ALBERT

Description: The blockade of Bcr-Abl signaling suppresses cellular growth and induces cell death in Bcr-Abl+ cells. While they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we assessed the regulatory mechanisms for cellular survival and death of Bcr-Abl+ leukemias more precisely, using a novel Bcr-Abl tyrosine kinase inhibitor, INNO-406 (formerly NS-187) which is more selective and 25-55-fold more active than imatinib (Kimura S, Blood 2005), in four CML-derived Bcr-Abl+ cell lines (K562, KT-1, BV173 and MYL), Ba/F3 harboring wild type bcr-abl (Ba/F3/wt bcr-abl), and in vivo CML mouse model. INNO-406 induces apoptosis in all lines examined, as were demonstrated by typical apoptotic morphology, loss of mitochondrial outer membrane potential (reduction of DiOC6 uptake), increase of cells in subG1 fraction by propidium iodide (PI) staining, DNA fragmentation and caspase-3 activation. However, when we inhibit caspase activity by zVAD-fmk (zVAD), a pan-caspase inhibitor, two modes of cell death execution were observed. In K562, KT-1 and BV173 cells treated with INNO-406, zVAD almost completely prevented apoptosis (i.e. showing atypical feature for apoptosis, no DNA fragmentation and no accumulation of subG1 fraction), with cell death resulting from morphologically non-apoptotic, so-called caspase-independent necrosis-like cell death (CIND). While, in MYL and Ba/F3/wt bcr-abl cells, despite the sufficient inhibition of caspases’ activity, the inhibition of the cell death by zVAD was only partial and these cell lines still underwent apoptosis (i.e. showing DNA fragmentation and the accumulation of subG1 population), suggesting the presence of alter cell death pathway which is caspase-independent apoptosis (CIA) in MYL and Ba/F3/wt bcr-abl. The propensity towards CIND or CIA in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity, that is CIND with a high apoptosome activity potential while CIA with low. Freshly isolated leukemic cell samples from Bcr-Abl+ leukemia patients also had either low or high apoptosome activity potential. Moreover, cells undergoing CIND exhibited hallmarks of autophagy (i.e. the autophagosome formation, punctate formations of LC3 and the accumulation of LC3-II isoform), suggested the participation of autophagy in response to Bcr-Abl blockade. Inhibition of autophagy with chloroquine enhanced INNO-406-induced cell death, which indicates that the autophagic response of the tumor cells is protective. While, in vivo CML model, INNO-406 treatment increased apoptotic cells regardless of the caspase-3 activation, further implicating the involvement of caspase-independent cell death regulatory pathway in vivo in primary Bcr-Abl+ leukemic cells. These findings suggest new insights into the biology and therapy of Bcr-Abl+ leukemias.

Description: Central nervous system (CNS) is one of the major cites for extramedullary relapse of Ph+ leukemias, which have been treated with imatinib mesylate (IM). The reason for this is that IM is a substrate for P-glycoprotein (P-gp) at the blood brain barrier and effluxed by it. We have already shown in the last annual meeting that INNO-406 had much stronger anti-tumor effects against the murine CNS leukemia model compared with IM, and INNO-406 is also effluxed from the murine CNS by P-gp. In this study, we investigated the combination effect of INNO-406 and P-gp inhibitors, verapamil or cyclosporin-A (CsA). First, we examined the growth-suppressive effect of INNO-406 and the combination with the P-gp inhibitors against the BCR-ABL positive leukemic cell line, K562 and the P-gp-overexpressing K562, K562/D1-9 cell line. K562/D1-9 showed 10 times higher resistant to both IM and INNO-406 compared with K562. Furthermore, both verapamil and CsA synergistically augmented the effect of INNO-406. Next, we investigated the pharmacokinetics of INNO-406 when orally administrated with CsA to mice. Mice were administrated p.o. with 50mg/kg of CsA 2 hours before INNO-406. We found that the concentration of INNO-406 in the CNS elevated by 2 times when combined with CsA, while the plasma concentration was decreased to two thirds of that when singly administrated with INNO-406. It was suggested that the decreased plasma concentration of INNO-406 seen here resulted from the increased uptake into the CNS by CsA inhibiting P-gp at the blood brain barrier. These changes of the drug distribution to the murine tissues may alter the anti-leukemic effect of INNO-406, thus we planned to investigate the combination effect of INNO-406 and CsA in the murine models of both CNS and systemic leukemia. We found that CsA significantly augmented the anti-tumor effect of INNO-406 in the CNS leukemia model. Moreover, in spite of the decreased plasma concentration of INNO-406, the combination with CsA also prolonged the survival phase of the mice in the systemic leukemia model, more significantly than single treatment of INNO-406 (Figure). This may be explained by which CsA increased the intracellular uptake of INNO-406, resulted from the direct inhibition of drug efflux via P-gp expressed in the leukemic cells. Phase I clinical study on INNO-406 is now underway in MD Anderson Cancer Center and in Frankfurt University. From the results of this study, we expected the effective application of INNO-406 in combination with P-gp inhibitor to the patients suffering from refractory Ph+ leukemia as well as CNS relapse. Figure Figure

Description: Many tumor cells become resistant to commonly used cytotoxic drugs due to the overexpression of ATP-binding cassette (ABC) transporters. Specifically, p-glycoprotein (MDR-1) is frequently up-regulated in chemotherapy-resistant tumor cells, which is associated with poor prognosis. On the other hand, human leukocyte antigen (HLA) class I molecules are known to be significantly down-regulated in advanced tumor cells to escape from immune surveillance. However, the relationship between MDR-1 expression and HLA expression is not fully understood. Recently, we have developed a recombinant single-chain Fv diabody specific to HLA-A and demonstrated that this agent mediates cell death in HLA-overexpressing lymphoid tumor cells but not in normal cells (Cancer Res2007; 67:1184). Here, we investigated the expression levels of HLA class I in chemo-resistant leukemic cells and evaluated the therapeutic potential of single-chain Fv diabody specific to HLA-A, C3B3-DB (Chugai Pharmaceutical Co. Ltd., Tokyo, Japan). Chemotherapy-resistant cells were established by subculturing of myeloid leukemia cell line HL60 and Burkitt’s lymphoma cell line BL-TH in increasing doses of vincristine (VCR), and named HL60/VCR and BL-TH/VCR, respectively. MDR-1 is strongly expressed in HL60/VCR and BL-TH/VCR cells both at the mRNA and protein levels, but not in the parental cells. Interestingly, expression levels of HLA class I molecules are 8 times higher in HL60/VCR and BL-TH/VCR cells than in the parental cells, suggesting that MDR-1 modulates cell surface expression of HLA by its transporter function. Next, we examined the cytotoxic activity of C3B3-DB on these chemo-resistant cell lines. C3B3-DB induced apoptosis in HL60/VCR and BL-TH/VCR cells and these chemo-resistant cell lines were more sensitive to C3B3-DB than the parent cells. Combination of C3B3-DB with chemotherapeutic agents such as VCR and daunorubicin (DNR) resulted in enhanced cytotoxicity against HL60/VCR and BL-TH/VCR cells. Importantly, pretreatment of these chemo-resistant cell lines with C3B3-DB reduced expression levels of MDR-1 and increased drug retention in these cells as detected by flow cytometry and confocal microscopy. Furthermore, combination of C3B3-DB with VCR significantly blocked the cell cycle at the G2 phase compared with VCR alone. Similar results were obtained with primary acute myeloid leukemia cells from 2 patients, resulting in up-regulation of both HLA class I and MDR-1 molecules at relapse phase compared at diagnosis. These results suggest that C3B3-DB enhances cytotoxicity of chemotherapeutic agents and provides a novel approach for overcoming drug resistance in hematological malignancies.

Description: ß-catenin is the downstream effector of the Wnt signaling pathway and has cyclin D1 and c-myc as its target genes. Activation of Wnt signaling is greatly involved in the process of carcinogenesis, and its overexpression has reported to be positively correlated with prognosis in many types of malignant tumors, as colorectal cancer and hepatocellular carcinoma, or hematological malignancy, as acute myeloid leukemia and chronic myeloid leukemia. Multiple myeloma (MM) is a relatively common hematological malignancy and remains incurable with conventional treatments. Here, we assessed the expression of ß-catenin in MM and growth inhibitory effect of ß-catenin siRNA on MM. We first examined the expression of ß-catenin on human MM cell lines: AMO-1, EJM, IM-9, KMS-12-BM, LP-1, NCIH929, OPM-2, RPMI8226, and U266. All cell lines examined expressed a significantly higher level of ß-catenin than normal human mononuclear cells. Moreover, ß-catenin was also overexpressed in myeloma cells from patients. We then assessed the in vivo growth inhibitory effects of ß-catenin siRNA in mouse model. Six -week-old male Balb/c nu/nu mice were subcutaneously inoculated in the right flank with 5 × 106 RPMI8226 myeloma cells in 100μl PBS. Three to four weeks later, when palpable tumors (100 mm3 in diameter) developed, mice (n = 5 for all groups) were treated with either subcutaneous (around tumors) injections of group A: ß-catenin siRNA (2.5μM) / atelocollagen complex (final atelocollagen concentration 0.5%), group B: control siRNA (2.5μM) /atelocollagen complex, group C: ß-catenin siRNA (2.5μM)/PBS, group D: PBS/atelocollagen twice a week for a total of eight injections. Tumor size was measured in two dimensions using a caliper, and tumor volume (mm3) was calculated as a2 × b /2 mm3 (a;minor axis,b;major axis). The mean tumor volume of each group after two weeks treatment was 412.2 mm3 in groupA, 1317.9 mm3 in group B, 2075.9 mm3 in group C, and 1802.3 mm3 in group D, respectively, and the treatment with ß-catenin siRNA (2.5μM) / atelocollagen complex significantly reduced tumor burdens and retarded tumor growth, measured as tumor volumes (p

Description: Background: The dose of imatinib 400 mg is considered to be standard for chronic-phase chronic myeloid leukemia (CML) based on the IRIS study. However, the optimal dose of imatinib is not yet settled and the response of lower dose imatinib has not been investigated. Lower dose of imatinib might be enough in Asian patients since the results of PK in IRIS study showed weak correlation between imatinib trough level and body weight. We showed the results of the interim analysis of JALSG CML202 study and analyzed the efficacy of imatinib according to the mean daily dose level. Methods: The objectives of CML202 study are to determine the efficacy of imatinib monotherapy in patients with newly diagnosed chronic-phase CML, and to test whether combining imatinib with IFN or cytarabine ocfosfate overcome the resistance of imatinib and to estimate the frequency and severity of toxicities. Primary end point of imatinib monotherapy was overall survival, and that of combination therapy was cytogenetic response after 9 months. Newly diagnosed patients with chronic phase CML will receive imatinib at a dose of 400mg daily. At 9 months, if patients don’t achieve major cytogenetic response (MCR), they were randomized to the combination therapies, imatinib plus conventional IFN or imatinib plus cytarabine ocfosfate. Results: From 2002 to 2006, 489 patients were enrolled to this study. On July 2007, 36 months median follow-up data were analyzed. Only 2 patients were enrolled to the combination therapy because a majority of patients achieved good response. 13 patients underwent HSCT. The estimated cumulative rates of complete hematologic response (CHR), MCR and complete cytogenetic response (CCR) were 97%, 97% and 91%, respectively. The estimated major molecular response (MMR) rate at 24 months was 55%. The estimated survival without AP/BC progression rate at 60 months was 94%. The overall estimated survival at 60 months was 94%. 95% of low risk patients achieved CCR significantly as compared with intermediate and high-risk patients according to Sokal risk. The mean daily dose of imatinib for the first 2 years was 400mg or more in 54% patients (400mg group), 300–399mg in 27% (300mg group), 200–299mg (200mg group) in 11%, and 100–199mg in 9% (100mg group), respectively. The cumulative rate of MCR and CCR at 60 months was 99% and 97% in 400mg group, 99% and 98% in 300mg group, 92% and 85% in 200mg group, and 81% and 48% in 100mg group, respectively (p=0.003 and p

Description: Toll-like receptors (TLRs) and complement are 2 components of innate immunity that are critical for first-line host defense and elicitation of adaptive immune responses. Many pathogen-associated molecular patterns activate both TLR and complement, but whether and how these 2 systems, when coactivated in vivo, interact with each other has not been well studied. We demonstrate here a widespread regulation of TLR signaling by complement in vivo. The TLR ligands lipopolysacharride (TLR4), zymosan (TLR2/6), and CpG oligonucleotide (TLR9) caused, in a complement-dependent manner, strikingly elevated plasma interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and IL-1β, and/or decreased plasma IL-12 levels in mice deficient in the membrane complement inhibitor decay-accelerating factor (DAF). A similar outcome was observed in wild-type mice cotreated with the TLR ligands and cobra venom factor, a potent complement activator. The regulatory effect of complement on TLR-induced cytokine production in vivo was mediated by the anaphylatoxin receptors C5aR and C3aR. Additionally, changes in lipopolysaccharide (LPS)–induced cytokine production in DAF-deficient mice correlated with increased mitogen-activated protein kinase and nuclear factor-κB activation in the spleen. These results reveal a strong interaction between complement and TLR signaling in vivo and suggest a novel mechanism by which complement promotes inflammation and modulates adaptive immunity.

Description: Umbilical cord blood (UCB) has been used as a potential source of various kinds of stem cells, including hematopoietic stem cells, mesenchymal stem cells, and endothelial progenitor cells (EPCs), for a variety of cell therapies. Recently, EPCs were introduced for restoring vascularization in ischemic tissues. An appropriate procedure for isolating EPCs from UCB is a key issue for improving therapeutic efficacy and eliminating the unexpected expansion of nonessential cells. Here we report a novel method for isolating EPCs from UCB by a combination of negative immunoselection and cell culture techniques. In addition, we divided EPCs into 2 subpopulations according to the aldehyde dehydrogenase (ALDH) activity. We found that EPCs with low ALDH activity (Alde-Low) possess a greater ability to proliferate and migrate compared to those with high ALDH activity (Alde-High). Moreover, hypoxia-inducible factor proteins are up-regulated and VEGF, CXCR4, and GLUT-1 mRNAs are increased in Alde-Low EPCs under hypoxic conditions, while the response was not significant in Alde-High EPCs. In fact, the introduction of Alde-Low EPCs significantly reduced tissue damage in ischemia in a mouse flap model. Thus, the introduction of Alde-Low EPCs may be a potential strategy for inducing rapid neovascularization and subsequent regeneration of ischemic tissues.

Description: Antibodies to neutrophil antigens have been implicated in neonatal alloimmune neutropenia, autoimmune neutropenia, and transfusion-related acute lung injury. Although FCM analysis with a typed panel of neutrophils has been used to detect such antibodies, this procedure has two major limitations. First, only freshly isolated neutrophils can be used for the assay. Second, the assay cannot identify an HNA antibody in the presence of anti-HLA class I antibodies that react to panel neutrophils. To overcome these limitations, we established a panel of cell lines in which the HNA-1a (−1b; polymorphisms of HNA-1), HNA-2a, HNA-4a (−4b), or HNA-5a (−5b) gene was transduced using a retrovirus vector to confer stable transgene expression in K562 cells that dose not express HLA or HNA and not exhibit background reactivity to normal human serum samples. We confirmed the specificity of the one of each HNA expression on the K562 panel cells by using mAbs. We further showed that several well-characterized human sera containing antibodies against several HNAs were unambiguously identified by our panel cell lines. We also observed a lower background reactivity of the K562 panel cell lines compared with isolated neutrophils, which have been used for the panel to identify antibodies against HNA in human sera. These results indicate that the K562 panel cell lines provide a good panel for detecting HNA-reactive neutrophil antibodies in human sera.

Description: In antibody-mediated non-hemolytic transfusion reactions, Transfusion-Related Acute Lung Injury (TRALI) tends to occur immediately after a blood transfusion. Leukocyte antibodies or immune complexes have been frequently shown to be associated with the syndrome, although the mechanisms by which they induce TRALI are poorly understood. To explore the mechanism of TRALI, an in vitro whole blood cell culture assay was established in which cells were stimulated by human antibodies and the activation of neutrophils was monitored by a cell surface marker (Mac-1) with FCM. To identify soluble mediators that were released instantly following immune stimulation, we examined the release of soluble factors, including, Perforin, IL-6, TNF-a, and Heparin binding protein (HBP) using ELISAs. In addition, the involvement of two neutrophil FcγRs (FcγRIIIb and FcγRIIa, also known as CD16 and CD32, respectively) in release of HBP was examined during antibody-induced cell activation using anti-FcγR blocking antibodies. Substantial amounts of HBP were released within 30 min upon stimulation by human antibodies, although other soluble mediators were not released within such an eary period. Furthermore, the release of HBP was mediated via signals though both FcγRIIIb and FcγRIIa. HBP that have been known as a multifunctional molecule in early inflammation appears to be a one of the primary effector molecules of antibody-mediated non-hemolytic transfusion reactions including TRALI.

Description: Chronic myelogenous leukemia (CML) is characterized by its refractoriness to various apoptotic insults by Bcr-Abl tyrosine kinase (TK)-mediated signalling. Although imatinib mesylate (IM), a Bcr-Abl TK inhibitor, has markedly improved the therapeutic outcomes of CML, additional or alternative molecular targeting strategies are still needed. Since the interplay of anti-apoptotic Bcl-2 proteins and BH3-only proteins, such as Bim and Bad, is crucial for regulating the cellular fate of Bcr-Abl+ leukemic cells (Kuroda J et al, PNAS , 2006; Cell Death Differ, 2007), the direct targeting of anti-apoptotic Bcl-2 proteins by the use of a BH3-only protein mimetic is an attractive approach for treating CML. We here investigated the activity of ABT-737, a mimic of BH3-only proteins that inhibit anti-apoptotic Bcl-2, Bcl-XL and Bcl-w, but not Mcl-1 or A1, against CML. The Annexin-V-staining study, the assay for mitochondrial outer membrane potential and the internucleosomal fragmentation assay revealed that ABT-737 potently induced apoptosis in CML cell lines (BV173, K562, KCL22, KT-1, MEG-01 and MYL) with the IC50 for induction of cell death by 48 h of treatment ranging from 0.04 to 4.06 μM. ABT-737 was also effective in killing primary CML samples in vitro. ABT-737 prolonged the survival of mice xenografted with a CML cell line, BV173, demonstrating its in vivo bioactivity. Higher expression of Bcl-2, Bcl-XL, or Mcl-1 reduced cell killing by ABT-737 in each cell line, but we found no correlation between the sensitivities to ABT-737 and the specific expression patterns of Bcl-2 family proteins among different cell lines. The levels of Bcr-Abl and Lyn, a member of Src kinase family associated with apoptosis resistance in CML, also varied among the cell lines, and we found no consistent relationship between the sensitivity to ABT-737 and the expression levels of these proteins Thus, the cell killing effect of ABT-737 in CML must be determined in part by other drug resistance mechanisms, such as high expression of Bcr-Abl, overexpression of P-glycoprotein (P-gp), a drug-efflux pump, and/or their combination. Importantly, ABT-737 augmented the cell killing effect of IM in CML cell lines with high levels of anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-XL, or even Mcl-1), Bcr-Abl, P-gp, or Lyn, unless leukemic cells harboured IM-insensitive Abl kinase domain (KD) mutations. Moreover, the combination of ABT-737 with homoharringtonine, an herbal-derived anti-CML therapeutic that potently reduces Mcl-1 within 6 hours in vitro, dramatically enhanced the killing by ABT-737 in CML cells with diverse drug resistance mechanisms, including IM-insensitive Abl KD mutations, such as T315I mutation. These results suggest that ABT-737 could be a useful component of combinatory chemotherapies for CML.