Inhibition of FLT3 and PIM Kinases by EC-70124 Exerts ... · apoptosis. EC-70124 is orally bioavailable and displays higher metabolic stability and lower human protein plasma binding

Small Molecule Therapeutics

Inhibition of FLT3 and PIM Kinases by EC-70124Exerts Potent Activity in Preclinical Models ofAcute Myeloid LeukemiaNoelia Puente-Moncada1,2, Paula Costales3, Isaac Antolín1,2, Luz-Elena N�u~nez3,Patricia Oro3, Maria Ana Hermosilla3, Jhudit P�erez-Escuredo3, Nicol�as Ríos-Lombardía3,Ana M. Sanchez-Sanchez1,2, Elisa Lu~no4, Carmen Rodríguez1,2, Vanesa Martín1,2, andFrancisco Morís3

Abstract

Internal tandem duplication (ITD) or tyrosine kinase domainmutations of FLT3 is the most frequent genetic alteration in acutemyelogenous leukemia (AML) and are associated with poordisease outcome. Despite considerable efforts to develop sin-gle-target FLT3 drugs, so far, the most promising clinical responsehas been achieved using the multikinase inhibitor midostaurin.Here, we explore the activity of the indolocarbazole EC-70124,from the same chemical space as midostaurin, in preclinicalmodels of AML, focusing on those bearing FLT3-ITD mutations.EC-70124 potently inhibits wild-type and mutant FLT3, and alsoother important kinases such as PIM kinases. EC-70124 inhibitsproliferation of AML cell lines, inducing cell-cycle arrest andapoptosis. EC-70124 is orally bioavailable and displays highermetabolic stability and lower human protein plasma binding

compared with midostaurin. Both in vitro and in vivo pharmaco-dynamic analyses demonstrate inhibition of FLT3-STAT5, Akt-mTOR-S6, and PIM-BAD pathways. Oral administration of EC-70124 in FLT3-ITD xenograft models demonstrates high efficacy,reaching complete tumor regression. Ex vivo, EC-70124 impairedcell viability in leukemic blasts, especially fromFLT3-ITDpatients.Our results demonstrate the ability of EC-70124 to reduce pro-liferation and induce cell death in AML cell lines, patient-derivedleukemic blast and xenograft animalmodels, reaching best resultsin FLT3mutants that carry other molecular pathways' alterations.Thus, its unique inhibition profile warrants EC-70124 as a prom-ising agent for AML treatment based on its ability to interferethe complex oncogenic events activated in AML at several levels.Mol Cancer Ther; 17(3); 614–24. �2018 AACR.

IntroductionAcute myelogenous leukemia (AML) is the most common

myeloid malignancy in adults (1, 2) and internal tandem dupli-cation (ITD) or kinase domain mutations of FMS-like tyrosinekinase 3 (FLT3) represent the most frequent genetic alterationsaccounting for nearly 30%of cases andbeing associatedwith poordisease outcome (3). FLT3 activation results in the inductionof downstream prosurvival pathways, including MAPK/extracel-lular signal–regulated kinase, PI3K/Akt and STAT5, and causesincreased cell proliferation with suppression of apoptosis (4).FLT3 has been a target of choice for many years and, as a result,several drugs are currently in advanced clinical trials (3).However,

despite promising preclinical results, none of FLT3 inhibitorsreported so far sustained its activity as single-agent therapy for.prolonged time, often due to the emergence of resistance in FLT3-ITD (5). In fact, best clinical data, including significant survivalbenefit, have been reported in the RATIFY trial with the multi-kinase inhibitor midostaurin, a semisynthetic analogue of thepromiscuous kinase inhibitor staurosporine (6). This clinicallyvalidated success story signals that intensive research is needed todiscover novel multikinase inhibitors targeting not only FLT3 butalso alternative pathways that cover the complex oncogenic eventsactivated in AML and the potential resistance mechanisms thatcan emerge after clinical treatments. For instance, resistance toFLT3 inhibition has been associated with upregulation of PIMkinases (7, 8).

In this sense, PIM proteins (PIM1, PIM2, and PIM3) are serine-threonine kinases with increased expression in a variety of malig-nancies, including AML (9–11). PIM kinases play an importantrole in enhancing cell survival and suppressing apoptosis inhematopoietic cells (12, 13), particularly PIM1 and PIM2 whoseoverexpression has been reported in AML blasts (14). Theirexpression can be induced by activation of STAT5 (15), one ofthemost important targets in constitutively activated FLT3, estab-lishing a relationship between both pathways. In fact, constitu-tively activated FLT3 signaling upregulates PIM1 expression inleukemia cells and this upregulation contributes to the prolifer-ative and antiapoptotic pathways induced by FLT3 signaling (16).Moreover, PIM kinase inhibition has been described to enhance

1Departamento de Morfología y Biología Celular, Facultad de Medicina, c/JulianClaveria, University of Oviedo, Oviedo, Spain. 2Instituto Universitario de Onco-logía del PrincipadodeAsturias (IUOPA),Oviedo, Spain. 3EntreChemSL,Oviedo,Spain. 4Hospital Universitario Central de Asturias (HUCA), Oviedo, Spain.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

N. Puente-Moncada and P. Costales contributed equally to this article.

Corresponding Author: Francisco Morís, EntreChem SL, Vivero de Cienciasde la Salud, Calle Colegio Santo Domingo Guzman, 33011, Oviedo, Spain.Phone: 34-985-259021; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-0530

�2018 American Association for Cancer Research.

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FLT3 inhibitors activity (17). The feedback loop between the twoproteins makes the combination of FLT3 and PIM inhibition arational strategy for AML treatment (18).

EC-70124 is a hybrid indolocarbazole obtained by combina-tion of genes from rebeccamycin and staurosporine biosynthesispathways (19, compound #8). This compound displays a potentmultikinase inhibitor spectrum affecting key intracellular kinasesimplicated in prosurvival and proliferative pathways. Thus,EC-70124 induces senescence of glioblastoma-initiating cells byinhibition of the NF-kB pathway (20) and exerts antitumoralactivity in triple-negative breast cancer by the inhibition of bothPI3K/mTOR and JAK/STAT pathways (21). Similar antitumoralactivity has been also described in colorectal cancer mediated byPI3K/Akt pathway inhibition (22). Interestingly, EC-70124 alsoacts as a dual STAT3/NF-kB inhibitor, reverting both tumorigenicand stem cell properties in prostate cancer (23).

In this study, we characterize the pharmacologic and activityprofile of this unique multikinase inhibitor in preclinical AMLmodels. EC-70124 inhibits AML cell proliferation and inducedcell-cycle arrest and apoptosis. In vivo oral treatment causescomplete regression in AML tumor xenograft models, and ex vivoinhibits proliferation in AML patient blasts at biochemicallyrelevant doses. Collectively, these resultsmake EC-70124 a prom-ising new candidate for the treatment of AML.

Materials and MethodsCell culture and reagents

MV4-11 (catalog no. ACC-102), MOLM-13 (catalog. no.ACC-554), MOLM-16 (catalog no. ACC-555), KG1 (catalog no.ACC-14), OCI-M1 (catalog no. ACC-529), NB4 (catalogno. ACC-207), THP-1 (catalog. no. ACC-16), and HL-60 (catalogno. ACC-3) cell lines were purchased from DSMZ German col-lection andmaintained in RPMI supplementedwith 10%FBS and1% antibiotic–antimycotic mixture containing 5,000 U/mL pen-icillin and 5,000 U/mL streptomycin except for MOLM-16, whichwas maintained in 20% FBS. Cells were maintained at 37�C in ahumidified atmosphere of 5% CO2. Culture cells have beenmonitored to ensure that they are Mycoplasma-free using theLookOutMycoplasma qPCR Detection Kit (Sigma Chemical Co.).All experiments were performed between passage 5 and 20. Cellculture reagents were purchased from Sigma (Sigma ChemicalCo.) except for FBS, which was obtained from Gibco (InvitrogenLife Technologies). Culture flasks and dishes were acquired fromThermo Fisher Scientific.

EC-70124 was synthesized by a proprietary process by Entre-Chem S.L. Midostaurin was synthesized from staurosporine (Bio-mar Microbial Technologies) and the identity of the isolatedproduct verified by comparison with an authentic sample (HPLC,NMR).

Isolation of leukemic cells from patient samplesAML patient bone marrow aspirates, collected during routine

examination, were obtained from Hospital Universitario Cen-tral de Asturias (HUCA). Informed consent was obtained fromeach patient, and the protocol was approved by the Institutionand the Clinic Research Ethics Committee of the Health Insti-tute of the Principality of Asturias, as instructed by the Decla-ration of Helsinki. Mononuclear cells were isolated by Ficolldensity-gradient centrifugation and cultured as described forAML cell lines.

Metabolic stabilityIn vitro incubation with human liver microsomes was per-

formed to evaluate the disappearance of the parent compound(EC-70124) by LC-MS. Half-life is calculated by the equations:T1/2¼ 0.693/k, where k is the rate constant. The assay included acontrol without cofactors at 60 minutes to account for the dis-appearance of the drug by reasons unrelated to cytochromeactivity.

For the in vivo assessment of metabolic stability, CD1 micewere dosed at 24 mg/kg intravenously and their plasma ana-lyzed after 3 hours by HPLC-UV to detect indolocarbazole-related metabolites.

Protein plasma bindingUnbound fraction of EC-70124 andmidostaurin uponbinding

to plasma proteins were determined measured by commerciallyavailable TRANSILXL High Sensitivity Binding Kit (SovicellGmbH) per the manufacturer's instructions. Membrane affinity(MA) of each molecule was previously assessed using thealso commercially available TRANSILXL Membrane Affinity Kitto estimate the optimal plasma dilutions and kit suitability.Analysis of drug concentrations was carried out by LC/MS-MS inMRM mode.

Oral bioavailability by Caco-2 assayOral bioavailability was measured by bidirectional perme-

ability through Caco-2 cell monolayers (Absorption Systems).The assay determines the permeability through Caco-2 cellmonolayers in both the apical-to-basolateral and basolateral-to-apical direction. EC-70124 concentration was 5 mmol/L.Samples were taken from the donor and receiver chambers at120 minutes and assayed by LC/MS-MS using electrosprayionization to calculate the apparent permeability, Papp, andpercentage of recovery.

Kinase-binding assaysThe binding affinity of EC-70124 and midostaurin were deter-

mined by quantitative-binding affinity of compound–kinaseinteractions (KdELECT, Discoverex) using a 10-point dose–response curve.

Cell viability assayFor dose–response curves, cellswere plattedon96-well dish at a

density of 5,000 cells/well and viability was determined by theMTT assay as previously described (24). Data were analyzedusing CalcuSyn software (Biosoft) that calculates the IC50 valuefor each drug.

Annexin V bindingCells were seeded in 6-well plates at a density of 2 � 105 cells/

well. Once the treatments were completed, apoptosis was evalu-ated by the Annexin V-FICT Apoptosis Detection Kit (SigmaChemical Co.) as per themanufacturer's protocol. Apoptosis level(Annexin V–positive cells) was determined in 10,000 cells pergroup using a Beckman Coulter FC500 flow cytometer (BecktonDickinson).

Flow-cytometry analysis of cell-cycle distributionAfter treatment, harvested cells were incubated with a ribonu-

clease A (100mg/mL) solution for 10minutes. Then, sampleswerestained with propidium iodide (0.005%) for 10 minutes in the

Antileukemic Effect of Multikinase Inhibitor EC-70124

www.aacrjournals.org Mol Cancer Ther; 17(3) March 2018 615




dark. A Beckman Coulter FC500 flow cytometer (Beckton Dick-inson) was used for counting 2� 104 cells per sample. Analysis ofcell-cycle distribution was carried out using the software ModfitVer 5.2 (Verity Software House).

Real-time quantitative PCRQuantitative analysis of CYCLIN D1 (CCND1), PIM1, and

C-MYC mRNA levels was performed by the SYBR Green real-time PCR method using Green PCR Core Reagents (AppliedBiosystems) in an AB7700 Real-Time System (Applied Biosys-tems) as described previously (25). The primers used were thefollowing: 50-GCTGCGAAGTGGAAACCATC-30 (sense) and 50-CCTCCTTCTGCACACATTTGA-30 (antisense) for CCND1; 50-CGAGCATGACGAAGAGATCAT-30 (sense) and 50-TCGAA-GGTTGGCCTATCTGA-30 (antisense) for PIM1; 50-TGCTCCAT-GAGGAGACACC-30 (sense), 50-CTTTTCCACAGAAACAACA-TCG-30 (antisense) for C-MYC and 50-TTCCCCATGGTGTCT-GAGC-30 (sense) and 50-ATCTTCTTTTGCGTCGCCAG-30 (anti-sense) for GADPH. Each sample was tested in triplicate, andrelative gene expression data were analyzed by means of the2�DC

t method.

Western blot analysisCells were lysed with ice-cold lysis buffer (150 mmol/L NaCl, 1

mmol/L EDTA, 1mmol/L EGTA, 1%v/v Triton X-100, 2.5mmol/Lsodiumpyrophosphate, 1mmol/Lb-glycerophosphate, 1mmol/LNa3VO4, 1 mg/mL leupeptin, 10 nmol/L NaF, 1 mmol/L PMSF,20 mmol/L Tris-HCl pH 7.5). Between 30 and 50 mg of totalprotein were separated by SDS-PAGE and transferred to polyvi-nylidene difluoride membranes (Amersham Bioscience). Primaryantibodies were used against the phosphorylated forms of STAT5,p70S6k, S6, 4-EBP, BAD (1:1,000, Cell Signaling Technology),total PIM1, PIM2 (1:1,000, Cell Signaling Technology) andGAPDH—as a loading control (1:1,000, Santa Cruz Biotechnol-ogy). For signaling detection appropriated secondary (anti-rabbit IgG peroxidase–conjugated and anti-goat IgG peroxidase1:3,000, Calbiochem) antibodies were used and the reactionwas visualized by means of enhanced-chemiluminescencedetection reagents (Amersham Biosciences) following the man-ufacturer's protocol.

In vivo xenograft modelFemale immunodeficient mice were purchased from Janvier

Laboratories (CB17 SCID, 5-weeks-old) and maintained understerile and controlled conditions with food and water ad libitum.All animal research protocols were approved by the AnimalResearch Ethical Committee of the University of Oviedo. MV4-11cells (5 � 106 cells with 25% Matrigel) were injected subcutane-ously into the right flank. Once tumor size reached 200 mm3

animals were randomized in the different experimental groups of8 mice, unless otherwise noted. Tumor volume was measureddaily, and drug efficacy was expressed as the percentage tumorgrowth inhibition (%TGI).

For pharmacodynamic studies, immunodeficient mice weretreated orally once the mean tumor volume was 400 mm3.Tumors were dissociated into single cell suspensions using aMACS Tissue Dissociation Kit and the GentleMACS Dissociatorsystem (Miltenyi Biotec) for protein, RNA isolation and drugaccumulation analysis. Blood samples were also collected fromsamemice via cardiac puncture and EC-70124 concentration wasanalyzed by LC/MS.

Statistical analysisExperiments were repeated at least three times, and data were

calculated as the average � SE. One-way ANOVA followed by aStudent Newman–Keuls multiple range test were carried out andstatistical significance was accepted when P < 0.05.

ResultsEC-70124 is a potent and selective inhibitor for AML kinases

Structures of EC-70124 and midostaurin are shown in Supple-mentary Fig. S1. Competition binding assays has revealed that thiscompound was highly active against several human kinasesincluding some AML related kinases such us FLT3, JAK or PIMkinases (19–22). Here, we evaluated kinase inhibitor activity atseveral EC-70124 concentrations to evaluate the affinity to thetargets. Data were compared with those of midostaurin from thesame assay platform (KdELECT, Discoverex). Results indicatedthat EC-70124 potently inhibits several AML related kinases,including FLT3, JAK, SYK or PIM kinases (Kd < 10 nmol/L, Table 1). Importantly, 7 out of 9 FLT3 mutants tested areinhibited at even lower concentrations than wild-type FLT3.

Moreover, EC-70124 shows higher potency than midostaurinfor several AML-related kinases. Thus, EC-70124 is more potentthan midostaurin for FLT3 and its mutants, 10x more potent forSYK, 10–70x more potent for JAK kinases and 2–3 orders ofmagnitude more potent for PIM kinases (Table 1). Interestingly,EC-70124 is less potent than midostaurin for KIT, a kinaseregarded as anti-target, due to its involvement in the developmentof undesired myelosuppression in AML treatment (26).

EC-70124 inhibits AML cell-line growthEC-70124 was tested for antiproliferative activity in a panel of

8 AML cell lines (Fig. 1A). All cell lines were sensitive to EC-70124with IC50 values ranging from 13 to 520 nmol/L (Fig. 1A and B).FLT3-ITD cell lines MV4-11 and MOLM-13 show the lowest IC50

values whereas wild-type cells show the biggest ones. In agree-mentwith the superior profile as inhibitor of AML-related kinases,

Table 1. Comparison of the EC-70124 and midostaurin dissociation constantsfor selected kinases

Kd, nmol/LKinase EC-70124 Midostaurin

FLT3 3.2 11FLT3 (ITD) 2.5 11FLT3 (D835H) 0.8 6.8FLT3 (D835Y) 1.1 15FLT3 (D835V) 0.8 2.2FLT3 (N841I) 1.4 6FLT3 (K663Q) 7 2FLT3 (R834Q) 11 12FLT3 (ITD, D835V) 1.1 3.5FLT3 (ITD, F691L) 1.2 5JAK1 9.4 670JAK2 2.5 94JAK3 1.5 12PDPK1 16 190PIM1 2.9 560PIM2 3.1 3,100PIM3 0.4 560SYK 8 88KIT 420 220PDGFRA 140 380PDGFRB 29 110

NOTE: Assays run by Discoverex.

Puente-Moncada et al.

Mol Cancer Ther; 17(3) March 2018 Molecular Cancer Therapeutics616




EC-70124 also displayed greater inhibitory effect on FLT3wt celllines compared tomidostaurin, whichwas particularly active onlyin FLT3-ITD cell lines (Fig. 1B and C).

Sensitivity to EC-70124 can be related with expression of AML-related kinase-targets (Fig. 1D). Phosphorylated STAT5 isexpressed in the five of the eight most sensitive cell lines. On theother hand, the three least sensitive cell lines (NB-4, THP-1, andHL-60) did not express neither phosphorylated STAT5 nor anyPIM isoforms.

EC-70124 was also effective in mononuclear cells obtainedfrom bonemarrow aspirates from newly diagnoses AML patients.As showed in Fig. 1E, 5 out of 6 samples tested were sensitive tonanomolar concentrations of EC-70124 in adose-dependentway.

In fact, EC-70124 treatment results in an increase in cell death asdetermined by trypan blue exclusion assay (Fig. 1F). Interestingly,best results were observed in FLT3 ITD samples, which agree withthe results obtained in AML cell lines and could be related to thefact that FLT3 mutants are inhibited at even lower concentrationsthan wild-type FLT3.

EC-70124 can induce cell-cycle arrest and apoptosisCell-cycle analyses in MV4-11, MOLM-13, and MOLM-16

(treated at concentrations near the IC50 value for each line)showed dose-dependent increase inG0/G1 population, indicatingcell-cycle arrest in response to EC-70124, especially in the twoFLT3-mutant cell lines (Fig. 2A). Moreover, an increase in

Figure 1.

Effect of EC-70124 and midostaurin oncell viability in AML cell lines and patientsamples. Eight AML cell lines weretreated with EC-70124 (0–10 mmol/L;A) or midostaurin (0–10 mmol/L; B) for48 hours, and viability was determinedby MTT reduction. C, Results wereanalyzed using CalcuSyn software tocalculate IC50 values for each drug THP-1cell line showed a plateau effect, andtherefore the IC50 value could not bedetermined for midostaurin. FLT3 statusis indicated for each cell line. D,Westernblots of cell-free extracts of the 8 celllines showing protein levels forphosphorylated STAT5 and p70S6K,pBAD, and PIM kinases, and GAPDH as acontrol. E, Cell viability determined afterEC-70124 (0–100 nmol/L) treatment for48 hours of isolated blast from 6 AMLpatient samples. FLT3 status isindicated for each sample.F,Cell death inAML patient samples determined byTrypan blue staining after EC-70124treatment for 48 hours. FLT3 status isindicated for each sample. � , P < 0.01versus control group (vehicle-treatedcells). ND, not determined.






Figure 2.

Effect of EC-70124 on cell cycle and apoptosis in MV4-11, MOLM-13, MOLM-16, and HL-60 cell lines. A, Cell-cycle distribution after 24 or 48 hourstreatment as determined by propidium iodide staining. B, Percentage of cell at sub-G1 phase after EC-70124 treatment as indicator of apoptosis cell death.C, Effect of 24 to 48 hours treatment with EC-70124 on AML cell lines apoptosis determined by Annexin V staining. A midostaurin-treated group (50 nmol/Lfor MV4-11, 50 nmol/L for MOLM-13, 50 nmol/L for MOLM-16, and 1,000 nmol/L for HL60) has been represented for comparison. � , P < 0.01 versus controlgroup (vehicle-treated cells).






apoptotic response (sub-G1 population) was observed in thethree cell lines (Fig. 2B), although it was considerably higher inMOLM-16 cells. On the contrary, less sensitive HL-60 cells hardlyshowed variations neither in the cell cycle nor in sub-G1 popu-lation even at high concentrations (Fig. 2A and B).

Consistent with the increase in sub-G1 population, dose-dependent increase in apoptotic cells was also observed inMV4-11, MOLM-13, and MOLM-16, as determined by AnnexinV–binding assay. According to the cell–cycle distribution data,induction of apoptosis is much higher on MOLM-16 cells. Apo-ptotic cells were barely detected in less sensitive HL-60 cells aftertreatment with EC-70124. Midostaurin, according to the previ-ously described selectivity for FLT3-ITD mutant AML cells, onlyinduces apoptosis in MV4-11 and MOLM-13 cells (Fig. 2C).

EC-70124 modulates AML-related kinasesAbnormalities in receptor tyrosine kinases or receptor-associ-

ated intracellular kinases are extremely frequent in cancer, includ-ingAML(27). Thus, FLT3 alterations occurs in about 30%samplesand are associated with poor outcome (3). Receptor activationresults in the induction of downstream prosurvival pathwaysincluding STAT5 and Akt/mTOR. Modulation of several proteinsof these pathways was measured after EC-70124 treatment. Lowconcentrations of EC-70124 results in rapid inhibition of Akt/mTOR pathway—upon few hours of treatment—as determinedby the phosphorylation status of the downstream targets p70S6K

and its substrate S6 ribosomal protein and also p4EBP1 (Fig. 3A).High EC-70124 concentrations were needed in less sensitive HL-60 cells to obtain similar results in agreement to the IC50 data.

As mentioned above, STAT5 appears to be one of the maintargets in constitutively activated FLT3 cell lines (28). In this sense,we found EC-70124 to induce a rapid decrease in phosphorylatedSTAT5 in MV4-11 and MOLM-13 cells (Fig. 3A). No inhibitionwas found inMOLM-16 cells or less sensitiveHL-60 cells (nobasalSTAT5 phosphorylation detected, Fig. 1D).On the other hand, wefound a decrease in phospho-BAD in all cell lines treated withEC-70124 (Fig. 3A). Several intracellular pathways can inhibit thepro-apoptotic activity of BADviaproteinphosphorylation includ-ing PI3K/Akt and also PIM kinases. In fact, it has been welldescribed that PIM1 kinase promote AML cell survival via phos-phorylation of BAD at Ser 112. Thus, according to low bindingassay Kd value for PIM1 kinase (Table 1), proapoptotic effect ofEC-70124 could be related to the inhibition of PIM1 in cells withbasal expression, but not in the PIM1-lacking HL-60 cell line(Fig. 1D). Because high concentrations are needed for cell viabilitydecrease in HL-60 we cannot exclude that other intracellularpathways may be altered.

We found similar effects in phosphorylated STAT5 and Akt/mTOR targets in FLT3-ITD mutant AML cells treated with mid-ostaurin (Fig. 3B). Interestingly, we did not found a decrease inphospho-BAD in these cells after midostaurin treatment. Thissupports our hypothesis that the inhibition of BAD can be

Figure 3.

Effect of EC-70124 on FLT3 downstream signaling in vitro. A, Phosphorylation levels of key targets upon treatment with two concentrations of EC-70124(based on the IC50) at 2 time points in MV4-11, MOLM-13, MOLM-16, and HL-60 cell lines. B, Phosphorylation levels of key targets upon treatment with twoconcentrations of midostaurin (based on the IC50) at 8 hours after treatment in MV4-11, MOLM-13, MOLM-16, and HL-60 cell lines. C, mRNA levels ofSTAT5 target genes in MV4-11, MOLM-13, MOLM-16, and HL-60 cell lines determined by RT-qPCR. Dashed line represents basal level of expression.� , P < 0.01 versus control group (vehicle-treated cells).






mediated by inhibition of PIM1 since misdostaurin lacks activityon PIM1.

We also treated the AML cells with SGI-1776, a well-knownFLT3-PIM dual inhibitor, and found similar effects on cell via-bility (Supplementary Fig. S2A), cell cycle (Supplementary Fig.S2B) and apoptosis (Supplementary Fig. S2C). Moreover, SGI-1776 treatment also resulted in inhibition of phosphorylation ofsame key targets than EC-70124 (Supplementary Fig. S2D).

In agreement to the inhibition of STAT5 phosphorylation andactivation, we also showed a dose-dependent decrease in themRNA expression levels of STAT5 targets as cyclin D1, c-myc andPIM1 in MOLM-13 and MV4-11 cells (Fig. 3C). No changes inSTAT5 targets were found in MOLM-16 cells where EC-70124 didnot induce inhibition of STAT5 phosphorylation or inHL-60 cellsthat did not express basal STAT5 phosphorylation (Fig. 3C).

EC-70124 is orally bioavailable, metabolically stable, andshows lower protein plasma binding than midostaurin

Permeation through Caco-2 monolayers has been establishedas a valid preclinical model to correlate the oral bioavailabilitypotential of drugs in preclinical development (29). As showedin Table 2, we determined the permeability (Papp) of EC-70124through Caco-2 monolayers in both the apical-to-basolateral(A–B) and basolateral-to-apical (B–A) directions. The resultingPapp A–B, Papp B–A and efflux ratio, indicates a high potential fororal absorption to EC-70124, without significant drug efflux.

Incubation with human liver microsomes was carried out toassess half-life and intrinsic clearance (Clint). The results obtained,half-life >60 minutes and Clint < 10 mL/min/kg, indicate anelevated metabolic stability (Supplementary Fig. S3A). Also,incubationwithout cofactors (to address disappearance unrelatedto cytochrome activity) show no changes in EC-70124 concen-tration. In addition, an in vivo experiment provided further met-abolic stability data. Mice were dosed with EC-70124 (24 mg/kg,i.v.) and its plasma analyzed 3 hours after dosing by HPLC-UV.The HPLC-UV profile shows only one peak, corresponding to thenon-metabolized drug (see Supplementary Fig. S3B for a repre-sentative example).

We determine the unbound fraction (fu) of EC-70124 andmidostaurin upon binding to human plasma proteins using ahigh sensitivity binding assay. As it can be seen in Table 3, dataobtained for EC-70124 shows a 3.9% fu, as compared with 1.1%fu for midostaurin in human plasma. The human plasma data for

EC-70124 is in line with the fu obtained in rat and dog plasma(4.2% and 3.0%, respectively).

In agreement with binding assay results, co-incubation withhuman alpha-acid glycoprotein (which represents 1%–3% ofplasma proteins) induced a significant increase in IC50 for mid-ostaurin compared with EC-70124 (Supplementary Fig. S3C).

EC-70124 efficacy and pharmacodynamics in AML xenograftsIn vivo activity of EC-70124 was evaluated in a MV4-11 xeno-

graft model. Inhibition of tumor growth was observed using bothintravenous and p.o. administration routes although oral admin-istration results in a more sustained cytostatic effect (Fig. 4A, andSupplementary Fig. S4A). Both administration routes were welltolerated by all mice because no significant weight loss wasdetected (Supplementary Fig. S5A).

Oral administration efficacy was found to be dose dependent,as daily treatment at 20 mg/kg of EC-70124 led to a TGI of 91%after 28 days, whereas 80 mg/kg every other day resulted incomplete regression of tumor growth in all mice before the endof treatment (Fig. 4B, and Supplementary Fig. S4B), and withoutapparent toxicity as judged by monitoring mice weight (Supple-mentary Fig. S5B). None of the high dosemice had reached 2,000mm3 at the time all the mice in the low dose hit that mark(Supplementary Fig. S6A). It is of note that one mice from thehigh dose group was permanently cured (tumor free for >6months).

We also compared EC-70124 with midostaurin, using anequivalent total dose under different schedules. In both cases,complete regression was observed; however, midostaurin-treatedmice reached tumor suppression faster (day 20) than EC-70124(day 35; Fig. 4C). Once fully regressed, 7/8 tumors grew backabout 1 week later in both treatment groups. It is of note that 1/8were permanently cured in each drug (mice tumor free for >6months).Once tumors reached 500mm3,micewere treated againwith the corresponding drug at the same dose and schedule as inthe first treatment cycle. Despite the high tumor volume, regres-sionwas again faster with dailymidostaurin (�100% regression),while the EC-70124 group fell short of complete regression(�70% regression). Both drugs were well tolerated with no miceweight loss observed (Supplementary Fig. S5C). The longerschedule used for EC-70124, a positive sign of non-accumulativetoxicity, is likely to translate into a survival benefit (Supplemen-tary Fig. S6B).

EC-70124 andmidostaurin were also evaluated in aMOLM-16xenograft. Treatment with midostaurin (100 mg/kg daily) has nosignificant effect on tumor growth (45% TGI), whereas EC-70124(80 mg/kg every other day) resulted in 95% TGI at day 32(Supplementary Fig. S7A). Individual mice tumor volume plotat day 34 also shows better response of EC-70124 respect tomidostaurin in the MOLM-16 xenograft model (SupplementaryFig. S7B).

Oral pharmacokinetic profile of EC-70124 was obtained bymonitoringplasma levels upona single 80mg/kgdose.Maximumconcentration in plasma was detected after 2 hours, reaching apeak that declines slowly over 48 hours, when plasma circulatinglevels dropped to 100 nmol/L. Drug analysis in tumor tissueshows much higher accumulation in MV4-11 tumor cells than inplasma (almost 10 times higher levels after 2 hours administra-tion), and even at 48 hours concentration was high enough tomaintain target inhibition (Fig. 4D). In agreement with results incultured cells, a rapid inhibition of STAT5, 4EBP1, S6 and BAD

Table 2. Oral absorption potential by the Caco-2 assay for EC-70124

Papp (10�6 cm/s)Direction Recovery % R1 R2 Average Efflux ratio

A-to-B 49 12.2 13.6 12.9 1.6B-to-A 72 19.4 20.9 20.2

NOTE: Absorption potential classification: Low if (Papp A–B) < 1.0 � 10�6 cm/s;high if (Papp A–B)¼ 1.0� 10�6 cm/s; significant Efflux defined as: Efflux ratio¼2.0 and (Papp B–A) ¼ 1.0 � 10�6 cm/s.

Table 3. Protein plasma binding for EC-70124 and midostaurin

Species EC-70124, fu Midostaurin, fu

Human 3.9 1.1Rat 4.2 n.d.Dog 3 n.d.

NOTE: TRANSILXL high sensitivity binding kit.Abbreviation: fu, fraction unbound.






Figure 4.

EC-70124 in vivo efficacy and pharmacodynamic analysis in AML xenograft models. A, CB-17 SCID mice were implanted subcutaneously with MV4-11 cellsand treated with either vehicle or EC-70124 by oral gavage (po) daily or intravenous injection every 3 days to assess tumor growth. Forty mg/kg daily podoses for 2 weeks, followed by 20mg/kg daily oral doses for another 2 weeks (20 doses total), were compared with 18 mg/kg i.v. doses every 3 days (8 doses total).� , Significant differences between treated and control groups (P < 0.01, t test). #, Significant differences between intravenous and po groups (P < 0.01, t test).B, Tumor growth determined in mice treated by oral gavage with two different schedules and dose (20 mg/kg daily vs. 80 mg/kg every other day).� , Significant differences between treated and control groups (P < 0.01, t test). #, Significant differences between 20 mg/kg and 80 mg/kg groups (P < 0.01, t test).C, Comparison of EC-70124 versus midostaurin effect on tumor volume using an equivalent total dose under different schedules: 13 doses of 80 mg/kgevery other day for EC-70124 (1,040mg/kg total) and 10 doses of 100mg/kg daily formidostaurin (1,000mg/kg total). Because tumors grew back in both treatmentgroups, mice were retreated once tumors reached 500 mm3 using same dose and schedule as in the first treatment cycle (treatment windows are representedon the bottom of the figure). D, Oral pharmacokinetic profile of EC-70124 obtained by monitoring plasma levels upon a single 80 mg/kg dose. Each datarepresented the average of 3 mice. E, Phosphorylation levels of key targets in MV4-11 xenograft model at different time points. Mice were implanted as above andtreated orally once with 80 mg/kg of EC-70124 when the mean tumor volume was 400 mm3. Tumors were dissociated into single cell suspensions andharvested for immunoblotting and RNA at eight different time points between 15 minutes and 48 hours. Each time point represented the pool of 3 mice.F, mRNA levels of STAT5 target genes determined by RT-qPCR in MV4-11 tumor samples. Dashed line represents basal level of expression. � , P < 0.01 versuscontrol group (vehicle-treated cells).






phosphorylation was found in tumor tissue, indicating that samepathways identified in vitro are mediating the effects in vivo(Fig. 4E). Recovery profile of the proteins analyzed showed thatthe inhibitionof STAT5, 4EBP1, andBADwas sustained for at least48 hours after dosing, being S6 protein the only target that showsfaster recovery after treatment. In addition, pharmacodynamicRNA expression analysis shows a decrease in expression levels ofSTAT5 target genes CCND1, C-MYC and PIM1, and their recoveryprofile rendered C-MYC as the only repressed gene at 48 hours,indicating a correlation with STAT5 inhibition and recoveryprofile (Fig. 4F).

DiscussionSeveral kinase inhibitors have been tested for AML treatment

because deregulated tyrosine kinase activity has been implicatedin pathogenesis of hematologic malignancies (2). EC-70124 is anindolocarbazole natural product, with in vitro antiproliferativeactivity in solid tumors at IC50 values >100 nmol/L [20–23]. Herewe report antitumor activity of EC-70124 in AML. In a panelof 8 AML cell lines, 5 of them displayed IC50 < 100 nmol/L,indicating this hematological cancer could be a suitable indica-tion for EC-70124, further supported by its antitumor activity inAML subcutaneous xenograftmodels, and in patient-derived AMLprimary cells.

Kinase affinity assays revealed that EC-70124 presents anexcellent potential for AML, because its inhibition profile includestargets like FLT3—the most frequent mutated target in AML—both wild type, ITD, and kinase domain mutants, that have beenrelated to kinase inhibitors resistance (30). However, despitepromising preclinical results, FLT3 inhibitors have failed to meetexpectations in advanced clinical trials (3) and novel inhibitorstargeting not only FLT3 but also other pathways contributing todisease progression are needed. This perception is likely tobecome more attractive on the wake of the successful RATIFYtrial (and subsequent FDA approval) that demonstrates that thedirty inhibitor midostaurin prolongs survival in a subset of AMLpatients (6). In this line, EC-70124, unlike midostaurin, is apotent inhibitor of other important AML related kinases such asPIM kinases. Moreover, EC-70124, is 100 times more potentagainst FLT3 than against KIT, responsible of undesired myelo-suppression in patients (26). Ideally, FLT3 inhibitors should spareC-KIT activity, basedon the evidence that FLT3andC-KIT knockedout mice develop severe hematopoietic deficiencies (31).

In agreement to its kinase inhibition profile, EC-70124 exertsantitumor effects in several AML cell lines. Potent antiproliferativeeffect has been found in FLT3-mutated cells that also express highlevels of PIM kinases, whereas modest results have been found inFLT3 wild-type cells that do not express PIM kinases. Comparableresultswere also observed inAMLblasts derived frompatients andtreated with the drug, with best results in FLT3-mutated blast thanFLT3 wild type blast. According to the kinase inhibition data,EC-70124 abrogation of PIM kinase activity in FLT3-ITD couldhappen not only at the transcriptional level through STAT5 butalso by inhibiting PIM1 kinase itself, an important fact on thebasis of the relation between PIM activity and resistance devel-opment. Actually, this fact represents the basis of the design ofdual inhibitors as SGI-1776 (32), and more recently SEL24 (33).Moreover, patients often develop secondary point mutations onthe FLT3 receptor at the residue D835, leading to TKI resistance.Both midostaurin and EC-70124 inhibit those TK mutations, but

only EC-70124 inhibits JAK2 or SYK kinases, that have beenimplicated in other TKI resistance mechanism (34, 35).

Treatment with EC-70124 rapidly inhibits FLT3 downstreamsignaling pathways, inhibiting phosphorylation of STAT5,p70S6k and its substrate S6. Inhibition of p70S6k and its ligandoccurs in all cells, whereas STAT5 inhibition only occurs inFLT3 mutant cells. MOLM-16 presents the highest apoptosisinduction, with no reduction on STAT5 phosphorylation. Recent-ly, a novel TK gene fusion (ELAVL1-TYK2) that controls theactivation of STAT5 has been described for this cell line (36),which could explain the lack of inhibition of STAT5 phosphor-ylation in this case.

EC-70124also inhibits phosphorylationof pro-apoptotic BAD.Phosphorylation of BAD at Ser 112 could be mediated by severalintracellular pathways such us Akt, P90RSK and PIM kinases. Infact, it has beenwell described that PIM kinases promote AML cellsurvival via phosphorylation of BAD and constitutively activatedFLT3 phosphorylates BAD partially through PIM (37). Accordingto kinase binding assays, effects of EC-70124 on BAD phosphor-ylation can be related both to the inhibitory effect on FLT3 andPIM kinase activity. Moreover, the lack of activity in BAD phos-phorylation of midostaurin (that lacks activity over PIM1),together with its inhibition after treatment with SGI-1776 (dualFLT3-PIM1 inhibitor) reinforced our hypothesis about EC-70214affecting both FLT3 and PIM1. Importantly, Pim expression canbe upregulated by STAT5, indicating a close relationship betweenFLT3 and PIM. Moreover, Pim kinase overexpression has beendescribed to reduce the efficacy of FLT3 inhibitors (16). For thisreason, design of safe strategies for concomitant FLT3/Pim kinaseinhibition has been recently proposed as a promising strategy inAML (17, 33)

In addition to BAD, Pim kinases share other substrates with theAkt/mTOR pathway, modulating protein translation throughp70S6k and 4-EBP1 that represents another convergent point inthe FLT3 and Pimkinases pathway (38) that could be exploited byEC-70124. For example, 4EBP1 phosphorylation is downmodu-lated by EC-70124 treatment in AML cells, as well as pBAD. In theHL-60 cell line, phosphorylationof BADcouldbemediatedby theAkt/mTOR pathway because there is no basal PIM1 expression,leading to the higher IC50 obtained for EC-70124.

EC-70124 not only presents antileukemic effects in culturedcell lines but also reduces tumor growth in subcutaneous xeno-graft models without signs of toxicity. The compound is moreefficacious by oral frequent administration, which can inducecomplete regression of the tumor even before the end of thetreatment, than spaced intravenous injection.

Comparison between EC-70124 andmidostaurin showed sim-ilar response for both drugs in the MV-4-11 xenograft model.However, EC-70124 demonstrates a higher efficacy than midos-taurin slowing tumor growth in the FLT3wt MOLM-16 xenograftmodel, evidencing the benefits of dual PIM-FLT3 inhibition byEC-70124 beyond the FLT3-ITD tumor models. This in vivo resultis in agreement to the in vitro IC50 values and PIM1 Kd for bothdrugs. The fact that Pim knockouts in mice show minimal phe-notype changes highlights the suitability of PIM as a clean targetfor therapy (39). The more spaced schedule of EC-70124 allowslonger progression-free periods as compared with equivalentdoses of midostaurin.

Preclinical pharmacology studies demonstrate that EC-70124seems stable, without significant degradation or appearance ofnew species. Instead, several metabolites are described for






midostaurin (40). Moreover, protein plasma binding—typicallyvery high for kinase inhibitors and cited often as a potential sourceof clinical failure (41)—shows higher free unbound fraction inEC-70124 than midostaurin, a potential benefit for the clinicaltranslation of the drug. In this sense, presence of alpha-acidglycoprotein, known to bind midostaurin (42), caused a mod-erate reduction of the EC-70124 activity, as compared with mid-ostaurin. Finally, data obtained showed also a high potential forhuman gut absorption according to the Caco-2 assay.

In summary, the present report describes the antileukemicactivity and preclinical characterization of a novel multikinaseinhibitor, EC-70124, based on its ability to interfere the complexoncogenic events activated in AML at several levels. Becausetreatments directed against single targets such as FLT3 have notdemonstrated the expected effectiveness, the unique kinase inhib-itor profile exhibited by EC-70124makes it an ideal candidate forthe treatment of AML, and future studies arewarranted to evaluateits clinical efficacy.

Disclosure of Potential Conflicts of InterestF. Moris has ownership interest (including patents) in EntreChem S.L. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: P. Costales, C. Rodríguez, V. Martín, F. MorísDevelopment of methodology: N. Puente-Moncada, P. Costales, L.-E. N�u~nez,P. Oro, M.A. Hermosilla, N. Ríos-Lombardía, V. Martín

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N. Puente-Moncada, P. Costales, I. Antolín,L.-E. N�u~nez, P. Oro, M.A. Hermosilla, J. P�erez-Escuredo, N. Ríos-Lombardía,A.M. Sanchez-Sanchez, E. Lu~noAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): N. Puente-Moncada, P. Costales, I. Antolín,J. P�erez-Escuredo, A.M. Sanchez-Sanchez, C. Rodríguez, V. MartínWriting, review, and/or revision of the manuscript: P. Costales, L.-E. N�u~nez,P. Oro, M.A. Hermosilla, J. P�erez-Escuredo, N. Ríos-Lombardía, E. Lu~no,C. Rodríguez, V. Martín, F. MorísAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): P. Costales, F. MorísStudy supervision: P. Costales, F. Morís

AcknowledgmentsEntreChem thanks Dr. Atanasio Pandiella for valuable input during the

course of the project. P. Costales, J. P�erez-Escuredo, andN. Ríos-Lombardíaweresupported by Torres-Quevedo program from Ministry of Science and Innova-tion (Spain; references PTQ-11-04-507, PTQ-13-06-368, PTQ-12-05-407,respectively). C. Rodriguez was supported by Ministry of Science and Innova-tion (SAF2014-58468-R) and FICYT (GRUPIN14-081). N. Puente-Moncadawas supported by an FICYT fellowship (BP13-108).

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 June 19, 2017; revised October 19, 2017; accepted December 15,2017; published OnlineFirst January 16, 2018.

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Inhibition of FLT3 and PIM Kinases by EC-70124 Exerts ... · apoptosis. EC-70124 is orally bioavailable and displays higher metabolic stability and lower human protein plasma binding