Panel Of Leukemia Cell Lines Research Articles

Abstract A78: Simvastatin synergistically potentiates tipifarnib cytotoxic and apoptotic effects and disrupts Ras membrane localization in human leukemia cells.

Abstract Background: Tipifarnib, a farnesyl transferase inhibitor, was initially designed to prevent Ras farnesylation and its subsequent membrane localization, which is necessary for Ras mediated signaling. Although, the clinical effectiveness of tipifarnib as a single agent in solid tumors was limited, it was modest in hematologic malignancies. Alternative prenylation by geranylgeranyl transferase has been postulated as an escape mechanism by which Ras evades the effect of tipifarnib. Simvastatin, an anti-hyperlipidemic drug that inhibits 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase, has been shown in several studies to induce apoptosis in cancer cells through blockade of the geranylgeranylation pathway of small GTPases. Thus, we hypothesized that simvastatin can augment the cytotoxic effect of tipifarnib by blocking the alternative Ras prenylation pathway. Our studies were carried out in a panel of leukemia cell lines. Methods: Studies were carried out using five cell lines of varied leukemic origin including HL60, K562, Molt4, Jurkat and HSB2. Cells were exposed to increasing drug concentrations for 72 hours either as a single or combination treatment with simvastatin and tipifarnib. Cell viability was measured using AlamarBlue (Invitrogen) and the combination index values were calculated based on the Chou-Talalay equations. Apoptosis was evaluated using Annexin V assay. Biochemical assays assessing the expression of caspases, c-PARP and Ras, in both membrane and cytosolic fractions, were also used. Results: The combination of tipifarnib with simvastatin was shown to induce a synergistic cytotoxic effect in all cell lines tested. Notably, our results showed that coadministarion of simvastatin and tipifarnib resulted in the induction of apoptosis through the activation of several apoptotic markers including Caspase 3, 7 and 9 as well as the upregulation of cleaved PARP. These effects were accompanied by disruption of Ras membrane localization and its sequestration into the cytosol. Conclusions: These findings demonstrate that combining simvastatin with tipifarnib effectively disrupts Ras prenylation and induces apoptosis in human leukemia cell lines. These effects were observed with simvastatin and tipifarnib concentrations that can be achieved in the clinic. Thus, the effectiveness of this combination should be explored further in future clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A78.

An antiapoptotic BCL-2 family expression index predicts the response of chronic lymphocytic leukemia to ABT-737

AbstractThe antiapoptotic BCL-2 proteins regulate lymphocyte survival and are over-expressed in lymphoid malignancies, including chronic lymphocytic leukemia. The small molecule inhibitor ABT-737 binds with high affinity to BCL-2, BCL-XL, and BCL-W but with low affinity to MCL-1, BFL-1, and BCL-B. The active analog of ABT-737, navitoclax, has shown a high therapeutic index in lymphoid malignancies; developing a predictive marker for it would be clinically valuable for patient selection or choice of drug combinations. Here we used RT-PCR as a highly sensitive and quantitative assay to compare expression of antiapoptotic BCL-2 genes that are known to be targeted by ABT-737. Our findings reveal that the relative ratio of MCL-1 and BFL-1 to BCL-2 expression provides a highly significant linear correlation with ABT-737 sensitivity (r = 0.6, P < .001). In contrast, antiapoptotic transcript levels, used individually or in combination for high or low affinity ABT-737-binding proteins, could not predict ABT-737 sensitivity. The (MCL-1 + BFL-1)/BCL-2 ratio was validated in a panel of leukemic cell lines subjected to genetic and pharmacologic manipulations. Changes after ABT-737 treatment included increased expression of BFL-1 and BCL-B that may contribute to treatment resistance. This study defines a highly significant BCL-2 expression index for predicting the response of CLL to ABT-737.

Vinblastine sensitizes leukemia cells to cyclin-dependent kinase inhibitors, inducing acute cell cycle phase-independent apoptosis

The efficacy of many chemotherapeutic agents can be attenuated by expression of the anti-apoptotic proteins Bcl-2, Bcl-XL and Mcl-1. Flavopiridol and dinaciclib are cyclin-dependent kinase 7 and 9 inhibitors that transcriptionally inhibit expression of Mcl-1. We have investigated the ability of flavopiridol and dinaciclib to sensitize a panel of leukemia cell lines to vinblastine and paclitaxel. Both drugs acutely sensitized most of the leukemia lines to vinblastine, with 100% apoptosis in 4 h. Furthermore, dinaciclib sensitized freshly isolated chronic lymphocytic leukemia cells to vinblastine. This rapid induction of apoptosis was attributed to vinblastine-mediated activation of JNK because (a) flavopiridol and dinaciclib failed to induce apoptosis when combined with non-JNK activating concentrations of vinblastine; (b) JNK inhibitors suppressed JNK activity and prevented apoptosis; (c) flavopiridol did not potentiate apoptosis induced by paclitaxel which does not activate JNK in these cells; and (d) Jurkat cells failed to activate JNK in response to vinblastine and were not sensitive to combinations of vinblastine and flavopiridol or dinaciclib. The rapid induction of apoptosis by this combination in multiple cell systems but not in normal lymphocytes provides justification for performing a clinical trial to assess the efficacy in patients.

Multiple BH3 Mimetics Antagonize Antiapoptotic MCL1 Protein by Inducing the Endoplasmic Reticulum Stress Response and Up-regulating BH3-only Protein NOXA

BH3 mimetics are small molecules designed or discovered to mimic the binding of BH3-only proteins to the hydrophobic groove of antiapoptotic BCL2 proteins. The selectivity of these molecules for BCL2, BCL-X(L), or MCL1 has been established in vitro; whether they inhibit these proteins in cells has not been rigorously investigated. In this study, we used a panel of leukemia cell lines to assess the ability of seven putative BH3 mimetics to inhibit antiapoptotic proteins in a cell-based system. We show that ABT-737 is the only BH3 mimetic that inhibits BCL2 as assessed by displacement of BAD and BIM from BCL2. The other six BH3 mimetics activate the endoplasmic reticulum stress response inducing ATF4, ATF3, and NOXA, which can then bind to and inhibit MCL1. In most cancer cells, inhibition of one antiapoptotic protein does not acutely induce apoptosis. However, by combining two BH3 mimetics, one that inhibits BCL2 and one that induces NOXA, apoptosis is induced within 6 h in a BAX/BAK-dependent manner. Because MCL1 is a major mechanism of resistance to ABT-737, these results suggest a novel strategy to overcome this resistance. Our findings highlight a novel signaling pathway through which many BH3 mimetics inhibit MCL1 and suggest the potential use of these agents as adjuvants in combination with various chemotherapy strategies.

P-glycoprotein and breast cancer resistance protein in acute myeloid leukaemia cells treated with the Aurora-B Kinase Inhibitor barasertib-hQPA

BackgroundAurora kinases play an essential role in orchestrating chromosome alignment, segregation and cytokinesis during mitotic progression, with both aurora-A and B frequently over-expressed in a variety of human malignancies. Over-expression of the ABC drug transporter proteins P-glycoprotein (Pgp) and Breast cancer resistance protein (BCRP) is a major obstacle for chemotherapy in many tumour types with Pgp conferring particularly poor prognosis in acute myeloid leukaemia (AML). Barasertib-hQPA is a highly selective inhibitor of aurora-B kinase that has shown tumouricidal activity against a range tumour cell lines including those of leukaemic AML origin.MethodsEffect of barasertib-hQPA on the pHH3 biomarker and cell viability was measured in a panel of leukaemic cell lines and 37 primary AML samples by flow cytometry. Pgp status was determined by flow cytometry and BCRP status by flow cytometry and real-time PCR.ResultsIn this study we report the creation of the cell line OCI-AML3DNR, which over-expresses Pgp but not BCRP or multidrug resistance-associated protein (MRP), through prolonged treatment of OCI-AML3 cells with daunorubicin. We demonstrate that Pgp (OCI-AML3DNR and KG-1a) and BCRP (OCI-AML6.2) expressing AML cell lines are less sensitive to barasertib-hQPA induced pHH3 inhibition and subsequent loss of viability compared to transporter negative cell lines. We also show that barasertib-hQPA resistance in these cell lines can be reversed using known Pgp and BCRP inhibitors. We report that barasertib-hQPA is not an inhibitor of Pgp or BCRP, but by using 14[C]-barasertib-hQPA that it is effluxed by these transporters. Using phosphoHistone H3 (pHH3) as a biomarker of barasertib-hQPA responsiveness in primary AML blasts we determined that Pgp and BCRP positive primary samples were less sensitive to barasertib-hQPA induced pHH3 inhibition (p = <0.001) than samples without these transporters. However, we demonstrate that IC50 inhibition of pHH3 by barasertib-hQPA was achieved in 94.6% of these samples after 1 hour drug treatment, in contrast to the resistance of the cell lines.ConclusionWe conclude that Pgp and BCRP status and pHH3 down-regulation in patients treated with barasertib should be monitored in order to establish whether transporter-mediated efflux is sufficient to adversely impact on the efficacy of the agent.

Abstract 5477: Multiple BH3 mimetics antagonize anti-apoptotic MCL1 by inducing the endoplasmic reticulum stress response and upregulating the BH3-only protein NOXA

Abstract Many small molecules have been designed or discovered that mimic the binding of BH3-only proteins to the hydrophobic groove of anti-apoptotic BCL2 proteins and thus have potential as anticancer agents. For example, ABT-737 preferentially binds and inhibits BCL2 and BCL-XL but not MCL1. It is generally believed that specific inhibitors of anti-apoptotic BCL2 proteins should induce apoptosis in a BAX/BAK-dependent manner. While this is true for ABT-737, many other purported BH3 mimetics have been shown to kill cells in a BAX/BAK-independent manner leading to the conclusion that they function through alternative undefined targets. The selectivity of these BH3 mimetics for BCL2, BCL-XL, or MCL1 has been established in vitro; whether they inhibit these proteins in cells has not been rigorously investigated. In this study, we used a panel of leukemia cell lines to assess the ability of seven putative BH3 mimetics to affect the interaction of BCL2 family members in a cell-based system. We show that ABT-737 is the only BH3 mimetic that inhibits BCL2 in cells as assessed by displacement of BAD from BCL2. The other six BH3 mimetics activated the endoplasmic reticulum (ER) stress response and induced ATF3 resulting in induction of NOXA which binds to and inhibits the anti-apoptotic MCL1 protein. The fact that 6 different compounds, all of which are thought to be BH3 mimetics, can induce ER stress suggests that they may interact with a common target although whether this is a BCL2 family member remains to be established. In most cancer cells, inhibition of either BCL2/X or MCL1 does not acutely induce apoptosis. However, by combining two BH3 mimetics, one that inhibits BCL2 and one that induces NOXA, apoptosis is induced within 6 h in a BAX/BAK-dependent manner. As MCL1 is a major mechanism of resistance to ABT-737, these results suggest a novel strategy to overcome this resistance. In summary, these results identify a novel signaling pathway through which many BH3-mimetics inhibit MCL1 and suggest the potential use of these agents as adjuvants in combination with various chemotherapy strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5477. doi:10.1158/1538-7445.AM2011-5477

Reversible epigenetic fingerprint-mediated glutathione- S-transferase P1 gene silencing in human leukemia cell lines

Glutathione- S-transferase P1 (GSTP1) gene is commonly silenced by CpG island promoter hypermethylation in prostate, breast, and liver cancers. However, mechanisms leading to GSTP1 repression by promoter hypermethylation in leukemia and its relationship with pathological alterations of the chromatin structure remain poorly understood. A panel of leukemia cell lines was analyzed for their GSTP1 expression, revealing cell lines with high, moderate or no detectable GSTP1 expression. Bisulfite sequencing, methylation-specific PCR and combined bisulfite restriction analysis revealed that GSTP1 promoter was completely methylated in transcriptionally inactive RAJI and MEG-01 cell lines. In contrast, cell lines expressing GSTP1 exhibited an unmethylated and transcriptionally active promoter. Furthermore, histone marks and effector proteins associated with transcriptional activity were detected by chromatin immunoprecipitation in the GSTP1 expressing hypomethylated K-562 cell line. However, repressive chromatin marks and the recruitment of silencing protein complexes were found in the non-expressing hypermethylated RAJI and MEG-01 cell lines. Finally, we provide evidence that treatment of RAJI and MEG-01 cells with the DNA demethylating agent, 5-aza-2′-deoxycytidine, resulted in GSTP1 promoter demethylation, drastic changes of histone modifications and promoter associated proteins and GSTP1 gene activation. In contrast, treatments with HDAC inhibitors failed to demethylate and reactivate the GSTP1 gene. Our study extends the knowledge on leukemia-specific epigenetic alterations of GSTP1 gene. Furthermore, we are showing the correlation of DNA methylation and histone modifications with the positive/negative GSTP1 transcriptional expression state. Finally, these data support the concept of the dominance of DNA methylation over HDAC inhibitor-sensitive histone deacetylation in gene silencing.

Abstract A28: The effects of combined treatment with NF-κB inhibitors and arsenic trioxide on leukemic cell lines

Abstract Arsenic trioxide (ATO) induces growth inhibition and apoptosis in a wide variety of cancer cell lines. Some reports indicate that this effect can be enhanced by inhibiting the activity of transcription factor NF-κB. The aim of our study was to evaluate the efficacy of combined treatment with ATO and selected NF-κB inhibitors (sulindac, parthenolide, BAY 11-7082, MG-132 and gliotoxin) in inducing cell death of five leukemic cell lines: EL-4 mouse thymoma, Jurkat, HL-60, K562 and HPB-ALL. The cytotoxicity of the chemicals was evaluated using 72-hrs WST-1 reduction assay and apoptosis was assessed with Annexin V-FITC/propidium iodide staining (FACS). We observed that among the inhibitors tested, gliotoxin and MG-132 when used alone showed the highest cytotoxicity on all cell lines (effective concentrations less than 1 μM). Parthenolide and BAY11-7082 induced complete death of all cell lines at the concentration of 10 μM. All four inhibitors did not potentiated the effects of ATO used at 0.5 or 1 μM. Although sulindac was the least cytotoxic compound when used alone, it showed the highest potentiating effect on ATO induced cytotoxicity on all cell lines. The response of cells to the combination was rather diverse with Jurkat and HPB-ALL cells being the most sensitive. As a rule, all cell lines treated simultaneously with ATO (1 μM) and sulindac (100 μM) showed complete decrease in viability after 72 hrs. The results with annexin V/PI staining indicated apoptosis as the main cell death mechanism. This combination had much weaker effect on the viability of healthy blood lymphocytes. The results of our study reveal an interesting cytotoxic effect of ATO with combination with sulindac on a panel of leukemic cell lines. The work was supported by the Polish State Committee for Scientific Research (grant No PB 2806/B/PO1/2007/33). Citation Information: Clin Cancer Res 2010;16(7 Suppl):A28

Enhancement of arsenic trioxide cytotoxicity by dietary isothiocyanates in human leukemic cells via a reactive oxygen species-dependent mechanism

Although clearly effective in acute promyelocytic leukemia (APL), arsenic trioxide (ATO) demonstrates little clinical benefit as a single agent in the treatment of non-APL hematological malignancies. We screened a library of 2000 marketed drugs and naturally occurring compounds to identify agents that potentiate the cytotoxic effects of ATO in leukemic cells. Here, we report the identification of three isothiocyanates (sulforaphane, erysolin and erucin) found in cruciferous vegetables as enhancers of ATO cytotoxicity. Both erysolin and sulforaphane significantly enhanced ATO-mediated cytotoxicity and apoptosis in a panel of leukemic cell lines; erucin activity was variable among cell types. Cellular exposure to sulforaphane in combination with ATO resulted in a dramatic increase in levels of reactive oxygen species (ROS) compared to treatment with either agent alone. Sulforaphane, alone or with ATO, decreased intracellular glutathione (GSH) content. Furthermore, addition of the free radical scavenger N-acetyl-l-cysteine (NAC) rescued cells from ATO/isothiocyanate-mediated cytotoxicity. Our data suggest that isothiocyanates enhance the cytotoxic effects of ATO through a ROS-dependent mechanism. Combinatorial treatment with isothiocyanates and ATO might provide a promising therapeutic approach for a variety of myeloid malignancies.

In vitro and in vivo anti-leukemia activity of CHR-2845, a cell-targeted HDAC inhibitor for use in monocytic leukemia

e14579 Background: Histone deacetylase inhibitors alter gene expression and induce apoptosis in a wide range of cancer cells including those derived from human leukemias. CHR-2845 is a novel hydroxamic acid derivative histone deacetylase inhibitor (HDACi) which is a selective substrate for the intracellular carboxylesterase hCE-1, whose expression is restricted to cells of the monocyte- macrophage lineage. Methods: We studied the in vitro and in vivo anti-leukemia activity of CHR-2845 using cell proliferation assay, annexin V binding assay, cell cycle analysis, western blot and in vitro primary leukemia cell culture. Results: Both U937 and THP1 cells express high levels of hCE-1 whereas the myeloid cell line, HL60, does not. In comparison to vorinostat, CHR-2845 showed increased anti-proliferative potency (IC50) against monocytic cell lines (THP1, 30 nM vs 700 nM and U937, 30 nM vs 475 nM), compared to a myeloid cell line (HL60, 700nM vs 470 nM). In a broad panel of leukemic cell lines, the potency of CHR-2845 over vorinostat correlated completely with hCE-1 expression. In monocytic cell lines, CHR-2845 induced more apoptosis than vorinostat (THP1: 45±5% vs 11±1% and U937: 23±14% vs 6±1%), as measured by flow cytometry using Annexin V. Biochemical assessment of histone H3 and H4 protein acetylation by Western blot also indicateed that CHR-2845 is at least 10 times more potent than vorinostat in monocytic cell lines but not in HL-60 cells. This increase in histone acetylation was associated with increased phosphohistone H2AX, indicating formation of double-strand DNA breaks induced by this compound. These data for CHR-2845 and vorinostat on apoptosis and histone acetylation in THP1 and U937 versus HL60 cells, confirmed the selectivity of this novel compound for cells of the monocytic lineage. We also studied the anti-leukemia activity of CHR-2845 in primary leukemia cells from 8 patients with acute or chronic myelomonocytic leukemia. CHR-2845 decreased proliferation and induced apoptosis more than an equivalent dose of vorinostat in some of the patients we studied. Conclusions: These results indicated that CHR-2845 has potential to be efficacious in the treatment of patients with monocytic leukemia. [Table: see text]

Epigenetic Inactivation of Notch Signaling Target Genes HES in B Cell Acute Lymphoblastic Leukemia.

The Notch signaling pathway has been implicated in multiple functions during normal hemato-lymphoid development. It also plays critical roles in T-cell leukemogenesis through influencing T-cell proliferation, differentiation and survival. In contrast, we have previously reported a tumor suppressor role in B-cell leukemias, where Notch signaling leads to growth inhibition and apoptosis. The Notch target genes Hairy/Enhancer of Split (HES1-7) encode transcriptional repressors with basic helix-loop-helix (bHLH) domains. Functional and phenotypic analyses of some of the HES family members have been reported, however, expression and epigenetic regulation of the HES family in leukemia is largely unknown. Using Methylated CpG Island Amplification (MCA) / DNA promoter microarray, we identified several HES family genes as hypermethylated in B cell acute lymphoblastic leukemia (B ALL). We further investigated the comprehensive methylation profiles of HES family genes in a panel of leukemia cell lines and ALL patient samples by bisulfite pyrosequencing. Aberrant DNA methylation of HES2, HES4, HES5 and HES6 was detected in most B ALL cell lines including B-JAB, RS4:11, REH, Raji and Ramos but not in normal B cell controls. In contrast, in T cell leukemia cell lines such as Molt4, PEER, T-ALL1 and J-TAG, these genes were generally unmethylated. In B ALL patient samples, the frequencies of DNA methylation in the promoter regions of these genes were 25% for HES2, 50% for HES4, 76% for HES5 and 71% for HES6. Expression analysis of HES4, HES5 and HES6 in leukemia cell lines by real-time PCR further confirmed methylation associated gene silencing. Treatment of methylated/silenced cell lines with DNA methyltransferase inhibitor 5‘-aza-2‘-deoxycytidine resulted in HES gene re-expression. Finally, forced re-expression of HES5 and HES6 in methylation silenced Rs4 and REH cell lines inhibited cell growth. These results suggest that the Notch/HES signaling pathway is epigenetically-inactivated in B ALL. These data support the role of the HES family as tumor suppressors in pre-B ALL and establish epigenetic modulation as a novel mechanism of Notch pathway regulation. We anticipate that therapies capable of activating Notch/HES signaling may have therapeutic potential in B cell leukemias.

Generation of efficient human blood progenitor–targeted recombinant adeno-associated viral vectors (AAV) by applying an AAV random peptide library on primary human hematopoietic progenitor cells

Currently standard recombinant adeno-associated virus serotype 2(rAAV2)-based vectors lack the efficiency for gene transfer into primary human CD34(+) peripheral blood progenitor cells (PBPC). An advancement in vector development now allows the generation of rAAV capsid mutants that offer higher target cell efficiency and specificity. To increase the gene transfer into hematopoietic progenitor cells, we applied this method for the first time on primary human CD34(+) PBPC cells. On a panel of leukemia cell lines (CML/AML), significantly higher gene transfer efficiency of the rAAV capsid mutants (up to 100% gene transfer) was observed compared to standard rAAV2 vectors. A higher transduction efficiency in the imatinib-resistant cell line LAMA84-R than in their sensitive counterpart LAMA84-S and a pronounced difference in susceptibility for the capsid mutants vs rAAV2 in LAMA84-S were particularly striking. On solid tumor cell lines, on the other hand, rAAV2 was more efficient than the capsid mutants, suggesting an increased specificity of our capsid mutants for hematopoietic progenitor cells. On primary human CD34(+) PBPC significantly higher (up to eightfold; 16% green fluorescent protein-positive) gene transfer could be obtained with the newly generated vectors compared to standard rAAV2 vectors. These novel vectors may enable efficient gene transfer using rAAV-based vectors into primary human blood progenitor cells for a future clinical application.

Novel Efficient Primary Human Peripheral Blood Progenitor Cell-Targeted Recombinant Adeno-Associated Viral Vectors.

Introduction: G-CSF mobilized peripheral blood progenitor cells (PBPC) are an excellent target for the cure of acquired and inherited disorders of the hematopoietic system. AAV-based vectors have an advantageous safety profile, due to their low immunogenicity, the lack of any associated human disease and its episomal localization. Unfortunately, currently standard rAAV2-based vectors lack the required efficiency for gene transfer into human blood progenitor cells. To increase the gene transfer efficiency on primary hematopoietic cells we applied an AAV random peptide library on primary human CD34+ PBPC.Materials and Methods: The AAV random peptide library was applied on primary human CD34+ PBPC samples (3 selection rounds). Several mutant blood progenitor-targeted rAAV2 capsid clones were obtained and sequenced. Five new rAAV vectors with the most efficient clones were produced. Titers were verified using real time PCR-based titration. To determine efficiency and specificity, a panel of leukemia (CML and AML) and non-leukemia cell lines, as well as primary human CD34+ PBPC were transduced. GFP expression was determined against uninfected control cells.Results: On a panel of leukemia cell lines at least one capsid mutant was significantly more efficient (%GFP+; p<0.01) than standard rAAV2 vectors. On solid tumor cell lines rAAV2 was more efficient, suggesting the increased specificity of our capsid mutants for hematopoietic cells. Significantly higher gene transfer efficiency (up to 16% GFP+ cells) could be obtained with the newly generated vectors compared to standard rAAV2 (up to 9-fold higher; p<0.01). Using a different approach, up to 25% gene transfer into CD34+ PBPC was observed.Conclusion: These novel vectors may finally enable efficient (>20%) gene transfer using rAAV-based vectors into primary human PBPC and may offer a safe alternative to other vector systems which is applicable in vivo.

Investigation of the Efficacy of PXD101 (Belinostat) on Primary Leukaemic Cells and Cell Lines as a Novel Agent for Childhood Acute Lymphoblastic Leukaemia (ALL).

PXD101 (belinostat) is a novel hydroxamate-type of histone deacetylation inhibitor that has been reported to inhibit the growth of a panel of adult carcinomas and melanoma tumour cell lines both in vitro and as xenografts. Activity was associated with a rapid and sustained increase in acetylation of histone proteins H3 and H4 in vitro, and in some cell lines with the induction of apoptosis as measured by PARP cleavage. We have evaluated the in vitro and ex vivo activity of belinostat in a panel of leukaemic cell lines and in primary childhood acute lymphoblastic leukaemia (ALL) to determine the potential for taking this compound forward into early clinical trials in childhood leukaemia. The cell lines used in this study were CCRF-CEM (T-ALL), including steroid sensitive and resistant derivatives, MOLT4 and Jurkat (T-ALL), NALM6 (B-ALL), Daudi (Burkitt's lymphoma) and Kasumi (AML). In vitro cytotoxicity was measured using the Sulphorodamine B assay and cell viability and apoptosis was assessed by flow cytometry using annexin V and propidium iodide staining respectively. The effects of belinostat as a single agent and in combination with dexamethasone, vincristine and doxorubicin were evaluated. Belinostat was also studied in primary leukaemia cells from children with ALL and mononuclear cells from normal bone marrow. Western blotting measuring the acetylation of histones H3 and H4 was used to assess belinostat HDAC inhibitory activity. The time course for onset and duration of HDAC inhibition was investigated. The leukaemia cell lines demonstrated a wide range of in vitro sensitivity to belinostat, with LC50s ranging from 0.3 μM in the Jurkat cells to 10 μM in MOLT4 cells. Normal bone marrow was shown to be refractory to belinostat compared to primary leukaemia cells and the majority of leukaemia cells lines. Primary childhood ALL samples were notably more sensitive to belinostat than the cell lines studied. Interestingly, primary ALL samples observed to be resistant dexamethasone were sensitive to belinostat. Inhibition of histone deacetylation (H3 and H4) was demonstrated within 30 minutes of exposure to belinostat in all cell lines and persisted throughout continuous 24 hour exposure. Histone acetylation was restored between 4–8 hours following removal of belinostat. The induction of histone acetylation did not always correlate with induction of cell death. Rapid inhibition of histone deacetylation was observed in the AML cell line Kasumi, which was refractory to belinostat. The cytotoxic activity of belinostat was associated with markers of apoptosis, as demonstrated by PARP cleavage and Annexin V staining In conclusion, belinostat demonstrated dose dependent in vitro cytotoxicity against a panel of leukaemia cell lines and primary childhood ALL samples. Notably, belinostat was effective in steroid resistant cell lines and primary ALL samples. No synergy was observed between belinostat and drugs used in the treatment of ALL. Further investigation of the scheduling of belinostat when combined with conventional cytotoxic agents is on-going. In addition, the in vivo efficacy of belinostat in a xenograft model of childhood leukaemia is currently being evaluated.

Telomere attrition, and chromosome instability via downregulation of TRF2 contributes to arsenic trioxide-induced apoptosis of human T-cell leukemia cell line molt-4 cells

Overexpression of human telomere repeat binding factor 2 (TRF2), which may play an important role in the fate of cancer cells, has been observed in adult T-cell leukemia. Previous reports have shown that the inhibition of TRF2 results in the apoptosis of cancer cells. In this study, we demonstrated that arsenic trioxide (As2O3) induced in vitro growth inhibition and/or apoptosis of human T-cell leukemia cell line Molt-4 in a caspase-independent manner. Telomerase activity was not inhibited, although the level of the reverse transcriptase subunit of the human telomerase gene (hTERT) mRNA expression was down regulated during the early times and then recovered to the level found in untreated controls about 48 hours after treatment with As2O3. Furthermore, a remarkable telomere shortening related to exposure of As2O3 was observed in 50 population doubling. In contrast, the alteration of telomere length did not occur after exposure to higher concentration of As2O3 (10 µM) for 24 hours and 48 hours, respectively, suggesting that the shortening of telomeres induced by As2O3 is dependent of a series of cell division cycles. Chromosomal analysis showed that As2O3 exposure caused chromosomal end-to-end fusion in human T-cell leukemia cells while downregulation of TRF2 was observed. Finally, the inhibition of TRF2 protein expression and the sensitivity to As2O3 in a panel of leukemia cell lines were checked. The data revealed that inhibition of TRF2 rendered leukemia cells more susceptible to As2O3. In conclusion, the downregulation of TRF2 by As2O3 contribute to chromosomal end-to-end fusion, and apoptosis in leukemia cells, suggesting that TRF2 could be an attractive target for new therapies of leukemia.

Anti-Leukemic Potency of Stapled BH3 Helices Correlates with Their Capacity for Bifunctional Activation of Apoptotic Pathways.

Selective targeting of apoptosis in vivo is a promising pharmacologic strategy for subverting cancer. BCL-2 family protein interactions constitute a critical control point for the regulation of apoptosis. Whereas multidomain anti-apoptotic proteins such as BCL-2 guard against cell death, multidomain pro-apoptotic proteins such as BAX constitute a gateway to cell death through mitochondrial damage. The BH3-only proteins function as death sentinels situated throughout the cell, poised to transmit signals of cell injury to multidomain members. BH3-only proteins deliver death signals via their alpha-helical BH3 domains, which are either neutralized by anti-apoptotic proteins or delivered, directly or indirectly, to the mitochondrial executioners BAX and BAK. By inserting hydrocarbon “staples” into native BH3 peptide sequences, we have generated a chemical toolbox of stabilized alpha-helices of BCL-2 domains (SAHBs) to dissect apoptotic pathways and develop prototype therapeutics. We previously demonstrated that a stapled peptide corresponding to the BID BH3 domain is a helical, protease-resistant, and cell-permeable compound that binds to anti-apoptotic targets with high affinity and exhibits anti-leukemic activity in vitro and in vivo. Using our expanded panel of compounds, we find that a stapled BAD BH3 likewise displays high affinity binding to select anti-apoptotic targets; however, BID SAHB is uniformly more potent than BAD SAHB in inducing apoptosis of a panel of leukemia cell lines. To explore the molecular mechanism underlying the differential potencies of BID and BAD SAHBs, we evaluated a model in which select BH3-only proteins directly engage pro-apoptotic multidomain proteins to trigger mitochondrial apoptosis. We detect and measure, for the first time, direct binding between select SAHBs, such as BID, and BAX. The observed interaction between BID SAHB and BAX triggered functional activation of BAX in vitro, resulting in mitochondrial cytochrome c release and FITC-dextran release from liposomes. The specificity of the BID SAHB-BAX interaction and its biochemical consequences is highlighted by abrogation of BID SAHB activity by point mutation and by competitive binding to anti-apoptotic BCL-XL. We confirmed the physiologic relevance of our observed in vitro interaction between BID SAHB and BAX by their co-immunoprecipitation from BID SAHB-treated leukemia cells. In contrast, BAD SAHB does not bind or activate multidomain pro-apoptotic BAX. These data provide an initial mechanistic explanation for the relative potency of BID SAHB in activating leukemia cell apoptosis. Thus, bifunctional SAHBs that directly engage both pro- and anti-apoptotic multidomain proteins may be more robust pro-apoptotic therapeutics, compared to compounds that selectively target anti-apoptotic proteins.

Generation of Highly Efficient Recombinant Adeno-Associated Virus (rAAV) Vectors Targeted to Chronic Myelogenous Leukemia (CML) - and CD34+ Progenitor Cells Obtained by the „AAV Random Peptide Library“ Method.

Resistance of chronic myelogenous leukemia (CML) to imatinib is a serious clinical challenge. In vivo targeted gene therapy may be a strategy to overcome resistance. For gene therapy of CML standard rAAV2-based vectors lack the required gene transfer efficiency as well as the in vivo selectivity. An advancement in vector development (Muller et al., Nat. Biotech., 2003) now allows the generation of rAAV capsid mutants that offer higher target cell specificity and efficiency. We used the “AAV random peptide library” method to generate capsid mutants efficient and specific for CML cell lines, primary CML cells and primary human peripheral blood progenitor cells PBPC. After previously applying this method successfully on CML cell lines, an effective vector for CML cell lines could be obtained. On primary human CML and PBPC the new vector was more efficient than rAAV2 but still at an unsatisfying level. To increase the gene transfer on primary cells we applied the “AAV library” on primary human CML and PBPC. A total of 44 different mutant capsid clones were obtained, most of them at three or less copies. Four clones were observed >10x to 39x and were selected for further testing. To determine efficiency and specificity, a panel of leukemic (CML, imatinib-sensitive and -resistant, and AML) and other solid tumor cell lines were transduced (moi 100). On a panel of leukemic cell lines similar or higher gene transfer efficiency of the rAAV capsid mutants was observed compared to standard rAAV2 vectors. The higher transduction efficiency in imatinib-resistant cell line LAMA84-R than in their sensitive counterpart LAMA84-S was particularly striking, as the difference in susceptibility for the capsid mutants vs rAAV2 in LAMA84-S (table 1). On solid tumor cell lines rAAV2 was more efficient, confirming the higher specificity of our capsid mutants. Compared to our previous work (<1% GFP+ cells), the new capsid mutants now result in efficient gene transfer into the AML cell line KG1a, and to lesser extend the CML cell line EM3. For both cell lines gene transfer with the capsid mutants was readily more efficient than with standard rAAV2 vectors. Preliminary data on primary PBPC showed a uniformly higher transduction efficiency for all capsid mutants (%GFP_mut1-4: 13.6% ± 2.2% vs %GFP_AAV2: 5.3%). Experiments with primary human CML cells are ongoing. Using the “AAV random peptide library” method on primary cells, we were able to generate rAAV vectors with higher transduction efficiency on leukemic cell lines and primary PBPC than standard rAAV2 vectors. The development of these highly efficient AAV mutant capsid vectors holds promise for future development.Gene transfer efficiency (%GFP+ cells)Cell lineCapsid mutantStandard rAAV2LAMA84-S78.2 ± 7.99.2 ± 2.5LAMA84-R97.8 ± 0.393.0 ± 0.2HL-6054.0 ± 5.457.6 ± 8.2BV17348.4 ± 7.430.6 ± 1.8EM33.9 ± 0.70.2 ± 0.2KG1a24.7 ± 0.712.8 ± 5.0Data are given as mean ± SD

677. Generation and Application of an Efficient Chronic Myelogenous Leukemia (CML)-Targeted Recombinant Adeno-Associated Virus (rAAV) Vector: New Therapeutic Options?

Top of pageAbstract Introduction: Despite great advances in vector development, most standard rAAV-2 vectors still lack the target cell specificity required for an efficient in vivo application. In addition, the gene transfer efficiency in many promising target cells (e.g.: peripheral blood progenitor cells (PBPC)) is low. Thus, designing gene therapy protocols for diseases like chronic myelogenous leukemia (CML) remained difficult, as standard rAAV-2-based vectors lack both the required gene transfer efficiency and selectivity. A recent advancement in vector development (Muller et al., 2003) now allows the generation of rAAV capsid mutants that offers higher target cell specificity and transduction efficiency. This method was applied to obtain a CML-targeted rAAV vector and was tested on a panel of leukemic cell lines, as well on primary CML material. Material and Methods: Performing latter method on the CML cell line K562 (4 selection rounds), several mutant CML-targeted rAAV2 capsid clones were obtained, cloned into an rAAV2 helper plasmid and for each of the mutants rAAV2-eGFP vector stocks were produced. Titers were verified using our real time PCR-based titration assay. To determine efficiency and specificity, a panel of leukemic (CML and AML) and non-leukemic cell lines, as well primary human CML and PBPC cells were transduced with these vectors. Mock, normal rAAV2 and random mutant clone-transduced cells served as controls. Transduction of primary human CML and PBPC cells was confirmed using FACS-sorted GFP+ cells with subsequent bcr-abl-FISH or FACS analysis for GFP/CD34+ co- expression, respectively. Results: Transduction of a panel of six leukemic cell lines with the CML-targeted rAAV2 vectors (moi 100 iu) resulted in an over 10-fold increase of gene transfer efficiency compared to random mutant and standard rAAV2 vectors; gene transfer rates of >60% were obtained. In primary human cells, for both CML (n=4) and PBPC (n=2), an increase in gene transfer was observed (CML: AAV2: 2.4 |[plusmn]| 0.2 vs AAV2-cml: 5.1 |[plusmn]| 1.6 (p=0.04). CD34+: AAV2: 3.2 |[plusmn]| 0.9 vs AAV2-cml: 8.5 |[plusmn]| 4.3 (p>0.05)). All transduced cell were positive for either the BCR-ABL fusion gene (CML) or the PBPC marker CD34. In contrast, gene transfer of the mutant CML-targeted rAAV2 vectors into non-leukemic cell lines resulted in a reduction in gene transfer efficiency compared to standard rAAV2 vectors (<50%). Conclusion: In this study, we were able to generate a CML-targeted rAAV2 vector, which was able to transduce leukemic cell lines and primary human CML cells with significant higher efficiency and specificity than standard rAAV2 vectors. The development of these vectors holds promise for future clinical applications.

Generation and Application of a CML-Specific Recombinant Adeno-Associated Virus (rAAV) Vector.

Introduction: Chronic myelogenous leukemia can be controlled but in most patients not be cured by tyrosine kinase inhibition. Direct targeting using gene therapy vectors combined with vaccination strategies may allow to eradicate residual leukemic progenitors. Adeno-associated virus (AAV) vectors are stable DNA vectors which were proven to be effective in the clinical gene therapy for e.g. coagulation disorders. The various AAV serotypes lack specificity for BCR-ABL+ leukemia cells. Recently developed AAV-library techniques allow a retargeting of vectors. We generated a set of rAAV vectors specific for BCR-ABL+ cells.Material and Methods: After four selection rounds on BCR-ABL+ cells, the peptide sequences of the persisting clones were cloned into an AAV helper plasmid. rAAV-GFP stocks (2 K562-specific, 1 random) of each of the mutants were produced. Titers were determined using real time PCR-based titration assay. Both, a panel of leukemic cell lines and CML primary material were transduced with these vectors and gene transfer was determined by FACS analysis. Specificity was tested in a competitive transduction assay using BCR-ABL+ and BCR-ABL− leukemic cell lines. Transduction of primary CML cells was confirmed using FACS-sorted GFP+ cells and subsequent BCR-ABL-FISH.Results: Using the CML-specific rAAV clone on a panel of BCR-ABL+ cell lines, gene transfer rates of >60% could be obtained (random clone: <1%; rAAV-2: <5%), whereas the BCR-ABL− cell lines were not susceptible to these vectors (gene transfer < 1 %). Admixing BCR-ABL− to BCR-ABL+ cells did not result in a significant drop of the gene transfer rates in the BCR-ABL− cell lines, suggesting that vector particles were not blocked by unspecific binding. Using primary material, significant gene transfer was observed (>5%; 6x more efficient than rAAV-2). In those cells, the CML-genotype was confirmed by BCR-ABL-FISH.Conclusion: In this study, we were able to generate and apply a CML-specific rAAV vector on CML cell lines and primary material. Efficient and selective gene transfer in these cells could be obtained compared to standard rAAV-2 vectors and randomly generated clones. These data hold promise for future developments.

Malignant hematopoietic cell lines: in vitro models for the study of erythroleukemia

A panel of leukemia cell lines has been assembled over the last 30 years representing a spectrum of erythroid cells arrested at various stages of differentiation. The oldest cell line is K-562 which is one of the most prolific in use. Most cell lines have been established from acute myeloid leukemia M6 or chronic myeloid leukemia in blast crisis and generally express immunoprofiles typically seen in immature erythroid cells. Several cell lines are constitutively growth factor-dependent, responding proliferatively to a variety of cytokines. The predominant cytogenetic abnormalities are the t(9;22)(q34;q11) found exclusively in CML-derived cell lines, and rearrangements of chromosomes 5 and 7 which occur in all disease subtypes. Ph+ve cell lines consistently displayed structural and numerical changes associated with disease evolution, including +8, −17/17p−/i(17q), and +19. It is striking that many cell lines though committed to either the erythroid or megakaryocytic lineage tend to co-express features of the other lineage, consistent with the concept of a common erythroid–megakaryocytic progenitor. Several cell lines may be induced to differentiate along the erythroid, megakaryocytic or monocytic pathway by treatment with pharmacological or physiological reagents. Notable functional features include expression of various globin chains or the complete hemoglobins as erythroid attributes. Taken together, this class of cell lines is relatively well characterized and affords useful model systems for immature erythroid cells.