Overexpression of an anti-apoptotic protein cIAP1 caused by its genetic amplification was reported in certain cancers, such as hepatocellular carcinoma, esophageal squamous cell carcinoma, cervical cancer, and lung cancer, which confers resistance to chemotherapy and radiotherapy. Here we report cIAP1 to be selectively down-regulated by a class of small molecules ((؊)-N-[(2S,3R)-3-amino-2-hydroxy-4-phenyl-butyryl]-L-leucine methyl ester (ME-BS)), resulting in a sensitization of cancer cells to apoptosis. ME-BS directly interacts with the BIR3 domain of cIAP1, promotes auto-ubiquitylation dependent on its RING domain, and facilitates proteasomal degradation of cIAP1. Other IAPs such as XIAP and cIAP2 were not affected by ME-BS. These results suggest targeted destabilization of cIAP1 by small molecules as a novel method to treat cancers expressing cIAP1, which interferes with treatment. Manipulation of the intrinsic ubiquitinligase activity could be a novel strategy to develop small molecules for therapeutic purposes.IAPs (inhibitor of apoptosis proteins) are a family of antiapoptotic proteins containing one to three baculoviral IAP repeat (BIR) 2 domains (1-3), some of which are frequently overexpressed in malignant cells. Certain IAPs such as XIAP/ hILP/BIRC4, cIAP1/MIHB/hiap-2/BIRC2, cIAP2/MIHC/hiap-1/BIRC3, ML-IAP/Livin/BIRC7 and Apollon/BRUCE/BIRC6 directly interact with and regulate caspases (4 -9). The BIR domain plays an important role in the interaction with caspases (8, 10, 11). These IAPs also contain a domain involved in ubiquitin conjugation (RING finger domain or UBC domain), and facilitate proteasomal degradation of caspases and IAPs (8, 12, 13). cIAP1 (cellular inhibitor of apoptosis protein 1) is overexpressed in human cancers such as esophageal squamous cell carcinoma, hepatocellular carcinoma, cervical cancer, and lung cancer, because of its genetic amplification and is regarded as an oncogene (14 -17). cIAP1 overexpression in cervical cancers correlates with resistance to radiotherapy. In addition, a comparative study of the expression of IAP family proteins and the sensitivity to chemotherapeutic drugs in 60 cell lines revealed the level of cIAP1 significantly correlates with resistance against anti-cancer drugs such as carboplatin, cisplatin, etoposide, and cytosine arabinoside (18). This evidence suggests cIAP1 to be a promising target for cancer therapy.Bestatin, an inhibitor of aminopeptidase N, has an immunomodulatory activity and is approved in Japan to treat patients with adult acute nonlymphatic leukemia (19 -21). In a clinical trial with stage I squamous cell lung carcinoma patients, bestatin significantly prolonged survival of these patients (22). Bestatin induces apoptosis in human leukemia cells (23) and augments death ligand-induced apoptosis in human solid tumor cell lines (24). In this paper, we describe selective down-regulation of cIAP1 by esterified analogs of bestatin represented by bestatin-methyl ester (ME-BS) (see Fig. 1A), resulting in a sensitization of cancer cells to ...

Ligation of death receptors, such as Fas, triggers apoptosis in many types of cells (28), which is inhibited by cellular FLIP (cFLIP, also known as I-FLICE, FLAME-I, Casper, CASH, MRIT and Usurpin) (8,11,13,16,34,40,41,44). The long form of cFLIP (cFLIP-L) is highly homologous to caspase 8, containing two death effector domains (DED) and a caspaselike domain at the amino and carboxy termini, respectively. cFLIP-L, however, does not have caspase activity due to the lack of a conserved cysteine residue in the caspase-like domain. Upon death receptor ligation, cFLIP-L is recruited to the death receptor complex, together with FADD and caspase 8, and inhibits apoptosis signaling. cFLIP-L is expressed in various cancers (2,16,27,36,43,44), which suggests a role for cFLIP-L in protecting cancer cells from cellular immunity using the Fas system (5, 6, 25).cFLIP, however, does not always inhibit apoptosis signaling but also mediates growth signals in some cases. Under conditions in which the proliferation of CD3-activated human T lymphocytes is increased by recombinant Fas ligand, cFLIP-L interacts with tumor necrosis factor receptor-associated factors 1 and 2, as well as the kinases RIP and Raf-1, resulting in the activation of the NF-B and ERK signaling pathways (17). The ability of cFLIP-L to switch Fas-mediated glucose signaling from a death signal into a proliferation signal in human pancreatic ␤ cells was also reported (24).The Wnt signal transduction pathway plays a critical and evolutionarily conserved role during embryogenesis (3, 10, 46). The Wnt signal is mediated by ␤-catenin, a transcription factor that is normally degraded by the ubiquitin-proteasome system in cytosol. Phosphorylation of ␤-catenin by a large protein complex involving adenomatous polyposis coli (APC) protein, Axin, and glycogen synthase kinase 3␤ (GSK3␤) initiates the ubiquitylation and proteasomal degradation of ␤-catenin (15, 49). Upon Wnt signaling, phosphorylation of ␤-catenin is inhibited, which results in the accumulation and translocation of ␤-catenin into nuclei, thereby inducing the expression of several genes, such as c-myc and the cyclin D gene. Mutations in APC, Axin, and ␤-catenin genes resulting in abolished ␤-catenin ubiquitylation are found in many human cancers (4,9,20,26,30,32,35,37), indicating that inappropriate activation of Wnt signaling plays an important role in human cancers (31,33).In this paper, we report that cFLIP-L inhibits ␤-catenin ubiquitylation and enhances Wnt signaling, which suggests an additional mechanism involved in tumorgenesis, in addition to inhibiting apoptosis signaling. MATERIALS AND METHODSPlasmids. Human cFLIP-L and a short splicing variant of cFLIP (cFLIP-S) were amplified by PCR from a Jurkat cDNA library and subcloned into pcDNAbased mammalian expression vectors (Invitrogen). For deletion mutant constructs, DNA sequences corresponding to different regions of cFLIP-L were

Background: Recent genetic and pathological studies have suggested that a lipoprotein receptor, LR11, is intricately implicated in the pathogenesis of Alzheimer disease (AD). We have recently established a novel sandwich ELISA, which enabled the sensitive quantification of a soluble LR11 (sLR11). By this ELISA, we attempted to determine the difference in the levels of CSF sLR11 in AD patients. Methods: We examined CSF from 29 AD patients, 20 frontotemporal lobar degeneration patients and 27 age-matched control subjects. The CSF sLR11 level as well as the levels of tau and β-amyloid42 (Aβ42) were determined by sandwich ELISA. Results: The CSF tau level and tau/Aβ42 ratio were significantly increased (p < 0.01) in the AD patients. The CSF sLR11 level in the AD patients was significantly higher (p < 0.01) than that of the frontotemporal lobar degeneration patients and the controls. The APOE-Ε4-positive AD patients have higher sLR11 levels than the APOE-Ε4-negative patients (p < 0.01). Conclusions: These results suggest that the quantification of CSF sLR11 may serve as a biomarker of AD, although the diagnostic value for individual patients is limited. An elevated CSF sLR11 level in AD patients may be relevant to AD pathogenesis.

1581 Introduction: LR11 is a type I membrane protein from which a large extracellular part, soluble LR11 (sLR11), is released by proteolytic shedding. It plays a key role in the migration of undifferentiated vascular smooth muscle cells, and circulating sLR11 is a known biomarker of carotid intima-media thickness. We have recently found that LR11 is specifically and highly expressed on the cell surface of acute leukemia cells in addition to normal leukocytes. Furthermore, patients with various hematological malignancies showed significantly high serum sLR11 levels especially in B-cell acute lymphoblastic leukemia. Serum sLR11 level has a significant association with remission and survival rate in patients with acute leukemia (Ohwada et al. 2010 ASH annual meeting). Based on these findings, we have retrospectively evaluated the clinical importance of serum sLR11 in patients with diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). Patients and Methods: Fifty-one patients with DLBCL and 23 patients with FL treated at Chiba University Hospital between 2002 and 2011 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). Patient biopsy specimens were subjected to immunostaining using anti-LR11 antibody. Serum sLR11 levels were measured by ELISA. Patient laboratory data and treatment outcome were obtained retrospectively. Results: Immunostaining of paraffin-embedded lymphoma tissue revealed that the cytoplasm of lymphoma cells of both DLBCL and FL specifically reacted against the anti-LR11 antibody. Furthermore, serum sLR11 levels of patients with lymphoma were significantly increased (DLBCL: 17.4±14.7 ng ml−1, FL: 22.7±25.5 ng ml−1) compared with those of normal control subjects (8.8±1.79 ng ml−1, P <0.0001). Paired sample analysis of patients at complete remission after chemotherapy showed significantly decreased sLR11 levels compared with that at the time of diagnosis (DLBCL: 13.6 ± 8.4 ng ml−1 vs. 10.8 ± 5.9 ng ml−1, P =0.045; FL: 20.0 ± 26.4 ng ml−1 vs. 7.4 ± 1.8 ng ml−1, P =0.0019). Multiple stepwise linear regression analysis showed that the serum sLR11 level at diagnosis was independently associated with bone marrow invasion in patients with DLBCL and bone marrow invasion and elevated LDH levels in patients with FL (DLBCL: r2 = 0.33, P =0.037, FL: r2 = 0.51, bone marrow invasion: P =0.0067, elevated LDH levels: P =0.013). Four of 7 patients with FL and high (≥16 ng ml−1) sLR11 levels relapsed, while none of the 16 patients with FL and low (≤16 ng ml−1) sLR11 did. The relapse rate was significantly higher in patients with FL and serum sLR11 levels ≥16 ng ml−1 (0% vs. 57%, P<0.004). At the median follow-up period of 13.5 months, the probability of 5-year progression-free survival (PFS) was significantly higher in patients with sLR11 >16 ng ml−1 at diagnosis compared with those with ≤16 ng ml−1 (Figure 1, 38.1 % vs 100 %, P =0.005). Stepwise Cox regression analysis showed that high serum sLR11 at diagnosis was the only independent risk factor associated with PFS in patients with FL. Conclusions: sLR11 was demonstrated to be expressed in B cell lymphoma cells by immunohistochemistry. Elevated serum sLR11 levels were associated with bone marrow invasion, and they significantly decreased to normal range after the remission. These findings suggest that sLR11 is a novel biomarker derived from lymphoma cells, which reflects not only tumor burden but also its migration and attachment activities, translating into inferior PFS in patients with FL. Although the number of patients in this study was limited, serum sLR11 can be a promising biomarker in patients with B cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

4834 Introduction: LR11 (also called SorLA or SORL1) is a type I membrane protein, from which a large extracellular part, sLR11, is released by proteolytic shedding. LR11 plays a key role in the migration of undifferentiated vascular smooth muscle cells, and circulating sLR11 is a biomarker of carotid intima-media thickness. In accordance with sLR11 levels correlating with the fraction of immature vascular cells, human CD34+CD38- immature hematopoietic precursors display high levels of LR11 mRNA. We investigated the expression of LR11 in normal leukocytes, leukemia cell lines and acute leukemia cells. Methods: A2-2-3 anti LR11 monoclonal antibody was used for immunoblotting. Biotinylated or FITC-conjugated anti LR11 monoclonal antibodies, M3 and R15 were used for flow cytometric analysis and immunohistochemistry. Normal mononuclear cells were obtained from healthy volunteer donors. Leukemia cells were obtained from patients' bone marrow or peripheral blood. LR11 protein levels and sLR11 in the culture supernatant of human leukemic cell lines were examined by Western blotting and ELISA using specific monoclonal antibody against LR11. The expression of LR11 mRNA of the cells was examined by Real-Time PCR. Flow cytometric analysis of cell surface LR11 was performed with desktop cell sorter JSAN (Bay Bioscience). Results: Most human leukemia cell lines expressed high level of LR11 mRNA and protein. sLR11 was also detected in the culture supernatant. The levels of LR11 mRNA, the amount of cellular LR11 protein, and the amount of released sLR11 protein were significantly correlated with each other. Flow cytometric analysis of peripheral leukocytes using the anti-LR11 mAb M3, showed expression of LR11 in most CD14+ monocytes. LR11 was not significantly expressed on most T cells (CD4+, CD8+), B cells (CD19+), or granulocytes. However, the leukemia cell lines HL-60 (acute promyelocytic), CCRF-SB (lymphoblastic), and U937 (monocytic), but not K562 (chronic myelogenous) expressed LR11. Since LR11 is expressed by leukemia cells of different origins, we explored the expression of LR11 on the surface of patients' leukemia cells. We have examined 7 AML cases (M0, M1, M2, M3, M4, M5 and M6) and 3 ALL cases. Although expression level of LR11 differs among these cases, LR11 was detected in every case except one ALL case. The most dramatic M3-stained population was the clonally expanded CD19+ mononuclear fraction in MLL-AF4 positive early precursor B acute lymphoblastic leukemia (ALL). In addition, more than 50% blastic cells were positive for LR11 in a Philadelphia chromosome positive ALL patient. Over 50% of CD34+ mononuclear cells in AML (M0) were LR11-positive, whereas LR11-positive blasts predominated in the CD38- fraction. The majority of mononuclear cells in AML (M4) with high CD11b-expression were also LR11-positive. Thus, LR11 is specifically expressed on the surface of leukemic blasts in both ALL and AML. Furthermore, immunohistochemistry of bone marrow clot sections of AML and ALL revealed that cytoplasm of leukemia cells are specifically reacted against the anti-LR11 antibody. Thus, LR11 is expressed both in the cytoplasm and on the cell surface of acute leukemia cells. Conclusion: LR11 is specifically and highly expressed on cell surface of acute leukemia cells in addition to normal leukocytes. Together with our finding that sLR11 is a novel marker for acute leukemia, the identification of novel surface antigen sheds light on leukocyte biology and leukemia cell development. Disclosures: No relevant conflicts of interest to declare.