Cancer Therapy: Preclinical

Sorafenib Inhibition of Mcl-1 AcceleratesATRA-Induced Apoptosis in Differentiation-Responsive AML CellsRui Wang, Lijuan Xia, Janice Gabrilove, Samuel Waxman, and Yongkui Jing

Abstract

Purpose: All trans-retinoic acid (ATRA) is successful in treatingacute promyelocytic leukemia (APL) by inducing terminal differ-entiation-mediated cell death, but it has limited activity in non-APL acute myeloid leukemia (AML). We aim to improve ATRAtherapy of AML by enhancing apoptosis through repression of theantiapoptotic proteins Bcl-2 and Mcl-1.

Experimental Design: APL and AML cell lines, as well asprimary AML samples, were used to explore the mechanismsregulating differentiation and apoptosis during ATRA treatment.Stable transfection and gene silencing with siRNA were used toidentify the key factors that inhibit apoptosis during induction ofdifferentiation and drugs that accelerate apoptosis.

Results: In differentiation-responsive AML cells, ATRA treat-ment induces long-lasting repression of Bcl-2 while first upmo-dulating and then reducing the Mcl-1 level. The Mcl-1 level

appears to serve as a gatekeeper between differentiation andapoptosis. During differentiation induction, activation of MEK/ERK and PI3K/Akt pathways by ATRA leads to activation ofp90RSKand inactivationof glycogen synthase kinase 3b (GSK3b),which increase Mcl-1 levels by increasing its translation andstability. Sorafenib blocks ATRA-inducedMcl-1 increase by revers-ing p90RSK activation and GSK3b inactivation, maintains therepressed Bcl-2 level, and enhances ATRA induced apoptosis innon-APL AML cell lines and in primary AML cells.

Conclusions: Inhibition of Mcl-1 is required for apoptosisinduction in ATRA differentiation-responsive AML cells. ATRAand sorafenib can be developed as a novel drug combinationtherapy for AML patients because this drug combinationaugments apoptosis by inhibiting Bcl-2 and Mcl-1. Clin Cancer Res;22(5); 1211–21. �2015 AACR.

IntroductionAll trans-retinoic acid (ATRA) together with chemotherapy

used as the first-line treatment for the newly diagnosed acutepromyelocytic leukemia (APL) patients achieves approximately75% cures (1). However, all APL patients treated with ATRA alonerelapse, most likely because of the cells that escape terminaldifferentiation (2, 3) and also escape death. Although ATRA caninduce differentiation in most non-APL AML cell lines and pri-mary AML cells in culture, its clinical activity in non-APL AMLpatients is very limited (4). Past efforts have focused on the searchof agents that can enhance ATRA-induced differentiation (4, 5).These have been proven to be unsuccessful in clinical testing (6).

Themainpathway throughwhich chemotherapy kills leukemiacells is induction of apoptosis. Apoptosis is mediated throughintrinsic (mitochondrial) and extrinsic (death receptor) signalingpathways. The extrinsic pathway affected by TRAIL and deathreceptor 5 (DR5) is thought to be responsible for apoptosis of

terminally differentiated APL cells after ATRA treatment (3). Themitochondrial apoptotic pathway is controlled by Bcl-2 family ofantiapoptotic proteins. ATRA treatment was shown to have var-iable effects on the transcription of the Bcl-2 family of antiapop-totic proteins; it decreases Bcl-2, increases Bfl-1, while showing noeffect on the Mcl-1 mRNA level (7, 8). However, we and othershave shown that Mcl-1 protein is upregulated by ATRA and thatthis regulation is time dependent (9, 10). Mcl-1 is a key prosur-vival protein that protectsmature neutrophils fromcell death (11)and its ATRA-induced increase might save AMLs from apoptosis.We propose that a rapid AML cell death can be induced byblocking Mcl-1 in cells in which Bcl-2 is repressed by ATRA.Mcl-1 has a short half-life which is regulated through translationand protein degradation (12). We explored the mechanism ofMcl-1 upregulation by ATRA in APL cells and found that ATRAincreasedMcl-1 levels at translational and posttranslational levelsassociated with p90RSK activation and GSK3b inactivation. Wealso studied the role of Mcl-1 in differentiation and apoptosisusing cells in which Mcl-1 was either forced expressed or silencedand found that Mcl-1 plays a gate keeper role that protects cellsfrom apoptosis. We then looked for an agent that, by reducingMcl-1, will eliminate the block to apoptosis. We found thatsorafenib, a multikinase inhibitor, counteracts ATRA-inducedMcl-1 increase and augments apoptosis induction in non-APLAML cell lines and in primary cells.

Materials and MethodsReagents

ATRA, rapamycin, LY290024, SB216763, and cycloheximidewere purchased from Sigma Chemical Co.. U0126, PD184352,

The Division of Hematology/Oncology, Department of Medicine, TheTisch Cancer Institute, Icahn School of Medicine at Mount Sinai, NewYork, New York.

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

Corresponding Author: Yongkui Jing, Department of Medicine, Icahn School ofMedicine at Mount Sinai, One Gustave L. Levy Place, Box 1178, New York, NY10029. Phone: 212-241-6775; Fax: 212-996-5787; E-mail:yongkui.jing@mssm.edu

doi: 10.1158/1078-0432.CCR-15-0663

�2015 American Association for Cancer Research.

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and sorafenib were obtained from LC Laboratories (Woburn).Antibody toMcl-1, ERK1, Bcl-2,b-actin, andUSP9Xwere purchasedfrom Santa Cruz Biotechnology, Inc.; to PARP was from BD Bio-sciences; toDR5was fromProSci, Inc.; to p-MEK (Ser217/221), p-ERK(Thr202/Tyr204), Akt, p-Akt (Ser473), p-mTOR, p70S6K, p-p70S6K(Thr389), p-p70S6K (Thr421/Ser424), p-p90RSK (Ser380), p90RSK,p-rpS6 (Ser235/236), rpS6, GSK-3b, p-GSK-3b (Ser9), caspase-8, -9, -3and Trail were from Cell Signaling Technology, Inc.

Cell lines and treatments.APL-derivedNB4 and its ATRA resistanceclone R4 were obtained from Dr. Willson Miller Jr (McGillUniversity, Montreal, QC, Canada) (13), AML-derivedHL-60 andTHP-1 were obtained from ATCC, and ME-1 cells were obtainedfrom Dr. Shigeru Fujita (School of Medicine, Ehime University,Japan) (14). MOLM13 cells were obtained from DSMZ. All celllines were not authenticated by us once received in our laboratoryandwere cultured in RPMI1640medium supplemented with 100U/mL penicillin, 100 mg/mL streptomycin, 1 mmol/L L-gluta-mine, and 10% (v/v) heat-inactivated FBS (FBS). HL-60/Bcl2 cellstransfected with Bcl-2 were obtained from Dr. Michael Cleary(StanfordUniversity School ofMedicine, Stanford, CA) (15).Mcl-1–expressing HL-60/M15 cells were generated in our laboratory(16). These cells were seeded at 105/mL and treated with ATRAalone or in combination with other agents for 2 days. For the timecourse studies, the cell number was adjusted and new mediumwith ATRA was changed every 2 days.

Detection of apoptosis, differentiation, protein levels, and silencingof Mcl-1. Apoptotic cells were determined by the Annexin Vassay, according to the manufacturer's instructions [Annexin V–FITC Apoptosis Detection Kit (BD Biosciences)]. The nitrobluetetrazolium (NBT) reduction assay and CD11b expression wereused to evaluate differentiation using FACS. Western blotanalysis was used to measure protein levels. To silence Mcl-1we used siRNA. All these methods were done as we previouslyreported (8, 17).

Isolation of patient-derived leukemic blasts. Leukemic blastswere obtained from a bone marrow and three peripheralblood samples of AML patients with FAB subtypes of M2and M4. These studies have been sanctioned by the Investi-gation Review Board of Mount Sinai School of Medicine(New York, NY) and all patients provided informed consent.Whole blood or bone marrow collected in heparinized tubeswere subjected to Ficoll-Hypaque density gradient separation,the blasts were washed and resuspended in RPMI1640 with20% FBS at 1 � 107 cells/mL.

Animal survival study. Twenty NOD/SCI/IL-2Rg (NSG)mice (age,8 weeks) were injected with 5� 106 log-phase MOLM13 cells viathe lateral tail vein. Five days after leukemic cell injection, themicewere divided randomly into 4 groups. A Cremophor EL/ethanol/water solution (12.5%Cremophor EL/12.5%ethanol/75%water)containing sorafenib was prepared and 20 mg/kg was adminis-tered by oral gavage. ATRA was prepared in DMSO and dilutedwith PBS to containing 10% DMSO and 10 mg/kg was admin-istered intraperitoneally. Both drugs were given alone or as acombination daily for five continuous days a week until themouse died. Control mice received the vehicle through the sameroute and on the same schedule. Survival times were comparedwith Kaplan–Meier survival analysis.

Statistical analysisData were analyzed for statistical significance using the Student

t test (Microsoft Excel, Microsoft Corp.). A P value of <0.05 wasconsidered statistically significant.

ResultsMcl-1 protein is increased while Bcl-2 protein is decreased inATRA differentiation-responsive NB4 cells

Treatment of NB4 cells with 1 mmol/L ATRA for 2, 4, and 6 daysproduced on the respective days approximately 26%, 57%, and38% of differentiated cells (Fig. 1A) and approximately 8%, 15%,and 40% of apoptotic cells (Fig. 1B). The parallel reduction in thepercent of differentiated cells, accompanied by the rise in apo-ptotic cells, suggests that terminally differentiated cells eventuallyundergo apoptosis. Both the extrinsic and the intrinsic apoptoticpathways were examined by measuring the cleavage of caspase-8and caspase-9 during the 2- to 6-day treatment (Fig. 1C). Thecleaved fragments of caspase-3, -8, and -9 were not detected onday 2. PARP cleavage, detected on day 4, was associated with theextrinsic pathway caspase-8 cleavage but only minimally withcaspase-9 cleavage. Consistent with the previous report, we foundthat the levels of both TRAIL and DR5 were induced by ATRA onday 2 and 4 (3). Increased PARP cleavage associated with cleavedfragments of caspase-3, -8, and -9was detected in cells treatedwithATRA for 6 days (Fig. 1C), indicating activation of both apoptoticpathways after prolonged treatment.

We also tested the levels of Bcl-2 andMcl-1 protein in NB4 cellstreated with 1 mmol/L ATRA for 2, 4, and 6 days. The Bcl-2 proteinlevel was decreased after 2 days of ATRA treatment and remainedrepressed throughout the treatment (Fig. 1C). The level of Mcl-1was increased on day 2 and gradually declined during day 4 and 6,at the time when the proportion of the differentiated cellsdeclined and of the apoptotic cells rose. These data suggest thatincreased Mcl-1 protein protects differentiated cells from apopto-sis and that Mcl-1 protein needs to be reduced for maximal

Translational Relevance

Cytotoxic chemotherapy induces acute myeloid leukemia(AML) cell death by apoptosis through mitochondrial path-way which is blocked in part by Bcl-2 andMcl-1, the twomainantiapoptotic proteins. All trans-retinoic acid (ATRA) inducesterminal differentiation-mediated cell death causing completeremission in patients with acute promyelocytic leukemia(APL), but only limited effect in other AML subtypes. In APLcells, Bcl-2 is repressed while Mcl-1 is induced during ATRAinduced differentiation process. Inhibition of Mcl-1 acceler-ates apoptosis of differentiated cells. Increase in the ATRA-induced Mcl-1 protein is due to increased translation andstability associated with RSK activation and GSK3b inactiva-tion. Sorafenib, a multikinase inhibitor, currently in clinicaltrials in AML patients, counteracts Mcl-1 induction by ATRA,together with ATRA augments apoptosis in cell culture, andin vivo extends the survival time of mice xenografted withhuman AML cells. This work provides a rationale for utilizingATRA and sorafenib as a combination therapy for AMLpatients by repressing both Bcl-2 and Mcl-1 proteins andaugmenting apoptosis.

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apoptosis to occur. To test the role of Mcl-1 protein in ATRA-induced NB4 differentiation process, Mcl-1 was knocked downusing siRNA (Fig. 1D). Although silencing of Mcl-1 decreased theability of ATRA to induce differentiation at day 2 (Fig. 1E) itstrongly enhanced ATRA-induced apoptosis as determined byPARP cleavage (Fig. 1D) and Annexin V staining (Fig. 1F). Thesedata suggest that Mcl-1 protein is required for induction ofdifferentiation byATRAand that it functions as amainprosurvivalprotein protecting differentiated cells from death.

Increase in Mcl-1 protein levels by ATRA is associated withp90RSK activation and GSK3b inactivation

Utilizing ATRA differentiation-sensitive NB4 and its resistantR4 subclone, the protein levels of Mcl-1 and Bcl-2 were deter-mined during ATRA-induced differentiation defined as theability to reduce NBT. After 4 days of treatment with 1mmol/L ATRA there were 52.7% NBT-positive cells in NB4 andonly 8.7% in R4 cells (Fig. 2A). Treatment with ATRA for 2 daysincreased the level of Mcl-1 protein but reduced Bcl-2 in NB4

cells (Fig. 2B). Previously, it has been shown that ATRArepresses Bcl-2 at both mRNA and protein levels (7). ATRAdid not increase Mcl-1 mRNA level in NB4 cells as measuredusing RT-PCR (Fig. 2B), suggesting a posttranscriptional regu-lation. MCL-1 is an unstable protein with a very short-halflife. To distinguish between ATRA effect on translation andstability of Mcl-1 protein, NB4 cells were pretreated with orwithout ATRA for 2 days and then treated with cycloheximidefor 1 to 4 hours. ATRA treatment increased the Mcl-1 half-life inpresence of cycloheximide from approximately 20 minutes to80 minutes (Fig. 2C and D), indicating that ATRA stabilizesMcl-1 protein. In contrast, ATRA effect on Bcl-2 was not influ-enced by cycloheximide treatment (Fig. 2C).

Raf/MEK/ERK and Akt/mTOR signaling pathways are activatedduring ATRA-induced differentiation process (18, 19) and ATRAtreatment of NB4 cells increases the levels of phosphorylatedp70S6K and rpS6 (20). Both Raf/MEK/ERK and Akt/mTOR path-ways phosphorylate p70S6K and then rpS6, increasing proteintranslation (21). ERK phosphorylates p70S6K at Thr421/Ser424

A

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Figure 1.ATRA-induced NB4 cell differentiation coincideswith increase inMcl-1 protein and reduction in Bcl-2 protein. NB4 cells were treatedwith 1 mmol/L ATRA for 2, 4, and6 days. Percent of differentiated cells was determined using CD11b expression (A) and percent of apoptotic cells using Annexin-V-FITC staining (B). C, lysatesof cells treated with ATRA for 2, 4, and 6 days were analyzed for apoptosis associated proteins by Western blotting. Mcl-1 was knocked down in NB4 cellsusing siRNA and the cells were treated with 1 mmol/L ATRA for 2 days. Silencing of Mcl-1 increased apoptosis as determined by enhanced PARP cleavage (D) andincreased Annexin-V-FITC staining (F), but decreased the percent of differentiated cells as determined by NBT assay (E). Con, Control.

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sites while mTOR phosphorylates it at Thr389 site (22). Theexistence of a link between rise in Mcl-1 and these factorswas tested in ATRA-treated NB4 cells. NB4 cells treated with10�8

–10�6 mol/L of ATRA had elevated levels of phosphorylatedMEK, ERK, Akt, mTOR, p70S6K, and rpS6 as well as an increasedlevel of Mcl-1 protein. Importantly, we found that at the lower(10�8mol/L) concentration of ATRA there was an increase in ERKpathway phosphorylated Thr421/Ser424 p-p70S6K level, while p-p70S6K (Thr389; the target of mTOR pathway) was only inducedby ATRA at concentrations higher that 10�7 mol/L (Fig. 2E). Theupregulation of p-p70S6K (Thr421/Ser424) and p-rpS6, correlatedwith an increase inMcl-1 level, most likely through an increase inMcl-1 protein translation.

The stability of Mcl-1 protein is regulated by phosphoryla-tion by ERK and GSK3b and by ubiquitination. GSK3bincreases Mcl-1 degradation while deubiquitinase USP9X sta-bilizes Mcl-1 (23, 24). GSK3b has been found to be inactivatedby phosphorylation in ATRA-differentiated NB4 cells (25, 26).GSK3b can be phosphorylated by AKT and p90RSK, the down-

stream target of ERK (27). We found that ATRA treatment ofNB4 cells increased the phosphorylation of p90RSK, GSK3b,and ERK, and that those are associated with the increased levelsof Mcl-1 (Fig. 2E). Because the level of USP9X was decreased byATRA treatment (Fig. 2E), we concluded that the ATRA inducedincrease in Mcl-1 stability proceeds through activation ofp90RSK, ERK, and inactivation of GSK3b.

To further identify the specific ATRA upregulated kinase path-ways which mediate the Mcl-1 induction, we treated cells withMEK inhibitor U0126, or PI3K inhibitor LY294002, or mTORinhibitor rapamycin. Inhibition of MEK and PI3K blocked theincrease in phosphorylated -p90RSK, -GSK3b, and -rpS6 aswell asthe level ofMcl-1 protein (Fig. 3A andB). Rapamycin (40nmol/L)decreased the level of phosphorylated p70S6K at Thr389 (Fig. 3C)but neither blocked ATRA-induced phosphorylation of p90RSK,GSK3b, and rpS6 nor Mcl-1 increase. These data suggest thatATRA-activated MEK/ERK and PI3K/Akt increase Mcl-1 proteinby both enhanced translation and increased stability involvingactivation of p90RSK and inactivation of GSK3b (Fig. 3D).

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Figure 2.Increase in Mcl-1 protein level after ATRA treatment coincideswith activation of both PI3K/Akt/GSK3b andMEK/ERK/p90RSK signalings in NB4 cells. NB4 cells andATRA-resistant R4 cells were treated with 1 mmol/L ATRA for 4 days and percentage of differentiated cells was determined by NBT reduction assay (A).The data shown are the mean of three independent experiments � SD. Lysates of ATRA-treated (2 days, 1 mmol/L) NB4 and ATRA-resistant R4 cells wereanalyzed by Western blotting for Mcl-1 and Bcl-2. b-actin was used as a loading control (B). NB4 cells were pretreated with or without ATRA for 2 days andthen incubated with cycloheximide (CHX, 10 mg/mL) for the indicated time. The protein levels of Mcl-1 and Bcl-2 were analyzed by Western blot (C). Proteinband intensity was analyzed using Image J software. Mcl-1 protein level at time 0 hour was set at 100% and the decline in protein level with time wasexpressed as percent of time zero (D). NB4 cells were treated for 2 days with increasing concentrations (10�8

–10�6 mol/L) of ATRA, lysed and analyzed foractivation of MEK/ERK pathway (E, left) and Akt/mTOR pathway (E, right) using specific antibodies to the phosphorylated proteins.

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Mcl-1 level is crucial for ATRA-induced differentiation andapoptosis in a FAB-M2-HL-60 cells

We tested the effect of 1 mmol/L of ATRA for 2, 4, and 6 days indifferentiation-responsive FAB-M2 HL-60 cells. We found thatsimilar to NB4 cells the percent of differentiated cells rose from31% on day 2 to 60% on day 4 (Fig. 4A) and, like in NB4, itdropped to 40% on day 6; this drop was accompanied by a sharprise (to�43%) in apoptotic cells (Fig. 4B). Moreover, like in NB4,the Mcl-1 protein levels increased on day 2 and dropped on day 6to the level in untreated cells (Fig. 4C). ATRA treatment of HL60cells increased the levels of phosphorylated p90RSK, phosphor-ylated (inactive) GSK3b, and phosphorylated rpS6 (Fig. 4C).Compared with NB4 cells (Fig. 1C), there was a difference in theintensity of Mcl-1 induction by ATRA in HL-60 cells; this differ-ence is correlated with the levels of p-GSK3b. The declining level

of the phosphorylated GSK3b day 6, indicating its increasedactivity, led to a reduction in Mcl-1 in both HL-60 and NB4 cells(Fig. 4C and data not shown). As in NB4 cells (Fig. 1), Bcl-2protein was repressed on day 2 and 4, but this was not accom-panied by substantial degree of apoptosis. Silencing ofMcl-1 withsiRNA (Fig. 4D) produced increased PARP cleavage (Fig. 4D), andAnnexin V staining (Fig. 4E), which rose to 48.8% in cells treatedwith ATRA for only 2 days. The fact that this profound apoptosis-inducing effect was not observed in absence of ATRA treatment(Fig. 4D and E), suggests that both, the repressed Bcl-2 and thesilenced Mcl-1, were crucial.

To conclusively test the role of Mcl-1 and Bcl-2 in differen-tiation and apoptosis, we used an Mcl-1–overexpressing cloneof HL-60 (HL60/M15 cells) and a clone with overexpressedBcl-2 (HL-60/Bcl2 cells) and, respectively, compared ATRA-

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Figure 3.The effects ofMEK inhibitor U0126, PI3K inhibitor LY294002andmTOR inhibitor rapamycin, onATRA-inducedMcl-1 increase inNB4 cells. NB4 cellswere treatedwith1.25 mmol/L MEK inhibitor, U0126 (A), 20 mmol/L PI3K inhibitor, LY294002 (B), and 40 nmol/L mTOR inhibitor, rapamycin (C). The treatment was for2 days with inhibitor alone or with 1 mmol/L ATRA. The protein levels of the indicated phosphorylated and unphosphorylated proteins were determined usingspecific antibodies in Western blot analysis. D, working model of ATRA-induction of Mcl-1. ATRA activates the MEK/ERK/p90RSK pathway which increasesMcl-1 translation and stability. ATRA also activates PI3K/Akt/GSK3b which increases Mcl-1 stability. p90RSK can directly inactivate GSK3b by phosphorylation andAkt can activate p90RSK by phosphorylation. Both pathways contribute to the Mcl-1 protein increase after ATRA treatment.

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induced differentiation and apoptosis to those in HL-60 clonestransfected with empty vector (HL-60/V5 and HL-60/neo)(Supplementary Fig. S1A and S1D). ATRA treatment of HL-60/M15 cells enhanced differentiation but blocked apoptosis(Supplementary Fig. S1B and S1C). Bcl-2 overexpression hadno effect on differentiation (Supplementary Fig. S1E), butprotected HL-60 cells from apoptotic cell death (Supplemen-tary Fig. S1F). These data show that although both Bcl-2 andMcl-1 block apoptosis, probably only Mcl-1 participates inATRA-induced differentiation.

Enhancement of ATRA-induced apoptosis by MEK inhibitors isassociated with blockade of Mcl-1 induction in FAB-M2 HL-60cells

MEK inhibitors can enhance ATRA-induced apoptosis in AMLcells but the mechanism has not been elucidated (28). Thecombined effects of ATRA with either of the two MEK inhibitors,

U0126 and PD184352, were tested in HL-60 cells. ATRA com-binedwith eitherU0126or PD184352 inducedmore than50%ofthe treated cells to undergo apoptosis (Supplementary Fig. S2A).The increased levels of p-ERK, p-p90RSK, p-rpS6, p-GSK3b,and Mcl-1 proteins by ATRA were blocked by either U0126 orPD184352 (Supplementary Fig. S2B). ATRA-mediated repressionof Bcl-2 protein was not reversed either by U0126 or byPD184352. At the concentrations used, these agents alone didnot induce apoptosis in HL-60 cells, even with repressed Bcl-2 orMcl-1, respectively (Supplementary Fig. S2B). These data suggestthat ATRA-induced Mcl-1 could be counteracted by inhibitingMEK/ERK signaling.

Sorafenib augments ATRA-induced apoptosis in AML cell linesand primary cells

Sorafenib, an inhibitor of B-raf, the upstream activator of theMEK/ERK pathway, was approved for clinical use (29) and it

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(Mcl

-1)

Figure 4.Mcl-1 plays an important role in ATRA-induced differentiation and apoptosis in HL-60 cells. HL-60 cells were treated with 1 mmol/L ATRA for the indicated timesand the percent of differentiated cells was determined by measuring CD11b expression (A) and of apoptotic cells using Annexin-V-FITC (B). HL-60 cells weretreated with 1 mmol/L ATRA for the indicated times and the levels of Mcl-1, Bcl-2, cleaved caspase-9, -8, -3, PARP and b-actin were determined usingspecific antibodies with Western blot (C). HL-60 cells transfected with Mcl-1 siRNA or control siRNA were treated with 1 mmol/L ATRA for 2 days. The proteinlevels of PARP, Mcl-1, Bcl-2, and b-actin were determined using specific antibodies with Western blot (D) and the percent of apoptotic cells was determinedusing FACS of cells stained with Annexin V-FITC (E).

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has been reported to inhibit Mcl-1 translation in leukemia cells(30, 31). We tested the combined effects of ATRA and sorafenibin FAB-M2 HL-60 cells, additional AML cell lines, FAB-M4ME-1cells and FAB-M5 THP-1 cells as well as primary AML cells fromfour AML patients. Like HL-60 cells (Fig. 4A), both ME-1 andTHP-1 cells are responsive to ATRA-induced differentiation(Supplementary Fig. S3). ATRA increased the levels of p-MEK,p-90RSK, p-GSK3b, and p-rpS6 as well as Mcl-1 protein whilerepressing Bcl-2 protein in HL-60 cells (Fig. 5A), ME-1 cells (Fig.5C), and THP-1 cells (Fig. 5E). Sorafenib at 2.5 mmol/L blockedthe ATRA-induced rise in the levels of p-MEK, p-GSK3b, p-p90RSK, and p-rpS6 as well as in Mcl-1 protein (Fig. 5A, C andE) and together with ATRA induced apoptosis in more than40% to 50% of cells; each agent alone induced only 5% to 20%apoptosis (Fig. 5B, D and F). The four primary samples wereisolated from two FAB-M2 AML patients and two FAB-M4 AML

patients. Three cases had normal genetic phenotype and onecase had inv (16) like ME-1 cells. Neither of the patient'ssample had FLT3 and (or/) NPM mutations. Treatment of thesesamples showed that ATRA or sorafenib alone induced less than10% apoptosis above the untreated control; the combination ofATRA with sorafenib induced apoptosis in more than 30% to40% of cells, suggesting a synergistic effect (Fig. 6A). ATRAincreased the levels of p-MEK, p-90RSK, p-GSK3b, and p-rpS6as well as Mcl-1 proteins in one case of tested primaryAML cells (Fig. 6B); these effects were blocked by the additionof sorafenib. We found that sorafenib blocking of ATRA-induced Mcl-1 levels always coincided with inhibition ofATRA-induced phosphorylation of p90RSK, GSK3b, and rpS6(Figs. 5A, 5C, 5E, and 6B). Although the regulation on p-ERKlevels by either ATRA or sorafenib in the different cell lineswas inconsistent, the combination of ATRA with sorafenib

Annexin V

4.3%

10.8%

Sorafenib 2.5 mmol/LSorafenib 2.5 mmol/L

Sorafenib 5 mmol/L

15.5%

57.1%

Sorafenib+ ATRA

Con

ATRA 1 mmol/L ATRA 1 mmol/LATRA 1 mmol/L

B

ME-1C

+ + + + +

+ + Sorafenib 2.5 mmol/L

Mcl-1

Bcl-2

PARP

p-rpS6(Ser235/236)

p-GSK-3b(Ser9)

p-p90RSK(Ser380)

p-ERK(Thr202/Tyr204)

p-MEK (Ser217/221)

5.7% 16.2%

19.9%

Con

ATRA +Sorafenib

Annexin V

DME-1

Annexin V

p-MEK (Ser217/221)

p-p90RSK(Ser380)

E+ +

+ + Sorafenib 5 mmol/L

Mcl-1

Bcl-2

PARP

THP-1

p-rpS6(Ser235/236)

p-GSK-3b(Ser9)

p-ERK(Thr202/Tyr204)

58.5%

ATRA 1 mmol/L ATRA 1 mmol/L ATRA 1 mmol/L + -

- - - - -

- - - - -

- Sorafenib 2.5 mmol/L

Mcl-1

p-MEK(Ser217/221)

A

b-Actinb-Actin b-Actin

PARP

Bcl-2

p-rpS6(Ser235/236)

p-GSK-3b(Ser9)

p-p90RSK(Ser380)

p-ERK(Thr202/Tyr204)

HL-60

HL-60Con

5.6%

5.8%

ATRA + Sorafenib

F THP-1

41.7%

Figure 5.Sorafenib enhances ATRA-induced apoptosis in HL-60, ME-1 and THP-1 cells and this effect is linked with repression of both Bcl-2 and Mcl-1 proteins.Sorafenib blocked ATRA-induced Mcl-1, p-p90RSK, p-GSK3b, and p-MEK and enhanced ATRA-induced apoptosis in HL-60 (A and B), ME-1 (C and D) and THP-1(E and F) cells. HL-60, ME-1, and THP-1 cells were treated with 1 mmol/L ATRA plus 2.5 or 5 mmol/L sorafenib for 2 days. The levels of the indicatedproteins were determined using Western blot analysis (A, C, E) and the apoptotic cells were determined using FACS after staining with Annexin V (B, D, F).

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consistently augmented apoptosis in all cell lines and primarycells tested and this was always associated with downregulationof Bcl-2 and Mcl-1 protein (Figs. 5A, 5C, 5E, and 6B).

Sorafenib is currently being tested as a FLT3-ITD inhibitor forAML treatment (32) and FLT3-ITD AML cells are responsive tosorafenib-induced apoptosis at lower concentrations (33).Recently, sorafenib togetherwithATRAwasused to treat 3patientswith FLT3-ITD AML, achieving durable responses (34). InMOML13 cells, which have FLT3-ITD, sorafenib, at lower con-centrations, was able to induce apoptosis (35). This cell lineundergoes differentiation in response to ATRA (36). We foundthat ATRA-induced differentiation ofMOLM13 cells (Supplemen-tary Fig. S3) increased the levels of phosphorylated pRSK, phos-phorylated GSK3b, and Mcl-1 (Fig. 6D). ATRA combined withlower concentrations of sorafenib wasmore effective in apoptosisinduction than sorafenib alone (Fig. 6C). The effects of ATRA,

sorafenib, and their combination were tested in vivo usingMOML13 cells xenografted into NSG mice. ATRA alone did notincrease survival while, as compared with control, sorafenibprolonged the survival of mice xenografted with MOLM13.Importantly, ATRA significantly enhanced the sorafenib-inducedsurvival (Fig. 6E and F). These data show that the antileukemiaeffect of the two drugs is mediated by sorafenib but greatlyimproved by the addition of ATRA.

DiscussionWe found that, similar to normal neutrophils (37), APL cells

terminally differentiated by ATRA treatment, die through apo-ptosis, and that this process is controlled, at least in part, byMcl-1 protein. Silencing of Mcl-1 appears to accelerate theapoptosis of the differentiation-responsive AML cells. This

A

0

10

20

30

40

50

60

70

80

An

nex

in V

–po

siti

ve c

ells

(%

)

#1

#2

#3

#4

** **

Cont ATRA Sorafenib ATRA/sorafenib

Primary cells

Primary cells BATRA 1 mmol/L +-

--+

+ +-

- ---

Sorafenib 2.5 mmol/L

PARP

Bcl-2

Mcl-1

p-rpS6(Ser235/236)

b-Actin

p-GSK-3b(Ser9)

p-MEK (Ser217/221)

p-p90RSK(Ser380)

p-ERK(Thr202/Tyr204)

P < 0.01, compared with control group. ⃰ P < 0.05, compared with sorafenib alone group. ILS, increase in life span over control.

Time (days)

4.6%

13.7%

18.1%

73.7%

Sorafenib 15 nmol/LCon

ATRA 1 mmol/L Sorafenib+ATRA

Annexin V

Per

cen

t su

rviv

al

C MOLM13

D

E

F

Mcl-1

PARP

Bcl-2

p-rpS6(Ser235/236)

p-ERK(Thr202/Tyr204)

b-Actin

ATRA 1 mmol/L ++++Sorafenib 15 nmol/L

p-p90RSK(Ser380)

p-GSK-3b(Ser9)

MOLM13

TreatmentNumber of

miceSurvival time (d) ILS (%)

18.2±1.85Control 0

16.4±0.95ATRA -9.9

35.4±0.95Sorafenib 94.5

44.6±4.1 5ATRA+Sorafenib 145.1

110100

9080706050403020100

0 5 10 15 20 25 30 35 40 45 50

Control

ATRA ATRA+sorafenib

Sorafenib

Figure 6.ATRA combined with sorafenib has enhanced apoptosis induction ability in primary AML cells and MOLM13 cells. The combined effect of ATRA and sorafenib onapoptosis induction in primary cells isolated from 4 cases of AML patients was examined after treatment with 1 mmol/L ATRA and 2.5 mmol/L sorafenib for2 days (A). The apoptotic cells were detected with Annexin V-FITC assay using flow cytometry. �� , P < 0.01 compared with cells treated with either ATRA orsorafenib alone. The combined effects of ATRA and sorafenib on Mcl-1 protein and its potential regulators in primary AML cells isolated from #2 patientwere examined using specific antibodies with Western blot analysis (B). The combined effect of ATRA and sorafenib on apoptosis induction (C) and proteinlevels (D) in MOLM13 cells was examined after treatment with 1 mmol/L ATRA and 15 nmol/L sorafenib for 2 days. Mice survival after treatment with sorafeniband ATRA, as described in Material and Methods, is shown in Kaplan–Meier survival plot (E) and the average survival times of each group and increase oflifespan over control was calculated (F). Five mice were used in each group.

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observation provides a rationale for achieving an acceleratedapoptosis induction by combining ATRA with an Mcl-1 inhib-itor in ATRA differentiation-responsive AML cells.

Mcl-1 has been found to be essential for the development andsurvival of AML (38–40).We found thatMcl-1 protein levels weredifferently regulated in differentiated and apoptotic APL NB4cells. The ratio of differentiated to apoptotic cells was approxi-mately 4:1 on day 4 with elevated Mcl-1; while on day 6 of ATRAtreatment, the ratio dropped to approximately 1:1 and Mcl-1 wasstrongly reduced (Fig. 1C). Although it is unclear how Mcl-1 isdecreased in the terminally differentiated cells undergoing apo-ptosis, the observation that Mcl-1 knockdown increased apopto-sis of ATRA-treated cells (Figs. 1D and 4D) indicates that Mcl-1protein is needed for survival of differentiated cells. Knockdownof Mcl-1 in control cells, which had high levels of Bcl-2, increasedapoptosis, but only slightly (Figs. 1F and 4E), indicating that bothantiapoptotic proteins might play an important role in protectingthese cells from death. This is supported by the observations thatoverexpression of either Bcl-2 or Mcl-1 blocks apoptosis of HL-60cells treated with ATRA for 6 days (Supplementary Fig. S1C andS1F). However, our results show that it is the upregulated Mcl-1that is the crucial prosurvival protein in NB4 and HL-60 cellsduring ATRA treatment as Bcl-2 level is suppressed by this treat-ment (Figs. 1C and 4C).

Mcl-1 is regulated by a large number of pathways at transla-tional and posttranslational levels (12, 41). We found that bothMEK/ERK and Akt/mTOR pathways are activated in ATRA-treatedAPL cells (Fig. 2E) and that this leads to increase in phosphor-ylation of p70S6K, p90RSK, and p-rpS6 levels and in Mcl-1protein (Fig. 2E). Pathway-specific inhibitors showed that bothMEK/ERK/RSK and PI3K/Akt, but not mTOR, pathways regulateMcl-1 protein levels inNB4 cells (Fig. 3A–C).On the basis of theseobservations, we propose that ATRA increases the rate of Mcl-1translation by activating p90RSK and rpS6 and stabilizes Mcl-1protein by inactivating GSK3b and/or activating ERK (summa-rized in Fig. 3D). However, it is possible that other unexploredfactors also participate in Mcl-1 protein regulation during ATRAtreatment.

Could targeting Mcl-1 improve the ATRA therapy for otherAMLs? In an earlier publication (28), ATRA/MEK inhibitor–treated AML cells were shown to undergo accelerated apoptosiswithout signs of differentiation and through an unknownmechanism. We propose that the apoptotic effect of this com-bination is due to dual inhibition of Bcl-2 andMcl-1. Treatmentof HL-60 cells with either of the two MEK inhibitors, U0126 orPD184352, inhibited both the basal level and the ATRA-induced level of phosphorylated ERK, p90RSK, GSK3b, andrpS6 as well as Mcl-1, while also maintaining Bcl-2 repressionby ATRA, leading to a profoundly increased apoptosis (Sup-plementary Fig. S2). As a preclinical approach, we tested acombination of ATRA with sorafenib, a multikinase inhibitorwith a transient effect in resistant and relapsed AML patients(29, 42). We showed that in FAB-M2 HL60 cells, a short (2days) treatment with sorafenib was almost as effective as MEKinhibitors in reversing ATRA-induced Mcl-1 increase whileallowing for ATRA repression of Bcl-2 to continue (Fig. 5A)and strongly increasing apoptosis (Fig. 5B). Relevant to ourgoal of expanding this treatment to other AMLs, sorafenibreversed ATRA-induced Mcl-1 level and maintained the lowATRA-repressed Bcl-2 level in FAB-M4 ME-1 cells and FAB-M5THP-1 cells, in a sample of primary human AML cells from a

FAB-M2 patient and in MOLM13 cells with FLT3-ITD mutation(Figs. 5 and 6). The pathways regulating Mcl-1 by ATRA in thesecells were in general similar to those in APL with induced p-p90RSK/p-rpS6 and p-GSK3b. Moreover, like in APL, the levelof Bcl-2 was reduced by ATRA. The only difference from thatobserved in APL was the effect on ERK phosphorylation. Whilein the primary AML and HL-60 cells, ATRA treatment increasedp-ERK level and sorafenib reduced it by approximately 50%, theeffect in ME-1 and THP-1 was unexpected. ATRA induced pERKin ME-1 cells but sorafenib did not block it, while in THP-1 cellsATRA strongly reduced the level of pERK (Figs. 5 and 6). All thekinases in this pathway, including ERK, are subject to multipleregulatory controls and rapid phosphorylation and dephos-phorylation (43, 44). We did not further study the reasons forthe differences in phosphorylation status of ERK after treatmentwith ATRA and/or sorafenib but the kinetics of ERK phosphor-ylation and dephosphorylation might be different in these cellslines which would require more detailed kinetic study to proveit (45). We also did not resolve the question of p90RSKupregulation in absence of pERK upregulation but possibly,the active p90RSK persists longer. Importantly, the apoptosisaugmenting effect of sorafenib when added to ATRA is presentin all 8 samples tested; 4 non-APL AML cell lines and 4 primaryAMLs (Fig. 6) and, it is consistently accompanied by reductionof both Mcl-1 and Bcl-2 proteins. Several reports showed thatATRA repressed Bcl-2 mRNA in differentiated AML cells (7, 46)and we found the cell lines used to be responsive to ATRA-induced differentiation (Fig. 4A, Supplementary Fig. S3). There-fore, the downregulation of Bcl-2 by ATRA might be transcrip-tional and secondary to differentiation. ATRA increases Mcl-1protein, but not its mRNA (Fig. 2B). The Mcl-1 protein has ashort half-life and it can be regulated by many factors such ascytokines, growth factors, and reactive oxygen species as well asactivated caspase due to spontaneous apoptosis of cells (11).Therefore, the regulation of Mcl-1 by ATRA may be cell linespecific and influenced by the culture conditions. However,differentiated AML cells with repressed Bcl-2, like normalneutrophils without Bcl-2 expression, rely on Mcl-1 to survive(47). Although we found that ATRA-increased phosphorylationof GSK3b and p90RSK, which contributes to Mcl-1 inductionby ATRA (Fig. 3D), is inhibited by sorafenib, this inhibitoralone also represses the basal level of Mcl-1 in the tested celllines. The inhibition of basal Mcl-1 levels by sorafenib alonemight be due to other mechanisms such as inhibition of FLT3-ITD and/or eIF4E phosphorylation, which are known toincrease Mcl-1 translation (31, 48–50). Overall, we found thatATRA together with sorafenib augments apoptosis in vitro andprolongs survival of AML-xenografted mice, providing a trans-lational rationale for studying this combination in AML cellsand patients.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: R. Wang, J. Gabrilove, Y. JingDevelopment of methodology: R. Wang, L. Xia, Y. JingAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Xia, J. Gabrilove, Y. JingAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Wang, Y. Jing

Combined Apoptosis Induction of ATRA with Sorafenib in AML

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Writing, review, and/or revision of the manuscript: R. Wang, J. Gabrilove,S. Waxman, Y. JingAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. WaxmanStudy supervision: Y. Jing

AcknowledgmentsThe authors thank Dr. Liliana Ossowski for editing of this article.

Grant SupportThis work was supported by NIH grant R01-CA93533 and The Samuel

Waxman Cancer Research Foundation.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 18, 2015; revised September 15, 2015; accepted October 6,2015; published OnlineFirst October 12, 2015.

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Combined Apoptosis Induction of ATRA with Sorafenib in AML

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