BCR-ABL Positive Cell Lines Research Articles

Activity of the Aurora Kinase Inhibitor, MLN8237 (alisertib) Alone or in Combination with Ponatinib Against Imatinib-Resistant BCR-ABL-Positive Cells

Abstract 1333Chronic myeloid leukemia (CML) is characterized by cytogenetic aberration (Philadelphia chromosome: Ph) and chimeric tyrosine kinase BCR-ABL. ABL tyrosine kinase inhibitor, imatinib has demonstrated the potency against CML patients. However, resistance to imatinib can develop in CML patients due to BCR-ABL point mutations. One of T315I mutation is resistant to currently available ABL tyrosine kinase inhibitors. Therefore, new approach against T315I mutant may improve the outcome of Ph-positive leukemia patients. Aurora kinases are serine/threonine kinases and upregulated in many malignancies including leukemia, and play an important role in cell cycle control and tumor proliferations. Because Aurora kinases are overexpressed in leukemia cells, Aurora kinases may present attractive targets for leukemia treatment. One of Aurora kinase inhibitor, MLN8237 (alisertib) is an oral and selective Aurora kinase A inhibitor and is currently being investigated in a pivotal phase 3 clinical trial against hematological malignancies. We suggested that alisertib mediated inhibition Aurora kinase activity and in combination with ponatinib, also known as AP24534 may abrogate the proliferation and survival of Ph-positive cells including T315I mutation. In this study, we investigated the combination therapy with a ponatinib and an alisertib by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line which was transfected with T315I mutant, ponatinib resistant Ba/F3 cells and T315I primary sample. Protein expression of Aurora A and B were increased in Ph-positive leukemia cells. 72 hours treatment of alisertib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. Alisertib also induced cell cycle arrest. The treatment of ponatinib exhibits cell growth inhibition partially against K562 cells in the presence of feeder cell (HS-5) conditioned media. We found that the treatment of alisertib abrogated the protective effects of HS-5 conditioned media in K562 cells. We investigated the alisertib activity against T315I positive cells. Alisertib potently induced cell growth inhibition of Ba/F3 cells ectopically expressing T315I mutation and induced cell cycle arrest. We investigated the efficacy between ponatinib and alisertib by using these cell lines. Combined treatment of Ba/F3 T315I cells with ponatinib and alisertib caused significantly more cytotoxicity than each drug alone. Ponatinib and alisertib were also effective against T315I primary samples. We examined the intracellular signaling of alisertib. Phosphorylation of Aurora A was inhibited in a time dependent manner. We also found the phosphorylation of histone H3 was also reduced in a dose dependent manner suggested that high concentration of alisertib also inhibits Aurora B activity. We next investigated by using ponatinib resistant Ba/F3 cells. In the ponatinib resistant cell lines, IC50 of ponatinib was up to 200 nM. BCR-ABL triple point mutations (T315I, E255K and Y253H) were detected by direct sequence analysis. The treatment of alisertib exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells in the dose dependent manner. Alisertib induced cell cycle arrest in ponatinib resistant cells. Combined treatment of Ba/F3 ponatinib resistant cells with ponatinib and alisertib caused significantly more cytotoxicity. To assess the activity of alisertib and ponatinib, we performed to test on CML tumor formation in mice. We injected nude mice subcutaneously with 1×107 Ba/F3 T315I cells. A dose of 30 mg/kg/day p.o of ponatinib and 30 mg/kg/day p.o of alisertib inhibited tumor growth and reduced tumor volume compared with control mice. The treatments were well tolerated with no animal health concerns observed indicating the feasibility of alisertib combination strategies in the clinic. Data from this study suggested that administration of the ponatinib and Aurora inhibitor, alisertib may be a powerful strategy against BCR-ABL mutant cells including T315I. Disclosures:No relevant conflicts of interest to declare.

Combining ABL1 Kinase Inhibitor, Imatinib and the Jak Kinase Inhibitor TG101348: A Potential Treatment for Residual BCR-ABL Positive Leukemia Cells

Abstract 3737ABL kinase inhibitor, imatinib is highly effective therapy against chronic myeloid leukemia (CML) patients and eliminates disease progression and transformation. However, imatinib is not curative for most CML patients. Residual CML cells are present in bone marrow microenvironment. Bone marrow microenvironment is a source of soluble factors and regulates the proliferation of leukemia cells. These leukemia cells are contained within a niche in the bone marrow and are often impervious to current treatments, thus maintaining their proliferative activity when the treatment is ceased, suggests that the new therapeutic strategies designed to override stroma-associated drug resistance are required to treat against Philadelphia (Ph)-positive leukemia patients. The hematopoietic cytokine receptor signaling is mediated by tyrosine kinases termed Janus kinases (Jaks) and downstream transcription factors, signal transducers and activators of transcription (STATs). Jak-STAT signaling is also activated in CML cells. One of the Jak kinase inhibitor, TG101348 (SAR302503) is an orally available inhibitor of Jak2 and developed for the treatment of patients with myeloproliferative diseases. Therefore, combination therapy using a BCR-ABL tyrosine kinase inhibitors and a Jak inhibitor, TG101348 may help prevent stroma-associated drug resistance and these approaches may be expected to improve the outcomes of CML patients. In this study, we investigated the ABL tyrosine kinase inhibitor, imatinib and TG101348 efficacy by using the BCR-ABL positive cell lines, K562 and primary CML samples when leukemic cells were protected by the feeder cell lines (HS-5 and S9). 72 hours treatment of imatinib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. However, the treatment of imatinib exhibits cell growth inhibition partially against K562 cells in the presence of HS-5 conditioned media. We found that the treatment of TG101348 did not exhibit cell growth inhibition against K562 cells directly, but the combination treatment with imatinib and TG101348 abrogated the protective effects of HS-5 conditioned media in K562 cells. We next investigated the intracellular signaling of imatinib and TG101348. Phosphorylation of BCR-ABL, Crk-L was not reduced after TG101348 treatment. However, phosphorylation of BCR-ABL, Crk-L was significantly reduced and increased apoptosis after combination treatment with imatinib and TG101348. We next investigated the efficacy between imatinib and TG101348 by using CD34 positive primary CML samples. The treatment of imatinib exhibits cell growth inhibition partially against CD34 positive CML samples in the presence of feeder cells. Combined treatment of CD34 positive primary samples with imatinib and TG101348 caused significantly more cytotoxicity and induced apoptosis. We also found that mitogen-activated protein kinase (MAPK) was also inhibited by imatinib and TG101348 treatment. We next investigated the intracellular signaling of imatinib and TG101348 by using the CD34 positive primary samples. Phosphorylation of BCR-ABL, Crk-L was significantly reduced and increased apoptosis after treatment with imatinib and TG101348. Moreover, combination of imatinib and TG101348 inhibited the colony growth of Ph-positive primary samples. We also investigated the TG101348 activity against feeder cell. Phosphorylation of STAT5 was reduced by TG101348 in a dose dependent manner. The cytokine production was analyzed by using cytokine array systems. The cytokine production such as granulocyte macrophage colony-stimulating factor (GM-CSF) from HS-5 was also reduced by TG101348 treatment. Data from this study suggested that administration of the imatinib and Jak inhibitor, TG101348 may be a powerful strategy against stroma-associated drug resistance of Ph-positive cells and enhance cytotoxic effects of imatinib in those residual CML cells. Disclosures:No relevant conflicts of interest to declare.

Microvesicles Transcripts As Hallmark and Vector From Leukemic Parental Cells

Abstract 1459Microvesicles (MVs) are nano-sized lipid bodies (100–1000 nm in diameter) that are released by cells, both in vitro and in vivo. MVs are secreted in the extracellular space and as carriers of proteins, mRNA and miRNA represent vectors from donor to target cells involved in intercellular communication. MVs modulate the functional state of receiving cells through fusion with the target cell. In leukemia MVs were suggested to modulate the hematopoietic niche.In this study, we investigated the transcriptome of MVs released from leukemic cell lines. In particular, we analyzed K562, a BCR-ABL positive human erythromyeloblastoid leukemia cell line, and we collected RNA both from cells and MVs released in culture. Since many different methods have been described for microvesicles isolation and description of MV populations are often ambiguous, an accurate protocol has been developed in order to select a defined MVs population. In detail, for MVs isolation cell culture medium was centrifuged at 2500g for 15 minutes. These centrifugations allowed to delete cells and bigger bodies. Then supernatant was filtered by means of a 1.2um filter, in order to keep only vesicles of defined physical measure and to eliminate residual bigger vesicles, such as apoptotic bodies (>1000nm). The filtration allowed an accurate selection of a well defined MVs population. The filtered medium was then centrifuged at 18000g for 1h at 4°C. MVs were resuspended in Trizol for RNA isolation. Also RNA of cells, from which MVs have been released, was extracted using the Trizol method, according to manufacturer instructions. Cell line RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies) and quantified by means of NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc.). To check MVs RNA integrity we determined the ratio of 5’ amplicons to 3’ amplicons of housekeeping transcripts by Real Time PCR. We screened the presence of several housekeeping transcripts in MVs and selected GAPDH as reference. The GAPDH 5’:3’ amplicon ratio should equal 1 to ensure MVs RNA quality. Afterwards, reverse transcription–polymerase chain reaction (RT-PCR) amplification was performed. cDNA was synthesized from 1mg of total RNA.We analyzed the gene expression profile of K562 cell line and MVs, released from these cells, using GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix). Gene expression data have been compared between cells and MVs. Ninety-one percent of probe sets showed a similar expression (fold-change lower than 1.5) between cell line and MVs. Thirteen percent of these probes were recognized as present call probe sets in both cell line and MVs. Analysis of probe sets using DAVID Functional Annotation Bioinformatics Microarray Analysis revealed a conservation of MVs gene transcripts involved in pathways of cell function such as RNA processing, protein translation, aminoacid metabolism and cell respiration. Remarkably, in MVs we observed a high presence of gene transcripts coding for protein belonging to the Chronic Myeloid Leukemia pathway that are expressed downstream of the BCR-ABL tyrosin kinase fusion protein. The maintenance of this pathway in MVs highlights the intrinsic peculiarity of BCR-ABL positive K562 cells apparently also conserved in MVs mRNA outfit representing a hallmark of the parental cell from which they have been released.Furthermore, 3.8% of the probe sets resulted to be up-regulated in MVs compare to the cell line (fold-change higher than 1.5). In MVs, we observed a higher expression of genes belonging to cell communication pathways, adhesion and migration processes, membrane and ionic channels signals.In conclusion, we isolated MVs released by K562 leukemic cells using an accurate selection of the MV population based on physical measures and for the first time a whole transcriptome gene expression analysis has been performed comparing K562 cells and MVs. Moreover, we identified an enrichment of transcripts coding for proteins involved in several essential pathways in the MVs supporting the hypothesis of a functional selection from the parental cell transcriptome and underlining the relevant role of MVs as vehicles of messages to target cells. Disclosures:No relevant conflicts of interest to declare.

Targeting STAT5 Expression Resulted in Molecular Response Improvement in Patients with Chronic Phase CML Treated with Imatinib

AbstractAbstract 696 Background.We have previously demonstrated that PPAR-γ agonists (such as pioglitazone) negatively regulate the level of Stat5A and Stat5Bgene expression in normal CD34+ bone marrow progenitors (Prost, Le Dantec et al. 2008). Aim.We investigated whether targeting Stat5 expression with pioglitazone may impact clonogenic activity of Chronic Myelogenous Leukemia (CML) cells in vitro and result in molecular response improvement in vivo. Patients and methods.Preliminary in vitro studies tested the ability of pioglitazone to impact viability and clonogenicity of CD34+ primary cells from CML patients (pts). We conducted clonogenic and LTC-IC studies. Cultivated Ph+ -CD34+cells were characterized by facs and analyzed by CSFE assay. BCR-ABL and Stat5 expression were quantified by real time PCR. Control experiments were conducted using lentiviral vectors and siRNA assay for Stat5 and PPAR-g.CML pts were eligible in the ACTIM trial (EudraCT 2009–011675-79) if they were treated by imatinib for at least 2 years with a stable daily dose for at least 3 months and in major molecular response without having achieved CMR4.5 (defined by a BCR-ABL/ABL IS ratio ≤ 0.0032%). After inclusion, pts received imatinib (no dose modification) and started pioglitazone (Actos®) 30 mg/d during 2 months and 45 mg/d thereafter for 12 months. BCR-ABL transcript level was monitored every 3 months during the study period. Primary objective was the proportion of patients achieving a confirmed undetectable level of BCR-ABL transcript. Secondary objectives included cumulative incidence of CMR4.5 and safety. A companion biologic study evaluated imatinib through levels, Stat5 expression in bone marrow at baseline, months 6 and 12 and clonogenic activity of bone marrow mononuclear cells at baseline, months 6 and 12. Results.From the in vitro studies we first demonstrated that pioglitazone at pharmacological doses inhibited cell growth of the Bcr-Abl positive cell line K562 through the activation of the PPAR-g/STAT5 pathway. We next showed that PPAR-g / STAT5 pathway induced a clonogenic defect in CD34+ cells from CML patients. Moreover, the activation of the PPAR-g / STAT5 pathway also induced a clonogenic and a proliferative defect in CML LTC-IC. We then confirmed that imatinib induced a selection of insensitive quiescent CML cells and showed that this effect was abrogated by the activation of the PPAR-g / STAT5 pathway.Twenty seven pts were enrolled in the clinical trial and 24 were evaluable (1 was excluded in CMR4.5, 1 pt was not in MMR and 1 pt had consent withdrawal). Median age was 61.6 years (24.1–79) and median follow-up after inclusion was 13 months (9.8–21). All evaluable pts started pioglitazone as planned. Seven pts (29.2%) discontinued pioglitazone before 12 months, 6 following investigator decision after the warning of the French ministry of health regarding the risk of bladder cancer and 1 after its own decision. No pt discontinued due to adverse events. Discontinuations occurred between month 3 and month 9. Median duration of pioglitazone therapy was 11.2 months (2.6–15.4) median daily dose was 39.9 mg. No interaction was observed between imatinib and pioglitazone in term of through level before (median 850 ng/ml) and after (median 927 ng/ml) pioglitazone initiation (p=ns). Main adverse events were weight gain and worsening fluid retention in 3 pts. Three pts (14%) obtained a confirmed undetectable level of BCR-ABL transcript. The one year cumulative incidence of CMR4.5 was 57%. Stat5 mRNA quantification was significantly diminished in pt samples at M6 and M12 compared to the baseline values and a reduction of the clonogenic potential was also observed in bone marrow cells at M6 and M12. We collected “control pts” with similar characteristic (n=20). None of these pts obtained a confirmed CMR and the cumulative incidence of CMR4.5 in this control group was 27% as compared to 57% in the pioglitazone group (p=0.02). Conclusion.We have extended our in vitro results showing that PPAR-γ agonists resulted in Stat5 down regulation in CML CD34+ cells and preferentially reduced their clonogenic and long term potency in CFCs and LTC-IC assays. We now demonstrated that these effects translate in vivo by the achievement of MMR in more than half of the pts treated with the combination of pioglitazone and imatinib suggesting that it may be possible to target quiescent CML cells in vivo and supporting the concept of stem cell pool erosion. Disclosures:Off Label Use: pioglitazone in CML. Roy:Novartis, BMS: Speakers Bureau. Legros:Novartis, BMS: Research Funding, Speakers Bureau. Coiteux:Novartis, BMS: Speakers Bureau. Mahon:Novartis, BMS: Consultancy, Research Funding.

Efficacy of PRT060318, a Novel Highly Specific SYK Inhibitor, in Acute Lymphoblastic Leukemia (ALL)

Abstract 3532B cell acute lymphoblastic leukemia (B-ALL) arises by transformation of progenitor (pre-B) cells. Cure rates in adults remain low and treatment is complicated by microenvironment-mediated resistance to cytotoxic drugs, indicating an urgent need for development of new, more targeted treatment approaches. Spleen tyrosine kinase (Syk), a B cell receptor (BCR)-associated tyrosine kinase, recently was identified as a novel therapeutic target in mature B cell malignancies, such as chronic lymphocytic leukemia (CLL). Besides its role in BCR signaling in mature B cells, Syk also plays an important role in maintenance and expansion of immature B cells. Syk-deficient mice display a severe defect of B lymphopoiesis, with a block at the pro-B to pre-B transition, consistent with a key role for Syk in pre-BCR signaling. We therefore hypothesized that pre-B ALL cells which express pre-BCRs might be more sensitive to Syk inhibition than other ALL subtypes, which either do not have functional pre-BCRs (pro-B ALL) or display modifying genetic lesions (Ph+/BCR-ABL+ B-ALL).PRT060318, a highly specific, ATP-competitive, small molecular inhibitor of Syk, has preclinical activity in CLL and DLBCL models (Hoellenriegel J et al., Leukemia 26:1576–83, 2012). Syk specificity was confirmed by cellular and non-cellular kinase inhibition assays. We performed metabolic assays using the tetrazolium dye XTT to screen B-ALL cell lines (RS4;11, REH, TOM-1, Nalm-20, Nalm-21, Nalm-6, 697, Kasumi-2, KOPN-8, RCH-ACV, SMS-SB) for responses to PRT060318. ALL cells were incubated with increasing concentrations of PRT060318 (100 nM – 10 μM) for 72 hours. Based on the XTT assays, we determined half maximal inhibitory concentration (IC50), and separated B-ALL cells into responsive (IC50 < 4.5 μM) and non-responsive (IC50 > 4.5 μM) groups. Interestingly, the responsive group consisted only of pre-B ALL cells (CD10+, TdT+, cytoIgμ+), whereas the resistant group comprised only ALL cell lines with a pro-B cell phenotype (CD10+/−, TdT+, cytoIgμ-). All BCR-ABL positive cell lines tested exhibit the pro-B cell phenotype and, similar to their BCR-ABL negative counterparts, were resistant to PRT060318. The Figure depicts XTT assay results for two PRT060318-sensitive pre-B (grey lines) and two resistant pro-B (black lines) B-ALL cell lines. Western Blot analysis demonstrated SYK protein expression in all B-ALL cell lines, excluding lack of target protein expression as possible cause for different response rates between pre-B and pro-B ALL cells. Cell proliferation assays revealed a significant, dose-dependent inhibition of pre-B ALL cell proliferation by PRT060318. Concordantly, dose depended S phase reduction was detected in all PRT060318-sensitive cell lines. In apoptosis assays, in which sensitive pre-B ALL lines were incubated with increasing concentrations of PRT060318 (up to 5 μM) we did not find any significant induction of apoptosis, suggesting anti-proliferative, rather than cytotoxic effects as a main mechanism of action of PRT060318. Preliminary data of primary ALL patient samples, cultured with KUSA-H1 marrow stroma cells in the presence or absence of PRT060318 demonstrate induction of apoptosis in 3 out of 10 samples, other assays are ongoing. In conclusion, we provide evidence that the Syk inhibitor PRT060318 thwarts pre-B ALL cell proliferation, providing a first rationale for clinical testing of PRT060318 in selected patients with B cell ALL. [Display omitted] Disclosures:Coffey:Portola Pharmaceuticals Inc.: Employment. Sinha:Portola Pharmaceuticals Inc.: Employment. Pandey:Portola Pharmaceuticals Inc.: Employment.

1093P - BCR ABL Fusion Protein Detection by a Modern Approach: Fluroscent Immuno Bead Assay

ABSTRACT Introduction Chronic Myeloid Leukemia (CML) is probably the most extensively studied human malignancy. CML patients (90-95%) harbor the Philadelphia (Ph) chromosome, a shortened chromosome 22, resulting from a reciprocal translocation t (9; 22) (q34; q11) between the long arms of chromosome 9 and 22, fusion ABL proto-oncogene on chromosome 9 with the BCR gene on chromosome 22. BCR-ABL fusion gene plays a key role in pathogenesis of the disease. Approximately 15-30% of adult and 2-5% of childhood ALL and Materials and methods Study Period: June 2009- February 2012 Test Specimen Peripheral blood or Bone marrow from 242 CML patient (from hospital registry of a tertiary cancer institute of Eastern India) Normal (as negative control): Peripheral blood from 20 individuals BCR-ABL positive cell line: K562 FISH was performed using (for 100 samples) directly labeled dual color prob for detection of BCR-ABL as standard laboratory protocol. Isolation of WBC were done by lysis buffer. Then lysate treatment were done to release the intracellular BCR-ABL protein. After fluorescent immune bead assay were performed by captured bead and detector reagent. After forming a sandwich complex (if sample contain targeted protein), they were analysed by flowcytometry method. Result In 242 cases 166 (68.6%) were positive for BCR ABL fusion protein by Flowcytometric Bead Assay. Approximately 96% cases were observed as positive by both techniques FISH and Flowcytometric Bead Assay. Conclusion We conclude that the Flowcytometry Immuno Bead Assay is a faster, easier and reliable technique for detection of BCR ABL protein in leukemic cells and shows promise for serial evaluation of patients undergoing treatment. The main advantage of immune bead assay is not dependence on the breakpoint position in the BCR gene. Moreover this technique is based on protein translation label. Disclosure All authors have declared no conflicts of interest.

Abstract 278: Post-transciptional activation of anti-oxidant enzymes by imatinib

Abstract Patients with Chronic Myelogenous Leukemia (CML) can be treated with targeted bcr-abl kinase inhibitors, such as imatinib. Recently, we reported that mononuclear cells obtained from individuals treated with imatinib often exhibit an increase in the enzyme activity of the antioxidant selenoprotein, glutathione peroxidase (GPx). Treatment of the bcr-abl positive cell line KU812 resulted in increased GPx enzyme and GPx1 protein levels in a dose (100-150 nM) and time (0-7 days) dependent manner, with a maximal increase of 3-fold. Imatinib-induced changes in other anti-oxidant enzymes were also assessed. Of these, no change in the levels of either catalase or GPx-4 were detected, although the levels of manganese superoxide dismutase (MnSOD) increased by a maximal level of 4-fold in imatinib-treated cells. Similar results were obtained in another bcr-abl positive, CML cell line, MEG-01. The elevation in the levels of these proteins occurred via a post-transcriptional mechanism as no increase in the steady state levels of the corresponding mRNA was detected by RT-PCR. The effect of imatinib was likely to be dependent on the presence of the bcr-abl fusion product as no changes in either GPx-1 or MnSOD were detected in either the immortalized lymphocyte cell line GM10832 or the prostate cancer-derived LNCaP human cell lines, neither of which contain bcr-abl. Since the mammalian Target of Rapamycin (mTOR) is a known protein substrate for the abl kinase, and mTOR activity can regulate protein translation, it was investigated whether inhibiting mTOR with rapamycin (1ng/ml) affected GPx-1 and MnSOD levels. Exposure of the KU812 and MEG-01 cells, as well as LNCaP and GM10832 with rapamycin resulted in increased GPx-1 protein and enzyme levels (2- to 4-fold), but had no measurable effects on MnSOD. These results indicate that imatinib, likely by the inhibition of bcr-abl, can post-transcriptionally enhance both GPx-1 and MnSOD levels, although by different mechanisms. These data may have therapeutic significance as both of these anti-oxidant enzymes may influence several different clinically relevant biological processes, including DNA damage susceptability, cell signaling and apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 278. doi:1538-7445.AM2012-278

Evaluation of deoxyhypusine synthase inhibitors targeting BCR-ABL positive leukemias

Effective inhibition of BCR-ABL tyrosine kinase activity with Imatinib represents a breakthrough in the treatment of patients with chronic myeloid leukemia (CML). However, more than 30 % of patients with CML in chronic phase do not respond adequately to Imatinib and the drug seems not to affect the quiescent pool of BCR-ABL positive leukemic stem and progenitor cells. Therefore, despite encouraging clinical results, Imatinib can still not be considered a curative treatment option in CML. We recently reported downregulation of eukaryotic initiation factor 5A (eIF5A) in Imatinib treated K562 cells. Furthermore, the inhibition of eIF5A by siRNA in combination with Imatinib has been shown to exert synergistic cytotoxic effects on BCR-ABL positive cell lines. Based on the structure of known deoxyhypusine synthase (DHS) inhibitors such as CNI-1493, a drug design approach was applied to develop potential compounds targeting DHS. Here we report the biological evaluation of selected novel (DHSI-15) as compared to established (CNI-1493, deoxyspergualin) DHS inhibitors. We show that upon the compounds tested, DHSI-15 and deoxyspergualin exert strongest antiproliferative effects on BCR-ABL cells including Imatinib resistant mutants. However, this effect did not seem to be restricted to BCR-ABL positive cell lines or primary cells. Both compounds are able to induce apoptosis/necrosis during long term incubation of BCR-ABL positive BA/F3 derivates. Pharmacological synergism can be observed for deoxyspergualin and Imatinib, but not for DHSI-15 and Imatinib. Finally we show that deoxyspergualin is able to inhibit proliferation of CD34+ progenitor cells from CML patients. We conclude that inhibition of deoxyhypusine synthase (DHS) can be supportive for the anti-proliferative treatment of leukemia and merits further investigation including other cancers.

Effects of NVP-BSK805, a Novel JAK2 Inhibitor, on BCR-ABL and JAK2V617F Positive Cell Lines

Abstract 5002 Background:Janus kinases are critical components of cytokine signaling pathways that regulate hematopoiesis, growth, immunity, inflammation, and development. Oncogenic mutations of the non-receptor tyrosine kinase JAK2 are found in many Philadelphia chromosome negative myeloproliferative neoplasms. The V617F mutation in JAK2 occurs in 95% of patients with polycythemia vera, 50% of those with essential thrombocythemia and 50% of primary myelofibrosis patients. Preclinical results strongly support that JAK2 inhibitors could be effectively used in these three indications. Replacement of valine 617 with phenylalanine upregulates the tyrosine kinase activity of JAK2, causing constitutive activation of the JAK-STAT pathway and growth factor-independent cell proliferation. JAK2 has also been postulated to play an important role in BCR-ABL signal transduction. Therefore, inhibitors of the tyrosine kinase activity of JAK2 are under investigation as new therapy strategies for CMPNs. In this study the role of the novel JAK2 inhibitor, NVP-BSK805 (Novartis Pharmaceuticals), has been investigated in cells expressing either BCR-ABL or mutant JAK2. Possible synergistic effects between NVP-BSK805 and the already established tyrosine kinase inhibitors imatinib and nilotinib were assessed. Methods:The in vitro activity of NVP-BSK805 was analyzed in 12 hematopoietic cell lines, including 7 BCR-ABL positive (K562, KCL22, KU812, Lama87, BV173, EM3, SUP-B15), 4 JAK2 mutated (CHRF288, SET2, UKE1, HEL), the T-cell leukemia cell line Jurkat, and the neuroendocrine colonic tumour line LCC-18. Concentration kinetics from 0 up to 25 μM were established using XTT proliferation assays and flow cytometry for measuring apoptosis. Protein levels of JAK2, phospho-JAK2, STAT5, phospho-STAT5 and BCR-ABL were analyzed using Western blotting. NVP-BSK805 was also tested in combination with imatinib and nilotinib. JAK2 was sequenced in all cell lines in order to detect possible mutations in the gene. Results:Of the JAK2 mutated cell lines tested, 3 of 4 (CHRF288, SET2, UKE1) showed a significant reduction of proliferation, as well as viability, compared to the other cell lines. CHRF288 responded best to NVP-BSK805 with an IC50 value of 0.22 ± 0.04 μM. UKE1 and SET2 had similar values of 0.35 ± 0.03 μM and 0.37 ± 0.05 μM. Interestingly, HEL (V617F positive) cells showed only an IC50 value (1.8 ± 0.17 μM) for NVP-BSK805, comparable with that of the non-mutated BCR-ABL positive cell lines (1.5 to 2.7 μM). LCC-18 showed the weakest response of all cell lines tested, with an IC50 value of 9.93 ± 0.202 μM. Each cell line responded to concentrations higher than 5 μM with a strong reduction of proliferation due to inhibition of various kinases. Combination of the JAK2 inhibitor with imatinib and nilotinib showed no significant additive or synergistic effects, although all BCR-ABL positive cell lines responded well to both CML therapeutic agents. Western blotting of proteins of the JAK-STAT pathway confirmed the results of the proliferation and apoptosis tests showing a strong reduction of phoshorylated STAT5 in CHRF288 cells after a 30 min incubation even with NVP-BSK805 concentrations as low as 0.01 μM. UKE-1 and SET-2 showed reduction of pSTAT5 from 0.1 μM. Levels of total STAT5 were not affected. In all the other cell lines no changes were detected in any of the proteins tested. Conclusions:Here, we tested a novel JAK2 inhibitor in cells carrying the V617F mutation. Interestingly, not every cell line with the JAK2 V617F mutation showed a good response upon JAK2 inhibition, indicating that there are additional factors determining response. On the other hand, clinical trials with JAK inhibitors in myelofibrosis have shown responses in V617F-mutated and non-mutated patients, warranting further research to identify predictors of response. In BCR-ABL mutant cells not harbouring JAK2 mutations no significant inhibition of proliferation or apoptosis was detected following JAK2 inhibition, indicating that there are JAK2 independent signal transduction pathways of BCR-ABL to avoid apoptosis. Disclosures:le Coutre:Novartis Pharmaceuticals: Honoraria, Research Funding.

Pharmacologic Doses of Amiloride Preferentially Induce Apoptosis and Growth Inhibition of Flt3-ITD Mutation Positive Acute Myeloid Leukemia Cell Lines

Abstract 5004 Introduction:Amiloride is an FDA approved potassium-sparing diuretic which targets Na+/H+ exchanger isoform 1 (NHE1). NHE1 is responsible for the regulation of the intracellular pH, as well as cell-cycle and apoptosis. In supra-pharmacologic concentrations, amiloride non-specifically inhibits protein kinases. Recent study demonstrated that proapoptotic effect of amiloride in CML cell lines is linked to the modulation of the alternative splicing of Bcl-x, HIPK3, and BCR/ABL genes and is independent of pHi.Here, we demonstrate that pharmacologic doses of amiloride preferentially induce growth inhibition, cell cycle arrest and apoptosis in Flt3-ITD positive acute myeloid leukemia cell lines as compared to Bcr-Abl positive leukemia cell line. Our data suggests that amiloride may have an effect on Flt3 signaling and that its treatment potential for Flt3-ITD positive acute myeloid leukemia needs to be explored. Methods:MV4-11, MOLM13 and K562 cells lines in log-phase growth were used for the experiments. Analysis of the baseline Flt3 expression and phosphorylation status was assessed via Flt3 immunoprecipitation and Western blotting for Flt3 and phosphotyrosine. Cells were incubated with various amiloride concentrations; equal volume dilutions of DMSO were used for control. Cell counting and trypan blue exclusion viability was performed on TC10 Bio-Rad automated cell counter. The cell cycle analysis was performed applying propidium iodide staining. To assess for apoptosis and cell death, we used annexin V/PI staining kit and flow cytometry. Results:MOLM13 and MV4-11 cell lines carry activating Flt3-ITD mutation. We confirmed the expression and constituative activation of Flt3 in MOLM13 and MV4-11 cells with Western blotting. Flt3 protein was not detectable in K562 cell line. Amiloride at 0.025 mM and 0.05 mM completely inhibited the growth of MV4-11 cells after 24 hrs of treatment with no significant increase in total or live cell numbers at 72 hrs, but only mildly affected K562 cell proliferation. While the above amiloride concentrations caused cell death in MV4-11 and MOLM13 cell lines, there was no increased cell death in K562 cells.Incubation of MOLM13 and MV4-11 cell lines with 0.05 mM amiloride for 20 hrs induced cell cycle arrest. In MV4-11 cell line, the proportion of S phase cells after amiloride treatment was 15.4% (SD=5.4%) as compared to 31.3% (SD=1.4%) in control. MOLM13 cell line demonstrated 15.3% (SD=4.7%) of cells in S after amiloride treatment as compared to 35.3% (SD=2.4%) cells in S phase in control treatment. In K562 cell line, there was less effect with 52% (SD=4.2%) of cells in S phase in control as compared to 37% (SD=8.9%) in amiloride treatment.MV4-11 and MOLM 13 cell lines were more sensitive than K562 cells to amiloride induced apoptosis with 28.8% (control 12.7%) of MV4-11 cells, 11.4% (control 7.4%) of MOLM13 cells, and 11.4% (control 8.6%) of K562 cells being apoptotic after 20 hr treatment with 0.05mM amiloride. At 72 hrs of amiloride treatment 34% (control 1.5%) of MV4-11 cells, 17% (control 5%) of MOLM13 cells and 11% of K562 cells (control 8.9%) were apoptotic. Amiloride had similar effect on the number of dead cells with no increase in total cell death in K562 cell line. Upon treatment with increasing amiloride concentrations, there was dose-dependent increase in cell death and apoptosis in all three cell lines with K562 line showing relative resistance to amiloride. Discussion:Our results demonstrate that amiloride induces cell cycle arrest and inhibits proliferation of Flt3-ITD positive cell lines MV4-11 and MOLM13 as well as K562 cell line at a pharmacologic concentration of 0.05 mM. Both, cell cycle arrest and antiproliferative effect are more pronounced in Flt3-ITD positive cells lines while it is mild in Bcr-Abl positive K562 cell line. Pharmacologic doses of amiloride induce cell death and apoptosis in Flt3-ITD positive cell lines but not in K562 cell line. Both, Bcr-Abl and Flt3 signaling stimulates proliferation and inhibits apoptosis in myeloid leukemia cells. Our study suggests that amiloride may induce cell cycle arrest and apoptosis via modulating Flt3 signaling cascade. We are currently investigating the effects of amiloride on Flt3 phosphorylation. In conclusion, our data suggests that amiloride presents a potential treatment option for Flt3-ITD positive acute myeloid leukemia. Disclosures:No relevant conflicts of interest to declare.

MiR-125b Cooperates with BCR-ABL and Enhances Therapy Resistance in Childhood Ph+ ALL

Abstract 78The treatment improvements in the past two decades led childhood acute lymphoblastic leukemia (ALL) cure rates reach over 80%, but children who carry the Philadelphia (Ph+) chromosome still have high risk of relapse due to therapy resistance. Different results with large series of patients have shown that an earlier remission after induction with glucocorticoids and intrathecal methotrexate is correlated to a better outcome. Besides, the minimal residual disease (MRD) risk measured after induction is also correlated to the therapy response, indicating that an early response can predict Ph+ ALL overall outcome. Several studies have shown that Ph+ ALL is heterogeneous in terms of clinical parameters even if all patients carry BCR-ABL and most of them carry the oncogenic isoform of IKAROS. They respond differently to the treatment, which suggests the presence of additional mechanisms involved in leukemogenesis. In an attempt to find secondary genetic abnormalities that may be responsible for the Ph+ ALL chemo resistance and heterogeneity, we studied the miRNA expression profile in two patient groups discriminated by the initial therapy response.We included samples from 78 consecutive Ph+ ALL children diagnosed in Italy between 2000 and 2010. The miRNA signature was analyzed by miRNA array using Applied Biosystems Array-Cards comparing two patient groups differentiated according to MRD and prednisone response. A particular miRNA profile was found in the poor responder group and it was confirmed using specific miRNA single assays from AB. Specially the miR-125b was up to ten-fold overexpressed compared to the good responder group (p =.006). To investigate the functional role of miR-125b in Ph+ ALL we used BCR-ABL positive cell lines (3 ALL and one CML in blast-crisis) to create a xenograft leukemia model and induced miRNA upregulation by direct inoculation of synthetic miR-125b (premiR). Tumors injected with premiR-125b (n=14) were significantly bigger than the scrambled oligonucleotide (n=10) and mock controls (n=4) (p=.04). After two weeks the premiR-125b tumors grew six-fold more than controls and average tumor weights for the scrambled oligonucleotides and the premiR-125b inoculated mice were 0.63 g and 1.56 g, respectively (p =.01). Further, we determined whether miR-125b protects the tumor cells from apoptosis after treatment with Dexamethasone. While tumors injected with scrambled molecules were 70% reduced after 10 days of corticoid treatment, tumors injected with premiR-125b were only 30% reduced (p=.05).Together, these results suggest that miR-125b acts as an oncogene in Ph+ ALL and its overexpression accelerates the role of BCR-ABL, increasing therapy resistance and leukemia aggressiveness. Additional studies will be necessary to understand why this miRNA is overexpressed in some patients and how it acts in cooperation with BCR-ABL to induce leukemia. In the future, novel therapies using miRNAs as targets can emerge as strategies to be added to the anti-tyrosine kinase drugs in order to improve treatment response and survivor in Ph+ ALL. Disclosures:No relevant conflicts of interest to declare.

Combining ABL1 Kinase Inhibitor, Ponatinib and the Histone Deacetylase (HDAC) Inhibitor Vorinostat: A Potential Treatment for BCR-ABL Positive Leukemia Cells

Abstract 1403▪▪This icon denotes a clinically relevant abstractResistance to imatinib is most commonly explained by acquired point mutations in the kinase domain of BCR-ABL, which impair drug binding. The more potent ABL1 inhibitors nilotinib and dasatinib have proven largely successful in imatinib-resistant chronic myeloid leukemia (CML) patients with the key exception of the T315I BCR-ABL1 mutant. Ponatinib, also known as AP24534, is an oral, the multi-targeted tyrosine kinase inhibitor (TKI) and highly activity against ABL kinase point mutation included T315I. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial (PACE trial). However, ABL1 inhibitor-resistant patients already harboring mutations have a higher likelihood of developing further mutations under the selective pressure of ABL1 TKIs. The challenge for development of an effective Philadelphia chromosome (Ph) positive leukemia therapy is therefore to develop an alternative treatment strategy that does not rely solely on kinase domain inhibition but rather results in degradation of the offending BCR-ABL1 protein regardless of its mutation status. Histone acetyltransferases (HAT) and histone deacetylases (HDAC) control the acetylation of histones and intracellular proteins, and regulate the transcription and function of the proteins. Several small molecule HDAC inhibitors such as vorinostat (suberoylanilide hydroxamic acid: SAHA) are also evaluated for significant clinical activity in hematological malignancies. Therefore, combination therapy using a ponatinib and an HDAC inhibitor, vorinostat may help prevent development of ABLTKI resistance and may improve their long-term outcome. In the present study, we analyzed the ponatininb and vorinostat efficacy by using the BCR-ABL positive cell line, K562 and murine Ba/F3 cell line which was transfected with imatinib resistant BCR-ABL random mutagenesis and T315I mutant cells. 72 hours treatment of ponatinib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. We also found that ponatinib potently induced cell growth inhibition of Ba/F3 cells ectopically expressing T315I mutation. We found that phosphorylation of BCR-ABL, Crk-L was decreased and poly (ADP-ribose) polymerase (PARP) was activated in a dose dependent manner in these cells. Combined treatment of Ba/F3 T315I mutant cells with vorinostat and ponatinib caused significantly more cytotoxicity than each drug alone. We investigated the intracellular signaling of ponatinib and vorinostat. Although phosphorylation of BCR-ABL, Crk-L was not reduced after vorinostat treatment for 24 hours, acetylation of histone H4 was increased. Caspase 3 and PARP activation were increased after combination of ponatinib and vorinostat. Moreover, an increase in phosphorylation levels of γ-H2A.X was observed after treatment with ponatinib and vorinostat, compared to ponatinib or vorinostat alone suggests that combination of ponatinib and vorinostat induced DNA damage against T315I mutant cells. We next established ponatinib resistant cells by using Ba/F3 BCR-ABL with resistant random mutants. In the ponatinib resistant cell lines, IC50 of ponatinib was 200nM. BCR-ABL triple point mutations (T315I, E255K and Y253H) were detected by direct sequence and invader analysis. Ponatinib resistant Ba/F3 cells were also resistant to imatinib (IC50: more than 10μM) or nilotinib (IC50: 7.5μM) and also resistant to dasatinib (IC50: more than 100nM). We investigated the efficacy between ponatinib and vorinostat by using these cell lines. Combined treatment of Ba/F3 ponatinib resistant cells with ponatinib and vorinostat caused significantly more cytotoxicity. Moreover, acetylation of histone H4, caspase 3 and PARP activation were found after combination of ponatinib and vorinostat. Data from this study suggested that administration of the ponatinib and HDAC inhibitor, vorinostat may be a powerful strategy against BCR-ABL mutant cells and enhance cytotoxic effects of ponatinib in those BCR-ABL mutant cells. Disclosures:No relevant conflicts of interest to declare.

Efficacy of Ponatinib Against ABL Tyrosine Kinase Inhibitor Resistant Leukemia Cells

Abstract 4413Dasatinib (Sprycel®) and nilotinib (Tasigna®) have each shown superior efficacy as front line treatment for patients with chronic myeloid leukemia (CML)-chronic phase (CP) in comparison with imatinib. Dasatinib and nilotinib are also used for the treatment of CML patients resistant or intolerant to imatinib therapy. However, a substantial number of patients are acquired resistance to nilotinib or dasatinib, the management of CML following the development of ABL tyrosine kinase inhibitor (TKI) resistance remains a challenge. Ponatinib, also known as AP24534, is an oral, the multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial (PACE trial) in patients with resistant or intolerant CML and Philadelphia positive acute lymphoblastic leukemia (Ph+ ALL). However, the molecular and functional consequences of ponatinib against ABL TKI resistant cells are not fully known. In this study, we investigated the ponatinib efficacy by using the BCR-ABL positive cell line, K562 and ABL TKI resistant (K562 imatinib resistant (K562IR), K562 nilotinib resistant (K562NR), K562 dasatinib resistant (K562DR) cells and murine Ba/F3 cell line which was transfected BCR-ABL random mutation, and established the new imatinib and nilotinib resistant Ba/F3 BCR-ABL point mutant (E334V) cells. We first examined the cell proliferation by using resistant cell lines. The proliferation of K562IR and K562 NR and K562DR did not decrease after imatinib (10 μM) or nilotinib (2 μM) or dasatinib (1 μM) treatment compared with parental cell line, K562. The BCR-ABL kinase domain mutation was not found. Point mutant Ba/F3 cell (E334V) was also highly resistant to imatinib (IC50: 15μM) and nilotinib (IC50: 7.5μM). We next examined the intracellular signaling by using these cell lines. Phosphorylation of BCR-ABL and Crk-L was not decreased by ABL TKIs in K562IR, K562NR and Ba/F3 BCR-ABL point mutant cells (E334V). We found the one of src family kinase, Lyn was activated in K562IR and K562NR cells. Co-treatment src kinase inhibitor, PP2 and imatinib or nilotinib significantly reduced the cell proliferation of K562IR and K562NR cells. We also found the phosphorylation of Lyn was reduced and poly (ADP-ribose) polymerase (PARP) was activated. We next examined the efficacy of ponatinib against imatinib and nilotinib resistant cell lines. 72 hours treatment of ponatinib exhibits cell growth inhibition against K562 (IC50: 0.02nM), K562IR (IC50: 15nM), and K562NR (IC50: 3.5nM) cells. We also found the phosphorylation of BCR-ABL, Lyn and Crk-L was reduced and PARP was activated after ponatinib treatment. We next examined the imatinib and nilotinib resistant Ba/F3 cells with point mutant (E334V). We found the cell proliferation was significantly decreased after ponatinib treatment (IC50: 3nM). We also found the phosphorylation of BCR-ABL, Crk-L was reduced and PARP was activated after ponatinib treatment. We next investigated the ponatinib activity against dasatinib resistant cells. We found K562DR cells were highly resistant to ponatinib. IC50 was 400nM. These results suggest that the expression and protein activation signatures identified in this study provide insight into the mechanism of resistance to ABL TKIs. We also demonstrate ponatinib has anti-leukemia effect by reducing ABL and Lyn kinase activity and development of ponatinib resistance and suggests that this information may be of therapeutic relevance. Disclosures:No relevant conflicts of interest to declare.

The Gene Expressions of hOCT1 and ABCB1 Change During the Course of CML in Relation to Imatinib Therapy

Abstract 2495 Background.The intracellular concentration of imatinib (IM) in patients with chronic myeloid leukemia (CML) is supposed to be influenced by the expression of its main cellular transporters, hOCT1 (influx) and ABCB1 (efflux). The assessment of the genes expression may be potentially important in clinical practice to effectively manage the therapy. Our recent results showed the necessity to cautiously interpret results from gene expression measurements of both transporters as their detected mRNA levels are affected by the proportion of different cell types in the sample analyzed (Racil et al. 2010 Am J Hem 85:525, Racil et al. 2011 Leuk & Lymph 52:331). Aims. In this report, we aimed to comprehensively assess differences in the expression of hOCT1 and ABCB1 in total leukocytes of peripheral blood (PB) in relation to the blood cell lineage in CML patients with different responses to IM. Methods. Kruskall Wallis’s and Dunn’s multiple comparison tests were applied to calculate differences in transcript levels of hOCT1 and ABCB1 in patients at diagnosis (Dg=43), in major molecular response (MMR=27), complete molecular response (BCR-ABL log 4.5 reduction; CMR4.5 =15), therapy failure (TF=13), accelerated phase (AP=12) and in 75 healthy controls. CMR4.5 is defined here as either a detectable disease ≤0.0032% BCR-ABLIS or as undetectable by nested PCR. TF is defined as non CCgR achievement. Additionally, we used the Spearman’s correlation test to investigate relationship between expressions and percentage of immature cells and neutrophils in patients with non-physiological blood count (Dg and AP). In patients with normal blood count (CMR4.5, MMR, TF), we calculated correlation with BCR-ABL transcript level. Finally, we performed in vitro experiments with BCR-ABL negative (SKM-1, MOLM-13) and positive cell lines (K562, MOLM-7) treated with IM to study its effect on ABCB1 expression. Results. We found a significantly lower expression of hOCT1 and ABCB1 at Dg (P<0.001) and in AP (P<0.001 and P<0.01, respectively) in comparison to controls. The lower expression probably depends indirectly on the immature cells count (hOCT1 r = −0.4850, P=0.0002; ABCB1 r = −0.6451, P< 0.0001). Interestingly, while a significant positive correlation of neutrophil count was found with hOCT1 mRNA levels (r=0.6090, P< 0.0001), no correlation was observed with ABCB1. In patients with physiological blood count, we observed significantly elevated expression of ABCB1 in MMR and CMR4.5 in comparison to controls (P< 0.01). As MMR and CMR4.5 samples are characterized by the BCR-ABL transcript level <0.1%, this result led us to perform in vitro experiments to test the effect of IM on ABCB1 expression in BCR-ABL negative cell lines showing an elevated expression after incubation with IM. We assume this effect to be a natural defense of BCR-ABL negative cells to get rid of IM. By contrast, we found a drop in ABCB1 expression after BCR-ABL positive cell lines incubation with IM, suggesting inhibitory effects of IM. A significantly reduced expression of hOCT1 was found in TF in comparison to MMR and CMR4.5 (P< 0.01). These samples did not differ in blood count, but we observed a significant negative correlation of hOCT1 with BCR-ABL transcript levels (r= − 0.5117; P< 0.0001). Conclusion. Cell composition in PB changes during the CML treatment with IM which influences the measurement of hOCT1 and ABCB1 mRNA levels in total leukocytes. However, hOCT1 expression was found to be significantly lower in patients who failed to achieve CCgR but had normal blood count. Interestingly, our data suggest that ABCB1 is differently expressed in BCR-ABL positive and negative cells due to the inhibitory and enhancing effects of IM, respectively. Supported by IGA NT11555 and MZOUHKT2005 Disclosures:Machova Polakova:Novartis: travel grant. Ondrackova:BMS: travel grant.

International Standardization of Minimal Residual Disease Assessment for in Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ALL) Expressing m-BCR-ABL Transcripts: Updated Results of Quality Control Procedures by the EWALL and ESG-MRD-ALL Consortia

Abstract 2535 Background:Minimal residual disease is well established as an important prognostic parameter in a number of hematologic diseases and is used for stratification and treatment decisions in adult and childhood acute lymphoblastic leukaemia (ALL). DNA- based PCR analysis of Ig-/TCR-rearrangements have been well standardised during the last decade, resulting in robust systems for MRD analysis with an intra- and inter-assay variability of less than half a log and low false positivity. In Ph+ALL, MRD analysis relies on RNA-based RT-PCR techniques that are highly sensitive but as yet lack standardisation between laboratories. Since laboratories differ substantially in both their methodology and analysis strategy, interpretation and meaningful comparison of results between laboratories and from different studies is difficult or impossible. Moreover, and in contrast to CML, there are no generally accepted definitions of molecular response that could identify thresholds on which to base therapeutic decisions. Aims:To assess i) the variability of BCR-ABL quantification between laboratories with recognized expertise in bcr-abl analysis, ii) identify optimal laboratory methods and iii) to standardize analysis and interpretation of RT-PCR results for Ph+ALL, the EWALL and ESG-MRD-ALL consortia initiated a multinational project for quality assurance involving more than 30 laboratories from 14 countries worldwide. We here report the results of the first six m-BCR-ABL laboratory control rounds, performed between march 2008 and august 2011, that focussed on inter-intraassay variability of rt-PR techniques, comparison of housekeeping genes, plasmids, platforms and reagents. Methods:Serial dilutions of the BCR-ABL positive cell line Sup B15 with the BCR-ABL negative cell line Nalm 6 covering 5 logs were produced. In the 6 lab rounds, 1355 aliquots, each generated from with a total amount of 5 × 10E+06 cells in a final volume of 1 ml were produced, stabilized in TRIZOL or buffer RLT and frozen at −20°C until shipment or centralized isolation of RNA and reverse transcribed. Participants were asked to process the material using predefined procedures. Results:In the first QC round a major finding was the high variability in RNA-yield between laboratories despite using the same extraction method, with an up to 3 log difference in ABL copy numbers. In addition, there was rare false-positivity and false negativity (specifity 94.8%). With centrally produced cDNA the variability improved and there was a lower frequency of false-positivity reaching 100% specifity in QC 2. By using standardized assays, in 12 of 33 laboratories no conversion factor is needed, 20 of 33 laboratories are at the moment within one log difference when comparing the BCR-ABL/ABL ratios. Comparison of housekeeping genes (ABL/GUS) revealed non-linearity at high transcript levels resulting in an underestimation of intraindividual log reductions and may be relevant when assessing disease prognosis. The quantitative range was generally above 10E-04. Conclusion:Initiation of laboratory control rounds and implementation of standardized methodology improved the variability and reduced the frequency of false-positive results. Sensitivity of current techniques is still unsatisfactory for diseases with aggressive growth kinetics such as Ph*ALL. Further standardisation of technical and analytical methodology will provide the basis for defining levels of molecular remission and relapse and help to ensure comparability between laboratories and clinical trials at the European level. Disclosures:Goekbuget:Micromet: Consultancy. Ottmann:Novartis Corporation: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding.

Long Circulating Lectin Conjugated Paclitaxel Loaded Magnetic Nanoparticles: A New Theranostic Avenue for Leukemia Therapy

Amongst all leukemias, Bcr-Abl positive chronic myelogenous leukemia (CML) confers resistance to native drug due to multi drug resistance and also resistance to p53 and fas ligand pathways. In the present study, we have investigated the efficacy of microtubule stabilizing paclitaxel loaded magnetic nanoparticles (pac-MNPs) to ascertain its cytotoxic effect on Bcr-Abl positive K562 cells. For active targeted therapy, pac-MNPs were functionalized with lectin glycoprotein which resulted in higher cellular uptake and lower IC50 value suggesting the efficacy of targeted delivery of paclitaxel. Both pac-MNPs and lectin conjugated pac-MNPs have a prolonged circulation time in serum suggesting increased bioavailability and therapeutics index of paclitaxel in vivo. Further, the molecular mechanism pertaining to pac-induced cytotoxicity was analyzed by studying the involvement of different apoptotic pathway proteins by immunoblotting and quantitative PCR. Our study revealed simultaneous activation of JNK pathway leading to Bcr-Abl instability and the extrinsic apoptotic pathway after pac-MNPs treatment in two Bcr-Abl positive cell lines. In addition, the MRI data suggested the potential application of MNPs as imaging agent. Thus our in vitro and in vivo results strongly suggested the pac-MNPs as a future prospective theranostic tool for leukemia therapy.

Leptin Reverts Pro-Apoptotic and Antiproliferative Effects of α-Linolenic Acids in BCR-ABL Positive Leukemic Cells: Involvement of PI3K Pathway

It is suspected that bone marrow (BM) microenvironmental factors may influence the evolution of chronic myeloid leukaemia (CML). In this study, we postulated that adipocytes and lipids could be involved in the progression of CML. To test this hypothesis, adipocytes were co-cultured with two BCR-ABL positive cell lines (PCMDS and K562). T cell (Jurkat) and stroma cell (HS-5) lines were used as controls. In the second set of experiments, leukemic cell lines were treated with stearic, oleic, linoleic or α-linolenic acids in presence or absence of leptin. Survival, proliferation, leptin production, OB-R isoforms (OB-Ra and OB-Rb), phosphoinositide 3-kinase (PI3k) and BCL-2 expression have been tested after 24h, 48h and 72h of treatment. Our results showed that adipocytes induced a decrease of CML proliferation and an increase in lipid accumulation in leukemic cells. In addition, CML cell lines induced adipocytes cell death. Chromatography analysis showed that BM microenvironment cells were full of saturated (SFA) and monounsaturated (MUFA) fatty acids, fatty acids that protect tumor cells against external agents. Stearic acid increased Bcl-2 expression in PCMDS, whereas oleic and linoleic acids had no effects. In contrast, α-linolenic acid decreased the proliferation and the survival of CML cell lines as well as BCL-2 and OB-R expression. The effect of α-linolenic acids seemed to be due to PI3K pathway and Bcl-2 inhibition. Leptin production was detected in the co-culture medium. In the presence of leptin, the effect of α-linolenic acid on proliferation, survival, OB-R and BCl-2 expression was reduced.

HTERT Promotes Imatinib Resistance in Chronic Myeloid Leukemia Cells: Therapeutic Implications

Imatinib mesylate has shown remarkable efficacy in the treatment of patients in the chronic phase of chronic myeloid leukemia. However, despite an overall significant hematological and cytogenetic response, imatinib therapy may favor the emergence of drug-resistant clones, ultimately leading to relapse. Some imatinib resistance mechanisms had not been fully elucidated yet. In this study we used sensitive and resistant sublines from a Bcr-Abl positive cell line to investigate the putative involvement of telomerase in the promotion of imatinib resistance. We showed that sensitivity to imatinib can be partly restored in imatinib-resistant cells by targeting telomerase expression, either by the introduction of a dominant-negative form of the catalytic protein subunit of the telomerase (hTERT) or by the treatment with all-trans-retinoic acid, a clinically used drug. Furthermore, we showed that hTERT overexpression favors the development of imatinib resistance through both its antiapoptotic and telomere maintenance functions. Therefore, combining antitelomerase strategies to imatinib treatment at the beginning of the treatment should be promoted to reduce the risk of imatinib resistance development and increase the probability of eradicating the disease.

GDC-0449, the Small Molecule Inhibitor of Hedgehog-Gli Pathway for the Treatment of BCR-ABL Positive Leukemia Cells and In Combination with Dasatinib

AbstractAbstract 2136Imatinib has shown clinical efficacy against Philadelphia chromosome (Ph) positive leukemia cells and it is now the standard care for initial therapy. However, recent studies reported imatinib are not effective in quiescent primitive chronic myeloid leukemia (CML) stem cells. Moreover, many Ph-positive leukemia patients develop resistance or fail to respond to imatinib by mutation in the ABL kinase domain in clinically. These results indicated that alternative combination therapy such as BCR-ABL targeting tyrosine kinase inhibitors (TKIs) and nontoxic agents are required to cure the Ph positive leukemia patients. Hedgehog (Hh)- Glioma-associated oncogene homolog (Gli) signaling regulates self-renewal of stem cells and implicates in a large number of human cancers. One of the Hh inhibitor, GDC-0449 is a potent small molecule inhibitor of Hedgehog-Gli pathway. It has been reported GDC-0449 showed high target specificity and demonstrated antiproliferative activity against tumors and it is now in clinical trial. Therefore, combination therapy using a BCR-ABL tyrosine kinase inhibitors and a Hedgehog-Gli inhibitor, GDC-0449 may help prevent CML relapse and these approaches may be expected to improve the outcomes of Ph-positive leukemia patients. In this study, we investigated the GDC-0449 efficacy by using the BCR-ABL positive cell lines, OM9;22, K562 and primary samples when leukemic cells were protected by the feeder cell line, S9 cells. We examined a comprehensive drug combination experiment using GDC-0449 and dual Src/ABL tyrosine kinase inhibitor, dasatinib. Gli proteins (Gli1, Gli2 and Gli3) were existed in Ph-positive cell lines. We found the cell numbers of OM9;22 were significantly increased with the feeder cell line, S9 cells compared to without S9 cells. The treatment of dasatinib exhibits cell growth inhibition partially against OM9;22 cells in the presence of feeder cell line, S9 cells. Caspase-3 activity by 100 nM dasatinib treatment was also reduced in the presence of S9 cells. 72 h of combined treatment of Ph-positive leukemia cells with 10 μM of GDC-0449 and 100 nM of dasatinib in the presence of feeder cell line, caused significantly more cytotoxicity than each drug alone. We next investigated the efficacy and intracellular signaling of GDC-0449. The treatment of GDC-0449 exhibits cell growth inhibition and induced apoptosis against OM9;22 cells in a dose and time dependent manner. Expression of Gli1 and Gli2 proteins were reduced after GDC-0449 treatment. 10 μM of GDC-0449 also inhibited the growth of Ph-positive primary samples by colony assay. Another Hh inhibitor, SANT-2 also exhibits cell growth inhibition against OM9;22 cells in a dose dependent manner. Data from this study suggested that administration of the Hh inhibitor, GDC-0449 may be a powerful strategy against Ph-positive leukemia cells and enhance cytotoxic effects of dasatinib in the presence of feeder cell. Disclosures:Ohyashiki: Nippon Shinyaku Co., Ltd.: Research Funding.

The Elevated Expression of FBP1, a Key-Enzyme of Gluconeogenesis Gene, Is Associated to High Sokal Risk In Chronic Myeloid Leukemia Patients.

AbstractAbstract 3399The oncogenic transformation of Chronic Myeloid Leukemia (CML) cell of origin has been associated to an increased glucose metabolism, where the glycolytic pyruvate is directed away from the mitocondria, converted into lactate and secreted from the cell. This metabolic conversion is termed aerobic glycolysis, or Warburg effect and very essentially, it serves to support the tumor cell proliferation, by providing nucleotides, aminoacids and lipids enough to replicate all of cellular content. CML patients are historically stratified according to their Sokal risk score, which for more than 30 years has been regarded as the most significant prognostic factor in this hematological malignancies. The putative genetic and/or genomic basis driving this stratification are still not known.Aim of the present study was to explore the molecular mechanisms associated to the CML patients stratification according to the Sokal risk score, by analyzing the transcriptome of the CD34+ cell fractions obtained at diagnosis from a cohort of high and non-high Sokal risk CML patients.Overall, 67 patients with previously untreated CML in chronic phase (CP) entered the study; all of them have been enrolled in GIMEMA CML protocols and provided highly enriched CD34+ cell fractions from peripheral blood. Gene expression profiling (GEP) was performed, in order to identify genes most significantly and most differentially expressed between high and non-high Sokal risk patients. All other cases were used in Real-time experiments, in order to validate the GEP data.By GEP, 82 probe-sets, corresponding to 78 genes resulted significantly differentially expressed between high and non-high Sokal risk patients in a supervised analysis of gene profiles. A gene enrichment analysis of this profile showed that genes involved in the Wnt, in the Notch signaling pathways and in the response to hypoxia and oxidative stress resulted significantly overexpressed in the comparison between high and non-high risk patients. We focused our attention on genes involved in the glycolysis and gluconeogenesis metabolic pathways (FBP1, SEPP1, GSTM3, GSTT1, PRDX2, MPO). We validated by Real-time the de-regulated expression of these genes in a different set of newly diagnosed CP-CML patients, thus confirming that they are differentially expressed between high and non-high risk patients, with trends similar to those observed by GEP. Particularly interesting resulted a significantly higher expression in high Sokal risk patients of FBP1 (fructose 1,6 bisphosphatase), a key-enzyme of gluconeogenesis, together with a significant over-expression of genes coding for enzyme involved in gluthatione biosynthesis (GSTM3, GSTT1, and PRDX2). These data suggest that CD34+ cells obtained from high Sokal risk patients might exhibit an unexpected moderation of the glycolytic flux, mainly due to the over-expression of FBP1, which might cause a re-direction of the pathway into the pentose phosphate shunt. A similar metabolic reprogramming has been already described in imatinib resistant bcr-abl positive cell lines and is supported also by the over-expression in high risk patients of G6PDH (Glucose-6-phosphate 1-dehydrogenase) and TK (Transketolase), which are key enzyme of the pentose phosphate shunt.Overall, our data demonstrate for the first time, that the expression at diagnosis of sugar metabolic enzymes, might drive the evolutive Sokal risk of CML patients.Supported by: European LeukemiaNet, BolognAIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrate program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures:Rosti:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Baccarani:NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.