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The EOL-1 cell line as an in vitro model for the study of FIP1L1-PDGFRA-positive chronic eosinophilic leukemia

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Jan Cools at KU Leuven
Jan Cools
Hilmar Quentmeier
Hilmar Quentmeier
Hilmar Quentmeier
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Brian J P Huntly at University of Cambridge
Brian J P Huntly
Peter Marynen at KU Leuven
Peter Marynen
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Abstract and Figures

We recently identified the chimeric kinase FIP1L1-platelet-derived growth factor receptor alpha (PDGFRalpha) as a cause of the hypereosinophilic syndrome and of chronic eosinophilic leukemia. To investigate the role of FIP1L1-PDGFRA in the pathogenesis of acute leukemia, we screened 87 leukemia cell lines for the presence of FIP1L1-PDGFRA. One cell line, EOL-1, expressed the FIP1L1-PDGFRA fusion. Three structurally divergent kinase inhibitors--imatinib (STI-571), PKC412, and SU5614--inhibited the growth of EOL-1 cells. These results indicate that the fusion of FIP1L1 to PDGFRA occurs rarely in leukemia cell lines, but they identify EOL-1 as an in vitro model for the study of FIP1L1-PDGFRA-positive chronic eosinophilic leukemia and for the analysis of small molecule inhibitors of FIP1L1-PDGFRalpha.
Fusion of FIP1L1 to PDGFRA in the EOL-1 cell line. Detection of the FIP1L1-PDGFRA fusion transcript in the EOL-1 and EOL-3 cell lines (A), detection of FIP1L1 expression in the EOL-1 cell line (B), and ampli fi cation of the FIP1L1-PDGFRA fusion gene on DNA from the EOL-1 cell line (C). Different transcripts are observed for FIP1L1 and FIP1L1- PDGFRA (A-B) because of alternative splicing. Expressions of PDGFRA- FIP1L1 and native PDGFRA were not detected (B). The sequence of the fusion gene surrounding the deletion is shown at the DNA level, at the RNA level (after splicing), and at the protein level (D). Splice donor and acceptor sites are underlined. A cryptic splice site is used in exon 12 of PDGFRA . Schematic representation of FIP1L1, PDGFR ␣ , and FIP1L1-PDGFR ␣ proteins (E). Points at which the proteins are interrupted by the deletion are indicated by arrowheads.
Fusion of FIP1L1 to PDGFRA in the EOL-1 cell line. Detection of the FIP1L1-PDGFRA fusion transcript in the EOL-1 and EOL-3 cell lines (A), detection of FIP1L1 expression in the EOL-1 cell line (B), and ampli fi cation of the FIP1L1-PDGFRA fusion gene on DNA from the EOL-1 cell line (C). Different transcripts are observed for FIP1L1 and FIP1L1- PDGFRA (A-B) because of alternative splicing. Expressions of PDGFRA- FIP1L1 and native PDGFRA were not detected (B). The sequence of the fusion gene surrounding the deletion is shown at the DNA level, at the RNA level (after splicing), and at the protein level (D). Splice donor and acceptor sites are underlined. A cryptic splice site is used in exon 12 of PDGFRA . Schematic representation of FIP1L1, PDGFR ␣ , and FIP1L1-PDGFR ␣ proteins (E). Points at which the proteins are interrupted by the deletion are indicated by arrowheads.
… 
Inhibition of cell growth, induction of apoptosis, and inhibition of FIP1L1-PDGFR kinase activity in EOL-1 cells treated with various kinase inhibitors. (A) Dose-response curves of EOL-1 cells treated with imatinib, PKC412, or SU5614. The percentage of growth relative to untreated cells is plotted for increasing drug concentrations (48-hour incubation). (B) Detection of apoptotic EOL-1 cells after 24-hour incubation with imatinib (10 nM), PKC412 (100 nM), or SU5614 (100 nM). Apoptotic cells (lower right quadrant), necrotic cells (upper right quadrant), and viable cells (lower left quadrant) were detected. (C) Phosphorylation of FIP1L1PDGFR was analyzed using antiphosphotyrosine antibody on immunoprecipitated FIP1L1-PDGFR. Phosphorylation of STAT5 was analyzed using anti-phosphoSTAT5 antibody, which recognizes phosphorylated STAT5a (upper band) and STAT5b (lower band). Membranes were blotted with anti-PDGFR or anti-STAT5a antibodies as loading control.
Inhibition of cell growth, induction of apoptosis, and inhibition of FIP1L1-PDGFR kinase activity in EOL-1 cells treated with various kinase inhibitors. (A) Dose-response curves of EOL-1 cells treated with imatinib, PKC412, or SU5614. The percentage of growth relative to untreated cells is plotted for increasing drug concentrations (48-hour incubation). (B) Detection of apoptotic EOL-1 cells after 24-hour incubation with imatinib (10 nM), PKC412 (100 nM), or SU5614 (100 nM). Apoptotic cells (lower right quadrant), necrotic cells (upper right quadrant), and viable cells (lower left quadrant) were detected. (C) Phosphorylation of FIP1L1PDGFR was analyzed using antiphosphotyrosine antibody on immunoprecipitated FIP1L1-PDGFR. Phosphorylation of STAT5 was analyzed using anti-phosphoSTAT5 antibody, which recognizes phosphorylated STAT5a (upper band) and STAT5b (lower band). Membranes were blotted with anti-PDGFR or anti-STAT5a antibodies as loading control.
… 
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doi:10.1182/blood-2003-07-2479
Prepublished online November 20, 2003;
2004 103: 2802-2805
and D. Gary Gilliland
Jan Cools, Hilmar Quentmeier, Brian J. P. Huntly, Peter Marynen, James D. Griffin, Hans G. Drexler
positive chronic eosinophilic leukemia−
The EOL-1 cell line as an in vitro model for the study of FIP1L1-PDGFRA
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NEOPLASIA
Brief report
The EOL-1 cell line as an in vitro model for the study of
FIP1L1-PDGFRA–positive chronic eosinophilic leukemia
Jan Cools, Hilmar Quentmeier, Brian J. P. Huntly, Peter Marynen, James D. Griffin, Hans G. Drexler, and D. Gary Gilliland
Werecently identified the chimeric kinase
FIP1L1–platelet-derived growth factor re-
ceptor ␣ (PDGFR␣) as a cause of the
hypereosinophilic syndrome and of
chronic eosinophilic leukemia. To investi-
gate the role of FIP1L1-PDGFRA in the
pathogenesis of acute leukemia, we
screened 87 leukemia cell lines for the
presence of FIP1L1-PDGFRA. One cell
line, EOL-1, expressed the FIP1L1-
PDGFRA fusion. Three structurally diver-
gent kinase inhibitors—imatinib (STI-
571), PKC412, and SU5614—inhibited the
growth of EOL-1 cells. These results in-
dicate that the fusion of FIP1L1 to
PDGFRA occurs rarely in leukemia cell
lines, but they identify EOL-1 as an in
vitro model for the study of FIP1L1-
PDGFRA–positive chronic eosinophilic
leukemia and for the analysis of small
molecule inhibitors of FIP1L1-PDGFR␣.
(Blood. 2004;103:2802-2805)
© 2004 by The American Society of Hematology
Introduction
The hypereosinophilic syndrome (HES) is a hematologic disease
characterized by prolonged eosinophilia, exclusion of reactive
eosinophilia, and organ damage.
1,2
HES is reclassified as chronic
eosinophilic leukemia (CEL) when clonality is demonstrated.
3
We
recently identified the kinase FIP1L1–platelet-derived growth
factor receptor ␣ (PDGFR␣) as the cause and the therapeutic target
of imatinib in 56% of HES cases.
4
These results demonstrate that
most HES cases are clonal in origin and could be reclassified as
FIP1L1-PDGFRA–positive CEL. The FIP1L1-PDGFRA fusion
gene is created by an interstitial chromosomal deletion on chromo-
some 4q12 that is not apparent using standard karyotypic analysis.
4
Expression of the FIP1L1-PDGFRA fusion is under control of the
ubiquitous FIP1L1 promoter, suggesting the possibility that FIP1L1-
PDGFR␣ may be involved in the pathogenesis of other hemato-
logic malignancies. To get insight into this, we screened 87
leukemia cell lines for the presence of the FIP1L1-PDGFRA fusion
gene. Leukemia cell lines have been proven to be a valuable
resource for the study of hematologic malignancies,
5
and our
results now identify the EOL-1 cell line as an in vitro model for the
study of FIP1L1-PDGFRA–positive CEL.
Study design
PCR and RT-PCR
FIP1L1-PDGFRA fusion was amplifiedfrom DNAwith primers FIP1L1-F9
(5⬘-tggggcaattgatgttatcg) and PDGFRA-RI12 (5⬘-gtgcaagggaaaagggagtct).
RNA was isolated from cell lines from the DSMZ collection (http://
www.dsmz.de), as described.
6
Reverse transcription–polymerase chain
reaction (RT-PCR) was performed with primers FIP1L1-F7 (5⬘-acctggtgct-
gatctttctgat) and PDGFRA-R14 (5⬘-tgagagcttgtttttcactgga) for the detection
of FIP1L1-PDGFRA and primers PDGFRA-F11 (5⬘-ggtgctgttggtgattgtga)
and FIP1L1-R10 (5⬘-cagctcctggagggaaaaac) for the detection of PDGFRA-
FIP1L1. Primers PDGFRA-F11 and PDGFRA-R14 were used to detect
PDGFRA expression, and primers FIP1L1-F7 and FIP1L1-R10 were used
to detect FIP1L1 expression.
Cell culture and dose-response curves
The EOL-1 cell line (DSMZ ACC386) was grown in RPMI 1640
medium with 10% fetal bovine serum. Imatinib and PKC412 were
kindly provided by Novartis; SU5614 was purchased from Calbiochem
(San Diego, CA). Kinase inhibitors were stored in water (imatinib) or
dimethyl sulfoxide (DMSO) (PKC412, SU5614) and diluted in RPMI
1640 medium. For dose-response curves, EOL-1 cultures were initiated
at 3 ⫻ 10
5
cells/mL, and viable cell numbers were determined at the
beginning and after 48 hours using the Celltiter AQueousOne solution
(Promega, Madison, WI). Dose-response curves were fitted using Origin
(OriginLab, Northampton, MA).
Detection of apoptosis
Apoptotic cells were detected byflow cytometricanalysis usinga FACSCali-
bur Cytometer (Becton Dickinson, Mountain View, CA) after staining with
annexin V–fluorescein and propidium iodide (Roche, Indianapolis, IN).
Western blotting and immunoprecipitation
EOL-1 cellswere treatedwith kinase inhibitors for 3hours andthen lysed inlysis
buffer (Cell Signaling Technology, Beverly, MA) for immunoprecipitation or in
sodium dodecyl sulfate (SDS) sample buffer (Cell Signaling Technology) for
whole-cell lysates. Immunoprecipitation was performed using anti-PDGFR␣
(C-20) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and Protein A
agarose (Roche). Antibodies used were anti-phospho–signal transducer and
From the Division of Hematology and the Howard Hughes Medical Institute,
Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; Dana-
Farber Cancer Institute, Harvard Medical School, Boston, MA; Center for
Human Genetics–Flanders Interuniversity Institute of Biotechnology (VIB),
University of Leuven, Belgium; and the DSMZ–German Collection of
Microorganisms and Cell Cultures, Department of Human and Animal Cell
Cultures, Braunschweig, Germany.
Submitted July 23, 2003; accepted November 16, 2003. Prepublished online as
Blood FirstEdition Paper, November 20,2003; DOI 10.1182/blood-2003-07-2479.
Supported by National Institutes of Health grant DK50654 and CA66996 and
the Leukemia and Lymphoma Society (D.G.G), and by research funding from
Novartis PharmaAG (J.D.G.).
Reprints: Jan Cools, Center for Human Genetics, Campus Gasthuisberg O&N 06,
Herestraat 49,B-3000 Leuven,Belgium; e-mail:jan.cools@med.kuleuven.ac.be.
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.
© 2004 by The American Society of Hematology
2802 BLOOD, 1APRIL 2004
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activator of transduction 5 (STAT5) (Cell Signaling), anti-PDGFR␣ (951),
anti-STAT5a (Santa Cruz Biotechnology), antiphosphotyrosine (4G10) (Upstate
Biotechnology, LakePlacid, NY), antimouse-peroxidase (PO),and antirabbit-PO
(APBiotech, Piscataway, NJ).
Results and discussion
We performed RT-PCR on RNA obtained from 67 acute myeloid
leukemia (AML) and 20 acute lymphoid leukemia (ALL) cell lines,
with a primer combination that would detect all known FIP1L1-
PDGFRA fusion variants.
4,7
In 1 AML cell line, EOL-1 (and
derivative EOL-3), a fusion between FIP1L1 and PDGFRA was
detected (Figure 1). No other cell lines harbored this fusion gene
(data not shown).
In consonance with our previous observations in patients with
FIP1L1-PDGFRA–positive CEL,
4,7
the EOL-1 cell line expressed
in-frame FIP1L1-PDGFRA fusion transcripts, with the fusion of
exon 9 of FIP1L1 to a truncated exon 12 of PDGFRA. Because of
splice variation within FIP1L1, different fusion transcripts were
observed in the EOL-1 cells (exon 8a can be present or absent
between exon 8 and exon 9, but in both variants an open-reading
frame is present) (Figure 1). Cloning of the fusion gene at the DNA
level confirmed that the breakpoint in FIP1L1 was located in intron
9 and that the breakpoint in PDGFRA was located in exon 12. No
reciprocal PDGFRA-FIP1L1 fusion gene could be detected (Figure
1). Taken together, these data indicate that FIP1L1-PDGFRA
fusion in the EOL-1 cell line is the consequence of the
del(4)(q12q12) interstitial chromosomal deletion, as observed in
FIP1L1-PDGFRA–positive CELpatients.
4
The EOL-1 cell line did
not express wild-type PDGFRA but did express wild-type FIP1L1
(Figure 1).
EOL-1 was originally derived from the blasts of a 33-year-old
man with CEL, when disease progressed to AML (54% blasts;
karyotype, 48,XY,⫹6,⫹8,9q⫺).
8
We recently discovered that this
cell line harborsa partial tandem duplication within the MLL gene.
9
Translocations involving MLL and partial tandem duplication
within the MLL gene were described as leukemogenic mutations
involved in the pathogenesis of AML.
10,11
Mouse models have
demonstrated that MLL fusion proteins are necessary but insuffi-
cient for leukemogenesis.
10
Based on these and other observations,
it has been proposed that AML cells harbor at least 2 mutations, 1
that confers a proliferative or survival advantage, or both, and 1
that results in impaired differentiation of hematopoietic progeni-
tors.
12
Taken together, these findings suggest that FIP1L1-
PDGFR␣ and mutated MLL may cooperate to cause the progres-
sion of CEL to AML.
We next tested for dose-dependent inhibition of the growth of
EOL-1 cells by the kinase inhibitors imatinib
13
and PKC412,
14
both
known to inhibit FIP1L1-PDGFR␣,
4,15
and SU5614,
16
known to
inhibit FMS-like tyrosine kinase 3 (FLT3).
17,18
The growth of
EOL-1 cells was inhibited by these drugs, with cellular IC
50
of
approximately 0.8 nM for imatinib, 20 nM for PKC412, and 50 nM
for SU5614 (Figure 2A). Twenty-four hours after drug treatment,
most EOL-1 cells were apoptotic (Figure 2B), indicating that
growth inhibition by these 3 drugs was caused by apoptosis.
FIP1L1-PDGFR␣ has recently been identified as the major phos-
phorylated protein in EOL-1 cells.
19
Our results suggest that all 3
inhibitors directly affect FIP1L1-PDGFR␣ activity, as indicted by
Figure 1. Fusion of FIP1L1 to PDGFRA in the EOL-1 cell line. Detection
of the FIP1L1-PDGFRAfusion transcriptin the EOL-1and EOL-3celllines
(A), detection of FIP1L1 expression in the EOL-1 cell line (B), and
amplification of theFIP1L1-PDGFRA fusion gene onDNAfrom the EOL-1
cell line (C). Different transcripts are observed for FIP1L1 and FIP1L1-
PDGFRA (A-B) because of alternative splicing. Expressions of PDGFRA-
FIP1L1 and native PDGFRA were not detected (B). The sequence of the
fusiongene surroundingthe deletionis shownat theDNAlevel,atthe RNA
level(after splicing),and atthe proteinlevel (D). Splicedonor andacceptor
sites are underlined. A cryptic splice site is used in exon 12 of PDGFRA.
Schematic representation of FIP1L1, PDGFR␣, and FIP1L1-PDGFR␣
proteins (E). Points at which the proteins are interrupted by the deletion
are indicated by arrowheads.
FUSION OF FIP1L1TO PDGFRA INTHE EOL-1 CELLLINE 2803BLOOD, 1APRIL 2004
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the dose-dependent decrease of phosphorylation of FIP1L1-
PDGFR␣ and STAT5, a downstream effector of FIP1L1-PDGFR␣–
mediated signal transduction. A 50% reduction of phosphorylation
of FIP1L1-PDGFR␣ was reached at approximately 5 nM imatinib,
100 nM PKC412, and 50 nM SU5614 (Figure 2C). Because these 3
inhibitors also inhibit KIT and because PKC412 and SU5614 also
inhibit FLT3, we investigated whether the inhibition of FLT3 or
KIT could be involved in the growth inhibition of EOL-1 cells.
However, we did not find evidencefor the phosphorylation of FLT3
or KIT. Although we cannot exclude that the inhibition of other
native tyrosine kinases may contribute to the inhibition of cell
growth, our results suggest that growth inhibition and induction of
apoptosis are primarily the result of direct FIP1L1-PDGFR␣
inhibition.
In conclusion, although CEL has been observed to progress to
AML,
4,8,20
our results suggest that FIP1L1-PDGFR␣ is not fre
-
quently involved in the pathogenesis ofAML. We identified EOL-1
as the first cell line expressing the FIP1L1-PDGFRA fusion gene
and as a valuable in vitro model for the screening for new
FIP1L1-PDGFR␣ inhibitors in the context of a human cell line
expressing the fusion protein from its endogenous promoter.
EOL-1 cells may provide a unique reagent for understanding
lineage involvement in FIP1L1-PDGFRA–positive CEL, FIP1L1-
PDGFR␣–mediated signaling, and the transcriptional targets of
FIP1L1-PDGFR␣ that contribute to the CEL phenotype.
Acknowledgments
J.C. is a “postdoctoraal onderzoeker” of the “Fonds voor Weten-
schappelijk Onderzoek–Vlaanderen.” B.J.P.H. is a senior clinical
fellow of the Leukaemia Research Fund (United Kingdom).
D.G.G. is an associate investigator of the Howard Hughes Medical
Institute.
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... A number of the additional targets captured by these clusters include pathways that have been implicated in disease pathogenesis and drug resistance in AML. These include targeting of PDGFRα in the rare FIP1L1-PDGFRα positive chronic eosinophilic leukemia form of AML (21) and of CSF1R to interrupt a paracrine signal between monocytes and AML cells (8,9). In addition, mutation of TP53 is a strong driver of drug resistance in both AML, including resistance to FLT3 inhibitors (6) and venetoclax (10,22,23). ...
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ATG5 promotes eosinopoiesis, but inhibits eosinophil effector functions
Article
  • Feb 2021
Eosinophils are white blood cells contributing to the regulation of immunity and they are involved in the pathogenesis of numerous inflammatory diseases. In contrast to other cells of the immune system, no information is available about the role of autophagy in eosinophil differentiation and functions. In order to study the autophagic pathway in eosinophils, we generated conditional knockout mice in which Atg5 is deleted within the eosinophil lineage only (designated Atg5eoΔ mice). Eosinophilia was provoked by crossbreeding Atg5eoΔ mice with Il5 (IL-5) overexpressing transgenic mice (designated Atg5eoΔIl5tg mice). Deletion of Atg5 in eosinophils resulted in a dramatic reduction in the number of mature eosinophils in blood and in an increase of immature eosinophils in the bone marrow. Atg5-knockout eosinophil precursors exhibited reduced proliferation under both in vitro and in vivo conditions, but no increased cell death. Moreover, reduced differentiation of eosinophils in the absence of Atg5 was also seen in mouse and human models of chronic eosinophilic leukemia. Atg5-knockout blood eosinophils demonstrated augmented levels of degranulation and bacterial killing in vitro. Moreover, in an experimental in vivo model, we observed that Atg5eoΔ mice achieve better clearance of the local and systemic bacterial infection with Citrobacter (C.) rodentium. Evidence for increased degranulation of ATG5low-expressing human eosinophils was also obtained in both tissues and blood. Taken together, mouse and human eosinophil hematopoiesis and effector functions are regulated by ATG5 which controls the amplitude of overall antibacterial eosinophil immune responses.
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... Seven partner genes of PDGFRA have been described hitherto, including FIP1L1 and FOXP1 [8]. The eosinophilic AML cell line EOL-1 expresses the fusion gene FIP1L1-PDGFRA and serves as a unique model for revealing leukemic functions of this oncogene [9]. In addition, EOL-1 contains a partial tandem duplication of the KMT2A gene [10], thus combining two genetic characteristics from different subgroups of myeloid malignancies. ...
Aberrant expression of NKL homeobox genes HMX2 and HMX3 interferes with cell differentiation in acute myeloid leukemia
Article
Full-text available
The NKL-code describes normal expression patterns of NKL homeobox genes in hematopoiesis. Aberrant expression of NKL homeobox gene subclass members have been reported in several hematopoietic malignancies including acute myeloid leukemia (AML). Here, we analyzed the oncogenic role of the HMX-group of NKL homeobox genes in AML. Public expression profiling data–available for HMX1 and HMX2—indicate aberrant activity of HMX2 in circa 2% AML patients overall, rising to 31% in those with KMT2A/MLL rearrangements whereas HMX1 expression remains inconspicuous. AML cell lines EOL-1, MV4-11 and MOLM-13 expressed both, HMX2 and neighboring HMX3 genes, and harbored KMT2A aberrations, suggesting their potential functional association. Surprisingly, knockdown experiments in these cell lines demonstrated that KMT2A inhibited HMX2/3 which, in turn, did not regulate KMT2A expression. Furthermore, karyotyping and genomic profiling analysis excluded rearrangements of the HMX2/3 locus in these cell lines. However, comparative expression profiling and subsequent functional analyses revealed that IRF8, IL7- and WNT-signalling activated HMX2/3 expression while TNFa/NFkB- signalling proved inhibitory. Whole genome sequencing of EOL-1 identified two mutations in the regulatory upstream regions of HMX2/3 resulting in generation of a consensus ETS-site and transformation of a former NFkB-site into an SP1-site. Reporter-gene assays demonstrated that both mutations contributed to HMX2/3 activation, modifying ETS1/ELK1- and TNFalpha-mediated gene regulation. Moreover, DMSO-induced eosinophilic differentiation of EOL-1 cells coincided with HMX2/3 downregulation while knockdown of HMX2 induced cell differentiation, collectively supporting a fundamental role for these genes in myeloid differentiation arrest. Finally, target genes of HMX2/3 were identified in EOL-1 and included suppression of differentiation gene EPX, and activation of fusion gene FIP1L1-PDGFRA and receptor-encoding gene HTR7, both of which enhanced oncogenic ERK-signalling. Taken together, our study documents a leukemic role for deregulated NKL homeobox genes HMX2 and HMX3 in AML, revealing molecular mechanisms of myeloid differentiation arrest.
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... Furthermore, the proliferation of the shLCP1 knockdown cell lines was moderately but significantly reduced in comparison to 32D-F/P scr cells ( Figure 1D). Reduction of STAT1 Y701 and AKT Ser473 phosphorylation was confirmed in the human eosinophilia-derived F/P-positive cell line Eol-1 35 when LCP1 messenger RNA (mRNA) expression was targeted by shRNA ( Figure 1E, shLCP1 #2). Although reduction of LCP1 protein was not effective in shLCP1 #2 cells, phosphorylation of LCP1 Ser5 was reduced, probably explaining the reduction of p-STAT1 and pS-AKT ( Figure 1E). ...
LCP1 triggers mTORC2/AKT activity and is pharmacologically targeted by enzastaurin in hypereosinophilia
Article
Full-text available
  • Nov 2019
Hypereosinophilia (HE) is caused by a variety of disorders, ranging from parasite infections to autoimmune diseases and cancer. Only a small proportion of HE cases are clonal malignancies, and one of these, the group of eosinophilia‐associated tyrosine kinase fusion‐driven neoplasms, is sensitive to tyrosine kinase inhibitors, while most subtypes lack specific treatment. Eosinophil functions are highly dependent on actin polymerization, promoting priming, shape change, and infiltration of inflamed tissues. Therefore, we investigated the role of the actin‐binding protein lymphocyte cytosolic protein 1 (LCP1) in malignant and nonmalignant eosinophil differentiation. We use the protein kinase C‐β (PKCβ) selective inhibitor enzastaurin (Enza) to dephosphorylate and inactivate LCP1 in FIP1L1‐platelet‐derived growth factor receptor α (PDGFRA)‐positive Eol‐1 cells, and this was associated with reduced proliferation, metabolic activity, and colony formation as well as enhanced apoptosis and impaired migration. While Enza did not alter FIP1L1‐PDGFRA‐induced signal transducer and activator of transcription 3 (STAT3), STAT5, and ERK1/2 phosphorylation, it inhibited STAT1Tyr701 and AKTSer473 (but not AKTThr308) phosphorylation, and short hairpin RNA knockdown experiments confirmed that this process was mediated by LCP1 and associated mammalian target of rapamycin complex 2 (mTORC2) activity loss. Homeobox protein HoxB8 immortalized murine bone marrow cells showed impaired eosinophilic differentiation upon Enza treatment or LCP1 knockdown. Furthermore, Enza treatment of primary HE samples reduced eosinophil differentiation and survival. In conclusion, our data show that HE involves active LCP1, which interacts with mTOR and triggers mTORC2 activity, and that the PKCβ inhibitor Enza as well as targeting of LCP1 may provide a novel treatment approach to hypereosinophilic disorders.
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... The EOL-1 cell line is characterized by the expression of the FIP1L1-PDGFRA fusion gene, which encodes the 110-kDa oncogenic variant of PDGFRα. This genetic background makes this cell line a commonly used in vitro model of FIP1L1-PDGFRA-positive chronic eosinophilic leukaemia [11]. The high sensitivity of the EOL-1 cell line to our compounds encouraged us to describe their mechanism of action in this cellular model in more detail. ...
Activity of 2,6,9-trisubstituted purines as potent PDGFRα kinase inhibitors with antileukemic activity
Article
  • Aug 2019
Receptor tyrosine kinase PDGFRα is often constitutively activated in various tumours and is regarded as a drug target. Here, we present a collection of 2,6,9-trisubstituted purines with nanomolar potency against PDGFRα and strong and selective cytotoxicity in the human eosinophilic leukaemia cell line EOL-1 that expresses the FIP1L1-PDGFRA oncogene. In treated EOL-1 cells, the example compound 14q inhibited the autophosphorylation of PDGFRα and the phosphorylation of STAT3 and ERK1/2. Interestingly, we observed pronounced and even increased effects of 14q on PDGFRα and some of its downstream signalling pathways after drug washout. In accordance with suppressed PDGFRα signalling, treated cells were arrested in the G1 phase of the cell cycle and eventually underwent apoptosis. Our results show that substituted purines can be used as specific modulators of eosinophilic leukaemia.
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... The origin was established at diagnosis from the peripheral blood of a 33-year-old man with acute myeloid (eosinophilic) leukemia following hypereosinophilic syndrome and is described to carry the fusion FIP1L1-PDGFRα gene [8]. The EoL-1 cell line is used as an in vitro model for the study of FIP1L1-PDGFRA-positive chronic eosinophilic leukemia [9] and is particularly useful for analyzing leukemic cell differentiation and the properties of malignant eosinophils [10]. EoL-1 cells differentiate not only phenotypically but also functionally into eosinophils by a number of stimuli (including alkaline pH, dimethylsulfoxide, TNF-α, G-CSF + TNF-α, HIL-3-derived factor, dibutyryl cAMP, IFN-γ). ...
Docosahexaenoic Acid Inhibits Proliferation of EoL-1 Leukemia Cells and Induces Cell Cycle Arrest and Cell Differentiation
Article
Full-text available
  • Mar 2019
Τhe effect of docosahexaenoic acid (DHA, an omega-3 polyunsaturated fatty acid) upon the proliferation of EoL-1 (Eosinophilic leukemia) cell line was assessed, while additional cellular events during the antiproliferative action were recorded. DHA inhibited EoL-1 cells growth dose-dependently by inducing growth arrest at G0/1 phase of the cell cycle. After DHA addition to the cells, the expression of MYC oncogene was decreased, PTAFR-mRNA overexpression was observed which was used as a marker of differentiation, and PLA2G4A-mRNA increase was recorded. The enzymatic activities of phospholipase A2 (PLA2), a group of hydrolytic enzymes, whose action precedes and leads to PAF biosynthesis through the remodeling pathway, as well as platelet activating factor acetylhydrolase (PAFAH) which hydrolyses and deactivates PAF, were also measured. DHA had an effect on the levels of both the intracellular and secreted activities of PLA2 and PAFAH. The inflammatory cytokines IL-6 and TNF-α were also detected in high levels. In conclusion, DHA-induced EoL-1 cells differentiation was correlated with downregulation of MYC oncogene, overexpression of PTAFR and PLA2G4A-mRNAs, increase of the inflammatory cytokines production, and alteration of the enzymatic activities that regulate PAF levels. DHA is a natural substance and the understanding of its action on EoL-1 cells on molecular level could be useful in further investigation as a future therapeutic tool against F/P + hypereosinophilic syndrome.
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... The etiology of the primary disease often specifies the best therapeutic strategy. For example, a subset of patients with HES have an 800kb interstitial deletion on chromosome 4 (4q12) that results in the fusion of an unknown gene FIP1-L1 with PDGFRA (Cools et al. 2003(Cools et al. , 2004a. This fusion gene produces a constitutively active tyrosine kinase (PDGFRA) that is exquisitively sensitive to the inhibitor imatinib mesylate, which is now approved for the treatment of HES (Gleevec). ...
Eosinophils: Biological Properties and Role in Health and Disease
Article
Full-text available
  • May 2008
  • CLIN EXP ALLERGY
Eosinophils are pleiotropic multifunctional leukocytes involved in initiation and propagation of diverse inflammatory responses, as well as modulators of innate and adaptive immunity. In this review, the biology of eosinophils is summarized, focusing on transcriptional regulation of eosinophil differentiation, characterization of the growing properties of eosinophil granule proteins, surface proteins and pleiotropic mediators, and molecular mechanisms of eosinophil degranulation. New views on the role of eosinophils in homeostatic function are examined, including developmental biology and innate and adaptive immunity (as well as their interaction with mast cells and T cells) and their proposed role in disease processes including infections, asthma, and gastrointestinal disorders. Finally, strategies for targeted therapeutic intervention in eosinophil-mediated mucosal diseases are conceptualized.
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Synthesis and biological activity evaluation of novel 2,6,9-trisubstituted purine conjugates as potential protein kinases inhibitors
Article
  • Mar 2022
  • BIOORG MED CHEM LETT
Deregulation of protein kinases is often associated with uncontrolled cell proliferation in various tumours and the inhibition of kinase activity remains an important target for anti-tumour drug development. Here, we report a novel series of 2-aminocyclohexylamino-6-(substituted benzylamino/anilino)-9-cyclopentylpurine derivatives conjugated with putrescine, spermidine or spermine moiety in an effort to expand library of highly potent 2,6,9-trisubstituted purine kinase inhibitors. Presented aniline-type conjugates exhibit significant cytotoxic activity in MV4-11 and EOL-1 cell lines which correlates with FLT3-ITD and PDGFRα inhibition. Furthermore, 6-anilinopurines affected MAPK and STAT pathways in the treated MV4-11 cells and induced cell cycle arrest in the G1 phase. 6-Benzylaminopurines showed comparable CDK2 inhibitory activity to 6-anilinopurines, however, the PDGFRα and FLT3-ITD inhibition was strongly suppressed. Our results show that novel compounds containing aniline in the structure can be involved in the development of potent tyrosine kinase inhibitors with strong activity toward acute myeloid leukemia or chronic eosinophilic leukemia.
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Discovery of 4-((N-(2-(dimethylamino)ethyl)acrylamido)methyl)-N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)benzamide (CHMFL-PDGFR-159) as a highly selective type II PDGFRα kinase inhibitor for PDGFRα driving chronic eosinophilic leukemia
Article
  • Mar 2018
Through exploration of the non-highly conserved allosteric hydrophobic pocket generated by DFG-out shifting in the inactive conformation, we discovered a highly selective type II PDGFRα kinase inhibitor 15i (CHMFL-PDGFRα-159), which exhibited strong potency against purified PDGFRα (IC50: 132 nM) but not structurally similar PDGFRβ, ABL, c-KIT and VEGFR2 kinases. In addition, it displayed a high selectivity profile (S score (10) = 0.02) at the concentration of 1 μM among 468 kinases/mutants in the KINOMEscan profiling. X-ray crystal structure of 15i in complex with PDGFRα revealed a distinct binding feature in the allosteric hydrophobic pocket which might help to expand the diversity of type II kinase inhibitors. Compound 15i potently inhibited the proliferation of PDGFRα driving Chronic Eosinophilic Leukemia (CEL) cell line EOL-1 through strong blockage of PDGFRα mediated signaling pathways, arresting cell cycle progression, and induction of apoptosis. Furthermore, compound 15i effectively suppressed the EOL-1 tumor progression in the xenograft model and increased the survival rate in the engraftment tumor model.
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Trisubstituted purine inhibitors of PDGFRα and their antileukemic activity in the human eosinophilic cell line EOL-1
Article
  • Oct 2017
  • BIOORGAN MED CHEM
Inhibition of protein kinases is a validated concept for pharmacological intervention in cancers. Many kinase inhibitors have been approved for clinical use, but their practical application is often limited. Here, we describe a collection of 23 novel 2,6,9-trisubstituted purine derivatives with nanomolar inhibitory activities against PDGFRα, a receptor tyrosine kinase often found constitutively activated in various tumours. The compounds demonstrated strong and selective cytotoxicity in the human eosinophilic leukemia cell line EOL-1, whereas several other cell lines were substantially less sensitive. The cytotoxicity in EOL-1, which is known to express the FIP1L1-PDGFRA fusion gene encoding an oncogenic kinase, correlated significantly with PDGFRα inhibition. EOL-1 cells treated with the compounds also exhibited dose-dependent inhibition of PDGFRα autophosphorylation and suppression of its downstream signaling pathways with concomitant G1 phase arrest, confirming the proposed mechanism of action. Our results show that substituted purines can be used as platforms for preparing tyrosine kinase inhibitors with specific activity towards eosinophilic leukemia.
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Establishment and characterization of a new human eosinophilic leukemia cell line
Article
Full-text available
  • Jan 1986
A human eosinophilic leukemia cell line, designated as EoL, was established from the peripheral blood of a patient with Philadelphia chromosome-negative eosinophilic leukemia (EL). The EoL cell line grows in single cell suspension with a doubling time of 48 hours for about one year. The reactivity of these cells was tested with a panel of monoclonal antibodies; they were found to express surface IA antigen, myeloid antigen (IF10, MY9) and membrane receptors for interleukin 2 (IL-2, Tac antigen). Under standard culture conditions, a small percentage of cells having more typical eosinophilic characteristics was present. These cells had cytoplasmic granules and were positive for Luxol-fast-blue and eosinophil peroxidase. Under culture conditions to induce the maturation of myeloid cells, such as alkaline medium or addition of dimethyl sulfoxide (DMSO), the frequency of cells with typical eosinophilic features increased to about 40%. In addition, cytogenetic studies showed that cultured cells and original leukemic blasts presented similar chromosome abnormalities. EoL seems to be a unique leukemic line committed to the eosinophilic lineage and can provide a useful in vitro model for the study of malignant eosinophilic properties.
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Partial Tandem Duplication of ALLÍas a Recurrent Molecular Defect in Acute Myeloid Leukemia with Trisomy II1
Article
Full-text available
  • Apr 1996
Gains of a single chromosome are frequent cytogenic findings in human cancer, but no molecular rearrangement has been consistently associated with any trisomy. In acute myeloid leukemia (AML), trisomy 11 (+11) occurring as a sole abnormality is the third most common trisomy. We have shown that the ALL1 gene, located at 11q23, can be rearranged as a result of a partial tandem duplication in two such cases of AML. To test the hypothesis that the partial tandem duplication of ALL1 is the recurrent molecular defect in cases of AML presenting with +11 as a sole cytogenic abnormality, we performed Southern analysis and PCR for defects of ALL1 in 17 cases of AML and one case of myelodysplastic syndrome with +11 or +11q but without cytogenic evidence of a structural abnormality involving 11q23. Twelve cases (67%) had rearrangement of ALL1, including 10 of 11 patients (91%) with +11 as a sole abnormality and 2 of 7 cases (29%) with +11 and other aberrations; all were classified as FAB M1 or M2. In 10 of the 12 cases, material was available for additional characterization; a partial tandem duplication of ALL1 was detected in each of these 10 cases (100%). Four cases demonstrated previously unreported duplications, two of which were detectable only by reverse transcription-PCR. Four patients with the ALL1 duplication also displayed a loss of material from 7q, suggesting an association between these two findings. We conclude that the partial tandem duplication of ALL1 is present in most, if not all, cases of AML with +11 as a sole abnormality, and can be found in cases of AML with +11 or +11q accompanied by other cytogenic abnormalities. The duplication is more prevalent in AML than was recognized previously in part because its size and location vary considerably, requiring a variety of molecular probes for detection. Our finding of the ALL1 duplication as a consistent defect in patients with +11 represents the first identification of a specific gene rearrangement associated with recurrent trisomy in human cancer.
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Establishment and characterization of a new human eosinophilic leukemia cell line
Article
  • Dec 1985
A human eosinophilic leukemia cell line, designated as EoL, was established from the peripheral blood of a patient with Philadelphia chromosome-negative eosinophilic leukemia (EL). The EoL cell line grows in single cell suspension with a doubling time of 48 hours for about one year. The reactivity of these cells was tested with a panel of monoclonal antibodies; they were found to express surface IA antigen, myeloid antigen (IF10, MY9) and membrane receptors for interleukin 2 (IL-2, Tac antigen). Under standard culture conditions, a small percentage of cells having more typical eosinophilic characteristics was present. These cells had cytoplasmic granules and were positive for Luxol-fast-blue and eosinophil peroxidase. Under culture conditions to induce the maturation of myeloid cells, such as alkaline medium or addition of dimethyl sulfoxide (DMSO), the frequency of cells with typical eosinophilic features increased to about 40%. In addition, cytogenetic studies showed that cultured cells and original leukemic blasts presented similar chromosome abnormalities. EoL seems to be a unique leukemic line committed to the eosinophilic lineage and can provide a useful in vitro model for the study of malignant eosinophilic properties.
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The hypereosinophilic syndrome. Analysis of fourteen cases with review of the literature
Article
  • Feb 1975
Several closely related disease entities make up the idiopathic hypereosinophilic syndrome (HES). The syndrome is manifest by persistent and prolonged eosinophilia with organ damage. A group of 14 patients had hematologic, cardiac, and neurologic abnormalities attributable to this disease. Patient survival and response to chemotherapy was significantly better in this group than in previously reported patients. The etiology of HES remains unknown, as does the mechanism of tissue damage.
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Differential Inhibition of the Epidermal Growth Factor-, Platelet-derived Growth Factor-, and Protein Kinase C-mediated Signal Transduction Pathways by the Staurosporine Derivative CGP 41251
Article
  • Nov 1992
The microbial alkaloid staurosporine is a potent but nonselective inhibitor of protein kinases. The derivative CGP 41251 has been shown to exert a high degree of selectivity for inhibition of protein kinase C activity. Both compounds are powerful inhibitors of proliferation of both normal and transformed cells in vitro and exert antitumor efficacy in vivo. In this work we have studied the mode of action of these compounds by analyzing their effects on early events in the induction of proliferation by different growth stimuli. Both drugs blocked the phorbol ester-induced expression of the c-fos proto-oncogene. The effect of CGP 41251 was reversible, since its removal led to a normal expression of c-fos mRNA in response to phorbol 12-myristate 13-acetate. Submicromolar concentrations of CGP 41251 and staurosporine directly inhibited both the platelet-derived growth factor (PDGF) receptor autophosphorylation and the c-fos mRNA expression induced by PDGF stimulation of intact BALB/c 3T3 cells. In contrast, ligand-induced epidermal growth factor receptor autokinase activity in A431 carcinoma cells and epidermal growth factor-dependent c-fos mRNA expression were relatively insensitive to inhibition by CGP 41251. Staurosporine suppressed signal generation by the epidermal growth factor receptor by reducing overall levels of the receptor. We conclude that CGP 41251 is a potent reversible inhibitor of protein kinase C and PDGF-mediated signal transduction. It inhibits the kinase activity of both protein kinase C and the PDGF receptor tyrosine kinase and the subsequent signaling cascade. The broad inhibition of kinases by staurosporine is also reflected at the cellular level and might contribute to the high toxicity of this compound, in comparison to CGP 41251.
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Eosinophilic leukemia and the idiopathic hypereosinophilic syndrome
Article
  • Nov 1996
Hypereosinophilic syndromes may result either from eosinophilic differentiation of a clone of neoplastic cells or from reactive eosinophilia. In other patients HES is idiopathic. It appears likely that in many patients the "idiopathic' hypereosinophilic syndrome is actually a chronic myeloproliferative disorder. Those cases showing an increase of blast cells or a demonstrable clonal cytogenetic abnormality should be classified as eosinophilic leukaemia. In other cases the neoplastic nature of the disease can be recognized only in retrospect when a granulocytic sarcoma or AMI, develops. A few cases of idiopathic HES are consequent on cytokine secretion whereas others remain idiopathic at the time of death. When eosinophilia occurs as a feature of an acute or chronic myeloid leukaemia or a chronic myeloproliferative disorder the eosinophils are usually part of the leukaemic clone. However, eosinophilia in association with acute lymphoblastic leukaemia is usually reactive. Rare cases have a biphenotypic leukaemia/lymphoma with both eosinophils and lymphoid cells arising from a mutant pluripotent stem cell.
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Synthesis and Biological Evaluations of 3-Substituted Indolin-2-ones: A Novel Class of Tyrosine Kinase Inhibitors That Exhibit Selectivity toward Particular Receptor Tyrosine Kinases
Article
  • Aug 1998
3-Substituted indolin-2-ones have been designed and synthesized as a novel class of tyrosine kinase inhibitors which exhibit selectivity toward different receptor tyrosine kinases (RTKs). These compounds have been evaluated for their relative inhibitory properties against a panel of RTKs in intact cells. By modifying the 3-substituted indolin-2-ones, we have identified compounds which showed selective inhibition of the ligand-dependent autophosphorylation of various RTKs at submicromolar levels in cells. Structure-activity analysis for these compounds and their relative potency and selectivity to inhibit particular RTKs has determined that (1) 3-[(five-membered heteroaryl ring)methylidenyl]indolin-2-ones are highly specific against the VEGF (Flk-1) RTK activity, (2) 3-(substituted benzylidenyl)indolin-2-ones containing bulky group(s) in the phenyl ring at the C-3 position of indolin-2-ones showed high selectivity toward the EGF and Her-2 RTKs, and (3) the compound containing an extended side chain at the C-3 position of the indolin-2-one (16) exhibited high potency and selectivity when tested against the PDGF and VEGF (Flk-1) RTKs. Recent published crystallographic data for two of these 3-substituted indolin-2-ones provides a rationale to suggest that these compounds may bind in the ATP binding pocket of RTKs. The structure-activity analysis supports the use of subsets of these compounds as specific chemical leads for the development of RTK-specific drugs with broad application for the treatment of human diseases.
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Continuous hematopoietic cell lines as model systems for leukemia-lymphoma research
Article
  • Dec 2000
  • LEUKEMIA RES
Along with other improvements, the advent of continuous human leukemia-lymphoma (LL) cell lines as a rich resource of abundant, accessible and manipulable living cells has contributed significantly to a better understanding of the pathophysiology of hematopoietic tumors. The first LL cell lines, Burkitt's lymphoma-derived lines, were established in 1963. Since then, more than 1000 cell lines have been described, although not all of them in full detail. The major advantages of continuous cell lines is the unlimited supply and worldwide availability of identical cell material, and the infinite viable storability in liquid nitrogen. LL cell lines are characterized generally by monoclonal origin and differentiation arrest, sustained proliferation in vitro under preservation of most cellular features, and specific genetic alterations. The most practical classification of LL cell lines assigns them to one of the physiologically occurring cell lineages, based on their immunophenotype, genotype and functional features. Truly malignant cell lines must be discerned from Epstein-Barr virus (EBV)-immortalized normal cells, using various distinguishing parameters. However, the picture is not quite so straightforward, as some types of LL cell lines are indeed EBV+, and some EBV+ normal cell lines carry also genetic aberrations and may mimic malignancy-associated features. Apart from EBV and human T-cell leukemia virus in some lines, the majority of wild-type LL cell lines are virus-negative. The efficiency of cell line establishment is rather low and the deliberate establishment of new LL cell lines remains by and large an unpredictable random process. Difficulties in establishing continuous cell lines may be caused by the inappropriate selection of nutrients and growth factors for these cells. Clearly, a generally suitable microenvironment for hematopoietic cells, either malignant or normal, cannot yet be created in vitro. The characterization and publication of new LL cell lines should provide important and informative core data, attesting to their scientific significance. Large percentages of LL cell lines are contaminated with mycoplasma (about 30%) or are cross-contaminated with other cell lines (about 15-20%). Solutions to these problems are sensitive detection, effective elimination and rigorous prevention of mycoplasma infection, and proper, regular authentication of cell lines. The underlying cause, however, appears to be negligent cell culture practice. The willingness of investigators to make their LL cell lines available to others is all too often limited. There is a need in the scientific community for clean and authenticated high-quality LL cell lines to which every scientist has access. These are offered by various institutionalized public cell line banks. It has been argued that LL cell lines are genetically unstable (both cytogenetically and molecular genetically). For instance, cell lines are supposed to acquire numerical and structural chromosomal alterations and various types of mutations (e.g. point mutations) in vitro. We present evidence that while nearly 100% of all LL cell lines indeed carry genetic alterations, these alterations appear to be stable rather than unstable. As an example of the practical utility of LL cell lines, the recent advances in studies of classical and molecular cytogenetics, which in large part were made possible by cell lines, are highlighted. A list of the most useful, robust and publicly available reference cell lines that may be used for a variety of experimental purposes is proposed. Clearly, by opening new avenues for investigation, studies of LL cell lines have provided seminal insights into the biology of hematopoietic neoplasia. Over a period of nearly four decades, these initially rather exotic cell cultures, known only to a few specialists, have become ubiquitous powerful research tools that are available to every investigator.
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