技术资料

Multiple myeloma is characterized by increased osteoclast activity that results in bone destruction and lytic lesions. With the prolonged overall patient survival achieved by new treatment modalities,additional drugs are required to inhibit bone destruction. We focused on a novel and more potent structural analog of the nonsteroidal anti-inflammatory drug etodolac,known as SDX-308,and its effects on osteoclastogenesis and multiple myeloma cells. SDX-101 is another structural analog of etodolac that is already used in clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Compared with SDX-101,a 10-fold lower concentration of SDX-308 induced potent (60%-80%) inhibition of osteoclast formation,and a 10- to 100-fold lower concentration inhibited multiple myeloma cell proliferation. Bone resorption was completely inhibited by SDX-308,as determined in dentin-based bone resorption assays. SDX-308 decreased constitutive and RANKL-stimulated NF-kappaB activation and osteoclast formation in an osteoclast cellular model,RAW 264.7. SDX-308 effectively suppressed TNF-alpha-induced IKK-gamma and IkappaB-alpha phosphorylation and degradation and subsequent NF-kappaB activation in human multiple myeloma cells. These results indicate that SDX-308 effectively inhibits multiple myeloma cell proliferation and osteoclast activity,potentially by controlling NF-kappaB activation signaling. We propose that SDX-308 is a promising therapeutic candidate to inhibit multiple myeloma growth and osteoclast activity and that it should receive attention for further study. View Publication

Neurofibromatosis type 1 (NF1) syndrome is caused by germline mutations in the NF1 tumor suppressor,which encodes neurofibromin,a GTPase activating protein for Ras. Children with NF1 are predisposed to juvenile myelomonocytic leukemia (JMML) and lethally irradiated mice given transplants with homozygous Nf1 mutant (Nf1-/-) hematopoietic stem cells develop a fatal myeloproliferative disorder (MPD) that models JMML. We investigated the requirement for signaling through the GM-CSF receptor to initiate and sustain this MPD by generating Nf1 mutant hematopoietic cells lacking the common beta chain (Beta c) of the GM-CSF receptor. Mice reconstituted with Nf1-/-,beta c-/- stem cells did not develop evidence of MPD despite the presence of increased number of immature hematopoietic progenitors in the bone marrow. Interestingly,when the Mx1-Cre transgene was used to inactivate a conditional Nf1 mutant allele in hematopoietic cells,concomitant loss of beta c-/- reduced the severity of the MPD,but did not abrogate it. Whereas inhibiting GM-CSF signaling may be of therapeutic benefit in JMML,our data also demonstrate aberrant proliferation of Nf1-/-myeloid progenitors that is independent of signaling through the GM-CSF receptor. View Publication

Overcoming imatinib mesylate (IM) resistance and disease persistence in patients with chronic myeloid leukemia (CML) is of considerable importance to the issue of potential cure. Here we asked whether autocrine signaling contributes to survival of BCR/ABL+ cells in the presence of IM and nilotinib (NI; AMN107),a novel,more selective Abl inhibitor. Conditioned media (CM) of IM-resistant LAMA84 cell clones (R-CM) was found to substantially protect IM-naive LAMA cells and primary CML progenitors from IM- or NI-induced cell death. This was due to an increased secretion of the granulocyte-macrophage colony-stimulating factor (GM-CSF),which was identified as the causative factor mediating IM resistance in R-CM. GM-CSF elicited IM and NI drug resistance via a BCR/ABL-independent activation of the janus kinases 2 (JAK-2)/signal transducer and activator of transcription 5 (STAT-5) signaling pathway in GM-CSF receptor alpha receptor (CD116)-expressing cells,including primary CD34+/CD116+ GM progenitors (GMPs). Elevated mRNA and protein levels of GM-CSF were detected in IM-resistant patient samples,suggesting a contribution of GM-CSF secretion for IM and NI resistance in vivo. Importantly,inhibition of JAK-2 with AG490 abrogated GM-CSF-mediated STAT-5 phosphorylation and NI resistance in vitro. Together,adaptive autocrine secretion of GM-CSF mediates BCR/ABL-independent IM and NI resistance via activation of the antiapoptotic JAK-2/STAT-5 pathway. Inhibition of JAK-2 overcomes GM-CSF-induced IM and NI progenitor cell resistance,providing a rationale for the application of JAK-2 inhibitors to eradicate residual disease in CML. View Publication

Chaetocin,a thiodioxopiperazine natural product previously unreported to have anticancer effects,was found to have potent antimyeloma activity in IL-6-dependent and -independent myeloma cell lines in freshly collected sorted and unsorted patient CD138(+) myeloma cells and in vivo. Chaetocin largely spares matched normal CD138(-) patient bone marrow leukocytes,normal B cells,and neoplastic B-CLL (chronic lymphocytic leukemia) cells,indicating a high degree of selectivity even in closely lineage-related B cells. Furthermore,chaetocin displays superior ex vivo antimyeloma activity and selectivity than doxorubicin and dexamethasone,and dexamethasone- or doxorubicin-resistant myeloma cell lines are largely non-cross-resistant to chaetocin. Mechanistically,chaetocin is dramatically accumulated in cancer cells via a process inhibited by glutathione and requiring intact/unreduced disulfides for uptake. Once inside the cell,its anticancer activity appears mediated primarily through the imposition of oxidative stress and consequent apoptosis induction. Moreover,the selective antimyeloma effects of chaetocin appear not to reflect differential intracellular accumulation of chaetocin but,instead,heightened sensitivity of myeloma cells to the cytotoxic effects of imposed oxidative stress. Considered collectively,chaetocin appears to represent a promising agent for further study as a potential antimyeloma therapeutic. View Publication

Drug resistance resulting from emergence of imatinib-resistant BCR-ABL point mutations is a significant problem in advanced-stage chronic myelogenous leukemia (CML). The BCR-ABL inhibitor,nilotinib (AMN107),is significantly more potent against BCR-ABL than imatinib,and is active against many imatinib-resistant BCR-ABL mutants. Phase 1/2 clinical trials show that nilotinib can induce remissions in patients who have previously failed imatinib,indicating that sequential therapy with these 2 agents has clinical value. However,simultaneous,rather than sequential,administration of 2 BCR-ABL kinase inhibitors is attractive for many reasons,including the theoretical possibility that this could reduce emergence of drug-resistant clones. Here,we show that exposure of a variety of BCR-ABL+ cell lines to imatinib and nilotinib results in additive or synergistic cytotoxicity,including testing of a large panel of cells expressing BCR-ABL point mutations causing resistance to imatinib in patients. Further,using a highly quantifiable bioluminescent in vivo model,drug combinations were at least additive in antileukemic activity,compared with each drug alone. These results suggest that despite binding to the same site in the same target kinase,the combination of imatinib and nilotinib is highly efficacious in these models,indicating that clinical testing of combinations of BCR-ABL kinase inhibitors is warranted. View Publication

Recent population studies provide clues that the use of metformin may be associated with reduced incidence and improved prognosis of certain cancers. This drug is widely used in the treatment of type 2 diabetes,where it is often referred to as an insulin sensitizer" because it not only lowers blood glucose but also reduces the hyperinsulinemia associated with insulin resistance. As insulin and insulin-like growth factors stimulate proliferation of many normal and transformed cell types� View Publication

Combined deletion of chromosomes 1p and 19q is associated with improved prognosis and responsiveness to therapy in patients with anaplastic oligodendroglioma. The deletions usually involve whole chromosome arms,suggesting a t(1;19)(q10;p10). Using stem cell medium,we cultured a few tumors. Paraffin-embedded tissue was obtained from 21 Mayo Clinic patients and 98 patients enrolled in 2 North Central Cancer Treatment Group (NCCTG) low-grade glioma trials. Interphase fusion of CEP1 and 19p12 probes detected the t(1;19). 1p/19q deletions were evaluated by fluorescence in situ hybridization. Upon culture,one oligodendroglioma contained an unbalanced 45,XX,t(1;19)(q10;p10). CEP1/19p12 fusion was observed in all metaphases and 74% of interphase nuclei. Among Mayo Clinic oligodendrogliomas,the prevalence of fusion was 81%. Among NCCTG patients,CEP1/19p12 fusion prevalence was 55%,47%,and 0% among the oligodendrogliomas,mixed oligoastrocytomas,and astrocytomas,respectively. Ninety-one percent of NCCTG gliomas with 1p/19q deletion and 12% without 1p/19q deletion had CEP1/19p12 fusion (P textless 0.001,chi(2) test). The median overall survival (OS) for all patients was 8.1 years without fusion and 11.9 years with fusion (P = 0.003). The median OS for patients with low-grade oligodendroglioma was 9.1 years without fusion and 13.0 years with fusion (P = 0.01). Similar significant median OS differences were observed for patients with combined 1p/19q deletions. The absence of alterations was associated with a significantly shorter OS for patients who received higher doses of radiotherapy. Our results strongly suggest that a t(1;19)(q10;p10) mediates the combined 1p/19q deletion in human gliomas. Like combined 1p/19q deletion,the 1;19 translocation is associated with superior OS and progression-free survival in low-grade glioma patients. View Publication

Recognition of tumor-associated Ags (TAAs) on tumor cells by CTLs and the subsequent tumor cell death are assumed to be dependent on TAA protein expression and to correlate directly with the level of peptide displayed in the binding site of the HLA class I molecule. In this study we evaluated whether the levels of Her-2/neu protein expression on human tumor cell lines directly correlate with HLA-A*0201/Her2/neu peptide presentation and CTL recognition. We developed a TCR mimic (TCRm) mAb designated 1B8 that specifically recognizes the HLA-A2.1/Her2/neu peptide (369-377) (Her2(369)-A2) complex. TCRm mAb staining intensity varied for the five human tumor cell lines analyzed,suggesting quantitative differences in levels of the Her2(369)-A2 complex on these cells. Analysis of tumor cell lines pretreated with IFN-gamma and TNF-alpha for Her2/neu protein and HLA-A2 molecule expression did not reveal a direct correlation between the levels of Her2/neu Ag,HLA-A2 molecule,and Her2(369)-A2 complex expression. However,compared with untreated cells,cytokine-treated cell lines showed an increase in Her2(369)-A2 epitope density that directly correlated with enhanced tumor cell death (p = 0.05). Although a trend was observed between tumor cell lysis and the level of the Her2(369)-A2 complex for untreated cells,the association was not significant. These findings suggest that tumor cell susceptibility to CTL-mediated lysis may be predicted based on the level of specific peptide-MHC class I expression rather than on the total level of TAA expression. Further,these studies demonstrate the potential of the TCRm mAb for validation of endogenous HLA-peptide epitopes on tumor cells. View Publication

We previously identified a rearrangement of mixed-lineage leukemia (MLL) gene (also known as ALL-1,HRX,and HTRX1),consisting of an in-frame partial tandem duplication (PTD) of exons 5 through 11 in the absence of a partner gene,occurring in approximately 4%-7% of patients with acute myeloid leukemia (AML) and normal cytogenetics,and associated with a poor prognosis. The mechanism by which the MLL PTD contributes to aberrant hematopoiesis and/or leukemia is unknown. To examine this,we generated a mouse knockin model in which exons 5 through 11 of the murine Mll gene were targeted to intron 4 of the endogenous Mll locus. Mll(PTD/WT) mice exhibit an alteration in the boundaries of normal homeobox (Hox) gene expression during embryogenesis,resulting in axial skeletal defects and increased numbers of hematopoietic progenitor cells. Mll(PTD/WT) mice overexpress Hoxa7,Hoxa9,and Hoxa10 in spleen,BM,and blood. An increase in histone H3/H4 acetylation and histone H3 lysine 4 (Lys4) methylation within the Hoxa7 and Hoxa9 promoters provides an epigenetic mechanism by which this overexpression occurs in vivo and an etiologic role for MLL PTD gain of function in the genesis of AML. View Publication

Granulocyte colony-stimulating factor (GCSF) administration has been linked to the development of monosomy 7 in severe congenital neutropenia and aplastic anemia. We assessed the effect of pharmacologic doses of GCSF on monosomy 7 cells to determine whether this chromosomal abnormality developed de novo or arose as a result of favored expansion of a preexisting clone. Fluorescence in situ hybridization (FISH) of chromosome 7 was used to identify small populations of aneuploid cells. When bone marrow mononuclear cells from patients with monosomy 7 were cultured with 400 ng/ml GCSF,all samples showed significant increases in the proportion of monosomy 7 cells. In contrast,bone marrow from karyotypically normal aplastic anemia,myelodysplastic syndrome,or healthy individuals did not show an increase in monosomy 7 cells in culture. In bone marrow CD34 cells of patients with myelodysplastic syndrome and monosomy 7,GCSF receptor (GCSFR) protein was increased. Although no mutation was found in genomic GCSFR DNA,CD34 cells showed increased expression of the GCSFR class IV mRNA isoform,which is defective in signaling cellular differentiation. GCSFR signal transduction via the Jak/Stat system was abnormal in monosomy 7 CD34 cells,with increased phosphorylated signal transducer and activation of transcription protein,STAT1-P,and increased STAT5-P relative to STAT3-P. Our results suggest that pharmacologic doses of GCSF increase the proportion of preexisting monosomy 7 cells. The abnormal response of monosomy 7 cells to GCSF would be explained by the expansion of undifferentiated monosomy 7 clones expressing the class IV GCSFR,which is defective in signaling cell maturation. View Publication