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Coltella et al.
EZN-2208 synergizes with ATRA to eradicate APL
Synergistic leukemia eradication by combined treatment with retinoic acid and
HIF inhibition by EZN-2208 (PEG-SN38) in preclinical models of PML-RARα
and PLZF-RARα driven leukemia
Nadia Coltella1,5*, Roberta Valsecchi1,2, Manfredi Ponente1,3, Maurilio Ponzoni3,4,5, &
Rosa Bernardi1,5*
1
Laboratory of Pre-clinical Models of Cancer, Division of Experimental Oncology,
IRCCS San Raffaele Scientific Institute, Milan, Italy, 2Department of Biomedical,
Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena,
Italy, 3Vita-Salute San Raffaele University, Milan, Italy, 4Pathology and 5Leukemia
Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy.
Running title: EZN-2208 synergizes with ATRA to eradicate APL
Keywords: APL, HIF-1a, mouse models, EZN-2208, leukemia stem cells
Financial support: This work was supported by the Giovanni Armenise-Harvard
Foundation with a Career Development Award to R. Bernardi, and grants by
Fondazione Cariplo, Italian Association for Cancer Research (AIRC, My First AIRC
Grant, and IG 12769) and the EU (Marie Curie International Reintegration Grant) to
R. Bernardi.
Correspondence to:
*Nadia Coltella; Phone number: +390226435604; Fax number: +390226435602;
Email: [email protected]; Address: Via Olgettina 60, 20132, Milan, Italy
*Rosa Bernardi; Phone number: +390226435606; Fax number: +390226435602;
Email: [email protected]; Address: Via Olgettina 60, 20132, Milan, Italy
Disclosure of potential conflicts of interest: The authors have no conflict of interest to
disclose.
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Coltella et al.
EZN-2208 synergizes with ATRA to eradicate APL
ABSTRACT
Purpose: Retinoic acid-arsenic trioxide (ATRA-ATO) combination therapy is the
current standard of care for acute promyelocytic leukemia (APL) patients carrying the
oncogenic fusion protein PML-RARα. Despite the high cure rates obtained with this
drug combination, resistance to arsenic is recently emerging. Moreover, APL patients
carrying the PLZF-RARα fusion protein are partially resistant to ATRA treatment.
HIF-1α activation has been recently reported in APL, and EZN-2208 (PEG-SN38) is
a compound with HIF-1α inhibitory function currently tested in clinical trials. This
study investigates the effect of EZN-2208 in different pre-clinical APL models, either
alone or in combination with ATRA.
Experimental design: Efficacy of EZN-2208 in APL was measured in vitro by
assessing expression of HIF-1α target genes, cell migration, clonogenicity and
differentiation, vis a vis the cytotoxic and cytostatic effects of this compound. In vivo,
EZN-2208 was used in mouse models of APL driven by PML-RARα or PLZFRARα, either alone or in combination with ATRA.
Results: Treatment of APL cell lines with non-cytotoxic doses of EZN-2208 causes
dose-dependent down-regulation of HIF-1α bona fide target genes and affects cell
migration and clonogenicity in methylcellulose. In vivo, EZN-2208 impairs leukemia
progression and prolongs mice survival in APL mouse models. More importantly,
when used in combination with ATRA, EZN-2208 synergizes in de-bulking leukemia
and eradicating leukemia initiating cells.
Conclusions: Our pre-clinical data suggest that the combination ATRA-EZN-2208
may be tested to treat APL patients who develop resistance to ATO or patients
carrying the PLZF-RARα fusion protein.
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Coltella et al.
EZN-2208 synergizes with ATRA to eradicate APL
Translational Relevance
Acute promyelocytic leukemia (APL) is caused in most patients by the oncogenic
fusion protein PML-RARα, which is targeted for degradation by retinoic acid
(ATRA) and arsenic trioxide (ATO). However, mutations that generate resistance to
these drugs are recently being described in PML-RARα. Also, among non PMLRARα-driven APL, patients carrying the oncoprotein PLZF-RARα constitute a
relevant clinical challenge as they do not fully respond to ATRA treatment.
We have recently shown that the PML-RARα and PLZF-RARα fusion proteins
cooperate with hypoxia inducible factors (HIFs) to induce HIF-mediated proleukemogenic functions. Here we explore the use of EZN-2208, a compound that
inhibits HIF factors and is of immediate therapeutic relevance as it is in clinical trials
for solid tumors, in combination with ATRA for APL treatment. Because of the
extraordinary synergism of EZN-2208 and ATRA towards leukemia eradication in
different APL models, this therapeutic combination may be translated into clinical
testing for APL patients resistant to current treatment regimens.
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EZN-2208 synergizes with ATRA to eradicate APL
Introduction
Acute promyelocytic leukemia (APL) represents a well-defined subtype of acute
myeloid leukemia (AML) characterized in about 95% of cases by the oncogenic
fusion protein PML-RARα resulting from the chromosomal translocation t(15;17) (1).
APL is characterized by a block of terminal differentiation at the promyelocytic stage
and by aberrant self-renewal of myeloid progenitors (2).
Although representing one of the most aggressive AML subtypes, APL has become a
curable disease following the introduction of all-trans retinoic acid (ATRA) and
arsenic trioxide (ATO) in its standard treatment regimen (1) (3) (4). ATRA and ATO
are believed to lead to APL remission by targeting the PML-RARα fusion protein for
degradation, acting on the RARα and PML moieties respectively (1). However,
mutations in PML-RARα that are responsible for ATRA and ATO resistance are
recently being described in patients (5) (6) (7). Moreover, APL patients who carry the
rare chromosomal translocation t(11;17) that gives rise to the oncogenic fusion
protein PLZF-RARα respond only partially to ATRA treatment, do not respond to
chemotherapy and have a poor prognosis (8) (9) (10). Thus for these patients new
therapeutic options are urgently required.
We have recently demonstrated that PML-RARα and other fusion proteins involved
in APL pathogenesis (i.e: PLZF-RARα and NPM-RARα) behave as transcriptional
co-activators of hypoxia inducible factors (HIFs), which are critical mediators of
adaptive response to hypoxia, are often de-regulated in solid tumors, and are more
recently being implicated in leukemia (11). In APL, HIF factors cooperate with PMLRARα to promote leukemia progression primarily by mediating APL cell migration,
homing to bone marrow, and bone marrow neo-angiogenesis (12) (13). Moreover, we
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Coltella et al.
EZN-2208 synergizes with ATRA to eradicate APL
found that HIF-1α expression is increased upon ATRA treatment, and mediates
ATRA-induced increase in clonogenic activity in APL cells (12) (14). Therefore,
HIF-1α silencing synergizes with ATRA treatment in vivo to eradicate leukemiainitiating cells (LICs) and prevent leukemia relapse (12).
Because of the emerging involvement of HIF factors in tumorigenesis, strong efforts
are been made to identify direct and indirect inhibitors of HIF-1α. Several compounds
with known chemotherapeutic function, including camptothecin analogs and
topotecan, have been identified as HIFs inhibitors in a high throughput cell-based
screen (15). Of particular interest, topoisomerase I inhibitors were shown to inhibit
HIF-1α protein accumulation even at non-cytotoxic concentrations both in vitro and
in pre-clinical settings (16) (15). Recently, improved formulations of these
compounds, such as EZN-2208 (a polyethylene glycol conjugate of SN38), have
shown prolonged in vivo stability and delivery, and sustained inhibition of HIF factors
(17) (18) (19).
By using a combination of in vitro assays and in vivo pre-clinical studies, we show
that EZN-2208 combines anti-angiogenic and leukemia de-bulking effects with
successful leukemia eradication in combination with ATRA in pre-clinical APL
models driven by both PML-RARα and PLZF-RARα, and can be thus investigated as
a therapeutic option for patients who develop resistance to ATRA or ATO and for
patients carrying the PLZF-RARα oncoprotein.
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EZN-2208 synergizes with ATRA to eradicate APL
Materials and Methods
Cell culture and reagents
The leukemic cell lines NB4 (wt, shCTRL and shHIF-1α) (12), U937-PR9 and U937MT (20) (kindly provided by S. Minucci and P. G. Pelicci), were maintained in RPMI
1640 supplemented with 10% FBS and standard antibiotics (Lonza) at 37°C in a
humidified atmosphere containing 5% CO2.
EZN-2208 was provided by Belrose Pharma and used in accordance with the
manufacturer’s instructions; ATRA, Propidium iodide (PI) and Zinc sulfate
monohydrate (Zn) were purchased from Sigma; ATRA pellets were from Innovative
Research of America.
Cell viability and EC50
EC50 was calculated by exposing NB4 cells (wt, shCTRL or shHIF-1α) to increasing
concentrations of EZN-2208 or ATRA (10-6M) for 48 hours. Cell viability was
measured using Cell Titer Blue (Promega), in a LB940 Mithras (Berthold
Technologies), according to the manufacturer’s instructions. Sigmoidal curves were
obtained with PRISM software.
Immunoblot
When indicated, NB4 cells were treated for 8 or 24 hours with 1, 5 or 10 nM EZN2208. Proteins were extracted with lysis buffer (150 mM NaCl, 50 mM Tris–HCl pH
7.5, 0.5% CHAPS) supplemented with protease and phosphatase inhibitors (Pierce)
and then briefly sonicated to extract nuclear proteins. Total lysates were resolved by
SDS–PAGE 4–15% and transferred to a PVDF membrane (Biorad). Non-specific
binding was blocked in 5% non-fat milk for 1 hour at RT and blotted with the
following antibodies: rabbit polyclonal phospho-ATR (Ser428) (Cell Signaling) and
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Coltella et al.
EZN-2208 synergizes with ATRA to eradicate APL
rabbit polyclonal phosphor-Chk1 (Ser317) (Abcam). Mouse β-actin (Sigma) was used
as internal loading control.
Real-time PCR
RNA was isolated with the RNeasy mini kit (Qiagen) and cDNA was obtained by
retro-transcription of 1 μg total RNA using Advantage RT for PCR Kit (Clontech).
All probes for TaqMan assays were purchased from Applied Biosystem. 18S was
used as internal control. The relative expression of different cDNAs was calculated
using the 2-
ΔΔCt
method.
For DNA Real-time PCR, DNA was extracted from bone marrow samples using
QIAamp DNA Micro kit (Qiagen). PML-RARΑ and ZBTB16-RARA (PLZF-RARΑ)
DNAs were quantified as previously described (10) by real-time PCR with TaqMan
assay using a 7900 Fast-Real-time PCR System (Applied Biosystem).
Cell cycle analysis
Analysis of cell cycle was performed using PI. Cells were counted, washed in
phosphate-buffered saline (PBS), and resuspended at a concentration of 8x105
cells/ml in a solution of PBS containing 0.02% NP40 (Sigma), 80 μg/ml RNase A
(Sigma) and 100 μg/ml PI (Sigma). Staining was performed at RT for 15 minutes in
the dark. Flow cytometry was performed using BD FACSCanto (BD Biosciences) and
linear emission of PI was collected for 30,000 cells. Analysis of the percentage of
cells in each phase of the cell cycle was performed using the Cell Cycle Platform in
FlowJo software.
Methylcellulose assays
For CFU-L experiments, 5x103 NB4, U937-PR9 or U937-MT cells were resuspended
in Human Methylcellulose Base Media (R&D) and plated in 35 mm culture dishes.
Cells were allowed to grow for 7-10 days, at the end of which colonies were blindly
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Coltella et al.
EZN-2208 synergizes with ATRA to eradicate APL
counted. In replating experiments colonies were disaggregated to obtain single cell
suspension, and 5x103 cells were re-seeded in methylcellulose. When indicated EZN2208 (1 nM) was added to the methylcellulose medium and washed out in serial
replating experiments. For CFU-L experiments in U937-PR9 and U937-MT cells,
EZN-2208 (1 nM), Zn (100 μM) or both were added to the methylcellulose medium
when indicated. When indicated, NB4 cells were pre-treated for 24 hours with EZN2208 (1 nM), ATRA 10-6M or a combination of both and than plated in
methylcellulose.
Migration assays
1x106 NB4 cells were seeded in the upper chamber of 6.5 mm diameter and 5 μM
pore transwells (Costar). When indicated, cells were pre-treated for 8 hours with
EZN-2208 (1 nM). Spontaneous migration was measured after 16 hours. Cells were
collected from the lower chamber and counted by flow cytometer as number of cells
acquired per minute (LSR II Beckton Dickinson). For U937-PR9 and U937-MT cells,
both spontaneous and SDF-1-induced migrations were measured after 2.5 hours.
When indicated, cells were pre-treated for 24 hours with EZN-2208 (1 nM), Zn (100
μM) or both.
Mouse APL models
CD-1 nude immunocompromised and 129/Sv immunocompetent mice were
maintained in a pathogen-free animal facility and treated in accordance to European
Union guidelines and as approved by the Institutional Animal Care and Use
Committee (IACUC).
APL leukemic mice were obtained as previously described (12) (10). Briefly, mice
bearing PML-RARα-driven APL were obtained by transduction of Lin- BM cells
from 129/Sv mice with a retroviral vector (pBABE backbone with ΔNGFR as a
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EZN-2208 synergizes with ATRA to eradicate APL
reporter) expressing PML-RARα (12). ΔNGFR-sorted cells (1 x 106) were inoculated
i.v. into lethally irradiated syngeneic mice. Mice were monitored periodically for
clinical signs of disease and leukocytosis. When terminally sick, animals were
sacrificed and 1 x 106 leukemic BM cells were inoculated into syngeneic recipients to
expand leukemia for further experiments. Mice were monitored periodically for
clinical signs of disease and leukocytosis. A similar transplantation model was used
for PLZF-RARα-driven APL. 3 x 106 leukemic BM cells from PLZF-RARA–RARAPLZF mice (21) were inoculated into CD-1 nude mice to expand leukemia for further
experiments. For mice treatment, EZN-2208 was administered at a dosage of 5 mg/Kg
or 10mg/Kg by tail vein injection every other day for a total of two or five
administrations as indicated. ATRA was administered by subcutaneous implantation
of 2 release pellets (5 mg each) into the lateral sides of the neck.
Hystopathology and Immunohistochemistry
Mouse tissues were fixed in 4% formalin, embedded and included in paraffin wax.
Sections of 4-5 mm were stained with hematoxylin and eosin according to standard
protocols. Immunohistochemistry for endothelial cells was performed using CD31
antibody (Thermo Scientific).
Flow cytometric analysis
Immunophenotypic analysis was carried out using the following fluorochromeconjugated antibodies: CD34 (FITC, eBioscience), CD16/CD32 (FcγRII/III, PE, BD
Pharmingen), CD117 (c-kit, APC, BD Pharmingen), CD11b, (Mac-1, APC, BD
Pharmingen), Gr-1 (PE-Cy7, BD Pharmingen), Ly-6A/E (Sca-1, FITC, BD
Pharmingen), Streptavidin (PerCP, BD Pharmingen). CD3ε chain, CD45R (B220),
TER-119, CD11b, Gr-1, CD127 and Ly-6A/E biotin rat anti mouse antibodies were
from BD Pharmingen. Anti-human CD13 (PE) was from Beckman Coulter; anti-
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EZN-2208 synergizes with ATRA to eradicate APL
human CD11b (PE) and CD33 (APC) were from BD Pharmingen. Staining was
performed at 4°C for 20 minutes.
Annexin V staining was performed using the PE Annexin V Apoptosis Detection Kit I
(BD Pharmingen) according to the manufacturer’s protocol.
For analysis of BM stroma, long bones from leukemic and EZN-2208 treated mice
were crushed using a porcelain mortar and pestle (Millipore). Cells localizing to the
inner and endosteal fractions of the BM were obtained as previously described (22).
Bone marrow stromal cells from the two fractions have been defined as negative for
the expression of the pan-hematopoietic marker CD45 and Lineages markers (CD19,
B220, CD3, Ter119) (CD45-Lin-).
Statistics
Two-sided t tests were used to validate the significance of the data analyzed and
considered statistically significant when P < 0.05. For survival experiments curves
were analyzed with the Mantel-Cox test.
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EZN-2208 synergizes with ATRA to eradicate APL
Results
EZN-2208 inhibits APL cell migration and self-renewal
We have previously demonstrated that in APL cells HIF-1α silencing impairs several
pro-tumoral functions including cell migration, chemotaxis, neo-angiogenesis and
self-renewal of clonogenic cells, thus impacting on leukemia progression (12).
Moreover, we found that ATRA induces HIF-1α expression, and HIF-1α silencing
synergizes with ATRA towards leukemia eradication (12). Based on these results, we
tested the feasibility of HIF-1α pharmaceutical inhibition as a therapeutic option for
APL.
EZN-2208 is a campthotecin analog in clinical trials for solid tumors and lymphoma
(ClinicalTrials.gov), which was previously shown to inhibit HIF factors in various
cell contexts (17) (18) (19). To test the efficacy of EZN-2208 as a HIF-1α inhibitor in
APL we treated human NB4 APL cells with increasing concentrations of EZN-2208
upon HIF-1α silencing. EZN-2208-induced toxicity was significantly reduced after
silencing of HIF-1α (Fig. 1A), indicating that the efficacy of this compound depends
at least in part on HIF-1α expression in NB4 cells. In addition, real-time PCR analysis
demonstrated that a number of HIF-target genes involved in metabolism (CAIX,
GLUT1), apoptosis (BNIP3) and migration/homing (CXCR4) were down-regulated
by EZN-2208 in a dose-dependent manner, indicating that this compound inhibits
HIF-1α-mediated transcription also in the context of APL (Fig. 1B). However,
because campthotecin derivatives are known topoisomerase I inhibitors that may
induce checkpoint activation, cell cycle arrest and cell death (23), we wished to
further characterize the effect of EZN-2208 treatment in APL cells. Consistent with
previous literature, treatment of NB4 cells with increasing concentrations of EZN2208 led to checkpoint activation (Fig. 1C) and induction of apoptosis in a dose-
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EZN-2208 synergizes with ATRA to eradicate APL
dependent manner, with over 50% of the cells undergoing apoptosis at 10 nM EZN2208 (Fig. 1D). At the intermediate concentration of 5 nM, apoptosis was
accompanied by cell cycle arrest and p21 upregulation (Fig. 1E and F), thus
demonstrating that EZN-2208 exerts typical functions of a topoisomerase inhibitor at
higher doses, while treatment with 1 nM EZN-2208 did not exert any significant
effect in NB4 cells (Fig. 1 C-F). We therefore chose to test whether treatment with a
non-cytotoxic dose of EZN-2208 still impacted on cell functions regulated by HIF-1α
in APL, like cell migration and self-renewal (12). Consistent with our previous data,
pre-treatment of NB4 cells with 1 nM EZN-2208 inhibited basal cell migration (Fig.
1G), and CFU-L formation in methylcellulose, also upon drug removal and re-plating
(Fig. 1H), thus recapitulating the effects of direct HIF-1α silencing (12).
To substantiate these findings in another APL cell line, we took advantage of the
U937-PR9 cell system where PML-RARα is expressed under the control of a zinc
(Zn)-inducible promoter (20). Treatment of U937-PR9 cells with Zn increased
CXCR4 mRNA levels (12) and cell migration towards SDF-1 (Fig. 2A). Interestingly,
treatment of U937-PR9 cells with non-cytotoxic doses of EZN-2208 inhibited basal
and SDF-1-directed cell migration only when PML-RARα was expressed (Fig. 2A).
Moreover, treatment of U937-PR9 cells with low doses of EZN-2208 significantly
reduced the number of CFU-L only when cells were co-treated with Zn (Fig. 2B).
Notably, treatment of control mock-transduced U937-MT cells with Zn or EZN-2208
did not have any effect on cell migration, chemotaxis or colony formation
(Supplementary Fig. S1A and B). Taken together, these data indicate that forced
expression of PML-RARα sensitizes leukemic cells to inhibition of HIF-1α-regulated
functions by EZN-2208, and suggest that in the context of wild-type U937 cells, cell
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EZN-2208 synergizes with ATRA to eradicate APL
migration and colony formation are regulated by genes that do not respond to EZN2208 treatment.
In conclusion, hampering HIF-1α functions with a therapeutically relevant compound
affects different aspects of APL biology, including cell migration and self-renewal.
EZN-2208 treatment limits progression of mouse APL
We next sought to test the therapeutic applicability of EZN-2208 in a relevant PMLRARα-driven APL mouse model. To this end, we took advantage of a serially
transplantable leukemia model where APL was initiated by retroviral transduction of
PML-RARα into lineage negative mouse BM cells (24) (12). In this model, we
observed that, similarly to recently characterized APL transgenic mice (25) (26), the
BM of leukemic mice shows an expansion of self-renewing c-kit+CD34+FcγRII/III+
leukemic promyelocytes and c-kit-CD34-FcγRIII/II+ leukemic granulocytes, both
expressing intermediate levels of Gr-1+ as compared to non-leukemic wild-type mice
(Fig. 3A). Having thus defined this leukemia model as similar to APL transgenic
mice, efficacy of EZN-2208 treatment was measured after one cycle low-dose drug
treatment (5 mg/Kg) administered every other day for a total of 5 administrations (q2d
x5 schedule) (18) (27). Treatment was started 12 days after leukemia transplantation,
when animals showed APL expansion in BM but no spleen enlargement nor
peripheral blood involvement (data not shown). EZN-2208 treatment significantly
prolonged the survival of leukemic mice upon drug discontinuation (Fig. 3B).
Separate cohorts of animals were sacrificed at the end of treatment to evaluate
leukemia progression, differentiation and drug cytotoxicity. Interestingly, leukemia
involvement in peripheral blood and spleen was significantly reduced in EZN-2208treated animals (Fig. 3C-D), although the percentage of leukemic promyelocytes and
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EZN-2208 synergizes with ATRA to eradicate APL
granulocytes was not affected by EZN-2208 treatment in BM (Fig. 3E), as EZN-2208
was not cytotoxic to APL BM cells (Fig. 3F). Moreover EZN-2208 did not induce
leukemia differentiation as assessed by co-expression of Mac-1 and Gr-1 (10) (Fig.
3G).
Because in vitro EZN-2208 exerted cytotoxic effects in a dose-dependent manner
(Fig. 1), we also examined the effect of a higher concentration of EZN-2208 (10
mg/Kg) in vivo. Similarly to the in vitro data, increasing EZN-2208 dosage resulted in
induction of apoptosis in the myeloid leukemic population in the BM, accompanied
by increased expression of Bax, which was not observed upon treatment with 5
mg/Kg (Supplementary Fig. S2A and B). Having thus fully characterized treatment
with 5 mg/Kg EZN-2208 as non-cytotoxic, we analyzed HIF-1α activity. As expected
based on previous literature (18), expression of bona fide HIF-1α target genes was
lower in the BM of mice treated with 5 mg/Kg EZN-2208 (Fig. 3H), as was BM neoangiogenesis (Fig. 3I).
Finally, we tested whether treatment with 5 mg/Kg EZN-2208 affected the leukemic
BM microenvironment. Percentages of CD19+ B cells, CD3+ T cells and Ter119+
erythroid precursors did not change, while CD45-Lin- stromal cells in inner BM
decreased upon treatment with EZN-2208 (Supplementary Fig. S3A and B). By
contrast, CD45-Lin- stromal cells in endosteal BM were not affected by EZN-2208
(Supplementary Fig. S3B). Taken together, these data indicate that besides inhibiting
HIF-1α function in leukemic cells, EZN-2208 also somehow impacts the leukemic
microenvironment, measured as BM endothelial cells and BM stroma, and this effect
may participate to reducing leukemia progression. Finally, analysis of the
hematopoietic stem cell (HSC) compartment of healthy mice showed that EZN-2208
is not toxic to HSCs (Supplementary Fig. S3C).
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EZN-2208 synergizes with ATRA to eradicate APL
Taken together, these data indicate that EZN-2208 impairs leukemia progression even
when used at doses that are not cytotoxic to leukemic cells, and do not induce
differentiation. Consistently with the inhibition of APL cell migration and selfrenewal that was observed upon in vitro EZN-2208 treatment (Fig. 1 and Fig. 2),
leukemia de-bulking by EZN-2208 may occur, at least in part, through inhibition of
bone marrow neo-angiogenesis and APL blasts migration to peripheral organs.
Combined ATRA and EZN-2208 treatment eradicates PML-RARα-driven APL
We have recently shown that besides inducing differentiation of leukemic cells,
ATRA also induces transcriptional expression of HIF-1α and increased clonogenicity
of APL cells in a HIF-dependent manner (12). Moreover acute inhibition of HIF-1α
with a specific RNA antagonist cooperates with ATRA in vivo to block leukemia
initiation by APL LICs (12).
To understand whether a pharmacological agent with HIF-inhibitory activity may
similarly cooperate with ATRA in vivo, we explored the combination of EZN-2208
and ATRA towards inhibiting leukemia relapse in PML-RARα-driven leukemia.
First, we observed that ATRA and EZN-2208 did not synergize in affecting cell
viability or inducing APL differentiation in vitro (Supplementary Fig. S4A and B).
Then, we tested the ability of EZN-2208 to abrogate ATRA induced clonogenicity in
vitro. NB4 cells were pre-treated with ATRA and/or EZN-2208 and plated in
methylcellulose after drug washout. While ATRA induced an increase in the number
of colonies and EZN-2208 alone had no effect on colony formation at a non-toxic
concentration, the combination ATRA+EZN-2208 abrogated ATRA-induced increase
in colony-forming cells in vitro (Fig. 4A), in accordance with our previous data
indicating that increased clonogenicity is HIF-1α-dependent (12).
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Coltella et al.
EZN-2208 synergizes with ATRA to eradicate APL
To test whether ATRA and EZN-2208 cooperated in targeting LICs in vivo, leukemic
mice were treated with short ATRA and EZN-2208 schedules to allow drug action
without complete disease eradication. 12 days after APL engraftment mice were
treated with ATRA for 4 consecutive days together with 2 administrations of 5 mg/Kg
EZN-2208. Animals were then divided in two cohorts: in the first group, treatment
was removed and animals were monitored for leukemia progression and sacrificed
when terminally sick (Fig. 4B); in the second group, animals were sacrificed at the
end of treatment and their bone marrow cells were used to challenge secondary
recipient animals to measure leukemia repopulating cells (Fig. 4C).
Of note, unlike the 5-doses experiment of Fig. 3B, two administrations of EZN-2208
did not prolong mice survival, either alone or in combination with ATRA, while
ATRA treatment generated leukemia de-bulking and increased survival upon
treatment discontinuation (Fig. 4B). Significantly, in the transplantation setting,
combination of EZN-2208 with ATRA resulted in leukemia eradication in the
majority of mice, with most of the animals never developing leukemia (Fig. 4C).
Molecular evaluation of APL expansion before transplantation revealed that ATRA
alone had significantly de-bulked leukemia, while EZN-2208 did not considerably
affect leukemia de-bulking at the molecular level (Fig. 4D). Therefore, these data
indicate that the ATRA+EZN-2208 treatment combination profoundly synergizes in
the eradication of LICs, thus affecting leukemia transplantation in a model of
leukemia relapse.
Combined ATRA and EZN-2208 treatment eradicates PLZF-RARα-driven APL
Among non PML-RARα-driven APL leukemia, patients carrying the oncogenic
fusion protein PLZF-RARα constitute a relevant clinical challenge as they do not
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EZN-2208 synergizes with ATRA to eradicate APL
fully respond to ATRA treatment (8). Because we have recently shown that besides
PML-RARα other APL fusion proteins, including PLZF-RARα, behave as HIFs
transcriptional co-activators (12), we wished to understand if HIF-1α inhibition could
improve current therapeutic regimens for PLZF-RARα APL. To this aim, the effect of
EZN-2208, either alone or in combination with ATRA, was analyzed in a
transplantable model of PLZF-RARα-driven leukemia (10) (21). Nude mice were
challenged intravenously with leukemic cells and 7 days later were treated with EZN2208 (5mg/Kg, q2d x 5 schedule) for a total of five administrations and/or with
subcutaneous implantation of 10 mg ATRA-release pellets for 7 days. The longer
administration schedule of ATRA and EZN-2208 as compared to PML-RARα-driven
leukemia (Fig. 4) was chosen because PLZF-RARα-driven leukemia is reportedly
more resistant to treatment (10) (28).
PLZF-RARα leukemic mice showed a marked increase in the percentage of immature
Gr-1+ myeloid cells as compared with wild-type aged-matched controls (Fig. 5A). As
previously reported, 7 days of ATRA treatment induced leukemia differentiation, as
measured by accumulation of Mac-1+/Gr-1+ cells (10) (Fig. 5B), but failed to restore
normal BM hematopoiesis (Mac-1-/Gr-1- cells; Fig. 5C), and did not considerably debulk leukemia as shown by substantial PLZF-RARα BM DNA (Fig. 5E).
Surprisingly, unlike what observed in PML-RARα-driven APL (Fig. 3G), five
administrations of EZN-2208 alone led to accumulation of Mac-1+/Gr-1+ cells,
similarly to treatment with ATRA (Fig. 5B). In addition, EZN-2208 partially restored
normal hematopoiesis in treated animals (Fig. 5C), normalized spleen weight (Fig.
5D) and strongly de-bulked BM leukemia at the molecular level (Fig. 5E). Taken
together, this data indicate that EZN-2208 exerts stronger anti-leukemia effects
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EZN-2208 synergizes with ATRA to eradicate APL
towards PLZF-RARα than PML-RARα-driven APL, particularly in terms of
leukemia de-bulking, where it acts more efficiently than ATRA.
Combined treatment with ATRA and EZN-2208 produced additive effects in all the
parameters that were measured (Fig. 5C-E), except in the number of Mac-1+/Gr-1+
cells, which reached 80% of bone marrow cells with either compound alone (Fig. 5B).
Consistently, combination of ATRA and EZN-2208 showed additive effect also on
mice survival after treatment discontinuation (Fig. 5F). Failure to observe EZN2208+ATRA additive effects on PML-RARα-driven leukemia (Fig. 4B) may be due
to shorter treatment with both agents, and a stronger effect of ATRA alone.
To assess whether also in the PLZF-RARα leukemia model EZN-2208 and ATRA
synergized in limiting LICs, a second cohort of mice was sacrificed at the end of
treatment, and their BM cells were injected into secondary recipient mice to measure
leukemia repopulating cells. As shown in Fig 5G, while either ATRA or EZN-2208
treatment only modestly increased mice survival and delayed leukemia progression in
transplanted animals, the combination of ATRA+EZN-2208 exquisitely synergized in
eradicating LICs, as the transplanted mice never developed leukemia (Fig. 5F).
In summary, these data provide the first experimental evidence that a HIF-inhibitory
agent successfully delays leukemia progression and synergizes with ATRA to
eradicate leukemia repopulating cells in a model of PLZF-RARα-driven APL.
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EZN-2208 synergizes with ATRA to eradicate APL
Discussion
Introduction of ATRA in the treatment of patients with acute promyelocytic leukemia
has turned this disease from a highly fatal to a curable leukemia. Because of the rapid
and effective induction of APL blasts differentiation and leukemia de-bulking, ATRA
treatment epitomizes differentiation therapy. Nonetheless, ATRA leaves LICs almost
unaffected, and rarely leads to prolonged disease remission. Conversely, combined
treatment with ATO synergizes with ATRA in causing PML-RARα degradation and
inducing LICs clearance in APL mouse models (1).
Notwithstanding these achievements, resistance to both ATRA and ATO through
PML-RARα mutations is being recently described (5) (6) (7), although so far
mutations have been identified in relapsed patients who had been treated sequentially
with combinations of ATRA, ATO and/or chemotherapy, and not in patients who
received ATRA+ATO combination as front-line therapy (7). In addition, rare APL
patients with the chromosomal translocation t(11;17) giving rise to the fusion protein
PLZF-RARα respond only partially to ATRA-based differentiation therapy, do not
respond to ATO treatment, and experience a poor clinical outcome (8) (9). Nowadays
the only therapeutic option for these patients is high-dose chemotherapy, which
however is ineffective, thus emphasizing the need of new therapeutic options.
We have recently defined a new pro-leukemogenic axis exploited in APL by the
oncogenic fusion protein PML-RARα through its synergistic cooperation with the
transcription factor HIF-1α. More importantly we have provided proof-of principle
experiments showing that ATRA strongly synergizes with a HIF-1α RNA antagonist
to eliminate LICs and prevent leukemia relapse (12). Here we explored the
therapeutic relevance of EZN-2208, a pharmaceutical agent with HIF-inhibitory
activity, in two different mouse models of APL driven by PML-RARα and PLZF-
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Coltella et al.
EZN-2208 synergizes with ATRA to eradicate APL
RARα. We demonstrate that similarly to other tumor contexts, in APL EZN-2208
inhibits the expression of HIF-target genes involved in critical pro-tumoral functions
like cancer metabolism, cell migration and protection from apoptosis. Consistently,
treatment of human APL cells with EZN-2208 impairs HIF-mediated functions like
cell migration, chemotaxis, clonogenicity and self-renewal in vitro. In vivo, EZN2208 inhibits expression of HIF-target genes and neo-angiogenesis, and delays
leukemia progression even when used at doses that do not induce apoptosis of
leukemic cells and do not affect HSCs numbers. Nonetheless, low doses of EZN-2208
reduce the number of stromal cells in leukemic BM, which may participate to
blunting leukemia progression. As it is increasingly being recognized that the
leukemia microenvironment importantly participates to regulating leukemia
progression and resistance to therapy, further investigation will be necessary to fully
characterize the APL leukemia microenvironment and the effect of HIF inhibition on
the different cell types that functionally interact with APL cells in the BM.
Our data extend beyond the effect of EZN-2208 in APL, and support the idea that
pharmacological compounds that act as chemotherapeutic agents while also inhibiting
pro-tumoral molecular pathways at non-toxic concentrations, may be tested in the
clinic at lower, more tolerable doses in specific disease contexts, either alone or in
combination with other pharmacological agents. Indeed, preclinical trials of EZN2208 treatment in APL showed promising efficacy not only in delaying leukemia
progression, but also in exquisitely cooperating with ATRA towards LICs eradication
and prevention of leukemia relapse.
Interestingly, besides synergizing with ATRA for LICs eradication, EZN-2208
appears to exhibit an additional anti-tumor effect on PLZF-RARα-driven APL, as it
leads to leukemia de-bulking and differentiation. The reason for such differential
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Coltella et al.
EZN-2208 synergizes with ATRA to eradicate APL
effects is at the moment unknown, and further experiments will be necessary to study
in detail the role of EZN-2208 in other leukemia sub-types. Moreover, future
experiments will be necessary to address the issue of inter-leukemia variability, as
with our experiments we have analyzed leukemia propagated from single animals.
From a therapeutic standpoint, our results lay the basis for proposing new clinical
trials aimed at evaluating the efficacy of HIF-targeting strategies in patients with
APL. It has been demonstrated that newly diagnosed APL patients may avoid
treatment with cytotoxic chemotherapy and undergo less harmful combination
treatments with ATRA and ATO, which reach similar cure rates without triggering
the long-term complications of chemotherapy (29) (4). However, ATO treatment
triggers degradation of the PML-RARα fusion protein while having no effect on
PLZF-RARα (9), and more importantly resistance to ATO is recently emerging. On
the basis of our data new therapeutic approaches with HIF-inhibiting agents may be
proposed for patients who do not respond to standard therapy. Furthermore, future
experiments will be required to evaluate the effect of ATO treatment on HIF
signaling, and test in pre-clinical settings the efficacy of combining ATO treatment
with HIF inhibition.
21
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Coltella et al.
EZN-2208 synergizes with ATRA to eradicate APL
Acknowledgments
The authors would like to thank: all current and previous members of the laboratory
of RB for collective thinking and support; Arianna Vino and Martina Rocchi for
technical support with tissue preparation and immunohistochemistry; Lee
Greenberger, Yixian Zhang and Belrose Pharma Inc. for supplying EZN-2208;
Saverio Minucci and Pier Giuseppe Pelicci for the U937-PR9 and U937-MT cell
lines; Hugues De The for PLZF-RARα mouse leukemic cells and insightful
discussion.
Financial support
This work was supported by the Giovanni Armenise-Harvard Foundation with a
Career Development Award to R. Bernardi, and grants by Fondazione Cariplo, Italian
Association for Cancer Research (AIRC, My First AIRC Grant, and IG 12769) and
the EU (Marie Curie International Reintegration Grant) to R. Bernardi.
Authors’ Contributions
Conception and design: N. Coltella, R. Bernardi
Development of methodology: N. Coltella
Acquisition of data (provided animals, acquired and managed patients, provided
facilities, etc.): N. Coltella, R. Valsecchi, M. Ponente, M. Ponzoni
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): N. Coltella, M. Ponzoni
Writing, review, and/or revision of the manuscript: N. Coltella, R. Bernardi
Study supervision: N. Coltella, R. Bernardi
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EZN-2208 synergizes with ATRA to eradicate APL
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Coltella et al.
EZN-2208 synergizes with ATRA to eradicate APL
FIGURE LEGENDS
Fig. 1 EZN-2208 treatment impairs cell migration and colony formation in APL
cells.
A, Cell survival (percentage of untreated cells) of shCTRL or shHIF-1α NB4 cells
treated with EZN-2208 at the indicated concentrations for 48 hours. Data are
expressed as mean values ± SD of triplicates from one representative experiment out
of two with similar results.
B, Real-time PCR analysis of the indicated HIF-1α-target genes in NB4 cells treated
for 24 hours with EZN-2208 at the indicated concentrations. Data represent mean
values ± SD of two independent experiments.
C, Western blot of pATR, pChk1 and β-actin in NB4 cells treated for 8 or 24 hours
with EZN-2208 at the indicated concentrations.
D, Flow-cytometric analysis of Annexin V in NB4 cells treated for 24 hours with
EZN-2208 at the indicated concentrations and expressed as percentage of Annexin V
positive cells. Data represent mean values ± SD of three independent experiments.
E, Cell cycle analysis in NB4 cells treated for 24 hours with EZN-2208 at the
indicated concentrations. Data are expressed as percentage of cells in different phases
of the cell cycle (G1, S and G2 phases) and represent mean values ± SD of three
independent experiments.
F, Real-time PCR analysis of p21 (CDKN1A) in NB4 cells treated for 24 hours with
EZN-2208 at the indicated concentrations. Data represent mean values ± SD of three
independent experiments.
G, Migration of NB4 cells after pre-treatment with 1 nM EZN-2208 for 8 hours and
drug washout. Cells were collected 16 hours post migration. Data are expressed as
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Coltella et al.
EZN-2208 synergizes with ATRA to eradicate APL
mean values ± SD of triplicates from one representative experiment out of two with
similar results.
H, Colony-forming assay of NB4 cells treated with 1 nM EZN-2208. EZN-2208 was
washed out in the second plating. Data are presented as percentage of mock-treated
cells. Data are expressed as mean values ± SD of triplicates from one representative
experiment out of two with similar results.
Fig. 2 EZN-2208 treatment impairs cell migration and colony formation in U937
cells upon expression of PML-RARα.
A, Basal and SDF-1-induced migration of U937-PR9 cells after treatment with Zn
(100 μM) or EZN-2208 (1 nM) for 24 hours.
B, Colony-forming assay of U937-PR9 treated with Zn (100 μM) and EZN-2208 (1
nM) as indicated. All data are expressed as mean values ± SD of triplicates from one
representative experiment out of at least two with similar results.
Fig. 3 In vivo treatment with EZN-2208 inhibits progression of PML-RARαdriven APL.
A, Flow-cytometric analysis of promyelocytic and granulocytic populations in the
bone marrow of one representative wt mouse and one mouse transplanted with APL
leukemic blasts and sacrificed when moribund (21 days after transplantation).
Selection indicates a cocktail of markers (CD3, B220, TER119, CD127, Sca-1). Red
boxes indicate accumulation of cells with intermediate Gr1+ expression.
B, Kaplan-Meier survival curve of 129/Sv mice injected with APL blasts and treated
with EZN-2208 at the indicated concentration starting at day 11 post-leukemia
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EZN-2208 synergizes with ATRA to eradicate APL
challenge, with a q2d x5 schedule (n=7). Survival curves were analyzed with the
Mantel-Cox test.
C, Peripheral white blood cells counts of leukemic mice treated as in B. Blood was
collected at the end of treatment (day 21); (n=11 for NT and n=10 for EZN-2208
treated mice).
D, Spleen weight of leukemic mice treated as in B and sacrificed at the end of
treatment (day 21); (n=21 for NT and n=28 for EZN-2208 treated mice).
E, Flow-cytometric analysis of promyelocytic and granulocytic populations in the
bone marrow of leukemic mice treated and sacrificed as in D. Graph shows mean
values of promyelocytic and granuclocytic populations expressed as percentages of
live cells (n=3).
F, Flow-cytometric analysis of Annexin V-positive promyelocytic and granulocytic
populations in the bone marrow of leukemic mice treated and sacrificed as in D. Data
are expressed as percentage of Annexin V-positive cells in the promyelocytic and
granulocytic populations (n=3).
G, Flow-cytometric analysis of Mac-1+/Gr-1+ cells in the bone marrow of leukemic
mice treated and sacrificed as in D. Graph shows percentage of double positive cells
(n=3).
H, Real-time PCR analysis of HIF-1α target genes Vegfa and Glut1 in the bone
marrow of leukemic mice treated and sacrificed as in D. Data represent mean values ±
SD (n=3).
I, CD31 immunostaining of bone marrow samples of leukemic mice treated and
sacrificed as in D. Graph on the left shows average number of microvessels per field.
Pictures on the right show representative images (arrows indicate microvessels).
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EZN-2208 synergizes with ATRA to eradicate APL
Fig. 4 EZN-2208 synergizes with ATRA towards LICs eradication in PMLRARα-driven APL.
A, Colony-forming assay of NB4 cells pre-treated for 24 hours with 1 nM EZN-2208
and/or 1 μM ATRA and plated in methylcellulose after drug washout.
B, Survival analysis of mice treated with 2 administrations of 5mg/Kg EZN-2208, 4
days ATRA or a combination of both 12 days after leukemia challenge. Kaplan-Meier
survival curve was obtained after treatment discontinuation (n=7). Survival curves
were analyzed with the Mantel-Cox test.
C, Survival analysis of recipient mice injected with 1x106 bone marrow cells from
leukemic mice at the end of 4-days treatment with ATRA and EZN-2208 performed
as in B (n=7). Survival curves were analyzed with the Mantel-Cox test.
D, PML-RARα DNA real-time PCR analysis of bone marrow cells extracted from
leukemic mice at the end of 4-days in vivo treatment with ATRA and EZN-2208
performed as in B and before transplantation to secondary recipients (n=3).
Fig. 5 EZN-2208 synergizes with ATRA towards LICs eradication in PLZFRARα-driven APL.
A, Flow-cytometric analysis of the myeloid markers Mac-1 and Gr-1 in the bone
marrow of one control nude mouse, one mouse transplanted with PLZF-RARα
leukemic blasts at 14 days post-transplantation, and one PLZF-RARα leukemic
mouse after 7 days in vivo treatment with 10mg ATRA.
B, Flow-cytometric analysis of Mac-1+/Gr-1+ cells in the bone marrow of PLZFRARα leukemic mice treated with 5 administrations of 5mg/Kg EZN-2208, 7 days
ATRA or a combination of both (ATRA+EZN-2208) starting at day 7 after leukemia
28
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Coltella et al.
EZN-2208 synergizes with ATRA to eradicate APL
challenge and sacrificed at the end of treatment. Graph shows percentage of double
positive cells (n=3).
C, Flow-cytometric analysis of Mac-1-/Gr-1- cells as readout of restoration of normal
hematopoiesis in the bone marrow of PLZF-RARα leukemic mice treated and
sacrificed as in B. Graph shows percentage of double negative cells (n=3).
D, Spleen weight of CD-1 nude mice challenged with 3x106 PLZF-RARα APL
leukemic blasts treated as in B and sacrificed at the end of treatment.
E, PLZF-RARα DNA real-time PCR analysis of bone marrow cells extracted from
leukemic mice treated and sacrificed as in B (n=3).
F, Survival analysis of leukemic mice treated as in B. Kaplan-Meier survival curve
was obtained after treatment discontinuation (n=7). Survival curves were analyzed
with the Mantel-Cox test.
G, Survival analysis of recipient mice injected with 1x106 bone marrow cells derived
from donor mice treated and sacrificed as in B (n=7). Survival curves were analyzed
with the Mantel-Cox test.
29
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Coltella et al. Figure 1
Relative expression
P= 0.0158
40
P= 0.034
20
0
0.9
0.6
0.3
0.0
1 nM
5 nM
10 nM
CAIX
BNIP3
D
C
8
5 nM
24
8
24
8
1nM
5nM
10nM
60
24 mock
pATR
pChk1
b-actin
GLUT1
CXCR4
mock
10 nM
Annexin V + cells (%)
1 nM
40
P< 0.0001
P< 0.0001
20
8
6
4
2
0
n.s.
mock 1 nM 5 nM 10 nM
E
F
mock
80
8
60
Relative expression
1nM
5nM
40
20
0
6
mock
1nM
5nM
4
2
0
G1
S
G
H
mock
EZN-2208
mock
EZN-2208
3000
Migrated cells/min
mock 1 nM 5 nM
G2
P= 0.0127
2000
1000
150
Percent of control
120
100
80
60
Author Manuscript Published OnlineFirst B
on April 30, 2015; DOI: 10.1158/1078-0432.CCR-14-3022
mock
shCTRL
Author
manuscripts have been peer reviewed and accepted
for publication but have not yet been edited.
shHIF-1a
1nM
5nM
n.s.
10nM
1.2
Cell cycle phases (%)
P< 0.0001
P= 0.0006
I plating
II plating
100
50
0
08
22
N-
m
oc
k
0
EZ
Percent survival
A
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Coltella et al. Figure 2
Author Manuscript Published OnlineFirst on April 30, 2015; DOI: 10.1158/1078-0432.CCR-14-3022
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
A
B
mock
EZN-2208
Zn
Zn+EZN-2208
P< 0.0001
25000
P< 0.0001
8
CFU-L/field (10X)
20000
15000
10000
1500
1000
500
0
P=0.0015
6
4
2
8
Zn
+E
ZN
-2
20
08
Zn
k
oc
22
N-
m
EZ
20
8
Zn
Zn
+E
ZN
-2
22
08
oc
N-
m
-2
ZN
+E
Zn
EZ
20
8
k
Zn
08
22
N-
m
oc
k
0
EZ
Migrated cells/min
30000
mock
EZN-2208
Zn
Zn+EZN-2208
P=0.0002
+SDF-1
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Coltella et al. Figure 3
Author Manuscript Published OnlineFirst on April 30, 2015; DOI: 10.1158/1078-0432.CCR-14-3022
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
A
250K
200K
10 5
10 5
58.7
10 5
20.2
4.15
10
4
10
4
10
4
150K
3
SSC-A
100K
10
50K
0
10
2
3
4
10
10
10
0
10
250K
10
2
3
4
10
10
10
<PE-A>
2
0
64.1
5
10
74.6
5
10
0
10 5
21
10
2
3
10
2
5
10
68.9
0
3.33
4
10
0
10 5
93.4
wild type BM
3
10
<PE-A>
2
0
0
200K
3
10
<APC-A>
10
2
3
10
5
10
10
10 5
18.7
10
4
80.7
4
10
4
10
4
leukemic BM
150K
3
10
3
4
10
10
5
10
Selection
0
10
2
3
10
4
10
C
25
0
5
10
15
20
25
30
11
87.9
0
10
2
3
0.27
4
10
5
10
10
10
3.5
0
10
2
3
10
D
P < 0.0001
60
40
20
4
10
5
10
1.0
P < 0.0001
0.8
0.6
0.4
0.2
0.0
0
35
2
0
Gr-1
80
WBC (103/mL)
NT
5 mg/Kg
50
0
10
2
0
Gr-1
P = 0.0009
75
5
10
CD34
100
Percent survival
24.4
10
<PE-A>
Spleen weight (g)
0
2
2
0
c-kit
SSC
0
3
10
<PE-A>
FcgRII/III
10
50K
B
3
10
<APC-A>
FcgRII/III
SSC-A
100K
NT
NT
EZN-2208
EZN-2208
Days after i.v. injection
10
5
0
n.s.
50
Mac-1/Gr-1 (%)
15
G
NT
EZN-2208
15
20
10
5
Pro
Pro
Gra
H
0.9
0.6
0.3
0.0
Vegfa
Gra
30
P =0.0002
N° vessels/field (40X)
P =0.0018
30
20
10
NT EZN-2208
I
NT
EZN-2208
1.2
40
0
0
Relative expression
Percentage (%)
25
F
NT
EZN-2208
Annexin V + cells (%)
E
Glut1
P < 0.0001
20
NT
10
0
NT EZN-2208
EZN-2208
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Coltella et al. Figure 4
Author Manuscript Published OnlineFirst on April 30, 2015; DOI: 10.1158/1078-0432.CCR-14-3022
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
A
P= 0.0006
Percent survival
6
4
2
60
P= 0.0457
20
0
20
40
60
days
EZ
A+
D
60
200
300
0
EZ
Days after transplantation
400 450
ZN
40
20
+E
0
40
RA
20
60
RA
40
80
AT
P= 0.0117
AT
60
100
08
80
P= 0.0002
22
E ZN-2208
ATR A
ATR A+E ZN
120
N-
100
NT
NT
20
P= 0.0002
40
N
A
EZ
C
0
P= 0.0291
AT
R
N-
22
AT
R
08
NT
E ZN-2208
ATR A
ATR A+E ZN
80
0
0
Percent survival
NT
100
PML-RARA DNA (%)
8
CFU-L/field (10X)
B
P< 0.0001
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Coltella et al. Figure 5
A
Author Manuscript Published OnlineFirst on April 30, 2015; DOI: 10.1158/1078-0432.CCR-14-3022
wild-typehave
BM been peer reviewedleukemic
Author manuscripts
and accepted
for publication but have
not yet been
BM
ATRA-treated
BM edited.
105
7.29
50.5
105
57.4
37
5.09
105
104
104
104
103
PE-Cy7-A
103
PE-Cy7-A
103
PE-Cy7-A
102
102
102
0
0
0
40.8
Gr-1
0 102
103
4.23
1.37
105
104
85.2
0 102
103
104
8.57
1.37
105
0 102
103
1.1
105
104
Mac-1
C
P=0.0005
25
40
20
0
ZN
D
1.0
120
0.8
100
0.6
0.4
P=0.0002
P=0.0003
P<0.0001
0.2
ZBTB16-RARA DNA (%)
AT
80
60
40
20
RA
EZ
F
AT
RA
+E
ZN
22
N-
+E
AT
RA
EZ
AT
NT
ZN
AT
RA
N-
22
08
NT
0
08
Spleen weight (g)
E
0.0
G
100
NT
P=0.0007
EZN
ATRA
P=0.0031
ATRA+EZN
60
40
20
Percent survival
100
80
Percent survival
RA
NEZ
AT
EZ
RA
22
+E
AT
08
NT
0
RA
5
22
n.s.
10
20
N-
P=0.0444
15
ZN
60
P=0.0010
+E
Mac-1-/Gr-1- (%)
80
RA
P=0.0001
08
P=0.0002
NT
Mac-1+/Gr-1+ (%)
100
AT
B
80
60
40
20
0
0
0
10
20
30
Days after leukemia challenge
40
NT
P=0.0025
EZN
P=0.0003
ATRA
P=0.0002
ATRA+EZN
0
20
40 80
120
160
Days after transplantation
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200
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Synergistic leukemia eradication by combined treatment with
retinoic acid and HIF inhibition by EZN-2208 (PEG-SN38) in
preclinical models of PML-RARα and PLZF-RARα driven
leukemia
Nadia Coltella, Roberta Valsecchi, Manfredi Ponente, et al.
Clin Cancer Res Published OnlineFirst April 30, 2015.
Updated version
Supplementary
Material
Author
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