ALBERT

Description: Abstract 2673 Introduction: Follicular Lymphoma International Prognostic Index 2 (FLIPI-2) is a widely accepted tool for risk assessment of follicular lymphoma (FL), which is based on age, hemoglobin level, presence of bone marrow (BM) invasion, tumor size, and b2-microgloblin levels. Although it is easy to evaluate in clinical practice, it is a combination of tumor burden and patient physical condition, and a simple and powerful biomarker reflecting the tumor burden and its character is still not established. LR11 (also called SorLA or SORL1) was identified and characterized as a regulator of uPAR function through complex formation with uPAR. We have identified that serum soluble LR11 (sLR11) levels are significantly elevated in patients with acute leukemia and B cell lymphomas, and are associated with tumor burden and BM invasion (Sakai et al 2012). We have also found that high sLR11 levels had a significant negative prognostic impact on progression-free survival (PFS) in FL. Therefore, we have retrospectively evaluated the clinical characteristics of sLR11 and its prognostic impact on FL, in a larger patient cohort. Patients and Methods: Sixty-one patients with FL treated at Chiba University Hospital and affiliated hospitals from 2002 to 2012 were evaluated. The majority of patients were treated by the R-CHOP regimen (rituximab 375 mg/m2 on day 1; cyclophosphamide, 750 mg/m2 on day 1; adriamycin, 50 mg/m2 on day 1; vincristine, 1.4 mg/m2 on day 1; and prednisolone, 100 mg/body on day 1–5). Serum sLR11 levels were measured by ELISA method. Patient laboratory data and treatment outcome were obtained retrospectively. Results: Serum sLR11 levels of patients with lymphoma were significantly increased (mean ± SD: 19.4 ± 17.1 ng/ml) compared with those of normal control subjects (8.8 ± 1.79 ng/ml, P

Description: Abstract 2409 Introduction: Membrane-anchored uPAR plays a role in regulating the engraftment and mobilization of murine hematopoietic stem/progenitor cells (HSPCs) to bone marrow (BM). Acute myeloid leukemia (AML) patients with higher proportions of uPAR-positive cells posed higher relapse risk after chemotherapy. LR11 (also called SorLA or SORL1) is a regulator of uPAR function through complex formation with uPAR., and the LR11 expression is highly induced in AML cells, and circulating soluble form of LR11 (sLR11) levels are significantly increased in AML patients and ameliorated by chemotherapy (Sakai et al. 2012). Although LR11 is over-expressed in AML, the role of over-expressed LR11 in AML leukemogenesis remains unknown. Recently we found that LR11 mRNA expression levels are drastically increased in murine transformed granulocyte macrophage progenitors (GMPs) with MLL-AF9 fusion gene. Here, we show the pathological significance of LR11 expressions in the progression of AML using mouse leukemia model. Materials and Methods: Stably LR11-knockdown (LR11-KD) U937 cells were generated by transfection of shRNA expression vector specific for LR11. For generation of mouse AML model, the GMPs transformed by MLL-AF9 (MA9-GMPs) or human leukemic cells were transplanted into syngeneic mice or immune-deficient mice, respectively. For colony replating assays, MA9-GMPs were cultured in methylcellulose medium and colonies were counted and pooled, and then 1×104 cells were replated in the same medium. For cell growth assays, 5th round MA9-GMPs were transferred to liquid culture. An adhesion assay was performed using human mesenchymal stromal cells (MSC)-coated plates. Results: U937 cells expressed high level of LR11 mRNA and protein, but there was no difference in cell growth between control and LR11-KD U937 cells in vitro. We transplanted LR11-KD or control U937 cells into recipient mice, and monitored the percent of peripheral blood (PB) leukemia cells by flow cytometry. All the recipient mice transplanted with control U937 cells were died of leukemia by 3 weeks after transplantation, whereas the survival of mice transplanted with LR11-KD U937 cells were prolonged significantly (the median survival: 17.5 days for control cells vs. 28 days for LR11-KD cells, Figure. 1). The percent of leukemia cells in PB of the mice transplanted with LR11-KD U937 cells on day 13 after transplantation were significantly lower than those of mice transplanted with control U937 cells (1.2 ± 1.1% vs. 10.2 ± 5.8%, respectively). We next examined the effect of LR11 on the malignant phenotype by cell growth assay and colony replating assay using the MA9-GMPs from Lr11+/+ and Lr11−/− mice. There was no difference in the cell growth and colony forming ability between Lr11+/+ and Lr11−/− MA9-GMPs. However, the donor chimerism of mice transplanted with Lr11−/− MA9-GMPs was significantly lower than that of mice transplanted with Lr11+/+ MA9-GMPs (the percentage of chimerism on day 28 and on day 35 after transplantation: 54.6 ± 2.1% and 66.3 ± 11.0% for Lr11+/+ donor cells vs. 0.7 ± 2.1% and 0.5 ± 0.1% for Lr11−/− donor cells). We finally examined the cell motility analysis. In the adhesion assay, the attached cell numbers to MSC-coated plates were significantly reduced to 0.7-fold in the LR11-KD U937 cells compared with control U937 cells. In homing assay, we transplanted LR11-KD U937 cells and control cells into recipient mice. The number of LR11-KD U937 cells in recipient BM 16 hours after transplantation was significantly decreased compared with those of control cells (0.011 ± 0.004% for control cells vs. 0.003 ± 0.001% for LR11-KD cells, Figure 2). Conclusion: These data showed that LR11 plays a critical role in the pathogenesis of AML by enhancing the cell adhesion and homing ability to BM. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction LR11, which is also known as SorLA or SORL1, is a type-I membrane protein from which a large extracellular part, soluble LR11 (sLR11), is released by proteolytic shedding. We have demonstrated that serum sLR11 levels are significantly elevated in patients with acute leukemia and that sLR11 levels are associated with the peripheral blast percentage (Sakai et al. Clin Chim Acta. 2012). In addition, we found that high sLR11 levels have a significant negative prognostic impact on progression-free survival in patients with follicular lymphoma (FL; Kawaguchi et al. Br J Haematol. 2013). In this FL analysis, the immunohistological intensity of LR11 in lymph nodes of FL patients did not show a significant association with serum sLR11 levels. Therefore, a shedding mechanism is presumed to play a key role in the functions of LR11. A disintegrin and metalloproteinase 17 [ADAM17, also known as tumor necrosis factor (TNF)-α converting enzyme (TACE)] has been identified as the enzyme that cleaves TNF-α from its transmembrane precursor form, and it has been found to cleave the ectodomains of other cell surface proteins. LR11 is also shed by ADAM17. Tetraspanin CD9 has been recently shown to inhibit the shedding activity of ADAM17. Therefore, we investigated the role of CD9 in sLR11 release. Materials and methods First, we examined the gene expression levels of LR11 by quantitative real-time PCR and the cell surface expression of LR11 and CD9 by flow cytometric analysis in normal human peripheral-blood mononuclear cells (PBMCs). Second, we investigated the cellular expression and the released levels of LR11 and cellular CD9 in THP-1 monocytes, phorbol 12-myristate-13-acetate (PMA)-induced THP-1 macrophages (PMA/THP-1), and the B lymphoblastoid cell line CCRF-SB by immunoblot analysis. Furthermore, to assess the relationship between LR11 and CD9 in PMA/THP-1, we performed double immunofluorescence staining of these molecules followed by confocal microscopy analysis. Third, we examined the effects of ectopic neoexpression of CD9, anti-CD9 mAbs, and CD9 silencing. THP-1 cells stably interfered with CD9 were generated by the transfection of the shRNA expression vector that is specific for CD9. Results The gene expression levels of LR11 were significantly higher in CD3+ T cells than in CD14+ monocytes, and there was no difference between monocytes and CD19+ B cells. However, LR11 was only expressed on the cell surface of monocytes in normal PBMCs. CD9 was broadly expressed on the cell surface of every PBMCs. Because it has been reported that CD9 associates with ADAM-17 on the cell surface, we hypothesized that CD9 associates with the shedding of LR11 on the cell surface. To verify this hypothesis, we chose the monocytic cell line THP-1 because monocytes have both LR11 and CD9 on their surface. In addition, because the gene expression levels of LR11 were significantly higher in human monocyte-derived macrophages than that in monocytes, we investigated the levels in PMA/THP-1. LR11 was not expressed or released in undifferentiated THP-1 cells, but it was expressed or released in differentiated PMA/THP-1. CD9 was poorly expressed in THP-1 cells, and the CD9 expression levels were increased in PMA/THP-1. The confocal microscopy analysis showed colocalization of LR11 and CD9 proteins in PMA/THP-1. We next investigated the effects of ectopic neoexpression of CD9 in CD9-negative cells, neutralizing anti-CD9 mAbs (clone ALB-6) in CD9-positive cells, and CD9 silencing in THP-1 on the shedding of LR11. When CCRF-SB, LR11-positive and CD9-negative cells were transiently overexpressed with CD9, the amount of sLR11 released from CD9-overexpressing cells was decreased compared with that from control cells. The amount of sLR11 released from LR11-transfected THP-1 cells that were cultured with ALB-6 was significantly increased compared with that cultured with an isotypic control antibody. The PMA-stimulated release of sLR11 was significantly increased in CD9 shRNA-interfered THP-1 cells compared with cells that were transduced with the control shRNA. The increase in CD9 shRNA-interfered THP-1 was nullified by treatment with the metalloproteinase inhibitor GM6001 (Figure 1). Conclusion These results suggested that the tetraspanin CD9 inhibits ADAM17-mediated LR11 shedding in leukemia cells and that it may have a role in controlling the function of LR11. Disclosures: No relevant conflicts of interest to declare.

Description: Recently, novel agents such as bortezomib and lenalidomide have been introduced for multiple myeloma (MM) treatment and have improved patients' survival drastically. However, dexamethasone remains a mainstay in the treatment of MM. Dexamethasone effectively induces tumor cell death when used for the initial treatment of MM. In addition, dexamethasone has a synergistic effect with novel agents and is hence used in combination with such agents. However, prolonged dexamethasone exposure may lead to drug resistance. To elucidate the mechanism of dexamethasone resistance, we generated a dexamethasone-resistant subline of the MM cell line RPMI8226. We cultured RPMI8226 cells with 1 µM dexamethasone for 7 weeks and established the dexamethasone-resistant cell line Dex-R. This cell line showed no difference in survival in the presence or absence of 1 µM dexamethasone. We then examined differences in gene expression between RPMI8226 and Dex-R cells using cDNA microarray. Expression of the FARP1 gene, which is a transforming growth factor beta (TGF-b) target gene in myeloma cells, was increased approximately 50-fold in Dex-R cells compared to that in RPMI8226 cells. In some myeloma patients who become chemoresistant, myeloma cells show high levels of FARP1 expression at the initial stage. FARP1 has a Rho-GEF domain and can associate with proteins on the cell membrane through the FERM domain. In the nervous system, FARP1 is involved in synaptogenesis via the activation of Rac1. Based on these observations, we hypothesize that Dex-R cells acquires dexamethasone resistance with an increase in the level of FARP1 expression via the activation of Rac1. To verify this hypothesis, we established inducible FARP1 knockdown Dex-R cells using the TET-ON lentiviral system. We cultivated these cells for 24 h with doxycycline and added 1 µM dexamethasone. A total of 48 h after adding dexamethasone, we measured cell viability using the MTS assay. We cultured Dex-R cells with a Rac1 inhibitor (NSC23766) and added dexamethasone 12 h later. FARP1 expression decreased to approximately 10% in FARP1 knockdown cells 24 h after the addition of doxycycline. Without dexamethasone, there was no difference in survival in the presence or absence of doxycycline. However, when cells were cultured with dexamethasone, the growth of FARP1 knockdown Dex-R cells was significantly inhibited compared with that of the control (Fig 1). Next, we examined the change in dexamethasone resistance on the addition of the Rac1 inhibitor. The number of cells increased after 96 h without dexamethasone. On the other hand, the number of cells significantly decreased when cultured with dexamethasone (Fig 2). These data suggest that resistance to dexamethasone in Dex-R cells was mitigated by the inhibition of Rac1. We conclude that the activation of Rac1 through FARP1 is one mechanism of dexamethasone resistance in MM. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4795 Introduction: We have recently reported that LR11 (also called SorLA or SORL1) is specifically expressed on the surface of acute leukemia cells, and the circulating levels of soluble form (sLR11) are highly associated with peripheral blast numbers in acute leukemia patients at diagnosis and decreased in remission (2012, Sakai et al). Although the functional role of sLR11 in the migration of undifferentiated vascular cells has been studied, the pathological significance of sLR11 has not been elucidated for the development of leukemia blast properties. The expressions of WT1, an oncogene for the various malignant tumors, are associated with poor prognosis of patients with acute leukemias, and the WT1 levels have been thought to indicate the blast properties. In this study, in order to know the significance of sLR11 for the oncogenic properties of leukemia cells, we first examined the correlations of circulating sLR11 levels with WT1 mRNA levels in the bone marrow of acute leukemia patients. Next, we analyzed the effects of sLR11 on WT1 mRNA expressions, particularly in association with GATAs, factors for the WT1 gene transcription, in leukemia cell lines. Materials and methods: Levels of serum sLR11 and bone marrow cell WT1 mRNA were measured by ELISA and real-time PCR, respectively, using samples of acute leukemia patients (AML n=6, ALL n=5). Statistical analyses were performed by Spearman rank correlation system. mRNA levels of GATA1, GATA2 and WT1 were analyzed using K562 cells with or without recombinant sLR11 and LR11-overexpressing K562 cells. Results: Figure 1 shows the relationship between sLR11 in serum and WT1 mRNA in bone marrow mononuclear cells in acute leukemia patients at diagnosis. The sLR11 levels were significantly positively correlated with the WT1 levels in the patients. In order to know the pathological link between sLR11 and WT1 in leukemia cells in detail, we then analyzed the effects of sLR11 on the WT1 mRNA expressions in K562 cells. WT1 mRNA levels were gradually increased after the incubation with 1 ng/ml sLR11, and the levels at 4 h were elevated to 2.5-fold of those before the incubation (Figure 2). The levels were then decreased to the levels equivalent to those before the incubation. The mRNA levels of GATA1 and GATA2 were also drastically elevated at 4 h and then sharply decreased after adding sLR11. GATA1 mRNA levels were particularly increased by 5-fold of those before the incubation. Thus, sLR11 were suggested to cause the activation of transcription factor network of GATAs, and induce the WT1 gene transcription in the cells. We finally investigated the effects of sLR11 overexpression on the elevation of WT1 mRNA in K562 cells. The LR11-overexpressing cells showed significantly increased expression levels of GATA1 and GATA2 in addition to WT1 mRNAs. Conclusions: Circulating sLR11 levels were significantly correlated with the WT1 mRNA expression levels in acute leukemia. sLR11 induces the WT1 expression, possibly through the activations of GATAs in leukemia cells. These results suggest that sLR11 may be a novel marker indicating the malignant properties of leukemia cells. Disclosures: No relevant conflicts of interest to declare.