ALBERT

Description: Functional deregulation of transcription factors has been found in many types of tumors. Transcription factor AML1/RUNX1 is one of the most frequent targets of chromosomal abnormalities in human leukemia and altered function of AML1 is closely associated with malignant transformation of hematopoietic cells. However, the molecular basis and therapeutic targets of AML1-related leukemia are still elusive. Here, we explored immediate target pathways of AML1 by in vitro synchronous inactivation in hematopoietic cells. We found that AML1 inhibits NF-κB signaling through interaction with IκB kinase complex in the cytoplasm. Remarkably, AML1 mutants found in myeloid tumors lack the ability to inhibit NF-κB signaling, and human cases with AML1-related leukemia exhibits distinctly activated NF-κB signaling. Furthermore, inhibition of NF-κB signaling in leukemic cells with mutated AML1 efficiently blocks their growth and development of leukemia. These findings reveal a novel role for AML1 as a cytoplasmic attenuator of NF-κB signaling and indicate that NF-κB signaling is one of the promising therapeutic targets of hematologic malignancies with AML1 abnormality.

Description: Dysfunction of AML1/Runx1, a transcription factor, plays a crucial role in the development of many types of leukemia. Additional events are often required for AML1 dysfunction to induce full-blown leukemia; however, a mechanistic basis of their cooperation is still elusive. Here, we investigated the effect of AML1 deficiency on the development of MLL-ENL leukemia in mice. Aml1 excised bone marrow cells lead to MLL-ENL leukemia with shorter duration than Aml1 intact cells in vivo. Although the number of MLL-ENL leukemia-initiating cells is not affected by loss of AML1, the proliferation of leukemic cells is enhanced in Aml1-excised MLL-ENL leukemic mice. We found that the enhanced proliferation is the result of repression of p19ARF that is directly regulated by AML1 in MLL-ENL leukemic cells. We also found that down-regulation of p19ARF induces the accelerated onset of MLL-ENL leukemia, suggesting that p19ARF is a major target of AML1 in MLL-ENL leukemia. These results provide a new insight into a role for AML1 in the progression of leukemia.

Description: Abstract 3033 In hematopoietic stem cell transplantation (HSCT), oral intake is severely impaired by preparative regimen-induced mucosal toxicity and nausea. Therefore, parenteral nutrition (PN) is usually administered. The vitamin preparation accompanying PN therapy is based on the FDA recommendation proposed in 2000. However, it remains unknown whether this preparation retains adequateness for HSCT recipients who undergo strenuous conditioning regimens and immunological reactions, which put them in a hyper catabolic state. Additionally, almost no study has been conducted to examine the relevance of standard regimen of mineral microelements supplementation during HSCT. In this prospective observational study, we measured the blood level of selected vitamins (vitamin B1, B6, C, K, E, and folate) and mineral microelements (iron, zinc, copper, and selenium) during the peri-HSCT period (measured weekly from 1 week before until 4 weeks after HSCT). Oral and parenteral total energy amounts, administration of multivitamin preparation, and mineral supplementation, were recorded. The subjects were 15 adult patients who received allogeneic HSCT for hematological malignancies (AML: 7, ALL: 1, CML: 1, MPN: 2, MDS: 2, NHL: 2) at The University of Tokyo Hospital. Eight of them received CY/TBI-based full conditioning and the others received fludarabine-based reduced intensity regimens. All the patients received PN with vitamin and mineral preparations when their oral intake decreased to less than 700 Kcal/day. PN was modified adequately in case of organ dysfunctions. Engraftment was observed in all but one patient, and one died before day 28 due to sepsis. The most striking finding was the marked deficiency of vitamin C, a major antioxidant vitamin. Vitamin C levels dropped just after conditioning regimens started, and reached a nadir (2.1+-1.49 microgram/mL, normal range: 5.5–16.8) at day 14. The deficiency levels at day 7 and day 14 were significantly correlated with increase of acute phase inflammatory proteins (p=0.03 for C-reactive protein and p=0.004 for ferritin, at day 7), which suggests that the current vitamin C complement regimen is insufficient for patients who suffer from intense inflammation. On the other hand, vitamin E, another antioxidant vitamin, remained within the normal level during the surveillance period. Selenium is a microelement that assists antioxidative effect and has attracted attention because emerging evidence suggests its relevance for critically ill condition. Although it is not contained in the standard PN preparations, our results indicated no sign of shortage. Selenium is abundant in normal diet, and short suspension of oral intake does not induce selenium depletion. Marked excess of vitamin K was another point of note. The average vitamin K level continued to rise after the start of the conditioning regimen, and exceeded 10 times of the normal upper level (0.15 – 1.25 ng/mL) at day14 (12.8 +- 5.6 ng/mL), day 21 (17.4 +- 17.9), and day 28 (12.1+- 25.0). These abnormal rises were observed especially in patients with prolonged administration of PN, suggesting overdosing of vitamin K in the standard vitamin preparations. Vitamin K excess would be problematic because it causes hypercoagulability; however, it was not clinically correlated with the onset of thrombotic microangiopathy in our cohort. Vitamin B1 was slightly but significantly below the normal lower limit. Its deficiency started before the conditioning regimen and persisted through day14. Considering increased requirement of vitamin B1 during a debilitating state, this shortage should be corrected with active supplementation before the patients undergo a stressful conditioning process. Other substances (folate, zinc, iron, and copper) remained almost within a normal range. In conclusion, the nourishment status during peri-HSCT period is characterized by marked depletion of vitamin C and excess of vitamin K, and other aberrations with less degree of deviation. These results indicate that normal PN is not adequate for HSCT patients. Development of vitamin and microelement preparation that is optimized for HSCT setting should be discussed. Disclosures: Nannya: Otsuka Seiyaku Koujyou: Research Funding.

Description: Abstract 48 Elevated expression of BAALC (brain and acute leukemia, cytoplasmic) is an adverse prognostic factor in patients with cytogenetically normal acute myeloid leukemia (CNAML). However, its precise role in normal hematopoiesis and leukemogenesis remains to be elucidated. To address this issue, we evaluated the effect of BAALC overexpression upon hematopoiesis and leukemogenesis and generated BAALC-deficient mice. First, we examined the effect of BAALC overexpression upon hematopoiesis. BAALC-transduced bone marrow (BM) cells had decreased colony-forming capacity. Bone marrow transplantation (BMT) assay demonstrated that the donor chimerism was lower in transplanted mice with BAALC-overexpressed cells than with control cells. To examine the mechanism, we performed gene expression analysis. This revealed that p53 pathway is up-regulated in BAALC-overexpressed cells. Colony-forming assay and BMT assay using p53-deficient BM cells demonstrated that the blockage of proliferation by BAALC overexpression is canceled in p53-deficient cells in vitro and in vivo, suggesting that the inhibition of proliferation by BAALC overexpression is dependent on p53 pathway. Next, we generated BAALC-conventionally deficient mice. Most BAALC-deficient female mice died between E11.5 and E12.5 because of defects of placental development. BAALC-deficient E11.5 yolk sac cells had increased colony-forming capacity. Transplantation assay showed that the donor chimerism at 4 weeks after transplantation was higher in transplanted mice with BAALC-deficient E12.5 fetal liver cells than with wild fetal liver cells. Moreover, the frequency of G0/G1 phase cells was decreased in BAALC-deficient yolk sac. Thus, the proliferation is activated and the frequency of quiescent cells is reduced in BAALC-deficient embryonic hematopoietic cells. In addition, we examined the role of BAALC in adult hematopoiesis using BAALC-conditionally deficient mice with Mx-Cre system. In BAALC-deficient (Mx-Cre+BAALC flox/flox) BM, long-term hematopoietic stem cells (HSCs), identified as CD34−Flt3−cKit+Sca1+Lin− cells, were reduced as compared with control (Mx-Cre−BAALC flox/flox) BM. BAALC-deficient adult BM had impaired long-term repopulating ability in serial competitive BMT. Moreover, BAALC-deficient mice showed higher mortality rates after weekly 5-FU injections. On the other hand, the ability of colony forming unit spleen (CFU-S) was enhanced and the recovery after myelosuppression induced by single 5-FU injection was faster in BAALC-deficient mice, indicating that BAALC-deficient HSCs have enhanced short-term repopulating ability. Next, to address the relationship between BAALC and p53 protein levels, we performed intracellular flow cytometric analysis and immunocytochemical staining. These experiments revealed that p53 protein is reduced in BAALC-deficient HSCs. Immunoprecipitation revealed that BAALC interferes with the binding of MDM2 to p53, which leads to stabilization of p53 protein. Finally, we tested the effect of BAALC overexpression in leukemia mouse models. BAALC overexpression promoted leukemia in mice in cooperation with TEL/PDGFBR-AML1/ETO (TPAE). BM cells transduced with TPAE and BAALC had increased colony-forming capacity as compared with TPAE and mock-transduced cells. By contrast, BAALC cannot accelerate myc/BCL2-induced leukemia. Western blot showed that p53 is inactivated in TPAE leukemic cells but not in myc/BCL2 leukemic cells. BM cells transduced with myc/BCL2 and BAALC had decreased colony-forming capacity as compared with myc/BCL2 and mock-transduced cells. However, BAALC overexpression promoted colony-forming capacity of p53-deficient cells transduced with myc/BCL2. Similarly, BAALC overexpression promoted the colony-forming capacity of BM cells transduced with BCR/ABL which is known to inactivate p53. Promoted proliferation of BCR/ABL-transduced cells by BAALC overexpression was canceled by the MDM2 antagonist, Nutlin-3. Taken together, BAALC overexpression accelerates leukemia with impaired p53 pathway. In conclusion, these data suggest that BAALC activates p53 pathway and maintains the stemness in normal HSCs. Once p53 pathway is impaired, however, BAALC promotes proliferation of leukemic cells. Our data for the first time uncovered a novel function of BAALC, which may lead to novel therapeutic strategy for CNAML with poor prognosis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1605 Reactive oxygen species (ROS) are small molecules containing oxygen with unpaired electron. ROS are always generated as the products of cellular metabolism, and cells have several antioxidant systems to avoid their harmful effect induced by their high chemical reactivity. While studies about biological effects of ROS have been mainly focused on the harmful aspects, a growing body of evidence suggests that ROS are critical mediators of several signaling pathways in cellular homeostasis. In hematopoiesis, for example, it was reported that the generation of intracellular ROS functions as the initiation signal in the development of Drosophila hematopoietic cells and that ROS control self-renewal of hematopoietic stem cells. However, little is known about the functions of ROS in regulating hematopoietic progenitors. In this study, we show a critical role of ROS in lineage decision of myeloid progenitor cells. Firstly, we measured the intracellular ROS level of hematopoietic cells from murine bone marrow by flow cytometry with H2-DCFDA (2’,7’-dichlorofluorescin diacetate) staining. It was found that intracellular ROS level of megakaryocyte-erythrocyte progenitor cells (MEP) was kept equal to or lower than that of lineage marker negative, Sca-1 positive, c-Kit positive (KSL) cells. On the other hand, that of granulocyte-monocyte progenitor cells (GMP) was significantly elevated. Additionally, mRNA expression of NADPH oxidase 2 (cytochrome b-245) and NOXA2 (neutrophil cytosolic factor 2), both of which are major components of the membrane-bound oxidase complex generating ROS in cells, was significantly suppressed in MEP and KSL cells, whereas it was up-regulated in GMP. Thus, intracellular ROS level of MEP is kept lowest in hematopoietic cells. Next, we investigated the effect of ROS on the differentiation of myeloid progenitors. In liquid culture assay, loading of ROS with low dose hydrogen peroxide inhibited the differentiation of progenitor cells into MEP, whereas removal of ROS with catalase accelerated the differentiation of those into MEP. Similarly, in the colony-forming assay with semisolid culture medium, we observed that loading of ROS inhibited the formation of megakaryocyte-erythrocyte colonies. To investigate the effect of intrinsic ROS on the colony-forming capacity, we sorted ROS-high and low common myeloid progenitor cells (CMP) individually and cultured them. It was shown that ROS-low CMP had high colony-forming capacity of megakaryocyte-erythrocyte, whereas ROS-high CMP had high colony-forming capacity of granulocyte-monocyte. There is inverse correlation between intracellular ROS level of CMP and colony-forming capacity of megakaryocyte-erythrocyte. In order to confirm that ROS can control the differentiation of CMP into MEP or GMP in vivo, we injected lipopolysaccharide (LPS), which can increase intracellular ROS level of bone marrow cells, into mice. We found that MEP were decreased in LPS-injected mice. Finally, we performed gene expression microarray analysis to compare gene expression profiles between ROS-low and high CMP. In terms of gene expression profiles, ROS-low CMP were similar to MEP and magakaryocyte whereas ROS-high CMP were similar to GMP. In conclusion, these findings suggest that intracellular ROS play a critical role in lineage decision of myeloid progenitor cells, especially in the generation of MEP. In several diseases with anemia or thrombocytopenia, such as myelodysplastic syndrome, Fanconi anemia and autoimmune diseases with chronic inflammation, it was reported that intracellular ROS level of hematopoietic cells was up-regulated. Thus, up-regulated intracellular ROS may be involved in their symptoms via differentiation disorder of MEP. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4560 Introduction: Infectious disease following hematopoietic stem cell transplantation (HSCT) is a major cause of transplantation-related mortality. The main manifestation of infectious diseases is fever. However, fever appears with several transplantation-related complications besides infectious diseases, such as acute graft-versus-host-disease (aGVHD), engraftment syndrome, thrombotic microangiopathy (TMA), veno-occlusive disease (VOD), and relapse of the underlying diseases. The more valuable markers to distinguish infections disease from non-infectious complications are needed. Methods: A total of 28 consecutive patients who underwent HSCT in our institution between September and December 2010 were included in the study. Procalcitonin (PCT) and C-reactive protein (CRP) were measured periodically throughout the clinical course. The febrile episodes were classified in four groups: systemic bacterial or fungal infections, immunological reactions such as engraftment syndrome and aGVHD, intracellular infections such as viral infection and tuberculosis, and the other febrile episodes including VOD, TMA, mucositis, enterocolitis not ascribed to infectious microorganisms, relapse of the underlying disease, and unidentified causes. Results: The median follow-up period for survivors was 101 days (range: 11–178 days). The diagnoses of 103 febrile episodes were classified as follows: systemic bacterial or fungal infections (n=30), intracellular infections (n=7), immunological reactions (n=19), and other febrile episodes (n=47). PCT and CRP level on the first day of fever (O-PCT and O-CRP, respectively) significantly increased in systemic bacterial or fungal infection (p

Description: Ecotropic viral integration site-1 (Evi-1) is a nuclear transcription factor that plays an essential role in the regulation of hematopoietic stem cells. Aberrant expression of Evi-1 has been reported in up to 10% of patients with acute myeloid leukemia and is a diagnostic marker that predicts a poor outcome. Although chromosomal rearrangement involving the Evi-1 gene is one of the major causes of Evi-1 activation, overexpression of Evi-1 is detected in a subgroup of acute myeloid leukemia patients without any chromosomal abnormalities, which indicates the presence of other mechanisms for Evi-1 activation. In this study, we found that Evi-1 is frequently up-regulated in bone marrow cells transformed by the mixed-lineage leukemia (MLL) chimeric genes MLL-ENL or MLL-AF9. Analysis of the Evi-1 gene promoter region revealed that MLL-ENL activates transcription of Evi-1. MLL-ENL–mediated up-regulation of Evi-1 occurs exclusively in the undifferentiated hematopoietic population, in which Evi-1 particularly contributes to the propagation of MLL-ENL–immortalized cells. Furthermore, gene-expression analysis of human acute myeloid leukemia cases demonstrated the stem cell–like gene-expression signature of MLL-rearranged leukemia with high levels of Evi-1. Our findings indicate that Evi-1 is one of the targets of MLL oncoproteins and is selectively activated in hematopoietic stem cell–derived MLL leukemic cells.

Description: Abstract 4064 Introduction: Chronic graft-versus-host disease (cGVHD) is one of the main obstacles for long-term survival after allogeneic hematopoietic stem cell transplantation (HSCT). Although the important role of antigen-presenting cells in cGVHD and the association between cGVHD and increased monocytes after HSCT were reported, it is unclear whether the number of peripheral blood (PB) monocytes influences the outcome after HSCT. Patients and methods: We retrospectively reviewed 198 consecutive patients who underwent HSCT for hematologic malignancies and bone marrow failure syndrome in our institution between 1997 and 2009 and survived at least 1 year without relapse. The association between the absolute number of PB monocytes at 1 year after HSCT and transplant outcome was assessed. Results: Median follow-up period from HSCT was 61.9 months (range: 12.3–180.6 months). Patients with cGVHD at 1 year after HSCT showed increased PB monocytes compared with those without cGVHD (median 0.54×109/L vs 0.40×109/L, P =0.0008). While relapse rate was not significantly different according to PB monocyte count, patients with PB monocyte count less than 0.50×109/L at 1 year after HSCT had significantly better disease free survival (DFS), better overall survival (OS) and lower non-relapse mortality (NRM) rate compared with those with more than 0.50×109/L (10-year DFS 86.0 vs 63.6%; P =0.0070, 10-year OS 88.8 vs 63.8%; P =0.0031, and 10-year NRM rate 5.8 vs 21.4%; P =0.011). Eighteen of 198 patients (9.1%) died from non-relapse causes after 1 year post-HSCT; 14 of 91 in the high PB monocyte count group (15.3%) and 4 of 107 in the low PB monocyte count group (3.7%) (P =0.0057). Among the 18 patients, 12 died of cGVHD (9 in the high monocyte count group and 3 in the low monocyte count group). By Kaplan-Maier estimates, risk factors of higher NRM rate were age older than 35 (P =0.043), female donor to male recipient (P =0.020), high disease risk (P =0.0033), unrelated donor (P =0.044), human leukocyte antigen (HLA) mismatch (P =0.00023), steroid use at 1 year after HSCT (P =0.00013), immunosuppressant use at 1 year after HSCT (P =0.010), and PB monocyte count more than 0.50 × 109/L at 1 year after HSCT (P =0.0081). The factors with P ≤0.1 in the univariate analyses, i.e. grade 2–4 acute GVHD (P =0.078), extensive type cGVHD at 1 year after HSCT (P =0.057) and PB platelet count less than 1.0×1011/L at 1 year after HSCT (P =0.054) were also included in the following multivariate analysis. Multivariate analysis with competing risk regression model revealed that NRM rate was associated with age older than 35 (HR=3.99, P =0.010), HLA mismatch (HR=7.96, P =0.040), steroid use at 1 year after HSCT (HR=5.55, P =0.010), and PB monocyte count more than 0.50×109/L at 1 year after HSCT (HR=3.65, P =0.023). Cox multivariate analysis for DFS also showed that PB monocyte count more than 0.50×109/L at 1 year after HSCT was an independent prognostic factor (HR=2.22, P =0.028). Other risk factors for DFS were high disease risk (HR=2.14, P =0.032) and extensive type cGVHD at 1 year after HSCT (HR=2.09, P =0.040). Increased PB monocytes also predict future onset of cGVHD. Among patients without cGVHD at 1 year after HSCT (n=68), those with increased PB monocytes had higher rates of future cGVHD onset (10-year 6.5 vs 42.6%; P =0.013) and NRM (10-year 0 vs 21.6%; P =0.0086). Conclusion: Increased PB monocytes after HSCT were associated with cGVHD, future cGVHD onset, higher NRM rate and poor survival. Our data suggest the importance of monocytes in the etiology of cGVHD. Disclosures: No relevant conflicts of interest to declare.

Description: Familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML) is an autosomal dominant disorder and is characterized by inherited thrombocytopenia and a lifelong risk of development of hematological malignancies. Although inherited RUNX1 mutation is the cause of thrombocytopenia, additional genetic events may be responsible for the tumor development as only 40 % of FPD/AML patients develop leukemia. Because of the rarity of this disorder, underlying mechanisms for malignant transformation in FPD/AML have not been elucidated. Thus we conducted a nationwide survey in Japan to collect samples and make a diagnosis of patients with familial thrombocytopenia or hematological malignancies. As a result, 56 pedigrees were extracted, and seven pedigrees with RUNX1 mutation were diagnosed as having FPD/AML, in which eight out of 14 patients had developed hematological malignancies. To systematically identify additional genetic alterations, we utilized whole exome sequencing in two patients with FPD/AML who had RUNX1_F303fsX566 mutation and developed MDS (Patient 1) or myelofibrosis (Patient 2) followed by AML. We identified 12 and 10 somatically acquired nonsynonymous mutations in these patients, respectively. Intriguingly, the two patients had common CDC25C mutation at codon 234 (D234G). Further genomic screening of other pedigrees revealed that four out of eight FPD/AML patients who developed hematological malignancy harbored somatic mutations in CDC25C (CDC25C_D234G in three patients and CDC25C_H437N in one patient). Recurrent CDC25C mutation in FPD/AML with subsequent hematological malignancy implies that it forms common genetic foundation of transformation in this disease. CDC25C is a phosphatase that prevents premature mitosis in response to DNA damage at the G2/M checkpoint, while it is constitutively phosphorylated at Ser216 throughout the interphase by c-TAK1. When phosphorylated at Ser216, CDC25C binds to 14-3-3 protein, which sequestrates CDC25C in the cytoplasm and inactivates it. In all of the mutated forms of CDC25C that we found in FPD/AML, their binding capacity with c-TAK1 and 14-3-3 protein was reduced, resulting in decreased phosphorylation status of CDC25C at Ser216. As a consequence, those CDC25C mutants led to enhanced mitosis entry, which was exaggerated by radiation-induced DNA damage. These results demonstrate that CDC25C mutation results in disruption of DNA checkpoint machinery. It is known that FPD/AML-associated RUNX1 mutations evokes DNA damage and induces cell cycle arrest in hematopoietic cells, suggesting that the DNA checkpoint mechanism is activated in the presence of those types of RUNX1 mutation. We found, however, that introduction of CDC25C mutation results in the marked enhancement of mitosis entry in spite of co-expression of RUNX1 mutation in Ba/F3 cells. Thus, premature mitosis by loss of DNA checkpoint mechanisms in the presence of mutated CDC25C may contribute to malignant transformation of RUNX1-mutated cells. Interestingly, analysis of clonal evolution during leukemic transformation revealed that a clone defined by CDC25C mutation was dominant in the early phase of disease progression in both patients, which supports the idea that CDC25C mutation is associated with establishment of the founder clone during the leukemic progression of FPD/AML. In Patient 1, the founder clone with CDC25C mutation acquired FAM22G and COL9A1 (group 1) mutations, followed by occurrence of GATA2 and LPP (group 2) mutations to become a dominant clone in the AML phase, whereas another subclone of the founder defined by CHEK2 and DTX2 (group 3) mutations regressed. Similar hierarchical progression was observed in Patient 2. An additional single cell genomic sequencing of bone marrow cells from Patient 1 in the AML phase revealed that group 1/2 mutations and group 3 mutations were mutually exclusive, which supports our predicted model. Collectively, these results indicate that somatic mutation in CDC25C is a recurrent event in the early phase of leukemic progression of FPD/AML, which induces premature mitosis and genetic instability in hematopoietic cells with germline RUNX1 mutation. Disclosures: Usuki: Alexion Pharmaceuticals, Inc.: Speakers Bureau. Kurokawa:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding.