Preclinical and Early Clinical Evaluation of the Oral AKT Inhibitor, … · AKT, as indicated by...

Cancer Therapy: Clinical

Preclinical and Early Clinical Evaluation of the Oral AKTInhibitor, MK-2206, for the Treatment of Acute MyelogenousLeukemia

Marina Y. Konopleva1, Roland B. Walter3,4,5, Stefan H. Faderl1, Elias J. Jabbour1, Zhihong Zeng1,Gautam Borthakur1, Xuelin Huang2, Tapan M. Kadia1, Peter P. Ruvolo1, Jennie B. Feliu1, Hongbo Lu1,LaKiesha Debose1, Jan A. Burger1, Michael Andreeff1, Wenbin Liu2, Keith A. Baggerly2, Steven M. Kornblau1,L. Austin Doyle6, Elihu H. Estey3,4, and Hagop M. Kantarjian1

AbstractPurpose:Recent studies suggested that AKTactivationmight confer poor prognosis in acutemyelogenous

leukemia (AML), providing the rationale for therapeutic targeting of this signaling pathway. We, therefore,

explored the preclinical and clinical anti-AML activity of an oral AKT inhibitor, MK-2206.

Experimental Methods: We first studied the effects of MK-2206 in human AML cell lines and primary

AML specimens in vitro. Subsequently, we conducted a phase II trial of MK-2206 (200mg weekly) in adults

requiring second salvage therapy for relapsed/refractory AML, and assessed target inhibition via reverse

phase protein array (RPPA).

Results: In preclinical studies, MK-2206 dose-dependently inhibited growth and induced apoptosis in

AML cell lines and primary AML blasts. We then treated 19 patients withMK-2206 but, among 18 evaluable

participants, observed only 1 (95% confidence interval, 0%–17%) response (complete remission with

incomplete platelet count recovery), leading to early study termination. Themost common grade 3/4 drug-

related toxicity was a pruritic rash in 6 of 18 patients. Nevertheless, despite the use ofMK-2206 atmaximum

tolerated doses, RPPA analyses indicated only modest decreases in Ser473 AKT (median 28%; range, 12%–

45%) and limited inhibition of downstream targets.

Conclusions: Although preclinical activity of MK-2206 can be demonstrated, this inhibitor has

insufficient clinical antileukemia activity when given alone at tolerated doses, and alternative approaches

to block AKT signaling should be explored. Clin Cancer Res; 20(8); 2226–35. �2014 AACR.

IntroductionIn adults, acute myelogenous leukemia (AML) remains

difficult to treat (1, 2). The need for novel therapies is thusunquestioned.With increasing availability of specific small-molecule inhibitors, considerable effort now focuses ontargeting signaling pathways that are pivotal for leukemiccells as ameans to improve therapeutic outcomes. Emerging

evidence suggests that the phosphoinositide 3-kinase(PI3K)/protein kinaseB (AKT)pathway (3, 4) couldprovidea rational drug target in AML. Constitutive activation ofAKT, as indicated by phosphorylation at Thr308 and/orSer473, is observed in 50% to 85%of patients with AML (5–12) and has been correlated with adverse cytogenetics aswell as inferior relapse-free and overall survival (11, 13, 14).ActivatedAKThas been implicated inproliferation,myeloidtransformation, cell survival, and chemoresistance of AMLcells (6, 9, 10); in fact, induction of PI3K/AKT signalingmaybe one of the mechanisms by which bone marrow stromacells confer protection from chemotherapy (15). Converse-ly, inhibition of the PI3K/AKT signaling pathway restoresapoptosis in AML cells in vitro (8).

MK-2206 is an oral, highly specific allosteric inhibitor forall three isoforms of human AKT (16). Preclinical studiesconfirmed efficient AKT dephosphorylation and demon-strated growth inhibition of various human solid tumor celllines (17–22); consequently, MK-2206 has entered clinicaltesting for patients with solid tumors (23). On the otherhand, the effects of MK-2206 on malignant hematopoieticcells are poorly explored so far except for recent studies,which indicated significant cytotoxic activity against diffuse

Authors' Affiliations: 1Department of Leukemia; 2Division of QuantitativeSciences, The University of Texas MD Anderson Cancer Center, Houston,Texas; 3Clinical Research Division, Fred Hutchinson Cancer ResearchCenter, Seattle,Washington; 4Division ofHematology/Department ofMed-icine; 5Department of Epidemiology, University of Washington, Seattle,Washington; and 6National Cancer Institute, Rockville, Maryland

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

M.Y. Konopleva and Roland B. Walter shared first authorship.

Corresponding Author:Marina Y. Konopleva, Department of Leukemia,The University of Texas MD Anderson Cancer Center, 1515 HolcombeBoulevard, Unit 428, Houston, TX 77030-4009. Phone: 713-794-1628;Fax: 713-745-4612; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-1978

�2014 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 20(8) April 15, 20142226

on June 4, 2021. © 2014 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 28, 2014; DOI: 10.1158/1078-0432.CCR-13-1978

http://clincancerres.aacrjournals.org/

large B-cell lymphoma and T-cell acute lymphoblastic leu-kemia (ALL) cells in vitro (24, 25). In this study, we haveinvestigated the antitumor activity of MK-2206 againsthuman AML cell lines and primary AML blasts. To begintesting this compound clinically, we then conducted aphase I/II trial in adults with poor prognosis AML todetermine the tolerability of the drug and obtain prelimi-nary data on its efficacy of AKT inhibition. Weekly (26)rather than every-other-day (23) dosing of MK-2206 wasexplored following recommendations of the Cancer Ther-apy Evaluation Program at the National Cancer Institute(CTEP/NCI; see Treatment plan).

Materials and MethodsIn vitro investigationsMaterials. All reagents were purchased from commer-

cial sources unless otherwise stated. MK-2206 was partiallyprovided by Merck & Co., Inc. and partially obtained fromLC Laboratories.AML cell lines and primary AML cells. OCI-AML3 cells

were kindly provided by M.D. Minden (Ontario CancerInstitute, Toronto, ON, Canada). HL60, U937, andMOLM13 cells were obtained from the Leibniz InstituteDSMZ-German Collection of Microorganisms and CellCultures (Braunschweig, Germany). THP-1 andMO7ewerepurchased from the American Type Culture Collection.MOLM14 cells were kindly provided by Dr. Mark Levis(Johns Hopkins University, Baltimore, MD). Cells weremaintained in RPMI-1640 supplemented with 5% FBS and5% bovine calf serum at 37�C in 5%CO2. Peripheral bloodspecimens containing >40% blasts were obtained frompatients with newly diagnosed or recurrent AML. Informedconsent was obtained following institutional guidelines.Mononuclear cells were isolated via Ficoll density gradients(Sigma-Aldrich). Samples from healthy bone marrowdonors were selected for CD34þ cells using a MiniMacsSeparator (Miltenyi Biotec) according to themanufacturer’sinstructions.

Analysis of cell viability and apoptosis. Cells were treatedwith various doses of MK-2206 for up to 72 hours. Cellviability and cell numbers were quantified by Trypan bluedye exclusion assay using a Vicell. To determine the mech-anism of cell death, cells were washed in PBS, and resus-pended in binding buffer containing Annexin V (RocheDiagnostics). Apoptotic cells were identified by positiveAnnexin V staining using a BD LSR II flow cytometer (BDBiosciences).

Western blot analysis. OCI-AML3, MOLM13, or prima-ry AML blasts were sonicated in lysis buffer (62.5 mmol/LTris (pH8.0), 2%SDS, 10%glycerol, 100mmol/L AEBSF, 80nmol/L aprotinin, 5 mmol/L bestatin, 1.5 mmol/L E-64, 2mmol/L leupeptin, 1 mmol/L pepstatin, 500 mmol/L sodiumorthovanadate, 500 mmol/L glycerol phosphate, 500 mmol/L sodiumpyrophosphate, and50mmol/LDTT), andprotein(5 � 105 cell equivalents) was subjected to electrophoresisusing 10% to 14%acrylamide/0.1% SDS gels. Proteinsweretransferred onto nitrocellulose, and membranes wereprobed with monoclonal antibodies against pAKT Thr308and Ser473, phospho-S6, S6 (all from Cell Signaling Tech-nology), and Tubulin (Sigma-Aldrich).

Clinical trialStudy population. A phase II study with MK-2206 was

conducted at the MD Anderson Cancer Center (MDACC;Houston, TX) and the Fred Hutchinson Cancer ResearchCenter (FHCRC; Seattle, WA) between October 2010 andOctober 2012. Patients�18 years of age were eligible if theyhad persistent or relapsing AML (other than acute promye-locytic leukemia; ref. 27) requiring second salvage therapy(i.e., treatment for second or higher relapse or for primaryrefractory disease after failure of two prior treatment regi-mens) provided they had a prior complete remission (CR)duration

Review Boards at MDACC and FHCRC approved this study(ClinicalTrials.gov: NCT01253447), and patients gave con-sent in accordance with the declaration of Helsinki.

Treatment plan. Treatment consisted of MK-2206 200mg orally once weekly, the maximum tolerated dose(MTD) in patients with solid tumors (26). The weeklydosing was recommended by CTEP based on phase I datademonstrating pAKT suppression in platelet-rich plasmaup to 96 hours after dose (26). With the exception ofhydroxyurea during the first month of therapy, concom-itant cytotoxic medications were prohibited. Patientswere assessed for response after every 28-day cycle andwere allowed to continue study treatment if there was noevidence of progressive disease or clinically significanttoxicities. Patients achieving either CR, CR with incom-plete platelet count recovery (CRp), or partial remission(PR) after three cycles of therapy could continue studytreatment for up to 12 cycles. Patients not achieving anobjective response but obtaining clinical benefit from thetrial drug could also remain on study beyond 3 months ifno significant toxicities occurred.

Statistical considerations. The primary objective of thestudywas to determine the proportion of patients achievingCR, CRp, or PR as best response within three cycles oftherapy; secondary objectives included the estimation ofdisease-free survival of patients who achieved CR/CRp, andthe establishment of the toxicity profile of MK-2206. ASimon two-stage optimum design (30) was used to test thenull hypothesis that the response rate (i.e., CR, CRp, or PR)at week 12 was �10% versus the alternative that theresponse rate is�25% at ana level of 0.05with 80%power.Patients who were removed from study therapy for reasonsother than disease progression (e.g., noncompliance, fatalinfections) before completion of three cycles of therapywere not included in the efficacy assessment, and additionalpatients were enrolled. With three or more responses in thefirst 18 patients, the trial would continue to accrue up to 43patients; otherwise, the trial would stop early and the drugwould be rejected for further study. With �8 responsesamong 43 patients enrolled, the drug would be consideredeffective. With this design, the probability of early termi-nationwas 73% if the true response ratewas only 10%.Withthe concern of treatment-related toxicity, the nonhemato-logic toxicity (�grade 3, with the exception of hyperglyce-mia) would also be continuously monitored during thestudy using established Bayesian-based methods (31). Thestopping rulewas set such that the trial would be halted if, atany time during the study, there was a >90% chance that thetoxicity rate exceeded 30%.

Biomarker analyses. Biomarkers studies were optionaland only performed in samples from patients consenting tothese studies. Peripheral blood samples were collectedbefore and 24 hours after the first administration of MK-2206 during the initial treatment cycle, and bone marrowsamples were collected at baseline and before the secondtreatment cycle. Mononuclear cells were separated by Ficollgradients, and cell lysates were subjected to reverse phaseprotein array (RPPA) analysis following previously

described and validated methods (32–34). Slides wereprobed with 157 primary antibodies, including two recog-nizing pAKT (Thr308 and Ser473; both Cell SignalingTechnology), a secondary antibody for signal amplification,and a stable dye for precipitation (35). The slides werescanned, and images quantified using Microvigene Version2.9 software (Vigene Tech). Raw signal intensity data wereprocessed with SuperCurve to estimate relative proteinconcentrations, with further data normalization to adjustloading bias by median centering each marker and mediancentering each sample (36, 37). We used paired t tests usingthe Benjamini–Hochberg method for adjustment to controlthe false discovery rate (38) to detect differential proteinexpression or phosphorylation of each protein in the follow-ing three subsets: (i) the target itself (pAKT Ser473 andThr308); (ii) selected proteins associatedwithAKT inhibition(4E-BP1-pS65, 4E-BP1-pT37-T46, Bim, cyclin D1, FOXO3apS318-S321, GSK3a-b pS21-S9, IRS1, Mcl1, mTOR pS2448,NF-kB-p65pS536,p70S6K-pT389,PRAS40-pT246,PTEN,S6pS235-S236, S6 pS240-S244, Survivin, and XIAP); and (iii)all 157proteins. In subsets (i) and(ii),weusedone-sided testsbecause we could specify the expected directions of change apriori. Heatmaps of selected markers and samples wereplotted in Supplementary Fig. S4, in which the data weremedian centered for each marker.

ResultsIn vitro investigations of MK-2206 in human AML cells

In line with previous investigations in solid tumorcancer as well as ALL cells (18, 20, 39–41), treatment ofhuman AML cell lines (OCI-AML3 and U937) with MK-2206 dose-dependently reduced Ser473 AKT phosphor-ylation but left total AKT levels unchanged (Fig. 1A).Reflective of loss of AKT activity, both cell lines exhib-ited reduced phosphorylation of PRAS40 and themTOR targets, S6 kinase (at Ser240), and 4EBP1(Thr37/45) in response to MK-2206, albeit inhibitionof these downstream targets required higher concentra-tions of MK-2206. Similar findings were seen in FLT3-mutated MOLM13 and MOLM14 cells (SupplementaryFig. S1). Consistent with these cell line data, treatmentwith MK-2206 suppressed Ser473 AKT phosphorylation,pPRAS40, and p4EPBP1 (Thr37/46) in three primaryAML samples tested (Fig. 1B).

Having confirmed target inhibition with MK-2206, wethen investigated the effects of the drug on viability ofvarious human AML cell lines. As shown in Table 1 andSupplementary Fig. S2A, MK-2206 at 1 mmol/L effectivelysuppressed cell growth in all cell lines except Mo7e cellsafter 72 hours, with OCI-AML3, MV4;11, MOLM14,THP1 but not U937 or MOLM13 cells exhibiting growthinhibition with as little as 0.1 to 0.2 mmol/L of MK-2206.In turn, induction of apoptosis was only seen at higherdoses (5–10 mmol/L) of MK-2206 (Supplementary Fig.S2A). Cell-cycle analyses in OCI-AML3 cells indicatedthat MK-2206 induced G1 arrest, particularly at higherdrug concentrations (Supplementary Fig. S2B). Finally,we assessed the effects of MK-2206 in two primary AML

Konopleva et al.

Clin Cancer Res; 20(8) April 15, 2014 Clinical Cancer Research2228




specimens harboring FLT3 mutations and found block-ade of AKT signaling, as indicated by inhibition of 4EBP1phosphorylation and S6 (Fig. 2). Furthermore, as shownin Fig. 2, MK-2206 induced apoptosis in both samplesafter 48 hours. Together, these in vitro studies indicate thatMK-2206 causes cell-cycle arrest, growth inhibition,and—at higher drug concentrations—apoptosis inhuman AML cell lines and primary AML blasts.

Clinical trialGiven the above findings, we conducted a phase II trial of

MK-2206 in adults with poor prognosis AML to begintesting its clinical efficacy and tolerability in this disease.Although initial clinical studies explored alternate dosingschedules (23), preclinical data indicated that weekly dos-ing would be at least as efficacious. Thus, later clinicalstudies investigated MK-2206 given once weekly (26).

Figure 1. Effects of MK-2206 onAKT signaling in human AML cells.A, OCI-AML3 and U937 cells wereexposed to the indicatedconcentrations of MK-2206 for6 hours. Whole-cell lysates weresubjected to immunoblot analyseswith the indicated antibodies. B,mononuclear cells from patients(PT) with AML with high (>50%)blast count were treated withMK-2206 (5 mmol/L) for 24 hours.Expression of AKT signalingproteins was analyzed byimmunoblotting (left) andquantified by densitometry (right).PT#1, refractory AML, N-Rasmutated, del(12p); PT#2, refractoryAML, diploid; and PT#3, newlydiagnosed AML arising fromantecedent MDS, N-Ras mutated,del(5q) and -7.

MK-2206 in AML

www.aacrjournals.org Clin Cancer Res; 20(8) April 15, 2014 2229




Building upon this experience,we administeredMK-2006 at200 mg weekly, the MTD established in patients with solidtumors (26).

Over a periodof 2 years, 19 adultswith amedian age of 70years were enrolled in this study and received at least one

dose of MK-2206 (Table 2). The median number of cyclesadministered was two, with 4 patients completing �3cycles. Eighteen patients were eligible for efficacy and tox-icity assessments. One patient was not included in theseanalyses because of study removal after one cycle due tononcompliance; at that time, however, this patient hadstable disease and no evidence of drug-associated toxicities.Among the 18 eligible patients, only one CRp was observed(5.6%; exact 95% confidence interval, 0%–17%). Thispatient was a 77-year-old male with normal karyotypesecondary AML without FLT3, KIT, or RAS mutations thatarose fromantecedent chronic myelomonocytic leukemia.He initially achieved CR with clofarabine/low-dose cytar-abine but then relapsed and failed a first salvage therapywith twice-daily fludarabine and cytarabine. He achieveda CRp after the first cycle of MK-2206 with a decrease inbone marrow blasts from 13% to 5MV4;11 t(4;11), MLL-rearranged 3.92

FLT3-MutantMOLM14 FLT3-Mutant 0.42

NOTE: AML cells were cultured with increasing concentra-tions of MK-2206 (from 40 nmol/L up to 10 mmol/L, intriplicates) for 48 hours, after which effects on cell growthwere determined by viable cell counts. IC50 values werecalculated using CalcuSyn software.Abbreviation: MLL, myeloid/lymphoid, or mixed lineagesleukemia.

PT#1 (90% blast, PB) PT#2 (>50% blast, PB)Absolute Annexin V+ cells (%) Absolute Annexin V+ cells (%)

60

50

40

30

20

10

00.1 0.2 0.6 1.7 5.0 0.1 0.2 0.6

MK2206 (mmol/L)MK2206 (mmol/L)

p4EBP1(Thr37/46)

t4EBP1

Co

ntro

l

Co

ntro

l

MK

2206 (0.6 mmo

l/L)

MK

2206 (1.7 mmo

l/L)

MK

2206 (5 mmo

l/L)

MK

2206 (5 mmo

l/L)

b-Actin

pS6K(Ser240/244)

tS6K

b-Actin

1.7 5.0

25

20

15

10

5

0

Figure 2. Effects ofMK-2206 oncellgrowth and survival of primaryAML cells. Primary AML cells from2 patients with AML were treatedwith MK-2206, and effects onapoptosis induction as examinedby Annexin V staining. Bothpatients had diploid karyotype;PT#1 had FLT3-ITD mutation, andPT#2 FLT3 D835 gene mutation.The extent of drug-specificapoptosis was assessed by theformula: [Percentage - specificapoptosis ¼ (test � control) �100/(100 � control) ref. 48].

Konopleva et al.





reduction in 3patients (135mg/wk) anddiscontinuation ofdrug in 1. Besides milder (grade 1/2) rashes in another 4patients (22.2%), other adverse events were hyperglycemia[n ¼ 12 (66.7%), all grade 1/2 except for grade 4 in onepatient], and grade 1 QTc prolongation [n ¼ 4 (22.2%)],respectively (Table 3). Ultimately, the study was terminated

early after inclusion of 18 eligible patients because ofinsufficient drug efficacy, as per predefined stopping rules.

Finally, we conducted proteomic analyses to investigatethe degree of target inhibition withMK-2206 using RPPA inpaired peripheral blood specimens—obtained before and24 hours after the first dose of MK-2206—in 8 patients, aswell as in paired bonemarrow specimens—obtained beforeand 28 days after the first dose of MK-2206—in 5 patients,respectively. Two patients (#1 and #17, the latter withextramedullary disease only) who had low pAKT levels inthe baseline samples (less than �4.0 on log2 scale) wereexcluded from this analysis and, unfortunately, the soleresponding patient did not consent to these optional bio-marker studies. Considering AKT alone, the decrease inSer473 AKTwas significant (P¼ 0.018). However, the largersets (i.e., testing direct AKT targets or the entire 157 pro-teins) showed no significant changes after adjusting formultiple testing. Of note, a decrease in Ser473 AKT (median28%; range, 12%–45%) was seen in five of seven peripheralblood and three of four bonemarrow specimens, whereas areduction in Thr308AKTphosphorylationwas found infiveof seven peripheral blood as well as three of four bonemarrow samples, respectively (Fig. 3A and B). In turn,changes in AKT targets were less evident (downregulationof pFOX3A in 3/7, pPRAS40 in 2/7, pS6K in 4/7, andp4EBP1 in 2/7 peripheral blood samples), possibly due toincomplete AKT inhibition. In samples with sufficientamounts of protein available, confirmatory immunoblot-ting was performed, which showed findings consistent withRPPA. Specifically,�50%downregulation of pAKT-473wasseen in peripheral blood samples from subjects #2, 3, 7 andin bone marrow sample #6, with lesser decreases in themTOR targets, pS6 and p4EBP1 (Fig. 4A and B), withchanges in pAKT being highly concordant between the twotechniques (P ¼ 0.01; Supplementary Fig. S3). With regardto downstream targets or other proteins, including phos-phorylated mitogen-activated protein kinase (MAPK), nosignificant changes were found after adjustment for multi-ple testing by RPPA (Fig. 3A, last column). We furtheranalyzed a subset of proteins recently reported to be upre-gulated in a compensatory fashion upon PI3K/AKT block-ade. RPPA analysis indeed demonstrated upregulation of asubset of these proteins, such as Bcl-2 (P¼0.036), Smad3 (P¼ 0.028), HER2 (P¼ 0.064), pY705 Stat-3 (P¼ 0.033), p38MAPK (P ¼ 0.039) and MEK1 (P ¼ 0.01), although thesechanges did not remain significant after accounting formultiple comparisons (Supplementary Fig. S4).

DiscussionThe in vitro data presented in this study showing that AKT

inhibition impairs the survival of AML cells provide furtherrationale for selecting this signaling pathway as pharmaco-logic target in AML.However, our findings also indicate thatthe oral AKT inhibitor, MK-2206, has insufficient activity asa single agent inpatientswith relapsed/refractoryAMLat thechosen dose. Although initial studies with MK-2206 wereconducted using every-other-day drug dosing, a weekly

Table 2. Characteristics of study cohort

Parameter n ¼ 19Median age (range), y 70 (31–86)Sex (male/female), n 12/7Disease stage, n (%)First relapse 10 (52.6%)Second relapse 9 (47.4%)

Disease manifestation, n (%)Bone marrow relapse 18 (94.7%)Isolated extramedullary relapse 1 (5.3%)

Performance status, n (%)1 17 (89.5%)2 2 (10.5%)

Cytogenetic risk-group, n (%)a

Favorable 0Intermediate 13Unfavorable 6

Secondary AML, n (%) 9 (47.4%)Laboratory findings at baseline, median (range)WBC (�103/mL)a 1.7 (0.5–11.5)Hemoglobin (g/dL) 9.6 (8.4–13.2)Platelets (�103/mL) 39 (11–1,887)LDH (U/L) 455 (153–1,222)Creatinine (mg/dL) 0.91 (0.6–1.69)Total bilirubin (mg/dL) 0.6 (0.3–1.3)AST (U/L) 22 (11–42)ALT (U/L) 27 (12–64)

aOn the basis of revised MRC prognostic classification (28).

Table 3. Tolerability and safety of study therapy

Parameter Grade 1–2 Grade 3–4

Fever, infectionNeutropenic fever 2Documented infection 6Pneumonia 2Fever of unknown origin 2

CardiacQTc prolongation 4

Rash maculopapular 4 6MetabolicHyperglycemia 11 1

OtherPRES syndrome 1

Abbreviation: PRES, posterior reversible encephalopathysyndrome.

MK-2206 in AML





dosing schedulewas exploredbecauseof the longhalf-life ofthe drug (60–80 hours) and an attempt to amelioratefeedback activation of alternative signaling pathways inresponse to continuous AKT inhibition. Despite the factthat MK-2206 was reasonably well tolerated in our studycohort, the frequent occurrence of grade 3/4 rash suggeststhat dose escalation at the weekly schedule may not befeasible. Skin toxicity was also a prominent toxicity inpatients with solid tumor who received MK-2206 at doseshigher than 60 mg every-other-day (23), a finding thatdisabled escalation of drug dosing in those patients.

In our study population, an objective response wasdocumented in only one patient, indicating suboptimalactivity of MK-2206. Failure of targeted anticancer agents

has been attributed to multiple causes, including (i) usein the "wrong" patient population, for example, becausetumor cells do not depend on the specific pathway; (ii)rapid activation of the compensatory survival pathways;and (iii) incomplete target inhibition. In AML, activationof the AKT pathway is generally attributed to mutation(s)and hyperactivation of the upstream receptor tyrosinekinases (RTK) such as KIT, FLT3, or RAS, without acti-vating mutations in PI3K or AKT itself. If activatingmutations in AKT are present, cells may become hyper-sensitive to MK-2206 (42). Because of the present studydid not include patients with such mutations, potentialactivity of MK-2206 in selected patient populations mayhave been missed.

Figure 3. Modulation of proteinexpression in AML cells by MK-2206 in peripheral blood (PB; A) orbone marrow (BM; B). PB or BMmononuclear cells were collectedbefore treatment ("1") or at selectedtime points after initiation ofMK-2206 ("2"), which was 24 hoursfor PB samples and at thecompletion of the first cycle oftherapy for BM samples. Proteinlysates were subjected to RPPAanalysis. Of note, y-axis,normalized log2-transformedrelative values of proteinexpression for the indicatedproteins.

Konopleva et al.





To assess the response toAKT inhibition and the potentialfor compensatory activation of other pathways, we inter-rogated intracellular signaling in AML blasts using RPPA.Recent evidence suggests that the acquired resistance toPI3K/AKT inhibition stems from the upregulation andactivation of RTKs through mechanisms that involveFOXO-regulated transcription (43) and of other cellular

survival proteins, including antiapoptotic proteins (Bcl-2,XIAP) and transcription factors (p-STAT3, p-STAT6, p-c-Jun,p-SMAD3), in part through cap-independent translation(44). RPPA analysis indeed demonstrated upregulation of asubset of prosurvival proteins, including Bcl-2, Smad3,pY705 Stat-3, p38 MAPK and MEK1. However, MK-2206failed to substantially downregulate p-FOXO3Aanddid not

Figure 4. Immunoblot analysis ofPB samples before and afterMK-2206 exposure in selectedpatients treated on the trial. PBsamples were collected beforeand 24 hours after the firstadministration of MK-2206 duringthe initial treatment cycle.Mononuclear cells were separatedby Ficoll-Hypaque (Sigma-Aldrich)density gradient centrifugation,cells were lysed in Laemmli samplebuffer (Bio-Rad Laboratories),transferred to Hybond-Pmembranes (GE Healthcare),probed with the appropriateantibodies (AKT, Ser 473–phosphorylated AKT,S6 ribosomal protein,Ser240/244-phosphorylated S6ribosomal protein, 4E-BP1, andThr37/46-phosphorylated 4E-BP1from Cell Signaling Technology),and visualized using the LI-COROdyssey system (LI-CORBiosciences). A, examples ofWestern blot analysis for selectedAKT targets. B, intensity of theimmunoblot signals was quantifiedand the relative intensity comparedwith total protein (AKT and S6K,respectively) calculated. �, patientswith�50% decrease in the densityof the protein.

MK-2206 in AML





significantly modulate other FOXO targets, indicatingFOXO-independent upregulation of these proteins. Furtherstudies with MK-2206 or other PI3K/AKT inhibitors mayhelp characterize AML-specific compensatory pathwaysinduced in response to AKT blockade, paving the way forfurther combination strategies. Our correlative studies indi-cate that the main clinical limitation of MK-2206 may beincomplete target inhibition, with

12. Muranyi AL, Dedhar S, Hogge DE. Combined inhibition of integrinlinked kinase and FMS-like tyrosine kinase 3 is cytotoxic to acutemyeloid leukemia progenitor cells. Exp Hematol 2009;37:450–60.

13. Min YH, Cheong JW, Kim JY, Eom JI, Lee ST, Hahn JS, et al. Cyto-plasmic mislocalization of p27Kip1 protein is associated with consti-tutive phosphorylation of Akt or protein kinase B and poor prognosis inacute myelogenous leukemia. Cancer Res 2004;64:5225–31.

14. KornblauSM,WombleM,Qiu YH, JacksonCE,ChenW, KonoplevaM,et al. Simultaneous activation ofmultiple signal transduction pathwaysconfers poor prognosis in acute myelogenous leukemia. Blood2006;108:2358–65.

15. Tabe Y, Jin L, Tsutsumi-Ishii Y, Xu Y, McQueen T, Priebe W, et al.Activation of integrin-linked kinase is a critical prosurvival pathwayinduced in leukemic cells by bone marrow-derived stromal cells.Cancer Res 2007;67:684–94.

16. Yan L. MK-2206: a potent oral allosteric AKT inhibitor [abstract]. In:ProceedingsAmericanAssociation forCancerResearch; 2009Apr 18–22; Denver, CO: 2009. Abstract nr DDT01-1.

17. Hirai H, Sootome H, Nakatsuru Y, Miyama K, Taguchi S, Tsujioka K,et al. MK-2206, an allosteric Akt inhibitor, enhances antitumor efficacyby standard chemotherapeutic agents or molecular targeted drugsstro and in vivo. Mol Cancer Ther 2010;9:1956–67.

18. Meng J, Dai B, Fang B, Bekele BN, Bornmann WG, Sun D, et al.Combination treatment with MEK and AKT inhibitors is more effectivethaneachdrugalone in humannon–small cell lungcancer in vitro and invivo. PloS ONE 2010;5:e14124.

19. Chen KF, Chen HL, Tai WT, Feng WC, Hsu CH, Chen PJ, et al.Activation of phosphatidylinositol 3-kinase/Akt signaling pathwaymediates acquired resistance to sorafenib in hepatocellular carcinomacells. J Pharmacol Exp Ther 2011;337:155–61.

20. Liu R, Liu D, Trink E, Bojdani E, Ning G, Xing M. The Akt-specificinhibitor MK2206 selectively inhibits thyroid cancer cells harboringmutations that can activate the PI3K/Akt pathway. J Clin EndocrinolMetab 2011;96:E577–85.

21. Pant A, Lee II, Lu Z, Rueda BR, Schink J, Kim JJ. Inhibition of AKT withthe orally active allosteric AKT inhibitor, MK-2206, sensitizes endo-metrial cancer cells to progestin. PloS ONE 2012;7:e41593.

22. Sangai T, Akcakanat A, Chen H, Tarco E, Wu Y, Do KA, et al. Bio-markers of response to Akt inhibitor MK-2206 in breast cancer. ClinCancer Res 2012;18:5816–28.

23. Yap TA, Yan L, Patnaik A, Fearen I, Olmos D, Papadopoulos K, et al.First-in-man clinical trial of the oral pan-AKT inhibitor MK-2206 inpatients with advanced solid tumors. J Clin Oncol 2011;29:4688–95.

24. Petrich AM, Leshchenko V, Kuo PY, Xia B, Thirukonda VK, UlahannanN, et al. Akt inhibitors MK-2206 and nelfinavir overcome mTORinhibitor resistance in diffuse large B-cell lymphoma. Clin Cancer Res2012;18:2534–44.

25. Simioni C, Neri LM, Tabellini G, Ricci F, Bressanin D, Chiarini F, et al.Cytotoxic activity of the novel Akt inhibitor, MK-2206, in T-cell acutelymphoblastic leukemia. Leukemia 2012;26:2336–42.

26. Biondo A, Yap TA, Yan L, Patnaik A, Fearen I, Baird RD, et al. Phase Iclinical trial of an allosteric AKT inhibitor, MK-2206, using a onceweekly (QW) dose regimen in patients with advanced solid tumors.J Clin Oncol 2011;29:3037.

27. Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A,et al. The 2008 revision of the World Health Organization (WHO)classification of myeloid neoplasms and acute leukemia: rationale andimportant changes. Blood 2009;114:937–51.

28. Grimwade D, Hills RK, Moorman AV, Walker H, Chatters S, GoldstoneAH, et al. Refinement of cytogenetic classification in acute myeloidleukemia: determination of prognostic significance of rare recurringchromosomal abnormalities among 5876 younger adult patients trea-ted in the United Kingdom Medical Research Council trials. Blood2010;116:354–65.

29. Cheson BD, Bennett JM, Kopecky KJ, B€uchner T, Willman CL, EsteyEH, et al. Revised recommendations of the International working groupfor diagnosis, standardization of response criteria, treatment out-

comes, and reporting standards for therapeutic trials in acute myeloidleukemia. J Clin Oncol 2003;21:4642–9.

30. Simon R. Optimal two-stage designs for phase II clinical trials. ControlClin Trials 1989;10:1–10.

31. Thall PF, Simon RM, Estey EH. Bayesian sequential monitoringdesigns for single-arm clinical trials with multiple outcomes. Stat Med1995;14:357–79.

32. Tibes R, Qiu Y, Lu Y, Hennessy B, Andreeff M, Mills GB, et al. Reversephase protein array: validation of a novel proteomic technology andutility for analysis of primary leukemia specimens and hematopoieticstem cells. Mol Cancer Ther 2006;5:2512–21.

33. Kornblau SM, Tibes R, Qiu YH, Chen W, Kantarjian HM, Andreeff M,et al. Functional proteomic profiling of AML predicts response andsurvival. Blood 2009;113:154–64.

34. Kornblau SM, Qiu YH, Zhang N, Singh N, Faderl S, Ferrajoli A, et al.Abnormal expression of FLI1 protein is an adverse prognostic factor inacute myeloid leukemia. Blood 2011;118:5604–12.

35. Hunyady B, Krempels K, Harta G, Mezey E. Immunohistochemicalsignal amplification by catalyzed reporter deposition and its appli-cation in double immunostaining. J Histochem Cytochem 1996;44:1353–62.

36. Hu J, He X, Baggerly KA, Coombes KR, Hennessy BT, Mills GB.Nonparametric quantification of protein lysate arrays. Bioinformatics2007;23:1986–94.

37. Coombes KR, Liu W, Ju Z, Roebuck P. SuperCurve 2012; Version1.4.3. Available from: http://bioinformatics.mdanderson.org/main/SuperCurve:Overview

38. Benjamini Y, Hochberg Y. Controlling the false discovery rate: apractical and powerful approach to multiple testing. J R Statist SocB 1995;57:289–300.

39. Cheng Y, Zhang Y, Zhang L, Ren X, Huber-Keener KJ, Liu X, et al. MK-2206, anovel allosteric inhibitor ofAkt, synergizeswith gefitinib againstmalignant glioma via modulating both autophagy and apoptosis. MolCancer Ther 2012;11:154–64.

40. Bressanin D, Evangelisti C, Ricci F, Tabellini G, Chiarini F, Tazzari PL,et al. Harnessing the PI3K/Akt/mTOR pathway in T-cell acute lym-phoblastic leukemia: eliminating activity by targeting at different levels.Oncotarget 2012;3:811–23.

41. Stegeman H, Kaanders JH, Wheeler DL, van der Kogel AJ, VerheijenMM, Waaijer SJ, et al. Activation of AKT by hypoxia: a potential targetfor hypoxic tumors of the head and neck. BMC Cancer 2012;12:463.

42. Gorlick R, Maris JM, Houghton PJ, Lock R, Carol H, Kurmasheva RT,et al. Testing of the Akt/PKB inhibitor MK-2206 by the PediatricPreclinical Testing Program. Pediatr Blood Cancer 2012;59:518–24.

43. Chandarlapaty S, Sawai A, Scaltriti M, Rodrik-Outmezguine V, Grbo-vic-Huezo O, Serra V, et al. AKT inhibition relieves feedback suppres-sion of receptor tyrosine kinase expression and activity. Cancer Cell2011;19:58–71.

44. Muranen T, Selfors LM,Worster DT, Iwanicki MP, Song L, Morales FC,et al. Inhibition of PI3K/mTOR leads to adaptive resistance in matrix-attached cancer cells. Cancer Cell 2012;21:227–39.

45. Floc'h N, Kinkade CW, Kobayashi T, Aytes A, Lefebvre C, MitrofanovaA, et al. Dual targeting of the Akt/mTOR signaling pathway inhibitscastration-resistant prostate cancer in a genetically engineeredmousemodel. Cancer Res 2012;72:4483–93.

46. Grabinski N, Ewald F, Hofmann BT, Staufer K, Schumacher U, NashanB, et al. Combined targeting of AKT and mTOR synergistically inhibitsproliferationof hepatocellular carcinomacells.MolCancer 2012;11:85.

47. Ewald F, Grabinski N, Grottke A, Windhorst S, Norz D, Carstensen L,et al. Combined targeting of AKT and mTOR using MK-2206 andRAD001 is synergistic in the treatment of cholangiocarcinoma. Int JCancer 2013;133:2065–2076.

48. Kojima K, Konopleva M, McQueen T, O'Brien S, Plunkett W, AndreeffM. Mdm2 inhibitor Nutlin-3a induces p53-mediated apoptosis bytranscription-dependent and transcription-independent mechanismsand may overcome Atm-mediated resistance to fludarabine in chroniclymphocytic leukemia. Blood 2006;108:993–1000.


MK-2206 in AML




2014;20:2226-2235. Published OnlineFirst February 28, 2014.Clin Cancer Res Marina Y. Konopleva, Roland B. Walter, Stefan H. Faderl, et al. MK-2206, for the Treatment of Acute Myelogenous LeukemiaPreclinical and Early Clinical Evaluation of the Oral AKT Inhibitor,

Updated version

10.1158/1078-0432.CCR-13-1978doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2014/03/07/1078-0432.CCR-13-1978.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/20/8/2226.full#ref-list-1

This article cites 46 articles, 22 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/20/8/2226.full#related-urls

This article has been cited by 2 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/20/8/2226To request permission to re-use all or part of this article, use this link


http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-13-1978http://clincancerres.aacrjournals.org/content/suppl/2014/03/07/1078-0432.CCR-13-1978.DC1http://clincancerres.aacrjournals.org/content/20/8/2226.full#ref-list-1http://clincancerres.aacrjournals.org/content/20/8/2226.full#related-urlshttp://clincancerres.aacrjournals.org/cgi/alertsmailto:[email protected]://clincancerres.aacrjournals.org/content/20/8/2226http://clincancerres.aacrjournals.org/

Date post:	26-Jan-2021
Category:	Documents
Upload:	others
View:	0 times
Download:	0 times

Preclinical and Early Clinical Evaluation of the Oral AKT Inhibitor, … · AKT, as indicated by...

Documents