Scientific Resources

Without effective therapy,chronic-phase chronic myeloid leukemia (CP-CML) evolves into an acute leukemia (blast crisis [BC]) that displays either myeloid or B-lymphoid characteristics. This transition is often preceded by a clinically recognized,but biologically poorly characterized,accelerated phase (AP). Here,we report that IKAROS protein is absent or reduced in bone marrow blasts from most CML patients with advanced myeloid disease (AP or BC). This contrasts with primitive CP-CML cells and BCR-ABL1-negative acute myeloid leukemia blasts,which express readily detectable IKAROS. To investigate whether loss of IKAROS contributes to myeloid disease progression in CP-CML,we examined the effects of forced expression of a dominant-negative isoform of IKAROS (IK6) in CP-CML patients' CD34(+) cells. We confirmed that IK6 disrupts IKAROS activity in transduced CP-CML cells and showed that it confers on them features of AP-CML,including a prolonged increased output in vitro and in xenografted mice of primitive cells with an enhanced ability to differentiate into basophils. Expression of IK6 in CD34(+) CP-CML cells also led to activation of signal transducer and activator of transcription 5 and transcriptional repression of its negative regulators. These findings implicate loss of IKAROS as a frequent step and potential diagnostic harbinger of progressive myeloid disease in CML patients. View Publication

The transcription factor Tal1 is a critical activator or repressor of gene expression in hematopoiesis and leukaemia. The mechanism by which Tal1 differentially influences transcription of distinct genes is not fully understood. Here we show that Tal1 interacts with the peptidylarginine deiminase IV (PADI4). We demonstrate that PADI4 can act as an epigenetic coactivator through influencing H3R2me2a. At the Tal1/PADI4 target gene IL6ST the repressive H3R2me2a mark triggered by PRMT6 is counteracted by PADI4,which augments the active H3K4me3 mark and thus increases IL6ST expression. In contrast,at the CTCF promoter PADI4 acts as a repressor. We propose that the influence of PADI4 on IL6ST transcription plays a role in the control of IL6ST expression during lineage differentiation of hematopoietic stem/progenitor cells. These results open the possibility to pharmacologically influence Tal1 in leukaemia. View Publication

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular,primitive leukemia cells,often termed leukemia stem cells,are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins,presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation,CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise,we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly,these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism,which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1),as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism,an intrinsic property of primary human AML cells. View Publication

Erythropoietin (Epo) is a cytokine that binds and activates an Epo receptor (EpoR) expressed on the surface of erythroid progenitor cells to promote erythropoiesis. While early studies suggested EpoR transcripts were expressed exclusively in the erythroid compartment,low-level EpoR transcripts were detected in nonhematopoietic tissues and tumor cell lines using sensitive RT-PCR methods. However due to the widespread use of nonspecific anti-EpoR antibodies there are conflicting data on EpoR protein expression. In tumor cell lines and normal human tissues examined with a specific and sensitive monoclonal antibody to human EpoR (A82),little/no EpoR protein was detected and it was not functional. In contrast,EpoR protein was reportedly detectable in a breast tumor cell line (MCF-7) and breast cancer tissues with an anti-EpoR polyclonal antibody (M-20),and functional responses to rHuEpo were reported with MCF-7 cells. In another study,a functional response was reported with the lung tumor cell line (NCI-H838) at physiological levels of rHuEpo. However,the specificity of M-20 is in question and the absence of appropriate negative controls raise questions about possible false-positive effects. Here we show that with A82,no EpoR protein was detectable in normal human and matching cancer tissues from breast,lung,colon,ovary and skin with little/no EpoR in MCF-7 and most other breast and lung tumor cell lines. We show further that M-20 provides false positive staining with tissues and it binds to a non-EpoR protein that migrates at the same size as EpoR with MCF-7 lysates. EpoR protein was detectable with NCI-H838 cells,but no rHuEpo-induced phosphorylation of AKT,STAT3,pS6RP or STAT5 was observed suggesting the EpoR was not functional. Taken together these results raise questions about the hypothesis that most tumors express high levels of functional EpoR protein. View Publication

BACKGROUND Mortality rates for leukemia are high despite considerable improvements in treatment. Since polyphenols exert pro-apoptotic effects in solid tumors,our study investigated the effects of polyphenols in haematological malignancies. The effect of eight polyphenols (quercetin,chrysin,apigenin,emodin,aloe-emodin,rhein,cis-stilbene and trans-stilbene) were studied on cell proliferation,cell cycle and apoptosis in four lymphoid and four myeloid leukemic cells lines,together with normal haematopoietic control cells. METHODS Cellular proliferation was measured by CellTiter-Glo(®) luminescent assay; and cell cycle arrest was assessed using flow cytometry of propidium iodide stained cells. Apoptosis was investigated by caspase-3 activity assay using flow cytometry and apoptotic morphology was confirmed by Hoescht 33342 staining. RESULTS Emodin,quercetin,and cis-stilbene were the most effective polyphenols at decreasing cell viability (IC50 values of 5-22 μM,8-33 μM,and 25-85 μM respectively) and inducing apoptosis (AP50 values (the concentration which 50% of cells undergo apoptosis) of 2-27 μM,19-50 μM,and 8-50 μM respectively). Generally,lymphoid cell lines were more sensitive to polyphenol treatment compared to myeloid cell lines,however the most resistant myeloid (KG-1a and K562) cell lines were still found to respond to emodin and quercetin treatment at low micromolar levels. Non-tumor cells were less sensitive to all polyphenols compared to the leukemia cells. CONCLUSIONS These findings suggest that polyphenols have anti-tumor activity against leukemia cells with differential effects. Importantly,the differential sensitivity of emodin,quercetin,and cis-stilbene between leukemia and normal cells suggests that polyphenols are potential therapeutic agents for leukemia. View Publication

Developing novel therapies that suppress self-renewal of leukemia stem cells may reduce the likelihood of relapses and extend long-term survival of patients with acute myelogenous leukemia (AML). AML1-ETO (AE) is an oncogene that plays an important role in inducing self-renewal of hematopoietic stem/progenitor cells (HSPCs),leading to the development of leukemia stem cells. Previously,using a zebrafish model of AE and a whole-organism chemical suppressor screen,we have discovered that AE induces specific hematopoietic phenotypes in embryonic zebrafish through a cyclooxygenase (COX)-2 and β-catenin-dependent pathway. Here,we show that AE also induces expression of the Cox-2 gene and activates β-catenin in mouse bone marrow cells. Inhibition of COX suppresses β-catenin activation and serial replating of AE(+) mouse HSPCs. Genetic knockdown of β-catenin also abrogates the clonogenic growth of AE(+) mouse HSPCs and human leukemia cells. In addition,treatment with nimesulide,a COX-2 selective inhibitor,dramatically suppresses xenograft tumor formation and inhibits in vivo progression of human leukemia cells. In summary,our data indicate an important role of a COX/β-catenin-dependent signaling pathway in tumor initiation,growth,and self-renewal,and in providing the rationale for testing potential benefits from common COX inhibitors as a part of AML treatments. View Publication

Extrinsic signaling cues in the microenvironment of acute myelogenous leukemia (AML) contribute to disease progression and therapy resistance. Yet,it remains unknown how the bone marrow niche in which AML arises is subverted to support leukemic persistence at the expense of homeostatic function. Exosomes are cell membrane-derived vesicles carrying protein and RNA cargoes that have emerged as mediators of cell-cell communication. In this study,we examined the role of exosomes in developing the AML niche of the bone marrow microenvironment,investigating their biogenesis with a focus on RNA trafficking. We found that both primary AML and AML cell lines released exosome-sized vesicles that entered bystander cells. These exosomes were enriched for several coding and noncoding RNAs relevant to AML pathogenesis. Furthermore,their uptake by bone marrow stromal cells altered their secretion of growth factors. Proof-of-concept studies provided additional evidence for the canonical functions of the transferred RNA. Taken together,our findings revealed that AML exosome trafficking alters the proliferative,angiogenic,and migratory responses of cocultured stromal and hematopoietic progenitor cell lines,helping explain how the microenvironmental niche becomes reprogrammed during invasion of the bone marrow by AML. View Publication

The mixed lineage leukemia (MLL) proto-oncogenic protein is a histone-lysine N-methyltransferase that is produced by proteolytic cleavage and self-association of the respective functionally distinct subunits (MLL(N) and MLL(C)) to form a holocomplex involved in epigenetic transcriptional regulation. On the basis of studies in Drosophila it has been suggested that the separated subunits might also have distinct functions. In this study,we used a genetically engineered mouse line that lacked MLL(C) to show that the MLL(N)-MLL(C) holocomplex is responsible for MLL functions in various developmental processes. The stability of MLL(N) is dependent on its intramolecular interaction with MLL(C),which is mediated through the first and fourth plant homeodomain (PHD) fingers (PHD1 and PHD4) and the phenylalanine/tyrosine-rich (FYRN) domain of MLL(N). Free MLL(N) is destroyed by a mechanism that targets the FYRN domain,whereas free MLL(C) is exported to the cytoplasm and degraded by the proteasome. PHD1 is encoded by an alternatively spliced exon that is occasionally deleted in T-cell leukemia,and its absence produces an MLL mutant protein that is deficient for holocomplex formation. Therefore,this should be a loss-of-function mutant allele,suggesting that the known tumor suppression role of MLL may also apply to the T-cell lineage. Our data demonstrate that the dissociated MLL subunits are subjected to distinct degradation pathways and thus not likely to have separate functions unless the degradation mechanisms are inhibited. View Publication

Oligomerization is an important regulatory mechanism for many proteins,including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity,suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges,resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations,which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl,reduced cell proliferation,and increased caspase-3/7 activity and DNA segmentation. Importantly,the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention. 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

Recent evidence suggests that enhanced aldehyde dehydrogenase (ALDH) activity is a hallmark of cancer stem cells (CSC) measurable by the aldefluor assay. ALDH1A1,one of 19 ALDH isoforms expressed in humans,was generally believed to be responsible for the ALDH activity of CSCs. More recently,experiments with murine hematopoietic stem cells,murine progenitor pancreatic cells,and human breast CSCs indicate that other ALDH isoforms,particularly ALDH1A3,significantly contribute to aldefluor positivity,which may be tissue and cancer specific. Therefore,potential prognostic application involving the use of CSC prevalence in tumor tissue to predict patient outcome requires the identification and quantification of specific ALDH isoforms. Herein we review the suggested roles of ALDH in CSC biology and the immunohistological studies testing the potential application of ALDH isoforms as novel cancer prognostic indicators. View Publication

4-Hydroxy-5-methoxy-2,3-dihydro-1H-[1,3]benzodioxolo[5,6-c]pyrrolo[1,2-f]-phenanthridium chloride (NK314) is a benzo[c] phenanthridine alkaloid that inhibits topoisomerase IIα,leading to the generation of DNA double-strand breaks (DSBs) and activating the G(2) checkpoint pathway. The purpose of the present studies was to investigate the DNA intercalating properties of NK314,to evaluate the DNA repair mechanisms activated in cells that may lead to resistance to NK314,and to develop mechanism-based combination strategies to maximize the antitumor effect of the compound. A DNA unwinding assay indicated that NK314 intercalates in DNA,a property that likely cooperates with its ability to trap topoisomerase IIα in its cleavage complex form. The consequence of this is the formation of DNA DSBs,as demonstrated by pulsed-field gel electrophoresis and H2AX phosphorylation. Clonogenic assays demonstrated a significant sensitization in NK314-treated cells deficient in DNA-dependent protein kinase (DNA-PK) catalytic subunit,Ku80,ataxia telangiectasia mutated (ATM),BRCA2,or XRCC3 compared with wild-type cells,indicating that both nonhomologous end-joining and homologous recombination DNA repair pathways contribute to cell survival. Furthermore,both the DNA-PK inhibitor 8-(4-dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one (NU7441) and the ATM inhibitor 2-(4-morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one (KU55933) significantly sensitized cells to NK314. We conclude that DNA-PK and ATM contribute to cell survival in response to NK314 and could be potential targets for abrogating resistance and maximizing the antitumor effect of NK314. View Publication