ALBERT

Description: CD26 is a transmembrane glycoprotein with intrinsic dipeptidyl peptidase IV (DPPIV) activity as well as costimulatory activity of mitotic signals triggered by the CD3/TCR complex. Based on the expression level of CD26, CD4+ and CD8+ T cells can be divided into 3 (high/intermediate/low or negative) subsets. The significance of CD26 has been studied mainly on CD4+ T cells, and CD26highCD4+ T cells are considered to represent effector memory T cells of a typical Th1 phenotype producing IL2 and IFNg. Furthermore, we reported a significant decrease of this subset in CML patients under imatinib therapy in comparison to those under IFNa therapy and normal volunteers. In contrast, the role of each subset of CD8+ T cells has not yet been clarified. Multi-parameter flow cytometry analysis was performed to characterize CD8+ T cells differentially expressing CD26 in combination with intracellular detection of effector molecules such as perforin (P) and granzyme B (Gr). The capacity to secrete effector cytokines such as IFNg following short-term stimulation was also assessed. As a result, according to the expression level of CD26, we could clearly categorize CD8+ T cells as follows: CD26highCD8+ T cells are defined as central memory T cells which has a phenotype of CD45RO+CD28+CD27+ IFNg+Gr−P+/−, CD26intCD8+ T cells as naïve T cells of CD45ROCD28+ CD27+ IFNg−Gr−P−, and CD26lowCD8+ T cells as effector memory/effector T cells of CD45RO−/+ CD28−CD27−IFNg++Gr++P++, respectively. We next investigated the effects of imatinib on 3 distinct subsets during CD8+ T cell differentiation program. Peripheral blood mononuclear cells were primed with anti-CD3/CD28 MAb and subjected to the grading doses of imatinib for short term culture, followed by flow cytometory. CFSE labeling was used for monitoring cell proliferation. Intriguingly, we found that imatinib dose-dependently inhibits activation, cytokine production and proliferation of CD26highCD8+ central memory T cell subsets in a differentiation stage-specific manner. Finally, we compared the absolute number of peripheral blood CD26highCD8+ T cell subsets between 20 patients with CML in imatinib-induced CCR and 20 normal volunteers, clearly indicating a significant decrease of this subset in CML patients (22.30/ml vs 45.60/ml, p

Description: This study found that MS-275, a novel synthetic benzamide histone deacetylase inhibitor (HDACI), blocked Akt/mTOR signaling in acute myelogenous leukemia (AML) HL60 and acute promyelocytic leukemia (APL) NB4 cells, as assessed by decreased levels of the phosphorylated (p)-Akt, p-p70 ribosomal S6 kinase (p70S6K), and p-S6K by Western blot analysis. Interestingly, further inactivation of mTOR by rapamycin analogue RAD001 (everolimus) significantly enhanced MS-275-mediated growth inhibition and apoptosis of these cells in parallel with enhanced upregulation of p27 kip1 and downregulation of c-Myc. In addition, RAD001 potentiated the ability of MS-275 to induce differentiation of HL60 and NB4 cells, as measured by expression of CD11b cell surface antigens, as well as reduction of nitroblue tetrazolium. Importantly, RAD001 potentiated the ability of MS-275 to induce expression of the myeloid differentiation-related transcription factor CCAAT enhancer binding protein e in these cells in association with enhanced acetylation of histone H3 on its promoter. Furthermore, RAD001 (5 mg/kg) significantly enhanced the effects of MS-275 (10 mg/kg) to inhibit proliferation of HL60 tumor xenografts in nude mice without adverse effects. Taken together, concomitant administration of a HDACI and a mTOR inhibitor may be a promising treatment strategy for the individuals with a subset of human leukemia.

Description: Introduction: Inotuzumab ozogamicin (CMC-544) is an antibody-targeted chemotherapeutic agent composed of a monoclonal antibody which targets the CD22 antigen, conjugated to calicheamicin, a potent cytotoxic antitumor antibiotic. Since CD22 is expressed on more than 90% of B-lymphoid malignancies, CMC-544 may be useful for treating patients (pts) with B-cell non-Hodgkin lymphoma (B-NHL). In a phase I study in the US and EU, CMC-544 showed definite clinical activity in pts with relapsed/refractory B-NHL (both follicular and diffuse large) with clinically manageable thrombocytopenia as the main toxicity. In this phase 1 study, the safety, tolerability, efficacy and pharmacokinetics (PK) of CMC-544 was evaluated in Japanese pts with relapsed or refractory B-cell NHL. Methods: CMC-544 was administered IV once every 28 days ± 2 days (1 cycle). A dose escalation was planned using 1.3 mg/m2 and 1.8 mg/m2, the maximum tolerated dose (MTD) which was previously determined in non-Japanese pts. Pts were allowed to enroll in the study if they had relapsed or refractory CD22+ B-NHL. Tumor responses were evaluated by investigator’s assessment according to the International Workshop Criteria for NHL. Results: Enrollment for this study is complete and included 13 pts (6 women, 7 men, median age [range] of 49 yrs [43–72]). All of the 13 pts who were enrolled had follicular lymphoma and had received at least one regimen of rituximab alone or rituximab-containing chemotherapy in their prior treatments. The median number of prior treatment regimens was 1 (range: 1–13). In dose escalation, no pts had dose limiting toxicities and the tolerability in the MTD previously determined in non-Japanese pts (1.8mg/m2) was confirmed for Japanese pts. 3 pts and 10 pts were treated with 1.3 mg/m2 and 1.8 mg/m2 of CMC-544, respectively. The median number of CMC-544 treatment cycles was 3 (range: 2–8). The most common drug-related adverse events (AEs, all grades ≥ 35% pts) included thrombocytopenia (100%), leukopenia (92%), neutropenia (85%), elevated AST (85%), anorexia (85%), lymphopenia (85%), nausea (77%), elevated ALT (54%), malaise (46%), and headache (46%). Grade 3/4 AEs ≥ 15% pts were: thrombocytopenia (54%), lymphopenia (31%), neutropenia (31%), and leukopenia (15%). 7 pts discontinued treatment due to AEs; 1 pt because of grade 2 rash, 1 pt because of grade 2 urticaria and 5 pts because of AEs which required treatment delays of 〉3 wks (2 pts with prolonged thrombocytopenia, 1 pt with prolonged thrombocytopenia and neutropenia, 1 pt with neutropenia and elevated alkaline phosphatase, and 1 pt with prolonged neutropenia and elevated total bilirubin). PK analyses demonstrated that maximum serum concentration (Cmax) and area under the curve (AUC) of CMC-544 increased in a dose dependent manner. Both parameters increased with the second dose in the second cycle. At all dose levels, terminal half-life (t1/2) was prolonged, and total clearance (CL) was decreased in the second cycle. Overall, the PK profile was similar to that of the previous study with non-Japanese pts. 7 pts had CRs (CR + CRu), 4 pts had PRs, and 2 pts had stable disease. The objective response rate (ORR) was 85% (11/13). Conclusions: The tolerability of CMC-544 for Japanese pts with relapsed or refractory follicular B-NHL who had been pretreated with rituximab was confirmed at 1.8 mg/m2 administered once every 28 days. This is the same dose level as the MTD for non-Japanese pts. The PK profile of CMC-544 in Japanese pts was similar to that in non-Japanese pts. Based on the acceptable safety profiles and a high preliminary ORR, CMC-544 should be considered for further investigations in Japanese pts with relapsed or refractory B-NHL who have been pretreated with rituximab.

Description: CD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in allograft- and self-tolerance and thus have the potential for therapeutic application in immunological and allergic disorders. However, the frequencies of Treg in peripheral blood are very low. Here we attempted the ex vivo expansion of Treg to enable the adaptive immunoregulatory therapy in humans. CD4+ T cells from peripheral blood of healthy donors or patients with chronic graft-versus-host disease (GVHD) were isolated by anti-CD4 monoclonal antibody (MAb)-conjugated magnetic beads, and cultured using a plastic plate coated with anti-CD28 and anti-CD3-MAbs in the medium containing recombinant human (rh) IL-2 and rhTGF-b. After one week of culture, expanding cells were once detached from the plate and subjected to the fresh medium including rhIL-2 and rhTGF-b but not MAbs. After 2-weeks of culture, phenotypic and functional analyses were performed. Mixed lymphocyte reaction was done using CFSE-labeled responder T cells and autologous or allogeneic dendritic cells (DC) with or without expanded Treg-rich populations. Xenogeneic -GVHD in NOD-Scid mice was induced by the injection of human T cells expressing luciferace transgene, followed by in vivo bioluminescence imaging (BLI) analysis using a CCD camera. Our expansion procedure with TGF-b yielded 45-83% purity of Foxp3+CD25+CD4+Treg co-expressing CTLA-4, CD54 and GITR, while 8-42% purity without TGF-b(p

Description: Animal models show infusions of donor NK cells given after allogeneic HCT can prevent GVHD while simultaneously mediating a graft-vs-tumor effect. However, it is unclear whether adoptively infused NK cells can mediate these beneficial effects in the presence of CSA, which is commonly given after HCT to prevent GVHD. In this study, we analyzed the in vitro effects of pharmacological concentrations of CSA on NK cell phenotype, cell proliferation, and tumor cytotoxicity. We also evaluated in vivo whether CSA administration would reduce the anti-tumor effects of adoptively infused NK cells in tumor bearing mice. PBMCs collected from healthy donors were labeled with CFSE then were stimulated in vitro with IL-2 for 7 days in the presence or absence of CSA (1000ng/ml). CFSE proliferation assays on fresh PBMC showed CSA inhibited IL-2 stimulated CD3+ T-cell proliferation more than CD3−/CD56+ NK cell proliferation (mean percentage inhibition of proliferation 49.4% vs. 22.2% for T cells and NK cells respectively; p

Description: Aurora B kinase is one of the key regulators of mitosis and has been reported to be over-expressed in a various malignancies including acute myelogenous leukemia; however, expression of Aurora B in non-Hodgkin’s lymphoma (NHL) and its role in lymphoma genesis remain to be fully elucidated. This study explored the levels of Aurora B in seventy four lymph nodes and tumor specimens excised operatively from individuals with various types of NHLs including diffuse large B cell lymphoma (n=22, DLBCL), follicular lymphoma (n=12, FL), Burkitt’s lymphoma (n=10, BL), mantle cell lymphoma (n=4, MCL), mucosa associated lymphoid tissue (n=4, MALT), adult T-cell leukemia/lymphoma (n=11, ATL), peripheral T-cell lymphoma (n=8, PTCL), and Hodgkin’s lymphoma (n=3, HL). Immunohistochemical examination found that DLBCL (11/22, 50 %) and BL (8/10, 80 %) cells highly (percentage of positive cells, 〉20 %) expressed Aurora B in their nucleus. On the other hand, FL (11/12, 92 %), MCL (1/4, 25 %), and MALT (1/4, 25 %) cells slightly (percentage of positive cells, 5–20 %) expressed Aurora B and none of them, except for one case of FL, highly expressed this protein kinase, suggesting that expression of Aurora B correlated with histological grade in B cell NHLs (p=0.0004). We next examined whether Aurora B could be a molecular target for treatment of NHLs utilizing BALM-14, -18, and -27, Daudi, Ramos, and Raji Burkitt’s lymphoma/leukemia cells. Exposure of these cells to AZD1152, a selective inhibitor of Aurora B kinase, potently induced apoptosis in association with activation of caspase 3, as measured by annexin V staining and Western blot analysis, respectively. Moreover, we found that AZD1152 synergistically enhanced the effects of vincristine (VCR), a tubulin depolymerizing agent, to induce growth arrest and apoptosis of these cells. Further experiments found that VCR increased levels of the phosphorylated forms of Aurora B (p-Aurora B) via activation of c-Jun N-terminal kinase (JNK), which was blocked in the presence of AZD1152 in BALM-27 cells. Taken together, Aurora B kinase is a promising molecular target for treatment of individuals with aggressive types of NHLs. The combined administration of AZD1152 and the conventional chemotherapeutic agents to patients with NHL warrants further investigation.

Description: The Ly49 natural killer (NK)–cell receptor family comprises both activating and inhibitory members, which recognize major histocompatibility complex (MHC) class I or MHC class I–related molecules and are involved in target recognition. As previously shown, the Ly49E receptor fails to bind to a variety of soluble or cell-bound MHC class I molecules, indicating that its ligand is not an MHC class I molecule. Using BWZ.36 reporter cells, we demonstrate triggering of Ly49E by the completely distinct, non–MHC-related protein urokinase plasminogen activator (uPA). uPA is known to be secreted by a variety of cells, including epithelial and hematopoietic cells, and levels are up-regulated during tissue remodeling, infections, and tumorigenesis. Here we show that addition of uPA to Ly49E-positive adult and fetal NK cells inhibits interferon-γ secretion and reduces their cytotoxic potential, respectively. These uPA-mediated effects are Ly49E-dependent, as they are reversed by addition of anti-Ly49E monoclonal antibody and by down-regulation of Ly49E expression using RNA interference. Our results suggest that uPA, besides its established role in fibrinolysis, tissue remodeling, and tumor metastasis, could be involved in NK cell–mediated immune surveillance and tumor escape.

Description: Ki11502 is a novel multitargeted receptor tyrosine kinase (RTK) inhibitor with selectivity against platelet-derived growth factor receptor alpha/beta (PDGFRα/β). Ki11502 (0.1-1 nM, 2 days) profoundly caused growth arrest, G0/G1 cell-cycle arrest, and apoptosis associated with down-regulation of Bcl-2 family proteins in the eosinophilic leukemia EOL-1 cells having the activated FIP1-like 1/PDGFRα fusion gene. Ki11502 decreased levels of p-PDGFRα and its downstream signals, including p-Akt, p-ERK, and p-STAT5, in EOL-1 cells. Of note, Ki11502 was also active against imatinib-resistant PDGFRαT674I mutant. In addition, Ki11502 inhibited proliferation of biphenotipic leukemia MV4-11 and acute myelogenous leukemia MOLM13 and freshly isolated leukemia cells having activating mutations in FMS-like tyrosine kinase 3 (FLT3). This occurred in parallel with the drug inhibiting FLT3 and its downstream signal pathways, as measured by fluorescence-activated cell sorting using the phospho-specific antibodies. In addition, Ki11502 totally inhibited proliferation of EOL-1 cells growing as tumor xenografts in SCID mice without any noticeable adverse effects. Taken together, Ki11502 has profound antiproliferative effects on select subsets of leukemia including those possessing imatinib-resistant mutation.

Description: MLL gene rearrangements are present in 5 to 10% of acute leukemias, which are generally associated with a poor prognosis. Chromosomal translocations at the MLL locus create MLL fusion genes that constitute 5’ portions of MLL and 3’ portions of partner genes. The resultant MLL fusion proteins transform myeloid progenitors by inappropriately activating a HOX-associated self renewal program. However, the molecular mechanisms underlying MLL oncoprotein function are not fully understood. Previously we identified menin as a component of the MLL macromolecular complex. Menin is a product of the MEN1 tumor suppressor gene, whose loss of function causes multiple endocrine neoplasia type 1 (MEN1). Despite its tumor suppressor role in endocrine tissues, menin functions as an essential oncogenic cofactor for MLL fusion protein-dependent leukemogenesis. This notion was supported by three lines of evidence: MLL fusion proteins lacking the menin binding motif do not transform myeloid progenitors; inactivation of menin causes growth arrest and subsequent differentiation of MLL oncogene transformed cells; and menin is required for MLL oncogene dependent transcriptional activation of HOX genes. These findings raised a fundamental question: how does menin contribute to MLL-dependent transcription? Because menin lacks known functional motifs, its molecular functions could not be deduced from its structure. We hypothesized that menin may function as an adapter that tethers MLL to unknown associated factors. To identify such associated factors, we performed affinity purification of the MLL-ENL/menin complex from nuclear extracts of cells that transiently over-expressed both MLL-ENL and menin. Mass spectrometry identified LEDGF, originally identified as a transcriptional co-activator, in the purified material as a novel associated factor. LEDGF associates conjointly with the MLL/menin complex but not with MLL or menin alone, supporting the hypothesis that menin plays an adapter role to bridge MLL and LEDGF. Further analysis revealed that LEDGF is critical for MLL fusion protein-dependent leukemogenesis. Fine mapping of the domain responsible for LEDGF binding determined MLL residues 109–153 as the LEDGF binding domain (LBD). Mutations in LBD resulted in loss of oncogenic activity of MLL fusion proteins. Moreover, knock down of LEDGF in MLL-transformed cells caused growth arrest and differentiation in the same manner as menin knock down. These results demonstrate that LEDGF is also required for the initiation and maintenance of MLL fusion protein-dependent transformation. In contrast to menin, LEDGF has a distinctive functional motif (the PWWP domain), which reportedly has chromatin binding activity. To further confirm that menin is an adapter that links MLL and LEDGF, we examined the oncogenic functions of an artificial MLL fusion protein whose menin binding motif is replaced by the PWWP domain of LEDGF. This PWWP-MLL-ENL fusion protein does not associate with menin because it lacks the menin binding motif, nevertheless transforms myeloid progenitors. Chromatin immunoprecipitation experiments show that the PWWP-MLL- ENL fusion protein localizes at the HOXA9 locus while menin is absent. Moreover, myeloid progenitors transformed by the PWWP-MLL-ENL fusion protein continue to proliferate after menin is genetically inactivated. Thus covalent tethering of the PWWP domain fully compensates for loss of menin’s cofactor function. Therefore, menin’s only role in MLL-associated leukemogenesis is to tether LEDGF to MLL fusion proteins. In summary, this study identifies a previously unknown essential oncogenic cofactor of MLL fusion proteins and proposes a stepwise association model in which the MLL fusion protein first associates with menin, then recruits LEDGF to its complex to become functionally active.

Description: Natural killer (NK) cells are innate immune cells that mediate resistance against viruses and tumors. They express multiple activating receptors that couple to immunoreceptor tyrosine-based activation motif (ITAM)–containing signaling chains for downstream cell activation. Ligation of activating NK-cell receptors induces NK-cell cytotoxicity and cytokine release. How these distinct events are selectively controlled is not well defined. Here we report the identification of a specific signaling pathway that operates downstream of the ITAM-coupled NK-cell receptors NK1.1, Ly49D, Ly49H, and NKG2D. Using primary NK cells from Bcl10−/−, Malt1−/−, Carma1−/−, and Card9−/− mice, we demonstrate a key role for Bcl10 signalosomes in the activation of canonical NF-κB signaling as well as JNK and p38 MAPK upon NK-cell triggering. Bcl10 directly cooperates with Malt1 and depends on Carma1 (Card11) but not on Card9 for NK-cell activation. These Bcl10-dependent cascades selectively control cytokine and chemokine production but do not affect NK-cell differentiation or killing. Thus, we identify a molecular basis for the segregation of NK-cell receptor–induced signals for cytokine release and target cell killing and extend the previously recognized roles for CARD-protein/Bcl10/Malt1 complexes in ITAM receptor signaling in innate and adaptive immune cells.