ALBERT

Description: Abstract 1274 Poster Board I-296 Introduction RCAN1 (also called DSCR1, calcipressin 1, MCIP1, Adapt78) is known to be a regulator of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, and has also been shown to regulate the activity of certain transcription factors, including NF-AT and NF-kB. RCAN1 expression is widely found in various tissues, including the brain, kidney, liver and heart, but the expression level of RCAN1 is extremely low in normal hematopoiesis-related organs (bone marrow and spleen). Although RCAN1 has been shown to act as a negative regulator of FceRI-induced mast cell activation and NF-AT signaling in megakaryocytes, the cellular function of RCAN1 in myeloid cells remains unknown. Recently, we found that RCAN1 was highly expressed in acute phase of chronic myeloid leukemia (CML) by DNA microarray analysis. In this study, to explore the role of RCAN1 in leukemogenesis, we compared RCAN1 expression in leukemia cells and that in normal hematopoietic cells. We also analyzed regulatory mechanisms of RCAN1 gene promoter function and searched for novel RCAN1-interacting molecules in myeloid leukemia cells. Methods Expression of RCAN1 was examined by quantitative PCR using normal human bone marrow cells, bone marrow cells from patients with acute myeloid leukemia (AML) and patients with CML, and various human hematological tumor cell lines. Promoter activity of the human RCAN1 gene was evaluated by a luciferase assay using various deletion mutants in KCL22 cells. To identify novel RCAN1-interacting proteins, a yeast two-hybrid assay was performed using a cDNA library from K562 leukemia cells. Results Fractions of CD34+CD38-, CD34+CD38+, CD33+CD14-CD16-, CD33+CD14+ and CD33+CD16+ cells from normal human bone marrow showed extremely low levels of RCAN1, indicating that RCAN1 is scarcely expressed in normal hematopoietic cells, including stem cells and progenitor cells. In contrast, bone marrow mononuclear cells (BMMC) from 15 of 16 patients with AML and all four patients with CML in acute phase showed substantial levels of RCAN1. Furthermore, RCAN1 was expressed in all human cell lines examined in this study that were derived from AML (THP-1, U937, HL60, KY821), CML in acute phase (KCL22,K562, KU812), acute lymphoblastic leukemia (Jarkat, Raji) and multiple myeloma (RPMI8226, U266). Interestingly, expression levels of RCAN1 were significantly decreased in BMMC from three of four AML patients when the patients were in complete remission. These results suggest that RCAN1 is extraordinarily expressed in a wide range of hematological tumor cells. To reveal the underlying mechanisms of ectopic expression of RCAN1, we next examined the promoter function of RCAN1 gene, which consists of seven exons. Although RCAN1 expresses several alternative isoforms that selectively include each of exons 1-4 and use distinct promoters, we found that only exon 1 isoform was expressed in these leukemia cells. Analysis of the function of human RCAN1 gene promoter upstream of exon 1 demonstrated that NKX2-5 binding site is important for basal activity of RCAN1 gene promoter in KCL22 cells. NKX2-5 is a homeobox-containing transcription factor and is involved in leukemogenesis of T-cell acute lymphoblastic leukemia. Our results raised the possibility that NKX2-5 plays a role in malignant transformation partly through expression of RCAN1 in myeloid leukemia cells. Finally, we searched for RCAN1-interacting proteins in leukemia cells. By screening a cDNA library from K562 cells using yeast two-hybrid assay, we found that RCAN1 interacts with HINT1, HINT2 and RINT1. HINT1 has been reported to be a tumor suppressor against hepatocellular carcinoma, and HINT2 and RINT1 have also been found to function as tumor suppressors. It would be interesting to clarify whether RCAN1 affects the activity of these molecules, and it is important to reveal the involvement of these molecules in leukemogenesis. Conclusion RCAN1 is extraordinarily expressed in a wide range of hematological malignant cells, and NKX2-5 might be important for its ectopic expression. It is possible that RCAN1 is biologically significant in leukemogenesis through affecting certain tumor suppressors. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1543 Background: CD25 (the alpha chain of the IL-2 receptor) has been shown to be expressed in hairy cell leukemia cells and a small subset of normal B cells. Recently, we reported that CD25 is expressed in a subset of diffuse large B-cell lymphoma (DLBCL) (ASH 2011 118:2666). However, its expression in follicular lymphoma (FL) has yet to be fully examined. The aim of this study was to clarify the clinical features of newly diagnosed CD25+ FL. Patients and Methods: Eighty-four patients with newly diagnosed CD25+ FL who were admitted to our hospital between 2002 and 2012 were retrospectively evaluated. Biopsy specimens from lymph nodes or related tissues were analyzed by flow cytometry (FCM) using a combined method of single- and two-color staining. CD25 expression was defined as positivity of 〉20% of clonal B cells with CD19 or 20 expression 〉70% in a gated region. Results were compared with patients with reactive lymphadenopathy (n=27), DLBCL at initial presentation (n=91), and FL at relapse (n=13). Results: CD25 expression in untreated patients with FL (mean±SD, 10.6±9.9%; n=84) was almost equivalent to that in patients with reactive lymphadenopathy (mean±SD, 9.1±4.4%; n=27, P =0.3), and lower than that in untreated patients with DLBCL (mean±SD, 28.5±31.1%; n=91, P

Description: Abstract 2762 Poster Board II-738 Introduction: Aurora kinases (Aurora-A, Aurora-B, Aurora-C) play an essential role in the regulation of mitosis. It has been shown that deregulation of aurora kinases is involved in tumorgenesis and that these kinases are overexpressed in a variety of tumor cells. Aurora kinase inhibitors are potential small-molecule agents for treatment of various kinds of tumors including leukemia, and clinical trials of several aurora kinase inhibitors against certain types of tumors are currently being carried out. However, mono-therapy with other small-molecule agents sometimes shows only limited clinical efficacy for treatment of leukemia, and the establishment of efficacious combination therapies therefore appears to be an attractive approach for making good use of aurora kinase inhibitors. Methods: We examined the cytotoxic effects of VE-465, a specific aurora kinase inhibitor, in combination with various conventional anti-leukemia agents, including doxorubicin, daunorubicin, idarubicin, mitoxantron, cytocine arabinoside, vincristine and etoposide, on AML cell lines (HL60, U937, THP-1, KY821), CML cell lines (KCL22, K562, KU812) and primary leukemia cells from a patient with AML in order to try to determine an effective therapeutic combination. Results: Steel and Peckham isobologram analyses demonstrated that a combination of VE-465 and vincristine had a synergistic/additive inhibitory effect on the growth of all leukemia cell lines as well as primary leukemia cells examined in this study. Flow cytometric analysis showed that the percentage of G2/M-phase cells was significantly increased at 12 h when VE-465 was added to THP-1 cells as a single agent. At 48 h, however, the percentage of sub-G1 cells was increased, with a decrease in the percentage of G2/M-phase cells, suggesting that VE-465 initially induced the cells into blockage of the cell cycle at M-phase, which may be caused by VE-465-mediated inhibition of aurora kinase activity, and that cells at G2/M arrest were subsequently induced to apoptosis. Importantly, vincristine enhanced VE-465-mediated induction of sub-G1 cells. Consistent with these results, vincristine enhanced VE-465-induced increase in the levels of cleaved caspase 3, cleaved caspase 7, cleaved caspase 9 and cleaved PARP. The level of Phospho-Chk2 was markedly increased by the combination, suggesting that Chk2-mediated activation of the G2/M checkpoint is involved in the induction of apoptosis. Interestingly, VE-465 alone and VE-465 in combination with vincristine markedly increased the level of phospho-ERK1/2, suggesting that the combination alters a network of cellular signaling pathways. Taken together, the results suggest that vincristine potentiated the effect of VE-465 by enhancement of apoptosis, resulting in effective inhibition of the growth of leukemia cells. In contrast to the combination of VE-465 and vincristine, however, combinations of VE-465 and other anti-leukemia agents showed no synergistic inhibition but rather had antagonistic effects on growth. Conclusion: Co-administration of VE-465 and most of the conventional anti-leukemia agents might have little clinical value. However, the results of this study indicate the utility of the combination of VE-465 and vincristine as a potential therapy for myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.

Description: Heme modulates various cellular functions as a component of hemoproteins. Recently, heme-mediated direct regulation of activity of transcription factors such as Bach1 has been shown. We reported in the last meeting that heme affects sensitivity to imatinib through, at least in part, regulation of Nrf2 transcription factor activity. In this study, we performed further analysis of the mechanisms of heme-mediated regulation of imatinib-sensitivity and the role of heme in the sensitivity to other small molecule compounds and conventional anti-leukemia drugs. IC50 values of imatinib against the BCR/ABL-positive cell line KCL22 were increased by addition of 1–100μM hemin, which eventually becomes heme in cells, in a dose-dependant manner. In the presence of hemin, imatinib-mediated increases in the levels of cleaved caspase 3, cleaved caspase 7, cleaved caspase 9 and cleaved PARP as well as the number of annexin V-positive cells were abrogated, suggesting that heme has an inhibitory effect on imatinib-induced apoptosis signaling. Luciferase reporter assay demonstrated that hemin increased MARE (Maf recognition element)-mediated γ-glutamylcystein synthetase (γ-GCS) light subunit gene promoter activity. Accordingly, the level of γ-GCS light subunit protein was increased after hemin treatment. We next examined the effect of suppression of Nrf2 expression on hemin-mediated change in imatinib sensitivity because Nrf2 might regulate MARE-mediated gene expressions. Transfection of Nrf2 siRNA resulted in partial abrogation of hemin-mediated upregulation of IC50 values of imatinib with suppression of hemin-mediated induction of γ-GCS light subunit expression, suggesting that suppression of Nrf2 partially restored the sensitivity to imatinib in hemin-treated cells. We also examined the effect of hemin on the sensitivity to conventional anti-leukemia drugs such as cytarabine, eoposide, hydroxyurea daunorubicin, idarubicin, doxorubicin and mitoxantrone in KCL22 cells. While IC50 values of cytarabine, etoposide and hydroxyurea were not changed by hemin treatment, addition of hemin resulted in significant increase in IC50 values of four anthracyclin anti-leukemia drugs. Furthermore, hemin also abrogated the sensitivity to U0126, which is a small molecule compound targeting MEK1/2. These results suggest that heme is an important molecule in the regulation of sensitivity to various anti-leukemia reagents.

Description: Heme has been recognized as an important modulator of the functions of various cellular molecules. Recent work showed that heme binds to Bach1 transcription factor, resulting in the loss of the repressor activity of Bach1. Because Nrf2 transcription factor and Bach1 may competitively recognize the common gene regulatory elements, heme may have an effect on the Nrf2-madiated gene expressions. We recently reported that Nrf2 is involved in the resistance to imatinib in BCR/ABL-positive cells. Here we investigated the role of heme in the sensitivity to imatinib in human BCR/ABL-positive cell lines. Addition of hemin to KCL22 and KU812 cells resulted in significant increases in the IC50 values of imatinib in accordance with the abrogation of imatinib-mediated induction of apoptosis-related molecules, cleaved caspase 3 and cleaved PARP. The levels of phosphorylated BCR/ABL and phosphorylated ERK1/2 MAP kinase (active form) were both decreased by imatinib even when hemin was added, indicating that hemin does not interfere with imatinib-mediated inhibition of BCR/ABL function and that mechanisms of hemin-induced acquisition of imatinib-resistance is not related to BCR/ABL kinase activity. Hemin increased Nrf2/DNA complex formation at the antioxidant response element of the human γ-glutamylcystein synthetase (γ-GCS) light subunit gene promoter. Because γ-GCS is a rate-limiting enzyme of the synthesis of glutathione (GSH), which has been shown to be involved in resistance to imatinib, we next examined the level of GSH and found that the intracellular amount of GSH was increased by hemin treatment of these cells. In addition, the protein level of heme oxygenase-1, which is important for survival of chronic myeloid leukemia cells and the gene expression of which can be also regulated by Nrf2, was increased after hemin treatment. Addition of ascorbic acid, which suppresses Nrf2-dependent gene expression, partially restored the sensitivity to imatinib in hemin-treated cells. We also showed that inhibition of intracellular heme synthesis by succinyl acetone increased the sensitivity to imatinib in imatinib-resistant cell lines, KCL22/SR and KU812/SR. The results suggest that heme is critically involved in the sensitivity to imatinib, at least in part, through regulation of Nrf2 function.

Description: Background AL(acute leukemias) with 11q23 abnormalities involving the MLL (mixed lineage leukemia) gene comprise one category of recurring genetic abnormalities in the WHO classification. However, the prognosis of adult AL with 11q23/MLL between fusion partners are controversial especially in Asia. Methods We analyzed data on 61 Japanese adult patients diagnosed as AL with 11q23/MLL abnormalities, including the translocations t(4;11), t(9;11), t(6;11), t(11;19) between 1990 and 2006. Results Median age of the 61 patients was 48 years (range, 17–79 years). 54 patients were AML (acute myeloid leukemia) and 7 patients were ALL (acute lymphoblastic leukemia).The incidence by fusion partners in AML were as follows; t(9;11) in 29.6%, t(11;19) in 24.0%, t(6;11) in 18.5%. The incidence of patients with t(11;19) was higher than that in Caucasian and t(4;11) was lower than that in childhood. As for the FAB (French-American-British) subtype, 68.5% of the 11q23/MLL patients were M4/M5. Organ involvement was observed commonly (46.2%) in AML with 11q23/MLL patients except for t(11;19) (7.6%).The CR (complete remission) rate of AML with 11q23/MLL was not low (78.7%), but the CR duration was very short (10 months).The DFS (disease free survival) at 1year and the OS (overall survival) at 2 years were 20% and 30% in AML with t(9;11), 10% and 19% in AML with t(11;19), 10% and 30% in AML with t(6;11). In total, the DFS at 1year and the OS at 2 years were 26% and 25% in AML patients, respectively, while 50% and 50% in ALL patients, respectively. The DFS and the OS at 2 years were 44% and 53% in AML patients with 11q23/MLL who were treated with HSCT(hematopoietic stem cell transplantations), while 14% and 21% in those without HSCT respectively. Conclusions Although there were reports that the prognosis of AML with t(9;11) was not bad in Caucasian, the present study showed poor prognosis of AML with 11q23/MLL in Japanese adult patients regardless of the fusion partners. Our study revealed the short CR duration of the AML patients with 11q23/MLL in the face of the high CR rate. AML patients with 11q23/MLL who were treated with HSCT had superior outcomes to those without HSCT (P=.04 for DFS and P=.04 for OS) .This findings suggest that treatment strategies including HSCT should be considered for AML with 11q23/MLL during the first CR.

Description: Abstract 1370 Background: The DNA methylation inhibitor 5-azacytidine (AZA), which is approved for treatment of myelodysplastic syndrome, is also a potential agent for treatment of leukemia; however, drug resistance is an ongoing problem, and mechanisms underlying developing resistance to AZA are poorly understood. Therefore, clarifying the resistance mechanisms is central to establish effective countermeasures. Methods: To probe the mechanisms of resistance to AZA and to develop an effective method for overcoming them, we first generated two AZA-resistant cell lines, THP-1/AR and HL60/AR, from the human acute myelogenous leukemia cell lines THP-1 and HL60. We then studied variations between the parental and resistant lines. Results: AZA increased the percentages of sub-G1 and G2/M-phase cells in the AZA-sensitive parental cell lines; whereas, it had no similar effect in the resistant lines. Consistent with these results, the AZA-induced increases in the levels of cleaved forms of caspase 3, caspase 7, caspase 9, and PARP seen in sensitive cells were diminished in resistant cells. Furthermore, AZA markedly elevated the level of phospho JNK/SAPK in sensitive cells, but not in resistant cells. These results suggest that AZA induced apoptosis as well as G2/M arrest due to activation of JNK/SAPK signaling, and that induction of these changes was prevented in resistant cells. We also found that the activity as well as protein levels of DNA methyltransferases (DNMTs), which are the main target molecules of AZA, were suppressed by AZA in sensitive cells. However, in resistant cells, this effect was abrogated; and accordingly, AZA-induced up-regulation of p16 gene expression was also negated. These findings thus suggest that resistance was acquired by a DNMT-dependent mechanism. There was no remarkable difference between resistant cells and sensitive cells in the levels of uridine-cytidine kinase 2 (UCK2), which is a key enzyme for conversion of AZA to active form. However, several point mutations were found restrictedly in exon 4 of the UCK2 gene in both resistant cells. These results raised the possibility that the AZA activation process was perturbed due to reduction of UCK activity; and consequently, AZA failed to suppress DNMT in resistant cells. In addition, by microarray analysis, we identified eleven genes that were expressed at significantly different levels in resistant cells versus sensitive cells. Finally, we showed that the histone deacetylase inhibitor romidepsin induced p16 gene expression and increased the levels of apoptosis-related molecules, while suppressing growth in both sensitive and resistant cell lines. An isobologram analysis demonstrated that simultaneous administration of AZA and romidepsin resulted in an additive inhibitory effect on both AZA-sensitive and AZA-resistant cell growth. These results suggest that romidepsin can overcome AZA resistance; therefore, the combination of AZA and romidepsin not only augments the anti-leukemia effect but also prevents acquisition of resistance to AZA. Conclusions: Newly established 5-azacytidine-resistant cell lines THP-1/AR and HL60/AR are good models to analyze the mechanisms of drug resistance to 5-azacytidine. Using these cell lines, we revealed that acquisition of resistance is primarily caused by a DNMT-dependent mechanism, which can be surmounted with addition of romidepsin. It is likely that the combination of AZA and romidepsin can prevent patients from acquiring resistance to AZA while augmenting its anti-leukemia therapeutic effect. Disclosures: No relevant conflicts of interest to declare.

Description: Background Interleukin 2 (IL-2) is an important cytokine that controls the proliferation and differentiation of not only T- but also B-lymphocytes. Recently, we reported that CD25 (IL-2 receptor alpha chain, IL-2R) is expressed in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), and high expression of CD25 in the two types of lymphoma is correlated with a poor prognosis following chemotherapy regimens containing rituximab (ASH annual meeting, 2011 118:2666, 2012 120:1543). We evaluated the clinical significance of CD25 expression in a larger series of different mature B-cell lymphomas (BCL). Patients and Methods Four hundred and thirty-seven newly diagnosed patients who were admitted to our hospital between 2002 and 2013 were retrospectively evaluated. Lymph node or related tissue biopsy samples of BCL were analyzed using flow cytometry, as follows: 182 patients, DLBCL; 92, FL; 48, chronic lymphocytic leukemia (CLL); 21, mantle cell lymphoma (MCL); 23, marginal zone lymphoma (MZL); 8, Burkitt lymphoma (BL); 18, B-cell lymphoma unclassifiable with features intermediate between BL and DLBCL (BL/DLBCL); 5, lymphoplasmacytic lymphoma (LPL); and 39, reactive lymphadenopathy with sufficient B-cells. CD25-positivity was defined as 〉20% of clonal B-cells in a gated region. Results CD25 expression in patients with MCL, CLL, MZL, and DLBCL was significantly higher than that in patients with reactive lymphadenopathy (P

Description: Background: Neutrophil recovery following allogeneic hematopoietic cell transplantation (allo-HCT) varies widely among patients. Some cases have a rapid increase in neutrophils and early engraft, while others show slow increase after neutrophils appearance and take time until engraftment. Such differences in neutrophil recovery may reflect the composition of the graft and affect the complications or prognosis after allo-SCT, though it has not been well documented in the literature. In this study, we retrospectively analyzed the influence of the slope of neutrophil recovery (N slope) following allo-HCT on post-transplant complications and prognosis. Methods: This study was a retrospective analysis of 120 patients with hematopoietic diseases who undergone first unrelated bone marrow transplantation at Jichi Medical University between January 2009 and December 2018. Patients who failed to achieve engraftment or did not receive granulocyte-colony stimulating factor were excluded. The N slope was defined as the increase of neutrophil counts from the last day of lowest neutrophil count after transplantation to the date of neutrophil engraftment. We evaluated the predictive value of N slope for acute graft-versus-host disease (GVHD) using the area under the receiver operating characteristic (ROC) curve and determined the cut-off value to maximize the sum of the sensitivity and specificity. Results: The median N slope was 205.5 /µL/day (range 26.4-7574). An ROC analysis showed that a cut-off value of N slope for grade II-IV acute GVHD was 207.5 /µL/day (sensitivity 0.73, specificity 0.6, AUC = 0.67, 95 % confidence interval (CI) = 0.59-0.79). Then, we classified patients into the low (n = 59) and high (n = 61) N slope groups according to the cut-off value of 200 /µL/day. The high N slope group correlated with older patients, RIC regimen, higher infused CD34+ cells ≥ 1.5 × 106/kg, and former transplantation between 2009 and 2013 compared with the low N slope group (p = 0.028, p = 0.003, p = 0.036 and p = 0.025, respectively). Cumulative incidence of grade II-IV acute GVHD at day 100 was significantly higher in the high N slope group than the low N slope group (44.3 % vs. 16.9%, p = 0.0007). With regard to the risk factors for grade II-IV acute GVHD, male, donor age ≥ 40 years, N slope ≥ 200 /µL/day and former transplantation between 2009 and 2013 were significant in univariate analysis. In multivariate analysis, donor age ≥ 40 years, N slope ≥ 200 /µL/day and former transplantation were identified as significant independent risk factors (hazard ratio (HR) 3.6, 95%CI 1.7-7.7, p = 0.001; HR 2.8, 95%CI 1.5-5.3, p = 0.002; HR 3.0; 95%CI 1.5-6.1, p = 0.02;, respectively). Cumulative incidence of grade III-IV acute GVHD at day 100 and chronic GVHD at 2 years did not differ in the high and low N slope groups (10.8 % vs. 5.1 %, p = 0.328; 63.3 % vs. 50.1 %, p = 0.12, respectively). In addition, there was no difference in relapse, non-relapse mortality and overall survival between the two groups (p = 0.76, p = 0.32, p = 0.65, respectively). Conclusion: The current study showed that a steeper slope of neutrophil recovery following HCT was associated with increased acute GVHD, though it did not affect relapse, NRM and OS. Early diagnosis and therapeutic intervention for acute GVHD may improve the outcome of patients with rapid neutrophil recovery. Disclosures Kanda: Chugai: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Astellas: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Ono: Consultancy, Honoraria, Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; Ono: Consultancy, Honoraria, Research Funding; Nippon-Shinyaku: Research Funding; Takara-bio: Consultancy, Honoraria; CSL Behring: Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; CSL Behring: Research Funding; Mochida: Consultancy, Honoraria; Eisai: Consultancy, Honoraria, Research Funding; Taisho-Toyama: Research Funding; Otsuka: Research Funding; MSD: Research Funding; Takara-bio: Consultancy, Honoraria; Shionogi: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Nippon-Shinyaku: Research Funding; MSD: Research Funding; Asahi-Kasei: Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Tanabe Mitsubishi: Research Funding; Celgene: Consultancy, Research Funding; Mochida: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Dainippon Sumitomo: Consultancy, Honoraria, Research Funding; Asahi-Kasei: Research Funding; Pfizer: Research Funding; Alexion: Consultancy, Honoraria; Sanofi: Research Funding; Celgene: Consultancy, Research Funding; Otsuka: Research Funding; Taiho: Research Funding; Novartis: Research Funding; Taiho: Research Funding; Taisho-Toyama: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; Dainippon Sumitomo: Consultancy, Honoraria, Research Funding; Tanabe Mitsubishi: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Eisai: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.