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BioAssay: AID 651745

Dose response of compounds that promote myeloid differentiation with Cherry Pick01

Project Title: Discovering small molecules that overcome differentiation arrest in acute myeloid leukemia ..more
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AID: 651745
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-11-01
Modify Date: 2012-11-30

Data Table ( Complete ):           Active    All
BioActive Compounds: 15
Depositor Specified Assays
588701Summary of HTS to identify compounds that promote myeloid differentiationsummarySummary of HTS to identify compounds that promote myeloid differentiation
UNMCMD Assay Overview:
Assay Support: 1 R03 DA032471-01
Project Title: Discovering small molecules that overcome differentiation arrest in acute myeloid leukemia
PI: David Sykes, PhD

Screening Center PI: Larry Sklar, PhD / UNMCMD
Screening Center Manager: Kristine Gouveia
Screening Lead: Mark Haynes, PhD
Assay Implementation: Mark Haynes, Stephanie Chavez

Chemistry Center PI: Stuart Schreiber, PhD / BIPDeC
Chemistry Center Manager: Patti Aha
Chemistry Lead: Tim Lewis

Assay Background and Significance:

The potential for successful differentiation therapy in acute leukemia was realized with the clinical development of all-trans retinoic acid (ATRA) and arsenic. ATRA and arsenic overcome the differentiation arrest imposed by the retinoic acid receptor alpha (RARa) fusion oncoprotein and treated leukemic promyelocytes terminally differentiate to mature neutrophils. These small molecules are remarkably well-tolerated by patients in comparison to traditional cytotoxic chemotherapy. Furthermore, incorporating ATRA into treatment regimens single-handedly improved the overall survival of patients with acute promyelocytic leukemia (APL) from 20% to 75% [1]. Unfortunately, differentiation therapy does not exist for the much larger fraction of non-APL acute myeloid leukemias where the standard of care results in an overall survival rate of only 25%.

The mammalian homeobox transcription factors contribute to lineage-specific hematopoietic differentiation, and their expression is tightly regulated during normal hematopoiesis. Though critical to early hematopoiesis, the expression of the HoxA cluster of genes is normally downregulated as cells mature [2]. The persistent, and inappropriate, expression of members of the HoxA cluster of genes has been described in the majority of acute myeloid leukemias. More specifically, HoxA9 has an important role in normal hematopoiesis and leukemogenesis. HoxA9 is directly involved in human leukemias as one partner of the fusion protein NUP98- HoxA9 [3]. In analyses of human AML, the level of HoxA9 expression has been correlated with poor prognostic karyotype [4] and inversely correlated with survival [5]. Furthermore, in patients with CML, a relatively higher level of HoxA9 expression was associated with transition from chronic phase to accelerated and blast phase [6]. More recently it has been shown that HoxA9 is critical to the small subset of lymphoid and myeloid leukemias that express fusion oncoproteins involving the mixed lineage leukemia (MLL) gene. Leukemias harboring MLL-rearrangements are a particularly poor prognosis subgroup of AML and depend on HoxA9 for proliferation and survival [7]. Overall, this suggests that HoxA9 dysregulation - via fusion with NUP98 or via inappropriate maintenance of HoxA9 expression - is a common pathway in the differentiation arrest in myeloid leukemia. This is an exciting possibility, as it suggests that by specifically targeting this pathway, one might be able to overcome differentiation arrest.

The identification of differentiation therapy has been hindered by the lack of a good model system of differentiation arrest in acute myeloid leukemia. Primary leukemic cells are difficult to isolate and culture, and their availability is limited. Leukemia cell lines (e.g. HL60, NB4, 32D) are readily available though their underlying mechanism of differentiation arrest is not known. Furthermore, these cell lines are only capable of incomplete differentiation and they differentiate in response to non-physiologic stimuli (e.g. DMSO, PMA). Finally, differentiation is cumbersome to assay in a high-throughput fashion, and previous studies have focused on complex screens using qPCR to monitor small changes in gene expression [8]. These problems have made it very difficult to adopt a system for the purpose of high-throughput screening to identify compounds which promote differentiation.

We have devised a novel cell-based assay with advantages over existing systems. The assay (1) provides an unlimited supply of cells, (2) the cells are derived from primary marrow, (3) the differentiation arrest is imposed by a single and clinically relevant oncoprotein, (4) the cells have a built-in marker of differentiation, and (5) when the oncoprotein is inactivated or inhibited, the cells are capable of recapitulating full and normal myeloid differentiation.
This assay will be used to identify small molecules that promote the differentiation of acute myeloid leukemia cells. Cells will be arrested in differentiation by the expression of HoxA9 from an estrogen receptor-HoxA9 construct. The screen will be conducted in the presence of estrogen so HoxA9 will be continuously expressed. The cells also contain a GFP construct regulated by the lysozyme promoter. Differentiated cells will express GFP. Small molecules that promote differentiation will be identified by green fluorescence. In order to eliminate compounds that are green fluorescent in nature or compounds that activate the expression of GFP, a MAC1-APC antibody will be used to detect true differentiation. True hits will be identified as those cells that are both green and red fluorescent.

Cells are cultured in RPMI supplemented with 10% fetal bovine serum, penicillin/streptomycin,L-glutamine, 5% conditioned media containing stem cell factor (SCF; approx 100ng/milliL), and beta-estradiol (0.5 microM). The conditioned media is generated by a Chinese Hamster Ovary (CHO) cell line which constitutively expresses and secretes SCF into the supernatant. On day 0, cells and inert polystyrene beads (5 micron; Spherotech CPX-50-10), and Pluronic (final 0.04%, Sigma P5556) suspended in media are dispensed into 384-well plates using an automated dispenser. Slow, continuous stirring using a sterile magnetic stir-bar keeps the cells dispersed during the dispensing process. The compounds (dissolved in DMSO) are pinned into the cells. The plates are incubated for four days under standard conditions (37 degrees-C, humidified, 5% CO2). On day 4, the MAC1-APC antibody will be added. After a twenty minute incubation with the antibody, the plates are analyzed using high-throughput flow cytometry via the HyperCyt autosampler (IntelliCyt, USA) connected to a Cyan flow cytometer. Cell viability and green fluorescence is stable for 24 hours after the day 4 time point. Gating on the inert bead population provides a measure of sampling quality, as the same number of beads are seeded into each well on day 0. The percent of GFP and APC positive live cells is used to determine whether the test compound has a differentiating effect.


The percent GFP positive values for the entire concentration range of a test compound were fitted by Prism(R) software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose response model with variable slope, also known as the four parameter logistic equation. Curve fit statistics were used to determine the following parameters of the model: EC50, microM - concentration of added test compound competitor that inhibited fluorescent ligand binding by 50 percent; LOGEC50 - the logarithm of EC50; TOP - the response value at the top plateau; BOTTOM - the response value at the bottom plateau; HILLSLOPE - the slope factor, or the Hill coefficient; STD_LOGEC50, STD_TOP, STD_BOTTOM, STD_HILLSLOPE - standard errors of LOGEC50, TOP, BOTTOM, and HILLSLOPE ; EC50_95CI_LOW, EC50_95CI_HIGH - the low and high boundaries of the 95% confidence interval of the EC50 estimate, RSQR - the correlation coefficient (r squared) indicative of goodness-of-fit.

Compounds with a 15 % GFP positive or greater at the maximum concentration and EC60 < 60 microM were deemed active. The calculation for the PUBCHEM_SCORE is based on the EC50 by the following equation:
PUBCHEM_SCORE = 100*(1-EC50/60)
All non-active compounds were scored as 0.
1. Fenaux, P., et al., Effect of all transretinoic acid in newly diagnosed acute promyelocytic leukemia. Results of a multicenter randomized trial. European APL 91 Group. Blood, 1993. 82(11): p. 3241-9.
2. Lawrence, H.J., et al., The role of HOX homeobox genes in normal and leukemic hematopoiesis. Stem
Cells, 1996. 14(3): p. 281-91.
3. Borrow, J., et al., The t(7;11)(p15;p15) translocation in acute myeloid leukaemia fuses the genes for nucleoporin NUP98 and class I homeoprotein HOXA9. Nat Genet, 1996. 12(2): p. 159-67.
4. Drabkin, H.A., et al., Quantitative HOX expression in chromosomally defined subsets of acute myelogenous leukemia. Leukemia, 2002. 16(2): p. 186-95.
5. Andreeff, M., et al., HOX expression patterns identify a common signature for favorable AML. Leukemia, 2008. 22(11): p. 2041-7.
6. Tedeschi, F.A. and F.E. Zalazar, HOXA9 gene expression in the chronic myeloid leukemia progression. Leuk Res, 2006. 30(11): p. 1453-6.
7. Faber, J., et al., HOXA9 is required for survival in human MLL-rearranged acute leukemias. Blood, 2009. 113(11): p. 2375-85.
8. Stegmaier, K., et al., Gene expression-based high-throughput screening(GE-HTS) and application to leukemia differentiation. Nat Genet, 2004. 36(3): p. 257-63.
Result Definitions
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OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ACTIVITY_TYPEQualifier for the value of EC50String
2EC50_MICROM*Effective concentration of half maximal event count as estimated by curve fitFloatμM
3EC50_95CI_LOWLower 95% confidence interval boundary for the EC50 curve fit estimateFloatμM
4EC50_95CI_HIGHUpper 95% confidence interval boundary for the EC50 curve fit estimateFloatμM
5BOTTOMResponse value at the bottom plateauFloat%
6TOPResponse value at the top plateauFloat%
7LOGEC50Log of the EC50 estimateFloat
8HILLSLOPEHill slope estimate for the fitted dose response curveFloat
9STD_BOTTOMStandard error for the response value at the bottom plateauFloat%
10STD_TOPStandard error for the response value at the top plateauFloat%
11STD_LOGEC50Standard error for the Log EC50 valueFloat
12STD_HILLSLOPEStandard error for the HILL SLOPEFloat
13RSQR Correlation coefficient for the fitted dose response curveFloat
14N_POINTSNumber of data points for each dose response curveInteger
15PercentGFPpositive_0.003microM (0.003μM**)Percent GFP Positive measured with 0.003 micromolar concentration of test compoundFloat%
16PercentGFPpositive_0.009microM (0.009μM**)Percent GFP Positive measured with 0.009 micromolar concentration of test compoundFloat%
17PercentGFPpositive_0.027microM (0.027μM**)Percent GFP Positive measured with 0.027 micromolar concentration of test compoundFloat%
18PercentGFPpositive_0.081microM (0.081μM**)Percent GFP Positive measured with 0.081 micromolar concentration of test compoundFloat%
19PercentGFPpositive_0.245microM (0.245μM**)Percent GFP Positive measured with 0.245 micromolar concentration of test compoundFloat%
20PercentGFPpositive_0.741microM (0.741μM**)Percent GFP Positive measured with 0.741 micromolar concentration of test compoundFloat%
21PercentGFPpositive_2.188microM (2.188μM**)Percent GFP Positive measured with 2.18 micromolar concentration of test compoundFloat%
22PercentGFPpositive_6.607microM (6.6μM**)Percent GFP Positive measured with 6.6 micromolar concentration of test compoundFloat%
23PercentGFPpositive_19.953microM (19.9μM**)Percent GFP Positive measured with 20 micromolar concentration of test compoundFloat%
24PercentGFPpositive_0.006microM (0.006μM**)Percent GFP Positive measured with 0.006 micromolar concentration of test compoundFloat%
25PercentGFPpositive_0.018microM (0.018μM**)Percent GFP Positive measured with 0.018micromolar concentration of test compoundFloat%
26PercentGFPpositive_0.055microM (0.055μM**)Percent GFP Positive measured with 0.055 micromolar concentration of test compoundFloat%
27PercentGFPpositive_0.166microM (0.166μM**)Percent GFP Positive measured with 0.166 micromolar concentration of test compoundFloat%
28PercentGFPpositive_0.49microM (0.49μM**)Percent GFP Positive measured with 0.49 micromolar concentration of test compoundFloat%
29PercentGFPpositive_1.514microM (1.514μM**)Percent GFP Positive measured with 1.5micromolar concentration of test compoundFloat%
30PercentGFPpositive_4.365microM (4.365μM**)Percent GFP Positive measured with 4.36 micromolar concentration of test compoundFloat%
31PercentGFPpositive_13.183microM (13.2μM**)Percent GFP Positive measured with 13.2 micromolar concentration of test compoundFloat%
32PercentGFPpositive_39.811microM (39.8μM**)Percent GFP Positive measured with 40.0 micromolar concentration of test compoundFloat%

* Activity Concentration. ** Test Concentration.

Data Table (Concise)