REH Cells Research Articles

Abstract 4545: Identifying neuroblastoma-specific ligands for cancer targeted therapy using a one-bead one-compound combinatorial approach.

Abstract Neuroblastoma is the most common extra-cranial tumor in children, with around 700 new diagnoses each year. High-risk patients have poor outcomes and only 30% survive despite the most intensive therapies available, which can also harm healthy cells. Thus, we are developing novel targeted therapies for neuroblastoma to increase treatment efficacy and limit damage to healthy cells. We utilized the One-Bead One-Compound (OBOC) combinatorial split-mix method developed by Kit Lam (Lam et al., 1991) to identify unique ligands against neuroblastoma cells for therapeutic delivery. The OBOC libraries consist of hundreds to millions of 90 μm Tentagel beads, and each bead displays 1013 copies of a peptide on its surface. Thus, each bead has one unique compound on its exterior. Twelve random OBOC libraries with varying peptide lengths (5 to 16 amino acids) were screened against three human neuroblastoma cell lines (SK-N-BE, SK-N-DZ, and IMR-32) to determine a library for more stringent screening of ligands. 37,500 beads from each library were screened against each cell line and the libraries averaged 2 positive candidates per 18,750 beads within 2 hours of incubation. The results also suggested preference for longer peptide libraries, but an intermediate length random decamer library was chosen for further screening due to both a good yield of positive beads and convenient sequencing. Each neuroblastoma line was screened against 150,000 beads from the decamer library to identify potential ligands. Positive beads were selected, retested three times, and decoded using Edman degradation. Several cancer cell lines (A549 lung cancer, and Reh and Jurkat leukemia cell lines) were used as negative controls. Ten beads per cell line displaying high binding affinity were selected. Candidate beads with positive binding to all three neuroblastoma cell lines are currently being sequenced and the ligands will be synthesized. We plan to characterize the ligands for high affinity and specificity, as well as internalization capacity, and to identify their receptors. Currently, there are very few targeted therapies for neuroblastoma. We believe our ligand discoveries can be developed for future novel targeted therapies in neuroblastoma. Citation Format: Andy Iskandar, Connie Duong, Cathy Chen, Eduardo Sanchez, David Olivos, Kit Lam, Noriko Satake. Identifying neuroblastoma-specific ligands for cancer targeted therapy using a one-bead one-compound combinatorial approach. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4545. doi:10.1158/1538-7445.AM2013-4545

Abstract 2770: Anti-leukemic response of a NSAID, tolfenamic acid.

Abstract Tolfenamic acid (TA), a non-steroidal anti-inflammatory drug is known to inhibit human cancer cells and mouse tumor growth in some cancer models; however its anti-leukemic response is not yet evaluated. Research from our laboratory and others showed that TA targets specificity protein (Sp) transcription factors which mediate the expression of several genes associated with cancer. We also showed that TA inhibits the expression of survivin, a key member of inhibitor of apoptosis family in several human cancer cells. Recent work from several laboratories revealed a strong association of survivin in leukemia. We hypothesize by targeting Sp proteins and survivin, TA can act as an anti-leukemic agent. The anti-proliferative response of TA was determined using four human leukemia cell lines, Jurkat (acute T-cell leukemia), Nalm-6 (pre-B cell leukemia), Molt-4 (acute lymphoblastic leukemia; T lymphoblast), and Reh (acute lymphoblastic leukemia, non-T; non-B) Cells were treated with increasing (25/50/75/100 μM) concentrations of TA and the cell viability was measured at 24, 48, and 72 h post-treatment using CellTiter-Glo kit. Results show a consistent decrease in cell viability in a dose and time-dependent manner. Confirmatory studies to elucidate the mechanism of action were conducted using selected cell lines, Jurkat and Nalm-6. Apoptosis and cell cycle analysis was performed using flow cytometry. The expression of c-PARP, Sp1, survivin, CDC2, CDC4, Cyclin D3 and pRb was determined by Western blot analysis and the caspases were measured by using Caspse-Glo kit(s). Results showed a significant increase in the apoptotic (annexin V positive) cell population following TA treatment, while cell cycle phase distribution analysis showed G0/G1 arrest. TA-induced cell apoptosis is supported by robust activation of caspases (3/7, 8 and 9), and the expression of c-PARP. TA down-regulated the expression of CDC2, CDC4, Cyclin D3 and pRb that mediate the early phases of cell cycle. In summary, TA modulated the expression of critical candidate genes associated with the early phases of cell cycle with validated efficacy in causing G0/G1 arrest. Western blot results reveal that TA significantly decreases Sp1 and survivin expression. Further work is needed to clarify the role of TA as a novel therapeutic agent for leukemia. Citation Format: Robert M. Sutphin, Sarah F. Connelly, Chris M. Lee, Umesh T. Sankpal, Don Eslin, Riyaz Basha. Anti-leukemic response of a NSAID, tolfenamic acid. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2770. doi:10.1158/1538-7445.AM2013-2770

Abstract 4606: Loss of PKR promotes leukemia progression by activating Bcl-2 to inhibit apoptosis.

Abstract The interferon-inducible, dsRNA-dependent protein kinase, PKR, is activated by diverse cellular stresses. Loss of PKR expression or activity has been found in tumors including various types of leukemia, suggesting that loss of PKR may contribute to malignancy. However, no convincing conclusion has been reached regarding the relationship between PKR and leukemia progression. We analyzed 511 AML primary bone marrow samples by reverse phase protein array (RPPA) and discovered that PKR protein expression is > 50% reduced in CD34+ blasts from AML patients compared to CD34+ cells from healthy donors. This finding prompted us to further study how PKR affected progression of leukemia. PKR expression was either knocked down (by 82%) by specific SiRNA or directly inhibited by specific PKR inhibitor in leukemia cell lines. Significantly, inhibition of PKR decreased the rate of apoptosis following treatment with hydrogen peroxide (H2O2) or doxorubicin for 48 hours. In addition, bone marrow mononuclear cells form wild type, PKR null (-/-) or PKR transgenic mice were harvested and subjected to growth factor withdrawal for up to 120 hours. Cells isolated from PKR null mice had decreased apoptosis while cells from PKR transgenic mice had increased apoptosis compared to cells from wild type littermate mice. Further investigation showed that PKR inhibition led to reduction of PP2A activity, and a 2.4 fold increase in Bcl-2 phosphorylation. In addition, okadaic acid (an inhibitor of PP2A) had a similar effect to PKR inhibition on both Bcl-2 phosphorylation (1.74 fold increase) and stress-induced apoptosis. By contrast, treatment of cells with a PP2A activator, FTY 720, resulted in decreased Bcl-2 phosphorylation (by 2.24 fold) and enhanced apoptosis in response to H2O2. Furthermore, our study showed that following PKR inhibition, phosphorylation of Bcl-2 stabilized Bcl-2/Bax complex following H2O2 treatment, preventing Bax from being released, anchoring and perforating the mitochondrial outer membrane, which would initiate apoptotic process. To confirm our in vitro findings, a leukemia xenograft model was established in NSG mice using REH cells expressing luciferase. Mice receiving REH SiPKR cells displayed significantly faster tumor progression compared to mice receiving Sicontrol cells. Furthermore, tumors from REH SiPKR cells were more resistant to doxorubicin treatment compared to tumors from REH sicontrol cells. Taken together our results demonstrate that loss of PKR function inhibits PP2A-dependent Bcl-2 dephosphorylation resulting in increased leukemia progression and survival. In addition, since our studies indicate that PKR activation enhances the effectiveness of chemotherapy, PKR level/function status may serve as a clinical predictor of prognosis, disease relapse and resistance to chemotherapy. Citation Format: Xiaodong Cheng, Richard L. Bennett, Xiangfei Liu, Stratford May. Loss of PKR promotes leukemia progression by activating Bcl-2 to inhibit apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4606. doi:10.1158/1538-7445.AM2013-4606

Collaboration Between MiR-125b, MiR-100 and MiR-99a Induces Vincristine Resistance in Childhood TEL-AML1-Positive Acute Lymphoblastic Leukemia

Abstract 1475MicroRNAs (miRNAs) are involved in the pathobiology of leukemia. We previously observed that miR-125b, miR-99a and miR-100 were highly expressed in ex vivo vincristine (VCR) resistant leukemic cells of children with precursor B-cell acute lymphoblastic leukemia (ALL) (Schotte et al. Haematologica 2011). VCR is a microtubulin-interfering drug applied in the treatment of children with newly diagnosed ALL. Leukemic cells of TEL-AML1-positive precursor B-cell ALL were found to be resistant to this drug (Ramakers-van Woerden et al. Blood 2000). The expression levels of miR-125b, miR-99a and miR-100 were highly correlated in childhood precursor B-ALL cells. MiR-125b, as the most well-known miRNA among these three, was shown to contribute to drug resistance in different types of solid tumors. Down-regulation of miR-125b was also shown to sensitize the TEL-AML1-positive Reh cell line to doxorubicin and staurosporine (Gefen et al. Leukemia 2010). However, recent studies suggest that miRNAs co-operate and the combination of multiple distinct miRNAs co-regulates expression of associated target genes. The current study addressed whether miR-125b alone or in combination with miR-99a and/or miR-100 contributed to VCR resistance in childhood TEL-AML1-positive ALL cells.MiRNAs were over-expressed in TEL-AML1-positive Reh cells using lentiviral particles and/or miRNA precursors. Expression level of mature (and hence processed precursor) miRNAs was analyzed by qRT-PCR. The cell cycle distribution and the amount of leukemic cells in apoptosis were determined by flow cytometry of propidium iodide stained nuclei and Annexin V-propidium iodide stained cells, respectively. Leukemic cells were incubated with 9 ng/mL VCR and after 3 days of exposure the cellular response to VCR was measured by an MTT-based cell survival assay.The expression level of mature miR-125b, miR-99a and miR-100 was raised >100-fold upon lentiviral and precursor miRNA transduction compared to basal expression levels of TEL-AML1-positive Reh cells. Over-expression of miR-125b, miR-99a or miR-100 as single miRNAs did not significantly affect cellular survival after 3 days of exposure to VCR (20±5%, 29±17% and 29±17% viable cells, respectively) compared to the cells transduced with a scrambled miR-control (10±4% viable cells, p>0.05 each) when cell viability in absence of VCR was set to 100%. Over-expression of the combination of miR-100 and miR-99a only had a limited protective effect on cell viability (30±7% viable cells compared to 10±4% viable cells for the scrambled miR-control, p<0.05). In contrast, co-expression of miR-99a or miR-100 together with miR-125b or the combination of miR-99a and miR-100 together with miR-125b strongly and significantly induced resistance to VCR in TEL-AML- positive Reh cells; 91±4% of miR-99a/miR-125b-transduced, 93±5% of miR-100/miR-125b-transduced and 82±17% of miR-99a/miR-100/miR-125b-transduced cells remained alive upon VCR exposure compared to only 38±13% of miR-125b-transduced cells (p<0.05). The combination of these miRNAs did not change the cell cycle distribution or the amount of apoptotic cells in the absence of VCR. This suggests that these 3 miRNAs synergize in the development of resistance to VCR. Computational miRNA target prediction algorithms (TargetScan and EIMMo3) predicted 14 possible protein-coding target genes for the combination of miR-125b, miR-99a and miR-100. Gene expression profiling of ex vivo VCR-resistant (n=20) and VCR-sensitive (n=10) leukemic cells of TEL-AML1-positive ALL patients revealed that the expression levels of these 14 predicted target genes did not differ between resistant and sensitive cases (p>0.05). Ongoing studies currently explore which other protein-coding genes can be directly targeted by the miR125b, miR-99a and/or miR100 combination using functional gene expression studies.In conclusion, miR-125b, miR-99a or miR-100 as single factors were not effective to induce VCR resistance. In contrast, miR-125b in combination with miR-99a and/or miR-100 strongly increased VCR resistance in leukemic cells of TEL-AML1-positive precursor B-ALL. Discovery of directly regulated protein-coding target genes of these 3 miRNAs may point to ways to modulate resistance to VCR in children with ALL. Disclosures:No relevant conflicts of interest to declare.

Down-Regulation of BMI-1 Is a New Marker of Sensitivity to Mdm2 Inhibition in B-Acute Lymphoblastic Leukemia.

AbstractAbstract 2522 Introduction:Although p53 gene mutations are relatively infrequent in cases of B-ALL, the CDKN2A locus is deleted or inactivated in nearly half of all cases, especially Ph+ B-ALL (Mullighan et al., 2008; Iacobucci et al., 2011), contributing to a worse prognosis. In testing novel therapeutic approaches activating p53, we investigated the preclinical activity of the MDM2 antagonist Nutlin-3a in leukemic cell line models and primary B-ALL patient samples. Methods:TP53 mutation screening was performed by Sanger sequencing of exons 4 to 11; copy number status of CDKN2A was determined by MLPA kit P335-A2 ALL-IKZF1 (MRC Holland); cellular viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche); apoptosis was assessed by use of Annexin V/Propidium Iodide (PI); gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix). Mdm2 inhibitor (Mdm2i) Nutlin-3a was provided by Roche. Results:BCR-ABL1-positive (BV-173, SUPB-15) and negative (NALM19, REH) ALL cell lines were investigated for TP53 mutations and CDKN2A deletion. A p53 mutation (R181C) was identified in REH cells, whereas all the remaining cell lines resulted p53 wild-type but they were deleted in the locus containing CDKN2A. Leukemia cell lines were incubated with increasing concentrations of Nutlin-3a (0.005–2 μM) for 24, 48 and 72 hours (hrs). Mdm2 inhibition resulted in a dose and time-dependent cytotoxicity with IC50 at 24 hrs ranging from around 1.5 μM for BV-173 and SUPB-15 to 3.7 μM for NALM-19. By contrast, no significant changes in cell viability were observed in RHE p53-mutated cells after incubation with Mdm2i. The time and dose-dependent reduction in cell viability were confirmed in primary blast cells from a Ph+ ALL patient with the T315I Bcr-Abl kinase domain mutation found to be insensitive to the available tyrosine kinase inhibitors and from a t(4;11)-positive ALL patient (IC50 at 24 hrs equal to 2 μM). Consistent with the results of cell viability, Annexin V/PI analysis showed a significant increase in apoptosis after 24 hrs in sensitive cell lines and in primary leukemia blasts, whereas no apoptosis was observed in REH cells. To examine the possible mechanisms underlying Mdm2i-mediated cell death, western blot analysis was performed. Protein levels of p53, p21 (an important mediator of p53-dependent cell cycle arrest), cleaved caspase-3 and caspase-9 proteins increased as soon as 24 hrs of incubation with Mdm2i. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis was next performed, comparing sensitive cell lines at 24 hrs of incubation with concentrations equal to the IC50 and their untreated counterparts (DMSO 0.1%). A total of 621 genes (48% down-regulated vs 52% up-regulated) were differentially expressed (p < 0.05). We found a strong down-regulation of GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) (fold-change −1.35 and −1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells. Both genes are repressors of INK4/ARF and p21 and their aberrant expression has found to contribute to stem cell state in tumor cells. Additionally, experimental reduction of BMI1 protein levels results in apoptosis in tumor cells and increases susceptibility to cytotoxic agents and radiation therapy (Wu et al., 2011). Given the importance of BMI in the control of apoptosis, we investigated by western blot its pattern in treated and untreated cells, confirming a marked decrease as soon as 24 hrs of exposure to MDM2i both in leukemia cell lines and primary blast samples. Noteworthy, the BMI-1 levels remained constant in resistant cells. Conclusions:Inhibition of Mdm2 efficiently activates the p53 pathway promoting apoptosis. BMI-1 expression is markedly reduced in sensitive cells and it may be used as a biomarker of response. Evaluation of its expression before and after treatment in clinical settings will better gain insight into its role. Supported by:ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione – Università 2007 – 2009, INPDAP. Disclosures:Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:BMS: Consultancy, Honoraria, Speakers Bureau; NOVARTIS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

The Role of MXD3 in Human Precursor B Cell Acute Lymphoblastic Leukemia

AbstractAbstract 3523Disregulation of the Hedgehog (Hh) signaling pathway is known to drive proliferation in several cancers, including hematopoietic malignancies. The role of the Hh pathway in precursor B cell (preB) acute lymphoblastic leukemia (ALL), the most common leukemia in children, is unclear; however, recent reports suggest that Hh signaling is involved in the development of B cell precursors as well as the self-renewal and survival of B cell malignancies.MXD3 is a transcription factor previously shown in our lab to be a novel member of the Hh pathway (Yun, Rust, Ishimaru, Diaz, Mol Bio Cell, 2007). We have previously shown that MXD3 is expressed in medulloblastoma (the most common brain tumor in children) and that its knockdown reduces proliferation of human medulloblastoma cell lines (Barisone et al., PLoS, 2012). In the current study, we investigated a possible role for MXD3 in preB ALL cell proliferation. Using quantitative real-time reverse-transcription PCR (qRT-PCR) we observed high levels of MXD3 mRNA expression in 8 primary preB ALL samples as well as in the preB ALL cell lines Reh and JM1. However, MXD3 levels were very low in mobilized peripheral blood mononuclear cells from healthy donors, mouse bone marrow and spleen. Indeed, MXD3 levels in the primary ALL cells and cell lines ranged between 13 and 35 fold increase when compared to the normal cells. Immunoblot analysis with anti-MXD3 monoclonal antibodies confirmed that the protein was present in the ALL samples but not in normal cells. We investigated the role of MXD3 in cell proliferation and survival, by silencing MXD3 in the Reh cell line. We used lentiviral delivery to knockdown MXD3 using an RNA interference approach. Briefly, lentiviruses were produced carrying a short hairpin RNA sequence specific for MXD3 (shMXD3) or a negative control sequence (shCTRL). Upon transduction by the viral vector, MXD3 knockdown was confirmed at both the RNA and protein level. Within 48 hours after transduction, MXD3 protein levels were reduced >90% in cells infected with the shMXD3 virus but not with the control virus. Interestingly, MXD3 knockdown resulted in decreased proliferation in Reh cells, supporting our hypothesis that it may be involved in the maintenance of this malignancy. As a first step toward understanding the mechanisms by which MXD3 exerts its function in leukemia cells, we analyzed cell cycle progression and apoptosis levels after knockdown using flow cytometry. We observed no significant differences in the G0/G1, S or G2/M populations between experimental and control samples. However, samples where MXD3 had been knocked down showed higher levels of apoptosis when compared to controls.Taken together, our results suggest that MXD3 is important for preB ALL cell proliferation, possibly by acting as an anti-apoptotic factor. Therefore, MXD3 may represent a suitable candidate for future efforts in developing targeted therapies against preB ALL. Disclosures:No relevant conflicts of interest to declare.

NF-κB Activation in Mesenchymal Stromal Cells Mediates Leukemia Cell Chemoresistance

Abstract 3518Within the bone marrow (BM) microenvironment, BM mesenchymal stromal cells (BM-MSC) provide leukemia cells with a rich environment that serves as a sanctuary and protects them from chemotherapeutic agents. Interactions between leukemia cells and BM-MSC are thought to change the behavior of both stroma and leukemia cells resulting in an increased resistance to standard drugs. To discover interaction-induced changes in BM-MSCs that might promote microenvironment-mediated chemoresistance we used genome-wide gene expression profiling to study normal-donor BM-MSCs co-cultured with REH cells, a pre-B acute lymphoblastic leukemia (ALL) cell line. Upregulated genes in normal-donor BM-MSCs by co-culture included many cytokines and chemokines and implicated activation of NF-κB in BM-MSCs. Similar results were obtained by co-culturing normal-donor BM-MSCs with OCI-AML3 cells, an acute myeloid leukemia (AML) cell line. Increased expression of NF-κB target genes in BM-MSCs was also induced by co-culture with primary cells from ALL and AML patients, suggesting that NF-κB activation is a common consequence of leukemia-stroma interaction. Blocking canonical-pathway NF-κB activation by overexpressing a super-repressor form of IκBα in BM-MSC, significantly reduced stroma-mediated chemoresistance to vincristine (VCR) of leukemia cells in vitro and in vivo using an extramedullary bone marrow model. When small-molecule IKKβ inhibitors (MLN3316 and CDDO-Me) were used in co-cultures, blocking canonical-pathway NF-κB activation in ALL cell lines (REH or RS4;11) as well as BM-MSCs, the apoptotic effects of VCR on leukemia cells were further increased. Conclusion:results indicate that leukemia cells activate NF-κB in stromal cells and that such activation is in turn essential to promote survival of leukemia cells during chemotherapy. Targeting NF-κB in BM-MSC may ameliorate stroma-mediated chemoresistance and help in eliminating BM-resident leukemia cells. Disclosures:No relevant conflicts of interest to declare.

Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

AbstractAbstract 1506Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs.From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL.On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above.This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL.This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures:No relevant conflicts of interest to declare.

Growth Inhibition of a Myeloid Leukemia Cell Line with 5q Deletion by Beta-Catenin Inhibition

AbstractAbstract 3608As the population ages, the incidence of AML with complex cytogenetics continues to increase. These patients have a poor prognosis with 5% percent of patients diagnosed surviving 5 years. New strategies are needed to improve survival in AML patients. 5q deletion is present in 6–10% of cases of AML, and is a common chromosome abnormality in therapy-related AML (t-AML). 5q deletion is associated with haplo-insufficiency of the APC gene in hematopoietic stem cells and progenitors, leading to deregulation of the Wnt pathway and stabilization of beta- catenin (Wang, J Blood 2010). Loss of APC causes rapid exhaustion of hematopoietic stem cells and progenitors in vivo and leads to the accumulation of beta-catenin (Qian, Z et al JEM 2008). Beta-catenin is required for the development of leukemia stem cells in AML, but it is not required for the self-renewal of normal hematopoietic stem cells (Wang, Y et al Science 2010). Development of treatment strategies aimed at blocking the Wnt pathway through suppression of beta-catenin may improve and prolong remission rates.The UoCM1 cell line maintains the unique characteristics of del(5q) leukemia (Qian et al, PNAS 2002). Western Blot shows that the expression of beta-catenin was higher in UoCM1 cells versus REH and MV4–11 cell lines, which do not have del(5q). To block the activation of Wnt signaling, through suppression of beta-catenin, we treated UoCM1, REH and MV4–11 cells with 100uM indomethacin. The UoCM1 cells showed 30% growth inhibition while REH and MV-4 cells displayed 10% growth inhibition. The down-regulation of beta-catenin in UocM1 is confirmed by Western Blot. Curcumin is a dietary polyphenol existing in Indian spice turmeric. We showed suppression of beta-catenin by curcumin in 293T cells by luciferase assay. Next, we treated UoCM1 and MV4–11 cells with 5uM or 10 uM curcumin. The growth of UoCM1 cells was significantly inhibited by curcumin as compared to MV4–11 cells. Treatment of UoCM1 cells with 10 uM curcumin produced 87% growth inhibition, while the growth inhibition was 29% for MV4–11 cells. 5uM concentrations of curcumin caused 48% growth inhibition in UoCM1 cells versus 16% in the MV4–11 cell line. Western Blot was performed to confirm that beta-catenin is down-regulated by curcumin, in the UoCM1 cell line.In summary, blockade of the Wnt pathway through inhibition of beta-catenin suppresses cell growth in UoCM1 leukemia cells, with 5q deletion. Suppression of beta-catenin in combination with cytotoxic therapy may provide a novel means of treating leukemia in patients with a 5q deletion that both improves remission rates and prevents relapse. Disclosures:No relevant conflicts of interest to declare.

L-Asparaginase Strongly Affects Bioenergetics in Leukemic Cells

AbstractAbstract 779L-asparaginase (L-asp) is an important component of childhood acute lymphoblastic leukemia (ALL) therapy. Its cytotoxic effect is based on the depletion of extracellular asparagine and glutamine. Leukemic cells are sensitive to this depletion due to the lower activity of asparagine synthetase compared to healthy cells. However, the mechanism of resistance development remains unclear. The aim of this study was to obtain further insights into the mechanisms underlying the cytotoxic effect of L-asp.We used two models: resistant preB ALL leukemic cells derived from the REH (TEL/AML1[+]; very sensitive) and NALM6 (TEL/PDGFRB[+]; medium sensitive) cell lines by long-term incubation with L-asp. As a second model we used REH and NALM6 incubated with L-asp for 24 hrs, mimicking acute phase of treatment.We performed GEP of REH, res-REH, NALM6 and res-NALM6. There was an overlap of 30 genes changed in both cases. Applying the pathway analysis we found that L-asp influences the regulation of lipid metabolism and apoptosis regulated by mitochondrial proteins. Next we merged our data with GEP data published by Holleman et al. (NEJM, 2004) of ALL patients′ samples sensitive/resistant to L-Asp. Two pathways, one regulating protein translation, the other metabolism, scored very highly. Next to glucose, glutamine is the other major cellular energy source, it is also important for activation of PI3K/Akt/mTOR pathway - the key regulator of translation. Since L-asp also depletes glutamine, we focused on the effect of L-asp treatment on bioenergetics and translation in leukemic cells. To confirm the effect of L-asp on PI3K/Akt/mTOR pathway we measured the amount of Akt protein and P70S6K/p-P70S6K (downstream target of mTOR) in REH and NALM6 treated for 24 hours with L-asp. Akt and p-P70S6K expression fell in both cell lines. Next we were interested if PI3K/mTOR inhibition affects the sensitivity of resistant cell lines. We measured the cytostatic effect of co-treatment: L-asp with mTORC1 inhibitor (rapamycin); with dual inhibitor of mTORC1 and PI3K (LY294002); and with specific inhibitor of PI3K (PX866) on REH, res-REH, NALM6 and res-NALM6. The MTS assay proved the L-asp/rapamycin combination the most effective. It strongly diminished viability of all cell lines, even in those resistant to L-asp.c-Myc as an activator of glutamine catabolism showed a significant decrease in REH and NALM6 after incubation with L-asp. In addition, c-Myc was also decreased in res-REH compared with REH. Importantly, c-Myc activates glycolysis which is a main energy generator in cancer cells rather than oxidative phosphorylation (OXPHOS). We showed diminishement of Glucose transporter type 1 protein expression and 2.5-times reduced level of lactate, the product of cancer cell glycolysis, in media after L-asp exposure. Next we measured cell respiration that mirrors oxidative fosforylation (OXPHOS). REH and NALM6 showed increased capacity of respiratory chain after L-asp exposure, which was observed as an increase of endogenous respiration in uncoupled state. REH boosted the capacity of the respiratory chain from 163 to 236 pmol O2/s/mg (p<0.01). NALM6 have increased the capacity of the respiratory chain from 78 to 155 pmol O2/s/mg (p<0.01). Interestingly, REH has a significantly higher capacity of respiratory chain compared with NALM6 and a lower ability to regulate it. Moreover, res-REH had elevated the level of the endogenous oxygen consumption whereas res-NALM6 levels did not change. Our results indicate that leukemic cells, which normally depend on glycolysis for energy generation rather than OXPHOS, are able to swap these two mechanisms of energy generation under amino acid deprivation stress.The data show that L-asp inhibits mTORC1 and strongly affects bioenergetics pathways of leukemic cells. It decreased c-Myc-regulated glycolysis and increased OXPHOS. We believe that these metabolic changes are mainly caused by the effort of the cells to mobilize another mitochondrial pathway as Krebs cycle and β-oxidation, with the aim to supply depleted amino acids. We conclude from these data that resistance of the cells is caused by better biochemical adaptability to the nutrient deprived environment.Support: GAUK 92710, IGA NT1249, UNCE204012 Disclosures:No relevant conflicts of interest to declare.

In Vitro and in Vivo Single-Agent Efficacy of Checkpoint Kinase 1 (Chk1) and 2 (Chk2) Inhibitor PF-0477736 (Pfizer) in B- and T-Acute Lymphoblastic Leukemia (ALL)

AbstractAbstract 1496 Introduction:Although progress in the treatment of ALL has been remarkable in children, in adults ALL still carries a dismal outcome. Thus, there is a need to improve therapeutic options. In the last years, selective inhibitors of Chk1 and/or Chk2 have been discovered, developed and entered in clinical trials. However, so far, they have not yet been investigated in leukemia. Chk1 and Chk2 are serine/threonine kinases that play a critical role in response to DNA damage both by halting the cell cycle through checkpoint activation and by actively repairing DNA. Here, we explored the in vitro and in vivo activity of single-agent inhibition of Chk1/2 by PF-0477736 in B- and T-progenitor ALL and we investigated potential biomarkers of functional inhibition. Methods:Human B (BCR-ABL1-positive: BV-173, SUPB-15; BCR-ABL1- negative: NALM-6, NALM-19, REH) and T (MOLT-4, RPMI-8402, CEM) leukemia cell lines were incubated with increasing concentrations of drug (5–2000 nM) for 24, 48 and 72 hours (hrs). Results:Inhibition of Chk1/2 resulted in a dose and time-dependent cytotoxicity with RPMI-8402 and BV-173 cells being the most sensitive (IC50 at 24 hrs: 57 nM and 82 nM, respectively), while NALM-6 cells the most resistant (IC50 at 24 hrs: 1426 nM)(WST-1 assay, Roche). Sensitivity did not correlate with p53 status (BV-173, SUPB-15, NALM-6 and NALM-19 cells were p53 wild-type whereas REH, MOLT-4, RPMI-8402 and CEM cells were p53 mutated) and with baseline levels of Chk1/2 and ATR/ATM phosphorylation, indicative of intrinsic genetic stress. Consistent with the viability results, Annexin V/Propidium Iodide (PI) staining analysis showed a significant increase of apoptosis at 24 and 48 hrs in a dose and time dependent manner coupled to increased proteolytic cleavage of PARP-1. In all sensitive cell lines in addition to the induction of apoptosis, Chk1/Chk2 inhibition induced DNA damage as demonstrated by the increased number of γH2AX foci (western blot and immunofluorescence analysis) and by a marked phosphorylation of Chk1 (ser317 and ser345). Moreover, PF-0477736 efficiently triggered the Chk1-Cdc25-Cdk1 pathway as soon as 24 hrs of treatment with a decrease of the inhibitory phosphorylation of Cdc25c (ser216) and Cdk1 (tyr15), leading to the abrogation of cell cycle arrest as confirmed by PI staining analysis at 6 and 24 hrs. The efficacy of PF-0477736 was thereafter demonstrated in primary leukemic blasts separated from 14 ALL patients. Based on the viability results at 24 hrs, 3 groups of patients were identified: very good responders, 5/14, 36% (IC50: 100–500 nM); good responders, 6/14, 43% (IC50: 600–1000 nM); poor responders, 3/14, 21% (IC50 > 1000 nM). By contrast, PF-0477736 did not show efficacy in primary cultures of normal bone marrow mononuclear cells, demonstrating its specificity for leukemia cells. We extended the in vitro and ex-vivo studies by assessing the efficacy of Chk inhibition in mice transplanted with T-lymphoid leukemia, demonstrating that PF-0477736 increases the survival of treated mice compared with mice treated with vehicle (p = 0.0016). Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated leukemia cells and their untreated counterparts (DMSO 0.1%) after 24 hrs of incubation with concentrations equal to the IC50. Treatment resulted in a differential expression (p < 0.05) of genes involved in chromatin assembly, nucleosome organization and DNA packaging (e.g. Histone H1-H2A, 2B family clusters), DNA damage (DDIT3, GADD34 and GADD45a) and apoptosis (e.g. CDKN1A, BAX, FAS, BTG1), confirming that PF-0477736 contributes to checkpoint replication abrogation, accumulation of DNA damage and subsequent apoptosis in leukemia cells. Interestingly, N-Myc and c-Myc expression strongly decreased after treatment, as also confirmed by western blot analysis, suggesting that a negative feedback loop may exist between Chk induction and Myc expression. Conclusions:Together, these results demonstrate the efficacy of PF-0477736 both in vitro and in vivo models of ALL, arguing in favor of its future clinical evaluation in leukemia. Supported by ELN, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer. Disclosures:Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Leukemia Microenvironment and Pathologic Hypoxia: Sensitivity to Hypoxia-Activated Cytotoxin TH-302

Abstract 1523We have recently demonstrated that the leukemic bone marrow (BM) niche is highly hypoxic and that hypoxia promotes resistance of leukemic cells to chemotherapy (Benito et al., PLoS One 2011, e23108). Our preliminary data indicate that AML cells surviving chemotherapy in the human xenograft mouse models of leukemia reside within hypoxic areas of BM microenvironment as documented by staining with the hypoxia marker CAIX. These findings support utility of hypoxia-activated pro-drugs with the goal to eliminate leukemic blasts and leukemic stem cells residing in hypoxic BM microenvironment. TH-302 is a 2-nitroimidazole linked bromo-isophosphoramide mustard cytotoxin that upon hypoxia-dependent activation induces DNA cross-linking. TH-302 exhibited potent hypoxia-selective anti-leukemia activity in pre-B ALL (REH, NALM6), AML (OCI-AML3, MOLM-13, KG-1) and CML cell lines (KBM5), with IC50s at 1% O2 ranging from 0.04μM to 2.3μM and hypoxia cytotoxicity ratio (HCR) ranging from 116 for KBM5 to 11 for REH cells. TH-302 at 5–7.5μM also exhibited hypoxia-dependent anti-leukemia activity in primary ALL and AML samples (N=3; normoxia, 2–8%; hypoxia, 28–65% apoptotic cells).To better recapitulate the multidimensional BM niche we utilized co-cultures of GFP-labeled leukemic cells with bone marrow-derived RFP-labeled mesenchymal stromal cells (MSC) immobilized within Matrigel. MSC and leukemic cells generated three-dimensional (3D) structures- “spheroids”- and co-proliferated over time with colonies of leukemic cells firmly attached to MSC, as monitored by confocal microscopy (Fig. 1). Pimonidazole staining shows that vast hypoxia is present in the MSC/AML spheroids grown at normal oxygen tension, in contrast to what is observed in plastic-based (2-D) stromal co-cultures. Anti-leukemia activity of TH-302 was next determined in 2D vs 3D co-cultures of MSCs plus MOLM-13 or OCI-AML3 cells. In 2D co-cultures, MSC protected MOLM-13 and OCI-AML3 cells from TH-302-induced cytotoxicity, while extensive apoptosis was documented in hypoxic spheroid co-cultures (at 50nM TH-302, reduction in viability 10–15% vs >60%, Fig. 1). These findings suggest that culture conditions faithfully mimicking BM microenvironment promote pathologic hypoxia generated by rapidly proliferating AML cells, which in turn leads to their increased sensitivity to hypoxia-activated cytotoxins. To validate these findings in vivo, we next tested anti-leukemia efficacy of TH-302 in the in vivo model of primary AML established in NSG mice. TH-302 (50 mg/kg IP 3 times a week for three weeks) reduced the number of circulating AML cells (control, 13.2+/−5.7 ×106/ml; TH-302, 2.5+/−2.1 ×106/ml) and prolonged survival of NSG mice engrafted with primary AML cells compared to the vehicle treated mice (median survival time: TH-302=75 days; Control=56 days; P=0.003, n= 8 mice/group). To test the ability of hypoxia-activated prodrug to target leukemia-initiating cells, secondary transplant experiments were performed in which BM cells from control or TH-302 treated mice (collected after two weeks of therapy) were serially diluted and injected into secondary NSG recipient mice at 0.01, 0.005 or 0.0001×106′ cells/mouse (N=5 mice/dilution). Although all mice transplanted with higher cell doses died from leukemia, we observed significantly prolonged survival of animals injected with 0.01×106 cells from TH-302-treated primary recipients compared with vehicle-treated controls (median survival control=68 days; TH-302=79 days; P=0.0031) or with 0.005×106 cells (control=79 days; TH-302=83 days; P=0.0462). In summary, our findings suggest that pathologic hypoxia is a prevalent condition of leukemic BM microenvironment that promotes survival of leukemic blasts and leukemia-initiating cells. The results support targeting hypoxia with hypoxia-activated cytotoxins such as TH-302 to enhance the efficacy of therapeutic regimens in AML. A Phase 1 single agent clinical trial of TH-302 in patients with relapsed/refractory hematologic malignancies is ongoing. [Display omitted] Disclosures:Handisides:Threshold Pharmaceuticals: Employment. Hart:Threshold Pharmaceuticals: Employment. Konopleva:Threshold Pharmaceuticals: Research Funding.

Antiproliferative Properties of the Serotonin Receptor Antagonist Ondansetron Correlate with Increased Nitric Oxides Release and Inducible Nitric Oxide Synthase Activity in the Acute Lymphoblastic Leukemia Cell Line REH

A recent report from our group described that the (serotonin receptor-3)-antagonist ondansetron exhibits antiproliferative effects in the B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell line REH. Furthermore, after each application of ondansetron to cultured REH cells, significant increases (+23%) in the concentration of nitric oxides (NO) were observed in the cell supernatants after 72 hours incubation in standard conditions, and this effect was found to correlate with the described antiproliferative activity. This feature was further confirmed by using mRNA dot blot hybridizations with a specific gene probe for the inducible NO-synthase (iNOS), yielding significant increases (+100%) of iNOS mRNA, which were found to widely correlate with the detected increases of NO release, and also with the previously described antiproliferative effects. The presented results are the first report on high specific pro-inflammatory features of a (serotonin receptor 3)-antagonist in a BCP-ALL cell line, which are associated with previously described antiproliferative properties.

Linger RMA, DeRyckere D, Brandão L, Sawczyn KK, Jacobsen KM, Liang X, Keating AK, Graham DK. Mer receptor tyrosine kinase is a novel therapeutic target in pediatric B-cell acute lymphoblastic leukemia. Blood. 2009;114(13):2678–2687.

The authors retract the 24 September 2009 article cited above, prepublished on 30 July 2009. They have recently learned that some of the cell lines used in their paper were inadvertently misidentified. Although the parental 697 and REH cell lines used to generate the Mer knockdown lines were authenticated by short tandem repeat (STR) analysis before publication, the transduced progeny were not analyzed until recently. The results of STR analysis indicate that the 697 shMer1A and 697 shMer1B cell lines are actually derived from the REH parental cell line. Importantly, the identities of the other 6 REH and 697 cell lines published in this study have been verified as authentic.Since this unfortunate discovery, the authors have generated new 697 Mer knockdown cell lines and authenticated their identity. These new cell lines are being used to replicate the original work. Data obtained to date support the overall findings and conclusions of the original report; these data will be described in a new manuscript. The authors sincerely apologize to the readers, reviewers, and editors of Blood for making this honest mistake.

Regulation of HIF-1α signaling and chemoresistance in acute lymphocytic leukemia under hypoxic conditions of the bone marrow microenvironment

Overcoming resistance to chemotherapy is the main therapeutic challenge in the treatment of acute lymphocytic leukemia (ALL). Interactions between leukemia cells and the microenvironment promote leukemia cell survival and confer resistance to chemotherapy. Hypoxia is an integral component of bone marrow (BM) microenvironment. Hypoxia-inducible factor-1α (HIF-1), a key regulator of the cellular response to hypoxia, regulates cell growth and metabolic adaptation to hypoxia. HIF-1α expression, analyzed by Reverse Phase Protein Arrays in 92 specimens from newly diagnosed patients with pre-B-ALL, had a negative prognostic impact on survival (p = 0.0025). Inhibition of HIF-1α expression by locked mRNA antagonist (LNA) promoted chemosensitivity under hypoxic conditions, while pharmacological or genetic stabilization of HIF-1α under normoxia inhibited cell growth and reduced apoptosis induction by chemotherapeutic agents. Co-culture of pre-B ALL or REH cells with BM-derived mesenchymal stem cells (MSC) under hypoxia resulted in further induction of HIF-1α protein and acquisition of the glycolytic phenotype, in part via stroma-induced AKT/mTOR signaling. mTOR blockade with everolimus reduced HIF-1α expression, diminished glucose uptake and glycolytic rate and partially restored the chemosensitivity of ALL cells under hypoxia/stroma co-cultures. Hence, mTOR inhibition or blockade of HIF-1α-mediated signaling may play an important role in chemosensitization of ALL cells under hypoxic conditions of the BM microenvironment.

Differential impact of structurally different anti-diabetic drugs on proliferation and chemosensitivity of acute lymphoblastic leukemia cells

Hyperglycemia during hyper-CVAD chemotherapy is associated with poor outcomes of acute lymphoblastic leukemia (ALL) (Cancer 2004; 100:1179–85). The optimal clinical strategy to manage hyperglycemia during hyper-CVAD is unclear. To examine whether anti-diabetic pharmacotherapy can influence chemosensitivity of ALL cells, we examined the impacts of different anti-diabetic agents on ALL cell lines and patient samples. Pharmacologically achievable concentrations of insulin, aspart and glargine significantly increased the number of ALL cells, and aspart and glargine did so at lower concentrations than human insulin. In contrast, metformin and rosiglitazone significantly decreased the cell number. Human insulin and analogs activated AKT/mTOR signaling and stimulated ALL cell proliferation (as measured by flow cytometric methods), but metformin and rosiglitazone blocked AKT/mTOR signaling and inhibited proliferation. Metformin 500 μM and rosiglitazone 10 μM were found to sensitize Reh cells to daunorubicin, while aspart, glargine and human insulin (all at 1.25 mIU/L) enhanced chemoresistance. Metformin and rosiglitazone enhanced daunorubicin-induced apoptosis, while insulin, aspart and glargine antagonized daunorubicin-induced apoptosis. In addition, metformin increased etoposide-induced and L-asparaginase-induced apoptosis; rosiglitazone increased etoposide-induced and vincristine-induced apoptosis. In conclusion, our results suggest that use of insulins to control hyperglycemia in ALL patients may contribute to anthracycline chemoresistance, while metformin and thiazolidinediones may improve chemosensitivity to anthracycline as well as other chemotherapy drugs through their different impacts on AKT/mTOR signaling in leukemic cells. Our data suggest that the choice of anti-diabetic pharmacotherapy during chemotherapy may influence clinical outcomes in ALL.

Abstract 4793: A novel laser ablation electrospray ionization mass spectrometry (LAESI-MS) platform for biomarker discovery in cancer cells

Abstract Introductory Sentence: LAESI is a novel technique for the direct and rapid interrogation of cancer cells by mass spectrometry (MS) that can be used to identify potential biomarkers in live cell populations. Experimental: Presented in this work, the human leukemic cell line, REH, was cultured under 3 different conditions to represent clinically relevant scenarios. These included media alone or co-culture with human bone marrow or osteoblast cells to represent conditions in which leukemic disease originates and environments in which the therapeutic response of tumors is known to differ. In previous work, REH cells co-cultured with stromal cells have shown increased resistance to chemotherapeutic treatments prompting our interest in evaluating differences in tumor cells exposed to bone marrow microenvironment conditions compared to those deprived of these survival cues. Subsequently, these unique signatures may identify therapeutic targets of interest. To identify potential therapeutic targets, the cells were pelleted using centrifugation, washed twice, and pipetted upon slides for direct LAESI-MS analysis. The LAESI-MS analysis was performed using a Protea DP-1000 LAESI system interfaced to a Waters Synapt G2 mass spectrometer. The mid-IR laser in the DP-1000 had a maximum output energy of 1 mJ, a 200 µm spot size, and an operational frequency of 10 Hz. The MS was continually calibrated using a lockmass of m/z 445.1206 resulting in mass differences typically less than 5 ppm. After LAESI-MS analysis, the data was analyzed using MarkerLynx software and subjected to a principle component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) for identification of potential biomarkers from each treatment. Five replicates of each cell population were analyzed for statistical significance and potential biomarkers were searched against online databases based on accurate mass and confirmed using MS/MS where possible. Summary of Data: The LAESI-MS analysis of the REH cell pellets produced several notable differences in the cell populations that could aid in the identification of biomarkers responsible for the increased resistance to chemotherapeutic treatments. The LAESI-MS analysis was very rapid, requiring less than five minutes of analysis time per cell population to get an average “fingerprint” mass spectrum in addition to MS/MS spectra to aid in metabolite verification. Conclusions: LAESI-MS is a rapid analysis technique that can be used for cancer research as applied here to cell populations. A LAESI-MS analysis requires minimal sample pretreatment with simple washing and pelleting of the cells being the only requirement for the analysis. This method allows the direct analysis of live cells, which can ultimately lead to the determination of biomarkers of disease through the comparison of different cell populations. 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 4793. doi:1538-7445.AM2012-4793

Abstract 1525: Regulation of bone marrow angiogenesis by CXCL12 in the leukemia tumor microenvironment

Abstract While it is well established that angiogenesis is essential for the progression of solid tumors, its role in hematological tumors is more recently becoming fully appreciated. In the current study we have investigated the potential role of leukemic cells in regulating local angiogenesis as well as the modulation of tumor cell CXCL12 expression subsequent to interaction with bone marrow derived osteoblasts as one mechanism by which local vascular rich niches may be created. Human REH acute lymphoblastic leukemia (ALL; ATCC #CRL-8286) increased vessel density when placed on the chorioallantoic membrane (CAM) assay. Conditioned media from these same tumor cells increased human brain microvessel endothelial cell (HBMEC) motility and tube formation suggesting leukemia cell influence on surrounding vasculature. Strikingly, while REH cells express negligible CXCL12 at baseline, the chemokine is consistently induced to easily detectable levels upon co-culture with human osteoblasts indicating one mechanism by which tumor cells within the bone marrow microenvironment may be poised to enhance vascular cell recruitment even more robustly than leukemic cells alone. Preliminary evaluation of primary, patient-derived ALL leukophoresis samples demonstrated a comparable increase in tumor cell CXCL12 indicating this modulation is not limited to an established tumor cell line. Increased expression of CXCL12 by REH leukemic cells was consistent with enhanced chemotaxis of HUVECs toward the co-culture of tumor and osteoblasts when compared to either cell type alone. The inclusion of 5-Aza-deoxycytidine (Aza-C) to REH cells alone resulted in expression of CXCL12 in the absence of osteoblast co-culture, suggesting methylation may play a regulatory role in the suppression of this chemokine. As a site in which metastatic disease is often refractory to standard therapy, in addition to the anatomical niche in which hematopoietic malignancies initiate and most frequently relapse, regulation of local angiogenesis in the bone marrow microenvironment requires further investigation. Our preliminary observations suggest CXCL12 may play an important role in regulating leukemic cell niche angiogenesis, and targeting it could potentially represent a novel application of antagonists currently in clinical trials in an effort to treat refractory leukemia. 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 1525. doi:1538-7445.AM2012-1525

Abstract 4264: NFκB activation in mesenchymal stromal cells induced by leukemia-stroma interaction plays a central role in stroma-mediated chemo-resistance of leukemic cells

Abstract Within the bone marrow (BM) microenvironment, BM mesenchymal stromal cells (BM-MSC) produce cytokines and chemokines and initiate cellular adhesion-mediated signals that tightly regulate normal and malignant hematopoietic cell development. This rich environment serves as a sanctuary for malignant hematopoietic cells, offering protection from chemotherapeutic agents and promoting residual disease with greater potential for developing acquired drug resistance. In this study we utilized gene expression profiling (GEP) as a genome-wide exploratory approach to characterize changes caused by the interaction between leukemic cells and BM-MSC, and how these changes can contribute to the observed microenvironment-mediated chemoresistance. Co-culture of REH, a pre-B-ALL cell line, with normal-donor BM-MSCs for 48 hours upregulated a variety of genes encoding cytokines and chemokines in BM-MSCs such as CXCR6, CCL5, CCL2, IL32, IL34, IL8, IL6, CXCL12, and the VLA4 ligand VCAM-1. These microarray-based GEP findings were confirmed by qRT-PCR. Gene Set Enrichment Analysis (GSEA) implicated activation of NFκB in BM-MSCs as a potential cause of these changes. Similar upregulation of NFκB target genes was found in MSC co-cultured with the AML cell line OCI-AML3, suggesting that NFκB activation is a common consequence of leukemia-stroma interation. Given known paracrine functions of NFκB-regulated target genes such as IL-8, we hypothesized that stromal NFκB activation contributes to stroma-mediated chemo-resistance of leukemic cells. Stable transduction of BM-MSC to overexpress a super-repressor form of IαBβ (IαBβ-SR), to block canonical-pathway NFκB activation in the stromal compartment, significantly reduced the stroma-mediated chemo-resistance to Vincristine (VCR) of REH cells. NFκB inhibition using a specific IKKα inhibitor (MLN120B), affecting both BM-MSC and tumor cells, highly increased the apoptotic effects in leukemia-stroma co-cultures of VCR in REH cells and NALM6, another pre-B-ALL cell line. Similar results were obtained with CDDO-Me, another NFκB inhibitor. Altogether these results indicate that the interaction of leukemic cells with BM stromal cells activates NFκB in the stromal compartment, with subsequent transcriptional upregulation of a number of cytokines and chemokines ultimately favoring the survival of leukemic cells. We propose that targeting NFκB may ameliorate stroma-mediated chemo-resistance and help in eliminating bone-marrow resident leukemic cells. 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 4264. doi:1538-7445.AM2012-4264

Abstract 4641: Hypoxia activated pro-drug TH-302 induces hypoxia-dependent anti-leukemia activity in vitro and in vivo

Abstract Hypoxia in tumors is generally associated with chemoresistance and radioresistance. Hypoxia-inducible factor-1α (HIF-1α) was expressed in the bone marrow in 58% of primary acute myelogenous (AML) cases by immunohistochemistry (n=156). We have recently demonstrated a marked expansion of hypoxia in leukemia bone marrow (Benito PLoSOne 2011) and demonstrated hypoxia promotes chemoresistance in acute leukemias. Normoxic stabilization of HIF-1α using inhibitor of prolyl hydroxylases DMOG, or overexpression through infection with lentiviral non-degradable mutant HIF-1α, significantly diminished apoptosis induced by vincristine and etoposide in REH and Nalm-6 cells. These findings suggest that hypoxia mediates chemoresistance at least in part through HIF-1a. TH-302 is a 2-nitroimidazole of the cytotoxin bromo-isophosphoramide mustard that upon hypoxia-dependent activation induces DNA alkylation, a novel approach to target leukemia. We report the in vitro and in vivo anti leukemia activity of TH-302. In vitro, TH-302 exhibited potent anti-leukemia activity in pre-B ALL (REH, NALM6), AML (OCI-AML3, MOLM-13, KG-1) and CML cell lines (KBM5), with IC50s at 1% O2 ranging from 0.04µM to 2.3µM. The hypoxic cytotoxicity ratios (aerobic IC50/hypoxic IC50) were 11 and 92 in ALL (REH, NALM6) and 14, 80 and 116 in AML (OCI-AML3, KG-1, KBM5). The drug at 5-7.5µM also exhibited specific hypoxia dependent anti-leukemia activity in primary ALL and AML samples (normoxic vs hypoxic specific apoptosis defined as %AnnV positive cells +/− SEM normalized to untreated control cells): ALL-1: 5.1+/−0.5% vs 65.3+/−4.2%; ALL-2: 8.3+/−1.9% vs 28.4+/−6.3%; AML 1: 2.6+/−0.2% vs 40.3+/−1.5%). In in vivo leukemia models TH-302 (50 mg/kg IP 3 times a week for three weeks) reduced the number of circulating AML cells (control, 13.2+/−5.7 x106/ml; TH-302, 2.5+/−2.1 x106/ml) and prolonged survival of NSG mice engrafted with primary AML cells compared to the vehicle treated mice (median survival time: TH-302=75 days; Control=56 days; P=0.003). Finally, anti-leukemia efficacy of TH-302 was tested in vitro in combination with chemotherapy commonly used for treatment of AML (AraC, doxorubicin) and with demethylating agents azacitidine (5-AZA) and decitabine (DAC). Combination TH-302 with AraC or with DAC were synergistic in killing OCI-AML3 and MOLM-13 AML cells under hypoxia (combination index values of 0.4 and 0.67 for AraC plus TH-302 in MOLM13 and OCI-AML3, respectively; 0.3 for DAC plus TH-302 in OCI-AML3). In summary, our findings suggest that hypoxia and HIF-1α constitute important factors in the survival of leukemic blasts within the BM microenvironment. The results support targeting hypoxia with a hypoxia-activated drug such as TH-302 to enhance the efficacy of therapeutic regimens in AML. A Phase 1 clinical study of TH-302 in patients with relapsed/refractory hematologic malignancies is ongoing. 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 4641. doi:1538-7445.AM2012-4641