Establishment of Acute Myeloid Leukemic Xenografts as
Preclinical Models of Epigenetic Therapies
Satyananda Patel, Yanyan Zhang, Audrey
Cras, Mallorie Mangean-D¨¦pond, Fabien Zassadowski, Victor Maud, Bruno Cassinat,
Christine Chomienne, Fawzia Louache
Satyananda
Patel, Yanyan Zhang, Mallorie Mangean-D¨¦pond, Fawzia Louache, Gustave Roussy, 114 rue Edouard
Vaillant, 94805 Villejuif, France
Satyananda
Patel, Yanyan Zhang, Mallorie Mangean-D¨¦pond, Fawzia Louache, Institut National de la Sant¨¦ et de la
Recherche M¨¦dicale (INSERM), U1170, 114 rue Edouard Vaillant, 94805 Villejuif,
France
Satyananda
Patel, Yanyan Zhang, Mallorie Mangean-D¨¦pond, Fawzia Louache, University of Paris Sud, 114 rue
Edouard Vaillant, 94805 Villejuif, France
Satyananda Patel, Audrey Cras, Fabien Zassadowski,
Victor Maud, Bruno Cassinat, Christine Chomienne,
UMRS1131, Hôpital
Saint Louis, 1 avenue Claude Vellefaux F-75475 Paris, France
Audrey
Cras, Fabien Zassadowski, Victor Maud, Bruno Cassinat, Christine Chomienne, Departement of Hematology, IUH,
University of Paris-Diderot, Sorbonne, Cit¨¦, Paris, France
Audrey
Cras, Fabien Zassadowski, Victor Maud, Bruno Cassinat, Christine Chomienne, AP-HP, Hospital Saint-Louis, Unit¨¦ de
Biologie Cellulaire, 1 avenue Claude Vellefaux, F-75475 Paris, France
Correspondence to: Christine Chomienne, UMRS1131,
Institut Universitaire d¡¯H¨¦matologie, Hôpital Saint Louis, 1 avenue Claude
Vellefaux, Department of Haematology, F-75475 Paris, France.
Email: christine.chomienne@sls.aphp.fr
Telephone: +33-0142499402
Fax: +33-0142395476
Correspondence to:
Fawzia Louache, Inserm U1170, Gustave Roussy, 114 Rue Edouard Vaillant, 94805, Villejuif
Cedex, France.
Email: Fawzia.LOUACHE@gustaveroussy.fr
Telephone: +33-142114233
Fax: +33-0142115240
Received: July 1, 2015
Revised: September 5, 2015
Accepted: September 9, 2015
Published online: October 20, 2015
Satyananda
Patel, Yanyan Zhang, contributed equally to this work.
ABSTRACT
Aim: There is currently little information
on epigenetic therapy in in vivo preclinical models of acute myeloid
leukemia. The study was designed to establish a preclinical model for the
validation and optimization of epigenetic therapies and other treatment
strategies.
Materials and Methods: We first characterized the response of
normal immunodeficient mice to different doses of 5-aza-2¡¯-deoxycytidine
valproic acid and all-trans retinoic acid. This allowed us to define an
optimized 28-days long protocol showing low toxicity and associated with
efficient global DNA demethylation and histone acetylation of bone marrow
mononuclear cells.
Results: AML xenografts of 5 non-promyelocytic
acute myeloid leukemia patients were treated with the defined protocol. One out
of 5 demonstrated reduced disease burden, cell differentiation (increase of
CD15, CD11b and to some extent CD14 positive cells and decrease of CD34+
population) and reduced capacity in reinitiating leukemia in secondary
recipients.
Conclusion: These findings mimic results of
clinical trials and indicate that acute myeloid leukemia xenografts may provide
useful preclinical models to optimize epigenetic therapies.
© 2015 ACT. All rights reserved.
Key words: Acute Myeloid
Leukemia; Xenografts; Epigenetic; All-trans retinoic acid;
5-aza-2¡¯-deoxycytidine; Valproic acid; Leukemia initiating cells
Patel S, Zhang Y, Cras A, Mangean-D¨¦pond M, Zassadowski
F, Maud V, Cassinat B, Chomienne C, Louache F. Establishment of Acute Myeloid
Leukemic Xenografts as Preclinical Models of Epigenetic Therapies. International Journal of Hematology Research 2015; 1(3): 79-89
Available from: URL: http://www.ghrnet.org/index.php/ijhr/article/view/1272
INTRODUCTION
The majority of acute myeloid leukemia (AML) patients are treated with
intensive chemotherapy with serious limiting side effects for older age
patients and unfortunately with short complete remissions. The persistence of a
subpopulation of cells called leukemia-initiating cells (LIC) resistant to
chemotherapy would be responsible for relapses[1]. Thus AML
treatment still requires improved efficacy while preserving quality of life and
alternative approaches and targets are currently being tested.
Aberrant DNA
methylation signatures are common in AML and myelodysplastic syndrome (MDS)
patients with mutations in genes coding for key proteins such as DNA methyl
transferase 3 (DNMT3), Tet methylcytosine dioxygenase 2 (TET2), Isocitrate
Dehydrogenase 1/2 (IDH1/2) and Histone deacetylase (HDAC)[2]. Unlike
deletions or mutations of tumor suppressor genes, altered chromatin
modifications are reversible and thus novel approaches such as chromatin
remodeling or epigenetic therapy has been investigated as potential anti-cancer
therapeutic approaches[3-6].
5-aza-2¡¯-deoxycytidine (AZA), a DNA methyl transferase inhibitor, is
currently in clinical practice for patients with MDS[7] and with
acute and chronic leukemia[8]. Of the HDAC inhibitors, the short
chain fatty acid, valproic acid (2-propylpentanoic acid; VPA), used since many
years in the treatment of epilepsy and mood stabilizer[9], was shown
to stimulate differentiation and/or inhibit cell growth of leukemia[10],
but has limited clinical activity on its own in MDS[11] or AML[12]
patients. All-trans retinoic acid (ATRA) can induce hypomethylation and its
activity is enhanced by AZA[13]. PLZF-RARA acute promyelocytic
leukemia (APL) variant resistance to ATRA can be reversed by the addition of
HDAC inhibitors (Trichostatin A or sodium phenyl butyrate) both in vivo
and in vitro[14]. VPA and AZA have been shown to sensitize
AML cells for ATRA[11,12,15]. The in vitro data showed that,
the combination had differentiation effect in leukemic cell line (HL-60 and
MOLT4)[16]. Such combinations are also effective in solid tumors[17,18].
Based on these
in vitro data, clinical trials have been initiated to investigate the
use of HDAC inhibitors, in combination with retinoic acid and/or DNMT
inhibitors, for the treatment of solid tumors[19] and myeloid
malignancies[20,21]. We have participated in a phase 2 clinical
trial study, where the combination of AZA, VPA and ATRA was studied in 65
patients (median age 72, Ranging from 50-87 years) with high risk AML and MDS[21].
Of the 65 patients that received the 6 cycles of the combination AZA, VPA and
ATRA, 17 (26%) responded (14 achieved complete remission and 3 partial
remission)[21], highlighting both the potential and limitation of
these therapies as heterogeneity in the responses are observed.
In order, to
increase the number of patients likely to respond to more effective DNMT
inhibitor and HDAC inhibitor combinations, preclinical models that reflect
patients¡¯ responses are mandatory. Indeed, In vitro cultures of fresh
AML cells are not sufficient, as they do not always predict in vivo
preclinical efficacy or toxicity. Furthermore, AZA¡¯s mechanism of action at the
DNA level requires promoter accessibility which is best obtained during DNA
synthesis and replication, and difficult to obtain in fresh AML cells in
conventional liquid cultures. Considering the important place of AML mouse
xenografts to study both human leukemogenesis and leukemic stem cells, we have
developed a set of AML xenografts as a preclinical platform to study the
efficacy of AZA, VPA and ATRA combination in AML. Acute promyelocytic leukemia (APL,
AML3 subtype) were excluded as ATRA induces leukemic cell differentiation and
complete remission in a high proportion of APL patients[22], as well
as shown by us in APL xenografts[23].
In this study,
we show for the first time that AML preclinical xenografts can be used to
identify the AML samples sensitive to the AZA, VPA and ATRA combination and
thus providing excellent models for assessing more potent epigenetic drug
combinations for all AML patients.
MATERIALS and METHODS
Patient samples
Peripheral blood (PB) and/or bone marrow (BM) samples from AML patients
were obtained at diagnosis after informed consent (Table S1). BM and PB
mononuclear cells were isolated using density-gradient centrifugation. Cells
were injected intravenously into NOD/shi-SCID IL2R¦Ã-/- (NOG) mice
either fresh or after storage in liquid-nitrogen.
NOG
xenotransplantation assays
NOG mice provided from M Ito[24], were bred in the
pathogen-free breeding facility at Gustave Roussy, Villejuif. All animal
experiments were performed in accordance with the French Ministry on animal
health care regulation. Human CD45 cells (1¡Á106 cells) per mouse were transplanted intravenously into
6¨C8-weeks-old NOG mice, conditioned with either sub-lethal irradiation (3 Gy)
or Busulfan treatment (25mg/kg/d twice 24 hours before)[23,25,26].
PB samples were taken once a month to validate the engraftment.
Determination
of the maximum tolerable dose (MTD) for AZA and VPA in NOG mice
Eight-week-old NOG mice (n=5 in each cohort) were injected intraperitoneally
(300 µL per injection) with AZA (0.1 or 0.4 mg/kg) or VPA (125, 250, 400 and
1200 mg/kg) daily over a period of 7 days. Measurements of various
hematological parameters of blood were performed using the ¡°Melet Schloesing
MS9-5V hematology analyzer (Melet Schloesing Laboratoires, Osny, France)¡±. The
MTD was defined as the dose at which all animals survived the 7 days treatment
period and the consecutive 21 days of observation and at which overt clinical
signs of toxicity, such as body weight loss and mortality were absent. In
addition, blood cell counts were measured before treatment, during the
treatment, and after the observation period to detect delayed effects of the
substances.
Flow cytometry
analysis and cell purification
A panel of FITC, PE, PC7, PerCP or APC-conjugated antibodies was used
against mouse CD45, human CD45, CD33, CD38, CD11b, CD15, CD14, CD34, CD44,
CXCR4, CD47, CD117, CD123 (Becton Dickinson, Le Pont-De-Claix, France). Human
CD45+ (hCD45+) cells from xenotransplanted mice were
purified by a double-positive magnetic cell sorting system (AutoMACS, Miltenyi
Biotec, France).
Experiments on
AML xenografts
Once AML engraftment was confirmed (hCD45>1%), mice were recruited
to the experiments. The experiments comprise 3 groups of xenografts: control
group (Placebo), ATRA group and AVA (AZA+VPA+ATRA) group. Mice were bled at
day-0, day-8 (before ATRA implantation), day-18 and on day 29. On day 29,
bleeding was followed by sacrifice and collection of BM cells and spleen cells.
Self-renewal capacity was assessed by secondary transplantations of equal
numbers of BM hCD45+ cells in conditioned mice.
Experiments on normal human hematopoiesis
For murine hematopoiesis, 8 weeks old C57BL/6 mice were used. For human
hematopoiesis, mononuclear cells were separated from human cord blood over a
ficoll density gradient. CD34 positive cells were isolated by a double positive
magnetic cell sorting system (AutoMACS; Miltenyi Biotec) according to the
manufacturer¡¯s instructions. CD34 positive cells (5¡Á104) were transplanted intravenously into 6¨C8-week-old
conditioned NOG mice. After validation of human engraftment, mice were treated
with PBS or 0.1mg/kg/day AZA and 250mg/kg/day VPA for 7 days and on day 8 ATRA
pellets (21day release) were implanted in treated group and untreated were
subjected to the same condition without drugs. At the end of the experiment,
mice were sacrificed and processed for various read outs in blood, BM and
spleen.
Western blot
For Western blot, BM cells were lysed in SDS lysis buffer (1% SDS, 10
mmol/L Tris/HCl (pH 7.4), 1 mmol/L vanadate) and proteins were denatured by the
addition of SDS-Gel loading buffer containing dithiothretol (DTT) followed by
heating in boiling water for 10 minutes. Equal amounts of proteins were
separated on a 10% SDS polyacrylamide gel and blotted onto polyvinylidene
difluoride membranes (Millipore). After blocking, the membranes were incubated
with rabbit anti-acetylated lysine (Cell signaling technology, Cat No #9441;
1:1000) antibodies diluted in 5% milk in PBST overnight at 4¡ãC. The
peroxidase-conjugated Donkey anti-rabbit IgG (Sigma; 1:20,000 in 5% milk in
PBST) served as secondary antibody. The bands of acetylated-H3 and
acetylated-H4 were found in the range 15KD-20KD. Signals were visualized using
the Supersignal West Dura detection system (Thermo Scientific). Signal
intensities were measured by densitometric analysis using the Scion Image
(Scion Corporation).
DNA methylation
The global methylated DNA was estimated in the BM hCD45+
cells of NOG mice according to the manufacturer¡¯s instruction {Epigentek
[MethylFlashTM methylated DNA Quantification kit (Colorimetric) #P-1034], NY,
USA}. Briefly genomic DNA was extracted by Qiagen kit according to the
recommendation of the manufacturer. DNA (200ng) was incubated with capture
antibody against methylated DNA. Addition of detection antibody was followed by
the addition of developing solution and than plate reading was performed in a
spectrophotometer. The quantity of methylated DNA was interpolated from the
standard curve processed at the same time and the percentage of methylated DNA
was calculated and compared to control mice.
Human myeloid
colony assays
Purified hCD45+ BM cells (105) from AML xenograft
cells were suspended in 500 ¦Ìl ¦Á-MEM, and cultured in 1.8 mL complete
methylcellulose medium allowing the selective proliferation of human progenitor
cells (0.8% methylcellulose medium H4100 Stem Cell Technologies, Grenoble,
France], 30% FBS, 1% deionized BSA, 10-4 mol/L ¦Â-mercaptoethanol) with the
addition of 25 ng/mL human Stem Cell Factor (Biovitrum AB, Stockholm, Sweden),
10 ng/mL interleukin-3 (Peprotech, France), 2 U/mL erythropoietin (Amgen,
Thousand Oaks, CA), and 10ng/ml rhu-GM-CSF (Peprotech, France). Plates were
incubated at 37¡ãC in an air atmosphere supplemented with 5% CO2 and
saturated with humidity. Progenitors were scored at day 14.
Statistics
Kaplan-Meier analyses were used to assay the survival. Unless otherwise
stated, data represent means¡ÀSEM of at least 3 mice. Statistically significant
differences between means were calculated using the non-parametric, unpaired,
two tailed, student t-test with a threshold of p¡Ü0.05, p¡Ü0.01, p¡Ü0.001 designated by *, $, #
respectively.
Results and discussion
To set up a preclinical model of DNMT inhibitor and HDAC inhibitor
combination with AZA, VPA and ATRA used in clinical trials, we first studied,
in two different mice strains NOD/shi-SCID IL2R¦Ã-/- (NOG) and
C57/BL6, the AZA and VPA doses that were well tolerated and effective to induce
DNA hypomethylation and histone acetylation (Figure 1). Different doses of AZA
(0.1 and 0.4 mg/kg/day) and VPA (125, 250, 400, 1200mg/kg/day) were injected
intraperitoneally for 7 consecutive days and mice were monitored for the 21
following days. Based on cumulative survival (Figure 1A and 1Bi) and body
weight (Figure 1Bii), the tolerated doses for VPA and AZA in NOG mice and in
C57/BL6 (not shown) were identified to be 250mg/kg/day and 0.1mg/kg/day respectively,
within the range of reported doses in SCID mice[27]. As expected,
these doses induced global DNA demethylation (Figure 1C) ($p¡Ü0.01 compared to the
control/untreated) and histone acetylation (Figure 1D) of bone marrow
mononuclear cells by spectrophotometric and western blotting assays,
respectively.
We then studied the effects of the AVA combination in 5 different AML
xenografts, according to the protocol described in Figure 2A. AML xenografts
were obtained as previously described by Patel et al[23] and
Zhang et al[28]. AML cell patients¡¯ characteristics at
diagnosis are shown in Table 1. Before initiation of treatment, confirmation of
leukemia development was determined by flow cytometry. The left panels of
figure 2B show the anti human and anti mouse CD45 antibody labeling performed
on the PB of xenografts for each patient sample. The lymphoid CD19 and myeloid
CD33 marker profile in the hCD45+ population shown in the right panels
indicated a robust leukemic engraftment in xenografted mice. Mice were then
randomized and injected intraperitoneally with VPA (250mg/kg/d) and AZA
(0.1mg/kg/d) for 7 consecutive days followed by sub-cutaneous implantation of
one 10mg ATRA 21 daylong releasing pellet. AVA-treated AML xenografts were
compared with untreated (Sham-operated) and ATRA treated xenografts. For these
comparisons, we analyzed the expression of specific hematopoietic cell surface
markers or Cluster of Differentiation (CD) proteins by flow cytometry. CD34 is
a surface marker of undifferentiated cells and its down regulation suggests
that the cells have progressed down one of the various hematopoietic
differentiation cell lineages. As cells differentiate to myeloid lineage, they
express various markers of mature cells including CD11b (myeloid marker), CD15
(granulocytic marker) and CD14 (monocytic marker).
Depending on
the leukemic aggressiveness and mice survival, the level of activity of each
treatment on leukemia development within the peripheral blood and bone marrow
was evaluated at either day 18 or day 29. Results are expressed as percentages
of hCD45 leukemic cells in PB (Figure 3A, first row, the columns from left to
right in each row represent the data of each AML xenograft of the 5 AML patient
sampels, P1, P2, P3, P4 and P5) and absolute numbers in PB (Figure 3A, second
row) and BM (Figure 3A, third row) of AML xenografts. In addition, the
percentage of hCD45+ leukemic cells expressing selected differentiation markers
was evaluated in PB at day 18 (Figure 3B, rows a, b, c, d correspond to the
percentage of CD34, CD11b, CD15 and CD14 positive cells within the hCD45+
population, respectively) and at day 29 (figure 4B). The same assessment was
performed on BM hCD45+ leukemic cells at either day 29 or day 18 (Figure 4A)
depending on mice survival.
We observed
three different groups of responses to AVA or ATRA treatment of AML patient
xenografts.
Non-responsive
group: this group includes AML patient 1 (P1) xenografts (n= 6) for
whom, only minor modifications were detected in the peripheral blood (PB)
(Figure 3A P1a, b) and bone marrow (BM) (Figure 3A P1c) in response to
treatment whether in the percentage (Figure 3A P1a) or absolute cell counts of
hCD45 cells (Figure 3A P1b). In addition, no significant modifications in the
expression of the differentiation markers CD34, CD11b, CD15 and CD14 within the
hCD45+ cells could be detected in the PB at day18 (Figure 3B, a, b,
c, d) or at day 29 in BM (Figure 4A) or PB (Figure 4B).
Partial
responsive group: this group includes AML patient P2, P3 and P4 xenografts for
whom partial effects of either AVA or ATRA alone were observed.
P2 AML: while
the PB of the treated AML xenografts showed no modification in the percentage
(Figure 3A P2a) or cell counts of hCD45 (Figure 3A P2b), the BM cells showed a
significant (*p¡Ü0.05) decrease in the percentages of hCD45 (Figure 3A P2c) and the
absolute numbers (data not shown) suggesting a partial anti leukemic effect of
AVA and ATRA treatment. In line with this observation, a close analysis of the
phenotype of hCD45+ cells in the PB showed at D18 an increase (*p¡Ü0.05 and $p¡Ü0.01 comparing control with AVA and
ATRA, respectively) in the percentage of CD15+ (Figure 3B P2c) cells compared
to control mice. Similar results were obtained at day 29 in BM (Fig 4A) or PB
(Fig 4B). However, the persistence of CD34 expression supports only partial
effects of treatment (Figure 3B P2a) and (Fig 4A and B) in this leukemia.
P3 AML:
Similar partial effects of AVA treatment were observed for P3 xenografts (n=3).
Interestingly, for this patient, a sharp increase in the percentage of hCD45+
cells was observed in the PB (Figure 3A P3a) and BM (Figure 3A P3c) of AVA
treated mice, while the percentage of CD34 expressing cells in hCD45+
population showed a decrease in PB (Figure 3B P3a) and BM (Fig 4A) of treated
mice (*p¡Ü0.05). This decrease is associated with an increase (*p¡Ü0.05) in the percentage of cells
expressing CD15 in the PB (Figure 3B P3c) with AVA treatment. These results
suggest an increase in leukemic cell differentiation.
P4 AML:
Partial effects of AVA treatment were also observed for P4 xenotransplants for
which the percentage (Figure 3A, P4a) and the absolute number (Figure 3A, P4b)
of hCD45+ cells was not modified in the PB by AVA treatment. However,
phenotypic analysis of the hCD45+ cells in PB of treated mice showed a decrease
tendency in the percentage of CD34 expressing (Figure 3B, P4a) associated with
an increase tendency in the percentage of cells expressing CD11b and CD15
(Figure 3B, P4b, P4c).
Responsive
Group: one AML xenograft (P5) showed complete antileukemic effects of AVA and
ATRA associated with differentiation towards the monocytic lineage. A two fold
decreased in the percentages (Figure 3A P5a, #p¡Ü0.001) and the absolute numbers
(Figure 3A P5b, *p¡Ü0.05) of hCD45+ cells in PB was detected upon either treatment. The
percentage of leukemic cells in the BM (Figure 3A P5c) was correlated to that
observed in PB. The anti leukemic effects of AVA and ATRA were accompanied by a
parallel decrease (#p¡Ü0.001) in the percentage of CD34+
cells (Figure 3B P5a). Concomitantly, we observed an enhancement in the
percentages of cells expressing markers of myeloid differentiation including CD11b
(Figure 3B P5b), CD15 (Figure 3B P5c) and CD14 (Figure 3B P5d). Of note, an
increase in the percentage of cells expressing the monocytic marker was noted
with AVA (#p¡Ü0.001) and to a minor extent with ATRA (*p¡Ü0.001) alone. Interestingly, the
kinetics of cell differentiation of leukemic cells in the PB showed that
changes in percentage of CD34+ (Figure 5A1), CD11b+ (Figure 5A2), CD15+ (Figure
5A3) and CD14+ (Figure 5A4) cells within the hCD45 population were detected as
soon as day 18 indicating that the antileukemic effects of AVA and ATRA were
relatively rapid (Figure 5A 1, 2, 3, 4). The impact of treatments in the BM of
treated animals was determined at the time of sacrifice on day 29. We observed
that the percentages (Figure 5B1, (#p¡Ü0.001) and the absolute numbers
(Figure 5B2, *p¡Ü0.05) of hCD45+ cells were decreased by almost two fold upon ATRA or
AVA treatment. As illustrated in figure 5C, the percentages of leukemic cells
(hCD45+) expressing the differentiation markers CD11b (Figure 5Ciii), CD15 (Figure
5Civ) and CD14 (Figure 5Cv) were increased while that of CD34+ cells was
decreased (Figure 5Cii, iv). These results were observed for all the mice
within each group (Figure 5D) and correlated to those observed in PB (Figure 3B
P5). To further document the antileukemic effect of the AVA combination in this
AML xenograft, hCD45+ cells from PBS and AVA treated mice were isolated from BM
at day 29 and transplanted in secondary recipient mice. The percentage of
leukemic cells in the PB (hCD45+) was monitored after 5 weeks. Leukemia
engraftment in the secondary transplants was obtained in the recipient of
PBS-treated mice (Figure 5E1) while hardly any hCD45+ leukemic cells were
detected in the recipient of AVA treated mice (Figure 5E2). These in vivo
data were corroborated by the reduction of leukemic myeloid colonies obtained
from the hCD45 positive cells of AVA treated mice compared to the controls
(Figure 5F).
To determine
whether normal hematopoiesis could be altered by AVA combination in mice,
C57BL/6 mice were treated with either ATRA or AVA using the same protocol used
for AML mice xenografts As shown in figure 6A, upon 8 days treatment, the total
number of BM cells was not modified. However, the distribution of myeloid and
lymphoid cells was altered. Indeed, the B-lymphoid (B220+) and
T-lymphoid (CD3+) cell numbers decreased and myeloid cell number
(Gr1+) increased (Figure 6A, ¥p¡Ü0.01, &p¡Ü0.01 and @p¡Ü0.01 for B220, CD3 and Gr1,
respectively). At D29, AVA combination effect is even more prominent as illustrated
in figure 6B with an important increase in Gr1 positive cells while B220
positive and CD3 positive cell numbers decreased (Figure 6B, 6C). The
importance of AVA treatment was evaluated by calculating the AVA
treated/untreated ratio of B-lymphoid (B220+) and myeloid (Gr1+). As shown in
figure 6C, in AVA treated mice, the ratio for Gr1 was 1.6 at day 8 and reached
more than 6 at day 29. To evaluate the effects of these treatments on more
immature cells, bone marrow cells from mice treated for 29 days were plated in
colony assay. We also observed that the numbers of myeloid colonies were
superior ($p¡Ü0.01) in the AVA group compared to ATRA alone or controls (Figure 6D).
Thus, AVA combination alters the myeloid and lymphoid compartment but
importantly spares immature progenitor cells.
Finally, the
myeloid differentiation effect of the AVA combination was also investigated on
human hematopoiesis in chimeric NOG mice (Figure 7). In these experiments,
irradiated NOG mice were injected with human CD34+ cells from cord
blood. Three months after injection, chimeric mice fully reconstituted with
human hematopoietic cells were treated with AVA according to the protocol
described in figure 2A. Strikingly, AVA treatment of reconstituted mice
resulted in a decrease in the percentage of human CD45 positive cells (Figure
7A). More comprehensive analysis using phenotypic markers of BM cells at day 29
of treatment revealed that AVA modified the distribution of myeloid and
lymphoid cells with an decrease in the percentage of B-lymphoid (CD19+)
and an increase of CD15 and CD14 myeloid cells (Figure 7B). We also observed an
increase in the percentage of immature CD34+ cells in BM treated
mice. Consistent with this finding, increased human myeloid colonies were
observed (Figure 7C) as already observed in normal C57BL/6 mice. Together,
these results suggest that AVA treatment leads to a specific loss of lymphoid
cells but spares myeloid cells and maintains immature CD34+ cells.
Altogether, our results indicate that AVA treatment may induce leukemic cell
differentiation at least in some selected patients while inducing only minor
deleterious effects on normal hematopoiesis further strengthening the potential
of these drugs for AML therapy. In the present study, we used drugs whose
mechanisms of action are beginning to be known. Indeed, the best known
mechanism of action of ATRA is through differentiation[29], AZA is
through DNA demethylation[30] (Figure 1C) and VPA¡¯s action is
through histone acetylation[31-32] (Figure 1D). These modifications
may lead to apoptosis and death of the leukemic cells[29-32]. It
remains to know why normal and leukemic cells respond differently to the same
epigenetic signals.
In conclusion,
our study has allowed establishing preclinical models to study combination
therapy with epigenetic drugs. Several preclinical models to explore the
effects of chemotherapeutic drugs are presently used. So far, most of the
studies used well-characterized AML cell lines including OCI-AML2 cells[33],
U937 cells[34], the RUNX1-mutated CG-SH AML line[35] and
AML1/ETO-positive Kasumi-1 cells[36]. When inoculated in severe
combined immunodeficient (SCID) mice, these cells produced leukemia that can be
targeted using chemotherapeutic drugs. These in vivo leukemic models are
also frequently used to evaluate the effect of gene modification and silencing.
In our study, we choose to use primary AML cells from different AML subtypes
(AML-M0, M2, M5), which represent the huge heterogeneity of AML disease. In
line with this, level of each marker of differentiation is different between
AML patient cells. In addition, AML samples are heterogeneous in their ability
to engraft in immunodeficient mice with less than 50% of successful engraftment[23,37].
This great heterogeneity may explain the variations in sensitivity to drugs.
This is further illustrated by the well-known heterogeneity of responses of AML
patients in clinical trials (Soriano et al[20], Raffoux et
al[21]). Strikingly, our small cohort is able to replicate the
heterogeneity of responses obtained in clinical trials. Thus, our models should
allow testing other combinations of HDAC and DNMT inhibitors and identifying
predictive biomarkers of response.
Acknowledgments
This work was supported by grants from Canc¨¦ropôle Ile de France,
Association de la Recherche Contre
le Cancer, INSERM and Universit¨¦ Paris Diderot, Universit¨¦ Paris XI, Association
Laurette Fugain, Association Cent pour Cent la vie, the Fondation Gustave
Roussy, Fondation de France. Satyananda Patel was a recipient of a
post-doctoral fellow ship from Canc¨¦ropôle Ile de France. We thank members of
the Gustave Roussy animal facility and the flow cytometry facility, Elisabeth
Savariau of the photography laboratory of the Institute of Hematology. We are
also grateful to M. Ito (Kawasaki, Japan) for the NOG mice.
Authorship: SP, YZ, FL performed research and analysed data; SP wrote
the paper; AC, MMD, FZ and BC performed research and analyzed data; FL, CC
designed research, analyzed the data and wrote the paper. All authors read,
commented on and approved the final version of the manuscript.
CONFLICT
OF INTERESTS
The authors have no conflicts of
interest to declare.
REFERENCES
1.
1. Bonnet,
D., Dick, J.E. Human acute myeloid leukemia is organized as a hierarchy that
originates from a primitive hematopoietic cell. Nat Med. 1997;3(7):730-737.
2.
Cazzola, M., Della, Porta, M.G.,
Malcovati, L. The genetic basis of myelodysplasia and its clinical relevance.
Blood. 2013;122(25):4021-4034.
3.
Herman, J.G., Baylin, S.B. Gene
silencing in cancer in association with promoter hypermethylation. N Engl J Med.
2003;349(21):2042-2054.
4.
4. Kitamura,
K., Hoshi, S., Koike, M., Kiyoi, H., Saito, H., Naoe, T. Histone deacetylase
inhibitor but not arsenic trioxide differentiates acute promyelocytic leukaemia
cells with t(11;17) in
combination with all-trans retinoic acid. Br J Haematol. 2000;108(4):696-702.
5.
Greenblatt, S.M., Nimer, S.D. Chromatin
modifiers and the promise of epigenetic therapy in acute leukemia. Leukemia.
2014;28(7):1396-1406.
6.
Kelly, T.K., De Carvalho, D.D., Jones,
P.A. Epigenetic modifications as therapeutic targets. Nat Biotechnol.
2010;28(10):1069-1078.
7.
Fenaux, P., Seymour, J.F., Santini, V.,
Silverman, L., Gore, S., List, A., Sanz, G., Mufti, G.J., Estey, E., Swern,
A.S., Beach, C.L., Hellstrom-Linderberg, E. Challenges of phase III trial
design for novel treatments in diseases with no standard treatment: the AZA-001
myelodysplasia study model. Leuk Res. 2014;38(2):258-262.
8.
Jones, P.A., Baylin, S.B. The
fundamental role of epigenetic events in cancer. Nat Rev Genet.
2002;3(6):415-428.
9.
Phiel, C.J., Zhang, F., Huang, E.Y.,
Guenther, M.G., Lazar, M.A., Klein, P.S. Histone deacetylase is a direct target
of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J
Biol Chem. 2001;276(39):36734-36741.
10.
Gottlicher, M., Minucci, S., Zhu, P.,
Kramer, O.H., Schimpf, A., Giavara, S., Sleeman, J.P., Lo Coco, F., Nervi, C.,
Pelicci, P.G., Heinzel, T. Valproic acid defines a novel class of HDAC
inhibitors inducing differentiation of transformed cells. EMBO J.
2001;20(24):6969-6978.
11.
Kuendgen, A., Strupp, C., Aivado, M.,
Bernhardt, A., Hildebrandt, B., Haas, R., Germing, U., Gattermann, N. Treatment
of myelodysplastic syndromes with valproic acid alone or in combination with
all-trans retinoic acid. Blood. 2004;104(5):1266-1269.
12.
Kuendgen, A., Schmid, M., Schlenk, R.,
Knipp, S., Hildebrandt, B., Steidl, C., Germing, U., Haas, R., Dohner, H.,
Gattermann, N. The histone deacetylase (HDAC) inhibitor valproic acid as
monotherapy or in combination with all-trans retinoic acid in patients with
acute myeloid leukemia. Cancer. 2006;106(1):112-119.
13.
Di Croce, L., Raker, V.A., Corsaro, M.,
Fazi, F., Fanelli, M., Faretta, M., Fuks, F., Lo Coco, F., Kouzarides, T.,
Nervi, C., Minucci, S., Pelici, P.G. Methyltransferase recruitment and DNA
hypermethylation of target promoters by an oncogenic transcription factor.
Science. 2002;295(5557):1079-1082.
14.
Cote, S., Rosenauer, A., Bianchini, A.,
Seiter, K., Vandewiele, J., Nervi, C., Miller, W.H., Jr. Response to histone
deacetylase inhibition of novel PML/RARalpha mutants detected in retinoic
acid-resistant APL cells. Blood. 2002;100(7):2586-2596.
15.
Cimino, G., Lo-Coco, F., Fenu, S.,
Travaglini, L., Finolezzi, E., Mancini, M., Nanni, M., Careddu, A., Fazi, F.,
Padula, F., Fiorini, R., Spiriti, M.A., Petti, M.C., Venditti, A., Amadori, S.,
Mandelli, F., Pelicci, P.G., Nervi, C. Sequential valproic acid/all-trans
retinoic acid treatment reprograms differentiation in refractory and high-risk
acute myeloid leukemia. Cancer Res. 2006;66(17):8903-8911.
16.
Yang, H., Hoshino, K., Sanchez-Gonzalez,
B., Kantarjian, H., Garcio-Manero, G. Antileukemia activity of the combination
of 5-aza-2'-deoxycytidine with valproic acid. Leuk Res. 2005;29(7):739-748.
17.
Ahuja, N., Easwaran, H., Baylin, S.B.
Harnessing the potential of epigenetic therapy to target solid tumors. J Clin
Invest. 2014;124(1):56-63.
18.
18 Cras,
A., Politis, B., Balitrand, N., Darsin-Bettinger, D., Boelle, P.Y., Cassinat,
B., Toubert, M.E., Chomienne, C. Bexarotene via CBP/p300 induces suppression of
NF-kappaB-dependent cell growth and invasion in thyroid cancer. Clin Cancer
Res. 2012;18(2):442-453.
19.
19. Kelly,
W.K., O'Connor, O.A., Marksm P.A. Histone deacetylase inhibitors: from target to clinical trials. Expert Opin
Investig Drugs. 2002;11(12):1695-1713.
20.
Soriano, A.O., Yang, H., Faderl, S.,
Estrov, Z., Giles, F., Ravandi, F., Cortes, J., Wierda, W.G., Ouzounian, S.,
Quezada, A., Pierce, S., Estey, E.H., Issa, J.P., Kantarjian, H.M.,
Garcia-Manero, G. Safety and clinical activity of the combination of
5-azacytidine, valproic acid, and all-trans retinoic acid in acute myeloid
leukemia and myelodysplastic syndrome. Blood. 2007;110(7):2302-2308.
21.
Raffoux, E., Cras, A., Recher, C.,
Boelle, P.Y., de Labarthe, A., Turlure, P., Marolleau, J.P., Reman, O., Gardin,
C., Victor, M., Maury, S., Rousselot, P., Malfuson, J.V., Maarek, O., Daniel,
M.T., Fenaux, P., Degos, L., Chomienne, C., Chevret, S., Dombret, H. Phase 2
clinical trial of 5-azacitidine, valproic acid, and all-trans retinoic acid in
patients with high-risk acute myeloid leukemia or myelodysplastic syndrome.
Oncotarget. 2010;1(1):34-42.
22.
Chomienne, C., Ballerini, P., Balitrand,
N., Daniel, M.T., Fenaux, P., Castaigne, S., Degos L.All-trans retinoic acid in
acute promyelocytic leukemias. II. In vitro studies: structure-function
relationship. Blood. 1990;76(9):1710-1717.
23.
Patel, S., Zhang, Y., Cassinat, B.,
Zassadowski, F., Ferre, N., Cuccuini, W., Cayuela, J.M., Fenaux, P., Bonnet,
D., Chomienne, C., Louache, F. Successful xenografts of AML3 samples in
immunodeficient NOD/shi-SCID IL2Rgamma(-/-) mice. Leukemia.
2012;26(11):2432-2435
24.
24. Ito,
M., Hiramatsu, H., Kobayashi, K., Suzue, K., Kawahata, M., Hioki, K., Ueyama,
Y., Koyanagi, Y., Sugamura, K., Tsuji, K., Keike, T., Nakahata, T.
NOD/SCID/gamma(c)(null) mouse: an
excellent recipient mouse model for engraftment of human cells. Blood.
2002;100(9):3175-3182.
25.
Ito, C.Y., Li, C.Y., Bernstein, A.,
Dick, J.E., Stanford, W.L. Hematopoietic stem cell and progenitor defects in
Sca-1/Ly-6A-null mice. Blood. 2003;101(2):517-523.
26.
Robert-Richard, E., Ged, C., Ortet, J.,
Santarelli, X., Lamrissi-Garcia, I., de Verneuil, H., Mazurier, F. Human cell
engraftment after busulfan or irradiation conditioning of NOD/SCID mice.
Haematologica. 2006; 91(10):1384.
27.
27. Feng,
W.H., Kenney, S.C. Valproic acid enhances the efficacy of chemotherapy in EBV-positive tumors by
increasing lytic viral gene expression. Cancer Res. 2006;66(17):8762-8769.
28.
Zhang, Y., Patel, S., Abdelouahab, H.,
Witnner, M., Willekens, C., Shen, S., Betems, A., Joulin, V., Opolon, P., Bawa,
O., Pasquier, F., Ito, M., Fujii, N., Gonin, P., Solary, E., Vainchenker, W.,
Coppo, P., De Botton, S., Louache, F. CXCR4 inhibitors selectively eliminate
CXCR4-expressing human acute myeloid leukemia cells in NOG mouse model. Cell
Death Dis. 2012;3:e396.
29.
Schenk, T., Stengel, S., and Zelent, A.
Unlocking the potential of retinoic acid in anticancer therapy. Br J Cancer.
2014; 111 (11), 2039-2045.
30.
Estey, E.H. Epigenetics in clinical
practice: the examples of azacitidine and decitabine in myelodysplasia and
acute myeloid leukemia. Leukemia. 2013; 27(9), 1803-1812.
31.
Bruserud, O., Reikvam, H., Fredly, H.,
Skavland, J., Hagen, K.M., van Hoang, T.T., Brenner, A.K., Kadi, A., Astori,
A., Gjertsen, B.T., Pendino F (2015). Expression of the potential therapeutic
target CXXC5 in primary acute myeloid leukemia cells - high expression is
associated with adverse prognosis as well as altered intracellular signaling
and transcriptional regulation. Oncotarget. 2015; 6(5), 2794-2811.
32.
Oike, T., Ogiwara, H., Amornwichet, N.,
Nakano, T., and Kohno, T. Chromatin-regulating proteins as targets for cancer
therapy. J Radiat Res. 2014; 55(4), 613-628.
33.
Yeung, M., Hurren, R., Nemr, C., Wang,
X., Hershenfeld, S., Gronda, M., Liyanage, S., Wu, Y., Augustine, J., Lee,
E.A., Spagnuolo P.A., P.A., Southall N., Chen C., Zheng W., Jeyaraju D.V.,
Minden M.D., Laposa R., Schimmer A.D. Mitochondrial DNA damage by bleomycin induces
AML cell death. Apoptosis. 2015; 20(6): 811-820.
34.
Yang, L., Weng, W., Sun, Z.X., Fu, X.J.,
Ma, J., and Zhuang, W.F. SphK1 inhibitor II (SKI-II) inhibits acute myelogenous
leukemia cell growth In vitro and in vivo. Biochem Biophys Res
Commun. 2015; 460(4): 903-908.
35.
Sivagnanalingam, U., Balys, M.,
Eberhardt, A., Wang, N., Myers, J.R., Ashton, J.M., Becker, M.W., Calvi, L.M.,
and Mendler, J.H. Residual Disease in a Novel Xenograft Model of RUNX1-Mutated,
Cytogenetically Normal Acute Myeloid Leukemia. PLoS One. 2015; 10(7): e0132375.
36.
Wei, S., Wang, Y., Chai, Q., Fang, Q.,
Zhang, Y., and Wang, J. and effects of heme oxygenase-1 silencing on the
survival of acute myelocytic leukemia-M2 cells. Exp Ther Med. 2015; 9(3):
931-940.
37.
Taussig, D.C., Vargaftig, J.,
Miraki-Moud, F., Griessinger, E., Sharrock, K., Luke, T., Lillington, D.,
Oakervee, H., Cavenagh, J., Agrawal, S.G.,Lister T.A., Gribben J.G., Bonnet D.
Leukemia-initiating cells from some acute myeloid leukemia patients with
mutated nucleophosmin reside in the CD34(-) fraction. Blood. 2010; 115(10):
1976-1984.
Peer reviewer: Ratana Banjerdpongchai, M.D., Ph.D., Associate Professor,
Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang
Mai 50200, Thailand.
Refbacks
- There are currently no refbacks.