Feedbacks and Adaptive Capabilities of the PI3K/Akt/Mtor Axis in Acute Myeloid Leukemia Revealed by Pathway Selective Inhibition and Phosphoproteome Analysis

ORIGINAL ARTICLE Feedbacks and adaptive capabilities of the PI3K/Akt/mTOR axis in acute myeloid leukemia revealed by pathway selective inhibition and phosphoproteome analysis

J Bertacchini1,2, M Guida1,6, B Accordi3,6, L Mediani1, AM Martelli2, P Barozzi4, E Petricoin III5, L Liotta5, G Milani3, M Giordan3, M Luppi4, F Forghieri4, A De Pol1, L Cocco2, G Basso3 and S Marmiroli1

Acute myeloid leukemia (AML) primary cells express high levels of phosphorylated Akt, a master regulator of cellular functions regarded as a promising drug target. By means of reverse phase protein arrays, we examined the response of 80 samples of primary cells from AML patients to selective inhibitors of the phosphatidylinositol 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) axis. We confirm that 460% of the samples analyzed are characterized by high pathway phosphorylation. Unexpectedly, however, we show here that targeting Akt and mTOR with the specific inhibitors Akti 1/2 and Torin1, alone or in combination, result in paradoxical Akt phosphorylation and activation of downstream signaling in 70% of the samples. Indeed, we demonstrate that cropping Akt or mTOR activity can stabilize the Akt/mTOR downstream effectors Forkhead box O and insulin receptor substrate-1, which in turn potentiate signaling through upregulation of the expression/phosphorylation of selected growth factor receptor tyrosine kinases (RTKs). Activation of RTKs in turn reactivates PI3K and downstream signaling, thus overruling the action of the drugs. We finally demonstrate that dual inhibition of Akt and RTKs displays strong synergistic cytotoxic effects in AML cells and downmodulates Akt signaling to a much greater extent than either drug alone, and should therefore be explored in AML clinical setting.

Leukemia (2014) 28, 2197–2205; doi:10.1038/leu.2014.123

INTRODUCTION membrane allowing PDK1 to phosphorylate Akt at T308.10 Acute myeloid leukemia (AML) is originated by a block in Phosphorylation at S473 by kinases such as mTOR complex 2 11 12 differentiation of hematopoietic stem cells resulting in growth of (mTORC2) or DNA-dependent protein kinase (DNA-PK) leads to a clonal population of neoplastic cells, associated to deregulation full activation of Akt. mTOR kinase is part of two complexes, of key signal transduction pathways. AML is a genetically mTORC1 and mTORC2, with different biochemical structures and 11 heterogeneous disease, and prognosis depends on a number of substrate specificity. Akt and mTORC1/2 are linked through a factors including gene translocations, age, white blood cells count complex relationship, in which each one of the factors regulate and additional cytogenetic aberrations.1 Most patients treated the other. Although mTORC2 phosphorylates Akt at S473, mTORC1 with chemotherapy achieve complete remission, nevertheless the is activated through direct phosphorylation of the tuberous relapse rate is very high, with only 20–30% survival at 5 years.2 sclerosis complex 1/2 by Akt. Moreover, Akt phosphorylates the Thus, efforts are being made to develop more effective therapies. inhibitor of mTORC1, proline-rich Akt-substrate-40 (PRAS40), The advances in the understanding of the molecular events preventing the ability of PRAS40 to suppress mTORC1 signaling. occurring during development of AML are leading to the mTORC1 is an energy sensor and a major regulator of protein identification of a growing number of druggable targets such as synthesis through the phosphorylation and activation of the S6 FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD), ribosomal protein kinase (S6K) and the phosphorylation and cKit, rat sarcoma Ras and the phosphatidylinositol 3 kinase (PI3K)/ inactivation of the repressor of mRNA translation initiation factor 13 Akt/mammalian target of rapamycin (mTOR) axis, frequently 4E-binding protein 1. mutated or aberrantly activated in AML.3–6 In particular, Aberrant activation of the PI3K/Akt/mTOR axis correlates with constitutive activation of the PI3K/Akt/mTOR pathway is poor prognosis or resistance to conventional chemotherapy in 8 detectable in 50–80% of AML patients and correlates with very several types of cancer, whereas Akt or mTOR inhibitors, such as poor prognosis.6,7 This pathway is central to many biological PP242, RAD001, perifosine, Akti 1/2 or its analog MK2206, 14–18 processes, including proliferation, differentiation, protein synthesis significantly inhibit cell proliferation and promote apoptosis. and apoptosis.8,9 Growth factor receptors activation recruits PI3K As a consequence, a number of specific inhibitors of these to the membrane where it converts PIP2 to PIP3. Binding of the signaling molecules are being developed, some of which are pleckstrin homology domain to PIP3 colocalizes Akt and the currently in clinical trials for solid or blood tumors in which the phosphoinositide-dependent kinase 1 (PDK1) to the plasma pathway is constitutively active.19,20

1Department of Surgery, Medicine, Dentistry and Morphology, University of Modena and Reggio Emilia, Modena, Italy; 2Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy; 3Department of Woman’s and Child’s Health, University of Padova, Padova, Italy; 4Section of Hematology, Department of Medical and Surgical Science, University of Modena and Reggio Emilia, Modena, Italy and 5Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA. Correspondence: Professor S Marmiroli, Department of Surgery, Medicine, Dentistry and Morphology, University of Modena and Reggio Emilia, Modena,Italy. E-mail: [email protected] 6These authors contributed equally to this work. Received 23 January 2014; revised 27 February 2014; accepted 7 March 2014; accepted article preview online 4 April 2014; advance online publication, 30 May 2014 Combined Akt and RTKs inhibition in AML J Bertacchini et al 2198 However, a better understanding of the molecular mechanisms Reverse phase protein arrays (RPPAs) comparison of sampled proteins underlying the response to these drugs is essential for their from bone marrow versus pheripheral blood displayed similar profiles, and effective clinical application. Here we demonstrate that the therefore these samples were used interchangeably (Supplementary allosteric Akt inhibitor Akti 1/218 and the ATP-competitive Figure 1). mTOR inhibitor Torin121 trigger paradoxical activation of Akt and downstream signaling in primary cells from AML patients. Apoptosis, cells growth and ELISA determination of growth factor Conversely, we demonstrate that inhibitors of receptor tyrosine receptors kinases (RTKs), such as sunitinib, linsitinib and quizartinib,3,22,23 Apoptosis was detected by Annexin V-fluorescein isothiocyanate staining. antagonize the hyperactivation of Akt. Remarkably, these drugs After 20 h of treatment, pellets (1.3 Â 105 cells) were washed twice with resensitize leukemic cells to Akti 1/2 inhibition, leading to massive phosphate-buffered saline and analyzed by Annexin V-fluorescein apoptosis, and should therefore be explored as a therapeutic isothiocyanate apoptosis detection kit I following manufacturer’s direc- strategy in AML patients with PI3K/Akt hyperactivation tions. A total of 10 000 cells per sample were analyzed on a FACSCalibur (BD Bioscences, Franklin Lakes, NJ, USA). downstream of constitutively active RTKs. Growth inhibition was determined by colorimetric 3-(4,5-dimethylthiazol- 2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay, as described previously.24 Where indicated, the combination index was calculated according to MATERIALS AND METHODS Chou–Talalay25 with the CompuSyn software (Biosoft, Cambridge, UK). Materials Total IRS-1, IR (b subunit), IGF-1r and PDGFr were evaluated with specific LY294002, AKTi 1/2, triciribine, perifosine and rapamycin were from Merck ELISA kits, following manufacturer’s instructions. Millipore (Darmstadt, Germany); Torin1, sunitinib, quizartinib and linsitinib were purchased from Selleck Chemicals (Houston, TX, USA). Insulin Western blot analysis and RPPAs receptor substrate-1 (IRS-1) and insulin receptor (IR; b subunit) enzyme- linked immunosorbent assay (ELISA) kits were from Invitrogen (Carlsbad, Samples were extracted in TPER reagent (Pierce, Rockford, IL, USA). For CA, USA); insulin-like growth factor-1 receptor (IGF-1R) and platelet-derived western blotting, 80 mg of total protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to growth factor receptor (PDGFR) ELISA kits were from Abcam (Cambridge, 26 UK). All primary and secondary antibodies were from Cell Signaling polyvinylidene difluoride membrane as previously described. Array assembly, printing, staining and analysis were performed as Technologies (Danvers, MA, USA). All cell culture reagents were from 27,28 EuroClone (Milan, Italy). Annexin V-fluorescein isothiocyanate was from BD described. The complete list of the 90 antibodies can be found in Biosciences (San Jose, NJ, USA). Supplementary Table 1.

Sample collection and cell culture Real-time PCR Archived primary blast cells samples, previously collected from deceased Total RNA was extracted using the Aurum Total RNA Fatty and Fibrous patients with newly diagnosed AML (excluding acute promyelocytic Tissue kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s leukemia), were retrospectively analyzed in vitro. The clinical characteristics instructions. Genomic DNA was eliminated by DNase treatment. Real-time of patients analyzed are listed in Table 1. Blast cells were obtained from ampliﬁcations of human IR and PDGFR were performed using Real-Time bone marrow or pheripheral blood. Mononuclear cells were isolated using PRC Primer Set (Biomol GmbH, Hamburg, Germany), whereas primers for the Ficoll–Hypaque system. Only samples with Trypan blue-positive cells human IGF1-R were as follows: F: 50-TGAAAGTGACGTCCTGCATTTC-30, 0 0 o20% and blast count 450% were used in this study. The human AML R: 5 -GGTACCGGTGCCAGGTTATG-3 ; detected with Eva Green detection cell lines MOLM-13 and THP1 were purchased from ATCC (American Type chemistry, and were run in triplicate on 96-well reaction plates with the Culture Collection, Manassas, VA, USA). Both primary AML cells and AML CFX96 machine (Bio-Rad). Baseline and threshold values were automati- 1 cally determined for all plates using the Bio-Rad CFX Manager Software 1.5. cell lines were cultured at 37 C in humidiﬁed atmosphere with 5% CO2,in RPMI 1640 with 10% fetal calf serum. The data are represented as fold expression±s.e.m., normalized with glyceraldehyde 3-phosphate dehydrogenase gene expression.

Table 1. Clinical characteristics of the patients included in this study Statistical analysis Comparisons between patient characteristics (French–American–British Variable Number or % subtype), were performed by Student’s t-test. Differences in basal No. of cases 80 phosphorylation of treated samples were compared by calculating the ratio of each sample’s signal intensity divided by vehicle (dimethylsulph- Age oxide) treated sample’s signal intensity. To determine the signiﬁcance of Median 63.8 the difference in mean of treated samples, we used a paired Student’s Minimum 21 t-test with a P-value ¼ 0.05. Maximum 86

FAB RESULTS M0 6 M1 16 Phosphoprotein analysis of AML blast cells reveals different M2 18 protein signatures M4 32 A broad survey of multiple signaling pathways was initially M5 28 performed on a population of 80 newly diagnosed AML patients, Mutations through RPPA analysis of 90 native and phosphorylated key end NPM 35 points related to cell growth, proliferation, survival and metabolism. FLT3-ITD 8 AML blast cells showed a marked increase in phosphorylation FLT3 D835V 2 of proteins involved in protein kinase C, signal transducer and activator of transcription, mitogen-activated protein kinases, PI3K/ Cytogenetics Akt/mTOR pathways (Figure 1). These data are in agreement with Favorable 36 6,7 Intermediate 44 recent reports that documented the activation of several Unfavorable 20 prosurvival pathways in AML and are an essential element to optimize molecular targeted therapies in cancer. Next, we Abbreviations: FAB, French–American–British; FLT3-ITD, FMS-like tyrosine measured whether expression/phosphorylation of the proteins kinase 3-internal tandem duplication. FAB subtypes, mutations and analyzed were associated with clinical parameters. Statistical cytogenetics are represented as percentage of the total population. analysis revealed that levels or activation state of signaling

Leukemia (2014) 2197 – 2205 & 2014 Macmillan Publishers Limited Combined Akt and RTKs inhibition in AML J Bertacchini et al 2199

Figure 1. Box plot analysis of signaling pathways activated in individual samples. White box length, interquartile range; black line, median level. Bars, 2 s.d. Outliers (O) and extremes (*) are shown. proteins were not significantly associated with gender, age, Table 2. Correlation of signaling protein phosphorylation with karyotype or nucleophosmin 1 mutations. As the French– American–British classification distinguishes AML cases based on M4–M5 versus M1–M2 FAB subtypes the degree of maturation, different expression signatures have Protein name Fold change Regulation P-value been reported for different French–American–British subtypes.7 For each sample and each antibody, the signaling architecture was Phosphorylation sites M4–M5 vs M4–M5 vs Po0.005 compared between patients classified as M1–M2 subtypes and M1–M2 M1–M2 those classified as M4–M5 subtypes. Of 90 proteins or phospho- p-Akt s473 2.35 Up 0.042 proteins analyzed, 32 display statistical significant subtype based p-Akt t308 2.21 Up 0.037 changes (Table 2). These molecules are associated with cell p-Fox01/03 T24-32 2.69 Up 0.12 proliferation, differentiation, metabolism and apoptosis. Activa- p-GSK3 s21-9 2.14 Up 0.036 tion/reduction of proteins involved in proapoptotic functions such p-mTor s2448 1.36 Up 0.051 as BCL2, BAD, FADD, Forkhead box O (FOXO), Cofilin, Arrestin and p-mTor s2441 1.47 Up 0.021 Caspases is higher in myeloid M1–M2 with respect to monocytic p-P70 1.89 Up 0.035 M4–M5 subgroups, whereas proteins involved in prosurvival and p-4EBP1 1.78 Up 0.014 differentiation such as protein kinase C, mitogen-activated protein p-MARCKS s152 156 2.87 Up 0.068 kinases and P3K/AKT/mTORs have significantly higher expression/ p-P38 4.26 Up 0.069 p-P90RSK 2.65 Up 0.021 phosphorylation in M4–M5 subtypes. These results are in good p-PKCpan 2.45 Up 0.014 agreement with the poorer prognosis associated to M4–M5 p-PKCa s257 4.69 Up 0.036 phenotypes and the better outcome associated to M1–M2 p-PKC d T505 2.36 Up 0.075 patients. pPKCt t538 1.37 Up 0.098 pPKCab T638-641 2.45 Up 0.068 p-ARRESTIN b1 S412 1.36 Down 0.098 Akt inhibition triggers paradoxical Akt phosphorylation as well as p-COFILIN s3 4.25 Down 0.26 apoptosis p-FADD s194 2.69 Down 0.17 The above-mentioned unsupervised clustering revealed two major p-Cleaved caspase 7 D198 4.36 Down 0.028 p-Cleaved caspase 9 D330 3.68 Down 0.047 groups of patients: one, representing 50 out of 80 samples, with p-Cleaved caspase 3 D175 3.75 Down 0.069 high PI3K/Akt signaling activation, and the other with a p-BCL2 t56 4.62 Down 0.014 comparatively low level of pathway signaling. This result was p-BCL2 s70 4.5 Down 0.035 not unexpected, as it is known that Akt phosphorylation at S473 or p-BAD s136 3.87 Down 0.046 T308 can be detected in most AML patients.6,7,29,30 Indeed, p-BAD s112 4.62 Down 0.026 this pathway is regarded as a drug target, and previous work by Abbreviation: FAB, French–American–British. Results are represented as other laboratories established that Akt inhibitors hamper cell 14–17,30–33 fold change of each end point versus mean value. The regulation (up or proliferation and trigger apoptosis of leukemia cells. In down) is indicated. All end points in the list show a P-value o0.05, by order to more deeply investigate the ability of PI3K/Akt inhibitors Student’s t-test. to diminish Akt phosphorylation and cause apoptosis, the signaling dynamics were studied in the 50 samples with aberrantly activated signaling. Blast cells were grown for 20 h in allosteric inhibitors of Akt,18,34 which prevent membrane binding a medium supplemented either with 0.01% dimethylsulphoxide of the pleckstrin homology domain and phosphorylation of the (DMSO; vehicle) or with the specific Akt inhibitor Akti 1/2, and with T308 site by PDK1. Thus, Akt phosphorylation can be monitored as the broad inhibitors perifosine and triciribine. These drugs are a direct readout of the ongoing Akt kinase activity. More than one

& 2014 Macmillan Publishers Limited Leukemia (2014) 2197 – 2205 Combined Akt and RTKs inhibition in AML J Bertacchini et al 2200 inhibitor was used to avoid off target effects. Moreover, samples However, this effect was followed at 20 h not only by complete were treated with the ATP-competitive inhibitor of PI3K-p110, recovery of the activation state, but also by an additional increase LY294002, which remains a very useful tool for biochemical and (more than twofold) of the phosphorylation of both Akt and its pharmacological analysis, although deleterious side effects and substrates (Figures 3a and b). The transient Akt inhibition may low solubility in aqueous solutions have hampered its clinical explain the ability of the drugs to initiate apoptosis in these development.34 The optimal concentration of each drug was samples. Furthermore, it suggested to us that protein network- previously established by MTT assays. Samples were then analyzed based feedback mechanisms may promote reinduction of the by RPPA and western blotting. Unexpectedly, upon overnight pathway. drug treatment in 35 out of 50 samples neither PI3K nor Akt inhibitors were able to abrogate phosphorylation of Akt at S473 and T308 (Figures 2a and b). Indeed, 35 samples showed Combined inhibition of mTOR and Akt enhances cell death but paradoxical increased phosphorylation of Akt as well as of its does not completely blunt Akt activation direct substrates glycogen synthase kinase 3 and PRAS40 far One such feedback mechanism may be mediated by a direct Akt above basal level. Remarkably, however, all drugs were able to kinase substrate: the FOXO family of transcription factors.35 activate caspases 9 and 6 (Figure 2c) and to trigger apoptosis Unphosphorylated/active FOXOs reside in the nucleus where (Figure 2d). Conversely, short-time administration (up to 4 h) of they regulate transcription of several target genes, including the Akti 1/2 to the 35 inhibitor-resistant samples completely blunted well-known activators of the PI3K/Akt/mTOR pathway IR, and Akt phosphorylation both at S473 and at T308 as well as IGF-1R.36 Conversely, phosphorylation by Akt determines nuclear phosphorylation of Akt effectors in all samples (Figures 3a and b). exclusion and degradation of FOXOs.37 Figure 3a shows that

Figure 2. Long-time administration of Akt inhibitors to primary blasts leads to paradoxical Akt activation. (a) Fifty samples of primary blast cells were incubated with the indicated PI3K/Akt inhibitors for 20 h, all at 10 mM, then lysates were analyzed by RPPA. All drugs trigger hyperphosphorylation of Akt, GSK, PRAS40 and FOXO in 35 samples (N ¼ 35, inhibitor-resistant samples), whereas they inhibit phosphorylation of the same end points in 15 samples (N ¼ 15, inhibitor-sensitive samples). Data are represented as mean fold change (Log2) of the treatment/vehicle ratio. (b) A western blot confirmation of the effect of the drugs in two representative inhibitor-resistant samples is shown. (c) Apoptosis-related caspases 6 and 9 activation was evaluated by RPPA in all samples shown in (a). (d) Cell viability of all the above samples was analyzed by MTT assay. Inhibitor-sensitive and inhibitor-resistant samples show a very similar profile with all drugs tested. All fold variations listed are statistically significant by paired t-tests, with P ¼ 0.05.

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Figure 3. Targeting Akt or mTOR upregulates RTKs in all inhibitor-resistant primary AML cells. (a) The inhibitor-resistant samples (n ¼ 35) were added with vehicle (dimethylsulphoxide (DMSO)) or with 10 mM Akti 1/2, followed by RPPA analysis of the indicated times and end points. (b) A western blot analysis of the effect of drug treatment in three representative inhibitor-resistant samples is shown. (c) The level of IR, IGF-1R and PDGFR protein was measured by ELISA in the 35 inhibitor-resistant samples treated as in (a). (d) Reverse transcriptase-PCR analysis of IGF1R, IRb and PDGFR mRNA expression in seven samples from the inhibitor-resistant group treated as in (a). The data are represented as fold expression±s.e.m., normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression. (e) Phosphorylation, measured by RPPA, of IGF1R, IRb and PDGFR in the 35 inhibitor-resistant samples treated as in (a). Data in panels a, c and e are represented as mean fold change (Log2) of the ratio treatment/DMSO (n ¼ 35).

FOXO3a phosphorylation at Akt sites T24/32 was markedly reduced reactivation of PI3K/Akt signaling. As a proof of concept, we in AML cells upon 2- or 4-h exposure to Akti 1/2. We therefore monitored IRS-1 levels in the above samples upon overnight analyzed by ELISA the amount of FOXO targets whose expression treatment with Akti 1/2 or with the well-known mTORC1 inhibitor has been shown to rise upon Akt inhibition in several cancer cell rapamycin.13 As expected, we observed that the amount of IRS-1 lines.35 The result was remarkably clear and showed a steady markedly increased after drug exposure (Figure 4a), concomitantly increase of IR, IGF-1R and PDGFR (Figure 3c). To confirm this with the boost of Akt phosphorylation (Figure 4b). Conversely, S6K observation, the expression of the above receptors was evaluated in activity was clearly inhibited as demonstrated by decreased the same setting by real-time PCR. Akt inhibition elicits a significant phosphorylation at T389 (Figure 4b). In addition, the decreased induction of IR, IGF-1R and PDGFR, equivalent to that observed by phosphorylation of 4E-binding protein 1 at S65 confirms the ELISA, in all sample tested (Figure 3d). In parallel, Akt inhibition also inhibition of mTORC1 by rapamycin (Figure 4b). Importantly, the evoked autophosphorylation of the same receptors (Figure 3e). We effects of Akt inhibition by Akti 1/2 on IRS-1 levels were conclude that inhibition of Akt enhances expression and phosphor- comparable to those evoked by rapamycin (Figure 4a). Taken ylation at least of this subset of RTKs, which in turn could activate together, these results confirm that, in AML primary cells, release and sustain PI3K/Akt/mTOR signaling. of mTORC1/S6K-dependent negative feedback either directly by A second regulatory circuit has been recently unraveled that rapamycin or indirectly through Akt inhibition, activates PI3K may explain the paradoxical Akt phosphorylation we observed in signaling. Therefore, we reasoned that an alternative way to blunt AML cells.38–41 Indeed, it has been reported that in solid tumor Akt activity in cells reliant on feedback circuits is inhibition of models, downstream of Akt, activation of mTORC1 drives S6K- rapamycin-insensitive S473-kinase mTORC2. A specific ATP-com- mediated degradation of the IR/IGF-1R adaptor protein IRS-1, and petitive mTORC1/2 kinase inhibitor, Torin1,21 was therefore is therefore a negative regulator of PI3K.41 Accordingly, drugs administered overnight to 35 primary blast samples. Of note, targeting mTORC1 block this feedback and trigger reactivation of phosphorylation of 4E-BP1 at S65, a residue known to be sensitive the pathway and phosphorylation of S473. This is in good to both rapamycin and Torin1,21 was reduced to a much greater agreement with previous reports indicating that in leukemia cells extent by Torin1 than by rapamycin (Figure 4b), confirming mTOR the rapamyicn derivative everolimus triggers an IGF-1/IGF-1R kinase inhibition. Nevertheless, phosphorylation of Akt at S473 autocrine loop, which in turn upregulates PI3K/Akt.42 It is thus and of Akt substrates was largely reduced but by no means conceivable that, in the above 35 primary AML samples, inhibition abolished by Torin1 used as single agent (Figures 4b and c). Of of Akt blocks the feedback loop causing IRS-1 stabilization and note, administered in combination with Akti 1/2, Torin1 triggered

& 2014 Macmillan Publishers Limited Leukemia (2014) 2197 – 2205 Combined Akt and RTKs inhibition in AML J Bertacchini et al 2202

Figure 4. (a) The inhibitor-resistant samples were treated with dimethylsulphoxide (DMSO), 10 mM AKTi 1/2 or 10 nM rapamycin, then IRS-1 levels were analyzed with a human IRS-1 ELISA kit. (b) The above samples were treated with either 10 mM Akti 1/2, 0.1 mM Torin1 or 10 nM rapamycin for 20 h, then the effects were monitored by RPPA analysis of S473 and T308 phosphoepitopes as a readout of Akt activity, and of S6K T389 and 4E-binding protein 1 (4EBP1) S65 as a readout of mTOR activity. (c) The effect of the drugs was conﬁrmed by western blot analysis. Three representative inhibitor-resistant samples are shown. (d) Cell viability was measured by MTT assays and is expressed as % cell survival (n ¼ 35). Where indicated, paired t-test was performed on each mean value. P ¼ 0.05. (e) The inhibitor-resistant samples were treated with DMSO, 10 mM Akti 1/2 or 0.1 mM Torin1, alone or in combination, followed by RPPA analysis of Akt phosphorylation. Data in panels a, b and e are represented as mean fold change (Log2) of the ratio treatment/DMSO (n ¼ 35).

apoptosis to a greater extent than either drug alone (Figure 4d), phosphorylation far below basal level in long-time treatment, and prevented the boost of Akt maintaining the activity around and prevented reactivation of downstream signaling (Figures 5b–d). basal levels (Figure 4e). However, basal Akt phosphorylation in Moreover, combined treatments significantly reduced cell these patients is aberrantly high, therefore this combination is not viability and cell proliferation, as documented by MTT and satisfactory. Annexin V assays (Figure 5e). Abrogation of Akt activity and induction of apoptosis were also confirmed by western blot analysis of cleaved caspase 6 and poly ADP-ribose polymerase Inhibition of RTK hampers the new steady state of Akt activity epitopes in AML THP1 and MOLM 13 cell lines (Figure 5f). Next, originated from drugs targeting Akt/mTOR THP1 cells, expressing c-Kit/PDGFR, were utilized to ascertain the As previously discussed, PI3K is activated downstream of insulin potential synergistic effects of the Akti 1/2 combination with and other growth factor receptors. Constitutive activation of such sunitinib, whereas co-treatment of either linsitinib or quizartinib RTKs would fuel PI3K signaling, counteracting the effect of drugs with Akti 1/2 was performed in MOLM 13 cells, which express Flt3 targeting PI3K/Akt.35,39–43 Accordingly, we profiled a panel of RTKs and IR/IGF-1 receptors. Cells were exposed to a range of drug known to activate PI3K in 28 samples of the inhibitor-resistant doses spanning the 50% of effective concentration for 20 h, then subgroup. We found that 21 out of 28 samples were characterized cell viability was analyzed. Remarkably, we observed that at all by high phosphorylation of the IGF-1R (Figure 5a), confirming that tested doses the combinations were synergistic, according to this receptor can indeed be regarded as an upstream trigger of Chou–Thalalay combination index calculations25 (Figures 6a–c). the signaling pathway, which maintains the signaling cascade on. Thus, combined treatments with these agents potentiate PI3K/ Surprisingly, however, we also discovered that most AML Akt/mTOR inhibition at lower doses of drugs. These findings primary blast cells displayed highly phosphorylated PDGFR and suggest that treatment of AML blasts displaying constitutive epidermal growth factor receptor (Figure 5a). Thus, PI3K/Akt activation of the above RTKs with selective RTK inhibitors signaling in these cells could be sustained also through combined to allosteric Akt inhibitors could be an effective strategy constitutive activation of these RTKs. Although the profile of the for breaking the adaptive circuits that take place in leukemia hyperphosphorylated RTKs analyzed differed among patients, primary cells and make them resistant to therapy. 20 out of 28 were characterized by a systemic pattern of high phosphorylation of all the receptors tested. We reasoned therefore that broad inhibitors of the above RTKs may contribute to DISCUSSION abrogate PI3K/Akt activation. All samples were thus treated with Outcome of adult AML patients remains very poor, and the need the PDGF receptor family inhibitor sunitinib or with the IGF-1R/IR for more effective and less toxic therapies is urgent. In AML, as in inhibitor linsitinib, alone or in combination with Akti 1/2. most cancer types, dysregulation of the Akt pathway and its Furthermore, although only seven patients carried the FLT3-ITD implication in resistance to chemotherapy is well documented. A phenotype, the inhibitor of Flt3 receptor quizartinib was large body of evidence suggests that targeting the PI3K/Akt/mTOR administered to all samples because of recent promising results pathway in AML may have significant therapeutic value.16,17,19,45–48 of this drug also in wild-type FLT3 AML cells.3,23,44 Remarkably, the Accordingly, we confirmed in this study that the above pathway is RTKs inhibitors used alone reduced phosphorylation of both Akt aberrantly activated in a large number of primary blast samples and its substrates PRAS40 and glycogen synthase kinase 3 at 20 h from AML patients. In accordance with previous work from several (Figures 5b–d). The effect of the RTK inhibitors together with Akti laboratories,42 our findings confirmed that most samples indeed 1/2 was also tested. All drug combinations resensitized the cells exhibit activation of IGF-1R. In addition, we demonstrated for the to the action of the Akt inhibitor, markedly reducing Akt first time that other upstream regulators of PI3K/Akt such as IR,

Leukemia (2014) 2197 – 2205 & 2014 Macmillan Publishers Limited Combined Akt and RTKs inhibition in AML J Bertacchini et al 2203

Figure 5. RTK inhibition resensitizes primary AML cells to Akti 1/2. (a) Box plot analysis of IR, IGF-1R, EGFR and PDGFR phosphorylation determined by RPPA analysis of autophosphorylation epitopes in 28 inhibitor-resistant samples. White box length, interquartile range; black line, median level. Bars, 2 s.d. (b, c, d) The same samples shown in (a) were treated with dimethylsulphoxide (DMSO), 10 mM Akti 1/2 alone or in combination with either 1 mM sunitinib, 1 mM linsitinib or 1 mM quizartinib for 20 h. The effect of the drugs was monitored by RPPA analysis of Akt, GSK and PRAS40 phosphorylation as a readout of Akt activity. Data are represented as mean fold change (Log2) of treatment/DMSO (n ¼ 28). (e) Cell viability in the above samples was assessed by MTT colorimetric assays (n ¼ 28) and by Annexin V staining (n ¼ 10). Results are ±s.d. (f) THP1 cells were treated with 1 mM sunitinib and/or 10 mM Akti 1/2 (left), whereas MOLM 13 cells were treated with either 1 mM linsitinib (center) or 1 mM quizartinib (right) and/or 10 mM Akti 1/2. Next, apoptosis was monitored by cleaved caspase 6 and poly ADP-ribose polymerase (PARP). Actin levels conﬁrmed equal loading.

Figure 6. Akti 1/2 has a synergistic cytotoxic effect in leukemia cell lines. THP1 cells were treated with Akti 1/2 and sunitinib (a), whereas MOLM 13 cells were treated with Akti 1/2 and linsitinib (b) or with Akti 1/2 and quizartinib (c) at ﬁve different concentrations. After 20 h, cell viability was analyzed by MTT assay. The combination index values (CI) were graphed for each effective dose (ED50, ED75, ED90 and ED95). The combination displays a synergistic cytotoxic effect, calculated with Calcusyn software (ComboSyn, Inc., Paramus, NJ, USA) as described under Materials and Methods section.

PDGFR or epidermal growth factor receptor are highly activated/ same panel of inhibitors the initial downmodulation of Akt phosphorylated in AML samples and that in principle each sample phosphorylation is followed by a second peak of phosphorylation may be selectively characterized by constitutive and systemic of Akt and its substrates in 35 out of 50 patients. A dual activation of multiple upstream RTKs concomitantly. Such activity mechanism can be devised through which Akt hyper- can account for the high basal Akt phosphorylation reported in phosphorylation after long-time treatment of unresponsive AML patients. We observed that short-time treatment with patients is not necessarily a paradox. First, we showed here that allosteric Akt inhibitors such as Akti 1/2 completely abrogates the initial inactivation of Akt, at 2 and 4 h of inhibitor treatment, phosphorylation of both Akt and its direct substrates, and initiates leads to a marked reduction of FOXO1/FOXO3a phosphorylation caspase 6- and caspase 9-regulated apoptosis. Unpredictably, at the Akt sites T24/T32. Interestingly, several RTKs have been however, we discovered that upon long-time treatment with the recently reported to be upregulated through FOXO transcriptional

& 2014 Macmillan Publishers Limited Leukemia (2014) 2197 – 2205 Combined Akt and RTKs inhibition in AML J Bertacchini et al 2204 activity, including HER3, IR, IGF1R, FGFR2 in breast cancer,49 PDGFR and can lead to paradoxical activation of signaling that would in neuroblastoma50 and IGF-1R, IR in breast, lung and ovarian ultimately mitigate the therapeutic activity of the drugs. cancer cells.41 In keeping with these reports, we observed a steady Conversely, our study revealed that drugs targeting upstream gain of IR, IGF-1R and PDGFR expression and phosphorylation after RTKs activity can resensitize resistant cells to Akt inhibition by Akti 16-h treatment of unresponsive samples with Akti 1/2. This finding 1/2, and trigger massive apoptosis. These studies highlight the can account for reactivation of PI3K and its downstream targets, power of broad-scale protein pathway activation mapping by which attenuates the action of the drug, and suggested that RPPA wherein phosphorylation analysis of the signaling architec- combination targeting of Akt together with RTKs may be a good ture can be quantifiably studied in detail. Such network-level therapeutic strategy for sustained shut off of driving PI3K/Akt/ detail can uncover the dominant signaling pathways in any given mTOR signaling. Indeed, we observed that co-treatment of Akti 1/ patient sample and also determine mechanisms of targeted 2 with either sunitinib, linsitinib or quizartinib resensitized cells to therapy response and resistance that are critical to understand in Akt inhibition, blunted Akt phosphorylation, preventing this new era of precision medicine. reactivation of the pathway on long-time exposure and triggered apoptosis to a much larger degree than Akti 1/2 alone. All drug combinations showed a combination index o1, CONFLICT OF INTEREST indicative of a synergistic action. The authors declare no conflict of interest. We further showed that an additional boost of signaling comes from perturbation of feedback mechanisms. Such feedbacks have been recently described in cell lines from solid tumors and in 35,38–43,51 ACKNOWLEDGEMENTS xenograft models. Activation of Akt drives S6K-mediated SM and JB acknowledge the ‘International short term mobility program’ from Italian IRS-1 downmodulation, which in turn attenuates PI3K/Akt CNR and Fondazione CaRiMo, respectively; MG was in part supported by Fondazione signaling. It follows that chronic inhibition of Akt or mTOR Angela Serra for Cancer Research, Modena, Italy. This work was supported by grants relieves the feedback, thus stabilizing IRS-1 and enhancing IRS-1- from Italian MIUR-Prin 2008, Istituto Superiore Sanita’ oncoproteome network, prot. mediated PI3K/Akt signaling. In good agreement with this scheme, 2011-527TR1, and MIUR/FIRB accordi di programma 2010. we observed a twofold increase of IRS-1 expression in non- responsive samples subjected to long-time treatment. 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