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NEOPLASIA

Brief report
Mammalian target of rapamycin (mTOR) inhibition activates phosphatidylinositol
3-kinase/Akt by up-regulating insulin-like growth factor-1 receptor signaling in
acute myeloid leukemia: rationale for therapeutic inhibition of both pathways
Jerome Tamburini,1-4 Nicolas Chapuis,1-3,5 Valérie Bardet,1-3,5 Sophie Park,1-4 Pierre Sujobert,1-4 Lise Willems,1-3
Norbert Ifrah,6 François Dreyfus,1-4 Patrick Mayeux,1-3, Catherine Lacombe,1,5 and Didier Bouscary1-4
1Institut
Cochin, Département d’Hématologie, Centre National de la Recherche Scientifique (Unité Mixte de Recherche 8104), Paris; 2Inserm U567, Paris;
3Université Paris Descartes, Faculté de Médecine René Descartes, Paris; 4Service de Médecine Interne, UF d’Hématologie, Assistance Publique-Hôpitaux de
Paris; Hôpital Cochin, Paris; 5Service d’Hématologie Biologique, Assistance Publique-Hôpitaux de Paris, Paris; and 6Service des Maladies du Sang, Centre
Hospitalier Universitaire, Angers, France

The phosphatidylinositol 3-kinase (PI3K)/
Akt and mTORC1 pathways are frequently
activated, representing potential thera-
peutic targets in acute myeloid leukemia
(AML). In 19 AML samples with constitu-
tive PI3K/Akt activation, the rapamycin
derivative inhibitor everolimus (RAD001)
increased Akt phosphorylation. This
mTOR C1-mediated Akt up-regulation was
explained by an insulin-like growth
factor-1 (IGF-1)/IGF-1 receptor autocrine
loop: (1) blast cells expressed functional
IGF-1 receptors, and IGF-1-induced Akt
activation was increased by RAD001, (2) a
neutralizing anti-IGF-1R ␣-IR3 monoclo-
nal antibody reversed the RAD001-
induced Akt phosphorylation, and
(3) autocrine production of IGF-1 was
detected in purified blast cells by quanti-
tative reverse transcription-polymerase
chain reaction and immunofluorescence.
This RAD001-induced PI3K/Akt up-
regulation was due to an up-regulated
expression of the IRS2 adaptor. Finally,
we observed that concomitant inhibition
of mTORC1 and PI3K/Akt by RAD001 and
IC87114 induced additive antiproliferative
effects. Our results suggest that dual
inhibition of the mTORC1 complex and
the IGF-1/IGF-1R/PI3K/Akt pathway in AML
may enhance the efficacy of mTOR inhibi-
tors in treatment of this disease. (Blood.
2008;111:379-382)
© 2008 by The American Society of Hematology

Introduction
Acute myeloid leukemia (AML) is associated with a low survival
rate. Therefore, new therapeutic strategies may prove effective in
addition to chemotherapy.
The deregulation of several signal transduction pathways is a
common feature in AML. The phosphatidylinositol 3-kinase (PI3K)/
Akt pathway is activated in AML blast cells.1-5 We showed
previously that the p110 ␦ isoform of PI3K is a potential
therapeutic target and that the p110 ␦-selective inhibitor IC87114
blocks AML cell proliferation.6,7 The mammalian target of rapamy-
cin (mTOR) is activated in response to stimuli activating the PI3K
pathway,8 and mTORC1 inhibitors may have therapeutic value in
the treatment of patients with AML9,10; however, rapamycin alone
led to modest antileukemic activity.10 We investigated the effect of
the mTORC1 inhibitor RAD001 (Everolimus; Novartis Pharmaceu-
ticals, Basel, Switzerland) in 19 bone marrow samples from
patients with newly diagnosed AML. We show that mTORC1
inhibition with RAD001 increased Akt activating phosphorylation,
as a result of up-regulated expression of the IRS-2 protein adaptor
that promoted insulin-like growth factor-1 (IGF-1) /IGF-1R signal-
ing. Moreover, we show that the enhanced activation of Akt was
dependent on the IGF-1 autocrine production by leukemic cells.
Our results provide a rationale for combined inhibition of both
mTORC1 and PI3K/Akt pathways in AML, and we observed an
additive effect of both RAD001 and IC87114 on blast cell
proliferation.
Methods
Bone marrow (BM) samples were obtained from 19 patients with newly
diagnosed AML, all treated in the AML2001 chemotherapy trial, initiated
by the French Multicenter Group, Groupe Ouest Est des Leucémies et
Autres Maladies du Sang (GOELAMS). All biologic studies were approved
by the GOELAMS Institutional Review Board, and signed informed
consent was obtained in accordance with the Declaration of Helsinki. The
clinical characteristics of patients are summarized in Table S1 (available on
the Blood website; see the Supplemental Materials link at the top of the
online article). All patients presented a constitutive activation of Akt at
diagnosis, as reported previously.5 Cells were incubated with the following
inhibitors: RAD001 (Everolimus) kindly provided by Novartis, IC87114
provided by ICOS (Bothell, WA), LY294002 from Sigma (St Louis, MO),
and ␣IR3 from Calbiochem (La Jolla, CA). IGF-1 was from Sigma.
Expression of total and phosphorylated proteins was detected by Western
blot (WB) analysis as reported previously.7 The references for the antibod-
ies are summarized in Table S2. Immunofluorescence staining for IGF-1
expression and quantitative reverse transcription-polymerase chain reaction
(RT-PCR) were performed on blast cells, sorted according to their CD45low
expression and side scatter (see Document S1 for details). Blast cell

Submitted March 17, 2007; accepted September 12, 2007. Prepublished The publication costs of this article were defrayed in part by page charge
online as Blood First Edition Paper, September 18, 2007; DOI 10.1182/blood- payment. Therefore, and solely to indicate this fact, this article is hereby
2007-03-080796. marked ‘‘advertisement’’ in accordance with 18 USC section 1734.

The online version of ths article contains a data supplement. © 2008 by The American Society of Hematology

BLOOD, 1 JANUARY 2008 䡠 VOLUME 111, NUMBER 1 379

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380 TAMBURINI et al BLOOD, 1 JANUARY 2008 䡠 VOLUME 111, NUMBER 1

Figure 1. mTORC1 inhibition with RAD001 induces Akt activation in primary AML samples by activation of the IGF-1/IGF1-R signaling pathway, dependent on
an IGF-1 autocrine loop. (A) Bone marrow (BM) blast cells from 19 patients with AML were starved for 4 hours in cytokine and serum-free minimal essential medium
(MEM), with or without the following kinase inhibitors: 25 ␮mol/L LY294002, 10 ␮mol/L IC87114, or 10 nmol/L RAD001, added during the last hour of starvation. Western
blots (WBs) were performed with anti-phospho-Akt (ser 473), anti-phospho-p70S6K (thr 389), and anti-Akt antibodies. Quantification of phospho-Akt signal intensity
was normalized to Akt signal intensity. Each histogram of the graph represents the phospho-Akt signal intensity in RAD001-treated blast cells, expressed as percentage
of signal intensity in control cells (M, medium without inhibitors). (B) BM blast cells from patients G192 and G194 were collected after Ficoll-Hypaque density gradient
separation, then washed once in PBS buffer. Blast cells (5 ⫻ 105/mL) from patient G192 were starved for 4 hours in cytokine and serum-free MEM, then incubated
without or with 10 nmol/L RAD001 for 1 hour. In independent experiments, 5 ⫻ 105/mL blast cells from patients G192 and G194 were incubated without or with RAD001
for 24 hours, in ␣-MEM with 10% fetal calf serum (FCS). RAD was used at 10 nmol/L for sample G192, and at 10, 50, or 100 nmol/L for sample G194. WBs were
performed with anti-phospho-Akt (ser 473), anti-phospho-p70S6 kinase (thr 389), anti-phospho-S6R (Ser 235/236), and anti-Akt antibodies. (C) RAD001 increases
IGF-1-stimulated Akt phosphorylation in AML blast cells. BM blast cells from patients G179, G194, and G149 were starved for 4 hours in serum-free MEM, without or with
10 nmol/L RAD001, then stimulated or not with 50 ng/mL IGF-1 for 10 minutes. WBs were performed with anti-phospho-Akt (Ser 473) and anti-Akt antibodies. (D) AML
blast cells express IGF-1 at the RNA and protein level. BM blast cells from 8 patients were highly purified by flow cytometry cell sorting according to CD45low expression
and side scatter. The MOLM-14 AML cell line was used as negative control, and the OPM2 myeloma cell line was used as a positive control for IGF-1 expression. IGF-1
mRNA expression was quantified in the purified blast cells by quantitative RT-PCR, and their levels were expressed relative to HPRT (hypoxanthine phosphoribosyl
transferase) mRNA levels. Similar results were obtained using another housekeeping gene, UBCV (C ubiquitin; data not shown). Immunofluorescence staining was
performed on purified blasts for the same 8 patients mentioned above, and on MOLM-14 and OPM2 cell lines, using a mouse monoclonal anti-IGF-1 antibody and
fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Images obtained from the
representative patient G102 and from MOLM-14 and OPM2 cell lines are presented. (E) The mTORC1-mediated positive feedback on PI3K/Akt activity involves the
IGF-1 receptor. BM blast cells from patients G179, G194, G199, and G205 were starved for 4 hours in serum-free MEM. Cells were incubated in the following conditions:
medium alone, 10 nmol/L RAD001 for 1 hour, 10 nmol/L RAD001 for 1 hour plus, and 5 ␮g/mL ␣IR3 (added 30 minutes before RAD001). WBs were performed with
anti-phospho-Akt (Ser 473) and anti-Akt antibodies. ␣IR3 is a blocking mouse monoclonal antibody directed against the alpha subunit of the IGF-1 receptor and was
obtained from Calbiochem. (F) mTORC1 inhibition by RAD001 increases the expression of the IRS2 adaptor. BM blast cells from patients G72, G99, G189, and G191
were starved for 4 hours in serum-free MEM then incubated with or without RAD001 (10 nmol/L) for 1 hour. WBs were performed with anti-IRS2 and anti-actin
antibodies.
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BLOOD, 1 JANUARY 2008 䡠 VOLUME 111, NUMBER 1 AKT UP-REGULATION AFTER MTORC1 INHIBITION IN AML 381

proliferation was assessed by [3H]thymidine incorporation as reported

previously.7

Results and discussion

This study was conducted in an attempt to inhibit Akt and
mTORC1 phosphorylation in AML samples. We compared the
effect of 3 kinase inhibitors, (1) IC87114,7 (2) LY294002, and
(3) the rapamycin derivative RAD001, in fresh BM blast cells from
patients with AML presenting constitutive PI3K activation.4,7 We
observed that IC87114 and LY294002 totally suppressed Akt
phosphorylation, whereas, in contrast, RAD001 substantially in-
creased Akt activation. The enhancement of Akt phosphorylation in
the presence of RAD001 (mean 86%) was detected in all 19 AML
samples (Figure 1A). Furthermore, increased Akt activation was
maintained after a 24-hour incubation with 10 nmol/L RAD001
(Figure 1B, sample G192) and also with RAD001 at higher
concentrations (Figure 1B, sample G194). Thus, RAD001 treat-
ment led to an increased Akt activation in all AML samples
presenting constitutive PI3K activation. Because mTORC1 has
been reported to inhibit Insulin/IGF-1 signaling,11 we hypothesized
that the IGF-1/IGF-1R pathway could play a role in the RAD001-
induced Akt activation in AML cells. We found that exogenous
IGF-1 stimulation increased Akt phosphorylation and that RAD001
increased IGF-1-stimulated Akt phosphorylation in AML samples
(Figure 1C). Because BM cells were cultured in serum-free
medium and harbored a functional IGF-1R, we analyzed the
production of IGF-1 inside the leukemic cells. To that end, we
purified the blast cell population by flow cytometry cell sorting as
described previously.4 IGF-1 expression was detected at the mRNA
level, and the IGF-1 protein was detected by immunofluorescence
analysis in all samples tested (Figure 1D). From these data, we
concluded that an autocrine production of IGF-1 was constantly
Figure 2. Concomitant inhibition of mTORC1 and p110␦ PI3K activity with
observed in primary AML blast cells. We inhibited the interaction RAD001 and IC87114, respectively, induces additive inhibition of blast cell
between IGF-1 and its receptor with a neutralizing monoclonal proliferation. (A) Blast cells (105/mL) from the 19 patients were incubated 48 hours
antibody ␣IR3, directed against the ␣-subunit of IGF-1 receptor.12 in duplicate in 5% FCS MEM under the following conditions: control (C), 10 nmol/L
RAD001 (R), 10 ␮mol/L IC87114 (IC), 10 nmol/L RAD001 and 10 ␮mol/L IC87114
As shown in Figure 1E, blocking the IGF-1/IGF-1R interaction (R ⫹ IC), and pulsed for 6 hours with [3H]thymidine (1 ␮Ci, [37 kBq]). A Student t test
reversed the RAD001-induced increase of Akt phosphorylation. was performed to compare proliferation rates between the different conditions.
These data strongly suggest that the enhancement of Akt phosphor- Significance was: C/R, P ⬍.001; C/IC, P ⬍.001; R/R ⫹ IC, P ⬍.001; IC/R ⫹ IC,
P ⬍.001. The WB analysis shows the effect of inhibitors on Akt (Ser 473) and P70S6K
ylation in response to RAD001 treatment involved the IGF-1R in (Thr 389) phosphorylation. (B) BM blast cells (105/mL) from 4 AML samples were
leukemic cells. incubated 48 hours in triplicate in 5% FCS MEM, with or without inhibitors as
mTORC1 activity down-regulates insulin/IGF-1 signaling described below. They were then pulsed 6 hours with [3H]thymidine 1 ␮Ci [37 kBq].
The amount of radioactivity incorporated was determined by trichloracetic acid
through proteasome-mediated decrease of IRS adaptor pro-
precipitation. In the top panel, increasing amounts (from 1.25 to 20 nmol/L) of
teins.13-15 To confirm the involvement of the IGF-1R in the RAD001 were used, without (top line) or with a constant concentration of IC87114
mechanism of Akt activation, a variation of IRS-2 expression after (10 ␮mol/L). In the bottom panel, increasing (from 1.25 to 20 ␮mol/L) amounts of
treatment with RAD001 was evaluated in AML samples. RAD001 IC87114 were used, without (top line) or with a constant concentration of RAD001
(10 nmol/L). Error bars indicate standard deviations.
treatment led to a significant increase of IRS2 protein expression,
which in each case paralleled the level of RAD001-induced Akt
phosphorylation (Figure 1F). compared with the effect of each compound alone (P ⬍ .001)
We have shown that IC87114, a PI3K p110␦-selective inhibitor, (Figure 2A). The additive effect of PI3K and mTOR inhibitors was
suppressed AML cell proliferation.7 On the other hand, we confirmed in dose response curves for the 2 compounds, in 4 AML
observed that the blockage of PI3K by IC87114 did not inhibit samples (Figure 2B).
mTORC1, as assessed by the persistence of P70S6K phosphoryla- In summary, our study shows that mTORC1 inhibition en-
tion (Figures 1A and 2A). Thus, these pathways seem to be hanced Akt activation in AML cells and may contribute to limit the
independent in AML, thereby allowing us to assess the rationale of efficacy of rapamycins when used in monotherapy, as described in
a treatment combining 2 inhibitors. We tested the effect of the other systems.16 It has been reported that a 24-hour exposure to
association of RAD001 and IC87114 on blast cell proliferation. rapamycin may impair mTORC2 assembly and, subsequently,
RAD001 and IC87114, when used alone, inhibited blast cell down-regulate Akt phosphorylation.17 Similar observations were
proliferation to the same level in our series of 19 patients. However, made by this group in primary AML samples treated with CCI-779
the concomitant inhibition of both pathways with RAD001 and (temsirolimus; Wyeth-Ayerst Pharmaceuticals, Princeton, NJ).18 In
IC87114 resulted in a significant additive antiproliferative effect, our hands, Akt activation was unchanged after 24-hour incubation
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382 TAMBURINI et al BLOOD, 1 JANUARY 2008 䡠 VOLUME 111, NUMBER 1

with RAD001, even at higher RAD001 concentrations (Figure
1B). The reasons for this discrepancy are unknown. It is possible
that the culture conditions for primary blast cells were different;
alternatively, CCI-779 could be more active on a prolonged
incubation period than RAD001 or could have additional side
inhibitory effects.
In conclusion, our results show that autocrine IGF-1 signaling is
present in primary AML blast cells and is responsible, at least in
part, for the effect of RAD001 on mTORC1-mediated PI3K/Akt
up-regulation. Our results provide a molecular basis for understand-
ing the increased Akt activation induced by mTORC1 inhibitors
and suggest that a combined therapeutic strategy targeting
both PI3K and mTORC1 pathways might be useful in patients
with AML.
Acknowledgments
We acknowledge the Novartis Institutes for Medical Research
Basel, Oncology for the supply of RAD001. We thank all participat-
ing investigators from the GOELAMS. We thank Dr David Fruman
(University of California-Irvine) for helpful criticism of the
manuscript.
This work was supported by grants from the Ligue Nationale
Contre le Cancer (LNCC, laboratoire associé), the Association pour
le Recherche contre le Cancer (ARC), the Institut National du
Cancer (INCA), the Association Laurette Fugain, and the Fonda-
tion pour la Recherche Medicale (FRM).
Authorship
Contribution: J.T. performed research, analyzed data, and wrote the
manuscript. N.C., P.S., V.B., S.P., and L.W. performed research and
analyzed data. N.I. and F.D. contributed AML patient samples and
analyzed clinical data. C.L. and P.M. analyzed data and wrote the
manuscript. D.B. designed research, analyzed data, and wrote the
manuscript.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Didier Bouscary, Département d’Hématologie,
Institut Cochin, 27 rue du Faubourg Saint-Jacques, F-75014 Paris,
France; e-mail: bouscary@cochin.inserm.fr.

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2008 111: 379-382

doi:10.1182/blood-2007-03-080796 originally published
online September 18, 2007

Mammalian target of rapamycin (mTOR) inhibition activates

phosphatidylinositol 3-kinase/Akt by up-regulating insulin-like growth
factor-1 receptor signaling in acute myeloid leukemia: rationale for
therapeutic inhibition of both pathways
Jerome Tamburini, Nicolas Chapuis, Valérie Bardet, Sophie Park, Pierre Sujobert, Lise Willems,
Norbert Ifrah, François Dreyfus, Patrick Mayeux, Catherine Lacombe and Didier Bouscary

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