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`NEOPLASIA
`
`Cell-cycle–dependent activation of mitogen-activated protein kinase kinase
`(MEK-1/2) in myeloid leukemia cell lines and induction of growth
`inhibition and apoptosis by inhibitors of RAS signaling
`Michael A. Morgan, Oliver Dolp, and Christoph W. M. Reuter
`
`the RAS–to–mitogen-
`Disruption of
`activated protein kinase (MAPK/ERK)
`signaling pathway,
`either directly
`through activating RASgene mutations
`or indirectly through other genetic aber-
`rations, plays an important role in the
`molecular pathogenesis of myeloid leu-
`kemias. Constitutive activation of ERK-
`1/2 and MEK-1/2, which elicit oncogenic
`transformation in fibroblasts, has re-
`cently been observed in acute myeloid
`leukemias (AML). In this study, the acti-
`vation of the RAS-to-MAPK cascade in
`14 AML and 5 chronic myeloid leukemia
`(CML) cell lines is examined and corre-
`lated with the effects of a panel of 9 RAS
`Introduction
`
`signaling inhibitors on cell viability,
`colony formation, cell-cycle progres-
`sion, and induction of apoptosis. Activa-
`tion of MEK, ERK, and the transcription
`factors CREB-1, ATF-1, and c-Myc is
`demonstrated in the majority of the cell
`lines (9 of 14 AML and 2 of 5 CML cell
`lines). Although activation of the ERK
`cascade did not always correlate with
`the presence of activating RAS muta-
`tions or BCR-Abl,
`it is linked to the
`G0/G1 and the G2/M phase of the cell
`cycle. In contrast to most inhibitors (eg,
`B581, Cys-4-Abs-Met, FPT-2, FTI-276,
`and FTS), a significant growth inhibition
`was only observed for FTI-277 (19 of 19),
`
`FPT-3 (10 of 19), and the MEK inhibitors
`U0126 (19 of 19) and PD098059 (8 of 19).
`Treatment of NB-4 cells with FTI-277
`primarily resulted in a G2/M block,
`whereas treatment with FPT-3 and U0126
`led to induction of apoptosis. FTI-277
`revealed strong toxicity toward normal
`purified CD341 cells. The results sug-
`gest differences in the mechanisms of
`action and support a potential therapeu-
`tic usefulness of these inhibitors in the
`treatment of myeloid leukemias. (Blood.
`2001;97:1823-1834)
`
`© 2001 by The American Society of Hematology
`
`The deregulation of RAS signal transduction has been implicated
`in the malignant growth of human cancer cells including myeloid
`leukemias.1,2 RAS proto-oncogenes (H-RAS, N-RAS, and K-RAS)
`encode 21-kd G-proteins that play key roles in signal transduction,
`proliferation, differentiation, and malignant transformation.3-5 RAS
`proteins are produced as cytoplasmatic precursors, which require
`several posttranslational modifications (eg, prenylation, proteoly-
`sis, carboxymethylation, and palmitoylation) for membrane bind-
`ing and full biologic activity.3-8 RAS functions as a biologic switch
`that relays signals from ligand-stimulated tyrosine kinase, cyto-
`kine, and heterotrimeric G-protein–coupled receptors to cytoplas-
`matic mitgen-activated protein kinase (MAPK) cascades. In its
`activated, GTP-bound state, RAS binds to and activates effector
`molecules such as Rafs, MEKK, PI-3K, and Ral-GEF.3-5,8-14 Raf
`kinases (A-Raf, B-Raf, c-Raf-1) selectively phosphorylate and
`activate MAPK kinases (MAPKK) MEK-1/2 in the MAPK/ERK
`pathway.13-17 MEK-1/2 are dual specificity kinases that activate
`MAPKs (ERK-1/2).18,19 The best-characterized ERK substrates are
`cytoplasmic phospholipase A2 (cPLA2), ribosomal protein S6
`kinases (RSKs), and transcription factors Elk-1 and CREB-1.18-20
`The importance of deregulation of ERK signaling in the
`molecular pathogenesis of myeloid leukemias is underscored by
`the positioning of several oncogene and tumor suppressor gene
`products on this pathway.5,21-23 RAS mutations are frequent genetic
`
`aberrations found in 20% to 30% of all human tumors, although the
`incidences in tumor type vary greatly.1,2 The most commonly
`observed RAS mutations arise at sites critical for RAS regulation,
`namely codons 12, 13, and 61.1,2,5,9 Additionally, mutations occur at
`codons 15, 16, 18, and 31.24,25 These mutations result in abrogation
`of normal intrinsic or GAP-stimulated GTPase activity of RAS,
`leading to increased half-lives of mutant RAS-GTP.5,9,26 Transfor-
`mation results, at least in part, from deregulated stimulation of
`mitogenic signal transduction pathways.1,2,5 The highest incidences
`of RAS mutations were detected in carcinomas of pancreas (90%),
`thyroid (50%), colon (50%), and lung (30%). RAS mutations are
`also frequently observed in myelodysplastic syndromes, acute
`myeloid leukemias (AML),
`juvenile myelomonocytic myeloid
`leukemia (JMML), and multiple myelomas (20%-40%).1,2,5,21-23
`N-RAS is mutated in the majority of cases and presence of the
`mutation is not associated with any particular FAB type, cytoge-
`netic abnormality, or clinical feature including prognosis.22
`In addition to activation by mutation, RAS is also deregulated in
`myeloid leukemias by constitutive activation of proto-oncogenes
`such as receptor or nonreceptor tyrosine kinases (RTKs and
`NRTKs) or inactivation of tumor suppressor genes.5,21-23 RTKs are
`constitutively activated by single point mutations (eg, colony-
`stimulating factor-1 [CSF-1] receptor and c-Kit receptor), duplica-
`tions of juxtamembrane domain-coding sequences (eg, FLT3
`
`From the Department of Hematology and Oncology, Section Molecular Biology,
`University of Ulm, Germany.
`
`Center for Internal Medicine, Hannover Medical School, Carl-Neuberg-Str 1,
`D-30625 Hannover, Germany; e-mail: reuter.christoph@mh-hannover.de.
`
`Supported by a grant to C.W.M.R. from the Deutsche Forschungsgemeinschaft
`(Re 864/4-1) and a grant from the University of Ulm (P.541).
`
`The publication costs of this article were defrayed in part by page charge
`payment. Therefore, and solely to indicate this fact, this article is hereby
`marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
`
`Reprints: Christoph W. M. Reuter, Department of Hematology & Oncology,
`
`© 2001 by The American Society of Hematology
`
`BLOOD, 15 MARCH 2001 z VOLUME 97, NUMBER 6
`
`1823
`
`Page 1 of 13
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`1824
`
`MORGAN et al
`
`BLOOD, 15 MARCH 2001 z VOLUME 97, NUMBER 6
`
`receptor), or deletions of the negative regulatory regions in ligand
`binding or transmembrane domains.5,21,27-31 Additionally, transloca-
`tions involving RTKs and NRTKs produce chimeric proteins in
`which varying N-terminal portions of either the ligand-binding or
`the transmembrane domain are replaced with novel protein se-
`quences (BCR-Abl in CML, Abl-TEL in AML, and TEL-PDGFRb
`in CMML).5,32-37 These fusion tyrosine kinases have been shown to
`activate RAS.32-37 The product of the NF-1 tumor suppressor gene,
`neurofibromin, encodes an RAS-GTPase–activating protein (GAP)
`and is mutated in the autosomal dominant type 1 neurofibromato-
`sis, which is associated with an increased tendency to develop
`myeloid leukemias, especially JMML.5,38-41 Leukemic cells from
`children with type 1 neurofibromatosis show a moderate elevation
`in the percentage of GTP-RAS.38-41
`Because the frequency of RAS mutations is among the highest
`for any gene in human cancers, development of inhibitors of the
`MAPK/ERK pathway as potential anticancer agents is a promising
`pharmacologic strategy.5,42-44 One strategy to impede oncogenic
`RAS function in vivo is the inhibition of RAS posttranslational
`modification (eg, inhibitors of FTase, GGTase, PPMTase, RE-
`Pase).5 Another strategy is the inhibition of downstream effectors
`of RAS (eg, geldanamycin, sulindac, and MEK inhibitors).5
`Inhibitors of RAS signaling revert RAS-dependent transformation
`and cause regression of RAS-dependent tumors in animal models.
`The most promising new class of these potential cancer therapeu-
`tics is the farnesyltransferase inhibitors (FTIs).42-44 In contrast to
`many conventional chemotherapeutics, FTIs are remarkably spe-
`cific and cause no gross systemic toxicity in animals. Some
`inhibitors (eg, R115777, SCH66336, and L-778123) are presently
`being evaluated in phase I and phase II clinical trials.5,45,46
`Based on the wealth of data reporting the effectiveness of RAS
`signaling inhibitors against human carcinomas harboring activated
`RAS, coupled with the implications of RAS in the pathophysiology
`of myeloid leukemias, the role of activated RAS signaling and the
`effects of these inhibitors on leukemia cell growth were investi-
`gated. Although we demonstrate an activation of MEKs, ERKs, and
`CREB-1/ATF-1 in myeloid leukemia cell lines, the presence of
`RAS mutations and BCR-Abl did not always correlate with an
`activation of the RAS-to-MAPK pathway. Interestingly, MEK-1/2
`activation coincided with the G0/G1 and G2/M phases of the cell
`cycle. Regardless of the presence of MAPK activation, incubation
`with various inhibitors (eg, FTI-277, FPT-3, U0126, and PD098059)
`of the RAS-to-MAPK cascade led to substantial growth inhibition
`of most myeloid leukemia cell lines tested. Incubation of NB-4
`cells with FPT-3 or U0126 induced apoptotic DNA fragmentation
`and exposure of phosphatidylserine on the outer leaflet of the
`plasma membrane. In contrast,
`treatment of NB-4 cells with
`FTI-277 primarily induced a G2/M block. Treatment with FTI-277
`also inhibited colony formation of purified human CD341 cells.
`These results suggest differences in the mechanisms of action and a
`potential role of some of these inhibitors in the treatment of
`myeloid leukemias.
`
`Materials and methods
`
`Cells and antibodies
`
`Cell lines were obtained from the German Collection of Microorganisms
`and Cell Cultures (Braunschweig, Germany). Antibodies against H-, K-,
`and N-RAS, ERK-1/2, MEK-1/2, monophospho- and diphospho-ERK-1/2
`(PP-ERK-1/2) were from Santa Cruz Biotechnology Inc (Santa Cruz, CA)
`and Sigma-Aldrich (Deisenhofen, Germany). Diphospho-MEK-1/2
`
`(PP-MEK-1/2), CREB-1, and phospho-CREB-1 antibodies were from New
`England Biolabs (Frankfurt, Germany). Inhibitors were purchased from
`Calbiochem-Novabiochem (Bad Soden, Germany).
`
`Trypan blue exclusion assay
`
`Cells (2.5 or 6.25 3 104 cells in 250 mL media) were seeded in 96-well
`plates and incubated 4 days with either solvent control or the stated
`concentration of inhibitor. Cell counts were evaluated using a 1:1 dilution of
`cell suspension in 0.4% trypan blue solution (Sigma-Aldrich). Viable and
`nonviable cells were counted in a Neubauer cell counting chamber.
`
`Colony-forming assays
`
`Colony-forming assays were performed essentially as described.47 Briefly,
`cells were seeded at 1.0 to 2.5 3 105/mL cell suspension in 96-well plates
`and treated with inhibitors as indicated. After 4 days, aliquots of the cell
`suspensions were plated in 400 mL methylcult H4230 (Cellsystems
`Biotech, St Katharinen, Germany) according to the manufacturer’s instruc-
`tions and incubated 7 to 14 days. Cell densities were evaluated and
`aggregates of more than 25 cells were scored as colonies.
`
`Western blot analysis
`
`Cells were cultured in RPMI medium containing 10% heat-inactivated fetal
`calf serum, glutamine (292 mg/mL), and antibiotics (penicillin 60.2 mg/mL,
`streptomycin 133 mg/mL). Cell extracts were prepared and Western blotting
`was performed as described.48,49 Cellular protein concentrations were
`determined using the Coomassie dye-binding assay according to Bradford50
`(Bio-Rad Laboratories, Hercules, CA).
`
`MAPK assays
`
`The MAPK assays were performed as previously described.48,49
`
`RAS-GTP pulldown assay
`
`The RAS-GTP pulldown assays were accomplished as described.51,52 The
`pGEX 2T-RBD construct (encoding a GST fusion protein containing amino
`acids 51-131 of c-Raf-1) was a gift from J. Bos. GST-RBD expression in
`Escherichia coli was induced with 1 mM isopropyl-b-D-thiogalactopyrano-
`side (IPTG) for 2 to 3 hours. Bacteria were sonicated in 50 mL
`phosphate-buffered saline (PBS) containing 0.5 mM DTT and 1 mg/mL
`aprotinin, leupeptin, and pepstatin A. After addition of 1% Triton X-100,
`clarified lysates were aliquoted and stored at 280°C as glycerol stocks
`(10%). The fusion protein was purified on glutathione-Sepharose beads (35
`mL/sample, Pharmacia, Uppsala, Sweden). For affinity precipitation, the
`beads were washed 3 times with lysis buffer (1 mL), incubated with fresh
`cell lysates (1 mg total protein) 30 minutes at 4°C, and collected by
`centrifugation (12 000g at 4°C). After washing 3 times with 100 to 200 mL
`lysis buffer and incubation in loading buffer (5 minutes at 95°C), samples
`were analyzed by 15% sodium dodecyl sulfate–polyacrylamide gel electro-
`phoresis (SDS-PAGE) and subjected to Western blotting using antibodies
`against H-, K-, and N-RAS to identify GTP-bound RAS.
`
`Cell-cycle analysis
`
`Cell permeabilization (1-2 3 106 cells) was performed using the GAS-002
`kit from Bio Research (Kaumberg, Austria) essentially according to the
`manufacturer’s instructions. After washing in PBS, fixation in 100 mL
`buffer A (15 minutes), and permeabilization in 100 mL buffer B (10
`minutes), 5 mL of the primary antibodies (aMEK-1, aPP-MEK-1/2,
`aERK-2, aPP-ERK-1/2) or negative control antibodies were added (1 hour
`at room temperature). Cells were washed with 3 mL 0.1% bovine serum
`albumin (BSA)–PBS, and 3 mL of the appropriate fluorescein isothiocya-
`nate (FITC)–conjugated secondary immunoglobulins were added (20
`minutes in the dark). After washing with 3 mL 0.1% BSA-PBS, cells were
`incubated in propidium iodide staining buffer (10 mM Tris-HCl pH 7.4,
`0.1% Triton-X-100, 15 mg/mL RNase A, 5 mM MgCl2, 50mg/mL
`propidium iodide, 15-30 minutes at 4°C in the dark). The cell-cycle profiles
`(10 000 cells) were analyzed on a FACScan flow cytometer (Becton
`
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`BLOOD, 15 MARCH 2001 z VOLUME 97, NUMBER 6
`
`TARGETING RAS SIGNALING IN MYELOID LEUKEMIA
`
`1825
`
`Dickinson, San Jose, CA) using the Modfit LT 2.0 software (Verity
`Software House, Topsham, ME).
`
`performance liquid chromatography-grade H2O and analyzed by an ABI
`Prism Sequence Detection System (PE Applied Biosystems).
`
`Immunocytochemical staining
`
`Detection of apoptosis
`
`Cells were washed twice in PBS and cytospin slides were prepared using
`standard techniques. Slides were air-dried for 2 to 24 hours and processed
`for staining or wrapped airtight and stored at 220°C. Immunocytochemical
`staining of the slides was done using the Dako LSAB1 kit (DAKO,
`Carpinteria, CA) according to the manufacturer’s instructions. After
`fixation in 3% paraformaldehyde (30 minutes, 4°C), cells were washed 3
`times with Tris-buffered saline containing 0.1% Tween-20 (TBS-T buffer; 5
`minutes) and once with TBS buffer (2 minutes). Slides were incubated in
`100% methanol (10 minutes, 220°C), washed in TBS-T buffer (3 times,
`each 5 minutes), and blocked with 5% horse serum in TBS-T buffer (1 hour,
`room temperature). Incubation with primary antibodies (aMEK-1, aPP-
`MEK-1/2, aERK-2, aPP-ERK-1/2) or negative control antibodies was
`done overnight at 4°C (1:400 in 5% BSA–TBS-T buffer). After washing in
`TBS-T (15 minutes) followed by 0.1% BSA–TBS-T (2 minutes) and
`incubation with appropriate biotinylated secondary immunoglobulins
`(DAKO) staining and counterstaining with hematoxylin was performed.
`
`For detection and quantification of apoptosis at the single cell level, the in
`situ cell death detection kit of Roche Diagnostics (Mannheim, Germany)
`was used. Labeling of DNA strand breaks was done according to the
`manufacturer’s instructions applying the TUNEL method. In addition, the
`annexin V-PE/7-amino-actinomycin (7-AAD) double-staining method was
`used to quantitatively determine the percentage of cells that are actively
`undergoing apoptosis. After washing once with PBS and twice with annexin
`V-binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2),
`cells were resuspended in annexin V-binding buffer (1 mL) and 5 mL
`annexin V-PE (BD Pharmingen/Biosciences, Heidelberg, Germany) was
`added. After 15 minutes at room temperature in the dark, washing twice
`with annexin V-binding buffer (1 mL) and resuspension in 200 mL, 40 mL
`7-AAD solution (0.2 mg/mL PBS) was added (20 minutes at 4°C in the
`dark). Finally, 300 mL annexin V- binding buffer was added to each tube
`and cells were analyzed by flow cytometry.
`
`Sequencing of RAS mutations
`
`Results
`
`Synthetic oligonucleotides were purchased from MWG-Biotech AG (Eber-
`sberg, Germany) for use as amplification primers to identify mutations in
`codons 12, 13, and 61 of N-RAS, H-RAS, and K-RAS.53,54 Genomic DNA
`(gDNA) samples were prepared using a kit from Qiagen (Hilden, Ger-
`many). Fragments of 103 and 109 base pairs (bp) containing the codons of
`interest were obtained by amplification of 200 ng gDNA with AmpliTaq
`Gold (PE Applied Biosystems, Langen, Germany). The DNA was purified
`by 3% agarose gel electophoresis, recovered with a Qiaex II gel extraction
`kit (Qiagen), and cloned using the TOPO-TA Cloning Kit from Invitrogen
`(Carlsbad, CA). M13 forward and reverse primers were used for sequencing
`400- to 600-ng plasmids using the Big Dye Sequencing kit from PE Applied
`Biosystems. The products of polymerase chain reaction were precipitated,
`washed with 70% ethanol, air-dried, and redissolved in 20 mL high-
`
`Activation of the RAS-to-MAP kinase cascade
`
`RAS mutations. The gDNA of several myeloid cell lines was
`analyzed for activating mutations in codons 12, 13, 15, 16, 18, and
`61 of H-, K- and N-RAS to correlate the frequency of RAS
`mutations with the presence of constitutive activation of the ERK
`pathway. Four of 14 AML cell lines (28.6%) contained activating
`RAS mutations (Table 1). As previously reported, a mutation of
`codon 12 of N-RAS was detected in THP-1 (GGT to GAT) and a
`N-RAS codon 61 mutation in HL-60 (CAA to CTA) resulting in
`G12D and Q61L.55 Two AML cell lines (Kasumi-1 and MV4-11)
`were found to harbor a K-RAS codon 12 mutation (GGT to GAT),
`
`Table 1. Activation of the RAS–to–mitogen-activated protein kinase signaling cascade in myeloid leukemia cell lines
`
`Cell line
`
`Leukemia
`
`RASmutation
`
`HL-60
`Kasumi-1
`Mutz-2
`NB-4 t15;17 PML /RARA
`THP-1
`PLB-985
`OCI-AML 2
`OCI-AML5
`Mutz-3
`ML-2 t6;11, MLL /AF6
`MV4-11 t4;11, MLL /AF4
`
`AML M2
`AML M2
`AML M2
`AML M3
`AML M4
`AML M4
`AML M4
`AML M4
`AML M4
`AML M4
`AML M5
`
`Mono-Mac-1
`KG-1
`M-07e
`RC2A
`EM-2
`JK-1
`K562
`MEG-01
`LAMA-84
`
`AML M5
`AML M6
`AML M7
`ALL
`CML blast crisis
`CML erythroid blast crisis
`CML blast crisis
`CML megakaryocytic blast crisis
`CML myeloid-megakaryocytic blast crisis
`
`N-RAS/61
`K-RAS/12
`None
`None
`N-RAS/12
`None
`None
`None
`None
`None
`K-RAS/12
`K-RAS/18
`None
`None
`None
`None
`BCR-Abl
`BCR-Abl
`BCR-Abl
`BCR-Abl
`BCR-Abl
`
`RAS
`
`(1)
`—
`—
`111
`(1)
`—
`—
`—
`—
`—
`—
`
`—
`—
`—
`—
`—
`—
`—
`—
`—
`
`Activation of
`
`ERK-1/2
`
`MEK-1/2
`
`CREB-1/ATF-1
`
`11
`(1)
`(1)
`111
`11
`11
`1
`(1)
`(1)
`1
`111
`
`111
`1
`(1)
`1
`
`—
`—
`1
`1
`
`—
`
`11
`11
`
`—
`111
`(1)
`—
`1
`
`—
`(1)
`1
`(1)
`
`11
`11
`1
`1
`1
`1
`1
`1
`
`11
`1
`1
`11
`
`—
`1
`111
`
`—
`—
`111
`1
`
`111
`1
`11
`1
`1
`
`11
`1
`111
`
`Among the myeloid leukemia cell lines used in this study are 14 AML cell lines and 5 CML cell lines (all from blast crisis). HL-60 and THP-1 harbor N-RASmutations in
`codons 61 and 12 (CAA 3 CTA and GGT 3 GAT resulting in Q61L and G12D, respectively). Kasumi-1 and MV4-11 contain a codon 12 K-RASmutation (GGT 3 GAT resulting
`in G12D). An additional codon 18 mutation was detected in MV4-11 (GCC 3 GAC resulting in A18D). A previously unknown silent H-RASmutation in codon 59 (GCC 3 GCT)
`was observed in MV4-11, Kasumi-1, and NB-4 (Ala). All cell lines were obtained from the German Collection of Microorganisms and Cell Cultures.
`AML indicates acute myeloid leukemia; CML, chronic myeloid leukemia.
`
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`1826
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`MORGAN et al
`
`BLOOD, 15 MARCH 2001 z VOLUME 97, NUMBER 6
`
`which also exchanges Gly12 with Asp. Interestingly, an additional
`K-RAS codon 18 mutation was found in the second allele of
`MV4-11 (GCC to GAC), which leads to replacement of A18D. No
`activating mutations of codons 15 and 16 or of the H-RAS gene
`were detected. Three cell lines (MV4-11, Kasumi-1, and NB-4)
`harbored a silent point mutation in codon 59 (GCC to GCT).
`RAS activation assays. The expression of all 3 RAS isoforms
`(H-, K- and N-RAS) was detected by Western blotting of the
`cellular lysates of all myeloid cell lines tested (not shown). To
`identify the presence of GTP-RAS, cell lysates were incubated with
`the minimal RAS-binding domain (RBD) of C-Raf-1. Western
`blotting with antibodies against H-, K- and N-RAS was performed
`to detect the binding of GTP-RAS with GST-RBD. Cellular lysates
`of H-RAS (L-61)–transformed NIH-3T3 fibroblasts were used as a
`positive control for activated H-RAS. As shown in Figure 1, only
`oncogenic H-RAS (L61) could be precipitated with GST-RBD
`from NIH-3T3 lysates, but not endogenous K- or N-RAS. THP-1
`and HL-60, which harbor N-RAS mutations in codon 12 and codon
`61, showed only a slight activation of N-RAS in some experiments
`but no activation of H-RAS or K-RAS. Surprisingly, lysates of
`Kasumi-1 and MV4-11, which harbor a K-RAS codon 12 mutation,
`did not contain substantial amounts of activated K-RAS. Although
`most lysates of cell lines with wild-type RAS did not contain
`significant amounts of GTP-RAS, high levels of activated H-, K-
`and N-RAS were found in NB-4 and ML-2 cell lysates. No
`apparent activation of RAS was detected in the CML cell lines,
`although all express the activated BCR-Abl fusion tyrosine kinase,
`which has been reported to induce RAS activation.5,39,40 Of the 5
`growth factor–dependent myeloid cell lines (AML-OCI-5, Mo-7e,
`
`Figure 1. Activation of RAS in myeloid leukemia cell lines. Lysates of myeloid
`leukemia cell lines were subjected to affinity precipitation (AP) with GST-RBD as
`described in “Materials and methods.” RAS proteins were detected by immunoblot-
`ting with H-, K-, and N-RAS antibodies. H-RAS (L61)–transformed NIH-3T3 fibro-
`blasts were used as a positive control for activated H-RAS.
`
`Figure 2. Activation of ERK-1/2 and MEK-1/2 in myeloid leukemia cell lines.
`Myeloid leukemia cell
`lysates were adjusted for protein concentration and equal
`amounts of total cell protein were subjected to SDS-PAGE. Western analysis was
`performed with antibodies against MEK-1, ERK-2, PP-MEK-1/2, and PP-ERK-1/2.
`L61-H-RAS–transformed NIH-3T3 cell lysates were used as positive controls for the
`RAS-induced activation of ERKs and MEKs.
`
`JK-1, Mutz-2, and Mutz-3), activation of H-, K-, and N-RAS was
`observed only in AML-OCI-5 on stimulation by interleukin 3 or
`conditioned medium of 5637 kidney cells (not shown).
`Activation of the MAPK cascade. To determine the frequency
`and the level of activation of signaling proteins downstream of
`RAS, lysates of myeloid cells were analyzed by Western blotting
`with antibodies against PP-MEK-1/2 and PP-ERK-1/2 (Figure 2).
`Cellular lysates of H-RAS (L-61)–transformed NIH-3T3 fibro-
`blasts were used as a positive control for activated MEK-1/2 and
`ERK-1/2. Significant activation of MEK-1/2 was detected in 8 of
`14 AML cell lines and 2 of 5 CML cell lines. ERK-1/2 activation
`was observed in 9 of 14 AML cell lines and 2 of 5 CML cell lines.
`MEK-1/2 activation correlated with ERK-1/2 activation in 7 of 14
`AML cell lines and 2 of 5 CML cell lines. Western blots for
`diphospho-ERK-1/2 were confirmed by immunocomplex MAPK
`assays using an ERK-2 antibody, which cross-reacts with ERK-1
`(Figure 3). The presence of PP-ERK-1/2 in Western blots of
`cellular lysates correlated closely with MAPK/ERK activity in
`immunocomplex kinase assays. Compared to leukemia cell lines,
`H-RAS (L-61)–transformed NIH-3T3 fibroblasts harbored at least
`50% higher amounts of PP-ERK-1/2. The presence of RAS
`mutations or BCR-Abl did not always correlate with significant
`ERK-1/2 activation (Table 1).
`Activation of transcription factors. Following activation, ERK-
`1/2 translocates to the nucleus resulting in phosphorylation and
`activation of transcription factors such as ELK-1, CREB-1, and
`Myc. To determine whether activation of ERK-1/2 induces activa-
`tion of these transcription factors, myeloid leukemia cell lysates
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`BLOOD, 15 MARCH 2001 z VOLUME 97, NUMBER 6
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`TARGETING RAS SIGNALING IN MYELOID LEUKEMIA
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`panels B and C, all cells were strongly positive for ERK-2 and
`MEK-1. Using anti-PP-ERK-1/2 and anti-PP-MEK-1/2 antibodies,
`most cells were only slightly positive (Figure 6D,E). Interestingly,
`PP-MEK-1/2 staining revealed 2 additional, strongly positive cell
`populations (Figure 6E) that corresponded to the G0/G1 and G2/M
`phases of the cell cycle. The difference in PP-ERK-1/2 and
`PP-MEK-1/2 staining might be due to rapid nuclear translocation
`of PP-ERK-1/2. Incubation with excess thymidine (2.5 mM)
`resulted in a G1/S-phase block with an increase of S-phase cells to
`60.1% and a concomitant reduction of PP-MEK-1/21 cells in G0/G1
`and G2/M (, 0.1% of the total cell population; Figure 6G,H). Cell
`viability was not significantly affected by this treatment. Treatment
`of cells with vinblastine (5 mg/mL, 10 hours), which induces
`depolymerization of mitotic interpolar microtubules and a cell
`metaphase block, increased the mitotic population of cells in G2/M
`to 80.9% and the amount of strongly positive PP-MEK-1/2 cells in
`G2/M to 13.1% (Figure 6I,J). These results demonstrate MEK-1/2
`activation in the G0/G1 and G2/M phases of the cell cycle of
`myeloid leukemia cells.
`
`Effect of inhibitors of RAS-to-MAPK signaling on myeloid
`leukemia cell growth
`
`Because activation of the ERK pathway has been implicated in the
`pathogenesis of myeloid leukemias, the effectiveness of several
`different
`types of inhibitors of RAS signaling was tested on
`viability and colony formation of myeloid leukemia cell lines. The
`cell lines tested are shown in Table 1 and included some that
`express mutationally activated N-RAS, K-RAS, or BCR-Abl.
`
`Figure 4. Activation of MAPK-dependent transcription factors in myeloid
`leukemia cell lines. Lysates of myeloid leukemia cell
`lines were subjected to
`SDS-PAGE followed by Western blotting with anti–c-Myc (A) and anti–CREB-1 (B)
`antibodies. Activated c-Myc and CREB-1/ATF-1 were detected with phosphospecific
`antibodies against activated c-Myc and CREB-1.
`
`Figure 3. Activation of ERK-2 in myeloid leukemia cell lines. Lysates of myeloid
`leukemia cell lines were subjected to immunoprecipitation with an antibody against
`ERK-2 (which also cross-reacts with ERK-1). After washing the immunoprecipitates,
`immunocomplex kinase assays were performed as described in “Materials and
`methods.” (A) Western blot of the immunoprecipitates with an antibody specific for
`ERK-2 and an antibody specific for activated PP-ERK-1/2. (B) Autoradiogram of
`kinase assay demonstrating MBP phosphorylation by immunoprecipitated ERK-1/2.
`N-RAS (L61)–transformed NIH 3T3 fibroblasts were used as a positive control for the
`RAS-induced activation of ERK-1/2. A nonspecific antibody was used as a nega-
`tive control.
`
`were subjected to Western blotting with specific antibodies against
`phospho-ELK, phospho-CREB, and phospho-Myc. Activated
`CREB-1/ATF-1 was found in 11 of 14 AML cell lines and 2 of 5
`CML cell lines and did not always coincide with the presence of
`phospho-ERK-1/2 (Figure 4 and Table 1).
`Intracellular localization of PP-ERK-1/2 and PP-MEK-1/2.
`To determine whether activation of ERK-1/2 and MEK-1/2 was
`limited to subpopulations of myeloid leukemia cells, cytospins
`were prepared and stained with specific antibodies against ERK-2,
`MEK-1, PP-ERK-1/2, and PP-MEK-1/2 (Figure 5). All cells were
`stained with anti–ERK-2 and anti–MEK-1 antibodies. However,
`only 1% to 5% of cells showed strong staining with anti-PP-ERK-
`1/2 and anti-PP-MEK-1/2 antibodies. Strong nuclear staining was
`found in approximately 50% of PP-ERK-1/21 and PP-MEK-1/21
`cells, whereas the other portion showed mainly cytoplasmic
`staining. These results were obtained for all samples tested and
`suggest activation of ERKs and MEKs in subpopulations (eg, cells
`in certain phases of the cell cycle).
`MEK activation during cell-cycle progression. To address the
`possibility of ERK-1/2 and MEK-1/2 activation during cell-cycle
`progression, a novel FACS method for cytoplasmic staining of
`activated kinases of the RAS pathway was developed. Cells were
`double stained with propidium iodide and antibodies specific for
`different members of the ERK signaling pathway (eg, ERK-2,
`PP-ERK-1/2, MEK-1, and PP-MEK-1/2). As shown in Figure 6,
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`BLOOD, 15 MARCH 2001 z VOLUME 97, NUMBER 6
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`colony formation. Only minor growth inhibitory effects on purified
`CD341 cells were observed in the presence of DMSO, or U0126
`and FPT-3 at concentrations up to 50 mM (Figure 7). FTI-277
`caused inhibition of stem cell colony formation with an inhibitory
`concentration of 50% (IC50) value of 10 mM. The NB-4, ML-2, and
`THP-1 cells showed significant leukemia selective inhibition of
`colony formation in the presence of FPT-3, U0126, and FTI-277.
`The IC50 values for FTI-277 were less than 5 mM for these cell
`
`Figure 5. Immunocytostaining of myeloid leukemia cells with PP-ERK-1/2 and
`PP-MEK-1/2 antibodies. Cytospins of myeloid leukemia cells were stained with
`antibodies specific for ERK-2, PP-ERK-1/2, MEK-1, and PP-MEK-1/2 as described in
`“Materials and methods.” Approximately 1% to 5% of the cells were strongly stained
`with anti–PP-ERK-1/2 and anti–PP-MEK-1/2 antibodies. Note the nuclear staining of
`some cells, whereas others are stained mainly in the cytoplasm.
`
`Purified CD341 cells were used as a control for inhibitor specific-
`ity. Inhibitors used in this study included (1) MEK inhibitors
`U0126 and PD098059, (2) farnesyl pyrophosphate (FPP)-based
`farnesyltransferase (FTase) inhibitors FPT-II and FPT-III, (3)
`CAAX-based FTIs FTI-276, FTI-277, B581, and Cys-4-Abz-Met,
`and the (4) prenylated protein methyltransferase (PPMTase) inhibi-
`tor FTS. To screen the compounds for growth inhibition of myeloid
`leukemia cells, liquid suspension cultures were incubated with 50
`mM inhibitor or an equal volume of solvent. After 24 to 96 hours of
`incubation, a decrease in cell viability was observed in trypan blue
`exclusion assays in some inhibitor-treated samples. Therefore, all
`myeloid cell lines were preincubated in liquid suspension cultures
`for 96 hours followed by incubation in methylcellulose to assay
`colony formation in the presence or absence of inhibitor. Limited
`growth inhibition was obtained by treatment with 50 mM FTI-276,
`B-581, and Cys-4-Abz-Met (Tables 2 and 3). Growth inhibition
`greater than 70% was observed with 50 mM FPP-competitive FTIs
`FPT-3 (10 of 19), FPT-2 (4 of 19), the CAAX-based FTase inhibitor
`FTI-277 (19 of 19), and the PPMTase inhibitor FTS (3 of 19; Tables
`2 and 3). Growth inhibition greater than 70% was also observed
`with MEK inhibitors PD098059 (8 of 19) and U0126 (19 of 19)
`(Table 3). In contrast to other inhibitors, FTI-277 elicited substan-
`tial toxicity toward purified CD341 human stem cells. Because it
`has previously been reported that this compound causes nonspe-
`cific toxicity at concentrations greater than 20 mM, we studied the
`concentration dependency of these inhibitors on cell viability and
`
`Figure 6. Activation of MEK-1/2 during cell-cycle progression. HL-60 cells were
`prepared for FACS analysis as described in “Materials and methods.” Graph A shows
`a representative control with unspecific mouse IgG antibodies. Similar results were
`obtained with rabbit and goat controls (data not shown). All cells were clearly positive
`when probed with ERK-2 or MEK-1 antibodies (graphs B,C), and only slightly positive
`when stained with phosphospecific antibodies against activated PP-ERK-1/2 and
`PP-MEK-1/2 (graphs D,E). Graph F shows the cell-cycle profile of untreated HL-60
`cells stained with propidium iodide. Additionally, PP-MEK-1/2 staining revealed 2
`strongly positive populations, which correlated with the G0/G1 and G2/M phases of the
`cell cycle (graphs E,F). Treatment of HL-60 cells with excess thymidine (2.5 mM) for
`10 hours: G, staining with anti–PP-MEK-1/2 antibodies; H, cell-cycle profile. Treat-
`ment of HL-60 cells with 5 mg/mL vinblastine for 10 hours:
`I, staining with
`anti–PP-MEK-1/2 antibodies; J, cell-cycle profile.
`
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`BLOOD, 15 MARCH 2001 z VOLUME 97, NUMBER 6
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`TARGETING RAS SIGNALING IN MYELOID LEUKEMIA
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`Table 2. Inhibition of colony formation and cell viability of myeloid leukemia cell lines by farnesyltransferase inhibitors
`
`Cell line
`
`Leukemia
`
`Ras-to-MAPK
`
`B581
`
`Cys-4 Abs-Met
`
`FPT2
`
`FPT3
`
`FTI277
`
`FTI276
`
`H