Primary Human Leukemia Cells Research Articles

Drug Screening of Primary Human Pediatric Pre-B Cell Acute Lymphoblastic Leukemia (pre-B ALL) Cells in Their Stromal Niche

AbstractAbstract 4295Pre-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common leukemia in children and is treatable. However, no in vitro nor in vivo models are available to investigate their pathophysiology other than a number of established cell lines that grow in the absence of any cytokine dependence or stromal interaction. We developed a serum-free MS-5 cell (a murine bone marrow stromal cell line) co-culture system that is capable of expanding human primary pre-B ALL CD34+CD19+ cells in vitro. To define a population of pre-B ALL initiating cells, our study reveals that a sorted CD34bright population displays a slow proliferation and maintains a high % of CD34+ cells. In contrast, CD34dim cells/CD34− cells fraction shows a higher proliferation but expanded cells lost CD34 antigens. A group of alkylating molecules (BO-1055, -1090, 1099, -1393 and -1509) was evaluated for proliferation of the pre-B ALL CD34+ cells, the pre-B ALL CD34− cells, human mesenchymal stem cells (hMSC), murine MSC (MS-5 cells and Op9 cells), human bone marrow derived endothelial cells (BMEC), and human cord blood (CB) CD34+ cells, as well as for a week 5 cobblestones area forming (CAFC) assay with CB CD34+ cells. BO-1055 molecule efficiently suppressed the growth of pre-B ALL CD34+ cells (IC50 = 0.29 μM) and CD34− cells (IC50 = 0.31 μM). In contrast, IC50 of BMEC, MSC, CB CD34+ cells and CAFC are >10, >25, 8, and >5 μM, respectively. Pre-B ALL cells expressing green fluorescent protein (GFP) and luciferase (GFP-Lu-pre-B ALL) were created, and a xenograft of the GFP-Lu-pre-B ALL cells to NOD/SCID IL2R gamma null (NSG) mice was established. The in vivo effect of BO-1055 to the GFP-Lu-pre-B ALL cells in NSG mice is under investigation. Our stromal culture system supports primary pre-B ALL cells and closely recapitulates the growth of primary human pre-B ALL cells in their niche in vivo. Based on this co-culture system, we identified BO-1055 as a potential therapeutic agent with an excellent toxicity window between pre-B ALL cells and normal tissues including BMEC, MSC and hematopoietic progenitor/stem cells. The in vitro stromal co-culture system combined with the xenograft model of GFP-Lu-pre-B ALL cells provides an efficient and powerful method to screen new drugs for pre-B ALL therapy. Disclosures:No relevant conflicts of interest to declare.

Inhibition of Mammalian target of rapamycin in human acute myeloid leukemia cells has diverse effects that depend on the environmental in vitro stress.

Effects of the mTOR inhibitor rapamycin were characterized on in vitro cultured primary human acute myeloid leukemia (AML) cells and five AML cell lines. Constitutive mTOR activation seemed to be a general characteristic of primary AML cells. Increased cellular stress induced by serum deprivation increased both mTOR signaling, lysosomal acidity, and in vitro apoptosis, where lysosomal acidity/apoptosis were independent of increased mTOR signaling. Rapamycin had antiproliferative and proapoptotic effects only for a subset of patients. Proapoptotic effect was detected for AML cell lines only in the presence of serum. Combination of rapamycin with valproic acid, all-trans retinoic acid (ATRA), and NF-κB inhibitors showed no interference with constitutive mTOR activation and mTOR inhibitory effect of rapamycin and no additional proapoptotic effect compared to rapamycin alone. In contrast, dual inhibition of the PI3K-Akt-mTOR pathway by rapamycin plus a PI3K inhibitor induced new functional effects that did not simply reflect a summary of single drug effects. To conclude, (i) pharmacological characterization of PI3K-Akt-mTOR inhibitors requires carefully standardized experimental models, (ii) rapamycin effects differ between patients, and (iii) combined targeting of different steps in this pathway should be further investigated whereas combination of rapamycin with valproic acid, ATRA, or NF-κB inhibitors seems less promising.

Efficacy of the investigational mTOR kinase inhibitor MLN0128/INK128 in models of B-cell acute lymphoblastic leukemia

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase whose activity contributes to leukemia proliferation and survival. Compounds targeting the mTOR active site inhibit rapamycin-resistant functions and have enhanced anticancer activity in mouse models. MLN0128 (formerly known as INK128) is a novel, orally active mTOR kinase inhibitor currently in clinical development. Here, we evaluated MLN0128 in preclinical models of B-cell acute lymphoblastic leukemia (B-ALL). MLN0128 suppressed proliferation of B-ALL cell lines in vitro and reduced colony formation by primary human leukemia cells from adult and pediatric B-ALL patients. MLN0128 also boosted the efficacy of dasatinib (DA) in Philadelphia Chromosome-positive (Ph+) specimens. In a syngeneic mouse model of lymphoid BCR-ABL+ disease, daily oral dosing of MLN0128 rapidly cleared leukemic outgrowth. In primary xenografts of Ph+ B-ALL specimens, MLN0128 significantly enhanced the efficacy of DA. In non-Ph B-ALL xenografts, single agent MLN0128 had a cytostatic effect that was most pronounced in mice with low disease burden. In all in vivo models, MLN0128 was well tolerated and did not suppress endogenous bone marrow proliferation. These findings support the rationale for clinical testing of MLN0128 in both adult and pediatric B-ALL and provide insight towards optimizing therapeutic efficacy of mTOR kinase inhibitors.

Selection for Evi1 activation in myelomonocytic leukemia induced by hyperactive signaling through wild-type NRas

Activation of NRas signaling is frequently found in human myeloid leukemia and can be induced by activating mutations as well as by mutations in receptors or signaling molecules upstream of NRas. To study NRas-induced leukemogenesis, we retrovirally overexpressed wild-type NRas in a murine bone marrow transplantation (BMT) model in C57BL/6J mice. Overexpression of wild-type NRas caused myelomonocytic leukemias ∼3 months after BMT in the majority of mice. A subset of mice (30%) developed malignant histiocytosis similar to mice that received mutationally activated NRas(G12D)-expressing bone marrow. Aberrant Ras signaling was demonstrated in cells expressing mutationally active or wild-type NRas, as increased activation of Erk and Akt was observed in both models. However, more NRas(G12D) were found to be in the activated, GTP-bound state in comparison with wild-type NRas. Consistent with observations reported for primary human myelomonocytic leukemia cells, Stat5 activation was also detected in murine leukemic cells. Furthermore, clonal evolution was detected in NRas wild-type-induced leukemias, including expansion of clones containing activating vector insertions in known oncogenes, such as Evi1 and Prdm16. In vitro cooperation of NRas and Evi1 improved long-term expansion of primary murine bone marrow cells. Evi1-positive cells upregulated Bcl-2 and may, therefore, provide anti-apoptotic signals that collaborate with the NRas-induced proliferative effects. As activation of Evi1 has been shown to coincide with NRAS mutations in human acute myeloid leukemia, our murine model recapitulates crucial events in human leukemogenesis.

Effect of phenylhexyl isothiocyanate on aberrant histone H3 methylation in primary human acute leukemia

BackgroundWe have previously studied the histone acetylation in primary human leukemia cells. However, histone H3 methylation in these cells has not been characterized.MethodsThis study examined the methylation status at histone H3 lysine 4 (H3K4) and histone H3 lysine 9 (H3K9) in primary acute leukemia cells obtained from patients and compared with those in the non-leukemia and healthy cells. We further characterized the effect of phenylhexyl isothiocyanate (PHI), Trichostatin A (TSA), and 5-aza-2’-deoxycytidine (5-Aza) on the cells.ResultsWe found that methylation of histone H3K4 was virtually undetectable, while methylation at H3K9 was significantly higher in primary human leukemia cells. The histone H3K9 hypermethylation and histone H3K4 hypomethylation were observed in both myeloid and lymphoid leukemia cells. PHI was found to be able to normalize the methylation level in the primary leukemia cells. We further showed that PHI was able to enhance the methyltransferase activity of H3K4 and decrease the activity of H3K9 methyltransferase. 5-Aza had similar effect on H3K4, but minimal effect on H3K9, whereas TSA had no effect on H3K4 and H3K9 methyltransferases.ConclusionsThis study revealed opposite methylation level of H3K4 and H3K9 in primary human leukemia cells and demonstrated for the first time that PHI has different effects on the methyltransferases for H3K4 and H3K9.

Abstract 1043: Combined targeting of chromatin modifying enzymes LSD1, histone deacetylases (HDACs) and EZH2 has superior efficacy against human acute myeloid leukemia cells.

Abstract LSD1 or KDM1A is a FAD-dependent demethylase, with homology to amine oxidases. LSD1 demethylates di- and mono-methylated lysine (K)4 on histone H3, reducing the permissive H3K4Me3 chromatin mark. LSD1 inhibition attenuates growth of pluripotent cancer cells with OCT4 and SOX2 expression. LSD1 complexes with HDAC1/2 and Co-REST, and high LSD1 expression confers poor prognosis in cancers. Previous studies have shown that HDAC inhibitors downregulate LSD1 thru Sp1 inhibition. Inhibition of LSD1 leads to increase in H3K4Me3-a permissive mark for gene expression. HCI2509 is an FAD-binding pocket, non-MAOA and MAOB LSD1 inhibitor. In the present studies, we determined the chromatin-modifying and cytotoxic effects of HCI2509 alone and in combination with the pan-histone deacetylase inhibitor, panobinostat (PS) in cultured (HL-60 and OCI-AML3) and primary human acute myeloid leukemia (AML) cells. Treatment with HCI2509 (100 to 500 nM), dose-dependently increased the levels of H3K4Me2 & Me3, p16 and p27, which was associated with inhibition of cell proliferation as measured by a decrease in Ki-67 expression. Chromatin immunoprecipitation followed by PCR demonstrated that treatment with HCI2509 increased the H3K4Me3 mark on the promoters of KLF4, HMOX1, and CDH1 in AML cells. Treatment with HCI2509 also induced C/EBPα expression and features of morphologic differentiation of cultured and primary AML cells. Treatment with HCI2509 (25 mg/kg B.I.W. via IP injection) significantly improved the survival of NOD/SCID mice bearing OCI-AML3- AML xenografts. We have previously reported that treatment with PS (Novartis Pharma) depleted PRC2 complex proteins EZH2, and SUZ12 but also modestly depleted LSD1 expression in AML cells. Co-treatment with PS enhanced the chromatin modifying effects of HCI2509 on K4 of Histone H3 in AML cells. Co-treatment with HCI2509 and PS synergistically induced apoptosis of the cultured AML cells (combination indices, CI <1.0). This was associated with greater induction of p16, and p27. Co-treatment with HCI2509 and PS also induced significantly greater loss of viability of primary AML cells but not of normal CD34+ cells. We have previously reported that treatment with the S-adenosylhomocysteine hydrolase and EZH2 inhibitor, DZNep, dose-dependently depleted EZH2, and SUZ12 levels in cultured and primary AML cells. Combined treatment with HCI2509 enhanced the apoptosis of AML cells induced by DZNep. Taken together, these findings indicate that combined targeted depletion of the level and activity of LSD1 and HDACs by PS and HCI2509, along with PS-mediated depletion of PRC2 proteins, exerts superior and selective cytotoxic activity against AML cells. These findings also support the in vivo testing of combined epigenetic therapies in the treatment of AML. 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 1043. doi:1538-7445.AM2012-1043

Disease‐stabilizing treatment with all‐trans retinoic acid and valproic acid in acute myeloid leukemia: Serum hsp70 and hsp90 levels and serum cytokine profiles are determined by the disease, patient age, and anti‐leukemic treatment

Heat shock protein (HSP) 70 and HSP90 are released by primary human acute myeloid leukemia (AML) cells during stress-induced spontaneous in vitro apoptosis. The AML cells also show constitutive release of several cytokines and the systemic serum levels of several soluble mediators are altered in patients with untreated AML. In the present study, we have investigated serum levels of HSP70/HSP90 and the serum cytokine profiles of patients with untreated AML and patients receiving AML-stabilizing palliative treatment based on all-trans retinoic acid (ATRA) plus valproic acid. Patients with untreated AML showed increased HSP90 levels and a distinct serum cytokine profile when compared with healthy controls, and low pre-therapy HSP90 levels were associated with a prolonged survival during treatment with ATRA + valproic acid + theophyllin. Hierarchical cluster analysis showed a close association between HSP70, HSP90, IL-1 receptor antagonist (IL-1ra), and hepatocyte growth factor (HGF) levels. Furthermore, disease-stabilizing therapy altered the serum-cytokine profile, but the correlations between HSP70/HSP90/IL-1ra/HGF were maintained only when ATRA + valproic acid were combined with theophyllin but not when combined with cytarabine. We conclude that both HSP levels and serum cytokine profiles are altered and may represent possible therapeutic targets or prognostic markers in human AML.

3, 3′-Diindolylmethane Exhibits Antileukemic Activity In Vitro and In Vivo through a Akt-Dependent Process

3,3′-diindolylmethane (DIM), one of the active products derived from Brassica plants, is a promising antitumor agent. The present study indicated that DIM significantly induced apoptosis in U937 human leukemia cells in dose- and time-dependent manners. These events were also noted in other human leukemia cells (Jurkat and HL-60) and primary human leukemia cells (AML) but not in normal bone marrow mononuclear cells. We also found that DIM-induced lethality is associated with caspases activation, myeloid cell leukemia-1 (Mcl-1) down-regulation, p21cip1/waf1 up-regulation, and Akt inactivation accompanied by c-jun NH2-terminal kinase (JNK) activation. Enforced activation of Akt by a constitutively active Akt construct prevented DIM-mediated caspase activation, Mcl-1 down-regulation, JNK activation, and apoptosis. Conversely, DIM lethality was potentiated by the PI3K inhibitor LY294002. Interruption of the JNK pathway by pharmacologic or genetic approaches attenuated DIM-induced caspases activation, Mcl-1 down-regulation, and apoptosis. Lastly, DIM inhibits tumor growth of mouse U937 xenograft, which was related to induction of apoptosis and inactivation of Akt, as well as activation of JNK. Collectively, these findings suggest that DIM induces apoptosis in human leukemia cell lines and primary human leukemia cells, and exhibits antileukemic activity in vivo through Akt inactivation and JNK activation.

Serum Heat Shock Protein Levels and Cytokine Profiles Are Altered In AML Patients Before Treatment and Following Disease Stabilizing Therapy,

Abstract 3599Heat shock proteins (HSPs) maintain cellular homeostasis and function as molecular chaperones assisting protein folding and translocation. Their intracellular levels are up-regulated in response to a wide variety of insults allowing the cell to survive otherwise lethal conditions. Heat shock proteins are also released to the extracellular space and lead to a more efficient cross-presentation to specific T cells and may also initiate release of proinflammatory cytokines, stimulate NK cells and facilitate DC maturation. Extracellular Heat shock protein (HSP) 70 and HSP90 are characteristics of immunogenic apoptosis, and primary human acute myeloid leukemia (AML) cells release these mediators during spontaneous in vitro apoptosis. Primary AML cells also show constitutive release of several cytokines; these heterogeneous mediators interact through local cytokine networks that regulate growth and differentiation of normal as well as malignant hematopoietic cells. In the present study we investigated serum levels of HSP70/HSP90 and the serum cytokine profiles of patients with untreated AML and patients receiving disease-stabilizing treatment based on all-trans retinoic acid (ATRA) plus valproic acid.Serum samples from 82 untreated AML patients, including 42 patients receiving AML-stabilizing therapy, and 20 healthy controls were analysed. HSP and cytokine levels were determined by ELISA and multiplex analyses.Patients with untreated AML showed significantly increased HSP90 levels compared to healthy controls (Mann Whitney U-test, p<0.0001). The serum levels of HSP70 were significantly lower than the HSP90 levels both for the patients and for the healthy controls (p<0.0001), but the HSP70 serum levels did not differ significantly between patients and controls (Fig. 1A). HSP70 and HSP90 serum levels were significantly correlated for the AML patients (Fig. 1B, Spearman, p<0.01, R=0.425). HSP70 levels were not altered during AML-stabilizing palliative treatment, whereas HSP90 levels decreased during treatment with ATRA, valproic acid and low-dose cytarabine. High age and low differentiation of AML cells according to the FAB classification predicted lower HSP90 levels. [Display omitted] AML patients showed an altered serum cytokine profile compared to healthy controls with significantly altered levels of several chemokines, interleukins, growth factors and immunomodulatory cytokines (Tab.1). These cytokine levels were usually increased compared to the healthy controls. Hierarchical cluster analysis of the untreated patients showed a close association between HSP70, HSP90 and Hepatocyte growth factor (HGF) levels, these mediators also showed a significant correlation (Spearman, p<0.01). Furthermore, disease-stabilizing therapy altered the serum cytokine profile, but the profiles differed when ATRA + valproic acid was combined with either theophyllin or low-dose cytarabine. The close association between HSP70, HSP90 and HGF levels was then maintained only in the cytarabine group.Tab.1CytokinePatientsControlsP-valueIMMUNOMODULATORY CYTOKINESTNF-α↑8.4 (<1.5–68.5)4.5 (2.3–13.4)0.016INTERLEUKINSIL-1α↓0.5 (<0.4–7.0)2.0 (2.0–2.9)<0.0001IL-4↓<4.5 (<4.5–53.9)11.8 (5–17.5)<0.0001IL-5↑<0.7 (<0.7–39.1)0.9 (<0.7–0.9)0.0001IL-6↑5.8 (<1.1–204.6)<1.1 (<1.1–7.7)<0.0001IL-10↑1.3 (<0.3–238.0)0.3 (<0.3–0.8)0.0005CHEMOKINESCXCL5↓128.4 (<4.1–3252)1283.5 (341–5307)<0.0001CXCL8/IL-8↑30.5 (<2.0–1103)10.7 (2.2–22.8)0.0003CXCL10↑56.7 (0.5–1958)15.25 (8.5–58.4)<0.0001GROWTH FACTORSHGF↑797.3 (1–9703)262.4 (117.3–729.3)<0.0001GM-CSF↑0.5 (<2.0–17.2)0.9 (0.9–3.6)0.0096We conclude that both HSP levels and serum cytokine profiles are altered in untreated AML, and the modulation of these serum levels by AML-stabilizing chemotherapy may reflect a possible mechanism for the clinical effect of this treatment strategy.The table shows serum levels for 82 AML patients and 20 healthy controls where significant differences were found (Mann-Whitney U-test, p<0.02). The results are presented as the cytokine analyzed, the alteration in the patients (↑ increased, ↓ decreased), levels in patients and healthy controls (median and range). All cytokine concentrations are presented as pg/ml, for cytokine levels not detected the values are set to < the detection limit. Disclosures:No relevant conflicts of interest to declare.

TTC5, a TPR Motif Cofactor Protein Required for Myeloid Leukaemogenesis

Abstract 59Identification of genes and cellular pathways that are critical for the function of leukemia stem cells (LSC) could lead to novel therapeutic approaches. We present data which demonstrates that TTC5 (also called Strap) is required in acute leukaemogenesis. In a targeted lentiviral shRNA knockdown (KD) screen of chromatin regulatory factors, we identified the EP300 co-factor TTC5 as required for survival of human THP-1 MLL-AF9 acute monoblastic leukaemia cells. TTC5 is a tetratricopeptide repeat (TPR) motif protein known to be a stress responsive co-factor and activator of the histone acetyltransferase EP300. Using two separate shRNAs targeting different parts of the transcript, TTC5 KD in THP-1 cells resulted in G1 cell cycle arrest and the initiation of myeloid differentiation (as evidenced by down regulation of KIT/CD117 and up regulation of the monocyte differentiation marker CD36 over 48–72 hours) prior to apoptosis. Colony formation in semi-solid culture was completely abolished. TTC5 KD also induced apoptosis and loss of colony forming potential in other myeloid leukaemia cell lines including Fujioka, Kasumi-1, NB4, HL60, K562 and Mono-Mac-1. In xenotransplantation assays, TTC5 KD THP-1 cells failed to initiate AML in contract to control cells infected with a non-targeting control (NTC) hairpin, which induced short latency AML (range 51–72 days). Likewise, lentivirally infected TTC5 KD primary human acute lymphoblastic leukaemia cells from a patient with a t(4;11) translocation, the cytogenetic hallmark of MLL-AF4, failed to induce lethal leukemia when xenografted into neonatal mice, in contrast to NTC cells which induced lethal ALL in recipients of median latency 117 days. Separately, TTC5 KD induced proliferative arrest, differentiation and death in in vitro analyses of primary human AML cells which exhibited MLL associated translocations or trisomy 11 (frequently associated with a partial tandem duplication of MLL) (n=4 patients). These data suggest a critical role for TTC5 in human leukaemic haematopoiesis.To confirm our conclusions, we extended our investigations in a murine model of human leukaemic haematopoiesis initiated by MLL-AF9. TTC5 KD MLL-AF9 AML cells formed significantly fewer colonies in methylcellulose versus control cells and failed to initiate AML in secondary transplantation experiments versus control cells which induced short latency disease (48 days).To investigate the mechanism by which TTC5 knockdown initiates the cell cycle arrest and myeloid differentiation phenotype observed in THP-1 cells, we performed exon array analyses 48 hours following lentiviral infection. Gene set enrichment analysis (GSEA) demonstrated co-ordinate down regulation of an MLL leukemia stem cell maintenance program as well as genes bound by the transcription factor MYB. In contrast there was co-ordinate up regulation of genes known to be positively regulated by PU.1 or IRF8, two myeloid transcription factors that promote differentiation. These significant transcriptional changes occurred in the absence of change in the levels of MYB or PU.1 transcripts, arguing that TTC5 may be critical in regulating the function of these transcription factors rather than modulating their expression. Published data from the FANTOM4 consortium identified MYB and, to a lesser extent, PU.1 as key transcriptional regulators of CD36 expression (Suzuki et al., 2008, Nature Genetics). We confirmed up regulation of CD36 upon MYB knockdown in THP-1 cells. CD36 up regulation was not observed when either MYC or EP300 was knocked down, suggesting that TTC5 is required for MYB function and loss of TTC5 perturbs MYB activity, initiating a differentiation program. The mechanism by which TTC5 regulates MYB activity is under investigation but appears independent of EP300. We propose that in addition to the role of TTC5 in the stress response, it is a critical cofactor protein that regulates haematopoietic transcription factors and is required for human leukaemogenesis. Disclosures:No relevant conflicts of interest to declare.

In Vitro Culture Reveals Microenvironment-Dependent Growth, Heterogeneity and Hierarchical Structure of Primary Human Pediatric Pre-B Cell Acute Lymphoblastic Leukemia (pre-B ALL) Cells

Abstract 1483In Vitro culture reveals microenvironment-dependent growth, heterogeneity and hierarchical structure of primary human pediatric pre-B cell acute lymphoblastic leukemia (pre-B ALL) cells.J.-H. Shieh1, P. Steinherz2, J Shieh3 and M. A.S. Moore1. 1Moore Laboratory. Cell Biology Program and 2Leukemia and Lymphoma Studies, Department of Pediatrics, Memorial-Sloan Kettering Cancer Center, New York, NY. 3Department of Biology, Brandeis Univ., Waltham, MAPre-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common leukemia in children. Although this pediatric pre-B ALLs are treatable, no in vitro nor in vivo models are available to investigate their pathophysiology other than a number of established cell lines that grow in the absence of any cytokine dependence or stromal interaction. To address this issue, we systemically evaluated the effects of various tissue culture parameters to the growth of primary pre-B ALL cells. A serum-free MS-5 cells (a murine bone marrow stromal cell line) co-culture system is capable of expanding the pre-B ALL CD34+CD19+ cells and supporting their differentiation to CD34−CD19+ B cells. This expansion requires a contact between the stromal cells and the pre-B ALL cells, and is inhibited by fetal bovine serum and IL-6 in a dose-dependent manner. c-Kit ligand and Flt3 ligand can reverse the IL-6 inhibition. Expansion of individual CD34+CD19+ cells revealed a hierarchical structure with respect to CD34 antigen expression and an heterogeneity in cell proliferation. When the pre-B ALL cells were sorted into CD34dim and CD34bright populations, the CD34dim cells were capable of a faster proliferation but gradually lost their CD34 antigen. In contrast, the CD34bright cells were more slowly proliferating and retained their CD34 antigen. We transduced the B-ALL cells with a fusion gene expressing green fluorescent protein (GFP) and luciferase (GFP-Lu-pre-B ALL). These GFP-Lu-pre-B ALL cells display the similar in vitro characteristics and in vivo xenograftment to NOD/SCID IL2R gamma null (NSG) mice as the non-transduced pre-B ALL cells. One hundred, 103, 104 or 105 GFP-LU-pre-B ALL CD34+ cells were i.v. transplanted to NSG mice. Both 104 and 105 cells resulted in the engraftment of the leukemia cells in limbs and cranium as judged by imaging after 6 weeks, and 103 cells engrafted after 13 weeks. When the 105 cells-transplanted mice were sacrificed after 14 weeks, the harvested peripheral blood, spleen (3–4×108cells/spleen) and bone marrow (5−10×106 cells/femur) displayed 2–3%, 51–55% and 75–81% of human CD34+CD19+ cells, respectively. Human CD34−CD19+ cells were 1–2%, 12–13% and 15–21%, respectively. Therefore, our stromal culture system supports leukemic stem cell/leukemia initiating cell proliferation and closely recapitulates the growth of primary human pre-B ALL cells in their niche in vivo, and reveals the heterogeneity and hierarchical structure of human pre-B ALL cells. The in vitro stromal co-culture system combined with the xenograft model of GFP-Lu-pre-B ALL cells provides powerful tools to dissect the pathophysiology of human pre-B ALL, and to screen new drugs for pre-B ALL therapy. Disclosures:No relevant conflicts of interest to declare.

Pharmacological Inhibition or Genetic Knockdown of Kdm1a (Lsd1 or Aof2) Induces Differentiation of MLL Acute Myeloid Leukaemia Stem Cells

Abstract 58Histone demethylases are candidate therapeutic targets in cancer. We analysed a previously published microarray expression dataset of murine acute myeloid leukemias (AML) experimentally initiated with human MLL fusion oncogenes to identify histone demethylases whose expression correlated with the frequency of leukemia stem cells (LSCs). Only expression of Kdm1a correlated positively and significantly (p<0.001) with LSC frequency, a finding confirmed by quantitative PCR of selected leukemias. To investigate whether Kdm1a has a critical role in MLL LSCs, we targeted Kdm1a transcripts for knockdown in murine MLL-AF9 AML cells using lentivirally expressed shRNAs. Knockdown AML cells formed significantly fewer colonies in semi-solid culture compared with AML cells expressing a non-targeting control shRNA (12±7% of control; n=4; p=0.01), and the extent of Kdm1a knockdown correlated significantly (six shRNAs tested) with loss of AML cell colony forming (CFC) potential (p<0.005). Residual Kdm1a knockdown colonies predominantly exhibited “Type 2” morphology (i.e. containing macrophages), a finding confirmed by examination of cytospin preparations. The phenotype was rescued by forced expression of human KDM1A. Kdm1a knockdown MLL-AF9 AML cells were unable to initiate leukemia in secondary transplant assays, in contrast to control cells which readily induced short latency AML. These data demonstrate that Kdm1a is required to prevent differentiation of murine MLL-AF9 AML LSCs.Tranylcypromine (TCP) is a licensed monoamine oxidase inhibitor that inhibits KDM1A, with an IC50 of 5–10uM. Treatment of murine MLL-AF9 AML cells, or human THP1 MLL-AF9 cells, with TCP phenocopied Kdm1a knockdown at a dose close to the KDM1A IC50; both murine and human MLL-AF9 cells exhibited reduced proliferation, fewer colonies and increased macrophage differentiation. Next, we tested the effect of KDM1A knockdown or TCP treatment (25uM) on primary human MLL leukemia cells. KDM1A knockdown reduced proliferation, induced morphological differentiation and induced apoptosis in the two samples of MLL-AF6 leukemia tested (5–20 fold fewer cells remained after a ten day culture versus control cells). Likewise, TCP treatment reduced proliferation 4-fold over five days (MLL-AF6 n=2; MLL-AF10 n=1), blocked CFC formation, reduced the proportion of blasts and increased the proportion of cells with features of monocyte/macrophage differentiation. Of note, addition of 25uM TCP to normal murine KIT+ BM stem and progenitor cells cultured in methylcellulose did not significantly reduce CFC frequency or size or induce macrophage differentiation, suggesting that MLL LSCs are more sensitive than normal BM cells to a given level of KDM1A inhibition.To investigate the mechanism by which KDM1A inhibits a differentiation programme in MLL-AF9 LSCs, we performed exon array analysis of Kdm1a knockdown versus control LSCs 48 hours following lentiviral infection, prior to appearance of morphologically differentiated cells. Down regulated transcriptional regulators included Id2, Hmgb3 and Egr1, but not Hoxa, Meis1 or Myb. Up regulated transcriptional regulators included Irf8. Knockdown of Id2 in murine MLL-AF9 AML cells reduced CFC frequency. In contrast, forced expression of Id2 enhanced CFC frequency both of control MLL-AF9 AML cells and of Kdm1a knockdown LSCs, the latter representing a partial phenotypic rescue of Kdm1a knockdown.These data demonstrate that KDM1A prevents differentiation of MLL LSCs by regulating the relative expression of pro- versus anti-differentiation transcriptional regulators and highlight it as a novel therapeutic target in MLL leukemias. Disclosures:No relevant conflicts of interest to declare.

Targeting Redox Homeostasis As a Means to Selectively Eradicate Primary Human Leukemia Cells,

Abstract 3506Many studies have indicated that induction of oxidative stress is one component of the mechanism by which anti-leukemia agents function. However, little is known about the differences between leukemic and normal redox homeostasis, and whether these agents achieve their selective toxicity to leukemia through specifically targeting leukemic redox homeostasis. Hence, the present study performed a detailed analysis of CD34+ cells derived from normal bone marrow (NBM) or primary acute myelogenous leukemia (AML) specimens. By evaluating the expression profiles of redox regulatory genes, we observed that glutathione peroxidase (GPX1), glutathione reductase (GSR), and several other genes are significantly dysregulated in AML cells. Consistent with these findings, we found a lower GSH/GSSG ratio as well as a lower amount of total glutathione in AML cells compared to normal. In addition, we also found that AML cells display a different spectrum of cell surface thiols. Together, these data suggest that leukemia cells have a distinct redox state maintained by a set of differentially regulated genes in comparison to normal cells.We next tested if parthenolide (PTL), a known ROS-inducer and an effective anti-leukemia agent, targets any of these differences to achieve its leukemia selectivity. We found that PTL directly depletes glutathione in a time and dose dependent manner. However, the amount of glutathione remaining after PTL treatment was less in primary CD34+ AML cells (∼1.5nmol/mg lysate) compared to CD34+ NBM cells (∼5nmol/mg). Thus, the net consequence on glutathione levels is much more severe in AML cells. Importantly, PTL, at the tested dose, selectively kills both AML bulk and stem/progenitor populations but spares normal cells. Given the central role of glutathione in redox homeostasis, we reason the selective toxicity of PTL to leukemia is at least partially because AML cells has a limited pool of glutathione compared to normal. Furthermore, the active moiety of PTL, a α-metheylene-γ-lactone group, predicts it will alkylate cysteine (Cys) residues required for the critical function and stability of several redox proteins. To identify these interactions, we used a biotinylated analogue of PTL to perform a streptavidin-based pull-down assays followed by liquid phase chromatography-mass spectrometry (LC-MS) analysis. We demonstrate that thioredoxin (TXN) and glutathione peroxidase (GPX1), key regulators of cell surface and intracellular redox balance, are direct binding targets of PTL. These binding events were found to at least partially inhibit the enzymatic function of these proteins, contributing to PTL-induced oxidative stress in AML cells. Knowing that GPX1 is already dysregulated in AML cells compared to normal, and considering the increased surface thiol level on AML cells, we hypothesize that these interactions are also important for the selectivity of PTL to AML cells. This is in addition to its ability to exploit the differential pool of glutathione in AML cells. Lastly, PTL induces ROS stress in AML cells as evidenced by redox dye staining, and robust Nrf2-mediated anti-oxidant responses in AML cells. Notably, SLC7A11, a gene that is required for importing cysteine into AML cells for glutathione synthesis, and GCLM which is directly involved in glutathione synthesis are also both up-regulated by PTL.In summary, our studies identify altered GSH/GSSG ratio, decreased glutathione levels, dysregulated expression of GPX1, GSR and other redox regulatory genes, and increased cell surface thiols as critical differences between leukemic and normal redox homeostasis. Our study of PTL indicates that leukemia-specific agents of this nature function by exploiting the differences between glutathione system in leukemic vs. normal cells, and further acting on redox proteins that are already dysregulated in AML cells. These findings have ramifications for the rational design of regimens that manipulate oxidative stress as a means to selectively kill malignant cells. Disclosures:No relevant conflicts of interest to declare.

Novel Dual E-Selectin-CXCR4 Inhibitors Mobilize Human Acute Myeloid Leukemia (AML) Cells in the NODscid IL2Rγc−/− Xenograft and Confer Susceptibility to Cytarabine

Abstract 579High expression of CXCR4, a chemokine receptor for CXCL12 (SDF1), is associated with poor prognosis in AML. CXCR4 inhibitors mobilize AML and enhance chemotherapy sensitivity in laboratory studies. They are currently under study as single inhibitors in combination with standard chemotherapy regimens for induction or salvage in AML. Antibody to CD44, an E-selectin ligand when properly glycosylated [HCELL (Sackstein et al 2004)], eliminates leukemia stem cell engraftment in NODscid animals (Jin et al 2006). High level expression of CD44 contributes to leukemia relapse (Quéré et al 2011). In addition, bcr-abl positive leukemia stem cells are dependent on CD44 for homing to the marrow, (Krause et al 2006) and Nalm6 ALL cell line homes to E-selectin+SDF1+ regions of the bone marrow microvasculature, dependent on both E-selectin and CXCR4 (Sipkins et al 2005).Our preclinical studies demonstrate very high level expression of CD44 (99.8% ± 1.7%) by AML blasts from patients, and by putative CD34+CD38−CD123+ leukemia stem cells (99.8% ± 0.6%). The mean fluorescence intensity (MFI) for CD44 expression by AML blasts is one to two logs higher than the MFI for 16 other adhesion receptors. We also have found that blocking of CD44 inhibits adhesion of AML blasts to a stromal cell line in vitro. Eighty percent of the AML blast samples tested demonstrated binding of E-selectin-IgG chimeric protein by flow cytometry, and nearly all AML blast samples exhibited functional adhesion to immobilized E-selectin. We then tested the hypothesis that functional inhibition of CD44 by an E-selectin inhibitor together with mobilization and inhibition of binding to CXCR4 would enhance chemotherapy sensitivity. To target these overlapping pathways, GMI-1215 and GMI-1257 were each rationally designed to inhibit both E-selectin and CXCR4. GMI-1215 and GMI-1257 inhibit E-selectin in vitro with IC50 values of 4.1μM and 3.6μM, respectively. Both molecules also target CXCR4 as determined by inhibiting antibody (12G5) binding to CXCR4 or inhibiting SDF-1-mediated transwell migration of CCRF-CEM cells (GMI-1215 IC50 = 31nM; GMI-1257 IC50 = 1.1μM, respectively). We have tested these novel dual inhibitors of both E-selectin and CXCR4 in a murine xenograft model. The inhibitor GMI-1257 inhibits E-selectin and SDF-1 mediated chemotaxis in a transwell-based assay for CXCR4 activity. Both E-selectin-CXCR4 bifunctional inhibitors GMI-1215 and GMI-1257 successfully mobilize primary human leukemia cells in the NODscid IL2Rγc−/− mouse. The peak rise occurs at 3 hours after IV injection of 5 mg/kg. After IV injection of GMI-1215, the total white blood count rose from 4.1 × 109/L ±1.1 to 11.4 × 109/L ±0.9 (2.8 fold), and human CD34+ leukemia count from 2.5 × 109/L ±0.8 to 7.7 × 109/L ±1.0 (3.1 fold), p=0.0024 and p=0.0045, respectively. After injection of GMI-1257, the human CD45+ leukemia count rose from 0.08 to 0.27 (3.4 fold). Administration of these inhibitors daily for 3 days, followed by cytarabine 300 mg/kg/day by intraperitoneal injection, 3 hours later each day, reduced circulating leukemia in the xenograft model without affecting recovery of normal mouse neutrophil counts. For example, at 7 days post treatment with GMI-1215, the human CD34+ leukemia count fell by 86%, and at 7 days post treatment with GMI-1257, it fell by 94%. For the control mice that received only cytarabine alone, but not GMIO-1257, the leukemia number fell by only 38% by day 7. Remarkably, by 15 days post treatment with the combination of GMI-1257 and cytarabine, human CD34+ leukemia cells were largely depleted from the bone marrow (by 90%) compared to treatment with cytarabine alone. Thus, these novel dual inhibitors of E-selectin and CXCR4 effectively mobilize leukemia from the marrow and reduce the bone marrow burden of AML in combination with chemotherapy. As both E-selectin and CXCR4 play roles in the sequestration of leukemic cells in the bone marrow, an antagonist that targets both adhesion mechanisms may have added benefits in blocking these interactions and enhancing the efficacy of chemotherapy. Disclosures:Patton:GlycoMimetics, Inc.: Employment, Equity Ownership. Smith:Glycomimetics, Inc.: Employment, Equity Ownership. Sarkar:Glycomimetics, Inc.: Employment, Equity Ownership. Magnani:GlycoMimetics, Inc.: Employment, Equity Ownership. Becker:GlycoMimetics, Inc.: Research Funding.

The surface molecule signature of primary human acute myeloid leukemia (AML) cells is highly associated with NPM1 mutation status

The surface molecule signature of primary human acute myeloid leukemia (AML) cells is highly associated with NPM1 mutation status

Novel copper complexes with potent/synergistic antitumor activity in vivo and promising clinical activity.

e13518 Background: The role of copper in cancer has been subject of intense investigations. Methods: In the current study, we investigated the anticancer properties of novel copper complexes in vitro in various cell lines (e.g., BJAB, Nalm-6, K562) determining DNA fragmentation, proliferation, mitochondrial membrane potential and calcium concentration. Furthermore, the in vivo efficacy of novel copper complexes in different syngenic (B16 melanoma, sarcoma 180, renal cancer RENCA) and xenograft (BJAB lymphoma) tumor models was investigated. Results: The novel copper complexes showed strong apoptotic and anti-proliferative effects in vitro. In comparison with standard cytotoxic drugs an at least two-fold increase in DNA fragmentation on primary human leukemia cells was observed. Synergistic effects up to +129% in lymphoma cells and potent activity in cell lines resistant towards standard cytotoxic drugs (vincristin, daunorubicin and doxorubicin) in cells of leukemia (Nalm-6) and lymphoma (BJAB) was demonstrated. The compounds target the mitonchdrial cyclosporine A-sensitive channels triggering the intrinsic apoptosis pathway. In the syngenic models (B16 melanoma, sarcoma 180) and in a lymphoma xenograft model a significant reduction of tumor weight/volume of 60-90% was shown. Significant anti-metastatic and anti-angiogenic activity was observed in the RENCA model. In investigator-initiated trials 43 patients with a variety of solid and hematological diagnoses (stage IV) were treated orally. Tumor regression of more than 50% was observed in more than 50% of the cases without causing severe side effects. Conclusions: The in vitro and in vivo data as well as the clinical cases and the safety profile position the novel copper complexes as exciting new cancer therapeutics.

Pro-apoptotic activity of α-bisabolol in preclinical models of primary human acute leukemia cells

BackgroundWe previously demonstrated that the plant-derived agent α-bisabolol enters cells via lipid rafts, binds to the pro-apoptotic Bcl-2 family protein BID, and may induce apoptosis. Here we studied the activity of α-bisabolol in acute leukemia cells.MethodsWe tested ex vivo blasts from 42 acute leukemias (14 Philadelphia-negative and 14 Philadelphia-positive B acute lymphoid leukemias, Ph-/Ph+B-ALL; 14 acute myeloid leukemias, AML) for their sensitivity to α-bisabolol in 24-hour dose-response assays. Concentrations and time were chosen based on CD34+, CD33+my and normal peripheral blood cell sensitivity to increasing α-bisabolol concentrations for up to 120 hours.ResultsA clustering analysis of the sensitivity over 24 hours identified three clusters. Cluster 1 (14 ± 5 μM α-bisabolol IC50) included mainly Ph-B-ALL cells. AML cells were split into cluster 2 and 3 (45 ± 7 and 65 ± 5 μM IC50). Ph+B-ALL cells were scattered, but mainly grouped into cluster 2. All leukemias, including 3 imatinib-resistant cases, were eventually responsive, but a subset of B-ALL cells was fairly sensitive to low α-bisabolol concentrations. α-bisabolol acted as a pro-apoptotic agent via a direct damage to mitochondrial integrity, which was responsible for the decrease in NADH-supported state 3 respiration and the disruption of the mitochondrial membrane potential.ConclusionOur study provides the first evidence that α-bisabolol is a pro-apoptotic agent for primary human acute leukemia cells.

Abstract 2157: Cell cycle dependent regulation of c-Myb target gene specificity

Abstract The c-Myb transcription factor is a critical regulator of proliferation and stem cell differentiation, and mutated alleles of c-Myb are oncogenic, but little is known about changes in c-Myb activity during the cell cycle. Previous studies have shown that the specificity of c-Myb depends in part on its DNA binding domain, but also on protein-protein interaction motifs in the C-terminal regulatory domains of the protein. These interactions are likely subject to regulation by post-translational modifications and are modified in oncogenic derivatives of c-Myb, which regulate different sets of target genes than the normal c-Myb protein. The analysis of cell cycle regulated target genes provides an ideal means of following the regulation of c-Myb activity in different subsets of cells that are in distinct cell cycle phases. Using Chromatin Immunoprecipitation (ChIP) assays, we found that c-Myb associated with some target genes only in specific cell cycle stages. For example, with the Cyclin E1 gene promoter only in G2/M phase cells. However, we also found that the levels of c-Myb protein were dramatically affected by treating with some cell cycle blocking agents. To map the association of c-Myb with specific target genes during the cell cycle, we developed a novel Fix-Sort-ChIP approach, in which asynchronously growing cells were fixed with formaldehyde, stained with Hoechst 33342 and separated into different cell cycle fractions by flow sorting, then processed for ChIP assays. This approach showed that the specificity of c-Myb is dramatically different in small subpopulations of cells, for example cells in the G2/M phase of the cell cycle, than in the bulk population. The repositioning of c-Myb during the cell cycle is not due to changes in its expression and also occurs with ectopically expressed, epitope-tagged versions of c-Myb. The repositioning occurs in established cell lines, in primary human CD34+ hematopoietic progenitors and in primary human acute myeloid leukemia cells. Our results suggest that c-Myb specificity is actively regulated during the cell cycle, probably through changes in protein-protein interactions. This sheds new light on the dynamic nature of gene regulation during the cell cycle and on the importance of post-translational modifications and/or oncogenic mutations that can dramatically alter the activity and the specificity of transcription factors like c-Myb. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2157. doi:10.1158/1538-7445.AM2011-2157

Phenethyl isothiocyanate exhibits antileukemic activity in vitro and in vivo by inactivation of Akt and activation of JNK pathways

Effects of phenethyl isothiocyanate (PEITC) have been investigated in human leukemia cells (U937, Jurkat, and HL-60) as well as in primary human acute myeloid leukemia (AML) cells in relation to apoptosis and cell signaling events. Exposure of cells to PEITC resulted in pronounced increase in the activation of caspase-3, -8, -9, cleavage/degradation of PARP, and apoptosis in dose- and time-dependent manners. These events were accompanied by the caspase-independent downregulation of Mcl-1, inactivation of Akt, as well as activation of Jun N-terminal kinase (JNK). Inhibition of PI3K/Akt by LY294002 significantly enhanced PEITC-induced apoptosis. Conversely, enforced activation of Akt by a constitutively active Akt construct markedly abrogated PEITC-mediated JNK activation, Mcl-1 downregulation, caspase activation, and apoptosis, and also interruption of the JNK pathway by pharmacological or genetically (e.g., siRNA) attenuated PEITC-induced apoptosis. Finally, administration of PEITC markedly inhibited tumor growth and induced apoptosis in U937 xenograft model in association with inactivation of Akt, activation of JNK, as well as downregulation of Mcl-1. Taken together, these findings represent a novel mechanism by which agents targeting Akt/JNK/Mcl-1 pathway potentiate PEITC lethality in transformed and primary human leukemia cells and inhibitory activity of tumor growth of U937 xenograft model.

Immunogenic apoptosis in human acute myeloid leukemia (AML): primary human AML cells expose calreticulin and release heat shock protein (HSP) 70 and HSP90 during apoptosis

Several previous studies have demonstrated that both conventional cytotoxic drugs as well as targeted therapeutics can induce apoptosis in primary human acute myelogenous leukemia (AML) cells. However, the apoptotic phenotype of dying AML cells has been less extensively characterized. Even though specific antileukemic immune reactivity is important in AML, especially for allotransplanted patients, it has not been investigated whether dying primary human AML cells show phenotypic characteristics consistent with immunogenic apoptosis [calreticulin exposure, heat shock protein (HSP) release]. We therefore investigated whether in vitro cultured primary human acute myeloid leukemia (AML) cells show calreticulin exposure and HSP70/HSP90 release during spontaneous (stress-induced) apoptosis when cultured in medium alone and when cultured in the presence of antileukemic drugs. Both surface exposure of calreticulin and release of HSP70 and HSP90 was detected but showed a wide variation between patients. This variation was also maintained when the AML cells were cultured in the presence of cytotoxic drugs (cytarabine, daunorubicin, mitomycin), all-trans retinoic acid (ATRA) and valproic acid. Finally, AML cells collected during in vivo ATRA therapy showed increased calreticulin exposure during spontaneous in vitro apoptosis, suggesting that in vivo pharmacotherapy can modulate the apoptotic phenotype. To conclude, apoptotic AML cells can show phenotypic characteristics consistent with immunogenic apoptosis, but there is a wide variation between patients and the level of calreticulin exposure/HSP release seems to depend on individual patient characteristics rather than the apoptosis-inducing agent.