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Therapeutics, Targets, and Chemical Biology

Adaptation to TKI Treatment Reactivates ERKSignaling in Tyrosine Kinase–Driven Leukemiasand Other MalignanciesJ. Kyle Bruner1, Hayley S. Ma1, Li Li1, Alice Can Ran Qin1, Michelle A. Rudek1,Richard J. Jones1, Mark J. Levis1, KeithW. Pratz1, Christine A. Pratilas1,2, and Donald Small1,2

Abstract

FMS-like tyrosine kinase-3 (FLT3) tyrosine kinase inhibitors(TKI) have been tested extensively to limited benefit in acutemyeloid leukemia (AML). We hypothesized that FLT3/internaltandem duplication (ITD) leukemia cells exhibit mechanismsof intrinsic signaling adaptation to TKI treatment that areassociated with an incomplete response. Here, we identifiedreactivation of ERK signaling within hours following treatmentof FLT3/ITD AML cells with selective inhibitors of FLT3. Whenthese cells were treated with inhibitors of both FLT3 and MEK

in combination, ERK reactivation was abrogated and anti-leukemia effects were more pronounced compared with eitherdrug alone. ERK reactivation was also observed followinginhibition of other tyrosine kinase–driven cancer cells, includ-ing EGFR-mutant lung cancer, HER2-amplified breast cancer,and BCR–ABL leukemia. These studies reveal an adaptive feed-back mechanism in tyrosine kinase–driven cancers associatedwith reactivation of ERK signaling in response to targetedinhibition. Cancer Res; 77(20); 5554–63. �2017 AACR.

IntroductionFMS-like tyrosine kinase-3 (FLT3) is one of themost commonly

mutated genes in acute myeloid leukemia (AML). The mostfrequently observed genetic alteration, the internal tandemdupli-cation (FLT3/ITD), occurs in approximately 23% of patients withAML and is associated with an inferior prognosis (1). FLT3/ITD isan established driver mutation in AML (2) and results in consti-tutive dimerization and activation of the receptor, thereby acti-vating downstream signaling pathways including STAT5, PI3K/AKT, and RAF/MEK/ERK (3, 4).

FLT3 tyrosine kinase inhibitors (TKI), including first-genera-tion (sorafenib and midostaurin) and second-generation (qui-zartinib and crenolanib) inhibitors, are being actively pursued as apromising therapeutic strategy in patients with FLT3/ITD AML.However, the extent of clinical responses to these therapies hasremained limited and is usually transient (5–7). Factors contrib-uting to the limited efficacy of these drugs include acquiredresistance mutations in the FLT3 kinase domain (2), acquiredresistance via activation of parallel pathways (8), upregulation ofFLT3 ligand (9), and bone marrow stromal cell–mediated acti-vation of ERK signaling (10).

In some cancers driven by mutated oncogenes, it has beenshown that small-molecule inhibition of the target results inrelief of feedback inhibition and resultant reactivation ofsignaling pathways. This response results in attenuation ofthe antitumor effects of targeted therapy and can be abrogatedwith combinatorial treatment strategies. This so-called "adap-tive resistance" has been described in the context of PI3K/AKT/mTOR (11–13) and BRAFV600E (14) inhibition. However, itremains unclear whether this phenomenon extends to cancersdriven by receptor tyrosine kinase (RTK) activation, such asFLT3/ITD AML.

Here, we describe, in FLT3/ITD leukemia cells, intrinsicmechanisms of signaling adaptation in response to FLT3inhibitor treatment, which result in reactivation of ERK sig-naling following maximal inhibition. The addition of submax-imal concentrations of a MEK inhibitor abrogates this ERKreactivation and sensitizes cells to FLT3 inhibitor treatment,resulting in a synergistic combination. A similar adaptiveresponse also occurs in response to TKI treatment in thecontext of BCR–ABL, EGFR-mutant, and HER2-amplified can-cer. These findings suggest that the addition of a low-dose MEKinhibitor to standard TKI treatment may improve outcomes forpatients with FLT3/ITD AML, and possibly other tyrosinekinase–driven malignancies.

Materials and MethodsCell lines and reagents

MV4;11, SKBR3, K562, andHL60 cellswere purchased from theATCC. Molm14 cells were obtained from the DSMZ. PC-9 cellswere obtained from Christine Hann (Johns Hopkins University,Baltimore,MD). Parental cell lineswere purchasedwithin the past10 years and authenticated by the ATCC or the DSMZ prior topurchase by the short tandem repeat method. Cell lines were notauthenticated after purchase. 32D/ITD cells were generated as

1Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, JohnsHopkins University School of Medicine, Baltimore, Maryland. 2Department ofPediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Donald Small, Johns Hopkins University School ofMedicine, Room253, 1650OrleansStreet, Baltimore,MD21231-1000. Phone: 410-614-0994; Fax: 410-955-8897; E-mail: [email protected]; and Christine A.Pratilas, Phone: 443-287-8623; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-2593

�2017 American Association for Cancer Research.

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previously described (15). All cells except SKBR3weremaintainedin RPMI 1640 supplemented with 10% fetal bovine serum (FBS).SKBR3 cells weremaintained inDMEM/F12medium supplemen-ted with the same. All cells were tested for mycoplasma contam-ination with the Southern BiotechMycoplasma Detection Kit lessthan 3 years prior to use. Sorafenib, quizartinib, lestaurtinib, andcrenolanib were obtained from LC Labs. PD0325901, trametinib,imatinib, and lapatinib were obtained from Selleckchem. TTT-3002 was a generous gift of Hanno Roder (TauTaTis Inc., Jack-sonville, FL). Drugs for in vitro studies were dissolved in DMSO toyield a 10 mmol/L or 1 mmol/L stock solution and stored at�80�C.

Immunoblot analysis and Ras-GTP assayCellular lysis was performed using Cell Lysis Buffer (Cell

Signaling Technology) following the manufacturer's protocol.Lysates were quantified by BCA assay (Pierce) and equal amountsof lysates were resolved by SDS-PAGE, transferred to a nitrocel-lulose membrane, and proteins probed with antibodies againstthe protein of interest. Antibodies against FLT3 were obtainedfrom Santa Cruz Biotechnology. All other antibodies wereobtained from Cell Signaling Technology. After incubation withhorseradish peroxidase–conjugated secondary antibodies, pro-teins were detected using a Chemidoc Touch Imaging System(Bio-Rad). GTP-bound Ras was measured using the Active RasDetection Kit (Cell Signaling Technology) following the manu-facturer's instructions. Briefly, GTP-bound Raswas captured usinga GST-tagged Raf1-Ras-binding domain peptide, followed byimmunoblot for total Ras.

Flow cytometry analysisFlow cytometry analysis was performed using a BD FACS-

Calibur machine (BD Biosciences). Apoptosis was measured byincubating treated cells with Annexin V-APC antibody for 10min-utes according to themanufacturer's instruction (BDBiosciences).Tomeasure leukemia burden, peripheral blood and bonemarrowwere isolated and, following red blood cell lysis, stainedwith anti-mouse CD45 and anti-humanCD45 for 30minutes. All datawereanalyzed by FlowJo analysis software (Tree Star).

Proliferation analysisCells were plated in 96-well plates at a density of 3,000 to 4,000

cells per well. Cells were analyzed 96 hours after drug treatmentusing Cell Proliferation Kit I (MTT, Roche) or Cell Counting Kit-8(Sigma-Aldrich) following the manufacturer's instructions, andplates were read using an absorbance spectrophotometer.

Animal studiesEight- to 10-week-old NOD/SCID g (NSG) female mice were

obtained from the Johns Hopkins Animal Core Facility. Experi-ments were carried out under an Institutional Animal Care andUse Committee–approved protocol, and institutional guide-lines for the proper and humane use of animals in research werefollowed. For transplant studies, 0.5 million Molm14 cells wereinjected into each mouse via tail vein injection. Treatment wasinitiated 3 days after injection. Mice were randomized to receiveDMSO, 8 mg/kg sorafenib, 5 mg/kg PD0325901, or the com-bination once daily for 3 weeks. All drugs were formulated incorn oil (Sigma-Aldrich) and administered by oral gavage. Micewere sacrificed by CO2 euthanasia.

Human samplesHuman AML and plasma samples were collected under a Johns

Hopkins Hospital Institutional Review Board–approved protocolwith patient informed consent in accordancewith theDeclarationof Helsinki. Mononuclear cells were isolated by Ficoll centrifu-gation and cryopreserved in liquid nitrogen until use. Cells werecultured in RPMI 1640 supplemented with 10% FBS for drugtreatments and lysed and analyzed via immunoblot, as described.Plasma was isolated following Ficoll centrifugation and frozen at�20�C until use. Plasma samples obtained during sorafenibtherapy were collected on study NCT01578109 and analyzed perpreviously described methods (16).

Statistical analysisStatistical analysis was performed with Student t test using the

GraphPad Prism software analysis program. Combination index(CI) values were calculated using the Chou–Talalaymethod (17).All data are presented as the mean � SD.

ResultsFLT3 inhibition in FLT3/ITD AML cell is associated withreactivation of ERK signaling

In studying the efficacy of FLT3 inhibitors, we and others havegenerally analyzed short-term drug exposures (in the range of 30minutes–2 hours) via immunoblot to assess maximal target anddownstream inhibition, either via addition of drug to in vitroculture (2, 18) or direct application of patient plasma via theplasma inhibitory activity (PIA) assay (19–21). We sought toinvestigate whether signaling downstream of FLT3 is effectivelyinhibited over a longer treatment course in the context ofFLT3-selective TKI treatment. We treated two FLT3/ITD AML celllines, Molm14 andMV4;11, with five different inhibitors of FLT3(18, 20, 22–24) for up to 24 hours at concentrations effectivefor >95% FLT3 and downstream target inhibition (25 nmol/Lsorafenib, 10 nmol/L quizartinib, 1 nmol/L TTT-3002,25 nmol/L lestaurtinib, and 25 nmol/L crenolanib). Despiteinhibition following 1 hour of treatment, rebound of ERK phos-phorylation (pERK) was observed after 24 hours, whereas nosuch rebound was observed in the phosphorylation of down-stream targets AKT or STAT5 (Fig. 1A and B). The same phenom-enon was also observed in murine 32D cells stably transfectedwith FLT3/ITD (Supplementary Fig. S1). The rebound in pERKbegan as early as 16 hours after treatment and was accompaniedby a time-dependent increase in phosphorylation of MEK andp90RSK. An increase in levels of active RAS was also prominentlyobserved in the MV4;11 cell line, suggesting a reactivation ofthe ERK signaling cascade (Fig. 1C and D). Aminimal rebound inAKT phosphorylation was observed in the Molm14 cell linestarting 8 hours after treatment, but was reduced by 24 hours.An increase in total FLT3 protein and, in some cases, FLT3phosphorylation was also observed, consistent with previousreports that demonstrate that FLT3 inhibition significantlydecreases the rate of its proteasomal degradation (25).

To investigate the possibility that the observed adaptiveresponse observedmay simply be a result of loss of drug exposure(due either to half-life or to drug efflux), cells were treated withsorafenib for 24 hours, followed either by no change, washoutand re-addition of drug, or replacement with culture mediumalone. Despite washout and re-addition of fresh drug, pERKremained at its 24-hour rebound level and continued to increase

ERK Signaling Is Reactivated following FLT3/ITD Inhibition

www.aacrjournals.org Cancer Res; 77(20) October 15, 2017 5555




over time, reaching pretreatment levels by 48 hours after initialtreatment (Fig. 2A). The rebound in pERKwas dose dependent, asthe degree of reboundwas reducedwith increasing concentrationsof sorafenib, crenolanib, and TTT-3002 (Fig. 2B; SupplementaryFig. S2A andS2B). Thisfinding suggests a role for FLT3 signaling inthe observed rebound; quizartinib and lestaurtinib treatment,however, elicited a low, persistent level of pERK rebound, evenat high drug concentrations (Fig. 2C; Supplementary Fig. S2C).Evidence of persistent adaptive increase in pERK despite effectiveinhibition of FLT3 suggests at least a partial contribution fromsignaling via alternate receptors and highlights the possibility thatoff-target effects may explain the dose-dependent nature of pERKrebound.

Rebound was also dependent on serum components, as areduction in serum concentration resulted in sustained ERKinhibition, and reintroduction of serum following treatment inlow serum conditions restored pERK rebound (Fig. 2D and E). Asexpected, 48 hours of persistent low-serum conditions resulted inreduced cell viability and increased protein degradation. Reduc-ing serum concentration for 1 hour following 24-hour sorafenibtreatment resulted in a profound decrease in MEK phosphoryla-tion (pMEK) and pERK (Fig. 2F), supporting the presence of

critical signaling molecules in serum that support pERK rebound.Importantly, FLT3 signaling was sustained over 24 hours in low-serum conditions in the absence of drug (Supplementary Fig. S3).

Together, these data demonstrate that FLT3/ITD leukemia cellsexhibit a mechanism of intrinsic signaling adaptation in responseto FLT3 inhibitor treatment, resulting in a reactivation of ERKsignaling despite persistent inhibition of FLT3 and other effectorpathways.

MEK inhibition overcomes rebound in phospho-ERK andsensitizes cells to FLT3 inhibition

We next sought to investigate whether FLT3 inhibitor–medi-ated pERK rebound could be overcome with the addition of aMEK inhibitor. The addition of allosteric MEK inhibitorsPD0325901 (26) or trametinib (27) at submaximal concentra-tions significantly reduced the degree of pERK rebound 24 hoursfollowing sorafenib or crenolanib treatment in bothMolm14 andMV4;11 cells (Fig. 3A and B; Supplementary Fig. S4A–S4D). Thisreduction was accompanied by decreased levels of the ERK targetcMyc. Combination treatment also resulted in a slight increase inPARP cleavage, indicating increased apoptosis as compared withFLT3 inhibition alone. Interestingly, MEK inhibitor treatment

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ERK signaling is reactivated over time in response to FLT3 inhibition.A andB,FLT3/ITDAML cell linesMolm14 (A) andMV4;11 (B)were treatedwith the indicated FLT3inhibitor for the indicated times. C and D, Molm14 (C) and MV4;11 (D) cells were treated with 25 nmol/L sorafenib for the indicated times. Protein lysateswere analyzed by immunoblot. Whole-cell lysates (WCL) were subjected to immunoprecipitation assays with GST-bound RAF1 Ras-binding domain. Whole-celllysate and pull-down (PD) products were immunoblotted with a pan-Ras antibody to detect levels of total and GTP-bound RAS, respectively.

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Cancer Res; 77(20) October 15, 2017 Cancer Research5556




alone resulted in an increase in pMEK and concurrent rebound inpERK in these cells, likely due to relief of negative feedback, as hasbeen described in the context of other RTK-driven cancers (28).

To evaluate whether MEK inhibition enhances the sensitivityof FLT3/ITD cells to FLT3 inhibition, cells were treated withvarious combinations for 48 or 96 hours and analyzed forapoptosis and cell proliferation. Although FLT3/ITD cells wereinsensitive to MEK inhibition alone, the simultaneous additionof even very low concentrations of PD0325901 sensitized cellsto sorafenib treatment, synergistically increasing apoptosis(Fig. 3C and D; Supplementary Fig. S5A–D) and reducing cellproliferation (Fig. 3E and F). The clinically approved MEKinhibitor trametinib was also evaluated in combination withsorafenib in FLT3/ITD cells. As with PD0325901, a low con-centration of trametinib sensitized cells to sorafenib treatment(Supplementary Fig. S5E–S5H).

One hypothesis for the efficacy of such a combination is thatsorafenib treatment together with MEK inhibition is a generallysynergistic combination in cells with high ERK signaling, irre-spective of FLT3 status. We, therefore, tested the effects of thesorafenib/MEK inhibitor combination in the FLT3-negative AMLcell line HL60, which harbors the NRAS-activating mutationQ61L. These cells showed intermediate sensitivity to MEK inhi-bition yet complete insensitivity to sorafenib treatment (Supple-mentary Fig. S6A and S6B). Notably, the addition of sorafenib toPD0325901 treatment failed to affect the extent of apoptosis or

proliferation of these cells. Additionally, K562 cells, which harborwild-type FLT3 and RAS, were insensitive to sorafenib,PD0325901, and the combination (Supplementary Fig. S6C).

These data support the notion that MEK inhibition, even atsubmaximal concentrations, is effective in overcoming the intrin-sic signaling adaptation in FLT3/ITD leukemia cells, therebysensitizing these cells to FLT3 inhibition and enhancing thebiological response to drug treatment.

The addition of a MEK inhibitor to sorafenib treatment reducesleukemia burden in vivo

MEK inhibition has been previously proposed as a means toovercome bone marrow stromal cell–mediated activation ofERK signaling in FLT3/ITD leukemia (10). This work providesevidence suggesting that MEK inhibition may increase efficacyof FLT3 inhibitors in vivo via a distinct mechanism. To evaluatethe efficacy of such a combination treatment, NSG mice wereengrafted with Molm14 cells and treated with 8 mg/kg sorafe-nib, 5 mg/kg PD0325901, or the combination once daily for 3weeks. Notably, the dose of PD0325901 used in these experi-ments is well below the reported maximum tolerated dose inmurine models (26). Compared with either treatment alone,the combination of sorafenib and PD0325901 significantlyreduced the leukemia cells in both the peripheral blood(Fig. 4A) and the protective environment of the bone marrow(Fig. 4B). These data provide further evidence for the in vivo

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Characterization of FLT3 inhibitor–mediated signaling adaptation. A, Molm14 cells were treated with 25 nmol/L sorafenib for 24 hours, followed by no change,re-addition of drug, or replacement with culture medium alone for an additional 1 or 24 hours. B and C,Molm14 cells were treated with the indicated concentrationsof sorafenib (B) or quizartinib (C) for up to 24 hours. D, Molm14 cells were treated with 25 nmol/L sorafenib cultured in medium with the indicatedconcentrations of FBS for up to 24 hours. E,Molm14 cells were initially treated with 25 nmol/L sorafenib for up to 24 hours and cultured in 1% FBS. After 24 hours oftreatment, FBS concentration was either increased to 10% or left at 1% for an additional 1 and 24 hours, as indicated. F, Molm14 cells were treated for up to24 hours in 10% FBS. After 24 hours of treatment, FBS concentration was decreased to 5%, 1%, or 0% for an additional hour as indicated. Protein lysateswere analyzed by immunoblot.






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MEK inhibitor treatment abrogates phospho-ERK rebound and sensitizes FLT3/ITD cells to FLT3 inhibitor treatment. A and B, Molm14 (A) and MV4;11 (B) cellswere treated with 25 nmol/L sorafenib, 5 nmol/L PD0325901, or both for the indicated times. Protein lysates were analyzed by immunoblot. Densitometricquantification of phospho-ERK relative to total ERK is shown.C andD,Molm14 (C) andMV4;11 (D) cells were treated for 48 hourswith 0 to 80 nmol/L sorafenib and 0to 80 nmol/L PD0325901 in combinations. Annexin V–positive (AnnVþ) cells were measured by flow cytometry as a readout of apoptosis. E and F,Molm14 (E) andMV4;11 (F) cellswere treated for 96 hourswith 0 to 80nmol/L sorafenib and0, 2.5, 5, or 10 nmol/L PD0325901, as indicated. Proliferationwasmeasured byMTT assay.Bar graphs, sorafenib IC50 as calculated using GraphPad Prism analysis software. Data are presented as the mean � SD. OD, optical density.

Bruner et al.





efficacy of combining a low dose of a MEK inhibitor with anFLT3 inhibitor in an FLT3/ITD leukemia model.

TKI-mediated signaling adaptation occurs in primary blastsand is elicited by human plasma

It is well recognized that several aspects of standard in vitroculture conditions do not purely reflect the kinetics and pharma-codynamics of drug targeting when dosing patients with small-molecule inhibitors of kinase signaling. This may be particularlyrelevant to our work given the dose-dependent nature of pERKrebound we observed in cultured FLT3/ITD leukemia cells (Fig.2B). We, therefore, sought to determine whether rebound in ERKsignaling following FLT3 inhibition occurs in patients as well. Wefirst examined whether primary leukemic cells from patients withFLT3/ITD AML (Supplementary Table S1) also exhibit the samephenomenon observed in FLT3/ITD cell lines. After 24 hours ofsorafenib treatment, the majority of primary blasts revealed a

similar rebound in pERK despite sustained inhibition of phos-pho-STAT5 (Fig. 5A). As observed with the cell lines, the additionof PD0325901 to sorafenib treatment resulted in sustained ERKinhibition after 24 hours despite profound pERK rebound whentreated with either drug alone (Fig. 5B).

Serum concentrations of orally dosed drugs are dependent onmultiple variables including absorption, metabolism, and pro-tein binding. Clinical trials have revealed extremely high inter-patient variability associated with sorafenib treatment, with max-imum concentrations ranging from 0.89 to 26.2 mmol/L (29, 30).Therefore, we next sought to determine whether signaling adap-tation observed in culture conditions of 10% FBS would berecapitulated by the concentrations of sorafenib present inhumanplasma frompatients being treatedwith this FLT3TKI. To replicatethese conditions, we cultured Molm14 cells in plasma samplesobtained from patients treated with sorafenib for a prolongedperiod (Supplementary Table S2). Using a variant of the PIA assay

Figure 4.

MEK inhibition improves efficacy ofFLT3 inhibition in vivo. A and B, 0.5million Molm14 cells were injected viatail vein injection into NSG mice. Micewere treated with 8 mg/kg sorafenib,5 mg/kg PD0325901, or both via oralgavage once daily for 3 weeks. Micewere sacrificed and peripheral blood(A) and bone marrow (B) cells wereanalyzed for human CD45 (hCD45)–positive (hCD45þ) leukemia cells byflow cytometry. n � 4 for each group.Error bars, SD.

Figure 5.

Phospho-ERK rebound is observed inpatient blasts and elicited by humanplasma. A, AML blasts from fourpatients (Supplementary Table S1)were treatedwith 25 nmol/L sorafenibin culture for the indicated times. B,AML blasts from one patient weretreated in culture with 25 nmol/Lsorafenib, 5 nmol/L PD0325901, orboth for the indicated times.Densitometric quantification ofphospho-ERK relative to total ERK isshown. C, Molm14 cells were culturedwith either healthy donor plasma for 1hour (ctrl) or plasma derived frompatients on active sorafenib treatment(Supplementary Table S2) for theindicated times. Protein lysateswere analyzed by immunoblot.






(19), Molm14 cells were treated in either normal human plasmafor 1 hour or plasma from patients treated with sorafenib foreither 1 or 24 hours. In all cases of cells exposed to plasma fromthese patients, pERK rebound was observed by 24 hours aftertreatment (Fig. 5C). These data support the notion that FLT3inhibitor–mediated signaling adaptation is a phenomenon thatoccurs in patients, is elicited at clinically relevant concentrationsof sorafenib in human plasma, and can be abrogated with theaddition of a MEK inhibitor.

TKI-mediated pERK rebound is observed in other tyrosinekinase–driven cancers

Finally, we sought to determine whether TKI-mediatedrebound in ERK activity was restricted to FLT3-mutant AML orwhether it also occurs in the context of other tyrosine kinase–driven cancers, such as those driven by EGFR mutation or HER2amplification. We treated the EGFR-mutant lung cancer cell linePC-9 with the EGFR/HER2 inhibitor lapatinib at concentrationssufficient for >95% EGFR inhibition. As was seen for FLT3/ITD–

driven AML, pERK rebound was observed following 24 hours ofTKI treatment despite persistent inhibition of the receptor anddownstream target AKT (Fig. 6A). Again, the rebound could beovercome with the addition of a submaximal concentration ofPD0325901 (Fig. 6B). A similar trendwas observed for the HER2-amplified breast cancer cell line SKBR3 (Fig. 6C and D) and theBCR–ABL cell line K562 (Fig. 6E and F). As with the FLT3/ITDcells, MEK inhibition increased sensitivity of K562 cells to ima-tinib treatment (Supplementary Fig. S7A). Unlike what wasobserved for FLT3/ITD cells, however, the addition of PD0325901failed tomarkedly increase sensitivity to TKI treatment in PC9 andSKBR3 cells (Supplementary Fig. S7B and S7C), consistent with alowdependency onERKoutput in these cell types (28). These datademonstrate that TKI treatment is associated with signaling adap-tation in a variety of tyrosine kinase–drivenmalignancies, includ-ing leukemia, lung cancer, and breast cancer.

DiscussionThe FLT3/ITD is an oncogenic driver in AML, and although

selective inhibitors of FLT3 thatmaximally and specifically inhibitthe target have now been studied (18, 20), clinical responsesremain limited (5–7). Several factors that may contribute to thislimited efficacy have been postulated and include both cellintrinsic and extrinsic processes, including acquisition of resis-tance mutations (2, 8), upregulation of FLT3 ligand (9), andstromal cell–mediated protection in the bone marrow microen-vironment (10). We sought to explore, however, whether thebiochemical response to FLT3/ITD inhibition might involvechanges in signaling networks that also contribute to their limitedefficacy.

Previous work has characterized either the short-term signalingresponse (<2 hours) or the long-term generation of resistance (>1month) following FLT3 inhibition in FLT3/ITD AML (8, 31).Here, we explored the signaling response to FLT3 inhibitionwithin 48 hours of treatment, finding reactivation of ERK signal-ing beginning as early as 16 hours after treatment despite persis-tent target inhibition. This effect was observed in standard cultureconditions as well as a modified PIA assay (19). The difference intiming of interrogation of the signaling pathways likely explainswhy this adaptive rebound was not previously observed. Thiswork reveals a limitation of the PIA assay in its current form and

highlights the need to analyze signaling over a greater treatmentduration to fully understand the biological response.

The concept of signaling adaptation in response to small-molecule inhibitors of tyrosine kinases has been recentlydescribed in the context of other targeted therapies (11–14). Inseveral cases, the adaptive changes in signaling suggest reactiva-tion of other nodes in critical mitogenic pathways that are,therefore, targets for combined therapies. In line with thesefindings, this report reveals a phenomenon by which FLT3/ITDAML selectively reactivates ERK signalingwithinhours in responseto targeted inhibition. These data contribute to the growingunderstanding of short-term signaling adaptation to oncoproteininhibitors, which is accompanied by attenuation of the antitumoreffects of these compounds (11–14). The reactivation of ERKsignaling described here may represent an extension of the pre-viously described phenomenon whereby attenuation of ERKfeedback inhibition allows for ligand-dependent signaling viawild-type receptors present on the cell surface (Fig. 7A–C). Thisidea is supported by the dependence of pERK rebound on asufficient concentration of serum, suggesting that signalingthrough receptors to mitogenic pathways is an essential compo-nent of the adaptation (Fig. 2B), though further work is needed tofully characterize the mechanism of ERK reactivation and todetermine what signaling molecule(s) may be responsible. How-ever, regardless of which receptors, if any, are involved in theenhanced signaling as an adaptive response, the measurement ofelevated levels of pERK suggest that addition of a MEK inhibitor,rather than specific inhibitors of cell surface receptors, is analternative approach for combination therapy that will likelyovercomemultiplemechanismsof adaptation through inhibitionof a shared terminal event.

RTK-driven cancers are, for the most part, insensitive to MEKinhibitors (28, 32), and clinical trials of single-agent MEKinhibitors in AML have shown little efficacy, with FLT3/ITDpatients achieving no responses (33). Combining FLT3 andMEK inhibitors as a treatment strategy for patients with FLT3/ITD AML has been proposed by others as a means to overcomebone marrow stromal cell–mediated resistance to FLT3 inhi-bition (10). Indeed, a dual FLT3/MEK inhibitor was recentlydescribed to be effective in preclinical models of FLT3/ITDAML, and a phase I clinical trial is ongoing (34). In this study,we have determined that MEK inhibition is an effective meansto overcome FLT3 inhibitor–mediated pERK rebound, and thatMEK inhibitor treatment sensitizes FLT3/ITD cells to FLT3inhibition, even in the absence of stromal cells. These datasupport the combination of MEK and FLT3 inhibitor treatmentas a means to improve treatment for patients with FLT3/ITDAML, and further suggest that a low-dose MEK inhibitor may besufficient for improved outcomes. This is supported by the lowconcentrations necessary to observe synergistic effects in vitro(Fig. 3) and by the reduced leukemia burden observed in vivousing a dose of PD0325901 well below the maximal tolerateddose previously reported for this compound in mice (Fig. 4;ref. 26). The advantage of this approach includes avoidance ofthe toxicities associated with full-dose MEK inhibitor whilemaintaining sufficient activity to allow for enhanced responseto receptor tyrosine kinase inhibition. Furthermore, this com-binatorial strategy could be readily applied to the clinic giventhe availability of inhibitors of FLT3 and MEK, includingclinically approved compounds such as sorafenib and trame-tinib. Indeed, a phase I clinical trial exploring sorafenib and

Bruner et al.





A B

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+- - - + + ++- + + - - +


Imatinib (750 nmol/L)

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Figure 6.

Phospho-ERK rebound is observed in other tyrosine kinase–driven cancers following exposure to TKIs. A, PC-9 (EGFR Del E746-A750) cells were treatedwith 500 nmol/L lapatinib for the indicated times. B, PC-9 cells were treated with 500 nmol/L lapatinib, 5 nmol/L PD0325901, or both drugs for theindicated times.C, SKBR3 (HER2 amplified) cells were treated with 75 nmol/L lapatinib for the indicated times.D, SKBR3 cells were treated with 75 nmol/L lapatinib,5 nmol/L PD0325901, or both drugs for the indicated times. E, K562 (BCR–ABL) cells were treated with 750 nmol/L imatinib for the indicated times. F, K562cells were treated with 750 nmol/L imatinib, 5 nmol/L PD0325901, or both drugs for the indicated times. Protein lysates were analyzed by immunoblot.Densitometric quantification of phospho-ERK relative to total ERK is shown.






trametinib in combination for the treatment of advancedhepatocellular cancer is ongoing (https://clinicaltrials.gov/show/NCT02292173).

We also explored the possibility that this phenomenonmay begeneralizable beyond FLT3/ITD AML. We demonstrate that asimilar phenomenon occurs in models of BCR–ABL leukemia,EGFR-driven lung cancer, and HER2-amplified breast cancer inresponse to TKI treatment. As in FLT3/ITD AML, pERK reboundcan be overcome with the addition of a MEK inhibitor (Fig. 5).Although further work is needed to validate the feasibility of sucha combination, these data suggest that the use of a MEK inhibitormay enhance the response of tyrosine kinase–driven cancers toTKI therapy, thereby improving outcomes for patients with can-cers carrying these genetic alterations.

Disclosure of Potential Conflicts of InterestC.A. Pratilas is a consultant/advisory board member for Genentech. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: J.K. Bruner, H.S. Ma, M.J. Levis, C.A. Pratilas, D. SmallDevelopment of methodology: J.K. Bruner, H.S. Ma, L. Li, M.A. Rudek,R.J. Jones, D. Small

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.K. Bruner, A.C.R. Qin, M.A. Rudek, R.J. Jones,M.J. Levis, K.W. Pratz, D. SmallAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.K. Bruner, H.S. Ma, M.A. Rudek, R.J. Jones,M.J. Levis, K.W. Pratz, D. SmallWriting, review, and/or revision of the manuscript: J.K. Bruner, H.S. Ma,M.A. Rudek, R.J. Jones, K.W. Pratz, C.A. Pratilas, D. SmallAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.C.R. Qin,Study supervision: C.A. Pratilas

AcknowledgmentsThe authors thank the patients and donors who contributed peripheral

blood and plasma specimens for these studies.

Grant SupportThisworkwas supported byNIHResearch Grant R01CA090668 (toD. Small),

NIH Project Grant P30CA006973, and the Giant Food Pediatric Cancer ResearchFund. D. Small is also supported by the Kyle Haydock Professorship.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 26, 2016; revised May 18, 2017; accepted August 16,2017; published OnlineFirst September 18, 2017.

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PP

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PP

PP

Alternatereceptor


Alternatereceptor


Alternatereceptor

PP

Raf

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2017;77:5554-5563. Published OnlineFirst September 18, 2017.Cancer Res J. Kyle Bruner, Hayley S. Ma, Li Li, et al.

Driven Leukemias and Other Malignancies−KinaseAdaptation to TKI Treatment Reactivates ERK Signaling in Tyrosine

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