技术资料

CD38,as a cell surface antigen is highly expressed in several hematologic malignancies including multiple myeloma (MM) and has been proven to be a good target for immunotherapy of the disease. CD38 is also a signaling enzyme responsible for the metabolism of two novel calcium messenger molecules. To be able to target this multifunctional protein,we generated a series of nanobodies against CD38 with high affinities. Crystal structures of the complexes of CD38 with the nanobodies were solved,identifying three separate epitopes on the carboxyl domain. Chromobodies,engineered by tagging the nanobody with fluorescence proteins,provide fast,simple and versatile tools for quantifying CD38 expression. Results confirmed that CD38 was highly expressed in malignant MM cells compared with normal white blood cells. The immunotoxin constructed by splicing the nanobody with a bacterial toxin,PE38 shows highly selective cytotoxicity against patient-derived MM cells as well as the cell lines,with half maximal effective concentration reaching as low as 10(-11) molar. The effectiveness of the immunotoxin can be further increased by stimulating CD38 expression using retinoid acid. These results set the stage for the development of clinical therapeutics as well as diagnostic screening for myeloma. View Publication

We recently reported that chronic lymphocytic leukemia (CLL) subgroups with distinct clonotypic BCRs present discrete patterns of TLR expression,function,and/or tolerance. In this study,to explore whether specific types of BCR/TLR collaboration exist in CLL,we studied the effect of single versus concomitant BCR and/or TLR stimulation on CLL cells from mutated (M-CLL) and unmutated CLL (U-CLL) cases. We stimulated negatively isolated CLL cells by using anti-IgM,imiquimod,and CpG oligodeoxynucleotide for BCR,TLR7,and TLR9,respectively,alone or in combination for different time points. After in vitro culture in the absence of stimulation,differences in p-ERK were identified at any time point,with higher p-ERK levels in U-CLL versus M-CLL. Pronounced p-ERK induction was seen by single stimulation in U-CLL,whereas BCR/TLR synergism was required in View Publication

Melanoma,a potentially lethal skin cancer,is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses,limited knowledge exists on the role of mature B cells. We describe an approach,including a cell-based ELISA,to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (Ptextless0.0001). Interestingly,we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (Ptextless0.0001). Overall,28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly,a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients,which is reduced with disease progression,adding to previous reports of tumor-reactive antibodies in patient sera,and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer. View Publication

We analyzed in B-chronic lymphocytic leukemia (B-CLL) whole blood assays the activity of therapeutic mAbs alemtuzumab,rituximab,and type II glycoengineered anti-CD20 mAb GA101. Whole blood samples were treated with Abs,and death of CD19(+) B-CLL was measured by flow cytometry. Alemtuzumab efficiently lysed B-CLL targets with maximal lysis at 1-4 h (62%). In contrast,rituximab induced a more limited cell death (21%) that was maximal only at 24 h. GA101 killed B-CLL targets to a similar extent but more rapidly than rituximab,with 19.2 and 23.5% cell death at 4 and 24 h,respectively,compared with 7.9 and 21.4% for rituximab. Lysis by both rituximab and GA101 correlated directly with CD20 expression levels (r(2) = 0.88 and 0.85,respectively). Interestingly,lysis by all three Abs at high concentrations was mostly complement dependent,because it was blocked by the anti-C5 Ab eculizumab by 90% in the case of alemtuzumab and rituximab and by 64% in the case of GA101. Although GA101 caused homotypic adhesion,it induced only limited (3%) direct cell death of purified B-CLL cells. Both rituximab and GA101 showed the same efficiency in phagocytosis assays,but phagocytosis was not significant in whole blood due to excess Igs. Finally,GA101 at 1-100 μg/ml induced 2- to 3-fold more efficient NK cell degranulation than rituximab in isolated B-CLL or normal PBMCs. GA101,but not rituximab,also mediated significant NK cell degranulation in whole blood samples. Thus,complement and Ab-dependent cellular cytotoxicity are believed to be the major effector mechanisms of GA101 in whole blood assays. View Publication

Patients with mantle cell lymphoma (MCL) typically respond to initial treatment but subsequently relapse. This pattern suggests that a population of MCL cells is both drug resistant and capable of clonogenic growth. The intracellular enzyme retinaldehyde dehydrogenase (ALDH) provides resistance to several toxic agents. ALDH can also identify stem cells in normal adult tissues and tumorigenic cancer stem cells in several human malignancies. We studied ALDH expression in MCL and found small populations of ALDH(+) cells that were highly clonogenic. Moreover,ALDH(+) MCL cells were relatively quiescent and resistant to a wide range of agents. Normal B cells can be activated by specific unmethylated cytosine-phosphate-guanosine (CpG) DNA motifs through toll-like receptor 9,and we found that the synthetic CpG oligonucleotide 2006 (CpG) reduced the frequency of quiescent ALDH(+) MCL cells,induced terminal plasma cell differentiation,and limited tumor formation in vitro and in vivo. Treatment with CpG also significantly enhanced the activity of the proteasome inhibitor bortezomib that was associated with induction of the unfolded protein response. Our data suggest that CpG may target clonogenic and resistant ALDH(+) cells as well as improve the activity of proteasome inhibitors in MCL. View Publication

Autophagy that is induced by starvation or cellular stress can enable cancer cell survival by sustaining energy homeostasis and eliminating damaged organelles and proteins. In response to stress,cancer cells have been reported to accumulate the protein p62/SQSTM1 (p62),but its role in the regulation of autophagy is controversial. Here,we report that the plant phytoalexin resveratrol (RSV) triggers autophagy in imatinib-sensitive and imatinib-resistant chronic myelogenous leukemia (CML) cells via JNK-dependent accumulation of p62. JNK inhibition or p62 knockdown prevented RSV-mediated autophagy and antileukemic effects. RSV also stimulated AMPK,thereby inhibiting the mTOR pathway. AMPK knockdown or mTOR overexpression impaired RSV-induced autophagy but not JNK activation. Lastly,p62 expression and autophagy in CD34+ progenitors from patients with CML was induced by RSV,and disrupting autophagy protected CD34+ CML cells from RSV-mediated cell death. We concluded that RSV triggered autophagic cell death in CML cells via both JNK-mediated p62 overexpression and AMPK activation. Our findings show that the JNK and AMPK pathways can cooperate to eliminate CML cells via autophagy. View Publication

The tumor suppressor gene phosphatase and tensin homolog (PTEN) is inactivated in many human cancers. However,it is unknown whether PTEN functions as a tumor suppressor in human Philadelphia chromosome-positive leukemia that includes chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL) and is induced by the BCR-ABL oncogene. By using our mouse model of BCR-ABL-induced leukemias,we show that Pten is down-regulated by BCR-ABL in leukemia stem cells in CML and that PTEN deletion causes acceleration of CML development. In addition,overexpression of PTEN delays the development of CML and B-ALL and prolongs survival of leukemia mice. PTEN suppresses leukemia stem cells and induces cell-cycle arrest of leukemia cells. Moreover,PTEN suppresses B-ALL development through regulating its downstream gene Akt1. These results demonstrate a critical role of PTEN in BCR-ABL-induced leukemias and suggest a potential strategy for the treatment of Philadelphia chromosome-positive leukemia. View Publication

Although Hodgkin and Reed-Sternberg (HRS) cells are B lymphoid cells,they are unlike any normal cells of that lineage. Moreover,the limited proliferative potential of HRS cells belies the clinical aggressiveness of Hodgkin lymphoma (HL). More than 20 years ago,the L428 HL cell line was reported to contain a small population of phenotypic B cells that appeared responsible for the continued generation of HRS cells. This observation,however,has never been corroborated,and such clonotypic B cells have never been documented in HL patients. We found that both the L428 and KM-H2 HL cell lines contained rare B-cell subpopulations responsible for the generation and maintenance of the predominant HRS cell population. The B cells within the HL cell lines expressed immunoglobulin light chain,the memory B-cell antigen CD27,and the stem cell marker aldehyde dehydrogenase (ALDH). Clonal CD27(+)ALDH(high) B cells,sharing immunoglobulin gene rearrangements with lymph node HRS cells,were also detected in the blood of most newly diagnosed HL patients regardless of stage. Although the clinical significance of circulating clonotypic B cells in HL remains unclear,these data suggest they may be the initiating cells for HL. View Publication

Some cases of pre-B cell acute lymphoblastic leukemia (pre-B-ALL) are caused by the Philadelphia (Ph) chromosome-encoded BCR-ABL oncogene,and these tend to have a poor prognosis. Inhibitors of the PI3K/AKT pathway reduce BCR-ABL-mediated transformation in vitro; however,the specific PI3K isoforms involved are poorly defined. Using a murine model of Ph+ pre-B-ALL,we found that deletion of both Pik3r1 and Pik3r2,genes encoding class IA PI3K regulatory isoforms,severely impaired transformation. BCR-ABL-dependent pre/pro-B cell lines could be established at low frequency from progenitors that lacked these genes,but the cells were smaller,proliferated more slowly,and failed to cause leukemia in vivo. These cell lines displayed nearly undetectable PI3K signaling function and were resistant to the PI3K inhibitor wortmannin. However,they maintained activation of mammalian target of rapamycin (mTOR) and were more sensitive to rapamycin. Treatment with rapamycin caused feedback activation of AKT in WT cell lines but not PI3K-deficient lines. A dual inhibitor of PI3K and mTOR,PI-103,was more effective than rapamycin at suppressing proliferation of mouse pre-B-ALL and human CD19+CD34+)Ph+ ALL leukemia cells treated with the ABL kinase inhibitor imatinib. Our findings provide mechanistic insights into PI3K dependency in oncogenic networks and provide a rationale for targeting class IA PI3K,alone or together with mTOR,in the treatment of Ph+ ALL. View Publication

Primary systemic amyloidosis (AL) is a rare monoclonal plasma cell (PC) disorder characterized by the deposition of misfolded immunoglobulin (Ig) light chains (LC) in vital organs throughout the body. To our knowledge,no cell lines have ever been established from AL patients. Here we describe the establishment of the ALMC-1 and ALMC-2 cell lines from an AL patient. Both cell lines exhibit a PC phenotype and display cytokine-dependent growth. Using a comprehensive genetic approach,we established the genetic relationship between the cell lines and the primary patient cells,and we were also able to identify new genetic changes accompanying tumor progression that may explain the natural history of this patient's disease. Importantly,we demonstrate that free lambda LC secreted by both cell lines contained a beta structure and formed amyloid fibrils. Despite absolute Ig LC variable gene sequence identity,the proteins show differences in amyloid formation kinetics that are abolished by the presence of Na(2)SO(4). The formation of amyloid fibrils from these naturally secreting human LC cell lines is unprecedented. Moreover,these cell lines will provide an invaluable tool to better understand AL,from the combined perspectives of amyloidogenic protein structure and amyloid formation,genetics,and cell biology. View Publication

In order to investigate the biologic processes underlying and resulting from the megakaryocytic hyperplasia that characterizes idiopathic myelofibrosis (IMF),peripheral blood CD34+ cells isolated from patients with IMF,polycythemia vera (PV),and G-CSF-mobilized healthy volunteers were cultured in the presence of stem cell factor and thrombopoietin. IMF CD34+ cells generated 24-fold greater numbers of megakaryocytes (MKs) than normal CD34+ cells. IMF MKs were also shown to have a delayed pattern of apoptosis and to overexpress the antiapoptotic protein bcl-xL. MK hyperplasia in IMF is,therefore,likely a consequence of both the increased ability of IMF progenitor cells to generate MKs and a decreased rate of MK apoptosis. Media conditioned (CM) by CD61+ cells generated in vitro from CD34+ cells were then assayed for the levels of growth factors and proteases. Higher levels of transforming growth factor-beta (TGF-beta) and active matrix metalloproteinase-9 (MMP9) were observed in media conditioned with IMF CD61+ cells than normal or PV CD61+ cells. Both normal and IMF CD61+ cells produced similar levels of VEGF. MK-derived TGF-B and MMP-9,therefore,likely contribute to the development of many pathological epiphenomena associated with IMF. View Publication

Infection with a recombinant murine-feline gammaretrovirus,MoFe2,or with the parent virus,Moloney murine leukemia virus,caused significant reduction in B-lymphoid differentiation of bone marrow at 2 to 8 weeks postinfection. The suppression was selective,in that myeloid potential was significantly increased by infection. Analysis of cell surface markers and immunoglobulin H gene rearrangements in an in vitro model demonstrated normal B-lymphoid differentiation after infection but significantly reduced viability of differentiating cells. This reduction in viability may confer a selective advantage on undifferentiated lymphoid progenitors in the bone marrow of gammaretrovirus-infected animals and thereby contribute to the establishment of a premalignant state. View Publication