技术资料

Most patients with acute myelogenous leukemia (AML) relapse and die of their disease. Increasing evidence indicates that AML relapse is driven by the inability to eradicate leukemia stem cells (LSC). Thus,it is imperative to identify novel therapies that can ablate LSCs. Using an in silico gene expression-based screen for compounds evoking transcriptional effects similar to the previously described anti-LSC agent parthenolide,we identified AR-42 (OSU-HDAC42),a novel histone deacetylase inhibitor that is structurally similar to phenylbutyrate,but with improved activity at submicromolar concentrations. Here,we report that AR-42 induces NF-κB inhibition,disrupts the ability of Hsp90 to stabilize its oncogenic clients,and causes potent and specific cell death of LSCs but not normal hematopoietic stem and progenitor cells. Unlike parthenolide,the caspase-dependent apoptosis caused by AR-42 occurs without activation of Nrf-2-driven cytoprotective pathways. As AR-42 is already being tested in early clinical trials,we expect that our results can be extended to the clinic. View Publication

Identification of agents that target human leukemia stem cells is an important consideration for the development of new therapies. The present study demonstrates that rocaglamide and silvestrol,closely related natural products from the flavagline class of compounds,are able to preferentially kill functionally defined leukemia stem cells,while sparing normal stem and progenitor cells. In addition to efficacy as single agents,flavaglines sensitize leukemia cells to several anticancer compounds,including front-line chemotherapeutic drugs used to treat leukemia patients. Mechanistic studies indicate that flavaglines strongly inhibit protein synthesis,leading to the reduction of short-lived antiapoptotic proteins. Notably though,treatment with flavaglines,alone or in combination with other drugs,yields a much stronger cytotoxic activity toward leukemia cells than the translational inhibitor temsirolimus. These results indicate that the underlying cell death mechanism of flavaglines is more complex than simply inhibiting general protein translation. Global gene expression profiling and cell biological assays identified Myc inhibition and the disruption of mitochondrial integrity to be features of flavaglines,which we propose contribute to their efficacy in targeting leukemia cells. Taken together,these findings indicate that rocaglamide and silvestrol are distinct from clinically available translational inhibitors and represent promising candidates for the treatment of leukemia. View Publication

Most forms of chemotherapy employ mechanisms involving induction of oxidative stress,a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However,recent studies have shown that relative redox levels in primary tumors can be heterogeneous,suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies,we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First,the majority of functionally defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed ROS-low"). Second� View Publication

ETV6-RUNX1 gene fusion is usually an early,prenatal event in childhood acute lymphoblastic leukemia (ALL). Transformation results in the generation of a persistent (> 14 years) preleukemic clone,which postnatally converts to ALL after the acquisition of necessary secondary genetic alterations. Many cancer cells show some expression of the erythropoietin receptor (EPOR) gene,although the functionality" of any EPOR complexes and their relevant signaling pathways in nonerythroid cells has not been validated. EPOR mRNA is selectively and ectopically expressed in ETV6-RUNX1(+) ALL but the presence of a functional EPOR on the cell surface and its role in leukemogenesis driven by ETV6-RUNX1 remains to be identified. Here we show that ETV6-RUNX1 directly binds the EPOR promoter and that expression of ETV6-RUNX1 alone in normal pre-B cells is sufficient to activate EPOR transcription. We further reveal that murine and human ETV6-RUNX1(+) cells expressing EPOR mRNA have EPO ligand binding activity that correlates with an increased cell survival through activation of the JAK2-STAT5 pathway and up-regulation of antiapoptotic BCL-XL. These data support the contention that ETV6-RUNX1 directly activates ectopic expression of a functional EPOR and provides cell survival signals that may contribute critically to persistence of covert premalignant clones in children. View Publication

We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting the human transferrin receptor 1 (TfR1,also known as CD71),which demonstrates direct in vitro cytotoxicity against malignant hematopoietic cells. This cytotoxicity is attributed to its ability to decrease the level of TfR1 leading to lethal iron deprivation. We now report that ch128.1Av shows the ability to bind the Fcγ receptors and the complement component C1q,suggesting that it is capable of eliciting Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-mediated cytotoxicity. In addition,in 2 disseminated multiple myeloma xenograft mouse models,we show that a single dose of ch128.1Av results in significant antitumor activity,including long-term survival. It is interesting to note that the parental antibody without avidin (ch128.1) also shows remarkable in vivo anticancer activity despite its limited in vitro cytotoxicity. Finally,we demonstrate that ch128.1Av is not toxic to pluripotent hematopoietic progenitor cells using the long-term cell-initiating culture assay suggesting that these important progenitors would be preserved in different therapeutic approaches,including the in vitro purging of cancer cells for autologous transplantation and in vivo passive immunotherapy. Our results suggest that ch128.1Av and ch128.1 may be effective in the therapy of human multiple myeloma and potentially other hematopoietic malignancies. View Publication

DNA methylation is one of the essential epigenetic processes that play a role in regulating gene expression. Aberrant methylation of CpG-rich promoter regions has been associated with many forms of human cancers. The current method for determining the methylation status relies mainly on bisulfite treatment of genomic DNA,followed by methylation-specific PCR (MSP). The difficulty in acquiring a methylation profiling often is limited by the amount of genomic DNA that can be recovered from a given sample,whereas complex procedures of bisulfite treatment further compromise the effective template for PCR analysis. To circumvent these obstacles,we developed degenerated oligonucleotide primer (DOP)-PCR to enable amplification of bisulfite-modified genomic DNA at a genome-wide scale. A DOP pair was specially designed as follows: first 3' DOP,CTCGAGCTGHHHHHAACTAC,where H is a mixture of base consisting of 50% A,25% T,and 25% C; and second 5' DOP,CTCGAGCTGDDDDDGTTTAG,where D is a mixture of base consisting of 50% T,25% G,and 25% A. Our results showed that bisulfite-modified DNAs from a cell line,cord blood cells,or cells obtained by laser capture microdissection were amplified by up to 1000-fold using this method. Subsequent MSP analysis using these amplified DNAs on nine randomly selected cancer-related genes revealed that the methylation status of these genes remained identical to that derived from the original unamplified template. View Publication

We have previously synthesized a series of 1,8-naphthyridine-3-carboxamide derivatives to identify potential anti-cancer/anti-inflammatory compounds. Three derivatives,7-chloro-N-(3-(cyclopentylamino)-3-oxo-1-phenylpropyl)-6-fluoro-4-oxo-1-(prop-2-yn-1-yl)-1,4-dihydro-1,8-naphthyridine-3-carboxamide (C-22),7-chloro-N-(2-hydroxy-3-oxo-1-phenyl-3-(phenylamino)propyl)-4-oxo-1-(prop-2-yn-1-yl)-1,4-dihydro-1,8-naphthyridine-3-carboxamide (C-31) and 7-chloro-6-fluoro-N-(2-hydroxy-3-oxo-1-phenyl-3-(phenylamino)propyl)-4-oxo-1-(prop-2-yn-1-yl)-1,4-dihydro-1,8-naphthyridine-3-carboxamide (C-34) demonstrated high cytotoxicity against a number of cancer cell lines and inhibited secretion of IL-1-β and IL-6. In the present study,C-22,C-31 and C-34 were assessed for modulation of pro-inflammatory cytokines,TNF-α and IL-8,chemokine RANTES and NO produced by lipopolysaccharide (LPS)-treated mouse Dendritic cells (DCs). Among the 3 compounds,C-34 showed the most potent inhibition of inflammatory markers in DC model at 0.2 and 2 μM. C-34 also significantly downregulated the secretion of TNF-α,IL-1-β and IL-6 by murine splenocytes and THP-1 cells against LPS induced levels. In vitro effects of C-34 on bone marrow toxicity were assessed in CFU-GM assay. Human CFU-GM population was comparatively more sensitive to C-34 (0.1-10 μM) than murine CFU-GM. IC50 values for murine and human CFU-GM were not attained. C-34 was further examined for in vivo suppression of LPS induced cytokines in a mice model. At doses ranging from 1.25 to 5 mg/kg,C-34 led to significant inhibition of TNF-α,IL-1-β,IL-6 and MIP-1-α. At the highest dose of 5 mg/kg,C-34 also protected LPS-treated mice against endotoxin-induced lethality. In conclusion,C-34 demonstrates anti-inflammatory activity in vitro and in vivo in addition to cytotoxic properties. This finding suggests its potential for further development as a synthetic naphthyridine derivative with dual anti-cancer and anti-inflammatory (cytokine inhibition) properties. View Publication