Positive Leukemia Cells Research Articles

Abstract B255: Discovery and validation of a novel class of small molecule inhibitors of the CDC7 kinase: Modulation of tumor cell growth in vitro and in vivo

Abstract High throughput screening of compounds comprising the Memorial Sloan Kettering chemical library resulted in several confirmed hits against the recombinant Cdc7:Dbf4 heterodimeric kinase, a key regulator in the initiation of DNA replication and the G1 to S phase transition. Chemoinformatic analysis of the hits revealed an enrichment in one chemical cluster made up of several naturally occurring compounds, of which the most potent compound, CKI-7, was selected for further investigation. First, CKI-7 was found to be a non competitive inhibitor for ATP and prompted us to prolife it against a panel of 200 known kinases in order to assess its selectivity profile. The results were as predicted and very few kinases were specifically affected. Second, CKI-7 cytotoxic activity was assessed against a panel of well established cancer cell lines representing both hematopoietic and solid tumor malignancies as well as against a panel of primary hematopoietic cells derived from leukemia patients (both chemotherapy naive and relapsed/refractory samples) and was found to be a very effective agent with potencies in the low nanomolar range. Subsequent studies using an isogenic pair of cell lines with one over expressing the Bcl_xL anti-apoptotic protein further confirmed the induction of the intrinsic apoptotic pathway via caspase-3 activation in the absence and attenuation of the activity in the presence of Bcl_xL. This was further demonstrated through standard cell cycle synchronization studies revealing that exposure to the Cdc7 inhibitor results in an S phase arrest, cell cycle dependent caspase-3 activation, and apoptotic cell death. This cell death is the direct result of Cdc7 kinase inhibition by CKI-7 as demonstrated using a Cdc7 substrate biomarker assay. Third, the physicochemical properties of this class of naturally occurring compounds also prompted us to investigate their effect on several multidrug resistence (MDR) over-expressing cell lines. We found that CKI-7 was not a substrate for the efflux pumps demonstrating that this novel compound can overcome a major mechanism of chemotherapy resistence in human tumor cells. Based of the above observations, in vivo dose-dependent anti-tumor activity of CKI-7 was subsequently demonstrated in a SCID-Beige mouse systemic tumor model utilizing a recently isolated Philadelphia chromosome positive acute lymphoblastic leukemia cell line (PhALL3.1) in addition to melanoma and non small cell lung cancer xenograft models. Taken together, our data confirm that Cdc7 is a new promising target for cancer therapy, and that the newly discovered inhibitor CKI-7, a naturally occurring selective small molecule inhibitor of this enzyme, is an equally promising novel cancer therapeutic agent. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B255.

Activation State of the Vascular Niche Regulates Homeostasis of the Normal and Malignant Hematopoietic Stem Cells.

Abstract 3624Poster Board III-560Hematopoietic stem cells (HSCs) reside in supportive microenvironment in bone marrow called niche. Growing evidence indicates that interaction between niche component cells and HSCs might be critical to balance the quiescence, expansion, and differentiation of HSCs. The osteogenic niche has been shown to be primarily important for the maintenance and modulation of the size of the HSCs mass. It has also been shown that vascular niche, which is composed of sinusoidal endothelial cells, plays a critical role in maintenance of HSCs and leukemic cells. Understanding the mechanism by which vascular niche controls fate and number of HSCs may be useful not only to identify as yet undetermined factors that control self-renewal or differentiation of HSCs but also to elucidate physiologically relevant molecular targets to treat malignant counterpart of HSCs. We have established a co-culture system of endothelial cells and hematopoietic stem cells mimicking the vascular niche by using primary endothelial cells (PECs) transduced with adenovirus gene E4ORF1, and succeeded in over 500-fold expansion of repopulating long-term (LT)-HSCs. In this study, we evaluated as to how activation state of endothelial cells changes the endothelial cell potential in supporting LT-HSCs or leukemic cell expansion.Akt or/and p42/44 MAPK pathway activated endothelial cells were generated by transducing PECs with lenti viruses expressing E4ORF1, constitutive active Akt, constitutive active c-Raf, constitutive active K-ras or polyoma virus middle T antigen. Microarray and qPCR analysis showed there are clear differences between Akt-activated and MAPK-activated PECs in expression profile of genes involved in self-renewal or differentiation of HSCs such as jagged-1, CXCR4 and IGFBP-2. After 20 days of co-culture with mouse hematopoietic lineage negative (Lin-) cells, Akt-activated PECs supported over 200-fold increase of Lin- cells, whereas MAPK-activated PECs only supported less than 5-fold increase and Akt plus MAPK-activated PECs supported around 40-fold increase. On the contrary, MAPK-activated PECs supported hematopoietic lineage positive cells generation or leukemic cells expansion to the same extent as Akt-activated PECs. Conditioned medium of MAPK-activated, but not Akt-activated, PECs inhibited Lin- cell expansion on PECs without affecting lineage positive cell generation. We also confirmed Lin- cells cultured on Akt-activated PECs maintained more LT-HSCs activity than that cultured on MAPK-activated or Akt plus MAPK-activated PECs by competitive transplantation assays.These results indicate that activation of Akt signaling in vascular niche can support HSCs self-renewal, whereas activation of p42/44 MAPK signaling can induce HSCs differentiation. Stable endothelial cells and angiogenic endothelial cells might differently regulate HSCs fate after injury such as myelosuppression in bone marrow. We are now trying to define growth factors that regulate HSC self-renewal and differentiation by using this co-culture system. Disclosures:No relevant conflicts of interest to declare.

Discovery and Validation of a Novel Class of Small Molecule Inhibitors of the CDC7 Kinase: Modulation of Tumor Cell Growth in Vitro and In Vivo.

Abstract 3771Poster Board III-707High throughput screening of compounds comprising the Memorial Sloan Kettering chemical library resulted in several confirmed hits against the recombinant Cdc7:Dbf4 heterodimeric kinase, a key regulator in the initiation of DNA replication and the G1 to S phase transition. Chemoinformatic analysis of the hits revealed an enrichment in one chemical cluster made up of several naturally occurring compounds, of which the most potent compound, CKI-7, was selected for further investigation. First, CKI-7 was found to be a non competitive inhibitor for ATP and prompted us to prolife it against a panel of 200 known kinases in order to assess its selectivity profile. The results were as predicted and very few kinases were specifically affected. Second, CKI-7 cytotoxic activity was assessed against a panel of well established cancer cell lines representing both hematopoietic and solid tumor malignancies as well as against a panel of primary hematopoietic cells derived from leukemia patients (both chemotherapy naïve and relapsed/refractory samples) and was found to be a very effective agent with potencies in the low nanomolar range. Subsequent studies using an isogenic pair of cell lines with one over expressing the Bcl_xL anti-apoptotic protein further confirmed the induction of the intrinsic apoptotic pathway via caspase-3 activation in the absence and attenuation of the activity in the presence of Bcl_xL. This was further demonstrated through standard cell cycle synchronization studies revealing that exposure to the Cdc7 inhibitor results in an S phase arrest, cell cycle dependent caspase-3 activation, and apoptotic cell death. This cell death is the direct result of Cdc7 kinase inhibition by CKI-7 as demonstrated using a Cdc7 substrate biomarker assay. Third, the physicochemical properties of this class of naturally occurring compounds also prompted us to investigate their effect on several multidrug resistence (MDR) over-expressing cell lines. We found that CKI-7 was not a substrate for the efflux pumps demonstrating that this novel compound can overcome a major mechanism of chemotherapy resistence in human tumor cells. Based of the above observations, in vivo dose-dependent anti-tumor activity of CKI-7 was subsequently demonstrated in a SCID-Beige mouse systemic tumor model utilizing a recently isolated Philadelphia chromosome positive acute lymphoblastic leukemia cell line (PhALL3.1). Taken together, our data confirm that Cdc7 is a new promising target for cancer therapy, and that the newly discovered inhibitor CKI-7, a naturally occurring selective small molecule inhibitor of this enzyme, is an equally promising novel cancer therapeutic agent. Disclosures:No relevant conflicts of interest to declare.

Downregulation of MiR-449a Is Essential for the Survival of EVI1 Positive Leukemic Cells through Modulation of NOTCH1 and BCL2.

Abstract 361▪▪This icon denotes an abstract that is clinically relevant.Chromosomal rearrangements involving the EVI1 gene are a recurrent finding in malignant myeloid disorders. These translocations or inversions contribute to ectopic expression or to the formation of fusion genes involving the EVI1 gene. EVI1 transcriptional activation has been reported in up to 10% of acute myeloid leukemia (AML) and is a prognostic marker of poor outcome. MicroRNA (miRNA) deregulation was recently identified as a major contributor to cancer initiation and progression. As miRNA genes were shown to be directly regulated by activated proto-oncogenes, we aimed to identify miRNAs under direct or indirect control of EVI1. To this purpose, we analyzed the expression of 366 miRNAs in 38 EVI1 rearranged/overexpressing patient samples, 6 normal bone marrow controls and 2 EVI1 knockdown model systems (siRNA mediated EVI1 knockdown in the EVI1 rearranged/overexpressing cell lines Kasumi-3 and UCSD-AML1). In total, 24 upregulated and 25 downregulated miRNAs (p<0.05) were shown to be related to the EVI1 expression status. Amongst these, miR-449a was selected for further study based on its homology to the known cancer associated miRNA miR-34a. Downregulation of miR-449a by EVI1 was further confirmed in the leukemic cell line U937 with tetracycline controllabel (tet-off) EVI1 overexpression. Next, direct transcriptional regulation of miR-449a expression by EVI1 was demonstrated by chromatin immunoprecipitation (ChIP). To test the functional consequences of downregulation of miR-449a in AML cells, reconstitution of the expression of miR-449a in the Kasumi-3 and UCSD-AML1 cell lines was performed, which resulted in significantly decreased cell viability, increased apoptosis and differentiation towards the megakaryocytic and monocytic lineages. Interestingly, siRNA mediated knockdown of EVI1 expression in Kasumi-3 or UCSD-AML1 almost completely abrogated the miR-449a induced reduction in cell viability, while electroporation of both cell lines with EVI1 siRNAs alone had essentially no effect on cell viability. These data strongly suggest that repression of miR-449a expression is essential for the survival and growth of EVI1 overexpressing cells and that this requirement is specifically imposed by EVI1 itself. We next demonstrated that the predicted miR-449a targets NOTCH1 and BCL2 were bona fide miR-449a targets using promoter reporter assays. To asses the contribution of these target genes to the observed phenotype upon miR-449a upregulation, knockdown of NOTCH1 and BCL2 was performed, revealing similar effects on cell viability and apoptosis. These results indicated that the effects seen upon treatment of cells with a precursor miR-449a are at least partly mediated through NOTCH1 and BCL2. In conclusion, we provided for the first time evidence that EVI1 mediated downregulation of miR-449a leads to NOTCH1 and BCL2 upregulation and is required for sustained proliferation and survival of EVI1 overexpressing cells. These data also open new perspectives for therapeutic intervention through modulation of miR-449a and/or its target genes. Disclosures:No relevant conflicts of interest to declare.

The Efficacy of Dual PI3K and mTOR Inhibitor, NVP-BEZ235 against BCR-ABL Positive Leukemia Cells Include ABL Kinase Domain Mutation in Combination with Nilotinib.

Abstract 3784Poster Board III-720Imatinib has shown clinical efficacy against chronic myeloid leukemia (CML) and it is now the standard care fore initial therapy of CML. However, a substantial number of patients are either primary refractory or acquire resistance to imatinib. Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance in patients with BCR-ABL-positive leukemia and this becomes a problematic in clinically. Phosphoinositide 3 kinase (PI3K)/Akt /mammalian target of rapamycin (mTOR) pathway regulates various processes including cell proliferation, survival and anti-apoptosis activity. Moreover, PI3K/Akt/mTOR pathway is desregulated in hematological malignancies and activated by upstream proteins such as BCR-ABL. NVP-BEZ235 is a dual inhibitor of PI3K and mTOR. It has been reported NVP-BEZ235 showed high target specificity and demonstrated antiproliferative activity against tumor. However, the molecular and functional consequences of NVP-BEZ235 against BCR-ABL expressing cells with BCR-ABL kinase domain mutation have not fully known. In this study, we investigated the NVP-BEZ235 efficacy by using the BCR-ABL positive cell line, K562 and murine Ba/F3 cell line which was transfected wild type (Wt) p210 BCR-ABL or imatinib resistant BCR-ABL mutants such as E255K, T315I and primary sample with T315I mutation. 48 hours treatment of NVP-BEZ235 exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. We also found that NVP-BEZ235 potently induced cell growth inhibition of murine Ba/F3 cells ectopically expressing wild type (Wt) p210 and imatinib resistant BCR-ABL mutants such as E255K and T315I mutation. We next examined the intracellular signaling by using these cell lines. We found that phosphorylation of Akt, eukaryotic initiation factor 4-binding protein 1 (4E-BP1) and p70 S6 kinase were decreased after NVP-BEZ235 treatment in a dose dependent manner. However, Mitogen-activated Protein Kinase (MAPK) activation was not reduced after NVP-BEZ235 treatment. Because Abl kinase inhibitor, nilotinib has shown efficacy against imatinib resistant BCR-ABL mutant, we investigated the efficacy between NVP-BEZ235 and nilotinib by using these cell lines. We found that combination of NVP-BEZ235 and nilotinib more potentially cell growth inhibition of K562, Ba/F3 Wt BCR-ABL, BCR-ABL mutations (E255K, T315I) Ba/F3 cells. We found that combination of NVP-BEZ235 and nilotinib more potentially induced apoptosis in 48 hours treatment. Phosphorylation of Akt, 4E-BP-1, S6 kinase was reduced after NVP-BEZ235 and nilotinib treatment in K562 cells. Cyclin D1 and BCL-XL were also decreased. PARP activation was synergistically increased after NVP-BEZ235 and nilotinib treatment. We next examined the affectivity of NVP-BEZ235 and nilotinib by using T315I positive primary sample. We found that NVP-BEZ235 and nilotinib potently induced cell growth inhibition and also increased apoptosis of primary T315I cells in 48 hours treatment. Data from this study suggested that administration of the a dual inhibitor of PI3K and mTOR, NVP-BEZ235 may be a powerful strategy against BCR-ABL mutant cells and enhance cytotoxic effects of nilotinib in those imatinib resistant BCR-ABL mutant cells. Disclosures:No relevant conflicts of interest to declare.

Downregulation of the Cell-Cycle Regulating Ubiquitin-Ligase APC/CCdh1 May Contribute to the Differentiation Block of AML1/Eto Positive AML.

Abstract 5045 IntroductionThe anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase regulating cell cycle progression by targeting various cell cycle regulators for proteasomal degradation. It is activated by the adaptor proteins Cdc20 in mitosis and by Cdh1 in late mitosis and G1/G0. Thereby, Cdh1 establishes a stable G1 phase enabling the cell to either exit the cell cycle and differentiate or to prepare for a new round of cell division. It has also been shown that Cdh1 plays a role in the differentiation of various cell types, such as neurons, myocytes, hepatocytes and lens epithelial cells. Methods and ResultsWe have examined the regulation of Cdh1 in several acute myeloid leukemia (AML) cell lines. We found that in the AML1/Eto positive leukemia cell lines SKNO-1 and Kasumi-1, Cdh1 protein and RNA levels are lower than in AML1/Eto negative cell lines KG-1 and HL-60. In addition, Cdh1 protein level in an AML1/Eto positive primary blast sample was lower than in AML1/Eto negative patient samples. The translocation t(8;21) is one of the most frequent chromosomal rearrangement in AML and results in an AML1/Eto fusion protein, which can act as a transcriptional repressor. Thus, our results are consistent with AML1/Eto mediated downregulation of Cdh1. To evaluate the potential role of APC/CCdh1 in myeloid differentiation, we established a stable Cdh1 knockdown (kd) in the AML1/Eto negative HL60 cell line with high Cdh1 expression by lentiviral vector mediated RNA interference. HL60 cells harbouring either a Cdh1 shRNA or a control shRNA against GFP were established simultaneously. We used PMA at concentrations of 0.5, 1, 2 and 50 nM to differentiate these cells into CD11b positive macrophage-like cells over 48h. Protein isolation and analysis of CD11b expression by flow cytometry were performed at 0, 6h, 12h, 24h and 48h to examine differentiation kinetics. Cdh1 and target proteins with a potential role in cell cycle arrest and differentiation, such as Skp2 (an activator of the SCF-ubiquitin ligase targeting p21 and p27) and ID2 (inhibitor of differentiation 2), were analyzed by Western blotting. We observed that kd of Cdh1 in HL60 cells resulted in 10% to 20% lower CD11b expression at any time, when PMA was used at concentrations 0, 0.5, 1nM over 48h. ID2 and Skp2 were stabilized in these Cdh1 kd cells compared to the control correlating with the less differentiated state. In addition, HL60 cells with a stable Skp2 kd showed a higher CD11b expression indicating a more differentiated status compared to the control. ConclusionThis is the first report that indicates a role for APC/CCdh1 in the differentiation of myeloid cells with SCFSkp2 being one of the relevant targets. Downregulation of Cdh1 may contribute to the differentiation block of AML1/Eto postive AML. DisclosuresNo relevant conflicts of interest to declare.

Non-Cycling Cells From Acute Myeloid Leukemia Patients Harbor the FLT3-ITD Mutation and Are Insensitive to TKI258, a Potent FLT3-Directed Inhibitor, in Vitro.

Abstract 479The identification of activating mutations in the FLT3 gene and their impact on prognosis has been crucial to the rationale behind the development of FLT3 inhibitors. While it has been shown that some leukemic cells with high tumorigenic potential exist mostly in a dormant state, it is unclear if this quiescent/non-cycling leukemia-initiating fraction carries the FLT3-ITD mutation and if it is successfully targeted by FLT3 inhibitors. As a paradigm, quiescent Ph+ stem cells in CML have been shown to exhibit resistance to bcr-abl targeted inhibitors. Additionally, results from clinical trials suggest that FLT3 inhibitors reduce the leukemic blast count in peripheral blood but are less successful in the bone marrow where factors regulating hematopoietic stem cell quiescence are active. In order to investigate the non-cycling and cycling human leukemic cell boundary, we devised a biological model that allowed us to distinguish non-cycling AML cells from cycling AML progenitors in human FLT3-ITD positive AML samples. CD34+ cells were isolated from AML samples using magnetic cell sorting, labeled with the cell membrane dye PKH26 to enable tracking of cell division, and cultured on murine stroma for 12 days. Non-cycling AML cells were then separated from cycling cells by FACS sorting and were found to retain a CD34+ primitive phenotype in contrast to expanding leukemic blasts. Fluorescence in situ hybridization analyses revealed that non-cycling cells carried leukemic gene rearrangements (trisomy 8, trisomy 13, t[3;21]and t[16;16] in our cases), and were therefore part of the original leukemic clone. PCR for the FLT3-ITD region showed that in four out of five cases, the FLT3-ITD mutation was present in the non-cycling fraction. To examine the distribution of FLT3-ITD to FLT3 wild type (WT) bearing cells, non-cycling AML cells were FACS sorted, DNA extracted and the PCR products subsequently cloned. Bacterial colonies were sequenced and colony-PCR used to determine the ratio of FLT3-ITD to WT bearing colonies for each patient. These data indicated that at least 25% of non-cycling cells (range 25%-100%) harbored the FLT3-ITD mutation. We then assessed the impact of a potent FLT3-directed inhibitor, TKI258 (Novartis), on leukemic cell expansion and the viability of non-cycling cells. TKI258 has been found to induce apoptosis of FLT3-ITD bearing cells of the human acute monocytic leukemia MV4;11 cell line. In our present study, CD34+ AML blasts from the same FLT3-ITD positive patient samples were grown in vitro in the presence of 0 μM, 0.3 μM (IC50 dose) and 1.25 μM TKI258. In stromal cultures, TKI258 significantly reduced leukemic cell expansion (range 2.13 to 20 fold for untreated cultures and 0.07 to 2.27 fold for 1.25 μM TKI258 treated cultures at day 12, p ≤ 0.05). In methylcellulose colony assays, TKI258 exposure resulted in dose-dependent suppression of colony formation of CD34+ FLT3-ITD positive leukemia cells (60% to 81% reduction in the mean plating efficiency of CD34+ AML cells at 0.3 μM TKI258). Despite this striking anti-proliferative effect, the majority of non-cycling cells from AML patients showed resistance to TKI258 (five out of six cases). In these samples, FLT3-ITD positive non-cycling cells could still be detected after treatment with the equivalent highest clinical dose (1.25 μM) of TKI258. Moreover, at a functional level, limiting-dilution experiments on non-cycling AML cells pre-treated with TKI258 showed no impairment in a modified leukemic cobblestone assay at four weeks compared to untreated non-cycling cells. These results suggest that the majority of non-cycling AML cells that harbor FLT3-ITD are unaffected by a FLT3 inhibitor and may constitute an as yet untargeted disease reservoir. Only one FLT3-ITD AML case showed exquisite sensitivity to TKI258 with elimination of the non-cycling fraction observed from 0.3 μM of TKI258 upwards. Possible explanations for this may include specific mutant receptor sensitivities or generic multi-drug resistance mechanisms operating in dormant cells. Interestingly, TKI258 selectively eradicated an ‘intermediate' dividing progenitor population in two of the insensitive cases, an indication that leukemic progenitors may be rendered sensitive to FLT3 inhibition on transiting the dormancy-cycling boundary. Further studies are needed to determine if these findings are representative of a generation of FLT3 inhibitors or specific for TKI258. Disclosures:No relevant conflicts of interest to declare.

Docetaxel enhances the cytotoxic effects of imatinib on Philadelphia positive human chronic myeloid leukemia cells

Chronic myelogenous leukemia (CML) results from a translocation between chromosomes 9 and 22 which generates BCR/ABL fusion protein and characterized by uncontrolled proliferation of immature white blood cells. Imatinib, a molecularly targeting anticancer agent, is used widely for the treatment of CML and showed significant activity in chronic and accelerated phases but much less in blast crisis phase. The resistance to imatinib especially in blast crisis phase is recognized as a major problem in the treatment of CML patients. Docetaxel is shown to arrest cells in G2/M phase of the cell cycle which makes cells more sensitive to chemo- and radiotherapy. In this study, we aimed to increase chemosensitivity of human K562 CML cells to imatinib in combination with docetaxel. Taken together, our results showed that the combination of imatinib and docetaxel decreased cellular proliferation and increased apoptosis in human K562 chronic myeloid leukemia cells as compared to any agent alone. Imatinib and docetaxel induced apoptosis through caspase-3 enzyme activity and mitochondrial membrane potential.

BAFF and APRIL expression in B-cell chronic lymphocytic leukemia: Correlation with biological and clinical features

B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) are involved in normal B cell survival and differentiation. We analyzed BAFF and APRIL plasma levels in patients with B-cell chronic lymphocytic leukemia (B-CLL). We also tested intracellular BAFF and APRIL expression in B-CLL and evaluated their prognostic relevance. The percentage of leukemic B cells with intracellular APRIL or BAFF expression correlated significantly with adverse prognostic factors such as ZAP-70 and CD38. Moreover, we found a close relationship between LPL protein expression or LPL/ADAM29 MFI ratio and proportion of B-CLL cells with intracellular BAFF or APRIL expression. Furthermore, patients with a low percentage of leukemic cells with intracellular BAFF or APRIL expression had a significantly longer overall survival than those with a high proportion of APRIL or BAFF positive leukemic cells.

Cross-talk between extracellular S1P/S1P2 and P42/44 MAPK in bcr/abl positive chronic myeloid leukemia cells

Objective BCR/ABL oncoprotein-expression is associated with uncontrolled cell growth. Sphingosine kinase 1 (SPK1) regulates the production of sphingosine 1-phosphate (S1P), a key lipid signal molecular in cell proliferation and survival. The objective of this study was to elucidate the roles of S1P and its receptors in bcr/abl positive chronic myeloid leukemia (CML) cells.

Mechanism of MK-0457 efficacy against BCR–ABL positive leukemia cells

Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance. MK-0457 is a small molecule inhibitor of the Aurora kinase family, but the mechanism of MK-0457 has not been evaluated. In this study, the gene expression profiles and intracellular signaling of chronic myeloid leukemia (CML) cell line K562 exposed to imatinib or MK-0457. MK-0457 induced cell growth inhibition in K562 cells. In gene expression profiles, there was an increase of 938 genes in imatinib and 895 genes in MK-0457 and 638 genes overlapped. In contrast, there was a decrease of 597 genes in imatinib and 582 genes in MK-0457 and 406 genes overlapped. These down-regulated genes include heat shock proteins (HSPs). These results indicate that MK-0457 is effective in CML cells by the down-regulation of HSPs which may relate to BCR–ABL stability, and offer new information regarding the molecular basis of strategy against to CML.

Inhibition of proteasome by bortezomib increase chemosensitivity of bcr/abl positive human k562 chronic myleoid leukemia cells to imatinib

Chronic myeloid leukaemia (CML) results from a translocation between chromosomes 9 and 22 which generates the BCR/ABL fusion oncopro-tein. BCR/ABL has constitutively tyrosine kinase activity resulting in leukemogenesis. Imatinib, a competitive inhibitor of the BCR/ABL tyrosine kinase, is the common treatment of CML. Despite the outstanding results of imatinib in the chronic phase of CML, cases of treatment failure have been reported, resulting in hetero-geneous molecular response. Bortezomib is a reversible inhibitor of the 26S proteasome in-ducing cell cycle arrest in G2/M phase, apop-tosis by inhibition of NF-kB. In this study, we examined the possible synergistic apoptotic effects of the imatinib/bortezomib combination and the responsible apoptotic mechanisms in-duced by this combination in K562 cells. The results of this study showed increased cyto-toxicity by XTT assay in combination of imatinib and bortezomib as compared to any agent alone. On the other hand, synergistic apoptotic affects of combination of these agents were also con-firmed by changes in caspase-3 enzyme activity and mitochondrial membrane potential. Taking together, all the results, confirming each other, showed that the combination of the imatinib and bortezomib has considerable synergistic effects on the apoptosis through increase in caspase-3 enzyme activity and decrease in mitochondrial membrane potential in human K562 CML cells.

Multidrug resistant P-glycoprotein positive L1210/VCR cells are also cross-resistant to cisplatin via a mechanism distinct from P-glycoprotein-mediated drug efflux activity

P-glycoprotein (P-gp, a drug transporter found in the plasma membrane)-mediated multidrug resistance of leukemia cells represents a real obstacle in the effective chemotherapeutic treatment of leukemia. While cisplatin (CisPt) is known to be a substance that is untransportable by P-gp, P-gp positive cells were often found to be resistant to CisPt. The aim of the current paper is to study this phenomenon using P-gp positive mouse leukemia cells L1210/VCR in which the overexpression of P-gp was induced by its ability to adapt to growth on vincristine (VCR). L1210/VCR cells are also resistant to CisPt. However, resistance to this substance could not be reversed by addition of the known P-gp inhibitor verapamil. CisPt induced more pronounced entry into apoptosis, as measured using the annexin V/propidium iodide kit, in sensitive L1210 cells than in resistant L1210/VCR cells. In addition, CisPt induced an increase in the proportion of L1210 cells that were in the g2 phase of the cell cycle when compared to L1210/VCR cells, as measured by staining with propidium iodide. Similarly, a higher release of cytochrome c from the mitochondria to the cytosol was induced by CisPt treatment in L1210 than in L1210/VCR cells. While similar levels of Bax and Bcl-2 proteins were observed in sensitive and resistant cells, CisPt induced a more pronounced decrease of the Bcl-2 levels in L1210 cells than in L1210/VCR cells. Consistent with this observation, CisPt induced a larger decrease of the Bcl-2 content in the Bcl-2:Bax heterooligomer in L1210 cells than in L1210/VCR cells. Moreover, CisPt induced a similar apoptotic DNA fragmentation pattern in both resistant and sensitive cells. All of the above observations indicated that L1210/VCR cells are also resistant to CisPt and that this resistance is related to the differences in the regulatory mechanisms responsible for CisPt-induced apoptosis in L1210/VCR cells without any contribution from the drug efflux activity of P-gp.

Anticancer Effects of Phenoxazine Derivatives Revealed by Inhibition of Cell Growth and Viability, Disregulation of Cell Cycle, and Apoptosis Induction in HTLV-1–Positive Leukemia Cells

Adult T-cell leukemia (ATL) is a malignant tumor of human CD4(+) T cells infected with a human retrovirus, T lymphotropic virus type-1 (HTLV-1). The aim of the present study was to investigate the apoptotic effects of phenoxazines, 2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one (Phx-1), 3-amino-1,4alpha-dihydro-4alpha,8-dimethyl-2H-phenoxazine-2-one (Phx-2), and 2-aminophenoxazine-3-one (Phx-3) on a T cell leukemia cell line from ATL patients, MT-1 cells; HTLV-1 transformed T-cell lines, HUT-102 cells and MT-2 cells; and an HTLV-1-negative rat sarcoma cell line, XC cells. Among these phenoxazines, Phx-3 at concentrations of less than 10 microg/ml extensively inhibited growth and cell viability; arrested cell cycles at sub G(0)/G(1) phase; and augmented apoptosis of MT-1, HUT-102, and MT-2 cells. However, these phenoxazines did not affect the cell viability of an HTLV-1-negative rat sarcoma cell line, XC cells, and phytohemaggutinin-activated human peripheral blood mononuclear cells, although they markedly inhibited the growth of these cells. The transmission of HTLV-1 from HTLV-1-positive cells (MT-2 cells) to HTLV-1-negative cells (XC cells) was considered to be prevented by Phx-1, Phx-2, or Phx-3 because the syncytium formation between these cells was inhibited markedly in the presence of these phenoxazines. The present results suggest that Phx-1, Phx-2, and, in particular, Phx-3 may be useful as therapeutic agents against ATL, which is extremely refractory to current therapies.

β-catenin is involved in N-cadherin–dependent adhesion, but not in canonical Wnt signaling in E2A-PBX1–positive B acute lymphoblastic leukemia cells

The t(1;19)(q23;13) translocation, resulting in the production of the E2A-PBX1 chimeric protein, is a common nonrandom translocation in pediatric B-lineage acute lymphoblastic leukemia (B-ALL). The E2A-PBX1 chimeric protein activates expression of several genes, including Wnt16. In the present study, we explored the role of Wnt16 and beta-catenin in t(1;19) B-ALL cells. Canonical Wnt signaling was measured by TOPflash activity. Localization of beta-catenin in the cell membrane and its involvement in leukemia-stroma interaction were studied by confocal microscopy. Adhesion to N-cadherin was analyzed by adding (3)H-thymidin-labeled cells to N-cadherin-coated wells. In contrast to previous reports, we detected no effects on cell viability or proliferation upon modulation of the Wnt16 levels. Moreover, despite high levels of Wnt16 and beta-catenin, the cells had very low levels of canonical Wnt signaling. Instead, beta-catenin was located in the cell membrane along with N-cadherin. E2A-PBX1-positive leukemia cells adhered strongly to bone marrow stroma cells, and we showed that adherence junctions stained strongly for both proteins. Moreover, knockdown of beta-catenin reduced the adhesion of E2A-PBX1-positive leukemia cells to N-cadherin, suggesting that beta-catenin and N-cadherin play a central role in homotypic cell-to-cell adhesion and in leukemia-stroma adhesion. Interestingly, knockdown of Wnt16 by small interfering RNA reduced the level of N-cadherin. Wnt16 does not activate canonical Wnt signaling in E2A-PBX1-positive cells. Instead, beta-catenin is involved in N-cadherin-dependent adherence junctions, suggesting for the first time that leukemia-stroma interactions may be mediated via an N-cadherin-dependent mechanism.

SRPX2 is overexpressed in gastric cancer and promotes cellular migration and adhesion

SRPX2 (Sushi repeat containing protein, X-linked 2) was first identified as a downstream molecule of the E2A-HLF fusion gene in t(17;19)-positive leukemia cells and the biological function of this gene remains unknown. We found that SRPX2 is overexpressed in gastric cancer and the expression and clinical features showed that high mRNA expression levels were observed in patients with unfavorable outcomes using real-time RT-PCR. The cellular distribution of SRPX2 protein showed the secretion of SRPX2 into extracellular regions and its localization in the cytoplasm. The introduction of the SRPX2 gene into HEK293 cells did not modulate the cellular proliferative activity but did enhance the cellular migration activity, as shown using migration and scratch assays. The conditioned-medium obtained from SRPX2-overexpressing cells increased the cellular migration activity of a gastric cancer cell line, SNU-16. In addition, SRPX2 protein remarkably enhanced the cellular adhesion of SNU-16 and HSC-39 and increased the phosphorylation levels of focal adhesion kinase (FAK), as shown using western blotting, suggesting that SRPX2 enhances cellular migration and adhesion through FAK signaling. In conclusion, the overexpression of SRPX2 enhances cellular migration and adhesion in gastric cancer cells. Here, we report that the biological functions of SRPX2 include cellular migration and adhesion to cancer cells.

The Analysis of HDAC Inhibitor, Vorinostat Efficacy against Wild Type and BCR-ABL Mutant Positive Leukemia Cells in Monotherapy and in Combination with a Pan-Aurora Kinase Inhibitor, MK-0457

The use of imatinib, an ABL tyrosine kinase inhibitor, has led to a dramatic change in the management of BCR-ABL positive leukemia patients. However, the resistance to imatinib mediated by mutations in the BCR-ABL domain has become a major problem in the treatment. Histone deacetylase (HDAC) inhibitors have been shown to mediate the regulation of gene expression, induce cell growth, cell differentiation and apoptosis of tumor cells. Vorinostat (suberoylamide hydroxamic acid:SAHA) is a hydroxamic acid based polar HDAC inhibitor. Vorinostat have shown efficacy in a wide range of cancers such as cutaneous T-cell lymphoma (CTCL). However, efficacy of vorinostat against the BCR-ABL mutants has fully not known. Here we report on the studies performed against murine Ba/F3 cell line which was transfected wild type (Wt) p210 and p185 BCR-ABL or imatinib resistant BCR-ABL mutants such as G250E, Q252H, Y253F, E255K, M294V, T315I, T315A, F317L, F317V, M351T, H396P and T315I(p185). 48 hours treatment of vorinostat exhibits cell growth inhibition and proapoptotic activity murine Ba/F3 cells ectopically expressing Wt and imatinib resistant BCR-ABL mutants including T315I mutation in a dose dependent manner. IC50 of these cell lines are Wt(720nM), G250E(625nM), Q252H(220nM), Y253F(525nM), E255K(685nM), M294V(785nM), T315I(500nM), T315A(715nM), F317L(560nM), F317V(565nM), M351T(375nM) and H396P(485nM). Aurora kinases play a pivotal role in the regulator of mitotic processes during cell division. MK-0457 is a small molecule inhibitor of the Aurora kinase family and was found to be active against the cells from BCR-ABL positive patients with T315I mutation in clinical trial. Because vorinostat also depleted BCR-ABL, as well as induced apoptosis and sensitized BCR-ABL-expressing leukemia cells, we examined whether vorinostat and MK-0457 enhances the apoptosis in imatinib resistant BCR-ABL-expressing cells. 48 hours treatment of MK-0457 exhibits cell growth inhibition of Ba/F3 cells ectopically expressing Wt and imatinib resistant BCR-ABL mutants including T315I mutation. IC50 of MK-0457 is Wt(215nM), G250E(205nM), Q252H(185nM), Y253F(245nM), E255K(185nM), M294V(238nM), T315I(205nM), T315A(165nM), F317L(200nM), F317V(200nM), M351T(225nM) and H396P(195nM). We examined the intracellular signaling by using these cell lines. We found that caspase 3, and poly (ADPribose) polymerase (PARP) were activated after MK-0457 treatment in a dose dependent manner. Phosphorylation of BCR-ABL and Crk-L which is downstream target of BCR-ABL was reduced after MK-0457 treatment. We found that combination of vorinostat and MK-0457 synergistically cell growth inhibition of Wt and BCR-ABL mutants Ba/F3 cells in 48 hours treatment. Phosphorylation of Crk-L was reduced after vorinostat and MK-0457 treatment. Caspase 3 and PARP activation were also synergistically increased after vorinostat and MK-0457 treatment. We evaluated the activity of MK-0457 and vorinostat in primary BCR-ABL positive acute lymphoblastic leukemia (ALL) cells with the T315I mutation. We found that MK-0457 potently induced cell growth inhibition of primary T315I cells in 48 hours treatment. Moreover, combination of vorinostat and MK-0457 synergistically increased the cell growth inhibition in primary T315I cells. This study demonstrate monotherapy of vorinostat and the combination of vorinostat and MK-0457 are more potent efficacy not only wild type BCR-ABL but also imatinib resistant BCR-ABL mutants cells and represents a promising new strategy for treatment of imatinib resistant BCR-ABL positive leukemias, including those harboring the T315I mutation.

DOCK180, a New Surrogate Marker to Distinguish CD34 Positive Acute Leukemia Cells from CD34 Positive Normal Hematopoietic Stem/Progenitor Cells.

DOCK180, which is an archetype of the CDM family protein, plays a pivotal role in the regulation of cell motility and phagocytosis through the activation of small GTPase Rac as a guanine nucleoside exchange factor (GEF). The expression of DOCK180 is ubiquitous except in hematopoietic cells, while the expression of another CDM family protein, DOCK2, is strictly limited in hematopoietic cells. However, DOCK180 was recently induced in two types of hematopoietic cells, namely, macrophages and dendritic cells, during their differentiation from CD14 positive monocytes (Fujimoto et al. ASH 2005, abstract #3077). Therefore, the expression of DOCK180 was analyzed in various sorts of normal hematopoietic cells, including CD34 positive hematopoietic stem/progenitor cells (HSC/P). Real time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine the relative levels of DOCK180 mRNA expression (defined in 293T cells as 1000). Consistent with the findings of a previous report, there was a negligible amount of DOCK180 mRNA and protein in the peripheral blood or bone marrow mononuclear cells from normal volunteers. On the other hand, purified CD34 positive cells from healthy human bone marrow (N=4) and G-CSF mobilized peripheral blood (N=4) expressed a high amount of DOCK180 mRNA (1302 ± 883 Units and 1455 ± 689 Units, respectively). Furthermore, the expression of DOCK180 protein was confirmed by Western blotting in these CD34 positive cells. To investigate the association of the DOCK180 expression and granulocytic differentiation, CD34 positive cells were cultured with stem cell factor, IL-3 and G-CSF in vitro to induce them to mature into a granulocytic lineage and thus lose their surface CD34 expression. Interestingly, there was a gradual reduction in the DOCK180 expression accompanied by a loss of the surface CD34 expression, thus suggesting that the DOCK180 expression may therefore be limited in CD34 positive, immature HSC/P. Since a large number of immature acute leukemia cells also express CD34 antigen on their surface, the expression of DOCK180 was investigated in CD34 positive leukemic cells from 25 acute leukemia patients. The subjects consisted of 16 patients with acute myeloid leukemia (AML: M0 2, M1 2, M2 4, M4 3, M7 1, MDS overt leukemia 2, secondary AML 2), 6 with acute lymphoblastic leukemia (ALL: Pro B 3, Ph1 positive 2, common 1), and 3 in blastic crisis of chronic myeloid leukemia (BC CML: 2 lymphoid BC and 1 myeloid BC). The leukemic cells were collected at the first onset of the disease from 19 patients and from 6 other patients at the time of relapse. While there was no difference in the expression of DOCK2 between leukemic and normal CD34 positive cells, the expression of DOCK180 in leukemic cells was significantly lower than that detected in normal CD34 positive cells (median 17 Units vs. 1096 Units, range 0–4746 Units and 279–2812 Units, respectively, p<0.01). In addition, the CD34 positive cells from four patients in remission after successful therapy expressed high levels of both DOCK180 mRNA and protein. Interestingly, three out of four patients whose leukemic cells had a compatible DOCK180 mRNA expression to that of normal CD34 positive cells showed a refractory response to either conventional induction therapy or bone marrow transplantation. Taken together, DOCK180 may therefore be useful as a new surrogate marker to distinguish most leukemic CD34 positive cells from those of normal HSC/P, thereby suggesting that DOCK180 may thus play a possible role as a marker for the detection of minimal residual disease. Moreover, further study to determine the significance of DOCK180 expression in the prognosis of acute leukemia is thus called for.

TAA/ecdCD40L VPP Vaccination Induces Robust Adaptive Immune Response Even in Individuals with Post Transplantation Lymphopenia

Individuals of advanced chronological age exhibit an impaired immune response to vaccines. This may be due to a reduction in the ratio of antigen naïve/memory CD4 and CD8 T cells and acquisition of functional defects in activated “helper” CD4 T cells (eg diminished CD40 ligand (CD40L) expression) during the aging process. The absence of the CD40L on activated CD4 helper T cells reduces the magnitude of expansion of antigen specific T and B cells induced by vaccination. In order to circumvent this defective response to vaccines among individuals in the fifth and sixth decades of life, our laboratory has developed an adenoviral vector (Ad-sig-TAA/ecdCD40L) vaccine which is designed to overcome the absence of CD40L expression in activated CD4 helper T cells in older individuals. The subcutaneous (sc) injection of this vector leads to the release of a fusion protein composed of a TAA linked to the extracellular domain (ecd) of the CD40L, which binds to the CD40 receptor on DCs, activates the DCs, and leads to the presentation of TAA fragments on Class I MHC. Two sc injections of the TAA/ecdCD40L protein as a booster following the sc administration of the Ad-sig-TAA/ecdCD40L vector (we call this the TAA/ecdCD40L VPP vaccine) expands the magnitude of the cellular and humoral immune response induced by the vector in 18 month old aged mice as well as in younger mice. In order to explore ways of further amplifying the immune response induced by this vaccine, we decided to test the feasibility of using this vaccine following treatments which reduce the number of T cells in the body of the test subject. We hypothesized that during states of chemotherapy or radiation induced lymphopenia, the number of negative regulatory CD4CD25FoxP3 T cells would be reduced, and all of the regulatory signals in the T cell compartment would be promoting expansion of T cells, thus creating an ideal state for vaccination. To test this hypothesis, we injected 100,000 cells from an established neoplastic cell line sc. Three days later, we administered myeloablative doses of total body irradiation (TBI) followed by a T cell depleted syngeneic bone marrow transplant (TCDBMT) to reconstitute neutrophil and platelet production. Three days following the TBI and TCDBMT, we intravenously infused donor lymphocytes (DLI) from a TAA/ ecdCD40L VPP vaccinated syngeneic donor. Four weeks later, we vaccinated the recipient mouse further with TAA/ecdCD40L sc injections. We tested this for a TAA composed of a junctional peptide from the p210Bcr-Abl protein of chronic myelogenous leukemia (CML) and for the E7 protein of the human papilloma virus (HPV). We found that in the case of the BcrAbl/ecdCD40L VPP vaccine, 50% of the mice treated with TBI, TCDBMT, ten million lymphocytes (DLI) from BcrAbl/ecdCD40L VPP vaccinated syngeneic donors followed in 4 weeks by 3 BcrAbl/ecdCD40L protein sc injections of the recipient test mouse, developed a complete response with the vaccination and that these mice remained disease free beyond 250 days after injection of the P210Bcr-Abl positive 32D leukemia cells, whereas C56BL/6J test mice treated with TBI and TCDBMT without DLI from vaccinated donors nor sc BcrAbl/ecdCD40L sc booster vaccination following injection with the p210Bcr-Abl positive 32D myeloid leukemia cell line all died by day 32. Mice treated with TBI, TCDBMT, DLI from unvaccinated donors followed by vaccination of the recipient with 3 sc BcrAbl/ecdCD40L protein injections exhibited a degree of leukemia suppression that was equal to mice receiving TBI, TCDBMT, DLI from a BcrAbl/ecdCD40L VPP vaccinated donor and BcrAbl/ecdCD40L vaccination. To test the effect of the TAA/ecdCD40L VPP vaccine against an antigen associated with an epithelial neoplasm, we injected 100,000 E7 positive TC-1 mouse cancer cells into syngeneic C57BL6J mice followed in 3–5 days by myeloablative doses of TBI and engrafting doses of TCDBMT. Three days later, the mice received 10 million spleen cells from syngeneic donor mice previously vaccinated with the E7/ecdCD40L VPP vaccine. Finally, 4 weeks later, the test mice received sc E7/ecdCD40L protein booster injections. The vaccinated mice achieved much greater degrees of tumor suppression than was seen following TBI and TCDBMT without DLI from vaccinated donors. These studies show that it is possible to induce a robust adaptive immune response by vaccination with the TAA/ecdCD40L VPP vaccine even in severely lymphopenic individuals.

Expression and Functional Analysis of Autotaxin, a Relevant Motility and Survival Factor, in FLT3-ITD Positive Acute Myeloid Leukemia and Primary Hematopoietic Stem Cells.

FLT3-mutations are among the most common abnormalities in adult acute myeloid leukemia. These mutations induce constitutive activation of the receptor and downstream signaling pathways, including RAS/ERK, PI3K and STAT5-signalling. The most frequent aberration is an internal tandem duplication (ITD) of the juxtamembrane domain coding sequence, which is present in up to 27% of the patients with AML and has been associated with inferior prognosis. We have recently demonstrated by microarray analysis that leukemic samples of patients with FLT3-ITD mutations have significantly upregulated expression levels of Autotaxin (ATX). The ATX protein acts as a secreted lysophospholipase D (lysoPLD) through generating lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). LPA has several important functions in cell migration and proliferation and acts via G-protein coupled receptors. It has been shown that ATX represents an aberrantly expressed motility and growth factor in a variety of cancer cells. However data on ATX in myeloid leukemias and especially in AML are missing. To study more deeply the role of ATX in leukemogenesis, we screened a series of human leukemic model cell lines for ATX expression. Using retroviral transduction, two alternatively spliced transcripts of ATX were expressed in several human leukemic cell lines without detectable levels of endogenous ATX. To investigate the ATX function and expression in normal haematopoiesis we used CD34+ human progenitor cells. The expression of ATX transcripts was confirmed at both mRNA and protein levels by RT-PCR and Western blotting, respectively. Transwell migration assays in the presence of LPC or LPA were performed to study effects of ATX on cell motility. Proliferation and clonogenic potential were investigated using MTT and colony forming assays. Moreover, we examined the effect of the FLT3 inhibitor N-benzoylstaurosporine (PKC412) on ATX expression and ATX mediated migration in MV4-11 cells. High ATX expression was found primarily in malignant cells. Western blot analysis showed that detectable levels of ATX are secreted into medium within 2 hours. In normal cells, highest ATX mRNA expression was found in purified CD34 cells, but could also be found at lower levels in T-cells. Stable overexpression of FLT3-ITD in OCI-AML3 cells induced an increased ATX expression (up to 6 fold). Vice versa, inhibition of FLT3-ITD by sublethal doses of PKC412 in MV4-11 cells resulted in a significant reduction of ATX expression down to 10% of the initial expression level. Furthermore, PKC412 treatment resulted in a complete loss of LPC or LPA induced specific migratory capacity in MV4-11 cells. The transduction of ATX increased the colony-forming capacity by 75% and significantly increased the short term proliferation. LPA increased chemotaxis in human leukemic cell lines and human CD34+ progenitors in a dose dependent manner and induced significantly higher migratory rates by at least 50%. LPC induced chemotaxis by 80–200% only in cells with high expression of endogenous or exogenous ATX, demonstrating the autocrine activity of ATX. Ongoing studies on the mechanism of ATX expression showed that ionomycin, an activator of the Ca2+–calcineurin–NFAT signalling pathway, upregulated ATX mRNA in several leukemic cell lines as well as in human primary progenitors up to 5-fold, which could be completely blocked by cyclosporine A treatment, indicating an involvement of NFAT in the regulation of ATX. Our data suggest that the production of bioactive LPA through ATX is involved in controlling proliferation and migration of hematopoietic stem cells and its deregulation may contribute to the pathogenesis of AML, especially in patients with FLT3-ITD mutations.