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The Runt domain transcription factor AML1/RUNX1 is essential for the generation of hematopoietic stem cells and is the most frequent target of chromosomal translocations associated with leukemia. Here, we present a new AML1 translocation found in a patient with acute myeloid leukemia M4 with t(8;21)(q24;q22) at the time of relapse. This translocation generated an in-frame chimeric gene consisting of the N-terminal portion of AML1, retaining the Runt domain, fused to the entire length of TRPS1 on the C-terminus. TRPS1 encodes a putative multitype zinc finger (ZF) protein containing 9 C2H2 type ZFs and 1 GATA type ZF. AML1-TRPS1 stimulated proliferation of hematopoietic colony-forming cells and repressed the transcriptional activity of AML1 and GATA-1 by 2 different mechanisms: competition at their cognate DNA-binding sites and physical sequestrations of AML1 and GATA-1, suggesting that simultaneous deregulation of AML1 and GATA factors constitutes a basis for leukemogenesis.

The mechanism by which paroxysmal nocturnal hemoglobinuria (PNH) clones expand is unknown. PNH clones harbor PIGA mutations and do not synthesize glycosylphosphatidylinositol (GPI), resulting in deficiency of GPI-linked membrane proteins. GPI-deficient blood cells often expand in patients with aplastic anemia who sustain immune-mediated marrow injury putatively induced by cytotoxic cells, hence suggesting that the injury allows PNH clones to expand selectively. We previously reported that leukemic K562 cells preferentially survived natural killer (NK) cell-mediated cytotoxicity in vitro when they acquired PIGA mutations. We herein show that the survival is ascribable to the deficiency of stress-inducible GPI-linked membrane proteins ULBP1 and ULBP2, which activate NK and T cells. The ULBPs were detected on GPI-expressing but not on GPI-deficient K562 cells. In the presence of antibodies to either the ULBPs or their receptor NKG2D on NK cells, GPI-expressing cells were as less NK sensitive as GPI-deficient cells. NK cells therefore spared ULBP-deficient cells in vitro. The ULBPs were identified only on GPI-expressing blood cells of a proportion of patients with PNH but none of healthy individuals. Granulocytes of the patients partly underwent killing by autologous cytotoxic cells, implying ULBP-associated blood cell injury. In this setting, the lack of ULBPs may allow immunoselection of PNH clones.

The outcome of acquired immunodeficiency syndrome–related lymphomas (ARLs) has improved since the era of highly active antiretroviral therapy, but median survival remains low. We studied dose-adjusted EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin) with suspension of antiretroviral therapy in 39 newly diagnosed ARLs and examined protein expression profiles associated with drug resistance and histogenesis, patient immunity, and HIV dynamics and mutations. The expression profiles from a subset of ARL cases were also compared with a matched group of similarly treated HIV-negative cases. Complete remission was achieved in 74% of patients, and at 53 months median follow-up, disease-free and overall survival are 92% and 60%, respectively. Following reinstitution of antiretroviral therapy after chemotherapy, the CD4+ cells recovered by 12 months and the viral loads decreased below baseline by 3 months. Compared with HIV-negative cases, the ARL cases had lower bcl-2 and higher CD10 expression, consistent with a germinal center origin and good prognosis, but were more likely to be highly proliferative and to express p53, adverse features with standard chemotherapy. Unlike HIV-negative cases, p53 overexpression was not associated with a poor outcome, suggesting different pathogenesis. High tumor proliferation did not correlate with poor outcome and may partially explain the high activity of dose-adjusted EPOCH. The results suggest that the improved immune function associated with highly active antiretroviral therapy (HAART) may have led to a shift in pathogenesis away from lymphomas of post–germinal center origin, which have a poor prognosis. These results suggest that tumor pathogenesis is responsible for the improved outcome of ARLs in the era of HAART.

Hypercalcemia is one of the most frequent and serious complications in patients with adult T-cell leukemia (ATL) and is due to marked bone resorption by accumulation of osteoclasts (OCLs). Although several cytokines such as interleukin 1 and parathyroid hormone–related protein are thought to be involved in the development of high serum Ca++ levels, its precise underlying mechanism remains unknown. This study analyzed the expression of various genes that are thought to regulate serum Ca++ levels in ATL and showed that the overexpression of the receptor activator of nuclear factor κB (RANK) ligand gene correlated with hypercalcemia. ATL cells from patients with hypercalcemia, which highly expressed the transcripts of the RANK ligand (RANKL) gene, induced the differentiation of human hematopoietic precursor cells (HPCs) into OCLs in vitro in the presence of macrophage colony-stimulating factor (M-CSF). In contrast, ATL cells from patients without hypercalcemia did not induce such differentiation, suggesting that the induction of the differentiation correlated with the expression of the RANKL gene in ATL cells. Cell differentiation was suppressed by osteoprotegerin/Fc, an inhibitor of RANKL, indicating that such differentiation occurred through the RANK-RANKL pathway. In addition, direct contact between ATL cells and HPCs was essential for the differentiation, suggesting that not the soluble form but membrane-bound RANKL played a role in this process. These results strongly suggested that ATL cells induce the differentiation of HPCs to OCLs through RANKL expressed on their surface, in cooperation with M-CSF, and ultimately cause hypercalcemia.

Acquired mutations of the PIG-A gene result in the hemolysis characteristic of paroxysmal nocturnal hemoglobinuria (PNH). Although the etiology of the mutation(s) is unclear, mutable conditions have been suggested by the coexistence of multiple clones with different mutations of PIG-A and by the appearance of leukemic clones in patients with PNH. This study sought to test this hypothesis by examining the frequency of hypoxanthine-guanine phosphoribosyl transferase (HPRT)gene mutations, identified by both resistance to 6-thioguanine (6-TG) and gene analysis. T-cell colonies resistant to 6-TG formed in methylcellulose culture were found in 8 (67%) of 12 PNH patients and 3 (18%) of 17 age-matched healthy volunteers (P

Normal B-lymphocyte maturation and proliferation are regulated by chemotactic cytokines (chemokines), and genetic polymorphisms in chemokines and chemokine receptors modify progression of human immunodeficiency virus-1 (HIV-1) infection. Therefore, 746 HIV-1–infected persons were examined for associations of previously described stromal cell-derived factor 1 (SDF-1) chemokine and CCR5 and CCR2 chemokine receptor gene variants with the risk of B-cell non-Hodgkin’s lymphoma (NHL). The SDF1-3′A chemokine variant, which is carried by 37% of whites and 11% of blacks, was associated with approximate doubling of the NHL risk in heterozygotes and roughly a fourfold increase in homozygotes. After a median follow-up of 11.7 years, NHL developed in 6 (19%) of 30 SDF1-3′A/3′A homozygotes and 22 (10%) of 202 SDF1-+/3′A heterozygotes, compared with 24 (5%) of 514 wild-type subjects. The acquired immunodeficiency syndrome (AIDS)-protective chemokine receptor variant CCR5-▵32 was highly protective against NHL, whereas the AIDS-protective variant CCR2-64I had no significant effect. Racial differences in SDF1-3′A frequency may contribute to the lower risk of HIV-1–associated NHL in blacks compared with whites. SDF-1 genotyping of HIV-1–infected patients may identify subgroups warranting enhanced monitoring and targeted interventions to reduce the risk of NHL.

PU.1 has previously been shown to be down-regulated in classical Hodgkin lymphoma (cHL) cells via promoter methylation. We performed bisulfite sequencing and proved that the promoter region and the -17 kb upstream regulatory element of the PU.1 gene were highly methylated. To evaluate whether down-regulation of PU.1 is essential for the growth of cHL cells, we conditionally expressed PU.1 in 2 cHL cell lines, L428 and KM-H2. Overexpression of PU.1 induced complete growth arrest and apoptosis in both cell lines. Furthermore, in a Hodgkin lymphoma tumor xenograft model using L428 and KM-H2 cell lines, overexpression of PU.1 led to tumor regression or stable disease. Lentiviral transduction of PU.1 into primary cHL cells also induced apoptosis. DNA microarray analysis revealed that among genes related to cell cycle and apoptosis, p21 (CDKN1A) was highly up-regulated in L428 cells after PU.1 induction. Stable knockdown of p21 rescued PU.1-induced growth arrest in L428 cells, suggesting that the growth arrest and apoptosis observed are at least partially dependent on p21 up-regulation. These data strongly suggest that PU.1 is a potent tumor suppressor in cHL and that induction of PU.1 with demethylation agents and/or histone deacetylase inhibitors is worth exploring as a possible therapeutic option for patients with cHL.

PU.1 is an Ets family transcription factor, which is necessary for differentiation of both myeloid and lymphoid lineages. It was previously reported that conditional knockout of the upstream regulatory element (URE) located in 14 kb 5' of the PU.1 gene resulted in down-regulation of PU.1 expression in granulocytes and B lymphocytes by 80% compared to that of wild type and induced acute myeloid leukemia and CLL-like diseases in mice. Since the URE contains a suppressor region for PU.1 expression in T cells, such mice express PU.1 in T cells and develop T cell lymphoma. Thus, the failure of proper expression of PU.1 in certain differentiation stages in certain cell lineages appears to result in hematological malignancies. We previously reported that PU.1 is down-regulated in various myeloma cell lines. In addition, PU.1 is expressed in normal plasma cells and PU.1 is down-regulated in myeloma cells of certain myeloma patients, who appear to have poor prognosis. In those myeloma cell lines, the promoter and URE of the PU.1 gene are highly methylated. A demethylation agent, 5-aza-2'-deoxycytidine, induced PU.1 up-regulation, growth arrest, and apoptosis in myeloma cell lines, KMS12PE and KHM11. In addition, conditionally expressed PU.1 induced cell growth arrest and apoptosis in PU.1-low-negative myeloma cell lines, U266 and KMS12PE, suggesting that PU.1 is a tumor suppressor for myeloma cells. To elucidate the mechanisms of the cell growth arrest and apoptosis in myeloma cells induced by PU.1, we performed DNA microarray analysis to compare gene expression levels before and after PU.1 expression. Among cell-cycle related genes, p21WAF1/CIP1 was found up-regulated in U266 cells, while among apoptosis related genes, TRAIL was highly up-regulated in both U266 and KMS12PE cell lines. With further investigation, we concluded that PU.1 directly transactivated the TRAIL gene in myeloma cells, leading to apoptosis. Based on the DNA microarray data generated, we found that IRF4 is downregulated in U266 myeloma cells after PU.1 induction. It has been reported that knockdown of IRF4 induces apoptosis in myeloma cell lines. Therefore, we examined whether IRF4 was down-regulated in three myeloma cell lines, U266, KMS12PE, and KHM11 following PU.1 induction. Conditional expression of PU.1 by tet-off system induced IRF4 down-regulation in U266and KMS12PE cells. With lentiviral transduction method, ectopic expression of PU.1 also induced IRF4 down-regulation, cell-cycle arrest, and apoptosis in KHM11 cells. To investigate the role of IRF4 in PU.1-expressing U266 cells, we stably expressed IRF4, partially rescuing U266 cells from apoptosis. IRF4 is known to directly bind to the IRF7 promoter and down-regulate IRF7 expression in activated B cell-like (ABC) subtype of diffuse large B-cell lymphoma cells. Therefore, we examined whether IRF4 bound to the IRF7 promoter in KMS12PEand U266cells using chromatin immunoprecipitation assays. We found that IRF4 directly bound to the IRF7 promoter in both myeloma cell lines. When we overexpressed PU.1, IRF4 levels were decreased and the IRF4 binding to the IRF7 promoter was significantly reduced in those cell lines. Moreover, knockdown of IRF7 significantly rescued PU.1-expressing U266cells from apoptosis. These data strongly suggest that PU.1-induced apoptosis is associated with IRF4 down-regulation and subsequent IRF7 up-regulation in myeloma cells. Since IRF4 is essential transcription factor for myeloma cell survival, up-regulation of PU.1 by demethylation agents, including 5-aza-2'-deoxycytidine may serve as a promising therapeutic modality of multiple myeloma by inducing down-regulation of IRF4. Disclosures No relevant conflicts of interest to declare.

PU.1 is an essential transcription factor for hematopoiesis and important for differentiation of both myeloid and lymphoid lineages. In mice conditionally knocked-out of 3.4 kb length of the enhancer region located in14 kb 5’ upstream of the PU.1 gene (URE), PU.1 is down-regulated in myeloid cells and B cells by 20% of that of wild type, and such mice develop acute myeloid leukemia and CLL-like diseases. These data strongly suggest that PU.1 has tumor suppressor activity in hematopoietic cells. We previously reported that human PU.1 is down-regulated in the majority of myeloma cell lines through the methylation of the promoter and the 17 kb upstream enhancer region (URE) of the PU.1 gene that is homologous to that in 14 kb 5’ upstream of the murine PU.1 gene. Conditionally expressed PU.1 with tet-off system induced cell growth arrest and apoptosis in two myeloma cell lines, KMS12PE and U266, suggesting that the down-regulation of PU.1 is necessary for myeloma cell growth. We have also reported that PU.1 is expressed in normal plasma cells and in contrast, PU.1 is down-regulated in primary myeloma cells from a subset of myeloma patients, who appear to have poor prognosis. In the present study, to test whether PU.1 has tumor suppressor activity in vivo, we generated xenograft mouse models. 0.6 - 1 x 107 KMS12PE cells were subcutaneously injected in 16 immunodeficient mice (Rag2-/- Jak3-/- bulb/c). The mice were then administered doxycycline through drinking water. Half of the mice (N=8) stopped taking doxycycline when the tumor sizes reached 1 cm in diameter, whereas the other half (N=8) kept taking doxycycline. Although the tumors in the mice taking doxycycline continued to grow, the tumor growth in the mice not taking doxycycline significantly slowed down. Flow cytometry analysis of the tumors in the mice that stopped taking doxycycline revealed that the cells from the tumor had completely lost PU.1 expression. Moreover, when U266 cells conditionally expressing PU.1 were subcutaneously injected to another 10 mice and the same experiment was conducted, although the tumors in the mice taking doxycycline (N=5) kept growing, the tumors in the mice not taking doxycycline (N=5), did not grow any further. The present data suggest that PU.1 serves as a tumor a suppressor in the multiple myeloma cell lines as examined in vivo. Disclosures No relevant conflicts of interest to declare.

[Introduction] Diagnosis of AL amyloidosis is dependent on the proof of light chains in amyloid lesions. However, immunostaining does not always successfully prove the presence of light chains in lesions in AL amylidosis patients. Here we report that the constant region of immunoglobulin lambda light chain (IGLC2) is seen in amyloid lesions where no positive signals are found with regular immunostaining. [Materials and Methods] Amyloid samples were stained with anti-human lambda light chain antibody (DAKO PO-0130) and analyzed with mass-spectrometry combining laser micro-dissection. Bone marrow samples were obtained from patients with amyloidosis, who gave written informed consent, and were subjected to plasma cell purification using CD138-immunomagnetic beads. Expression of immunoglobulin light chain mRNA was examined with RT-PCR. Anti-human IGLL5 antibody, capable of detecting immunoglobulin light chain constant region 2 (IGLC2) in paraffin embedded samples, was utilized. [Results and Discussion] We performed immunostaining for immunoglobulin light chains with 18 samples and found that six and eight cases were positive for kappa and lambda light chains, respectively, whereas light chains were not detected in remaining four cases (immunostaining-negative amyloidosis; INA). However, interestingly, mass spectrometry analysis revealed the presence of IGLC2 in all of the INA cases. RT-PCR analysis revealed the presence of IGLC2 mRNA in plasma cells from such INA cases. Surprisingly, amyloid lesions in all of the INA cases were positively stained with anti-IGLL5 antibody, whereas no staining was found in other samples positively stained with DAKO PO-0130. These observations suggest that the deposition of IGLC2 may cause AL amyloidosis, which otherwise could not be diagnosed with regular immunostaining. Although high dose chemotherapy produced hematological remission, half of such cases died within one year, suggesting irreversible and life-threatening amyloid fibril depositions in critical organs in IGLC2-related cases. We further examined additional twelve cases with AL amyloidosis to determine the incidence of IGLC2-related amyloidosis by immunostaining. With regular immunostaining, kappa and lambda chain were found in three and five cases, respectively. Interestingly, the remaining four cases were negative with regular immunostaining but positive with anti-IGLL5 antibody. Taken these observations together, eight IGLC2-related amyloidosis cases and thirteen lambda type amyloidosis were identified. Thus, the incidence of IGLC2-related amyloidosis should be approximately 38% (8/21) among lambda type AL amyloidosis. We conclude that diagnosis of IGLC2-related AL amyloidosis was possible only with the use of anti-IGLL5 antibody, but not with regular immunostaining. Given the relatively high incidence and often poor prognosis of IGLC2-related amyloidosis, it is important that this clinical entity is recognized to potentially improve outcomes of treatments. Analysis of mechanisms regulating amyloid formation with IGLC2 peptides is currently underway. Disclosures No relevant conflicts of interest to declare.

PU.1 is an Ets family transcription factor, which is essential for differentiation of both myeloid and lymphoid lineages. It was previously reported that conditional knockout of the upstream enhancer region (URE) located in 14 kb 5’ of the murine PU.1 gene resulted in down-regulation of PU.1 expression in granulocytes and B lymphocytes by 80% compared to that of wild type and induced acute myeloid leukemia and CLL-like diseases in mice. Therefore, down-regulation of PU.1 in myeloid and B cell lineages results in hematological malignancies. We previously reported that PU.1 is down-regulated in 5 out of 7 myeloma cell lines as well as primary myeloma cells from a subset of myeloma patients; that the promoter and the URE located in 17 kb 5’ of the human PU.1 gene that is homologous to that in 14 kb 5’ of murine PU.1 gene are highly methylated in these cell lines; and that conditionally expressed PU.1 with tet-off system induces cell growth arrest and apoptosis in myeloma cell lines, U266 and KMS12PE, suggesting that the down-regulation of PU.1 is necessary for myeloma cell growth. Here, to evaluate tumor suppressor activity of PU.1 in mature B and plasma cells in vivo, we generated Cγ1-Cre PU.1 knockout mice by crossing Cγ1-Cre and PU.1-loxP mice. We confirmed that PU.1 alleles were both conditionally deleted in the maturation stages of B cells from post germinal center B to plasma cells. By 18-24 months of age, about 77.7% (10 of 13) of the knockout mice had developed serum M proteins. To induce B cell differentiation to plasma cells, those mice were immunized with NP-CGG and 76.9% (20 of 26) of the mice developed serum M protein. ELISA of sera from those mice revealed that IgG was not elevated compared to those from the PU.1-loxP mice, which was thought because Cγ1-Cre locus fails to produce IgG1. Instead, a small number (5 of 20) of the mice showed relatively large amounts of IgM and/or IgA. When 11 such mice were sacrificed, 7 had developed splenomegaly and/or intestinal B cell lymphoma. Immunostaining revealed that B220+ cells had infiltrated into the tumors and various organs including the spleen, liver, and bone marrow. Those cells were monoclonal for κ chain and partly CD138 positive. When we transplanted those tumor cells into Rag2-/- Jak3-/- immunedeficient mice, all the mice died within 3 weeks. Thus, PU.1 apparently functions as a tumor suppressor in mature B cells and its deletion in late B cell maturation stages produces B cell lymphoma with M proteinemia. The remaining 4 mice developed high titer IgM and/or IgA levels and flow cytometry of bone marrow cells and splenocytes revealed that those cells were monoclonal for κ chain and positive for B220 and IgM and/or IgA, suggesting that those mice suffered from multiple myeloma or monoclonal gammopathy with undetermined sighnificance (MGUS). These data strongly suggest that conditional knockout of PU.1 in post germinal center B and plasma cells results in B cell lymphoma and plasma cell neoplasms related to multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Opportunistic infections frequently occur in patients with adult T-cell leukemia (ATL) and human T-cell leukemia virus type I (HTLV-I) carriers. However, the underlying mechanisms of such infections remain unknown. To clarify the mechanism of immunodeficiency in those infected with HTLV-I, this study analyzed the T-cell subsets in HTLV-I carriers and patients with HTLV-I–associated myelopathy/tropical spastic paraparesis and ATL using 3-color fluorescence with CD62L and CD45RA coexpression either with CD4+ or CD8+ T cells. The number of naive T lymphocytes was markedly suppressed in patients with ATL, particularly in those with acute form, compared with uninfected control individuals. The number of naive T cells was low in HTLV-I–infected individuals under 50 years old compared with uninfected individuals, whereas the number of memory T lymphocytes was greater in HTLV-I–infected individuals. Although the increase of memory T lymphocytes correlated with HTLV-I provirus loads, no relationship was found between naive T-cell counts and provirus loads. T-cell receptor rearrangement excision circles (TRECs), which are generated by DNA recombination during early T lymphopoiesis, were quantified to evaluate thymic function in HTLV-I–infected individuals. TREC levels were lower in HTLV-I–infected individuals than in uninfected individuals. In HTLV-I carriers less than 70 years old, an increase of Epstein-Barr virus DNA in peripheral blood mononuclear cells was observed in 6 of 16 (38%) examined, whereas it was detectable in only 1 of 11 uninfected controls. These results suggested that the low number of naive T lymphocytes was due to suppressed production of T lymphocytes in the thymus, which might account for immunodeficiency observed in HTLV-I–infected individuals.

Normal B-lymphocyte maturation and proliferation are regulated by chemotactic cytokines (chemokines), and genetic polymorphisms in chemokines and chemokine receptors modify progression of human immunodeficiency virus-1 (HIV-1) infection. Therefore, 746 HIV-1-infected persons were examined for associations of previously described stromal cell-derived factor 1 (SDF-1) chemokine and CCR5 and CCR2 chemokine receptor gene variants with the risk of B-cell non-Hodgkin's lymphoma (NHL). The SDF1-3'A chemokine variant, which is carried by 37% of whites and 11% of blacks, was associated with approximate doubling of the NHL risk in heterozygotes and roughly a fourfold increase in homozygotes. After a median follow-up of 11.7 years, NHL developed in 6 (19%) of 30 SDF1-3'A/3'A homozygotes and 22 (10%) of 202 SDF1-+/3'A heterozygotes, compared with 24 (5%) of 514 wild-type subjects. The acquired immunodeficiency syndrome (AIDS)-protective chemokine receptor variant CCR5-triangle up32 was highly protective against NHL, whereas the AIDS-protective variant CCR2-64I had no significant effect. Racial differences in SDF1-3'A frequency may contribute to the lower risk of HIV-1-associated NHL in blacks compared with whites. SDF-1 genotyping of HIV-1-infected patients may identify subgroups warranting enhanced monitoring and targeted interventions to reduce the risk of NHL.

Introduction It has been reported that the growth of certain cancer cells depends on the Warburg effect (aerobic glycolysis) for such cells to adapt to hypoxic environment and obtain ATP efficiently via glycolysis. We have previously reported that genes related to aerobic glycolysis are up-regulated in MM cells, and MM cells produce large amounts of lactate (Br J Cancer 2013, 108, 170-8). In the last ASH meeting, we reported that the expressions of lactate transporters were critical for the survival of MM cells. In this study, we further investigated the roles of lactate transporters in MM cells, focusing on the role of stromal cells as the supplier of lactate in microenvironment. Methods Six MM cell lines, RPMI8226, U266, KMS12BM, KMS12PE, KHM11, and KMM1 were utilized. Bone marrow samples were obtained from newly diagnosed MM patients. Bone marrow mononuclear cells were isolated by Ficoll-Hypaque density sedimentation. To obtain stromal cells, bone marrow mononuclear cells were cultured in RPMI1640 medium supplemented with 10% FCS and allowed to attach to the bottom of plastic wells. After 3-4 weeks incubation, adherent cells were harvested and utilized as stromal cells. The expression of three components of the lactate transporter complex [monocarboxyl transporter 1 (MCT1), MCT4, and CD147] were analyzed with western blotting or flow cytometry. Lactate uptake was quantified using [14C]-Lactate. Cellular ATP production was quanatified with the ATP Determination Kit (Molecular Probes). An inhibitor of MCT1, a-cyano-4 hydroxycinnamic acid (CHC), was utilized to analyze cytotoxic effects on MM cells under both normoxia ( O2 20% ) and hypoxia (O2 1% ). AnnexinV/PI staining was performed to quantify cytotoxicity. Knockdown of MCT1 was achieved using siRNA. Results Knockdown of MCT1, a molecule responsible for lactate incorporation into cytoplasm, induced apoptosis in MM cells, while lactate uptake and ATP production of MM cells were reduced, suggesting that MCT1 inhibition might induce apoptosis in MM cells by decreasing the lactate-derived ATP production. On the other hand, we found significant lactate production by stromal cells. We also found an abundant amount of MCT4, a molecule responsible for lactate excretion, in bone marrow-derived stromal cells. By contrast, lactate amount in the culture supernatant of stromal cells was significantly decreased when they were co-cultured with MM cells. Therefore, it appears that lactate produced by stromal cells is incorporated into MM cells as an energy source. Interestingly, we found that the expression of MCT1 is up-regulated in MM cells under an aerobic condition, while that of MCT4 is up-regulated under a hypoxic condition. The treatment of MM cells with a-cyano-4 hydroxycinnamic acid (CHC), a MCT1 inhibitor, induced apoptosis in MM cells, suggesting that lactate may be more preferentially incorporated into MM cells under a normoxic than hypoxic condition (Figure A). Conclusion Our results suggest that the growth of MM cells, at least in part, depends on lactate under a normoxic condition. The data also suggest that lactate may play a role as an energy interplay shuttle between stromal cells and MM cells (Figure B). Therefore, regulating lactate incorporation by MM cells may provide a new avenue for a therapeutic strategy for multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

Aims Mutations of the genes associating with cell differentiation or proliferation are recognized as factors of tumorigenesis or prognosis in hematological malignancies. In pediatric acute lymphoblastic leukemia (ALL), alterations of IKZF1 (a factor of lymphocyte differentiation), TP53 (a cell cycle regulator) and CREBBP (a histone modifier) are found as possible prognostic markers for stratification of treatments. On the other hand, in adult ALL, clinical significance of such alterations remains to be determined. In the present work, we examined whether the mutations in those genes affected the incidence and prognosis in adult ALL patients. Methods We investigated 87 adult patients with newly diagnosed ALL treated with JALSG protocols between 1986 and 2011. Age ranged from 15 to 86 years, with a median of 51 years. We obtained cDNA and genomic DNA from the peripheral blood or bone marrow mononuclear cells at diagnosis. CREBBP mutations are dominantly identified in the histone acetyltransferase (HAT) domain. HAT domain in the CREBBP gene was amplified by PCR using cDNA and was subjected to direct sequencing. Additionally other histone modifiers, EZH2, EED, and UTX, were sequenced as the same as in CREBBP. TP53 exons 5 – 8 and 10, in which mutations were commonly reported, were sequenced using genomic DNA. We amplified IKZF1 using RT-PCR for detecting aberrant dominant negative isoforms: Ik6 and Ik10. Genomic deletions of IKZF1 were assessed with RQ-PCR or genomic DNA PCR. We compared clinical profiles between patients with and without such gene mutations. The present study was approved by the Institutional Review Boards and informed consent was obtained from each patient according to guidelines based on the revised Declaration of Helsinki. Results In 87 adult patients with ALL, alterations of CREBBP, EED, TP53 and IKZF1 were detected in 7 (9.5%), 3 (4.8%), 6 (6.9%) and 42 (50%), respectively. None of EZH2 and UTX mutation was found. The alterations of CREBBP and IKZF1 at diagnosis in adult patients were more frequent than those in pediatric patients ever reported. Some gene mutations were not found frequently. Each gene mutation per se did not significantly affect prognosis. We tried to predict the prognosis by scoring gene mutations and chromosomal abnormalities. Philadelphia chromosome (Ph) has great impact to prognosis of patients with ALL. We scored the number of mutated genes and Ph for each patient. As the score was higher, adult patients with ALL had poorer relapse-free survival (P=0.0439) and OS (P=0.4819), but statistical significance was not detected in this small cohort. Conclusions and Discussion Single gene mutations, such as IKZF1, can predict the prognosis in pediatric ALL. In adult ALL, however, only few gene mutations are reported to be promising prognostic factors which have impacts to treatment outcomes. Scoring system may be a useful method for predicting prognosis and stratifying treatment in adult ALL. Our study implies the possibility that a variety and heterogeneity of genetic alterations in adult ALL are associated with the pathogenesis for treatment resistance and prognostic marker of adult ALL. Disclosures: No relevant conflicts of interest to declare.

Purpose Recurrent mutations in the genes coding the isocitrate dehydrogenases IDH1 and IDH2 have recently been identified in patients with acute myeloid leukemia (AML). However, the prognostic importance of IDH1 and IDH2 mutations is not consistent among several studies. In the present study, we examined the incidence and prognostic impacts of IDH1 and IDH2 mutations in Japanese adults with AML. Patients and Methods A total of 233 adults with AML were subjected to the study. Age ranged from 15 to 86 years, with a median of 57.0 years. Patients with aged 69 or less were treated with the protocols of Japan Adult Leukemia Study Group (JALSG). Patients with aged 70 or more were treated with low dose cytarabine and aclarubicin in combination with granulocyte colony-stimulating factor (G-CSF) (Suzushima et al. Leuk Res 2010). DNA was extracted from bone marrow or peripheral blood mononuclear cells of AML patients at diagnosis and subjected to PCR amplification and direct sequencing of the IDH1 and IDH2 genes. NPM1, FLT3 and CEBPA gene mutations were also analyzed. The study was approved by the Institutional Review Boards and informed consent was obtained from each patient according to guidelines based on the tenets of the revised Declaration of Helsinki. Results IDH1 R132 mutations were detected in 20 (8.6%) patients with AML. IDH2 mutations were found in 19 (8.2%; 17 R140 and two R172) patients. IDH1 and IDH2 mutations were mutually exclusive and were most frequent in the cytogenetic intermediate-risk group. In addition, IDH1 and IDH2 mutations were mutually exclusive with the CEBPA mutation. Moreover, IDH1 and IDH2 mutations were associated with NPM1 mutations. Of 39 patients with IDH mutations, 17 (44%) had the NPM1 mutation, whereas 36 of 194 (19%) lacking an IDH mutation had an NPM1 mutation (P = 0.001). There was no significant correlation of the IDH mutation with FLT3-ITD. Of 226 patients evaluable for treatment outcome, there was no significant difference in 5-year overall survival (OS) between patients with and those without IDH1 mutations (25.1% vs. 29.9%, P = 0.3089). In contrast, 5-year OS rates were significantly lower (6.2%) in patients harboring the IDH2 mutation than in patients lacking the IDH2 mutation (31.5%) in the entire cohort of AML (P = 0.0026). Because IDH1 and IDH2 mutations were associated with NPM1 mutations, we compared OS in patients with and those without IDH1 or IDH2 mutations among patients with and those without NPM1 mutations. Of 51 patients with NPM1 mutations, there was no significant difference in 5-year OS between patients with and those without the IDH1 or IDH2 mutation. In contrast, among 175 patients lacking the NPM1 mutation, 5-year OS in patients with IDH1 or IDH2 mutations was significantly lower than in those without such mutations (0% vs. 32.4%, P = 0.0023 and 0% vs. 32.6%, P = 0.0001, respectively). Moreover, among patients with the wild-type of NPM1, 5-year OS in patients with IDH1 or IDH2 mutations was significantly lower than that in those with the CEBPA mutation, FLT3-ITD, or the wild-type of CEBPA, FLT3, IDH1 and IDH2 (P=0.0002). Conclusions Frequent mutations in IDH1 and IDH2 are also found in Japanese adults with AML. Our data strongly suggest that IDH1 and IDH2 mutations confer adverse prognostic effect in NPM1 wild-type AML. On the other hand, an adverse effect of IDH1 and IDH2 mutations may be negated among patients with NPM1 mutations, because NPM1 mutations generally have a higher complete remission rate and more favorable OS. Disclosures: No relevant conflicts of interest to declare.

Background Despite of recent advances in therapeutic strategy for multiple myeloma (MM), MM still remains incurable and novel therapeutic approach is urgently needed. We have previously demonstrated that a natural small molecule, shikonin (SHK), induced both apoptosis and necroptosis (programmed necrosis) in MM cells. In this study, we attempted to elucidate biological mechanisms of SHK in inducing apoptosis and necroptosis. Methods Six MM cell lines, KMS-12-PE, RPMI 8226, U266, KMM1, KMS-11 and a bortezomib-resistant MM cell line, KMS-11/BTZ (obtained from Kyowa Hakko Kirin Co. Ltd.), were utilized. Inhibitors of pan-caspase and necroptosis, ZVAD-fmk and Nec-1 (necrostatin-1), were employed to distinguish apoptosis and necroptosis, respectively. Cell death was analyzed using the trypan blue dye exclusion method (WST-8 assay) and flow cytometry analysis using AnnexinV/PI staining. Morphological examinations of cells were performed with May Giemza staining. Caspases, RIP1, ubiquitinated proteins, and heat shock proteins were analyzed with western blot. Knockdown of RIP1, an essential molecule for necroptosis, was performed using siRNA. ER stress was assessed by detecting activated XBP-1, which was analyzed by digestion of PCR products with ApaLI. Because the ApaLI site in XBP-1 mRNA is spliced out upon activation, the activated XBP-1 shows one large band after ApaLI digestion, while inactivated XBP-1 shows two ApaI-digested bands. Thapsigargin, sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, was used as an ER stress inducer. Results By screening natural compounds libraries (provided by Institute of Natural Medicine, Toyama University, Japan), we found that SHK, a natural compound derived from the root of Lithospermum erythrorhizon, induced cell death in MM cells. Apoptosis was induced at a relatively low concentration (2.5∼5 µM) and was inhibited by a caspase-inhibitor, while necroptosis was promptly induced at higher concentrations (10∼ 20 µM) within 5 hours and was completely inhibited by Nec-1. Morphological analysis showed that SHK at low concentrations induced typical apoptotic changes, such as fragmented nucleus, while SHK at higher concentrations induced necrotic morphology, such as translucent cytoplasm and swelling of cell membranes. By contrast, SHK did not induce apoptosis or necrptosis in peripheral blood mononuclear cells from healthy donors at low concentrations. SHK activated caspase-8 and -3 at low concentrations but did not at higher concentrations. RIP1, an essential molecule for necroptosis, was cleaved after treatment with SHK at low concentrations, which leads to the inhibition of necroptosis, while it was not cleaved and remained active at higher concentrations, suggesting that SHK dynamically regulates the cleavage of RIP-1. At low concentrations, shikonin induced an accumulation of ubiquitinated proteins and activated XBP-1, suggesting SHK may have a property of proteasome inhibitor eventually inducing endoplasmic reticulum stress. Finally, SHK at low concentration killed bortezomib resistant cells with lower IC50 comparing to that of the parental cells (0.91 vs 1.56 µM, respectively). Conclusions We here report, for the first time, that SHK induces apoptosis and necroptosis in MM cells at low and high concentrations, respectively, by regulating proteasome function and RIP-1 cleavage. Given the fact that SHK efficiently induces cell death in bortezomib-resistant cell line, SHK may act as a novel proteasome inhibitor for bortezomib-resistant myeloma cells. Moreover, SHK at higher concentrations, which induces nectoptosis, should be an attractive future therapeutic option potentially to eradicate MM cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 3956 Introduction: Although CD138 expression is a hallmark of plasma cells and myeloma cells, decreased expression of CD138 is occasionally found. We previously reported in the last ASH meeting that 1) CD138 expression decreases in patients with relapsed/progressive disease compared with untreated MM patients and that 2) Patients with low levels of CD138 expression had a worse overall survival compared with patients with high levels of CD138 expression. However, the mechanisms of CD138 down-regulation in myeloma cells are still unclear. It is known that myeloma patient's bone marrow environment is hypoxic (Colla et al. Leukemia. 2010; 24: 1967–1970). It is also reported that tumor progression delivers hypoxic environment to MM cells in vivo (Azab et al. Blood. 2012; 119: 5782–5794). Based upon our and other reports, we hypothesized that CD138 expression may be down regulated by hypoxia. In the present study, we examined changes of CD138 and transcription factor expression in myeloma cells under hypoxic condition. Materials and methods: Two myeloma cell lines (RPMI 8226 and KMS-12-BM) were cultured under normoxic (20% O2) and hypoxic (1% O2) conditions for 24 hrs to 72 hrs. CD138 expression of these cell lines under normoxic and hypoxic conditions were analyzed by flow cytometry. Only viable cells were analyzed by excluding 7-AAD positive dead cells. Real time RT-PCR analysis was utilized to analyze gene expression between normoxic and hypoxic cells. Amounts of IRF4 at protein level were determined with western blotting and flow cytometry. Results: CD138 expression was found to decrease under hypoxic condition compared to normoxic condition in 24 hrs in culture of RPMI 8226 and in 48 hrs in KMS-12-BM when examined with flow cytometry. Real time RT-PCR analysis revealed that CD138 expression was down-regulated under hypoxic condition, indicating that CD138 was down-regulated at transcriptional level. Gene expressions of IRF4, PRDM1 and XBP1, known as plasma cell specific transcription factors, were down-regulated under hypoxic condition compared to those under normoxic condition. Western blot and flow cytometry analysis showed IRF4 was also down-regulated under hypoxic condition. Interestingly, the decreased CD138 expression rendered under hypoxic condition recovered when they were placed under normoxic condition along with an increase of IRF4 expression. Conclusions: We conclude that hypoxia induces down regulation of CD138 in myeloma cells, accompanying with decreased expressions of IRF4, PRDM1 and XBP1. The present data together with our previous finding that reduced CD138 expression is frequently seen in patients with relapsed/progressive diseases suggest that the microenvironment of myeloma cells in patients with relapsed/progressive diseases may be more hypoxic comparing to that at diagnosis. Since IRF4 is a master transcription factor required for the maturation of plasma cells, the hypoxia-induced down-regulation of IRF4 suggests phenotypic shift from mature to immature state, which resembles to the report demonstrating a shift of disease status by hypoxia to more aggressive and immature phenotypes in solid tumors (Axelson et al. Semin Cell Dev Biol. 2005; 16: 554–563). Since IRF4 is also important as a target of IMiDs, further analysis of CD138-negative myeloma cells might contribute not only to better understanding of disease progression but also to drug resistance mechanisms. Disclosures: No relevant conflicts of interest to declare.

Abstract 1821 Introduction It has been reported that cancer cells utilize glycolysis pathway (non-oxidative breakdown of glucose) even in the presence of adequate oxygen to provide cancer cells with energy, called the Warburg effect (aerobic glycolysis) that ultimately leads to produce lactate. We reported in the last ASH meeting that aerobic glycolysis is up-regulated in multiple myeloma (MM) cells in patients with high serum LDH levels and aerobic glycolysis itself could serve as a novel therapeutic target in MM patients. Here we report an importance of lactate transporter for the growth and survival of MM cells. Lactate, produced from pyruvate by lactate dehydrogenase A (LDHA), is known as an important energy source for solid tumor cells and is associated with tumor angiogenesis and chemo-resistance (Pinheiro, C., et al. J Bioenerg Biomembr. 44:127–139, 2012). On the other hand, LDHB converts lactate to pyruvate, thus negatively regulating lactate production. It is known that lactate is pumped out through monocarboxylate trasnporter, MCT4, while MCT1 mainly imports lactate to inside of cells. However, roles of MCT1 and MCT4 in MM cells remain to be elucidated. We here investigated the roles of these two molecules in the growth and survival of MM cells. CD147, a purported chaperone protein for MCT1, was also examined. Methods Six MM cell lines, RPMI8226, U266, KMS12BM, KMS12PE, KHM11, and KMM1 were employed. Six genes associated with glycolysis, i.e., LDHA, LDHB, MCT1-4, were examined using real time PCR analysis. Expressions of MCT1 and MCT4 were analyzed with western blotting. Expression of CD147 was investigated by flow cytometry. Lactate production into culture supernatants of MM cell lines were analyzed by using a lactate analyzer. An inhibitor of MCT1, a-cyano-4 hydroxycinnamic acid (CHC), was utilized to analyze cytotoxic effects on MM cells. AnnexinV/PI stained cells was analyzed by flow cytometry to quantify cytotoxicity. MCT1-expression was inhibited by using siRNA. Dichroloacetate (DCA), an inhibitor of PDK1, was utilized for inhibiting glycolysis. Results Accumulation of lactate was found in the supernatants of MM cell lines as cell density increased. Transporters of lactate, MCT1, MCT4 and CD147, were found in most MM cell lines at various levels, suggesting that transportation of lactate occurs through membrane of MM cells. To examine the role of lactate as a growth promotion factor, lactate was exogenously supplemented to KMS-12-PE cells. Interestingly, expressions of MCT1 and LDHB genes increased by the addition of lactate while those of MCT4 and LDHA only moderately changed (Fig. 1), suggesting that lactate was imported to cells through MCT1, then converted to pyrvate by LDHB. These results raised a possibility that lactate is utilized by MM cells as a growth factor. To examine the possibility, CHC, an inhibitor of MCT1, was supplemented to MM cell cultures. Interestingly, CHC induced apoptosis in MM cells in a dose dependent manner (Fig. 2). Moreover, inhibition of MCT1 gene by siRNA showed significant induction of apoptosis (Fig. 3), strongly suggesting that MCT1 plays a crucial role for survival of MM cells. Finally, we found a significant increase in the apoptosis of MM cells when CHC and DCA were simultaneously added in the culture (Fig.4), suggesting that MCT1 functions independently from glycolysis per se and that CHC and DCA act additively in starving lactate within MM cells. Conclusion Our results suggest that lactate is actively transported through monocarboxylate transporters. Given the results that exogenous lactate production increased MCT1 and LDHB expression, lactate should play a role as a regulator of lactate transportation and glycolysis as well as an important energy source. Because we found significant amount of lactate was produced from stromal cells obtained from MM patients, lactate may be supplied not only from MM cells themselves but also from micro-environment. Our finding that inhibition of MCT1 leads to cell death suggests that MCT1 could be a potential novel target molecule in MM therapy that could be stratified in combination with glycolysis inhibitor. Disclosures: No relevant conflicts of interest to declare.

Abstract 1419 Aims: Molecular pathogenesis of acute lymphoblastic leukemia (ALL) has largely been verified in pediatric patients and the identification of genetic alterations have contributed to stratifying therapeutic applications. In adult patients with ALL, cytogenetic and genetic abnormalities have not sufficiently been elucidated and therapeutic improvement has been hindered. CREB binding protein (CREBBP) is a transcriptional coactivator that interacts with a diverse range of transcription factors and regulates transcription by histone acetylation in hematopoiesis. Mutations of the CREBBP gene are recently found in approximately 2–4% of pediatric patients with ALL. Especially in relapsed cases, the mutations prevail (18–63%) and are possible markers for prediction of relapse in pediatric ALL. In adult patients with ALL, the clinical significance of CREBBP mutations remains to be determined. Here we examined adult ALL patients in an attempt to determine the incidence, clinical characteristics and prognostic impact of the CREBBP mutations. Methods: We investigated 71 adult patients with newly diagnosed ALL treated with JALSG protocols between 1986 and 2010. Age ranged from 15 to 86 years, with a median of 54 years. CREBBP mutations are dominantly identified in histone acetyltransferase (HAT) domain. HAT domain in the CREBBP gene was amplified with RT-PCR using RNA isolated from the peripheral blood or bone marrow mononuclear cells at diagnosis and was subjected to direct sequencing. We compared clinical profiles between patients with and without CREBBPHAT domain mutations. This study was approved by the Institutional Review Boards and informed consent was obtained from each patient according to guidelines based on the revised Declaration of Helsinki. Results: CREBBP HAT domain mutations were detected in 8 of 71 (11.3%) patients: one nonsense mutation, five insertion mutations with frameshifts, and five missense mutations. Two patients harbored biallelic mutations. The mutations at diagnosis in adult patients were seen more frequently than those in pediatric patients ever reported. Such mutations were not completely identical to those detected in pediatric ALL, but were seen in the region within the HAT domain, indicating that such mutations are loss-of-function mutations. The mutations were found in both B-cell (6/53: 11.3%) and T-cell (1/9: 11.1%) ALL, and distributed in patients harboring IKZF1 alterations (3/31: 9.7%) or the BCR-ABL fusion gene (2/19: 10.5%). There were no statistical difference in age, sex, leukocyte, platelet counts and complete remission rate between patients with and without the CREBBP HAT domain mutations. Patients with the mutations had a trend with worse cumulative incidence of relapse (P=0.4637), relapse-free survival (P=0.4195) and OS (P=0.2349) compared to patients lacking the mutations, but statistical significance was not detected in this small cohort. Conclusions: CREBBP HAT domain mutations at diagnosis in adult ALL are found more frequently than in pediatric ALL. This may be one of the mechanisms that adult ALL has been associated with poor OS compared with pediatric ALL. In this study, CREBBP HAT domain mutations were observed in various subtypes of ALL: both B-cell and T-cell ALL, and both Philadelphia chromosome positive and negative ALL. In pediatric ALL, CREBBP mutations were frequently seen in relapsed patients but not in previously untreated patients. These observations suggest that CREBBP mutations play an important role in an additional late event(s) leading to the development and progression of ALL. Our study implies the possibility that mutations of the CREBBP gene are associated with the pathogenesis and prognostic marker of adult ALL and represent specific epigenetic modifiers in adult ALL, serving as potential therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

Abstract 2538 Aims: Among acute myeloid leukemia (AML) patients with intermediate-risk cytogenetics, C/EBPa mutations represent a distinct disease entity with a favorable clinical outcome and is adopted in the current WHO classification of AML as a provisional disease entity in the category AML with recurrent genetic abnormalities. CEBPA encodes a transcription factor that is essential for neutrophil development. AML patients with CEBPA mutations can be separated into two subgroups with a single mutation in the CEBPA (CEBPA sm) and double mutations (CEBPA dm). Biallelic mutations consisted of an N-terminal frameshift mutation and a C-terminal inframe bZIP mutation were detected in the majority of CEBPA dm, whereas CEBPA sm occurs in either N-terminal or C-terminal regions. More recent data indicate that favorable outcome is mainly observed in AML patients with CEBPA dm but not with CEBPA sm. In addition, concurrent gene mutations may occur more frequently in AML with CEBPA sm than in CEBPA dm. In contrast, transcription factor GATA2 mutations are frequently identified in AML with CEBPA dm. In this study, we examined incidence, concurrent gene mutations and clinical significance of CEBPA dm and CEBPA sm in Japanese adults with cytogenetically intermediate-risk AML. Methods: To identify the prevalence and prognostic impact of CEBPA dm and CEBPA sm, we examined 111 patients with intermediate-risk AML who were mainly treated with the JALSG protocols. Age ranged from 16 to 86 years, with a median of 58.5 years. DNA was extracted from bone marrow or peripheral blood mononuclear cells at diagnosis and subjected to PCR amplification and direct sequencing of the CEBPA, FLT3, NPM1, IDH1, IDH2, DNMT3A and GATA2 genes. This study was approved by the Institutional Review Boards and informed consent was obtained from each patient according to guidelines based on the revised Declaration of Helsinki. Results: Of 111 cytogenetically intermediate-risk AML, we found 12 (10.8%) CEBPA dm and 7 (6.3%) CEBPA sm. In 7 CEBPA sm, one NPM1 mutation and one FLT3-ITD were detected. Two FLT 3-ITD and no concurrent mutation of NPM1 were found in CEBPA dm. No mutation in the IDH1, IDH2, DNMT3A exon 23 was identified in both patients with CEBPA sm and CEBPA dm. On the other hand, mutations in the GATA2 zinc finger domains were detected in 3 of 12 (25%) patients with CEBPA dm. No GATA2 mutations were found in 7 CEBPA sm. One of 21 patients with wild-type CEBPA (CEBPA wt) had a GATA2 mutation. Patients with CEBPA double or single mutations showed a better 5-year overall survival (OS) compared to CEBPA wt (51.3% vs 16.0%, P=0.0048). CEBPA dm AML was associated with a significant superior clinical outcome compared with CEBPA wt (5-year OS, 55.6% vs 16.0%, P=0.0025). However, no significant difference was identified between CEBPA dm and CEBPA sm AML (5-years OS, 55.6% vs 42.9%, P=0.1375) or between CEBPA sm and CEBPA wt AML (5-year OS, 42.9% vs 16.0%, P=0.4827). In addition, the presence of additional GATA2 mutations did not significantly influence the clinical outcome of AML patients with CEBPA dm. Conclusions: A total of 19 (17.1%) patients with cytogenetically intermediate-risk AML harbored CEBPA mutations. Our study indicates that the presence of the CEBPA dm but not CEBPA sm is associated with favorable outcome in Japanese patients with cytogenetically intermediate-risk AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 2519 Purpose: Recurrent mutations in the genes coding the isocitrate dehydrogenases, IDH1 and IDH2, have recently been demonstrated in patients with acute myeloid leukemia (AML). However, it remains to be determined how frequently IDH1 and IDH2 mutations occur in Asian AML patients and whether IDH1 and IDH2 mutations are associated with clinical profiles and outcome. In this study, we examined the incidence and prognostic impacts of IDH1 and IDH2 mutations in Japanese adults with AML. Patients and Methods: A total of 190 adults with AML were subjected to the study. Age ranged from 15 to 86 years, with a median of 57.0 years. Patients with aged 69 or less were treated with the protocols of Japan Adult Leukemia Study Group (JALSG). Patients aged 70 or more were treated with low dose cytarabine and aclarubicin in combination with granulocyte colony-stimulating factor (G-CSF) (Suzushima et al. Leuk Res 2010). DNA was extracted from bone marrow or peripheral blood mononuclear cells of AML patients at diagnosis and subjected to PCR amplification and direct sequencing of the IDH1 and IDH2 genes. NPM1, FLT3 and CEBPA gene mutations were also analyzed. The study was approved by the Institutional Review Boards and informed consent was obtained from each patient according to guidelines based on the tenets of the revised Declaration of Helsinki. Results: IDH1 R132 mutations were detected in 16 patients (8.4%). IDH2 mutations were found in 18 patients (9.5%; 17 with R140 and one with R172). IDH1 and IDH2 mutations had mutually exclusively occurred. IDH1 mutation was associated with higher platelet counts and was more frequent with M1 in FAB classification. IDH2 mutation was associated with ages, lower leukocyte counts and was more frequent with M0. Median age of patients with IDH2 mutation was 63.5 years, while that with wild type was 55.5 years (P=0.036). No IDH2 mutation was observed in patients aged 39 or less. Both IDH1 and IDH2 mutations were most frequent in cytogenetically normal AML and cytogenetic intermediate-risk group. Although IDH1 mutations were not significantly associated with mutations in NPM1 and FLT3, IDH2 mutations were associated with NPM1 mutations. Of 15 IDH2 mutations, 8 (53%) had NPM1 mutation, whereas 26 of 121 (21%) lacking IDH2 mutation had NPM1 mutation (P=0.012). Interestingly, both IDH1 and IDH2 mutations were mutually exclusive with CEBPA mutations. There was no significant difference in 5-year overall survival (OS) between patients with and without IDH1 mutation (32.6% vs. 26.7%, P=0.7377). In contrast, 5-year OS rates were 6.6% for patients harboring IDH2 mutation and 34.5% for patients lacking IDH2 mutation in the entire cohort of AML (P=0.0020). This unfavorable effect was also found in cytogenetically normal AML (P=0.0383). Of patients aged 59 or less, there was no significant difference in 5-year OS between IDH2 mutations (14.3%) and wild-type (43.3%) (P=0.1440). On the other hand, among patients aged 60 or older, 5-year OS rates were 0% for patients carrying IDH2 mutation and 21.6% for patients lacking IDH2 mutation (P=0.0100). Conclusions: Frequent mutations in IDH1 and IDH2 are also found in Japanese adults with AML. Our study indicates that IDH2 mutation has an unfavorable impact on the treatment outcome in adult patients with AML, especially in older (age ≥60 years) patients. These data suggest that IDH2 mutation confer a significant adverse impact on elderly patients with AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 1799 Introduction: A number of studies have shown that the high level of serum lactate dehydrogenase (LDH) serves as an indicator for poor prognosis in multiple myeloma (MM). LDH is a key enzyme for glycolysis converting pyruvate to lactate, which is eventually utilized as an energy source particularly in tumor cells. It has been reported that cancer cells utilize this glycolysis pathway even in the presence of adequate oxygen to provide cancer cells with energy, called the Warburg effect (aerobic glycolysis). Myc is known to regulate LDH and pyruvate dehydrogenase kinase 1 (PDK1), which are master regulators of glycolysis (Figure 1). Although myc is a well known gene expressed in MM cells, there has been no report analyzing its association with the glycolysis-regulating genetic system, which is located downstream to the myc gene, in MM cells. In the present study, we examined if the glycolysis system is directly or indirectly associated with the survival of MM cells. Methods: MM cells were purified from primary bone marrow samples from 54 patients using CD138-magnetic beads. Written informed consent was obtained from all cases. Seven MM cell lines, RPMI8226, U266, KMS12BM, KMS12PE, KHM11, KMM1 and KMS11, were employed. Five genes associated with glycolysis, i.e., c-MYC, GLUT1 (glucose transporter 1), LDHA (LDH-encoding gene), hypoxia induced factor-1 alpha (HIF1a) and PDK1, were examined using real time PCR analysis. Glucose consumption and lactate production in culture supernatants of MM cell lines were analyzed. Oxamate, a competitive inhibitor of LDHA, was utilized to quantify cytotoxic effects on MM cells. Cytotoxicity was evaluated with AnnexinV/PI staining. Results: Heterogeneous expression of LDHA gene was observed (Figure 2A). High LDHA mRNA expression levels significantly correlated with poor survival (Figure 2B, p Conclusion: Our results suggest that aerobic glycolysis (the Warburg effect) is up-regulated in MM cells of patients with high serum LDH levels and that the aberrant expression of LDHA, PDK1 and GLUT1 is critical for the survival of MM cells with high serum LDH levels. Thus, aerobic glycolysis itself could serve as a novel therapeutic target in MM patients. Since MM with high serum LDH is with poor prognosis even after the advent of new agents, the present data might have a clinical relevance and might open a new avenue to develop novel therapeutic modalities for treating MM with high serum LDH levels. Disclosures: No relevant conflicts of interest to declare.

Abstract 2628 PU.1 is an Ets family transcription factor, which is essential for differentiation of both myeloid and lymphoid lineage cells. We have previously shown that PU.1 is down-regulated in various myeloma cell lines and myeloma cells from a subset of myeloma patients. In such cell lines, the promoter and the upstream regulatory element (URE) located in 17 kb 5'-upstream of the PU.1 gene are highly methylated. Furthermore, conditionally expressed PU.1 induces both cell growth arrest and apoptosis in PU.1-low to -negative myeloma cell lines, U266 and KMS12PE. Therefore, we concluded that the down-regulation of PU.1 is necessary for myeloma cell growth. In another B cell malignancy, classical Hodgkin lymphoma, it has been reported that PU.1 is also down-regulated through methylation of its promoter. To evaluate whether down-regulation of PU.1 is essential for growth of classical Hodgkin lymphoma cells, we conditionally expressed PU.1 in two classical Hodgkin lymphoma cell lines, L428 and KMH2, using the tet-off system (designated as L428tetPU.1 and KMH2tetPU.1 cells, respectively). Up-regulation of PU.1 by tetracycline removal induced complete growth arrest in L428tetPU.1 and KMH2tetPU.1 cells. Annexin V staining revealed that up-regulation of PU.1 induced apoptosis in both cell lines. Furthermore, BrdU staining analysis revealed that PU.1 induced G0/G1 arrest in those cells. L428tetPU.1 and KMH2tetPU.1 cells expressing PU.1 showed morphological changes that included the enlargement cytosol and the appearance of various sizes of vacuoles. We next injected L428tetPU.1 and KMH2tetPU.1 cells to immunodeficiency mice (Rag2−/− Jak3−/− bulb/c) subcutaneously. Tumor formation was observed in all those mice with continuous administration of tetracycline (0.5 g/l) in the drinking water. After enlargement of tumor to 1–2 cm diameter, we removed tetracycline in half of the mice. Tetracyclin withdrawal resulted in tumor regression or stable disease, whereas all the mice continuously receiving tetracycline had continuous tumor growth and finally died. These data strongly suggest that PU.1 induced growth arrest and apoptosis of classical Hodgkin lymphoma cells both in vitro and vivo. We next performed DNA microarray analysis to compare gene expression levels of L428tetPU.1 cells before and after PU.1 expression to elucidate the mechanisms of growth arrest and apoptosis induced by PU.1. Among genes related to cell cycle and apoptosis, p21 (CDKN1A) was highly up-regulated in L428tetPU.1 cells after PU.1 induction, and this was also confirmed by mRNA and protein levels. Finally, to clarify the role of p21 up-regulation by PU.1, we stably introduced p21 siRNA in L428tetPU.1 cells. Such stably expressed p21 siRNA rescued L428tetPU.1 cells from growth arrest induced by PU.1, suggesting that the growth arrest in L428tetPU.1 cells by PU.1 should be at least partially dependent on p21 up-regulation. These data suggested that up-regulation of PU.1 by demethylation agents and/or HDAC inhibitors might serve as a possible treatment modality for classical Hodgkin lymphoma. Disclosures: No relevant conflicts of interest to declare.

Abstract 3939 Introduction: CD138 expression is a hallmark of plasma cells. However, decreased expression of CD138 is frequently found although its clinical significance is unknown. To evaluate the significance of decreased CD138 expression, we examined clinical phenotypes of cases with decreased CD138 expression (CD138-dim cases). Furthermore, we attempted to elucidate the mechanism(s) regulating CD138 expression by utilizing two MM cell lines, KYMM-1 and KYMM-2, established from a CD138-dim case. Materials and methods: CD138 expression levels were determined in 107 primary MM cases using flowcytomerty with the CD38 gating method. Cases with more than 20% of MM cells with CD138 negative population were categorized as CD138-dim cases. Real time RT-PCR was utilized to analyze gene expression. Two MM cell lines, KYMM-1 and KYMM-2, were established from pleural effusion of a CD138-dim case. Both KYMM-1 and KYMM-2 had high CD38 expression with cytoplasmic kappa light chains, while lacking CD19 and CD20 expression. EB virus infection was undetectable with PCR in both cell lines. Identical DNA fingerprinting profile (Cell ID™ System, Promega KK, JAPAN) was observed in patient's primary pleural effusion cells, KYMM-1 and KYMM-2. We also analyzed methylation status of the promoter region of CD138, IRF4 and Blimp-1 by treating KYMM-1 genomic DNA with sodium bisulfite. Results: Flowcytometry analysis of primary MM cells revealed a significant decrease of CD138 expression in those with relapse or progressive diseases compared to untreated MM patients (p Conclusions: Our observations suggest that low CD138 expression in MM cells could serve as an indicator for poor prognosis. Since reduced CD138 expression is preferentially seen in previously treated cases, the regulation of CD138 gene could be associated with disease progression or drug resistance. Previous reports have showed the significance of BCL6 in MM (Hideshima et al. Blood.2010; 115 (18): 3772–3775. Fuhler GM et al. Exp Cell Res. 2010; 316(11):1816–28). High expression of BCL6 in KYMM-1 suggests immature phenotype resulted from down-regulation of IRF4 and Blimp-1 by BCL6. The observed distinctly different expression profile of transcription factors regulating the differentiation of plasma cells in the two cell lines suggest that a transition of differentiation state may have occurred during disease progression. Disclosures: No relevant conflicts of interest to declare.