ALBERT

Description: Monocytes/macrophages exert a series of important functions in vivo. To facilitate detailed investigation of their functional capacity and the mechanism leading to their differentiation, several cell lines have been established from primary material. We present here a new human monoblastic cell line, designated UG3. UG3 cells are characterized by the following features. (1) UG3 cells harbor the t(9;11)(p22;q23) translocation that results in fusion of the MLL and the AF9 genes and produce the corresponding AF9-MLL and MLL-AF9 fusion transcripts. (2) UG3 cells rely on the presence of exogenous growth factors for viability and proliferation, such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), or macrophage colony-stimulating factor (M-CSF). (3) When cultured in the presence of G-CSF, UG3 cells differentiate along the granulocytic lineage, as evidenced by segmentation of nuclei and positive staining for neutrophilic alkaline phosphatase and peroxidase. (4) When cultured in the presence of GM-CSF or M-CSF, UG3 cells differentiate into mature macrophages while preserving surface expression of CD14 and CD68 and also start to release cytokines into cell-culture supernatants. Under these culture conditions, UG3 cells also take up acetylated LDL. (5) When cultured in the presence of M-CSF and IL-4, UG3 cells differentiate into osteoclast-like multinucleated giant cells capable of bone resorption and display tartrate-resistant acid phosphatase (TRAP) activity. UG3 cells thus provide features to qualify them as a useful model to further investigate the mechanism underlying these processes and also to further elucidate the functional role of mature monocytes/macrophages or osteoclasts.

Description: ABO incompatibility between donor and recipient is not a barrier for successful allogeneic hematopoietic stem cell transplantation, but conflicting data still exist concerning its influence on transplant outcome, graft-versus-host disease (GVHD), relapse, and survival. We retrospectively analyzed the data of patients who underwent UR-BMT through the Japan Marrow Donor Program between January 1993 and September 2005, with complete data on ABO-blood group compatibility, age, and gender in donors and recipients. A total of 4,970 patients were transplanted with marrow from ABO-matched (M; n=2,513, 50.6%), major incompatible (MA; n=1,254, 25.2%), minor incompatible (MI; n=1,081, 21.8%), and bidirectional incompatible donors (IA; n=122, 2.5%), and were followed up over a median period of 325 days. Among these four groups, excluding age, there was no significant difference in the gender of patients and donors, number of transplantations, conditioning regimen, GVHD prophylaxis, and performance status before transplantation by the likelihood ratio test. The 5-year overall survival of any ABO-incompatible group was significantly lower compared to an identical group (Wilcoxon test, p

Description: Introduction:Central nervous system invasion in multiple myeloma (CNS-MM) is extremely rare (approximately 1% of MM). Prognosis of patients with CNS-MM is generally poor, and the median overall survival (OS) time from its diagnosis (Dx) has been reported to be 2 to 7 months. However, because of its rareness, most available data concerning CNS-MM are based on anecdotal reports on small case series. In this study, we retrospectively collected clinical data of Japanese patients with CNS-MM and analyzed them to reveal clinical features and prognosis of this disease entity. Methods:We conducted a nationwide multicenter retrospective study involving 107 centers that consist of educational facilities authorized by Japanese Society of Hematology and its related hospitals. CNS-MM was defined based on the previously reported criteria (Br J Haematol 2013;162:483-48). Univariate and multivariate analyses were performed to explore prognostic factors and the suitable treatment of CNS-MM. Results:From December 1978 to February 2016, 75 patients with CNS-MM were identified. The median age was 58 (range: 32-77 years). CNS invasion was detected at initial Dx of MM in 4% and at relapse in 96%. The median time from Dx of MM to that of the secondary CNS-MM was 1.8 years. Concomitant plasma cell leukemia (18%), skull plasmacytoma (12%), and myeloma cells in the cerebrospinal fluid (63%) were observed at the Dx of CNS-MM. Common symptoms of CNS-MM included consciousness disturbance (37%), cranial nerve palsy (25%), diplopia (18%), headache (16%), spinal nerve disorder (13%), and nausea/vomiting (12%). Intrathecal chemotherapy (IT) was used in 37%, cranial and/or spinal irradiation therapies (RTx) in 45% in addition to various systemic therapies, which included immunomodulatory drugs (IMiDs) (21%), bortezomib (19%), alkylators (21%), dexamethasone alone (6%), autologous stem cell transplant (auto-SCT, 4%), and allogeneic stem cell transplant (allo-SCT, 3%). With a median follow-up of 3.4 months from Dx of CNS-MM and of 31.5 months from that of MM, the median OS time from Dx of CNS-MM was only 3.7 months. The median OS times for the 12 untreated and 61 treated patients were 0.2 and 5.1 months, respectively (P 〈 0.01), suggesting that the patients who did not receive any treatments (Txs) were in a severe condition and their prognoses were extremely poor. In univariate analyses for the entire group, absence of both atypical lymphocytes and plasma cells in the peripheral blood at Dx of MM and no Tx for MM before Dx of CNS-MM were significantly favorable prognostic factors (P = 0.008 and P 〈 0.0001, respectively). Regarding Tx for CNS-MM patients who received any Txs (conventional chemotherapies, bortezomib, IMiDs, IT, and RTx), only RTx was correlated with longer OS time (P = 0.047). In multivariate analyses, RTx and IT had a significant impact on longer OS time (〉6 months, OR = 3.80, 3.97, P = 0.002, 0.01, respectively). Although the prognosis of CNS-MM in this study cohort was very poor, seven patients could survive for 〉1 year from Dx of CNS-MM. Among them, RTx, IMiDs, IT, bortezomib, auto-SCT, and allo-SCT were given to 5, 4, 5, 3, 1, and 1 patients, respectively. Conclusions:Prognosis of CNS-MM is extremely poor in general. Although there must be a selection bias that patients who received various salvage Txs for CNS-MM were in a better condition, the results of our study suggest that multi-modality Txs with RTx, IT, and IMiDs/bortezomib may prolong the survival time in some cases. Prospective studies are needed to confirm our preliminary observations. Disclosures Takamatsu: Celgene: Honoraria; Janssen Pharmaceuticals: Honoraria. Sunami:Janssen Pharmaceutical: Research Funding; Sanofi: Research Funding; Daiichi Sankyo: Research Funding; Bristol-Myers Squibb K.K.: Research Funding; Novartis: Research Funding; Celgene: Honoraria, Research Funding; Takeda: Research Funding; Ono Pharmaceutical: Research Funding. Hagiwara:Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Kuroda:Janssen: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; Astra Zeneca: Research Funding; Celgene: Honoraria, Research Funding. Murakami:Bristol Meyers Squibb: Honoraria; Astellas: Honoraria; Mochida: Honoraria; Eisai: Honoraria; Takeda: Honoraria; MSD: Honoraria; Chugai: Honoraria; Celgene: Honoraria. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding. Tobinai:Celgene: Research Funding; Mundipharma KK: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; SERVIER: Research Funding; HUYA Bioscience: Honoraria; Kyowa Hakko Kirin: Research Funding; Zenyaku Kogyo: Honoraria; Chugai Pharma: Research Funding; Abbvie: Research Funding; GlaxoSmithKline: Research Funding; Ono Pharmaceutical: Research Funding; Daiichi Sankyo Co., Ltd.: Consultancy; Janssen Pharmaceuticals: Honoraria, Research Funding. Iida:Celgene: Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding.

Description: To better understand the biology of graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation (HSCT) and develop novel treatment strategies, accurate and clinically relevant experimental animal models are indispensable. The majority of our understanding of this potentially lethal complication is based on mouse models of bone marrow transplantation in major histocompatibility complex antigens (MHC) and/or minor histocompatibility antigen mismatched settings. These mouse models of GvHD provide us with an immeasurable wealth of information; however, many findings obtained from mouse models are not necessarily correlated with clinical GVHD in humans. In addition, these mouse models are not suitable for predicting the effectiveness of recent human-specific therapies such as monoclonal anti-human antibodies and adaptive cell immunotherapies. To overcome these limitations, xenogeneic GvHD models using severe immunodeficient mice are currently being used in worldwide and allow us to investigate in vivo human immune reactions. The major aim of our study is to clarify the precise mechanism of immune response in vivo and to determine the principle components responsible for xenogeneic GvHD. We first observed that immunodeficient NOG mice receiving either no or sublethal irradiation consistently showed gradual body weight loss and eventual severe GvHD following injection of human unmanipulated peripheral blood mononuclear cells (PBMCs). Histopathology performed at the late phase of GvHD showed extensive infiltration of human CD45+ mononuclear cells, most of which were T-cells, and tissue destruction in lungs, bone marrow, liver, and spleen, with a smaller number detected in gut and skin unlike in human GvHD. We identified the infiltration of human T-cells exclusively in lungs and spleen before the onset of GvHD. Flow cytometric analysis showed a marked reduce of naïve (CD45RA+CCR7+) and central memory (CD45RA-CCR7+) T-cells and an increase of effector memory (CD45RA-CCR7-) T-cells within CD4+ subset in lungs, suggesting that xenogeneic response by human cells in lungs occur preceding systemic inflammation. Of note, the distribution of human cells was similar between intraperitoneal and intravenous injection. These results indicated that the acute lung injury is not associated with cell trapping within mouse pulmonary microvasculature by intravenous delivery. We next subdivided human lymphocyte subsets by magnetic cell sorting before adoptive transfer to characterize human cells responsive for xenogeneic GvHD development. CD4+ T-cells mediated more rapid and severe GvHD than did an equal number of CD8+ T-cells, whereas neither natural killer cells nor γδT cells caused any symptoms of GvHD. All antigen presenting cells (APCs)-depleted PBMCs mediated GvHD as well, suggesting that human T-cells are activated independently of their own APCs in this model. CFSE cell division assay showed more rapid proliferation of CD4+ rather than CD8+ T-cells. It is a noteworthy that the proliferation of and the expression of early activation marker CD69 on CD8+ T-cells was accelerated in the presence of CD4+ T-cells, indicating that CD4+ T-cells are required for the rapid and more efficacious response of CD8+ T-cells. We also found a strong increase of human IL-2, IFN-γ, and TNF-α in serum of mice injected with whole T-cells. In contrast, we observed very low levels of these inflammatory cytokines in mice injected with isolated CD8+ T-cells alone. Taken together, these results suggest that the early activation of human naïve CD4 T-cells by recognition of foreign antigen on mouse APCs following cytokine production play a critical role in triggering systemic inflammation in xenogeneic GvHD model. This in vivo response was confirmed in vitro by demonstrating that human CD4 T-cells rapidly proliferated and expressed CD69 with significantly increase of IL-2 and IFN-γ in response to mouse dendritic cells. These findings are helpful for adequate evaluation of immune response and creation of optimal experimental conditions in xenogeneic GvHD model. Disclosures Kanda: Otsuka Pharmaceutical: Honoraria, Research Funding.

Description: Abstract 5027 Background Various factors such as malignant lymphoma cause abdominal lymph node enlargement. A histological diagnosis of malignant lymphoma is important in planning therapy, and although superficial lymph node biopsies are often obtained, target lesions occasionally arise only around the abdominal aorta. These require surgical, or more recently, endoscopic biopsies. To intraoperatively determine whether target lymph nodes are correctly resected during a surgical biopsy can be difficult, and therapy could be delayed due to postoperative complications. Objective To assess the safety and effectiveness of percutaneous biopsy of paraaortic lymph nodes under computed tomography (CT) fluoroscopic guidance. Subjects and Methods We retrospectively investigated puncture success rates based on findings of diagnostic imaging, size of target lymph nodes, rate of confirmed diagnosis, frequency of changes in therapeutic plans and incidence of complications among 36 patients after percutaneous needle biopsy using an 18G cutting needle between September 2002 and March 2009. Results The pre-biopsy diagnoses for the 36 subjects who underwent percutaneous needle biopsy included: malignant lymphoma (n = 26), lymph node metastasis of esophageal cancer (n = 3), lymph node metastasis of lung cancer (n = 3), lymph node metastasis of pancreatic cancer (n = 3) and unknown (n = 3). The median size of the target lymph nodes was 25 mm (range: 9–102 mm). The results of diagnostic imaging indicated that all punctures were successful. The diagnoses were confirmed in 33 of the 36 patients (91.7%). Histopathological findings confirmed malignant lymphoma (n = 21; Follicular Lymphoma in 11 patients, Diffuse Large B cell Lymphoma in 8, T-Cell Lymphoma in 1, Hodgkin Disease in 1), squamous cell carcinoma (n = 2), poorly differentiated adenoma (n = 2), other malignancies (n = 5), benign lesions (n = 3) and unknown in 3. Therapeutic strategies were altered based on the results of CT-guided percutaneous needle biopsies in 12 of the 36 patients (33.3%). Three patients developed 4 adverse events comprising grade 1 radiation dermatitis (n = 1), grade 1 hematoma (n = 2) and grade 1 pneumothorax (n = 1). Conclusions Target paraaortic lymph nodes can be accurately punctured using CT-guided percutaneous needle biopsy, and the specimens were too small to examine the flowcytometry or chromosome analysis, however it was enough to confirmed the diagnosis according to WHO classification of malignant lymphoma. Percutaneous biopsy of paraaortic lymph nodes under CT fluoroscopic guidance is useful for confirming diagnoses with a low risk of serious deep organ damage. Disclosures No relevant conflicts of interest to declare.

Description: The BCR/ABL fusion protein transforms hematopoietic stem cells and causes chronic myeloid leukemia (CML). An increasing number of mature neutrophils is a characteristic feature in the chronic phase of CML. However, the mechanism by which stem cells transformed by Bcr/Abl differentiate mainly to mature neutrophils remains obscure. To investigate this mechanism, we compared the gene expression profile of CML neutrophils with that of normal neutrophils by microarray analysis. The genes encoding neutrophil granule proteins were upregulated in CML neutrophils, and C/EBPα and C/EBPε, critical transcription factors that regulate granulocytic differentiation, were also upregulated in these neutrophils. On the contrary, the expression of c-jun, a transcriptional factor that contributes to monopoiesis, was downregulated in CML neutrophils. Differences in the expressions of these genes were confirmed by quantitative RT-PCR. A BCR/ABL tyrosine kinase inhibitor, imatinib, released the downregulation of c-jun expression in primary CML neutrophils, showing that Bcr/Abl inhibited the expression of c-jun. Next, to explore the roles of these transcriptional factors in the chronic phase of CML, we established sublines of KCL22, a cell line derived from CML blastic crisis, in which C/EBPα or C/EBPε expression was inducible (KCL22/α or KCL22/ε respectively). Overexpression of either C/EBP protein resulted in morphological changes, such as a reduction of the nuclear to cytoplasmic ratio, more condensed nuclear chromatin, and segmented nuclei, as well as the expression of differentiation specific markers including G-CSF receptor. These data indicate that C/EBPα/ε expression is sufficient to induce myeloid differentiation in BCR/ABL-positive CML cells. Imatinib treatment released the down regulation of c-jun in KCL22, KCL22/α, and KCL22/ε cells in a manner similar to that in primary cells. Interestingly, imatinib induces monocytic differentiation of KCL22/α cells instead of granulocytic differentiation. This effect of imatinib is independent from C/EBPα induction in KCL22/α cells. The monocytic differentiation and the inhibition of granulocytic differentiation in KCL22/α cells were accompanied by c-jun upregulation. To investigate whether these effects on differentiation of KCL22/α cells depend on releasing the downregulation of c-jun expression with imatinib, we knocked down c-jun expression with siRNA in KCL22/α cells after induction of C/EBPα protein and with imatinib treatment. In the fraction of KCL22/α cells with segmented nuclei, the G-CSF receptor increased when c-jun expression was inhibited with siRNA, indicating that the level of c-jun expression controlled the differentiation fate of CML cells. These findings suggest that Bcr/Abl promotes neutrophil differentiation through downregulation of c-jun accompanied by elevated expressions of C/EBPα/ε.

Description: Monocytes/macrophages exert a series of important functions in vivo. To facilitate detailed investigation of their functional capacity and the mechanism leading to their differentiation, several cell lines have been established from primary material. We present here a new human monoblastic cell line, designated UG3. UG3 cells are characterized by the following features. (1) UG3 cells harbor the t(9;11)(p22;q23) translocation that results in fusion of the MLL and the AF9 genes and produce the corresponding AF9-MLL and MLL-AF9 fusion transcripts. (2) UG3 cells rely on the presence of exogenous growth factors for viability and proliferation, such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), or macrophage colony-stimulating factor (M-CSF). (3) When cultured in the presence of G-CSF, UG3 cells differentiate along the granulocytic lineage, as evidenced by segmentation of nuclei and positive staining for neutrophilic alkaline phosphatase and peroxidase. (4) When cultured in the presence of GM-CSF or M-CSF, UG3 cells differentiate into mature macrophages while preserving surface expression of CD14 and CD68 and also start to release cytokines into cell-culture supernatants. Under these culture conditions, UG3 cells also take up acetylated LDL. (5) When cultured in the presence of M-CSF and IL-4, UG3 cells differentiate into osteoclast-like multinucleated giant cells capable of bone resorption and display tartrate-resistant acid phosphatase (TRAP) activity. UG3 cells thus provide features to qualify them as a useful model to further investigate the mechanism underlying these processes and also to further elucidate the functional role of mature monocytes/macrophages or osteoclasts.

Description: Introduction: Lenalidomide (LEN) plus low-dose dexamethasone (Rd) has been shown to be effective and to have a manageable safety profile in non-Japanese patients (pts) with newly diagnosed multiple myeloma (NDMM) (Rajkumar, Lancet Oncol 2010). Recently, the FIRST trial showed prolonged progression-free survival (PFS) and favorable overall survival (OS) benefits for pts who were administered continuous Rd until progressive disease (PD) vs. the standard of care melphalan-prednisolone-thalidomide (Facon, Blood 2013). In Japan, the combination of melphalan- prednisolone -Velcade®(MPV) is the current standard of care for transplant-ineligible NDMM pts. However, prolonged treatment (Tx) with MPV is associated with increased toxicity including peripheral neuropathy (PN) and bone marrow suppression. Consequently, continuous Tx with MPV may have limited benefits due to toxicity and related Tx discontinuation. MM-025 is a phase 2, multicenter, open-label, registration, single-arm trial. It aimed to evaluate the efficacy and safety of the continuous Rd regimen in Japanese NDMM pts who are transplant-ineligible. Methods: The study enrolled Japanese NDMM pts who were aged ≥ 65 years (yrs), or were not candidates for hematopoietic stem cell transplantation. Pts with an ECOG performance status (PS) score 〉 2 or with grade ≥ 2 PN were excluded from the study. Tx consisted of LEN (25 mg once daily on days 1–21 of each 28-day cycle) and dexamethasone (40 mg for pts aged ≤ 75 yrs or 20 mg for pts aged 〉 75 yrs, once daily on days 1, 8, 15, and 22 of each 28-day cycle) until PD or discontinuation. The dose of LEN was adjusted according to baseline renal function: 25 mg/day for pts with normal or mild renal impairment (RI) (creatinine clearance [CrCl] ≥ 60 mL/min); 10 mg/day for moderate RI (CrCl ≥ 30 to 〈 60 mL/min); and 15 mg every other day for those with severe RI (CrCl 〈 30 mL/min, not requiring dialysis). Pts who discontinued Tx were followed up every 2 months (mos) for ≥ 5 yrs from the start of Tx. The primary endpoint was overall response rate (ORR; defined as complete response [CR] + very good partial response [VGPR] + partial response [PR]) based on the IMWG criteria. The secondary endpoints included time to response (TTR), duration of response (DOR), PFS, OS, and safety. Statistical analyses included the one-sample binomial test for ORR and Kaplan-Meier analysis for DOR, PFS, and OS. The data cutoff date for this analysis was November 2013. Results: A total of 26 pts were enrolled. Of these, 46.2% of pts (n = 12) were aged 〉 75 yrs, 50.0% (n = 13) were male, 19.2% (n = 5) had ISS stage III disease, 23.1% (n = 6) had an ECOG PS score of 2, and 7.7% (n = 2) had severe RI (CrCl 〈 30 mL/min). With a median follow-up of 7.4 mos, the median Tx duration was 6.4 mos. The ORR was 83.3%, including VGPRs (12.5%) (n=3) and PRs (70.8%) (n=17). The median TTR was 2.0 mos. Due to the short follow-up DOR, PFS, and OS were not reached at data cutoff. The most common grade 3–4 adverse events (AEs; reported in 〉 10% of pts) were anemia (19.2%), neutropenia (15.4%), and rash (11.5%). The most frequently reported AEs (≥ 20% incidence) were rash, constipation, anemia, nasopharyngitis, and insomnia. A total of 8 pts (30.8%) discontinued the study: 4 due to AEs, 3 through the investigator’s decision and 1 due to protocol deviation. No deaths, thromboembolic events, or second primary malignancies were reported. Conclusion: Continuous Rd was effective and well tolerated by Japanese transplant-ineligible NDMM pts. These findings are consistent with those reported in the FIRST trial (Facon, Blood 2013) and support the use of the Rd combination regimen as a first-line Tx for this pt group. Disclosures Off Label Use: Lenalidomide used in newly diagnosed multiple myeloma patients. Taniwaki:Celgene Corporation: Honoraria, Research Funding. Iida:Celgene K.K.: Honoraria, Research Funding. Matsumura:Celgene: Honoraria. Ogaki:Celgene K.K.: Employment. Midorikawa:Celgene K.K.: Employment. Houck:Celgene Corporation: Employment. Ervin-Haynes:Celgene Corporation: Employment.

Description: Background Hepcidin (HAMP) is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. The expression of HAMP increased in patients with anemia of chronic disease. Previously, a synthesized compound K7174 (Kowa Company Ltd., Tokyo, Japan) was identified through chemical screening as a novel inhibitor for the adhision of monocytes to cytokine-stimulated endothelial cells (Umetani et al. BBRC 2000). Interstingly, K7174 restored anemia induced by inflammatory cytokines in mice (Imagawa et al. FASEB J 2003), implying that K7174 might modulate hepcidin level. In the present study, we assessed the impact of K7174 on hepcidin expression based on human hematoma cell line and in vivo mice. Method The HepG2 hematoma cells as well as K562 erythroid cells were used for the analyses. The cells were treated with K7174 at doses of 10 and 20 uM for 24 h. For transcription profiling, Human Oligo chip 25K (Toray) were used for K7174-treated HepG2 cells. Western blotting and quantitative chromatin immunoprecipitation (ChIP) analyses were performed with antibodies to GDF15 (abcam), C/EBPbeta (CEBPB) (C-19) (Santa Cruz), Smad1 and Phospho-Smad1-5-8 (CST). For GDF15 knockdown in HepG2 cells, anti-GDF15 siRNA (Thermo Scientific Dharmacon) and Lipofectamine RNAiMAX (Invitrogen) were used. GDF15 promoter assay was conducted with Dual Luciferase Reporter Assay system (Promega). Human GDF15 concentration in the K7174-treated media was evaluated with ELISA (R&D systems). For in vivo analysis, ICR mice were injected intraperitoneally with PBS (control) or 30 mg/kg K-7174, respectively, days 0, 1, 2, 3, 5, 6, 7 and 8, and the samples were taken on day 9. Serum hepcidin1 concentration was determined with LC-MS/MS method (MCProt Biotechnology, Kanazawa, Japan). Results We first demonstrated that K7174 treatment (20 uM) in HepG2 cells significantly decreased HAMP expression (〉 2-fold). Thus, we next conducted microarray analysis to reveal the molecular mechanism by which K7174 inhibits the HAMP expression. Transcriptional profiling confirmed the downregulation of HAMP. Interestingly, K7174 strongly induced GDF15 (10-fold), a negative regulator of HAMP expression (Tanno et al. Nat Med 2007). Quantitative RT-PCR, Western blotting as well as ELISA analyses confirmed the induction of GDF15 by K7174 treatment. Furthermore, siRNA-mediated GDF15 knockdown during K7174 treatment significantly re-activated HAMP expression, suggesting that the increase of GDF15 induced by K7174 was responsible for the HAMP downregulation. Noticeably, we also found that K7174 upregulates CEBPB (2.9-fold). Promoter assay and quantitative ChIP analysis demonstrated that K7174-mediated upregulation of CEBPB contributes to the transcriptional activation of GDF15. On the other hand, the microarray analysis and quantitative RT-PCR-based validation identified the significant K7174-mediated downregulation of BMP4 (2.6-fold), a positive regulator of HAMP expression (Babitt et al. Nat Genet 2006). However, the level of SMAD1-5-8 phosphorylation in K7174-treated cells remained unchanged, implying that BMP-SMAD signaling might not be essential in K7174-mediated HAMP suppression. Next, we assessed if K7174 inhibits hepcidin expression in mice. Quantitative RT-PCR analysis with liver sample from K7174-treated mice demonstrated significant upregulation of Gdf15 and downregulation of Hamp (n = 8, p〈 0.05). Furthermore, serum hepcidin concentration was also significantly decreased in K7174-treated mice (Average: 138.1 and 110.4 ng/mL for K7174-treated and control mice, respectively. n = 8, p〈 0.05). Beyond the regulation of Gdf15 in hepatocytes, erythroid cells have also been suggested to be one of the main sources of GDF15. Thus, we treated K7174 with K562 erythroid cells, and confirmed significant increase of GDF15, suggesting that systemic administration of K7174 may act on hepatocytes as well as erythroid cells to stimulate GDF15 production. Conclusion Our in vitro and in vivo analyses suggested that K7174 suppresses HAMP expression through modulating GDF15 expression. K7174 may be considered as a potential therapeutic option to treat anemia of chronic disease. Disclosures: No relevant conflicts of interest to declare.

Description: The BCR/ABL fusion protein transforms hematopoietic stem cells and causes chronic myeloid leukemia (CML). However, the mechanism leading to the expansion of mature myeloid cells in CML remains obscure. To investigate this mechanism, we compared the gene expression profile of CML neutrophils with that of normal neutrophils by microarray analysis. The genes upregulated in CML neutrophils include critical transcription factors that regulate granulocytic differentiation and proliferation, as well as genes encoding neutrophil granule proteins (Table 1). The protein levels of these upregulated genes were also elevated in the CML neutrophils. To investigate whether BCR/ABL regulates the expression of these upregulated genes, primary CML neutrophils were exposed to 1 μM imatinib (a BCR/ABL tyrosine kinase inhibitor) for 12–16 hours. C/EBPα protein expression was downregulated in primary CML neutrophils after treatment with imatinib, compared to control cultures. We further explored the function of these upregulated genes in KCL22 cells, which are derived from CML blastic crisis. To investigate the effect of C/EBPα or C/EBPε on myeloid differentiation, we established KCL22 cells in which C/EBPα or C/EBPε expression was inducible (KCL22/α or KCL22/ε, respectively). Overexpression of either C/EBP protein resulted in morphological changes, such as reduction of the nuclear to cytoplasmic ratio, more condensed nuclear chromatin, and segmented nuclei, as well as the expression of differentiation specific markers including the G-CSF receptor and macrophage adhesion molecule-1 (Mac-1). These data indicate that C/EBPα /ε expression is sufficient to induce myeloid differentiation in BCR/ABL positive CML cells. We examined whether BCR/ABL regulates the protein expression of c-Myb. The expression of c-Myb in KCL22 cells treated with 1 μM imatinib was downregulated and growth in these cells decreased in parallel. Knock-down of c-Myb expression with siRNA inhibited the growth of KCL22 cells and these data indicate that c-Myb expression is important for the growth of CML cells. To test the effect of c-Myb on differentiation of KCL22 cells, we knocked down c-Myb expression with siRNA in KCL22/α or KCL22/ε cells after induction of C/EBP protein. The knock-down of c-Myb in KCL22α /ε cells resulted in downregulation of differentiation specific genes, including G-CSF receptor and neutophil elastase. Morphologically, the fraction of KCL22α /ε cells with segmented nuclei decreased when c-Myb expression was knocked down. Our results suggest that elevated expression of C/EBPα /ε and c-Myb play a key role in proliferation and differentiation in CML cells. Table 1. Genes Upregulated in CML Neutrophils. Description Gene transcription factor C/EBP α transcription factor C/EBP ε transcription factor c-Myb neutrophil granule protein lactoferrin neutrophil granule protein neutrophil elastase neutrophil granule protein lysozyme