Glucocorticoid-Induced Proliferation and Differentiation of Untreated Pediatric AML Cells

Abstract Abstract 4873 The aim of the present study was to contribute to a more rational use of glucocorticoids (GC) in pediatric AML, by studying the relationship between in vitro GC responsiveness of AML cells, cell biological features and prognosis in 116 pediatric AML patients treated with GC-containing protocols (AML-BFM-87/93, DCOG-ANLL 87/93). In vitro GC resistance and GC-induced proliferation were determined by the 4-days MTT assay, GC-induced differentiation by morphology. Resistance to prednisolone (PRD) was expressed as the LC50 value, the concentration lethal to 50% of leukemic cells. Fifteen percent of the AML samples were somewhat sensitive to GCs (LC50<150 ug/ml), with an overrepresentation in that subgroup of FAB type M1 samples. Six out of 14 AML FAB type M1 samples were somewhat sensitive as compared to only 11 out of 101 non-FAB type M1 samples (p=0.002). However, none of the AML samples were highly sensitive to GC (LC50 < 0.1 ug/ml), in contrast to what is commonly observed in ALL cells (Kaspers et al., Blood 1998). Moreover, GC-induced AML cell proliferation was observed in 26% of samples, more frequently in FAB type M5 (p=0.01). Patients whose AML cells proliferated in vitro upon GC exposure had a lower 10-year event-free survival (32 % SE 5.5% versus 49% SE 8.5%, p=0.04). Such a difference was not found for patients with or without spontaneous proliferation. GC-induced differentiation was not observed in vitro. Multivariate analysis including BFM risk-group classification, FLT3 internal tandem duplications (FLT3/ITD) and GC induced proliferation, showed that only FLT3/ITD was an independent prognostic factor (P = 0.01). In conclusion, GCs are rarely highly cytotoxic towards AML cells, but do induce proliferation in a significant part of AML samples. That phenomenon is undesirable, and it is associated, though not independently, with a worse treatment outcome. We thus advice to use GC as antileukemic or anti-emetic drug with great reluctance in pediatric AML. Disclosures: No relevant conflicts of interest to declare.

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In Vitro Effect Of VPA Together With ATRA and Ara-C On Proliferation and Viability Of Pediatric AML Cell Line THP-1

Blood ◽

10.1182/blood.v122.21.4924.4924 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4924-4924

Author(s):

Sema Anak ◽

Ayca Gul Kanbay ◽

Cagri Gulec

Keyword(s):

Cell Line ◽

Animal Studies ◽

Conflicts Of Interest ◽

Bipolar Disorders ◽

Mood Stabilizer ◽

Leukemic Cells ◽

Pcr Method ◽

Pediatric Aml ◽

Vitro Effect

Abstract In addition to its HDAC inhibitory property, Valproic acid is also known as anticonvulsant agent and mood stabilizer in the treatment of bipolar disorders. Due to its HDAC inhibitory activity and its safety in long-term usage, VPA is presumed to be a good candidate for cancer treatment. It is known that VPA induces apoptosis in leukemic cells, while not in normal cells. VPA is reported as an effective agent in treatment of pediatric AML in clinical studies and is also well tolerated in children. In this study, the in vitro effect of the combination of HDAC inhibitor VPA with Ara-C and ATRA which are used in AML therapy, is investigated on AML cells. For this purpose, the effect of VPA, Ara-C and ATRA on proliferation of AML cell line THP-1 is tested in cell culture condition. To assess the effect on cell proliferation, p21 expression was measured by RT-PCR method. The use of VPA alone, did not affect the cell viability, while increasing the expression of the p21 gene. VPA in combination with Ara-C, increased the expression of p21 gene more than the other combinations. Thus it is determined that the p21 gene expression is higher as a result of known cell cycle stops. In this study, the understanding of how effective is VPA together with ATRA and/or Ara-C on AML cells, might be a good start for animal studies and clinical trials as a remarkable data for the development of new chemotherapeutic protocols. Disclosures: No relevant conflicts of interest to declare.

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Glucocorticoid-Induced Proliferation and Lack of Differentation in Untreated Pediatric Acute Myeloid Leukemic Blasts

Blood ◽

10.1182/blood.v126.23.4843.4843 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4843-4843

Author(s):

Kim Klein ◽

Eric Haarman ◽

Valerie De Haas ◽

C. Michel Zwaan ◽

Ursula Creutzig ◽

...

Keyword(s):

Cell Survival ◽

Leukemic Cell ◽

Tetrazolium Salt ◽

Induced Differentiation ◽

Highly Sensitive ◽

Flt3 Mutations ◽

Pediatric Aml ◽

Acute Myeloid

Abstract Background Although most pediatric acute myeloid leukemia (AML) protocols no longer prescribe glucocorticoids (GCs) as part of antileukemic treatment, several low-income countries may still rely on them. Furthermore these agents are globally being used in the prevention or treatment of chemotherapy-induced nausea, or in the treatment of acute respiratory distress syndrome in children with leukemia. Therefore, to study potential effects of GCs on AML cells remains important. The aim of this study was to evaluate the in vitro responsiveness of pediatric AML cells to GCs in relation to cell biological features. Methods In vitro drug resistance or potential proliferation upon GCs (prednisone) was determined using a methyl-thiazol-tetrazolium salt (MTT)-assay. Samples with more than 70% blasts at day 4 in the control wells and a control optical density (OD) >0.050 units were used to calculate the LC50-value, i.e. the PRD concentration lethal to 50% of the AML blasts. Samples were classified as highly sensitive (LC50 PRD ≤0.1 µg/ml), intermediately sensitive (LC50 PRD >0.1-150 µg/ml) or resistant (LC50 PRD ≥150 µg/ml). PRD-induced leukemic cell proliferation was defined as a leukemic cell survival (LCS=(OD drug-exposed well)/(mean OD control wells)) of >105% plus 2 times the standard deviation of the control cell survival in the PRD containing wells. Potential glucocorticoid (GC)-induced differentiation was evaluated using basic morphology. To validate the overall effects of GCs, a subset of randomly selected samples was tested for both prednisone and dexamethasone (DXM). Samples of patients treated according to (at that time) GC-containing protocols, provided by the BFM-Germany and the DCOG, were used to evaluate associations between in vitro effects and clinical outcome, both at an univariate and multivariate level. Within these protocols GCs were only administered during consolidation. Results Approximately 70% (n=117) of the 167 available pediatric AML samples could be evaluated for GC resistance and GC-induced proliferation. None of the samples were highly sensitive and only 15% (n=17) were intermediately sensitive to the lytic action of GCs. FAB type M1 samples were significantly more often intermediately sensitive than other FAB types (43% of the M1 samples versus 0-13% in FAB type M0, and M2-M6 samples) (P=0.006). FAB type M7 samples were more intermediately sensitive as well (67%, n=2), but sample numbers were small. GC-induced differentiation was not observed after evaluating 10 unselected samples. Contrary, 27% (n=32) of the samples showed proliferation upon GC-exposure. GC-induced proliferation occurred independently from spontaneous proliferation. Samples with FAB type M5 or activating FLT3 mutations were significantly more prone to this phenomenon: 63% (n=12) of the M5 samples compared to 0-33% of the other samples (P<0.001), and 60% (n=6) of the FLT3itd-positive samples versus 23% (n=22) of the negative samples (P=0.011) showed GC-induced proliferation. There were no significant differences in GC-induced proliferation between samples with favorable or other cytogenetics. The results for the subset of samples tested for both PRD and DXM were similar with regard to GC-resistance, GC-induced proliferation, and GC-differentiation. Probabilities of relapse free survival (pRFS) were lower for patients with GC-induced proliferation at a borderline significant level (P=0.045, univariately). However, in a multivariable COX model, including white blood cell count, cytogenetics, and FLT3 status an independent association between inferior pRFS and GC-induced resistance or GC-induced proliferation could not be confirmed. Conclusion Assuming that the proliferation induced in vitro is paralleled in vivo, it is tempting to speculate that GCs given during consolidation treatment may have an adverse effect on any present minimal residual leukemic cells, and that such exposure may increase the risk of relapse in a significant subset of patients. Although we did not study this in a prospective or in vivo design, given the lack of GC-induced differentiation effects, and the potential of GC-induced proliferation in this study - with at best unknown clinical consequences - the use of GCs in pediatric AML patients should be discouraged in general, and especially in subgroups with FAB type M5 or activating FLT3 mutations. Disclosures Kaspers: Galen Pharmaceuticals: Consultancy; Boehringer Ingelheim: Consultancy; Celgene: Consultancy.

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Leukemia of non-T lineage natural killer cells

Blood ◽

10.1182/blood.v72.5.1701.1701 ◽

1988 ◽

Vol 72 (5) ◽

pp. 1701-1707 ◽

Cited By ~ 35

Author(s):

W Sheridan ◽

EF Winton ◽

WC Chan ◽

DS Gordon ◽

WR Vogler ◽

...

Keyword(s):

Natural Killer ◽

Leukemic Cell ◽

High Rate ◽

Leukemic Cells ◽

Pokeweed Mitogen ◽

Intermittent Fever ◽

Electron Microscopic ◽

Cold Urticaria ◽

Spontaneous Proliferation

Abstract An unusual case of an aggressive leukemia of natural killer (NK) cells occurred in a 65-year-old male. Clinical characteristics of this case included hepatosplenomegaly, ascites, marrow infiltrate with leukemic cells, and a WBC up to 82.8 X 10(9) before therapy. One year before his presentation he had been noted to have a WBC of 12.1 X 10(9) with 78% lymphocytes, and 6 months before had noted intermittent fever and weight loss. He and his brother had well documented hereditary cold urticaria. The patient was treated with a modification of ProMACE CYTABOM regimen and had prompt regression of the leukemia with associated acute tumor lysis. Renal, hepatic, and marrow failure predominated during a terminal course that ended 22 days after therapy was commenced, and at autopsy there was no evidence for leukemic cell infiltrate in the liver, spleen or marrow. The leukemic cells were large granular lymphocytes by light and electron microscopic criteria, and had the following immunophenotype: CD2+, DR+, Leu7+, NKH1+, CD11+, CD3-, CD5-, CD4-, CD8-, CD16-. The cells displayed high antibody- dependent cell-mediated cytotoxicity (ADCC) and NK activity, and had a high rate of spontaneous proliferation in vitro that was not augmented by phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM). Southern analysis of DNA from leukemic cells revealed normal germline arrangements for the beta and gamma chains of the T cell antigen receptor and immunoglobulin heavy chain genes. The majority of metaphases were clonally abnormal revealing consistent rearrangements involving extra material attached to the long arms of chromosomes 5 and 11.

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Common and pre-B acute lymphoblastic leukemia cells express interleukin 2 receptors, and interleukin 2 stimulates in vitro colony formation

Blood ◽

10.1182/blood.v66.3.556.556 ◽

1985 ◽

Vol 66 (3) ◽

pp. 556-561 ◽

Cited By ~ 44

Author(s):

I Touw ◽

R Delwel ◽

R Bolhuis ◽

G van Zanen ◽

B Lowenberg

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Colony Formation ◽

B Lymphocyte ◽

Lymphoblastic Leukemia ◽

Interleukin 2 ◽

Colony Growth ◽

Leukemic Cells ◽

In Vitro Proliferation ◽

Proliferation And Differentiation

Abstract The role of interleukin 2 (IL 2) as a possible regulator of in vitro proliferation and differentiation of non-T acute lymphoblastic leukemia (ALL) cells was investigated. For this purpose, leukemic cells from the blood or bone marrow of eight untreated patients with common or pre-B ALL were analyzed using the anti-Tac monoclonal antibody (reactive with the IL 2 receptor) in indirect immunofluorescence. The receptors for IL 2, which were initially absent from the cell surface, were induced on high percentages of the ALL cells after the in vitro exposure to the lectin phytohemagglutinin or the phorbol ester 12-O- tetradecanoylphorbol-13-acetate in six patients, suggesting that the cells had become sensitive to IL 2. In colony cultures to which feeder leukocytes and IL 2 had been added, colony growth was obtained in five of eight cases. Whereas the cells from one patient formed colonies in the absence of exogenous stimuli, the cells from others were dependent on the addition of feeder leukocytes plus IL 2. In the latter cases, feeder leukocytes alone, releasing some IL 2, stimulated growth suboptimally at different cell concentrations. Their stimulative effect was significantly enhanced when leukocyte-derived IL 2 or pure recombinant IL 2 was supplemented. Alone, IL 2 (up to 500 U/mL) did not support colony formation. Apparently, IL 2 and feeder leukocytes are both required for the induction of colonies in these cases of ALL. From cell sorting of fluorescent anti-common ALL antigen (CALLA) stained cells it appeared that colonies descended from cells with high as well as low or negative CALLA expression. Immunophenotyping demonstrated the presence of the original leukemia markers on colony cells, but was not indicative of maturation of ALL toward more differentiated B cells. We suggest that IL 2 can stimulate the in vitro proliferation of certain neoplastic B lymphocyte progenitors.

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Predominant Complement Mediated Lysis of B-CLL Cells by Therapeutic MAbs Rituximab and Campath-1H in Whole Blood Assays.

Blood ◽

10.1182/blood.v114.22.3445.3445 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3445-3445 ◽

Cited By ~ 1

Author(s):

Josee Golay ◽

Luca Bologna ◽

Elisa Gotti ◽

Alessandro Rambaldi ◽

Renato Bassan ◽

...

Keyword(s):

Target Cell ◽

Whole Blood ◽

Conflicts Of Interest ◽

Leukemic Cells ◽

Human Igg ◽

Blood Samples ◽

Expression Levels ◽

Cd20 Expression

Abstract Abstract 3445 Poster Board III-333 The mechanism of action of unconjugated MAbs such as Rituximab and Campath-1H in vivo is still a matter of debate. Most in vitro assays with antibodies rely upon purified effector cells or proteins taken outside their natural context, and on target cell lines rather than patients cells. In order to analyse the activity of therapeutic MAbs on circulating leukemic cells in more physiological conditions and in a system the least manipulated as possible, we have set up a whole blood assays using Rituximab and Campath-1H. Peripheral blood samples were drawn from B-CLL patients or normal donors in sodium citrate and antibodies were directly added at different concentrations. We first demonstrated that neither apoptosis, induced by cross-linked anti-CD20 antibody, nor complement mediated cytotoxicity (CDC) induced by Campath-1H or Rituximab were significantly inhibited by citrate used at the standard concentration (0.1 M). We then performed a number of experiments using whole blood samples in citrate, into which increasing concentrations of Rituximab or Campath-1H were added. Lysis was analysed by FACS analysis after different incubation times at 37°C. We observed that Campath-1H very rapidly and efficiently lysed normal B cells or B-CLL targets in vitro in whole blood: maximal lysis was reached within 4 hours and was observed already with 1 and 10 μg/ml antibody (61 %), even though it was still more effective at 25 or 50 μg/ml (up to 90 % lysis). 25 μg/ml is known to be reached in the circulation after 30mg infusions of the antibody 3 times a week. Lysis by Campath-1H was fully complement dependent since it was inhibited by 90% in presence of excess blocking anti-C5 antibody Eculizumab (200 μg/ml). Eculizumab alone in contrast had no effect on cell viability. We then analysed the efficacy of increasing concentrations of Rituximab in the same assay conditions. We observed in general a much reduced lysis with Rituximab compared to Campath-1H, even using antibody up to 200 μg/ml, a concentration that is reached in the circulation after standard 375 mg/m2 administration of the antibody once a week. Lysis showed also slower kinetics, with limited lysis at 4 hours (mean 6.4%) and maximal lysis with Rituximab reached only after 24 hours incubation (mean 18.8%). Also in this case, target cell death was inhibited by at least 90% in presence of Eculizumab, suggesting a major role of complement. Lysis by Rituximab correlated directly with CD20 expression levels (R=0.8) in 13 B-CLL samples analysed, as expected for a mechanism complement dependent. Indeed a mean 29.3% and 73.2% killing could be observed in the two CD20 bright B-CLL, at 4 and 24 hours respectively, whereas a mean of 3.1% and 10.9% lysis was observed in the 11 low-intermediate CD20 samples analysed at the same time points. These data in whole blood confirm our previously published results on the role of CD20 expression levels in CDC of isolated B-CLL cells (Golay et al., Blood 98, 3383-3389, 2001). In contrast to CDC and apoptosis, ADCC was strongly inhibited by citrate as well as several anti-coagulants tested and therefore could not be analysed in this type of assay. Nonetheless in B-CLL samples, NK cells were below detection limit (<0.1%) in most cases analysed, suggesting that ADCC in the circulation is not a major mechanism of lysis in this disease subtype. Finally we determined the effect of citrate on phagocytosis mediated by Rituximab and in vitro differentiated human macrophages. Phagocytosis could be observed in presence of 0.1M citrate (31%, compared to 44% in absence of citrate). Phagocytosis of B-CLL in whole blood was therefore analysed by layering samples directly onto the macrophages. We observed that phagocytosis of B-CLL targets in whole blood was very low (less than 1% over background) compared to a mean of 47% for purified B-CLL targets phagocytosed in normal culture medium. Phagocytosis in whole blood was low presumably due to the presence of high concentration of human IgG in whole blood since as low as 50 μg/ml human IgG is known to inhibit phagocytosis by 90%. We conclude that the major activity of Campath-1H and Rituximab in the circulation is through complement. Apoptosis, ADCC and phagocytosis appear to play a marginal role in this context but may become more important in tissues. The method presented could be used to rapidly screen novel antibodies for their efficacy through either as apoptosis or CDC directly on unmanipulated patients material. Disclosures No relevant conflicts of interest to declare.

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Oral Supplementation with Omega3 Fatty Acids Inhibits NFkB Activation In Chronic Lymphocytic Leukemia (CLL) Cells.

Blood ◽

10.1182/blood.v116.21.4607.4607 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4607-4607

Author(s):

Oscar F. F Ballester ◽

Johannes Fahrmann ◽

Theodore Witte ◽

Gabriela Ballester ◽

W. Elaine Hardman

Keyword(s):

Conflicts Of Interest ◽

Early Stage ◽

Gradient Centrifugation ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Dependent Manner ◽

Red Cell ◽

Omega 3 ◽

Dose Dependent

Abstract Abstract 4607 Introduction: Nuclear factor kappa B (NFkB) is a critical transcription factor involved in the growth and survival of CLL cells. NFkB is recognized as an important target for the development of novel therapies for the treatment of various malignancies. In vitro and in experimental animal models, OMEGA-3 fatty acid (O3FA) supplementation has been shown to inhibit NFkB activity. Patients and Methods: Patients with early stage CLL (Rai stages 0-II) who required no therapy, where accrued to this phase I-II trial. O3FA supplements were given for a total of 12 months at doses ranging from 2250 mg (EPA plus DHA), escalated to 4500 mg and 6750 mg per day as tolerated. NFkB activity was measured in peripheral blood samples after separation of mononuclear cell by gradient centrifugation and expressed as luminescence units/μ g of protein. Baseline and multiple serial samples were obtained during the study period. In-vitro cytotoxicity assays to doxorubicin were conducted using standard LD50 methods. Compliance was monitored by analysis of red cell and lymphocyte membrane lipid composition by gas chromatography. Results: Fifteen patients have been accrued to the trial, 8 of them have currently completed the planned 12 months of the study period. No significant clinical changes in disease activity were noted. O3FA was well tolerated. Supplementation resulted in a dose-dependent increase of O3FA composition of red cell and lymphocyte membranes in a dose dependent manner. At baseline, CLL patients had NFkB above the range observed in normal controls (2.05 × 104 to 2.32 × 105 NFkB lum units/μ g). The median value in CLL patients at baseline was 11.60 × 106 NFkB lum units/μ g (range 0.9 × 105 to 23.12 × 106). Among 5 patients with the highest baseline levels of NFkB, a decrease in NFkB activity ranging from 0.02 to 0.19 of the baseline value, was noted at the 2 higher doses of O3FA supplementation. Similar results were seen in patients with relatively lower levels of baseline NFkB activity (0.9 × 105 to 2.96 × 106 lum units/μ g). In vitro, significant doxorubicin cytotoxicity (>50%) was noted in samples obtained during supplementation, at μ gM concentrations which produced no detectable cell kill in baseline samples. Conclusions: O3FA supplementation resulted in significant inhibition of NFkB activity in leukemic cells from patients with CLL. In-vitro, after O3FA supplementation CLL cells became more sensitive to doxorubicin. Preliminary analysis of whole genome micro arrays revealed significant down-regulation of multiple genes associated with O3FA supplementation. Disclosures: No relevant conflicts of interest to declare.

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Increased CD39 Expression on CD4+ T-Lymphocytes Has Clinical and Prognostic Significance in Chronic Lymphocytic Leukemia,

Blood ◽

10.1182/blood.v118.21.3895.3895 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3895-3895

Author(s):

Yair Herishanu ◽

Inbal Hazan-Hallevi ◽

Sigi Kay ◽

Varda Deutsch ◽

Aaron Polliack ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Prognostic Significance ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Anti Inflammatory

Abstract Abstract 3895 Chronic lymphocytic leukemia (CLL) cells depend on their microenvironment for proliferation and survival. Ectonucleotidase CD39 has anti-inflammatory properties as it hydrolyzes pro-inflammatory extra-cellular ATP, generates anti-inflammatory adenosine and also protects regulatory T cells from ATP-induced cell death. In this study we investigated the clinical significance of CD39 expression on CD4+T-cells in 45 patients with CLL as well as its compartmental regulation and explored the possible mechanisms for its induction. Compared to healthy individuals, CD4+CD39+ lymphocytes were increased in the peripheral blood of patients with CLL (4.6%±2.28 vs. 17.3%±12.49, respectively, p=0.004), and correlated with advanced stage of disease (9.72%±5.76, 18.15%±12.03 and 25.90%±16.34, of CD4+ lymphocytes, in patients with Rai stages 0, 1+2 and 3+4, respectively, p=0.019). CD4+CD39+ cells were also higher in patients with CLL who needed therapeutic intervention (untreated; 12.99%±10.63 vs treated; 22.21%±12.88, p=0.01) and in those who were ZAP70+ or had b2-microglobulin levels>3g/L. There were more CD4+CD39+ lymphocytes in the bone marrow compartment (22.25%±16.16) than in the peripheral blood (16.60%±15.84, p=0.009). In-vitro studies showed that CD39 can be induced on CD4+cells by exposure to ATP or indirectly, following B-cell receptor (BCR) engagement (CD4+CD39+ lymphocytes increased by 1.56 fold, in the BCR engaged samples compared to their paired controls; 20.27%±11.3 vs. 13%±9.42, respectively, p=0.0006). Conclusions: Increased CD39 expression on CD4+ T-lymphocytes in CLL associates with an aggressive disease. This may reflect the ability of the leukemic cells to suppress the surrounding immune environment, and contribute to a poorer prognosis. CD39+ may also serve as a future target for the development of novel therapies with immune modulating anti–tumor agents in CLL. Disclosures: No relevant conflicts of interest to declare.

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Enantiomer-Specific Transformation by 2HG Is Linked to Opposing Effects on α-Ketoglutarate-Dependent Dioxygenases

Blood ◽

10.1182/blood.v118.21.lba-4.bld0076_p2_lba-4 ◽

2011 ◽

Vol 118 (21) ◽

pp. LBA-4-LBA-4

Author(s):

Julie-Aurore Losman ◽

Sungwoo Lee ◽

Peppi Koivunen ◽

Ryan E. Looper ◽

William G. Kaelin

Keyword(s):

Growth Factor ◽

Cancer Cell ◽

Leukemic Cells ◽

Dependent Manner ◽

Hydroxylase Activity ◽

Induced Differentiation ◽

Gm Csf ◽

Mutant Idh1 ◽

Opposing Effects

Abstract LBA-4 Somatic mutations in IDH1 and IDH2 occur frequently in clonal myeloid disorders and result in the neomorphic ability of IDH to convert α-ketoglutarate (2-OG) to the R-enantiomer of 2-hydroxyglutarate (R-2HG) (Dang, et al Nature 462: 739, 2009). 2OG is an essential cofactor for many metabolic enzymes, including the TET family of 5-methylcytosine hydroxylases and the EglN family of prolyl-4-hydroxylases, and 2HG has been shown to inhibit several 2OG-dependent dioxygenases in vitro, including TET2 (Xu, et al Cancer Cell 19: 17, 2011; Figueroa, et al Cancer Cell 18: 1, 2010). We recently showed that the (S) enantiomer of 2HG (S-2HG), but not the (R) enantiomer of 2HG (R-2HG), inhibits the EglN prolyl-4-hydroxylases (Koivunen, et al. Submitted for publication). Moreover, we found that R-2HG can act as a cofactor to promote the hydroxylase activity of EglN1, EglN2 and EglN3. We hypothesized that the qualitatively different effects of R- and S-2HG on the EglN prolyl-4-hydroxylases might influence their transforming activities. In order to elucidate the role of mutant IDH, and R- and S-2HG, in myeloid leukemia, we developed a myeloid transformation assay using TF-1 cells. TF-1 is a human erythroleukemia cell line that requires GM-CSF for growth and undergoes erythrocytic differentiation when stimulated with erythropoietin (EPO). We expressed wild-type IDH1 (WTIDH1), a tumor-derived mutant IDH1 (IDH1R132H), or a catalytically inactive IDH1R132H variant (IDH1R132H/3DN) in TF-1 cells. As expected, cells expressing IDH1R132H, but not cells expressing WTIDH1 or IDH1R132H/3DN, had dramatically elevated levels of 2HG. Furthermore, we found that expression of IDH1R132H, but not WTIDH1 or IDH1R132H/3DN, conferred growth factor-independence to TF-1 cells (Figure 1a), and blocked their EPO-induced differentiation (Figure 1b). In order to determine whether transformation of TF-1 cells by IDH1R132H is mediated by 2HG, we treated TF-1 cells with cell-permeable esterified R-2HG or S-2HG. R-2HG recapitulated the growth and differentiation phenotypes of IDH1R132H expression in a dose-dependent manner. In contrast, S-2HG did not induce these phenotypes at any concentration tested. Next, we examined the effect of loss of TET2 on TF-1 cells. We infected TF-1 cells with shRNAs targeting TET1 or TET2 and found that knockdown of TET2, but not TET1, induced growth factor-independence and blocked EPO-induced differentiation similarly to expression of IDH1R132H or treatment with R-2HG. Interestingly, we found that transformation by IDH1R132H and TET2 knockdown were reversed by inhibition of EglN1 (Figure 2), suggesting that R-2HG, but not S-2HG, transforms leukemic cells by inhibiting targets such as TET2 while preserving, and possibly enhancing, EglN activity. These findings further suggest that therapeutic targeting of EglN prolyl-4-hydroxylase activity might be effective in the treatment of IDH1-mutant and TET2-mutant myeloid leukemias. Disclosures: Kaelin: Fibrogen: Consultancy, Equity Ownership.

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

Blood ◽

10.1182/blood.v120.21.1506.1506 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1506-1506

Author(s):

Marika Masselli ◽

Serena Pillozzi ◽

Massimo D'Amico ◽

Luca Gasparoli ◽

Olivia Crociani ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Map Kinases ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Significant Proportion ◽

Leukemic Cells ◽

K Channel

Abstract Abstract 1506 Although 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.

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Unique Effects of p53−/− Leukemic Cells On Mesenchymal Stromal Cell Gene Expression Profile in Vitro

Blood ◽

10.1182/blood.v120.21.3468.3468 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3468-3468

Author(s):

Xiaoyang Ling ◽

Ye Chen ◽

Peter P. Ruvolo ◽

Vivian Ruvolo ◽

Zhiqiang Wang ◽

...

Keyword(s):

Gene Expression ◽

Cell Biology ◽

Stromal Cell ◽

Conflicts Of Interest ◽

Leukemic Cells ◽

Bone Marrow Niche ◽

In Vivo Experiments ◽

Cell Gene Expression

Abstract Abstract 3468 Mesenchymal stromal cells (MSC) participate in the generation of the microenvironmental bone marrow niche which protects normal and leukemic stem cells from injuries, including chemotherapy. MSC produce numerous factors that aid in this function; however, little is known about how leukemic cells affect MSC. In this study, paired murine AML cells, MLL/ENL/FIT3-ITD/p53−/− and MLL/ENL/FIT3-ITD/p53wt, originally derived from C57BL/6 mice (Zuber et al. Genes & Dev. 2009), were co-cultured with MSC from the same strain. After 48 hrs, MSC were isolated by FACS sorting using CD45−/PDGFr+ as markers. Total RNA was profiled on Illumina WG6 mouse whole-genome bead arrays by standard procedures. The significance analysis of microarrays (SAM) method identified 429 differentially-expressed genes (DEG) whose expression in MSC differed significantly (false discovery rate, 10%) in co-cultures with p53−/− (C78) vs. p53wt (C147) leukemic cells. Differences in these DEG were highly consistent in replicates (Figure 1). The results demonstrate that: 1) p53 status (p53−/− vs. p53wt) of AML cells affects GEP patterns in co-cultured MSC. Comparison of the GEP in MSC co-cultured with p53−/− (78) or p53wt (147) (Fig 1) identified the following 5 genes that showed the most significant differences (up- or down-regulated): up-regulated: WNT16, WNT5, IGFBp5, GCNT1, ATP1B1; down-regulated: NOS2, DCN, CCL7, CCL2, CAR9, CCL4. These were selected for qPCR validation, and the results confirmed the array data. In addition, immunohistochemical staining showed that WNT16 was up-regulated in MSC co-cultured with p53wt leukemic cells. In addition, CXCL5 was found up-regulated in MSC co-cultured with p53−/− leukemic cells. These results were consistent with the GEP data. 2) Leukemic cells alter MSC Signaling proteins in vitro: Western blotting showed that Stat3, Akt, PTEN, CXCL5 and HIF-1α were up- regulated in MSC co-cultured with p53−/− leukemic cells as compared to p53wt leukemic cells (48 hrs). Additional analyses showed that the downstream targets of HIF-1α, VEGFa and VEGFc, but not VEGFb, were up-regulated. Taken together, these results suggest that 1) leukemic cells with different p53 genetic background co-cultured with normal MSC have profoundly differential effects on GEP of normal MSC; 2) MSC co-cultured with p53−/− leukemic cells resulted in increased levels of onco-proteins such as Akt and HIF-1α when compared to MSC co-cultured with p53wt leukemic cells. Results suggest, for the first time, that the genetics of leukemic cells determines gene expression in co-cultured MSC. In vivo experiments are in progress to provide in vivo evidence for the existence of a novel model of leukemia-stroma interactions where the genetics of the tumor cell impacts stromal cell biology. Disclosures: No relevant conflicts of interest to declare.

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