (MM) pathogenesis, novel models to study the MM cell in its neoplastic microenvironment are needed. Methods: To mimic the neoplastic BM microenvironment of MM patients, we have established a special hydrogel-based 3-dimensional (3-D) model by ex-vivo culturing MM patient-derived mesenchymal stem cells (MM-MSCs), the predominant cellular component of the marrow niche, which promotes greater mineralization and differentiation than a 2-dimensional (2-D) system. Results: To characterize MM-MSCs in different stages of MM, we utilized an 11 multi-color flow cytometry panel. The percentage of MSCs (CD73+CD90+CD105+lin-CD45-CD34-HLA-DR-) population in BM aspirate samples of 50 MM patients (MGUS, smoldering MM, newly diagnosed MM, and relapsed or relapsed/refractory MM) was evaluated, and correlated with the distribution of (CD38+ CD138+) plasma cells. MSCs were less frequent (10x) than plasma cells, and increased with disease progression to relapsed/refractory MM. We seeded MM-MSCs (N=34) which had been expanded by adhesion methods in 2-D versus 3-D models in order to create an ex-vivo MM niche-like structure. In the hydrogel-based 3-D model, MM-MSCs formed compact clusters with active fibrous connections and meshwork-like structures at day 3 to 7. Moreover, calcium mineralization of clusters was observed, associated with the capacity for differentiation towards the osteoblastogenic or adipogenic lineage when cultured with differentiation media. Furthermore, the production of osteopontin (OPN) and angiopoietin-2 (Ang-2) was significantly higher in 3-D vs. 2-D MM-MSCs, assessed by multiplex luminex technology. Phenotypic profiling of 3-D MM-MSCs clusters revealed high expression of CD73+CD90+CD105+ and lack of expression of CD45, CD34 and HLA-DR, as in to 2-D MM-MSCs. MSC-specific markers including CD166 and HLA-ABC did not reveal any significant changes in 3-D vs. 2-D MM-MSCs; however, 3-D MM-MSCs had significantly decreased expression of CD271 and CD146 compared to 2-D cultures. We also observed significantly higher expression of extracellular matrix (ECM) molecules including fibronectin, laminin, collagen I, and collagen IV (p Conclusions: This 3-D co-culture system closely mimics the myeloma BM niche, and therefore may be useful to identify and validate novel targeted therapies. Disclosures No relevant conflicts of interest to declare.

Bidirectional interaction between MM cells and accessory cells regulates tumor development on the one hand, while transforming the BM microenvironment into a tumor promoting and immune suppressive milieu on the other. Recent developments in targeted therapies have indicated that generation of the most effective therapeutic strategies requires not only targeting tumor or stroma, but also methods to overcome blockade of anti-tumor immune responses. Tumor associated immune suppressor cells such as Treg and myeloid derived suppressor cells (MDSC) can effectively block anti-tumor immune responses, thereby representing an important obstacle for immunotherapy. Co-inhibitory molecules programmed cell death-1 (PD-1) and its ligand (PD-L1) play a fundamental role in tumor immune escape by inhibiting immune effector functions. Since PD-1/PD-L1 signaling promotes tumor growth while inhibiting effector cell mediated anti-tumor immune responses, we here assessed the impact of PD-1/PD-L1 blockade, alone or in combination with lenalidomide (Len), on accessory and immune cells function, as well as tumor cell growth of MM in the BM milieu. Methods: PD-L1 gene expression was determined in IFM MM data set (n=170). Cell surface expression of PD-L1 in CD138+ MM cells, stroma, and MDSCs; as well as PD-1 expression on effector cells (CD4T, CD8T, NK, NKT and monocytes/macrophages) were determined in the bone marrow (BM) and peripheral blood (PB) of patients with MGUS (n=3), newly diagnosed (ND-MM, n=5) and relapsed/refractory (RR-MM, n=11) compared to healthy donors (HD, n=10). PD-1/PD-L1 signaling was assessed in the autologous cocultures of patient MM cells or MM cell lines with stroma (BMSC) or MDSCs and effector cells, in the presence or absence of PD-1/PD-L1 blockade and Len. Len effect on PD-1 expression on effector cells and PD-L1 expression on tumor, BMSC and MDSCs was determined by flow cytometry analysis. BMSC and MDSC induced MM growth/viability was measured by MTT, 3H-Thy and CFSE flow cytometry analysis. Effector cell-mediated MM cytotoxicity was measured by CFSE/PI flow cytometry. The effect of PD-1/PD-L1 blockade with or without Len on cytokine pattern was determined by intracellular cytokine flow cytometry analysis. Results: Statistical analysis of IFM MM data demonstrated that the majority of patient MM cells have increased PD-L1 mRNA compared to HD (p=0.0064). Cell surface expression of PD-L1 was also significantly increased in ND-MM cells (median 65%) and even higher in RR-MM cells. Correspondingly, there was a significant increase in PD-1 expression on CD8T and NK cells in ND-MM and RR-MM. Moreover, PD-L1 expression was significantly higher on both mMDSC and nMDSC than APCs in ND-MM and RR-MM. Coculture with BMSC significantly increased expression of PD-L1 on MM cells. PD-1/ PD-L1 blockade overcame BMSC-induced tumor cell growth in both patient MM cells and MM cell lines. Importantly, Len significantly reduced PD-L1 expression on MM cells; and combined blockade of PD-1/PD-L1 with Len further decreased BMSC-induced MM growth. Immunomodulatory effects of PD-1/PD-L1 blockade were also evaluated in autologous cocultures of immune effector cells with MM cells. Even though there was no change in effector cell proliferation, PD-1/PD-L1 blockade significantly induced cytotoxic activity of autologous T cells, NK cells, and macrophages cultured with MM cells; and Len further enhanced effector cell-mediated cytotoxicity. PD-1/PD-L1 blockade induced intracellular expression of cytotoxic cytokines IFNg and Granzyme B (Gzm B) in CD4T cells, CD8T cells, NK cells and Macrophages. Furthermore, MDSC-mediated MM growth was significantly decreased by PD-1/ PD-L1 blockade. Finally, PD-1/PD-L1 blockade induced intracellular expression of IFNg and Gzm B in T cells, NK cells and NKT cells cultured with autologous MDSC; and Len further enhanced this effector cell activation. Conclusion: Our data demonstrated that immune checkpoint signaling plays an important role in providing the tumor promoting, immune suppressive microenvironment in MM. Blockade of PD-1/PD-L1 signaling induces anti-MM immune responses that can be enhanced by Len. Targeting checkpoint signaling using PD-1 and PD-L1 blocking antibodies, particularly in combination with Len, therefore represents a promising novel immune-based therapeutic strategy to both inhibit tumor cell growth and restore host immune function in MM. Disclosures Kikuchi: The ITO Foundation for the Promotion of Medical Science: Research Funding. Hideshima:Acetylon Pharmaceuticals: Consultancy. Raje:novartis, Amgen, Celgene, Millenium, Onyx: Consultancy; Eli Lilly, Acetylon: Research Funding. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.

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

Abstract 4023 Background: mTOR kinase-targeted therapy is in the early phase of clinical evaluation in multiple myeloma (MM). Despite promising preclinical results with mTOR inhibitors, resistance to this class of drugs in MM patients may occur due to feedback Akt activation by mTORC1. This led to the development of mTORC1/2 inhibition strategies in the treatment for MM, predicated upon the rationale that mTORC2 inhibitors prevent inhibition of mTORC1 blockade-induced feedback AKT activation by mTORC1 inhibitors. Indeed, our previous studies using a novel dual mTORC1 and mTORC2 selective inhibitor AZD8055 show MM cell growth inhibition via apoptosis, associated with inhibition of mTORC1 and mTORC2 signaling, including rapamycin-resistant 4E-BP1 (downstream of mTORC1) and Akt as well as NDRG1 (an effector of mTORC2). Importantly, AZD8055 also inhibited PI3K/Akt signaling and related MM cell growth induced by cytokines (i.e., IL-6, IGF-1) or co-culture with bone marrow stromal cells (BMSCs). Recent studies, however, reveal that constitutively activated Akt signaling negatively regulates IGF-1 receptor (IGF-1R) at the transcriptional level, independent of mTOR activity. Moreover, AKT-induced IGF-1R down-regulation reduces sensitivity of IRS1 to IGF-1 stimulation. We have also shown that IGF-1R inhibitor triggers significant MM cell toxicity. Methods and Results: In this study, we therefore hypothesized that mTORC2 blockade may upregulate IGF-1R expression and/or activity via Akt modulation in MM cells, and that IGF-1R blockade may enhance the cytotoxic effects of mTOR kinase inhibition in MM cells. We first examined the tyrosine phosphorylation sites (Y1135/1136) in the activation loop of the IGF-1R kinase domain in three MM cell lines (MM.1S, OPM1 and RPMI8226) treated with AZD8055 or rapamycin. AZD8055 induced more pronounced upregulation of p-IGF-1R in MM.1S and OPM1 MM cells than rapamycin at earlier time periods. IGF-1 clearly upregulated Akt phosphorylation in MM cells; however, it had no effect on mTOR phosphorylation (Ser2481). Moreover, AZD8055-treated cells exposed to IGF-1 sustained p-Akt (Ser473) expression, while p-mTOR (Ser2481) remained fully inhibited. These results suggest that IGF-1/IGF-1R signaling may bypass mTORC2/Akt when mediating p-Akt (Ser473) upregulation. Moreover, reactivation of IGF-1 signaling in MM cells in the context of mTOR kinase inhibitors suggests that MM may survive in an IGF-1 R–dependent fashion. We therefore next treated MM.1S, OPM1 and RPMI8226 cells with AZD8055, in the presence or absence of IGF-1. MM.1S and OPM1 MM cells (with higher Akt baseline activity) partially escaped AZD8055 cytotoxicity, while RPMI8226 MM cells (with lower Akt activity) did not. Moreover, the addition of blocking IGF-1R antibody or of IGF-1R inhibitor enhanced AZD8055 cytotoxicity in MM.1S and OPM1 cells. Conclusions: Our study therefore shows interaction of mTOR/Akt and IGF-1R/Akt pathways in MM tumors with IGF-1-enabled Akt activation. Importantly, they suggest that combination treatment with AZD8055 and IGF-1R inhibitor is a promising strategy to mTOR kinase inhibition in MM with potential IGF-1R/Akt signaling mediated survival. Disclosures: Hideshima: Acetylon Pharmaceuticals, Inc.: Consultancy. Guichard:AstraZeneca: Employment, Shareholder Other. Anderson:Onyx: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Raje:Onyx: Consultancy; Celgene: Consultancy; Millenium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

Chronic graft versus host disease (cGVHD) is the most common cause of poor outcomes after hematopoietic stem cell transplantation (HSCT), while the pathophysiology of cGVHD remains poorly understood. We have revealed the association Fc receptor-like protein 3 (FCRL3) gene -169C/T single nucleotide polymorphism (SNP) and developing cGVHD. This gene is a member of Fc receptor-like protein gene family which contains 5 genes and well known as autoimmune disease associated gene. These 5 genes make cluster lesion on chromosome 1q21 to 23. In this current study, to investigate the relationship with another Fc receptor-like protein genes and cGVHD, we used 4 microsatellite polymorphisms belong to FCRL1, 2, 3 and 4 as a linkage analysis genetic marker. And we also investigated FCRL3 -169C/T SNP and another 3 SNPs to explore haplotype and the incidence of cGVHD. We analyzed 123 case of Japanese HLA-matched sibling recipients and their donor who underwent HSCT in the Japanese Red-Cross Nagoya First hospital. Before day 100, 11 patients died from severe acute GVHD, infection, thrombotic microangiopathy or disease relapse. Therefore, we analyzed 112 recipients and 108 donors for the relationship between gene polymorphisms and chronic GVHD. We analyzed microsatellite polymorphisms and SNPs by using TaqMan® assay. Chronic GVHD occurred in 54 out of 112 (48.2%) patients in this cohort. The nearest microsatellite marker from FCRL3 gene had significant different allele frequency between cGVHD and non-GVHD recipient (p=0.0034). There were no association with another microsatellites allele and developing cGVHD. FCRL3 gene haplotypes with frequency >0.01 were -TGGG-, -CACA- and -CGCA-. Chronic GVHD patients significantly less the frequency of -CACA- or -CGCA- allele than non GVHD patients (p=0.0040). There were no significant difference in donor allele frequency and incidence of cGVHD. In conclusion, our studies show FCRL3, which is autoimmune disease associated gene, is also the candidate of cGVHD associated gene and its protective haplotype may prevent from occurring cGVHD but not homologous another FCRL proteins.

The Dickkopf-1 (DKK-1) gene product is an extracellular Wnt inhibitor. Hypermethylation of the DKK-1 promoter results in transcriptional silencing and may play an important role in cancer development. Here, we investigated hypermethylation of the DKK-1 promoter in patients with acute myeloid leukaemia (AML), especially core-binding factor (CBF) leukaemia. The methylation status of DKK-1 was analyzed by methylation-specific polymerase chain reaction in 47 patients with AML. DKK-1 methylation was found in 14 (29.8%) of the patients, and more frequently in those with CBF leukaemia (6 of 12 patients), than in those with acute promyelocytic leukaemia (0 of 6 patients) (P = .03). In contrast, Wnt inhibitory factor-1 methylation was found in acute promyelocytic leukaemia (4 of 6 patients) but not in CBF leukaemia (0 of 12 patients) (P = .001). Multivariate analyses suggested that DKK-1 methylation was a risk factor for poorer overall survival. Sequential analysis using 4 paired samples obtained at diagnosis and relapse suggested that DKK-1 methylation was involved in the progression of leukaemia. DKK-1 methylation may therefore be involved in leukaemogenesis, especially in CBF leukaemia, and may be a useful prognostic marker in AML.

Acute myeloid Leukemia(AML) is known as heterogeneous disease, especially in adult AMLs. FLT3/ITD and NPM1 mutation are frequentry seen at the same time, and expected as a role of stratification factor. FLT3/ITDs are the most frequent genetic alterations in AML, found in approximately 20% of adult AMLs. FLT3/ITD is associated with leukocytosis and worse prognoses. ITD mutations vary in size and location, ranging in size from 3 to hundreds of nucleotides. Recently, it is reported that nucleophosmin gene(NPM1) is mutated in a high proportion of gene(NPM1) is mutated in a high proportion of adults with AML and associated with a normal karyotype and FLT3 mutations. In this study, we examined prognostic significance of FLT3/ITD and NPM1 mutation in de novo adult AMLs. We analyzed 51 AML patients(median age:48y/o, range:17–83y/o). Mainly almost patients underwent JALSG AML97 protocol therapy. Acute promyelocytic leukemia was also comprised in this analysis, and 18 of 54 cases were underwent bone marrow transplantation(BMT). Genomic DNA was amplified by means of a polymerase chain reaction, and agarose gel elecrophoresis was used for detection of ITD mutation. ITD size was determined by sequencing analysis. For NPM1 mutation detection, we amplified genomic DNA by PCR using primers as previously reported. We found 11(22%) patients with FLT3/ITD mutation. ITDs were associated with higher WBC count(median of 90.7×109/L versus 5.85×109/L; p

T-cell prolymphocytic leukemia (T-PLL), a rare type of peripheral T-cell leukemia, is characterized by marked splenomegaly with rapidly progressive lymphocytosis and a poor prognosis. Nine kinds of ABL1 chimeric genes have been identified in various kinds of hematological malignancies, such as chronic myeloid leukemia and B- or T-lymphoblastic leukemia. However, there have been no reports describing T-PLL cases with ABL1 rearrangements. We herein report a case of T-PLL with a novel SEPT9-ABL1 fusion gene which induced strong resistance to tyrosine kinase inhibitors such as imatinib and dasatinib., Highlights • We herein report a case of T-PLL with a novel SEPT9-ABL1 fusion. • Ten ABL fusions, including SEPT9-ABL1, have so far been reported. • The current case was resistant to tyrosine kinase inhibitors.

A 40-year-old male developed refractory acute promyelocytic leukemia (APL) after various treatments including all-trans retinoic acid, tamibarotene, arsenic trioxide (As2O3), conventional chemotherapy, and autologous peripheral blood stem cell transplantation. We attempted to use both tamibarotene and As2O3 as a combination therapy, and he achieved molecular complete remission. Grade 2 prolongation of the QTc interval on the electrocardiogram was observed during the therapy. The combination therapy of As2O3 and tamibarotene may be effective and tolerable for treating refractory APL cases who have no treatment options, even when they have previously been treated with tamibarotene and As2O3 as a single agent.