技术资料

BAFF plays a central role in B-lineage cell biology; however,the regulation of BAFF-binding receptor (BBR) expression during B cell activation and differentiation is not completely understood. In this study,we provide a comprehensive ex vivo analysis of BBRs in human B-lineage cells at various stages of maturation,as well as describe the events that drive and regulate receptor expression. Our data reveal that B-lineage cells ranging from naive to plasma cells (PCs),excluding bone marrow PCs,express BAFF-R uniformly. In contrast,only tonsillar memory B cells (MB) and PCs,from both tonsil and bone marrow tissues,express BCMA. Furthermore,we show that TACI is expressed by MB cells and PCs,as well as a subpopulation of activated CD27(neg) B cells. In this regard,we demonstrate that TACI is inducible early upon B cell activation and this is independent of B cell turnover. In addition,we found that TACI expression requires activation of the ERK1/2 pathway,since its expression was blocked by ERK1/2-specific inhibitors. Expression of BAFF-R and B cell maturation Ag (BCMA) is also highly regulated and we demonstrate that BCMA expression is only acquired in MB cells and in a manner accompanied by loss of BAFF-R expression. This inverse expression coincides with MB cell differentiation into Ig-secreting cells (ISC),since blocking differentiation inhibited both induction of BCMA expression and loss of BAFF-R. Collectively,our data suggest that the BBR profile may serve as a footprint of the activation history and stage of differentiation of normal human B cells. View Publication

Kinase inhibitors constitute an important new class of cancer drugs,whose selective efficacy is largely determined by underlying tumor cell genetics. We established a high-throughput platform to profile 500 cell lines derived from diverse epithelial cancers for sensitivity to 14 kinase inhibitors. Most inhibitors were ineffective against unselected cell lines but exhibited dramatic cell killing of small nonoverlapping subsets. Cells with exquisite sensitivity to EGFR,HER2,MET,or BRAF kinase inhibitors were marked by activating mutations or amplification of the drug target. Although most cell lines recapitulated known tumor-associated genotypes,the screen revealed low-frequency drug-sensitizing genotypes in tumor types not previously associated with drug susceptibility. Furthermore,comparing drugs thought to target the same kinase revealed striking differences,predictive of clinical efficacy. Genetically defined cancer subsets,irrespective of tissue type,predict response to kinase inhibitors,and provide an important preclinical model to guide early clinical applications of novel targeted inhibitors. View Publication

BACKGROUND AIMS: Previous studies have demonstrated that the combination of granulocyte-colony-stimulating factor (G-CSF) + plerixafor is more efficient in mobilizing CD34(+) hematopoietic stem cells (HSC) into the peripheral blood than G-CSF alone. In this study we analyzed the impact of adding plerixafor to G-CSF upon the mobilization of different HSC subsets. METHODS: We characterized the immunophenotype of HSC subsets isolated from the peripheral blood of eight patients with multiple myeloma (MM) before and after treatment with plerixafor. All patients were supposed to collect stem cells prior to high-dose chemotherapy and consecutive autologous stem cell transplantation,and therefore received front-line mobilization with 4 days of G-CSF followed by a single dose of plerixafor. Samples of peripheral blood were analyzed comparatively by flow cytometry directly before and 12 h after administration of plerixafor. RESULTS: The number of aldehyde dehydrogenase (ALDH)(bright) and CD34(+) cells was significantly higher after plerixafor treatment (1.2-5.0 and 1.5-6.0 times; both P textless 0.01) and an enrichment of the very primitive CD34(+) CD38(-) and ALDH(bright) CD34(+) CD38(-) HSC subsets was detectable. Additionally,two distinct ALDH(+) subsets could be clearly distinguished. The small ALDH(high) subset showed a higher number of CD34(+) CD38(-) cells in contrast to the total ALDH(bright) subpopulation and probably represented a very primitive subpopulation of HSC. CONCLUSIONS: A combined staining of ALDH,CD34 and CD38 might represent a powerful tool for the identification of a very rare and primitive hematopoietic stem cell subset. The addition of plerixafor mobilized not only more CD34(+) cells but was also able to increase the proportion of more primitive stem cell subsets. View Publication

The expression of an enzyme,GnT-V,that catalyzes a specific posttranslational modification of a family of glycoproteins,namely a branched N-glycan,is transcriptionally up-regulated during breast carcinoma oncogenesis. To determine the molecular basis of how early events in breast carcinoma formation are regulated by GnT-V,we studied both the early stages of mammary tumor formation by using 3D cell culture and a her-2 transgenic mouse mammary tumor model. Overexpression of GnT-V in MCF-10A mammary epithelial cells in 3D culture disrupted acinar morphogenesis with impaired hollow lumen formation,an early characteristic of mammary neoplastic transformation. The disrupted acinar morphogenesis of mammary tumor cells in 3D culture caused by her-2 expression was reversed in tumors that lacked GnT-V expression. Moreover,her-2-induced mammary tumor onset was significantly delayed in the GnT-V null tumors,evidence that the lack of the posttranslational modification catalyzed by GnT-V attenuated tumor formation. Inhibited activation of both PKB and ERK signaling pathways was observed in GnT-V null tumor cells. The proportion of tumor-initiating cells (TICs) in the mammary tumors from GnT-V null mice was significantly reduced compared with controls,and GnT-V null TICs displayed a reduced ability to form secondary tumors in NOD/SCID mice. These results demonstrate that GnT-V expression and its branched glycan products effectively modulate her-2-mediated signaling pathways that,in turn,regulate the relative proportion of tumor initiating cells and the latency of her-2-driven tumor onset. View Publication

The resistance of glioma cells to a number of antitumor agents and the highly invasive nature of glioma cells that escape the primary tumor mass are key impediments to the eradication of tumors in glioma patients. In this study,we evaluated the therapeutic efficacy of a novel PI3-kinase/mTOR inhibitor,PI-103,in established glioma lines and primary CD133(+) glioma-initiating cells and explored the potential of combining PI-103 with stem cell-delivered secretable tumor necrosis factor apoptosis-inducing ligand (S-TRAIL) both in vitro and in orthotopic mouse models of gliomas. We show that PI-103 inhibits proliferation and invasion,causes G(0)-G(1) arrest in cell cycle,and results in significant attenuation of orthotopic tumor growth in vivo. Establishing cocultures of neural stem cells (NSC) and glioma cells,we show that PI-103 augments the response of glioma cells to stem cell-delivered S-TRAIL. Using bimodal optical imaging,we show that when different regimens of systemic PI-103 delivery are combined with NSC-derived S-TRAIL,a significant reduction in tumor volumes is observed compared with PI-103 treatment alone. To our knowledge,this is the first study that reveals the antitumor effect of PI-103 in intracranial gliomas. Our findings offer a preclinical rationale for application of mechanism-based systemically delivered antiproliferative agents and novel stem cell-based proapoptotic therapies to improve treatment of malignant gliomas. View Publication

Aldehyde dehydrogenase (ALDH) is a candidate marker for lung cancer cells with stem cell-like properties. Immunohistochemical staining of a large panel of primary non-small cell lung cancer (NSCLC) samples for ALDH1A1,ALDH3A1,and CD133 revealed a significant correlation between ALDH1A1 (but not ALDH3A1 or CD133) expression and poor prognosis in patients including those with stage I and N0 disease. Flow cytometric analysis of a panel of lung cancer cell lines and patient tumors revealed that most NSCLCs contain a subpopulation of cells with elevated ALDH activity,and that this activity is associated with ALDH1A1 expression. Isolated ALDH(+) lung cancer cells were observed to be highly tumorigenic and clonogenic as well as capable of self-renewal compared with their ALDH(-) counterparts. Expression analysis of sorted cells revealed elevated Notch pathway transcript expression in ALDH(+) cells. Suppression of the Notch pathway by treatment with either a γ-secretase inhibitor or stable expression of shRNA against NOTCH3 resulted in a significant decrease in ALDH(+) lung cancer cells,commensurate with a reduction in tumor cell proliferation and clonogenicity. Taken together,these findings indicate that ALDH selects for a subpopulation of self-renewing NSCLC stem-like cells with increased tumorigenic potential,that NSCLCs harboring tumor cells with ALDH1A1 expression have inferior prognosis,and that ALDH1A1 and CD133 identify different tumor subpopulations. Therapeutic targeting of the Notch pathway reduces this ALDH(+) component,implicating Notch signaling in lung cancer stem cell maintenance. View Publication

RARA (retinoic acid receptor alpha) haploinsufficiency is an invariable consequence of t(15;17)(q22;q21) translocations in acute promyelocytic leukemia (APL). Retinoids and RARA activity have been implicated in hematopoietic self-renewal and neutrophil maturation. We and others therefore predicted that RARA haploinsufficiency would contribute to APL pathogenesis. To test this hypothesis,we crossed Rara(+/-) mice with mice expressing PML (promyelocytic leukemia)-RARA from the cathepsin G locus (mCG-PR). We found that Rara haploinsufficiency cooperated with PML-RARA,but only modestly influenced the preleukemic and leukemic phenotype. Bone marrow from mCG-PR(+/-) × Rara(+/-) mice had decreased numbers of mature myeloid cells,increased ex vivo myeloid cell proliferation,and increased competitive advantage after transplantation. Rara haploinsufficiency did not alter mCG-PR-dependent leukemic latency or penetrance,but did influence the distribution of leukemic cells; leukemia in mCG-PR(+/-) × Rara(+/-) mice presented more commonly with low to normal white blood cell counts and with myeloid infiltration of lymph nodes. APL cells from these mice were responsive to all-trans retinoic acid and had virtually no differences in expression profiling compared with tumors arising in mCG-PR(+/-) × Rara(+/+) mice. These data show that Rara haploinsufficiency (like Pml haploinsufficiency and RARA-PML) can cooperate with PML-RARA to influence the pathogenesis of APL in mice,but that PML-RARA is the t(15;17) disease-initiating mutation. View Publication

Ecotropic viral integration site-1 (Evi-1) is a nuclear transcription factor that plays an essential role in the regulation of hematopoietic stem cells. Aberrant expression of Evi-1 has been reported in up to 10% of patients with acute myeloid leukemia and is a diagnostic marker that predicts a poor outcome. Although chromosomal rearrangement involving the Evi-1 gene is one of the major causes of Evi-1 activation,overexpression of Evi-1 is detected in a subgroup of acute myeloid leukemia patients without any chromosomal abnormalities,which indicates the presence of other mechanisms for Evi-1 activation. In this study,we found that Evi-1 is frequently up-regulated in bone marrow cells transformed by the mixed-lineage leukemia (MLL) chimeric genes MLL-ENL or MLL-AF9. Analysis of the Evi-1 gene promoter region revealed that MLL-ENL activates transcription of Evi-1. MLL-ENL-mediated up-regulation of Evi-1 occurs exclusively in the undifferentiated hematopoietic population,in which Evi-1 particularly contributes to the propagation of MLL-ENL-immortalized cells. Furthermore,gene-expression analysis of human acute myeloid leukemia cases demonstrated the stem cell-like gene-expression signature of MLL-rearranged leukemia with high levels of Evi-1. Our findings indicate that Evi-1 is one of the targets of MLL oncoproteins and is selectively activated in hematopoietic stem cell-derived MLL leukemic cells. View Publication

Pim kinases are Ser/Thr kinases with multiple substrates that affect survival pathways. These proteins are overexpressed in acute myeloid leukemia (AML) blasts and we hypothesized that Pim kinase inhibition would affect AML cell survival. Imidazo[1,2-b]pyridazine compound,SGI-1776 inhibits Pim-1,Pim-2 and Pim-3,and was evaluated in AML-cell line,-xenograft model,and -primary blasts. Treatment of AML cells with SGI-1776 results in a concentration-dependent induction of apoptosis and we investigated its effect on Pim kinase functions. Phosphorylation of traditional Pim kinase targets,c-Myc(Ser62) and 4E-BP1 (Thr36/Thr47),were both decreased in actively cycling AML cell lines MV-4-11,MOLM-13 and OCI-AML-3. Levels of antiapoptotic proteins Bcl-2,Bcl-x(L),XIAP,and proapoptotic Bak and Bax were unchanged; however,a significant reduction in Mcl-1 was observed. This was correlated with inhibition of global RNA and protein synthesis and MCL-1 transcript decline after SGI-1776 treatment. These data suggest that SGI-1776 mechanism in AML involves Mcl-1 protein reduction. Consistent with cell line data,xenograft model studies with mice bearing MV-4-11 tumors showed efficacy with SGI-1776. Importantly,SGI-1776 was also cytotoxic in AML primary cells,irrespective of FLT3 mutation status and resulted in Mcl-1 protein decline. Pim kinase inhibition may be a new strategy for AML treatment. View Publication

Disruptor of telomeric silencing 1-like (Dot1l) is a histone 3 lysine 79 methyltransferase. Studies of constitutive Dot1l knockout mice show that Dot1l is essential for embryonic development and prenatal hematopoiesis. DOT1L also interacts with translocation partners of Mixed Lineage Leukemia (MLL) gene,which is commonly translocated in human leukemia. However,the requirement of Dot1l in postnatal hematopoiesis and leukemogenesis of MLL translocation proteins has not been conclusively shown. With a conditional Dot1l knockout mouse model,we examined the consequences of Dot1l loss in postnatal hematopoiesis and MLL translocation leukemia. Deletion of Dot1l led to pancytopenia and failure of hematopoietic homeostasis,and Dot1l-deficient cells minimally reconstituted recipient bone marrow in competitive transplantation experiments. In addition,MLL-AF9 cells required Dot1l for oncogenic transformation,whereas cells with other leukemic oncogenes,such as Hoxa9/Meis1 and E2A-HLF,did not. These findings illustrate a crucial role of Dot1l in normal hematopoiesis and leukemogenesis of specific oncogenes. View Publication

Acute basophilic leukemia (ABL) is a rare subtype of acute leukemia with clinical features and symptoms related to hyperhistaminemia because of excessive growth of basophils. No known recurrent cytogenetic abnormality is associated with this leukemia. Rare cases of t(X;6)(p11;q23) translocation have been described but these were sporadic. We report here 4 cases of ABL with a t(X;6)(p11;q23) translocation occurring in male infants. Because of its location on chromosome 6q23,MYB was a good candidate gene. Our molecular investigations,based on fluorescence in situ hybridization and rapid amplification of cDNA ends,revealed that the translocation generated a MYB-GATA1 fusion gene. Expression of MYB-GATA1 in mouse lineage-negative cells committed them to the granulocyte lineage and blocked at an early stage of differentiation. Taken together,these results establish,for the first time,a link between a recurrent chromosomal translocation and the development of this particular subtype of infant leukemia. View Publication

Little is known about the origin of basal-like breast cancers,an aggressive disease that is highly similar to BRCA1-mutant breast cancers. p63 family proteins that are structurally related to the p53 suppressor protein are known to function in stem cell regulation and stratified epithelia development in multiple tissues,and p63 expression may be a marker of basal-like breast cancers. Here we report that ΔNp63 isoforms of p63 are transcriptional targets for positive regulation by BRCA1. Our analyses of breast cancer tissue microarrays and BRCA1-modulated breast cancer cell lines do not support earlier reports that p63 is a marker of basal-like or BRCA1 mutant cancers. Nevertheless,we found that BRCA1 interacts with the specific p63 isoform ΔNp63γ along with transcription factor isoforms AP-2α and AP-2γ. BRCA1 required ΔNp63γ and AP-2γ to localize to an intronic enhancer region within the p63 gene to upregulate transcription of the ΔNp63 isoforms. In mammary stem/progenitor cells,siRNA-mediated knockdown of ΔNp63 expression resulted in genomic instability,increased cell proliferation,loss of DNA damage checkpoint control,and impaired growth control. Together,our findings establish that transcriptional upregulation of ΔNp63 proteins is critical for BRCA1 suppressor function and that defects in BRCA1-ΔNp63 signaling are key events in the pathogenesis of basal-like breast cancer. View Publication