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IL2RA promotes aggressiveness and stem cell-related properties of acute
myeloid leukemia
Chi Huu Nguyen1,2*, Angela Schlerka1,2, Alexander M. Grandits1,2, Elisabeth Koller3, Emiel van der Kouwe4, George S. Vassiliou5, Philipp B. Staber4, Gerwin Heller1,2,#, Rotraud
Wieser1,2,#*
1Division of Oncology, Department of Medicine I, Medical University of Vienna, Vienna, Austria. 2Comprehensive Cancer Center, Vienna, Austria. 33rd Medical Department, Hanusch Hospital, Vienna, Austria. 4Division of Hematology and Hemostaseology, Department of Medicine I, Medical University of Vienna, Vienna, Austria. 5Wellcome Medical Research Council Cambridge Stem Cell Institute, Department of Haematology, University of Cambridge, Cambridge, CB2 0AW, United Kingdom
#Equal contribution
*Corresponding authors: Chi Huu Nguyen, PhD Rotraud Wieser, PhD Medical University of Vienna Medical University of Vienna Department of Medicine I Department of Medicine I Division of Oncology Division of Oncology Waehringer Guertel 18-20 Waehringer Guertel 18-20 A-1090 Vienna, Austria A-1090 Vienna, Austria Phone: +43 1 40400 73535 Phone: +43 1 40400 73779 E-mail: [email protected] E-mail:[email protected]
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Running title: IL2RA promotes AML aggressiveness and stem cell properties Keywords: IL2RA, CD25, acute myeloid leukemia, leukemic stem cells.
Conflict of interest statement
The authors declare no potential conflicts of interest.
Funding statement
This work was funded by the Austrian Science Fund (FWF), projects P28256-B28 and
P28013-B28 to Rotraud Wieser.
Competing interests
None of the authors declares any conflict of interest with respect to this study. The FWF did
not play any role in the design of the study, the writing of the manuscript, or its submission to
“Cancer Research".
Abstract: 223 words
Main Text: 4649
Figures: 6
Tables: 1
References: 55
Supplemental Files: 3
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Abstract
Overexpression of IL2RA, which encodes the alpha chain of the interleukin-2 receptor, is associated with chemotherapy resistance and poor outcome in acute myeloid leukemia
(AML). The clinical potential of anti-IL2RA therapy is therefore being explored in early- stage clinical trials. Notwithstanding, only very limited information regarding the biological function of IL2RA in AML is available. Using genetic manipulation of IL2RA expression as well as antibody-mediated inhibition of IL2RA in human cell lines, mouse models, and primary patient samples, we investigated the effects of IL2RA on AML cell proliferation and apoptosis, and on pertinent signalling pathways. The impact of IL2RA on the properties of leukemic stem cells (LSC) and on leukemogenesis were queried. IL2RA promoted proliferation and cell cycle activity and inhibited apoptosis in human AML cell lines and primary cells. These phenotypes were accompanied by corresponding alterations in cell cycle machinery and in pathways associated with cell survival and apoptosis. The biological roles of IL2RA were confirmed in two genetically distinct AML mouse models, revealing that
IL2RA inhibits differentiation, promotes stem cell-related properties, and is required for leukemogenesis. IL2RA antibodies inhibited leukemic, but not normal, hematopoietic cells and synergised with other anti-leukemic agents in this regard. Collectively, these data show for the first time that IL2RA plays key biological roles in AML and underscore its value as a potential therapeutic target in this disease.
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Statement of Significance
This study identifies IL2RA as a potential therapeutic target in AML, where it is shown to regulate proliferation, differentiation, apoptosis, stem cell-related properties, and leukemogenesis.
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Introduction
Acute myeloid leukemia (AML) is a hematopoietic malignancy with an annual incidence of
5–8 cases per 100,000 population and a median age of 67 years at diagnosis (1-3). It is
organized in a hierarchical manner, i.e., bulk leukemic cells are derived from leukemic stem
cells (LSCs), which are thought to represent the source of disease emergence, therapy
resistance, and relapse (4,5). Standard treatment is based on chemotherapy with the
deoxycytidine analog cytarabine (araC) and an anthracycline, but both primary and secondary
resistance are frequent, so that only 20-30% of patients achieve long-term disease-free
survival (6,7).
AML is a genetically and prognostically heterogeneous disease. It is caused by various
recurrent chromosomal abnormalities and gene mutations, which accumulate in
hematopoietic stem and progenitor cells (HSPCs) and transform them into LSCs (8-12).
Additionally, gene expression changes contribute to AML formation (12-15). The
identification and characterization of such recurrent molecular and genetic abnormalities has significantly improved our understanding of the mechanisms of leukemogenesis, as well as
prognostication. Furthermore, certain aberrations represent potential targets for rationally designed novel therapeutics for the treatment of AML (16-18). Some of these have recently been approved for clinical use, among them Gemtuzumab-Ozogamicin, an antibody-drug conjugate directed against the cell surface molecule CD33 (16,17). Despite of these advances,
AML remains a deadly disease, and additional potential therapeutic targets are actively being searched for (16,19,20). Among the identified candidates is the cell surface molecule IL2RA
(19).
IL2RA (CD25) represents a low affinity receptor for its ligand, interleukin 2 (IL-2). Together
with IL2RB (CD122) and IL2RG (CD133), it forms the high-affinity IL-2 receptor (21,22).
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IL-2 binding to its receptor causes activation of JAK1 and JAK3, which in turn activate
several downstream pathways regulating cell survival and proliferation, namely, the
PI3K/AKT, RAS/RAF/MEK/ERK, and STAT5 pathways (21,23).
In healthy tissues, IL2RA is mainly expressed on activated T cells and regulatory T cells, as
well as some other (activated) hematopoietic cell types, but notably not on hematopoietic
stem cells (HSCs) (19,21,24). IL2RA expression was elevated in a variety of cancers, mostly
of the hematopoietic system (19,21,22,24,25). It is a diagnostic marker, and also used for
detection of minimal residual disease, in hairy cell leukemia (26). In chronic myelogenous leukemia (CML), IL2RA was specifically up-regulated in LSCs (24), but reports on its function were controversial, describing IL2RA as either a promoter or an inhibitor of CML cell proliferation and disease aggressiveness (24,25). In AML, IL2RA overexpression was
consistently associated with poor therapy response and adverse outcome (27-36) and scored
as the second top hit in an unbiased search for genes whose expression predicted survival
(37). IL2RA expression was also increased upon development of AML from myelodysplastic
syndrome or myeloproliferative disease and at relapse of AML (29). Despite this compelling
evidence for the role of IL2RA in disease aggressiveness, and even though phase I studies
exploring the possible use of IL2RA directed therapy in AML have been incepted
(NCT02588092, NCT00085150) (21), the biological functions of IL2RA in AML and the
underlying molecular mechanisms have not been addressed so far.
In the present study, we used human AML cell lines, Flt3-ITD/Npm1c and MLL-AF9 driven
mouse models of AML, and primary AML samples to show that IL2RA promoted
proliferation and inhibited apoptosis and differentiation of AML cells. Moreover, it
augmented LSC-related properties and was required for leukemogenesis. IL2RA antibodies
inhibited leukemic, but not normal hematopoietic cells, and synergized with araC and with
BCL2 and CDK4/CDK6
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inhibitors in this regard, suggesting effective therapeutic strategies for AML patients with
IL2RA overexpression.
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Methods
Ethics Statement
Animal experiments were approved by the Animal Ethics Committee of the Medical
University of Vienna and the Austrian Federal Ministry of Science, Research, and Economy
(GZ66.009/0308-WF/V/3b/2015). Federation of European Laboratory Animal Science
Associations and Austrian guidelines to minimize animal distress and suffering were followed. Experiments with primary AML samples were approved by the Ethics Committee of the Medical University of Vienna (EK 1394/2019) and conducted in accordance with the declaration of Helsinki.
Cell culture
The human myeloid cell lines HL60 and UCSD/AML1 were kindly provided by Dr. Peter
Valent (Department of Medicine I, Medical University of Vienna, Austria) in 2013, and by
Dr. Frank Speleman (Centre for Medical Genetics, Ghent University Hospital, Ghent,
Belgium) in 2012, respectively. The viral packing cell line Phoenix-GP was a gift from Dr.
Hannes Stockinger (Institute for Applied Immunology, Medical University of Vienna,
Austria) in 2009. Upon receipt, stocks of the cell lines were prepared and stored in liquid nitrogen. New aliquots from these stocks were thawed every 1-3 months, and cultured as
described in the Supplemental Methods. Authentication was performed via the specific
growth characteristics (adherent or suspension, doubling times), morphology, functional
properties (ability to produce infectious particles in the case of Phoenix-GP cells), and
expression of specific genes (EVI1, IL2RA, etc.). All cell lines were tested regularly (last test,
July, 2020) for mycoplasma contamination using MycoAlert mycoplasma detection kit
(Lonza), and showed negative results.
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Immunoblot analysis
Preparation of protein lysates, SDS-PAGE, transfer to PVDF membranes (Hybond-P;
Amersham), and incubations with antibodies (Supplemental Table 1) were performed using standard procedures. Blots were developed using SuperSignal West Femto or Pico
Chemiluminescent Substrates (both from Thermo Scientific) and scanned using a ChemiDoc
Touch Imaging System (Bio Rad). Densitometric analysis was performed with Image-J software (National Institutes of Health, Maryland, USA).
Ex vivo culture of cells from Flt3-ITD/Npm1c driven murine AML, Il2ra knock-down, and biological assays
Spleen cells from C57BL/6 mice that had succumbed to AML following transplantation with
Flt3-ITD/Npm1c transformed hematopoietic cells (38) were cultured in IMDM medium
containing 10% FBS, 1% L-Glutamine (all from Thermo Fisher Scientific), 50 ng/ml mSCF
(Peprotech), 10 ng/ml mIL-3 (Peprotech), and 10 ng/ml mIL-6 (BioLegend). To knock down
Il2ra, they were transduced with the lentiviral vector pRRL_SFFV_GFP_mirE_PGK_NeoR containing shIl2ra-1 (5'-CACAGCAGTTCTAAAGCTTTA-3'), shIl2ra-2 (5'-
AAGAGAGGTTTCCGAAGACTA-3'), or shRen (5'-AAGGAGGAAAGTTAAATAGAAT-
3') as a control. Generation of shRNA constructs and transduction of leukemic cells are
described in Supplemental Methods. Sorted GFP+ cells were expanded in the above described medium and used for in vitro assays. Proliferation, cell cycle, and apoptosis were measured as described for human cell lines (Supplemental Methods). To assess myeloid differentiation, cells were stained with the respective antibodies (Supplemental Table 1) and subjected to
flow cytometry (LSRFortessa, Becton Dickinson). For serial replating assays, 2,000 cells per
well of a six-well plate were seeded into methyl cellulose (MethoCult GF M3434; Stem Cell
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Technologies). Technical duplicates were performed. Colonies were counted after 7 days, and
2,000 cells per condition were used for replating.
Transplantation experiments
For transplantation, 6-8 week old female C57BL/6 recipient mice were sub-lethally irradiated
(5 Gy), anaesthesized on the next day, and injected retro-orbitally with GFP+ shRen- or
shIl2ra-transduced spleen cells from mice with Flt3-ITD/Npm1c-driven AML (600,000
cells/mouse). Mice were sacrificed when terminally ill. The significance of differences in
survival was probed using the log-rank test. Peripheral blood cell counts were determined
using a hematology analyzer Sysmex XN-350 (Sysmex), and the proportion of GFP+ cells in
BM and spleen was assessed by flow cytometry.
Primary human AML samples and healthy controls
Cryopreserved primary human AML and healthy bone marrow samples were provided by the
Hanusch Hospital, Vienna, Austria, and the General Hospital, Vienna, Austria, respectively.
BM CD34+ cells from healthy donors were purchased from Lonza. Primary cells were thawed
and cultured as described previously (39). Briefly, after thawing in a 37° waterbath, cells
were washed and incubated with RPMI medium containing 50 µg/ml DNAse (Sigma-
Aldrich) for 60 min to prevent cell clumping. Cells were cultured in RPMI medium supplemented with 10% FBS, 1% glutamine, 1% Penicillin/Streptomycin, and 100 ng/ml each of SCF, IL3, and G-CSF (all from Peprotech). For antibody treatment, cells were seeded
at a density of 2x105/ml and incubated with 3 µg/ml monoclonal human IL2RA (hIL2RA)
antibody (Clone BC 96, BioLegend), 6 µg/ml Basiliximab (Szabo Scandic), or the corresponding amounts of isotype control antibodies (BioLegend or Szabo Scandic) for 72 h.
For combination treatment, cells were incubated for 48 h with the indicated concentrations of
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hIL2RA antibody, araC (provided by the dispensary of the General Hospital of Vienna), the
BCL2 inhibitor Navitoclax (MedChemExpress), and/or the CDK4/CDK6 inhibitor
Abemaciclib (MedChemExpress). Incubation with human recombinant IL-2 (Sigma-Aldrich) employed a concentration of 100 ng/ml for 72 h. Cell viability, cell cycle distribution, and apoptosis were measured as described for cell lines (Supplemental Methods). For colony formation assays, cells were treated as described above and transferred to methyl cellulose
(MethoCult H4434, Stemcell Technologies) at a concentration of 1x105 cells per well of a 6- well plate. Technical duplicates were performed, and total colonies were counted after 14 days. To determine total viable cell numbers, colonies were harvested and cells were washed, re-suspended in PBS, and counted using a CASY Cell Counter (Roche Innovatis AG).
Statistical analyses
For experiments with cell lines and primary mouse cells, at least three independent biological replicates were performed; results are displayed as means ± SEM. For experiments with primary AML samples, technical replicates were performed whenever feasible in terms of cell numbers; results are displayed as means ± SD. Apoptosis and cell cycle assays with primary samples were performed as single measurements. Significance of differences between two independent groups was calculated using Student’s two-tailed t-test; significance of differences between multiple groups was determined by 2-way ANOVA followed by Bonferroni’s post-hoc test. The log-rank test was used to evaluate survival differences between groups of mice. p-values <0.05 were considered statistically significant.
Analyses were performed using GraphPad Prism 6 software (GraphPad Software, San Diego,
CA, USA).
Additional methods
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Additional and more detailed methods are available in Supplemental Methods.
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Results
High IL2RA expression is an independent prognostic parameter for poor outcome of
AML
Aberrant expression of IL2RA protein or mRNA was associated with unfavourable outcome
in AML (27-34,37). To confirm and extend these findings, we analysed the expression and potential prognostic relevance of IL2RA in several publicly available gene expression data
sets. IL2RA expression was significantly up-regulated in AML compared to healthy bone
marrow cells (GSE13159; Figure 1A). Its expression was also significantly higher in LSC
enriched cell populations as compared to LSC depleted cell populations (GSE76008; Figure
1B) or to healthy HSCs and progenitor cells (GSE63720, GSE30029; Figure 1C). In addition,
high IL2RA expression was an independent prognostic parameter for decreased overall
survival (OS) in 7 AML patient cohorts, contained in data sets GSE37642, GSE12417 (2
cohorts), GSE6891 (2 cohorts), GSE71014, and TCGA_LAML and comprising a total of
1272 patients (Figure 1D, Table 1, Supplemental Figure 1, the p-value was adjusted for multiple testing (padj) according to Altman et al.(40)). European LeukemiaNet (ELN) risk classification data were available for GSE37642. The favourable, intermediate, and adverse risk groups contained 107, 174, and 86 patients, respectively. High IL2RA expression had no
impact on OS in the adverse risk group, but was significantly associated with shorter OS in
the favourable (p=0.034) and intermediate (p=0.006) risk groups (Figure 1E). We therefore
asked whether IL2RA expression could refine the ELN risk classification. Based on median
survival times, ELN-favourable/IL2RAlow patients were assigned to a re-defined favourable
risk group, ELN-favourable/IL2RAhigh and ELN-intermediate/IL2RAlow patients were combined into an intermediate risk group, and ELN-intermediate/IL2RAhigh and ELN-adverse
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patients constituted the adverse risk group. The resulting ELN + IL2RA classification
substantially improved the ELN risk score (median survival, 4029, 292, and 215 days
according to ELN, and not reached, 493, and 113 days according to the ELN + IL2RA
classification; Figure 1F).
In summary, IL2RA expression was up-regulated in AML vs. normal BM, and in LSCs vs.
bulk leukemic cells and vs. HSCs. Moreover, its expression was an independent prognostic
parameter for poor outcome of AML, and could further refine the ELN risk classification.
IL2RA promotes proliferation and inhibits apoptosis of human AML cell lines
To address the functional role of IL2RA in AML, we transduced UCSD/AML1 cells, which
expressed high levels of IL2RA (Supplemental Figure 2A), with lentiviral vectors containing
two different shRNAs against IL2RA (shIL2RA-1, shIL2RA-2), or a non-targeting shRNA as
control (shCtrl). Down-regulation of IL2RA, confirmed by flow cytometry (Figure 2A), significantly reduced cell proliferation (Figure 2B). Accordingly, it decreased the proportion
of actively cycling cells (cells in S/G2/M), and increased the proportion of cells in G0/G1 as
well as in sub-G1 (apoptotic cells; Figure 2C, Supplemental Figure 2B). The increase in
apoptosis was confirmed by the Annexin V assay (Figure 2D; Supplemental Figure 2C; higher proportions of apoptotic cells than in Figure 2C are most likely due to the fact that
Annexin V labels also early apoptotic cells). Corroborating the specificity of these effects, re-
expression of a codon-optimized (i.e., shRNA insensitive) version of IL2RA in
UCSD/AML_shIL2RA-1 and UCSD/AML_shIL2RA-2 cells counteracted the effects of the
knock-down on cell proliferation, cell cycle distribution, and apoptosis (Supplemental Figure
2D-G).
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Next, we asked whether antibodies specifically targeting IL2RA would also reduce AML cell
proliferation. Indeed, treatment of UCSD/AML1 cells with two different hIL2RA antibodies,
including Basiliximab which was approved for the prevention of renal transplant rejection
(41), resulted in dose and time-dependent inhibition of cell proliferation (Supplemental
Figure 2H, I). In contrast, hIL2RA antibodies had no effect on the proliferation of the
IL2RAlow cell line HL60 (Supplemental Figure 2J).
HL60 cells were also used to investigate the consequences of lentiviral overexpression of
IL2RA. Expression of IL2RA on the surface of transduced cells was confirmed by flow cytometry (Figure 2E). It promoted cell proliferation (Figure 2F), increased the proportion of cells in S/G2/M, decreased the proportion of cells in G0/G1 and in sub-G1, and inhibited apoptosis as determined by Annexin V staining (Figure 2G, H; Supplemental Figure 2K, L).
In summary, our data show that IL2RA promoted proliferation and cell cycle progression and
inhibited apoptosis of human AML cell lines.
IL2RA affects the abundance and activity of regulators of proliferation, survival, cell
cycle activity, and apoptosis
To explore the mechanisms by which IL2RA exerts its above described effects, we determined the expression and activation status of key regulators of proliferation, survival, cell cycle progression, and apoptosis by immunoblot analysis. Knock-down of IL2RA in
UCSD/AML1 cells decreased the activating phosphorylations on the proliferation and
survival promoting kinases, AKT and ERK (Figure 3A, Supplemental Figure 3A). It also
resulted in reduced phosphorylation of STAT5, another central regulator of survival and proliferation in AML (Figure 3A, Supplemental Figure 3A). Accordingly, JAK-STAT signaling was among the pathways that were enriched in the list of genes co-expressed with
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IL2RA in the TCGA_LAML dataset (Supplemental Table 2A, B). Regarding the core cell
cycle machinery, the positive regulators of the G1/S transition, CDK6, CDK2, and cyclin E,
were down-regulated, while the cell cycle inhibitors p21 and p27 were up-regulated in
shIL2RA-transduced UCSD/AML1 cells (Figure 3A, Supplemental Figure 3A). Furthermore,
IL2RA knock-down reduced the level of the anti-apoptotic BCL2 protein, and increased
expression of the pro-apoptotic BAX protein (Figure 3A, Supplemental Figure 3A).
Overexpression of IL2RA in HL60 cells elicited molecular effects opposite to those observed
upon IL2RA knock-down in UCSD/AML1 cells (Figure 3B, Supplemental Figure 3B). Taken
together, the pro-proliferative effects of IL2RA on AML cells were associated with
corresponding effects on key regulators of proliferation, survival, cell cycle progression, and
apoptosis.
Knock-down of Il2ra promotes differentiation and apoptosis, decreases proliferation and LSC-related properties, and prevents leukemogenesis in murine AML
In human AML, aberrant IL2RA expression correlated positively with the presence of
activating internal tandem duplications in the FLT3 gene (FLT3-ITD) and with
nucleophosmin (NPM1) gene mutations (NPM1c) (32). A Flt3-ITD/Npm1c driven mouse
model of AML (38) was therefore employed to investigate the effects of Il2ra on leukemia
cell proliferation, differentiation, and apoptosis, LSC activity, and leukemogenesis.
BM cells from mice that had succumbed to AML after transplantation with Flt3-ITD/Npm1c
transformed hematopoietic cells contained higher levels of Il2ra mRNA than their healthy
counterparts (Supplemental Figure 4A). Monoclonal mouse IL2RA (mIL2RA) antibody inhibited proliferation, promoted apoptosis, and decreased serial replating activity
(considered a proxy of LSC activity) of spleen cells from these mice (Supplemental Figure
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4B-D). To further investigate the role of Il2ra in Flt3-ITD/Npm1c driven AML, leukemic
spleen cells were transduced with lentiviral vectors expressing shRNAs against Il2ra
(shIl2ra-1, shIl2ra-2), or an shRNA against Renilla luciferase (shRen) as a control (Figure
4A). Down-regulation of IL2RA in shIl2ra- vs. shRen-transduced cells was confirmed by flow cytometry (Figure 4B). Sorted GFP+ cells were used for in vitro assays and for transplantation of recipient mice. By analogy to the results obtained with the human AML cell lines, knock-down of Il2ra in murine leukemic cells significantly inhibited cell
proliferation (Figure 4C), decreased the proportion of cells in S/G2/M, and increased
apoptosis (Figure 4D, E; Supplemental Figure 4E, F). Knock-down of Il2ra also induced
myeloid differentiation, as shown by increases in the proportions of Gr-1+ among CD11b+
GFP+, and cKit- among GFP+ cells (Figure 4F, G; Supplemental Figure 4G, H). Furthermore,
Il2ra down-regulation reduced the serial replating activity of murine AML cells (Figure 4H).
Upon transplantation into recipient mice, both shRen- and shIl2ra-expressing cells caused
AML-like disease with increased white blood cell counts and spleen weight, and decreased
red blood cell counts and platelet numbers (Supplemental Figure 4I), yet down-regulation of
Il2ra significantly increased disease latency (median survival, shRen: 56 days, shIl2ra-2: 76
days, shIl2ra-1: 113 days; Figure 4I). Moreover, while large proportions of BM and spleen
cells from moribund mice maintained shRen expression (as measured by the proportion of
GFP+ cells), cells carrying shIl2ra were entirely outcompeted by shRNA-negative cells
(Figure 4J; Supplemental Figure 4J).
To confirm the role of Il2ra on the background of a different genetic driver lesion, a mouse
model based on the AML-associated fusion oncogene MLL-AF9 was employed (39,42,43).
As with the Flt3-ITD/Npm1c model, Il2ra mRNA expression was up-regulated in BM cells
from mice that succumbed to AML after transplantation with MLL-AF9 transformed hematopoietic cells as compared to healthy BM cells (Supplemental Figure 5A). mIL2RA
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antibody significantly inhibited cell proliferation, and induced differentiation and apoptosis
(Supplemental Figure 5B-D). Furthermore, mIL2RA antibody diminished LSC-related
properties: it reduced the abundance and quiescence of a cell population enriched for LSCs
(defined by the marker combination Venus+ lin− Sca1− c-Kit+ CD34+ CD16/CD32high in the
MLL-AF9 model (37,39,42)), and decreased the serial replating activity of MLL-AF9
expressing leukemic BM cells (Supplemental Figure 5E-G). Likewise, shRNA-mediated
knock-down of Il2ra in MLL-AF9 cells decreased proliferation, led to a strong depletion of
transduced cells over time, increased differentiation and apoptosis, and reduced LSC-related properties (Supplemental Figure 5H-N).
In summary, these data show that Il2ra inhibits differentiation and apoptosis, and augments
proliferation, LSC-related properties, and leukemogenesis in murine AML.
hIL2RA antibodies inhibit proliferation and clonogenic activity of primary human
AML cells, and enhance the anti-leukemic activity of araC and of targeted drugs
We next asked whether our findings in AML cell lines and murine AML models could be
translated to primary AML cells. Flow cytometry confirmed high and low IL2RA expression,
respectively, in four and two AML samples selected based on qRT-PCR data (Supplemental
Figure 6A). Clinical characteristics of the patients are summarised in Supplemental Table 3.
Exposure of IL2RAhigh, but not IL2RAlow, primary AML cells to hIL2RA antibody or
Basiliximab decreased viability, proliferation, and the proportion of cells in S/G2/M, and
enhanced apoptosis (Figure 5A-D, Supplemental Figure 6B-D). Pre-treatment with hIL2RA
antibodies also strongly reduced colony numbers and the total number of viable cells in
colonies upon plating in methyl cellulose, reflecting reduced stem cell/progenitor activity, in a manner dependent on IL2RA expression (Figure 5E; Supplemental Figure 6E, F).
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Furthermore, hIL2RA antibodies led to alterations of the levels of p-ERK, p-AKT, pSTAT5,
CDK6, CDK2, BCL2, and BAX in a manner consistent with their biological effects (Figure
5F). In support of the potential therapeutic utility of hIL2RA antibodies in IL2RAhigh AML, these agents had much weaker effects on the viability, apoptosis, and clonogenic growth of
CD34+ HSPCs from healthy donor BM (Supplemental Figure 6G-I). Also, treatment with
hIL2RA antibody had no impact on the relative abundance of different progenitor populations
in healthy BM cells (Supplemental Table 4).
In agreement with a previous report (44), araC treatment induced IL2RA expression both in
primary AML samples (Figure 6A) and in UCSD/AML1 cells (Supplemental Figure 7A).
Therefore, we asked whether the hIL2RA antibody would enhance the anti-leukemic activity
of araC. Indeed, the combination of these two agents inhibited viability of primary AML
samples (Figure 6B) and of UCSD/AML1 cells (Supplemental Figure 7B) in a synergistic
manner in the majority of dose combinations. These results were confirmed using the
Annexin V assay (Figure 6C, Supplemental Figure 7E).
To also explore potential combination therapies suggested by our molecular data (Figures 3 and 5F, Supplemental Figure 3), human AML cells were co-treated with hIL2RA antibody
and either the BCL2 inhibitor Navitoclax or the CDK4/CDK6 inhibitor Abemaciclib. Again,
synergistic inhibition of AML cell viability was observed (Supplemental Figure 7C-G).
In summary, hIL2RA antibody efficiently inhibited the proliferation and clonogenic activity
of primary IL2RAhigh AML samples, while healthy CD34+ BM cells were substantially less sensitive. hIL2RA antibody synergised both with araC and with molecularly targeted
inhibitors to cause cytotoxicity in AML cells, suggesting that these combinations have
therapeutic potential for patients with IL2RAhigh AML.
19
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Discussion
Several studies have highlighted the prognostic importance of IL2RA in AML (27-36), and
IL2RA is actively pursued as a therapeutic target in this disease (21). Nevertheless,
surprisingly little is known about the pathophysiological role of this gene in AML cells. Here,
we confirmed IL2RA as an independent prognostic parameter in several publicly available
gene expression datasets, which comprise AML patient populations with different genetic and age compositions and a total of almost 1300 patients. Moreover, IL2RA expression was able
to further refine the ELN classification. These analyses strengthen and extend previous findings, and further emphasise the need to understand the function of IL2RA in AML.
Using gene overexpression and knock-down approaches in, and/or antibody treatment of,
human AML cell lines, AML mouse models, and primary AML cells, we report here for the
first time that IL2RA promoted proliferation and cell cycle activity, and inhibited
differentiation and apoptosis, of AML cells. Interestingly, these effects likely occurred
independently of the IL2RA ligand, IL-2. Firstly, neither the primary AML samples nor the
AML cell lines used in our study secreted detectable levels of IL-2 (Supplemental Figure
8A). Secondly, in line with a previous report that suggested that IL-2 acted on AML cells in a non-cell autonomous, indirect manner (45), incubation with human recombinant IL-2 had no
effect on proliferation, apoptosis, or colony formation of either IL2RAhigh or IL2RAlow AML
cells (Supplemental Figure 8B-H).
The phenotypes elicited by manipulation of IL2RA expression were associated with
corresponding changes in proteins associated with proliferation/survival (p-ERK, p-AKT, p-
STAT5), cell cycle regulation (CDK6, CDK2, Cyclin E, p21 and p27), and apoptosis (BCL2
and BAX). Notably, IL2RA was previously described both as a downstream target and an
20
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upstream regulator of STAT5 (24,46,47), suggesting a positive feedback loop between these
two signaling components. This pivotal role in the JAK-STAT pathway, as well as its effect
on several other central signaling pathways, explain the strong impact of alterations in IL2RA
levels on numerous key features of AML. Most compellingly, knock-down of Il2ra
completely abolished the ability of Flt3-ITD/Npm1c-transformed cells to give rise to AML in
vivo.
Beyond its roles in the proliferation and survival of AML blasts, IL2RA was also relevant to
AML stem cells. We found that IL2RA expression was higher in AML LSCs compared to
bulk leukemic cells or healthy HSCs. Moreover, Il2ra augmented LSC-related properties in two independent AML mouse models, and inhibition of IL2RA by specific antibodies
inhibited stem cell/progenitor activity in primary human AML samples. The inability of
shIl2ra-transduced murine Flt3-ITD/Npm1c AML cells to contribute to leukemogensis in vivo
precluded bona fide stem cell assays like serial transplantation or in vivo limited dilution
assays, but per se suggested a requirement for Il2ra to maintain leukemia initiating potential.
In line with our data, IL2RA has previously been reported as an AML LSC associated gene
(19), and the gene expression signature of IL2RAhigh AML blasts was enriched for gene
expression profiles associated with AML LSCs (32). Similarly, studies on human CML found
that IL2RA was predominantly expressed in LSCs, but not in more mature cell fractions or in
normal HSCs (24,25,48). In a CML mouse model, IL2RAhigh cells were enriched for LSCs,
and therapeutic targeting of IL2RA reduced LSC numbers and improved animal survival
(25).
Taken together, our results provide, for the first time, strong experimental support for the
potential of IL2RA as a novel therapeutic target in AML. The fact that knock-down of Il2ra
in IL2RAhigh murine AML cells diminished LSC-related properties and entirely prevented
any contribution of the knock-down cells to leukemia formation suggests that IL2RAhigh
21
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AML may be "addicted" to this oncogene. Thus, even though numerous different driver
lesions are able to cause AML (8-15), a subset of patients may exhibit an actionable
dependency on the activity of IL2RA. Correspondingly, IL2RAhigh AML cells were sensitive to inhibition by hIL2RA antibodies. In contrast, healthy CD34+ BM HSPCs were hardly responsive to this treatment. In line with this, transplantation of healthy IL2RAlow HSCs into
NOD-SCID mice resulted in multilineage hematopoietic reconstitution, including the
generation of IL2RAhigh myeloid and lymphoid progeny (19). These data indicate that normal
HSC function is independent of IL2RA, and point towards the possible existence of a
therapeutic window. Furthermore, even though Il2ra knock-out mice suffered from a
lymphoproliferative disorder and autoimmune disease in adulthood, they were healthy and
indistinguishable from wild-type littermates up to 4 weeks of age (49), suggesting that even the complete absence of Il2ra function is tolerable for several weeks. Inhibition of IL2RA through specific antibodies, or elimination of IL2RAhigh AML cells through antibody-drug or antibody-radioisotope conjugates, may therefore represent an effective and safe treatment
strategy for patients with IL2RAhigh AML. Indeed, several early-phase clinical studies addressed the potential utility of IL2RA antibodies in hematological malignancies (21,50-54).
Efficacy and acceptable toxicity of IL2RA antibodies conjugated to radioisotopes or toxins were reported in various types of lymphoma (50,51,53). Also, a phase I study of the
pyrrolobenzodiazepine-conjugated hIL2RA antibody ADCT-301 in patients with
relapsed/refractory IL2RAhigh AML or ALL (NCT02588092) showed an acceptable safety profile (54).
Because efficacies of targeted therapies can be augmented by combination with
chemotherapeutic drugs (16), we queried the anti-leukemic activity of hIL2RA antibody and
of araC alone and in combination. Indeed, these two agents inhibited the viability of an AML cell line and of primary AML samples in a moderately synergistic manner. This synergy may
22
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be due to the fact that both drugs have different mechanisms of action, i.e., hIL2RA antibody
enhances anti-leukemic signalling pathways while araC causes DNA damage. As an
alternative or additional explanation, araC treatment resulted in higher levels of IL2RA on
AML cells (44) (and Figure 6A, Supplemental Figure 7A). This could be a defense
mechanism providing some protection from araC induced apoptosis that might be alleviated
by IL2RA inhibition. In line with our observations, the combined application of the anti-
IL2RA immunotoxin LMB-2 and the antimetabolite gemcitabine resulted in increased cytotoxicity and antitumor activity both in vitro and in a mouse xenograft model of a human
epidermoid carcinoma cell line, proposed as a model for adult T-cell leukemia (55). IL2RA antibodies also enhanced the activity of targeted drugs. Here, we show synergistic effects with inhibitors of the IL2RA downstream effectors BCL2 and CDK6 both in an AML cell line and in primary AML samples. In a previous report, an IL2RA antibody augmented the reduction in leukemic burden effected by the tyrosine kinase inhibitor nilotinib in a mouse model of CML (25).
In summary, we report here the first functional characterization of the previously proposed
therapeutic target, IL2RA, in AML. IL2RA promoted proliferation and stem cell-related
properties, and inhibited differentiation and apoptosis in different AML model systems. AML
cells with experimental down-regulation of Il2ra were unable to contribute to leukemia formation in recipient mice, possibly indicating oncogene addiction. IL2RA inhibition reduced the viability of AML cells alone and in synergy with araC and with targeted drugs, but had little effect on normal HSPCs. Together, these results provide a strong rationale for further development of therapeutic strategies directed at IL2RA for the treatment of AML.
23
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Author contributions
CHN, PBS, GSV, and RW designed experiments. CHN, AS, AMG, and EvdK performed
experiments. CHN and RW interpreted experimental data. GH and CHN performed
bioinformatics analyses of publicly available gene expression data. EK provided patient AML
samples. CHN, GH, and RW wrote the manuscript. CHN, RW, and GH designed and
supervised the study. All authors critically read the manuscript and approved of the final
version, as well as its submission to "Cancer Research".
Acknowledgements
This work was funded by the Austrian Science Fund (FWF), projects no P28256-B28 and
P28013-B28 to R.W. The authors gratefully acknowledge Dr. Andreas Spittler of the Core
Facility for Flow Cytometry, Medical University of Vienna, Vienna, Austria, who performed
cell sorts and gave invaluable advice for flow cytometry applications. We thank Dr. Klaus
Schmetterer and Marlene Gerner, Msc (Department of Laboratory Medicine, Medical
University of Vienna) for performing IL-2 ELISAs. Dr. Gregor Hoermann (Clinical
Department of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna,
Austria) kindly provided human IL2RA shRNA (pLKO.1_shIL2RA_mCherry and
pLKO.1_shCtrl_mCherry) and lentiviral overexpression constructs
(pLOC_IL2RA_IRES_GFP, pLOC_IRES_GFP). Dr. Johannes Zuber (Research Institute of
Molecular Pathology, Vienna, Austria) is acknowledged for providing
pRRL_SFFV_GFP_mirE_shRen713 and pMSCV_MLL-AF9_IRES_Venus. Dr. Karin
Nowikovsky, Erwin Tomasich, Msc, and Dr. Peter Valent (all from the Department of
Medicine I, Medical University of Vienna, Austria) shared BCL2, BAX, p21, ERK, p-
STAT5, and STAT5 antibodies. The dispensary of the General Hospital, Vienna, is gratefully
acknowledged for supplying araC. We thank Dr. Tobias Herold from the Acute Myeloid
24
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Leukemia Cooperative Group (AMLCG) Munich for providing clinical data to data set
GSE37642.
25
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References
1. Almeida A, Ramos F. Acute myeloid leukemia in the older adults. Leuk Res Rep
2016;6:1-7
2. Sanz M, Iacoboni G, Montesinos P, Venditti A. Emerging strategies for the treatment
of older patients with acute myeloid leukemia. Ann Hematol 2016;95:1583-93
3. Sanford D, Ravandi F. Management of Newly Diagnosed Acute Myeloid Leukemia in
the Elderly: Current Strategies and Future Directions. Drugs Aging 2015;32:983-97
4. Wiseman DH, Greystoke BF, Somervaille TC. The variety of leukemic stem cells in
myeloid malignancy. Oncogene 2014;33:3091-8
5. Zagozdzon R, Golab J. Cancer stem cells in haematological malignancies. Contemp
Oncol (Pozn) 2015;19:A1-6
6. Forman S, Rowe J. The myth of the second remission of acute leukemia in the adult.
Blood 2013;121:1077-82
7. Bryan J, Jabbour E. Management of Relapsed/Refractory Acute Myeloid Leukemia in
the Elderly: Current Strategies and Developments. Drugs Aging 2015;32:623-37
8. Schoch C, Kern W, Schnittger S, Buchner T, Hiddemann W, Haferlach T. The
influence of age on prognosis of de novo acute myeloid leukemia differs according to
cytogenetic subgroups. Haematologica 2004;89:1082-90
9. Grimwade D, Ivey A, Huntly B. Molecular landscape of acute myeloid leukemia in
younger adults and its clinical relevance. Blood 2016;127:29-41
10. Walker A, Marcucci G. Molecular prognostic factors in cytogenetically normal acute
myeloid leukemia. Expert Rev Hematol 2012;5:547-58
26
Downloaded from cancerres.aacrjournals.org on October 7, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 1, 2020; DOI: 10.1158/0008-5472.CAN-20-0531 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
11. Papaemmanuil E, Gerstung M, Bullinger L, Gaidzik V, Paschka P, Roberts N, et al.
Genomic Classification and Prognosis in Acute Myeloid Leukemia. N Engl J Med
2016;374:2209-21
12. Network CGAR. Genomic and epigenomic landscapes of adult de novo acute myeloid
leukemia. N Engl J Med 2013;368:2059-74
13. Valk P, Verhaak R, Beijen M, Erpelinck C, Barjesteh van Waalwijk van Doorn-
Khosrovani S, Boer J, et al. Prognostically useful gene-expression profiles in acute
myeloid leukemia. N Engl J Med 2004;350:1617-28
14. Ng S, Mitchell A, Kennedy J, Chen W, McLeod J, Ibrahimova N, et al. A 17-gene
stemness score for rapid determination of risk in acute leukaemia. Nature
2016;540:433-7
15. Metzeler K, Hummel M, Bloomfield C, Spiekermann K, Braess J, Sauerland M, et al.
An 86-probe-set gene-expression signature predicts survival in cytogenetically normal
acute myeloid leukemia. Blood 2008;112:4193-201
16. Bohl SR, Bullinger L, Rucker FG. New Targeted Agents in Acute Myeloid Leukemia:
New Hope on the Rise. Int J Mol Sci 2019;20
17. Lai C, Doucette K, Norsworthy K. Recent drug approvals for acute myeloid leukemia.
J Hematol Oncol 2019;12:100
18. Perl AE. The most novel of the novel agents for acute myeloid leukemia. Curr Opin
Hematol 2018;25:81-9
19. Saito Y, Kitamura H, Hijikata A, Tomizawa-Murasawa M, Tanaka S, Takagi S, et al.
Identification of therapeutic targets for quiescent, chemotherapy-resistant human
leukemia stem cells. Sci Transl Med 2010;2:17ra9
27
Downloaded from cancerres.aacrjournals.org on October 7, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 1, 2020; DOI: 10.1158/0008-5472.CAN-20-0531 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
20. Gluexam T, Grandits AM, Schlerka A, Nguyen CH, Etzler J, Finkes T, et al. CGRP
Signaling via CALCRL Increases Chemotherapy Resistance and Stem Cell Properties
in Acute Myeloid Leukemia. Int J Mol Sci 2019;20
21. Flynn MJ, Hartley JA. The emerging role of anti-CD25 directed therapies as both
immune modulators and targeted agents in cancer. Br J Haematol 2017;179:20-35
22. Kuhn DJ, Dou QP. The role of interleukin-2 receptor alpha in cancer. Front Biosci
2005;10:1462-74
23. Olejniczak K, Kasprzak A. Biological properties of interleukin 2 and its role in
pathogenesis of selected diseases--a review. Med Sci Monit 2008;14:RA179-89
24. Sadovnik I, Hoelbl-Kovacic A, Herrmann H, Eisenwort G, Cerny-Reiterer S, Warsch
W, et al. Identification of CD25 as STAT5-Dependent Growth Regulator of
Leukemic Stem Cells in Ph+ CML. Clin Cancer Res 2016;22:2051-61
25. Kobayashi CI, Takubo K, Kobayashi H, Nakamura-Ishizu A, Honda H, Kataoka K, et
al. The IL-2/CD25 axis maintains distinct subsets of chronic myeloid leukemia-
initiating cells. Blood 2014;123:2540-9
26. Maitre E, Cornet E, Troussard X. Hairy cell leukemia: 2020 update on diagnosis, risk
stratification, and treatment. Am J Hematol 2019;94:1413-22
27. Du W, He J, Zhou W, Shu S, Li J, Liu W, et al. High IL2RA mRNA expression is an
independent adverse prognostic biomarker in core binding factor and intermediate-
risk acute myeloid leukemia. J Transl Med 2019;17:191
28. Allan JN, Roboz GJ, Askin G, Ritchie E, Scandura J, Christos P, et al. CD25
expression and outcomes in older patients with acute myelogenous leukemia treated
with plerixafor and decitabine. Leuk Lymphoma 2018;59:821-8
28
Downloaded from cancerres.aacrjournals.org on October 7, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 1, 2020; DOI: 10.1158/0008-5472.CAN-20-0531 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
29. Fujiwara SI, Muroi K, Yamamoto C, Hatano K, Okazuka K, Sato K, et al. CD25 as an
adverse prognostic factor in elderly patients with acute myeloid leukemia.
Hematology 2017;22:347-53
30. Miltiades P, Lamprianidou E, Vassilakopoulos TP, Papageorgiou SG, Galanopoulos
AG, Vakalopoulou S, et al. Expression of CD25 antigen on CD34+ cells is an
independent predictor of outcome in late-stage MDS patients treated with azacitidine.
Blood Cancer J 2014;4:e187
31. Cerny J, Yu H, Ramanathan M, Raffel GD, Walsh WV, Fortier N, et al. Expression of
CD25 independently predicts early treatment failure of acute myeloid leukaemia
(AML). Br J Haematol 2013;160:262-6
32. Gonen M, Sun Z, Figueroa M, Patel J, Abdel-Wahab O, Racevskis J, et al. CD25
expression status improves prognostic risk classification in AML independent of
established biomarkers: ECOG phase 3 trial, E1900. Blood 2012;120:2297-306
33. Terwijn M, Feller N, van Rhenen A, Kelder A, Westra G, Zweegman S, et al.
Interleukin-2 receptor alpha-chain (CD25) expression on leukaemic blasts is
predictive for outcome and level of residual disease in AML. Eur J Cancer
2009;45:1692-9
34. Min WS, Kim HJ, Choi Y, Jeong HY, Kim CC. Interpretation of interleukin-2
receptor alpha positive cells during induction chemotherapy for adult acute
myelogenous leukaemia patients. Hematol Oncol 2007;25:76-83
35. Nakase K, Kita K, Kyo T, Ueda T, Tanaka I, Katayama N. Prognostic Relevance of
Cytokine Receptor Expression in Acute Myeloid Leukemia: Interleukin-2 Receptor
alpha-Chain (CD25) Expression Predicts a Poor Prognosis. PLoS One
2015;10:e0128998
29
Downloaded from cancerres.aacrjournals.org on October 7, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 1, 2020; DOI: 10.1158/0008-5472.CAN-20-0531 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
36. Kageyama Y, Miwa H, Arakawa R, Tawara I, Ohishi K, Masuya M, et al. Expression
of CD25 fluctuates in the leukemia-initiating cell population of CD25-positive AML.
PLoS One 2018;13:e0209295
37. Nguyen CH, Gluxam T, Schlerka A, Bauer K, Grandits AM, Hackl H, et al. SOCS2 is
part of a highly prognostic 4-gene signature in AML and promotes disease
aggressiveness. Sci Rep 2019;9:9139
38. Mupo A, Celani L, Dovey O, Cooper J, Grove C, Rad R, et al. A powerful molecular
synergy between mutant Nucleophosmin and Flt3-ITD drives acute myeloid leukemia
in mice. Leukemia 2013;27:1917-20
39. Nguyen CH, Bauer K, Hackl H, Schlerka A, Koller E, Hladik A, et al. All-trans
retinoic acid enhances, and a pan-RAR antagonist counteracts, the stem cell
promoting activity of EVI1 in acute myeloid leukemia. Cell Death Dis 2019;10:944
40. Altman DG, Lausen B, Sauerbrei W, Schumacher M. Dangers of using "optimal"
cutpoints in the evaluation of prognostic factors. J Natl Cancer Inst 1994;86:829-35
41. Nashan B, Moore R, Amlot P, Schmidt AG, Abeywickrama K, Soulillou JP.
Randomised trial of basiliximab versus placebo for control of acute cellular rejection
in renal allograft recipients. CHIB 201 International Study Group. Lancet
1997;350:1193-8
42. Krivtsov A, Twomey D, Feng Z, Stubbs M, Wang Y, Faber J, et al. Transformation
from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature
2006;442:818-22
43. Zuber J, Rappaport A, Luo W, Wang E, Chen C, Vaseva A, et al. An integrated
approach to dissecting oncogene addiction implicates a Myb-coordinated self-renewal
program as essential for leukemia maintenance. Genes Dev 2011;25:1628-40
30
Downloaded from cancerres.aacrjournals.org on October 7, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 1, 2020; DOI: 10.1158/0008-5472.CAN-20-0531 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
44. Guo Z, Wang A, Zhang W, Levit M, Gao Q, Barberis C, et al. PIM inhibitors target
CD25-positive AML cells through concomitant suppression of STAT5 activation and
degradation of MYC oncogene. Blood 2014;124:1777-89
45. Nakase K, Kita K, Katayama N. IL-2/IL-3 interplay mediates growth of CD25
positive acute myeloid leukemia cells. Med Hypotheses 2018;115:5-7
46. Lecine P, Algarte M, Rameil P, Beadling C, Bucher P, Nabholz M, et al. Elf-1 and
stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptor
alpha gene (vol 16, pg 6831, 1996). Molecular and Cellular Biology 1997;17:2351-
47. Malek TR, Castro I. Interleukin-2 receptor signaling: at the interface between
tolerance and immunity. Immunity 2010;33:153-65
48. Gerber JM, Gucwa JL, Esopi D, Gurel M, Haffner MC, Vala M, et al. Genome-wide
comparison of the transcriptomes of highly enriched normal and chronic myeloid
leukemia stem and progenitor cell populations. Oncotarget 2013;4:715-28
49. Willerford DM, Chen J, Ferry JA, Davidson L, Ma A, Alt FW. Interleukin-2 receptor
alpha chain regulates the size and content of the peripheral lymphoid compartment.
Immunity 1995;3:521-30
50. Janik JE, Morris JC, O'Mahony D, Pittaluga S, Jaffe ES, Redon CE, et al. Y-90-
daclizumab, an anti-CD25 monoclonal antibody, provided responses in 50% of
patients with relapsed Hodgkin's lymphoma. Proceedings of the National Academy of
Sciences of the United States of America 2015;112:13045-50
51. Dancey G, Violet J, Malaroda A, Green AJ, Sharma SK, Francis R, et al. A Phase I
Clinical Trial of CHT-25 a I-131-Labeled Chimeric Anti-CD25 Antibody Showing
Efficacy in Patients with Refractory Lymphoma. Clinical Cancer Research
2009;15:7701-10
31
Downloaded from cancerres.aacrjournals.org on October 7, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 1, 2020; DOI: 10.1158/0008-5472.CAN-20-0531 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
52. Przepiorka D, Kernan N, Ippoliti C, Papadopoulos E, Giralt S, Khouri I, et al.
Daclizumab, a humanized anti-interleukin-2 receptor alpha chain antibody, for
treatment of acute graft-versus-host disease. Blood 2000;95 (1):83-9
53. Goldberg AD, Tallman MS, Solh MM, Ungar D, Rizzieri DA, Walter RB, et al.
Results from an Ongoing Phase 1 Study Indicate ACDT-301 (Camidanlumab
Tesirine) Is Well-Tolerated in Patients with Relapsed or Refractory CD25-Positive
Acute Leukemia. Blood 2017;130
54. Horwitz SM, Hamadani M, Fanale MA, Feingold J, Spira AI, Fields PA, et al. Interim
Results from a Phase 1 Study of ADCT-301 (Camidanlumab Tesirine) Show
Promising Activity of a Novel Pyrrolobenzodiazepine-Based Antibody Drug
Conjugate in Relapsed/Refractory Hodgkin/Non-Hodgkin Lymphoma. Blood
2017;130
55. Singh R, Zhang YJ, Pastan I, Kreitman RJ. Synergistic Antitumor Activity of Anti-
CD25 Recombinant Immunotoxin LMB-2 with Chemotherapy. Clinical Cancer
Research 2012;18:152-60
32
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Table 1. Prognostic significance of IL2RA in publicly available AML gene expression data sets.
IL2RA , univariable IL2RA , multivariable Patient cohort HR 95% CI p-value HR 95% CI p-value GSE12417, cohort 1 2.34 1.54-3.55 0.00003 2.36 1.56-3.59 0.00005 GSE12417, cohort 2 2.62 1.46-4.7 0.0012 2.61 1.45-4.68 0.001 GSE6891, cohort 1 2.25 1.57-3.22 0.00001 1.56 1.04-2.33 0.032 GSE6891, cohort 2 1.73 1.19-2.52 0.004 1.66 1.13-2.44 0.011 GSE37642 1.81 1.28-2.56 0.0008 1.75 1.23-2.48 0.0017 GSE71014 3.92 1.94-7.88 0.0001 n.a. TCGA_LAML 2.61 1.41-4.8 0.002 1.98 1.02-3.84 0.044
HR, hazard ratio; CI, confidence interval; n.a., not applicable (no information on other prognostic parameters provided in data set). Characteristics of the data sets have been described previously (37).
33
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Figure legends
Figure 1: High IL2RA expression is associated with leukemic stem cells and poor
outcome of AML. (A) Expression of IL2RA in AML compared to normal bone marrow cells
(nBM), contained in data set GSE13159. (B) Expression of IL2RA in stem cell enriched
(LSC+) vs. stem cell depleted (LSC–) AML cell populations, contained in data set GSE76008.
(C) Left panel: expression of IL2RA in CD34+ cell populations from AML and from healthy
donors (nCD34+), contained in data set GSE30029. Right panel: expression of IL2RA in
surface-marker defined leukemic stem cells (LSC), normal hematopoietic stem cells (HSC),
and multipotent progenitors (MPP), contained in data set GSE63270. (A-C) Data were used
as provided in the respective data sets (log2 transformed or not). False discovery rates (FDR)
were determined using the lmFit function of the R package Limma. FDRs <0.1 were
considered significant. (D) Kaplan Meier curves for overall survival of 379 AML patients
contained in data set GSE37642, and classified as IL2RAlow and IL2RAhigh using maximally
selected rank statistics. Statistical significance was calculated using the log rank test, and the
p-value was adjusted for multiple testing (padj) according to Altman et al. (40). (E) Kaplan
Meier curves for overall survival of 107 ELN favourable, 174 ELN intermediate I/II, and 86
ELN adverse risk AML patients from GSE36742, stratified into IL2RAlow and IL2RAhigh
using the cutoff determined for the entire patient cohort (panel D). Significance was probed using the log rank test. (F) Kaplan Meier curves for overall survival of AML patients
(GSE37642) stratified into favourable, intermediate, and adverse risk groups based on the
European Leukemia Net (ELN) 2010 classification (left panel), or on a combination of the
ELN 2010 classification and IL2RA expression (right panel). The left panel was already shown in our previous paper (37) (published under a CC BY licence) and is presented here for comparison only.
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Figure 2: IL2RA promotes proliferation and cell cycle progression, and reduces apoptosis in human AML cell lines. (A, E) Cell surface expression of IL2RA in
UCSD/AML1 and HL60 derivative cell lines. (A) UCSD/AML1 cells transduced with shRNAs against IL2RA (UCSD_shIL2RA-1, UCSD_shIL2RA-2) or with a non-targeting control shRNA (UCSD_shCtrl). (E) HL60 cells transduced with IL2RA expression vector
(HL60_IL2RA) or empty vector as a control (HL60_vec). Left panels, representative flow cytometric analyses. Right panels, mean fluorescence intensities (MFI) of IL2RA. (B, F)
IL2RA promotes proliferation. UCSD/AML1 (B) and HL60 (F) derivative cell lines were maintained in suspension culture and counted on the indicated days. (C, G) IL2RA promotes cell cycle progression. Cell cycle distribution of UCSD/AML1 (C) and HL60 (G) derivative cell lines was determined by DAPI staining. (D, H) IL2RA inhibits apoptosis. The percentage of apoptotic cells among UCSD/AML1 (D) and HL60 (H) derivative cell lines was determined by Annexin V staining. Proportions of apoptotic cells are higher than in (C, G) because Annexin V stains also early apoptotic cells. (A-H) Mean ± SEM of three independent experiments; *, p<0.05; **, p<0.01; ***, p<0.001; ns, not significant; ANOVA followed by
Bonferroni's post-hoc test.
Figure 3: IL2RA affects the abundance and activity of regulators of proliferation, survival, cell cycle activity, and apoptosis. Immunoblot analyses for the abundance of key regulators of cell proliferation/survival, cell cycle progression, and apoptosis in
UCSD/AML1 (A) and HL60 (B) derivative cell lines. The experiments were performed 3 times with comparable results. Quantifications are shown in Supplemental Figure 3. p-ERK,
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ERK with activating phosphorylations on Thr202 and Tyr204; p-AKT, AKT with activating
phosphorylation on Ser473; p-STAT5, STAT5 with activating phosphorylation on Tyr694.
Figure 4: Il2ra promotes proliferation, reduces apoptosis and differentiation, augments
LSC activity, and is essential for leukemogenesis in Flt3-ITD/Npm1c driven murine
AML. (A) Schematic of experimental design. FC, flow cytometry. (B) Expression of IL2RA
on shRen- and shIl2ra-transduced spleen cells collected from mice that succumbed to AML
after transplantation with Flt3-ITD/Npm1c transformed hematopoietic cells. Left panel,
representative flow cytometric analysis. Right panel, mean fluorescence intensities (MFI) of
IL2RA. (C) Proliferation of shRen- and shIl2ra-expressing Flt3-ITD/Npm1c cells. Cells were
maintained in suspension culture and counted on the indicated days. (D) Cell cycle
distribution of shRen- and shIl2ra-expressing Flt3-ITD/Npm1c cells, analysed after staining with DAPI. (E) Percentage of apoptotic cells among shRen- and shIl2ra-expressing Flt3-
ITD/Npm1c cells, assessed through the Annexin V assay. Proportions of apoptotic cells are
higher than in (D) because Annexin V stains also early apoptotic cells. (F, G) Flow
cytometric analysis of myeloid differentiation markers (Gr1+ cells among CD11b+ GFP+ cells
(F), and cKit- cells among GFP+ cells (G)) on shRen- and shIl2ra-expressing Flt3-ITD/Npm1c
cells. (H) Serial replating in methyl cellulose. Numbers of colonies are presented as percent of shRen-expressing Flt3-ITD/Npm1c cells in each round of plating. (B-H) Mean ± SEM of three independent experiments (based on independent transductions of spleen cells from three different leukemic mice); *, p<0.05; **, p<0.01; ***, p<0.001; ns, not significant; ANOVA followed by Bonferroni's post-hoc test. (I) Kaplan–Meier plot of mice transplanted with
shRen- or shIl2ra-expressing Flt3-ITD/Npm1c cells (600,000 cells/mouse). One recipient of shIl2ra-1-, and two recipients of shIl2ra-2-transduced cells remained disease free and were terminated 190 days after transplantation. n = 5/group; *, p<0.05; **, p<0.01; log-rank test.
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(J) Proportions of GFP+ cells in BM (left panel) and spleen (right panel) of mice (n =
3/group) terminally ill after transplantation with shRen- or shIl2ra-expressing Flt3-
ITD/Npm1c cells. ***, p<0.001; ANOVA followed by Bonferroni's post-hoc test.
Figure 5: hIL2RA antibodies inhibit proliferation and colony formation and promote apoptosis in primary IL2RAhigh AML cells, and these effects are accompanied by corresponding molecular changes. (A) Relative viability (measured using metabolic
activity as a proxy). Primary AML samples were incubated with the indicated concentrations
of hIL2RA antibody (hIL2RA Ab), Basiliximab, or isotype control antibody (isotype ctrl) for
72 h and subjected to the Cell Titer Glo Assay. (B) Cell proliferation. Primary AML samples
were incubated with 3 µg/ml hIL2RA Ab, 6 µg/ml Basiliximab, or the corresponding
amounts of isotype control and counted on the indicated days. (A, B) Mean +/- SD from
triplicate measurements; *, p<0.05; **, p<0.01; ***, p<0.001; ns, not significant; ANOVA
followed by Bonferroni's post-hoc test. (C, D) Primary AML samples were incubated with 3
µg/ml hIL2RA Ab, 6 µg/ml Basiliximab, or the corresponding amounts of isotype control for
72 h. (C) Cell cycle distribution analysed by DAPI staining. (D) Percentage of apoptotic cells as determined by the Annexin V assay. (E) Colony formation in methyl cellulose. AML
samples were incubated with 3 µg/ml hIL2RA Ab or isotype control for 72 h prior to plating
in methyl cellulose. Colony numbers are presented as percent of isotype control-treated cells.
Mean +/- SD from duplicate measurements; *, p<0.05; **, p<0.01; ***, p<0.001; ns, not significant; ANOVA followed by Bonferroni's post-hoc test. (F) The abundance of key regulators of cell proliferation/survival, cell cycle progression, and apoptosis after a 72 h incubation with 3 µg/ml hIL2RA Ab or isotype control was determined by immunoblot
analysis. p-ERK, ERK with activating phosphorylations on Thr202 and Tyr204; p-AKT,
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AKT with activating phosphorylation on Ser473; p-STAT5, STAT5 with activating
phosphorylation on Tyr694.
Figure 6: hIL2RA antibody and araC exhibit synergistic anti-leukemic activity in
primary human AML cells. (A) Expression of IL2RA in primary AML samples treated with
80 nM araC for 72 h. Left panel, representative flow cytometric analyses. Right panel, mean fluorescence intensities (MFI) of IL2RA. Mean +/- SD from duplicate measurements; **, p<0.01, t-test. (B) Primary AML samples were incubated with the indicated concentrations of
hIL2RA antibody and araC for 48 h, and viability was measured using metabolic activity as a
proxy. Left panels, viability relative to controls treated with 6 µg/ml isotype control antibody
only, mean +/- SD from triplicate measurements. Right panels, combination index (CI)
according to the Chou-Talalay method. A CI of 1 indicates an additive effect, a CI below 1 a
synergistic interaction, and a CI greater than 1 an antagonistic effect. (C) Primary AML
samples were incubated with hIL2RA antibody, araC, or both for 48 h and apoptosis was
determined through the Annexin V assay.
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IL2RA promotes aggressiveness and stem cell-related properties of acute myeloid leukemia
Chi Huu Nguyen, Angela Schlerka, Alexander M. Grandits, et al.
Cancer Res Published OnlineFirst September 1, 2020.
Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-20-0531
Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2020/09/01/0008-5472.CAN-20-0531.DC1
Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.
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