Small Molecule Therapeutics

A Kinase Inhibitor with Anti-Pim Kinase Activity isa Potent and Selective Cytotoxic Agent TowardAcute Myeloid LeukemiaRonja Bjørnstad1,2, Reidun Aesoy1, �ystein Bruserud3, Annette K. Brenner3,Francis Giraud4, Tara Helen Dowling5, Gro Gausdal6, Pascale Moreau4,Stein Ove Døskeland7, Fabrice Anizon4, and Lars Herfindal1

Abstract

More than 40 years ago, the present standard inductiontherapy for acute myeloid leukemia (AML) was developed.This consists of the metabolic inhibitor cytarabine (AraC) andthe cytostatic topoisomerase 2 inhibitor daunorubucin(DNR). In light of the high chance for relapse, as well asthe large heterogeneity, novel therapies are needed to improvepatient outcome. We have tested the anti-AML activityof 15 novel compounds based on the scaffolds pyrrolo[2,3-a]carbazole-3-carbaldehyde, pyrazolo[3,4-c]carbazole,pyrazolo[4,3-a]phenanthridine, or pyrrolo[2,3-g]indazole.The compounds were inhibitors of Pim kinases, but couldalso have inhibitory activity against other protein kinases.Ser/Thr kinases like the Pim kinases have been identifiedas potential drug targets for AML therapy. The compound

VS-II-173 induced AML cell death with EC50 below5 mmol/L, and was 10 times less potent against nonma-lignant cells. It perturbed Pim-kinase–mediated AML cellsignaling, such as attenuation of Stat5 or MDM2 phos-phorylation, and synergized with DNR to induce AML celldeath. VS-II-173 induced cell death also in patients withAML blasts, including blast carrying high-risk FLT3-ITDmutations. Mutation of nucleophosmin-1 was associatedwith good response to VS-II-173. In conclusion newscaffolds for potential AML drugs have been explored.The selective activity toward patient AML blasts and AMLcell lines of the pyrazolo-analogue VS-II-173 make it apromising drug candidate to be further tested in preclin-ical animal models for AML.

IntroductionAcute myeloid leukemia (AML) is a highly heterogeneous

disease characterized by aberrant myeloid differentiation andsubsequent accumulation of immature myeloid cells in the bonemarrow (BM). The standard chemotherapy regimen is based onthemetabolic inhibitor cytarabin (AraC) in combinationwith thecytostatic topoisomerase 2 inhibitor daunorubucin (DNR), andhas changed little over the past 40 years (1). Intense researchefforts aim to identify drug targets that can help overcome AMLchemotherapy resistance and relapse, as well as the severe toxicside effects ofDNR (see ref. 2, for a recent review onpotential drugtargets). One of the important regulators of AML cell survival arefound in the family of three serine/threonine-protein kinases,

Pim1, 2, and 3 (3). They are involved in the regulation ofmultiple cellular processes, including cell-cycle progression andcell survival. The Pim kinases mediate signals from Abelsonmurine leukemia viral oncogene (ABL), Janus kinase 2 (JAK2),and fms like tyrosine kinase 3 (FLT3; refs. 4, 5), all oncogenes.Pim kinases lack regulatory domains, and are constitutivelyactive (6). Their activity is regulated by altering the intracellularlevels, that is by transcription and translation, or by degrada-tion (7). Together this makes them a likely Achilles heel for AMLcells dependent on a reliable communication of cytokine signalsto the nucleus in order to survive and multiply. Mice depleted ofall three Pim kinase proteins have a normal life span and arefertile, only exhibiting a slight deficient growth response with areduced body size (8). Consequently, no severe side effects frominhibition of selective Pim kinases are expected. Several Pimkinase inhibitors have been evaluated for therapeutic potentialagainst hematologic disorders, in particular multiple myelomaand AML, but also CML, ALL, and B-cell lymphoma (9, 10).Previously, we have reported the synthesis of a series of Pimkinase inhibitors exhibiting various N-containing heteroaro-matic scaffolds such as pyrrolo[2,3-a]carbazole-3-carbaldehydes,pyrazolo[3,4-c]carbazoles, pyrazolo[4,3-a]phenanthridines, andpyrrolo[2,3-g]indazoles (Fig. 1A; refs. 11–18). A broad selectionof molecular scaffolds is important in order to develop the besttherapeutic agents against a specific target. All the above-men-tioned compound classes showed acceptable IC50 values towardPim kinases (below 10 nmol/L in some cases, see Table 1). TheATP-binding pocket, along with the substrate-recognition resi-dues are identified as crucial for Pim kinase inhibitors (19).

1Department of Clinical Science, Centre for Pharmacy, University of Bergen,Bergen, Norway. 2Hospital Pharmacy in western Norway, Bergen. 3Departmentof Clinical Science, University of Bergen, Bergen, Norway. 4Universit�e ClermontAuvergne, CNRS, Sigma Clermont, ICCF, F-63000 Clermont-Ferrand, France.5Centre for Cancer Biomarkers, Department of Clinical Science, University ofBergen, Bergen, Norway. 6BerGenBio AS, Bergen, Norway. 7Department ofBiomedicine, University of Bergen, Bergen, Norway.

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

Corresponding Author: Lars Herfindal, University of Bergen, Laboratoriebyg-get, Jonas Lies vei 87, Bergen N-5021, Norway. Phone: 4755974628; E-mail:lars.herfindal@uib.no

doi: 10.1158/1535-7163.MCT-17-1234

�2019 American Association for Cancer Research.

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These compounds were reported to have activity toward AMLcells. We wanted to extend our investigation of these compoundsas potential drugs for AML, and investigate the possibilities forone or two selected compounds to enter further preclinicalinvestigations.

Materials and MethodsDescription of the kinase inhibitors

All compounds described in Fig. 1A are designed to bind inthe ATP-recognition site of Pim kinases. Their synthesis hasbeen described elsewhere (11–18). Compound B3 exhibited aselective profile when evaluated toward a panel of 66 proteinkinases (11). X-ray co-crystal structure with Pim1 (PDB code:3JPV) demonstrated that it inserted into the ATP-binding

pocket in a manner typical for non-ATP mimetic Pim inhibitors(Fig. 1B; ref. 11). A similar binding mode was observed bymolecular modeling for analogues of this series. CompoundLG-VII-126 is part of another Pim inhibitor series (Fig. 1A).Molecular modeling showed that the indazole ring insertedinto the ATP-binding pocket with high-affinity interactions inPim1 and Pim3 (16). The synthesis of a homolog seriesidentified VS-II-173 as a nanomolar Pim1 and Pim3 inhibi-tor (17). The study of its putative binding with Pim1/Pim3 bymolecular modeling demonstrated that VS-II-173 inserteddeeply into the ATP site of Pim1 and Pim3 (17).

The in vitro protein kinase screening was performed at theInternational Centre for Kinase Profiling (ICKP; Dundee, Scot-land). VS-II-173 was evaluated toward a panel of 50 proteinkinases in duplicate assays at a compound concentration of

Figure 1.

Kinase inhibitors used in this study. A, Structures of the compounds used in this study, B, X-ray co-crystal structure of compound B3 in Pim1 ATP-binding pocket(PDB code: 3JPV).

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1 mmol/L as described previously (20–22). The results wereexpressed as the percentage of residual kinase activity. Theprotein kinases were of human origin except Lck (mouse) andROCK2 (rat).

Cell culture conditionsThe following AML cell lines were utilized in this study; the

human acute monocytic leukemia cells Molm-13 (ACC, 554;ref. 23), Molm-13 cells with shRNA mediated silencing of p53(Molm-13 shp53; ref. 24), MV4-11 human monocytic leukemiacells (ATCC, CRL-9591), MV4-11 cells with silenced p53(MV4-11 shp53), the rat promyeloid leukemia wild-type celllines IPC-81 (25), IPC-81 with enforced expression of Bcl-2(both created by Dr. Michel Lanotte, Hopital St. Louis, Paris,France, and maintained by prof. Stein Ove Døskeland; ref. 26),and the human AML cell line OCI-AML3 (ATCC, ACC-582). TheMolm-13 and MV4-11 cells with silenced p53 were created byProf. Stein Ove Døskeland, Drs. Sjur Huseby, and Gro Gausdalby retroviral transfection for stable expression of shRNA againstp53 using the pRETRO SUPER-p53 vector (27). Selection was byincreasing puromycin concentration up to 400 mg/mL over 14days. All compounds were also tested on the chronic myeloidleukemia (CML) cell line K562 (ATCC, CCL-243), NRK ratkidney epithelial (ATCC, CRL-6509), and H9c2 cardio myoblasts(ATCC, CRL-1446). Molm-13 cells were cultured in RPMI medi-um with 10% FBS (Invitrogen). MV4-11 and K562 were culturedin Iscove medium added 8 mmol/L L-glutamine and 10% FBS.OCI-AML3, NRK, and H9c2 cells were cultured in DMEM,enriched with 10% FBS and the IPC cells were supplementedwith 10% horse serum. All culture media were from Sigma-Aldrich Inc., and were supplemented with 100 IU/mL penicillinand 100 mg/L streptomycin. Culturing of all cells was in ahumidified atmosphere (37�C, 5% CO2). The suspension cellswere kept at 10 to 80 � 104 cells/mL and diluted by adding freshmedium. The adherent cells were cultured until reaching 90%confluence. They were then detached by mild trypsination,centrifuged, and reseeded in fresh medium at 40% confluence.Adherent cells that had more than 12 passages were not used.The cells were tested for mycoplasma infection using MycoAlert

every second month. No positive tests were obtained in any cellline during the time of the experiments.

Assessment of cell deathCytotoxicity assays were performed in 96-well plates at 0.1mL

medium/well. Suspension cells were seeded at 0.5 � 106 cells/mL and used the day of seeding. The NRK and H9c2 cells wereseeded at 0.05�106 cells/mL, and left over night to attach beforeexperiments. The compounds were tested alone, or in combina-tions with the following cytostatics; DNR (Cerubidine;Sanofi Aventis Lysaker), Geldanamycin, Emetine (both pur-chased from Sigma-Aldrich), bortezomib (Velcade, Janssen),Etoposide (Eposin, Teva), Cisplatin (Accord Healthcare),and AraC (Cytarabin, Fresenius Kabi). The pan-Pim kinaseinhibitor AZD1208 was from Tocris Bioscience, the Bcl-2 inhib-itor Venetoclax / ABT-199 from Cayman Chemical, and thetyrosine-kinase inhibitor Midostaurin from Sigma-Aldrich. Theviability of the treated cells was assessed using a WST-1 CellProliferation Assay (Roche Applied Sciences). After recordingmetabolic activity, the cells were fixed by adding 2% buffered(PBS, pH 7.4) formaldehyde containing 0.1% of the DNA-specific Hoechst 33342 (0.01 mg/mL; Sigma–Aldrich) to visu-alize the nuclei. Cell death was assessed by UV-microscopicevaluation of nuclear morphology (28), and the data from themetabolic activity were used to confirm the data from micros-copy. EC50 values were estimated by nonlinear regression usingthe statistical software IBM SPSS statistics for Apple, ver. 24,(IMB Corp.):

Y ¼ minþ max �minð Þ1þ EC50

X

� �h ; ð1Þ

where Y is the response (cell death); max and min, the maximumand minimum cell death from the curve; X the concentration ofthe drug/compound; and h is the hill index. Determination ofcoefficient of drug interaction (CDI)was done by finding the ratiobetween the effect of treatment and control (R) and furthercalculates as shown in Eq. 2.

CDI ¼ RCombination

RDrug1 � RDrug2ð2Þ

A value around one indicates additive effect, values below 0.7demonstrate clear synergy, and higher than one antagonisticeffect.

Membrane permeability assayThe Corning Gentest pre-Coated parallel artificial membrane

permeability assay PAMPA Plate System (Corning DiscoveryLabware) was done according to manufacturer's protocol. Thecompounds were tested at 50 mmol/L in PBS at 21�C. Afterincubation for 5 hours, 50 mL of each sample were added 15 mLacetonitrile, and injected into a reversed phase HPLC column(Kromasil 100-5 C18 150-4.6 mm; Akzo Nobel) connected to aMerck-Hitachi LaChrome HPLC-system (VWR) with a L-7455diode array detector. Mobile phase A was 0.05% aqueousTFA, and mobile phase B was acetonitrile. The flow rate was1.6 mL/min and compounds eluted between 1 and 4.5 minutesduring a 4.5 minutes gradient from 70:30% mobile phase A:Bto 0:100% mobile phase A:B. The ratios of the compound con-centrations in the donor and acceptor compartments were calcu-lated after integration of the peaks at 248 nm. Peff was calculatedas described in ref. 29.

Table 1. Ability of the novel compounds to inhibit activity of Pim-1, 2, and 3kinases

IC50 (mmol/L)Pim1 Pim2 Pim3

B3 (11)a 0.12 0.51 0.01RAG-II-45 (11) 0.0068 0.131 0.0124RAG54 (12) 0.57 >10 0.04RAG-II-115 (13) 0.075 >1 0.08FG587 (14) 0.009 >1 0.026MB032 (14) 0.042 >1 0.05MB071 (15) 1.4 nd 0.38MB069 (15) 1.56 nd 0.6FG644B (15) 0.39 nd 0.19EE057 (15) 1.1 nd 0.7FG610 (15) 0.74 nd 0.34FG773 (15) >1 nd 2.9LG-VII-126 (16) 0.04 49% (1 mmol/L)b 0.10VS-II-173 (17) 0.07 46% (1 mmol/L)b 0.02LG-VI-142 (18) 0.46 >10 0.033

nd, not determined.aThe numbers in parentheses refer to the citation in the reference list.b% residual kinase activity at 1 mmol/L.

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Western blot analysisWestern blotting was conducted as described previously (30).

To ensure that we studied effects of kinase inhibition, we choseconcentrations that gave less than 30% apoptosis. This wouldavoid loss of proteins due to caspase cleavage or disintegratedcells. After treatment with Pim kinase inhibitors, the cells werewashed and lysed, and protein concentrations determined usingthe Quick Start Bradford protein assay (Bio-Rad Laboratories).SDS-polyacrylamide gels, 10% or 12% were loaded with 30to 50 mg protein, and the prelabeled "Precision Plus ProteinStandard All Blue" (Bio-Rad Laboratories). Proteins were trans-ferred to polyvinylidene difluoride membranes (GE HealthcareLife Sciences) by electrophoresis. The following primary antibo-dies were used: Pim1, Pim2, and Pim3 (all from Santa CruzBiotechnology, except for on OCI-AML3 cells, where Pim2 anti-body from Sigma-Aldrich was used), Bad, pBad phospho S112,4EBP1, p4EBP1 phospho T37/46, STAT5 and pSTAT5 phosphoY694, Akt and Akt phospho S473, p70 S6 kinase and p70 S6kinase phospho S371 (from Cell Signaling Technology), MDM2and pMDM2 phospho S166 antibody (Abcam plc), anti-b-actin(Sigma-Aldrich). The secondary antibodies were donkey anti-mouse and anti-rabbit antibodies conjugated to horse-radishperoxidase (Jackson Immunoresearch Laboratory). Membraneswere developed using Supersignal West Pico or West FemtoChemiluminiscence Substrate from Pierce Biotechnology Inc.,according to the manufacturers' protocols. The immunoblotswere imaged by a LAS-4000 image reader (Fujifilm). After imag-ing, the membranes were stripped using Restore PLUS WesternBlot Stripping Buffer (ThermoFisher Scientific) to reprobe themembranes with the antibody for the phosphorylated version ofthe proteins. Anti-b-actin was used for loading control.

Flow cytometric analysis of AML patient materialThe collection of patient cells was approved by the regional

research ethics committee (Health Region III, Bergen, Norway,REKapproval numbers for biobanking: III 060.02215.03, 231.06,and 2015/1410, and for experimental use of cells: REK Vest 2015/1759 and 2017/305) and conducted in accordance with theDeclaration of Helsinki. Samples were collected after writteninformed consent, and stored under liquid nitrogen in biobanksapproved by the Norwegian Royal Ministry of Health and theNorwegian Directorate for Health and Social Affairs. Blasts from37 patients were used in this study. The patients were not selectedafter response to chemotherapy. All patients had a high count ofAML blasts in peripheral blood, and blasts were isolated bydensity gradient separation of peripheral blood (Lymphoprep;Axis-Shield). After isolation, the samples contained at least 95%leukemic blasts. The BM aspirate was from a healthy donor andkindly provided by Professor BT Gjertsen (Health Region III; REKapproval number: 2012/2247). Peripheral blood mononuclearcells (PBMC) were obtained from freshly drawn peripheral blood(Department of Immunology and Transfusion Medicine, Hauke-land University Hospital, Bergen, Norway) and isolated by den-sity gradient separation (Lymphoprep; Axis-Shield). Erythrocyteswere lysed (BD Biosciences Pharm Lyse buffer) and leukocyteswere suspended in Minimum Essential Medium Eagle (MEME),enriched with 10% FBS and 2 mmol/L L-glutamine, and supple-mented with 100 IU/mL penicillin and 100 mg/L streptomycin(all from Sigma-Aldrich Inc.). Upon the experiment AML blasts ornormal BM cells were thawed and suspended in Stem Span SFEMmedium (Stem Span Technologies), whereas PBMCs were used

fresh, and suspended in MEME medium. Treatment with VS-II-173 and DNR were for 24 hours. Drug-induced cell death wasassessed by flow cytometric analysis of cells stained with Pacific-Blue-AnnexinV (#640918; BioLegend) and propidium iodide (PI;#421301; BioLegend). Between 10,000 and 30,000 events werecollected for each sample using a BD Fortessa flow cytometer. Thedata were analyzed with the software Flow Jo (Tree Star, Inc.).

Statistical analysisResponse to treatments were evaluated by Student t test, cor-

relation of data by two-tailed Pearson correlation test, and patientdata by cross-table analyses for chi-square, all by using IBM SPSSstatistics for Apple, ver. 24 (IMB Corp.). Significance was definedas P� 0.05. Heatmaps were created byMeV (31), analyses of Pimexpression was done using the J-Express 2012 software (32) afternormalization and log(10) transformation of data. Kinome treewas created using the Kinome Render software (33).

ResultsCharacterization of anti-AML activity of kinase inhibitors

We previously found that pyrrolo[2,3-a]carbazole-3-carbalde-hyde Pim kinase inhibitors (15) had promising activity againstAML cells. We therefore decided to test the efficacy of an in-houseselection of Pim inhibitors containing different hetero-aromaticscaffolds (Fig. 1A) against a panel of cell lines (Fig. 2A). Theinhibitor VS-II-173was found to be a potent and selective inducerof AML cell death. The cardiomyoblasts, epithelial cell lines, andthe chronic myeloid leukemia cell line K562 were unaffected atVS-II-173 concentrations inducing 100% death in the AML cells.The compounds MB032, LG-VI-142, LG-VII-126, RAG54, andFG587 also showed selectivity toward AML cell lines, but wereless potent than VS-II-173. The compound RAG-II-115 was apotent inducer of cell death, but did not discriminate betweenAML-derived and other cell lines (Fig. 2A). VS-II-173 was able toovercome the prosurvival effect of poor prognostic factors in AMLsuch as FLT3-ITD expression (MV4-11 and Molm-13 cells), p53silencing (Molm-13 shp53 and MV4-11 shp53) and, to someextent, Bcl-2 overexpression in IPC-81 cells (Fig. 2A). To find if thedifferences in efficacy was due to the compound's ability topenetrate the cellular membrane, we tested the membrane per-meability using the PAMPA assay. Membrane permeability wasclassified by the range defined by Bennion and colleagues (29),which divides compounds into four groups: high permeability(LogPeff>�5.33), intermediate permeability (LogPeff>�5.66 to<�5.33), low permeability (LogPeff > �6.14 to < �5.66) imper-meable (LogPeff < �6.14). The compounds RAG-II-115 andMB032 showed intermediate to high efficacy toward cells(Fig. 2A) and intermediate to high membrane permeability(Fig. 2B), whereas compound VS-II-173 had high efficacy towardcells, but low membrane permeability. There were also com-pounds with low efficacy toward cells, and low membrane per-meability, like RAG-II-45, FG587, RAG54, andMB071. However,the compounds MB069, FG644B, EE057, and FG610 all showedhigh membrane permeability (Fig. 2B), but low ability to inducecell death (Fig. 2A). We also checked whether there were largedifferences between microscopic assessment of nuclear morphol-ogy and metabolic conversion of the WST-1 reagent (Fig. 2C andD). The WST-1 assay gave a lower EC50 value compared withmicroscopic evaluation, presumably due to its ability to detect cellgrowth inhibition. Still there was a good correlation between the

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Figure 2.

Ability of different compounds to induce cell death in AML cell lines.A, The cells were incubated for 24 hours with the compounds (30 mmol/L) beforeassessment of metabolic activity using theWST-1 proliferation assay and then fixed in 2% buffered formaldehyde containing the DNA stain Hoechst 33342.Apoptotic cells were identified and quantified by microscopic evaluation of cell nuclei. B,Membrane permeability (LogPeff) of the different compounds,measured by the Corning Gentest precoated parallel artificial membrane permeability assay PAMPA Plate System. C and D, Dose–response curves of Molm-13cells with VS-II-173 over 24 hours. The same experiments were assessed by both microscopy (C) and metabolic activity byWST-1 assay (D) and EC50 valuesdetermined by nonlinear regression. The inset in C shows surface and nuclear morphology of Molm-13 cells after 24 hours of treatment with either solvent (upperimages) or 10 mmol/L VS-II-173. The bars indicate 9 mm. The inset inD shows correlation between theWST-1 signal and apoptotic morphology, using PearsonCorrelation statistics (two tailed, P > 0.05). E,Molm-13 cell death kinetics of different kinase inhibitors. The cells were incubated with the different compounds,and aliquots sampled and fixed at the given time-points and apoptotic cells quantified by microscopic evaluation of nuclear morphology. The data inA–E arefrom three to five separate experiments and SD (B–D). F, Kinase screen of the residual activity of different kinases after treatment with 1 mmol/L of VS-II-173. Thesize of the circles is inversely proportional to the residual activity of the kinases. The tree was generated using the Kinome Render software, and the illustrationreproduced courtesy of Cell Signaling Technology (www.cellsignal.com). A table with the values from F is available as supplementary information(Supplementary Table S1).

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two methods (Pearson's correlation test), as a decline in WST-1signal was always accompanied with increase in apoptosis (seeinset in Fig. 2D). The cell death induced by VS-II-173 had typicalapoptotic morphology like cell shrinkage, nuclear condensation,and fragmentation (inset in Fig. 2C).

We next assessed the time kinetics of the cytotoxic action byselected compounds at concentrations giving between 80% and100% death after 24 hours. The times to obtain 50% apoptoticcells were similar (ranging from 4.4 to 6 hours) for the AML-specific compounds (Fig. 2E). RAG-II-115 induced a much morerapid cell death (about 2 hours; Fig. 2E).

All compounds showed high activity toward the Pim kinases(Table 1), and we chose the most promising compound from theinitial cell experiments, VS-II-173, to be evaluated toward 50protein kinases at 1 mmol/L concentration (Fig. 2F; Supplemen-tary Table S1). This revealed that the compound had activitytoward other kinases as well. The most notable besides Pim1 and3 was the dual specificity tyrosine phosphorylation regulatedkinase 1A (DYRK1a), AMP-activated kinase (AMPKa1), home-odomain interacting protein kinase 2 (HIPK2), protein kinaseC-related protein kinase 2 (PRK2), and ribosomal protein S6kinase A1 (RSK1), all having less than 10% residual activity aftertreatment with 1 mmol/L of VS-II-173.

VS-II-173 acts synergistically with the anthracyclinedaunorubicin, and additivelywith anumber of other anticancerdrugs

To establishwhether our kinase inhibitors could have potentialin combination therapy,we chose the twomost potent of theAMLselective compounds, VS-II-173 andMB032.We found that VS-II-173 at subtoxic concentration increased the Molm-13 AML celldeath from 40% to 80% when combined with 50 nmol/L dau-norubicin (DNR) for 24 hours (Fig. 3A). The compound MB032did not synergize with DNR (Fig. 3A).

We next tested VS-II-173 in combination with other anticancerdrugs. The best synergy for VS-II-173 was with DNR (Fig. 3B andC), only amodest synergy being observed with cisplatin (Fig. 3B).It was also noted that high concentrations of the proteasomeinhibitor bortezomib antagonized the action of VS-II-173 (Fig. 3Band D), whereas lower concentrations gave an additive effect(Fig. 3D). The other drugs tested (etoposide, emetine, and gelda-namycin) exhibited additive effects in combination with VS-II-173 (Fig. 3B and E).

The first test on the panel of AML cells (Fig. 2A) revealed thatcells with silenced p53 had reduced, but not abolished sensi-tivity to the compounds tested, including VS-II-173. Wewanted to investigate if lack of p53 could affect the synergybetween VS-II-173 and DNR. We found that DNR synergizedwell with 4 mmol/L VS-II-173, both against Molm-13 WT andMolm-13-shp53 cells, but also that a higher concentration ofboth drugs was needed to achieve a substantial synergisticeffect (Fig. 3F and G).

Next, the anti-AML activity of three other compounds wastested. A total of 50 mmol/L of the pan-Pim kinase inhibitorAZD1208 induced around 60% cell death in MV4-11 cells after24 hours incubation, whereas the other cell lines were mostlyunaffected (Fig. 3H). The Bcl-2 inhibitor venetoclax was effi-cient toward Molm13 and MV4-11 in the low nanomolar range,but were nontoxic to the OCI-AML3 cells at the concentrationstested (Fig. 3I). Finally, the AML drug midostaurin was tested.This multitarget kinase inhibitor was efficient toward Molm13

and MV4-11 cells, but to a small degree toward OCI-AML3(Fig. 3J).

VS-II-173 attenuates phosphorylation of Pim kinase substratesPim kinases have been shown to act downstream of FLT3

signaling (5), and we compared the efficacy of VS-II-173 on AMLcell lines with different FLT3 mutation status. The VS-II-173 hadhigher efficacy toward cell lines harboring the FLT3-ITDmutation(Molm-13, FLT3-ITD heterozygous, and MV4-11 FLT3-ITDhomozygous), with EC50 values between 2 and 3 mmol/L(Fig. 4A and B, respectively). The OCI-AML3 cells (FLT3 WT)were less sensitive to VS-II-173, with EC50 about 16 mmol/L(Fig. 4C).

To study early events (the initiation phase) of the apoptosisprocess triggered by VS-II-173, we used concentrations that wouldproduce apoptosis after 5 to 10 hours, in line with previousstudies on for instance anthracycline and AML (34, 35). Whenexamining the expression of Pim1, 2, and 3 in the AML cell linestreated with VS-II-173, we noted a rapid, but transient down-regulation of all three kinases in Molm13 cells (Fig. 4D, seeSupplementary Fig. S1 for quantification). The response was lessprofound in MV4-11 and OCI-AML3 cells (Fig. 4E and F). Fur-thermore, theMolm13 andMV4-11 cells showed a rapid dephos-phorylation of Stat5 (Fig. 4G and H) upon treatment with VS-II-173. The MV4-11 cells showed a decrease of the prosurvivalphospho-Bad form of the otherwise apoptogenic BH3 only pro-tein Bad (Fig. 4H) and the OCI-AML3 cells showed a strongdecrease in p-4E-BP1 and pMDM2 (Fig. 4I).

Blasts frompatientswithAMLwithpoorprognosticmarkers aresensitive to the kinase inhibitor VS-II-173

To better assess the potential of VS-II-173 as a lead com-pound for the treatment of AML, it was tested on a panelof nonselected AML patient blasts. The blasts were treated withVS-II-173 alone, or in combination with DNR. Our experiencewith AML patient blasts is that they are generally more resistantto cytostatics compared with most AML cell lines, and in orderto ensure an effect, we increased the doses of both VS-II-173and DNR. The age of the patients were from 19 to 86 years, witha median of 69 years. They were classified using the French–American–British (FAB) classification (36), and cytogeneticsanalyzed based on the MRC classification (37). Elevenpatients had FLT3-ITD (high-risk characteristic), 21 had WTFLT3, and 3 were not tested. Ten patients had insertion muta-tion in NPM-1 (favorable prognostic factor), and 22 had wild-type NPM-1 (three not tested). See Fig. 5A for further patientinformation.

The results from theAMLpatient blast experiments showedonelarge group that responded well to treatment with VS-II-173,where doses between 6 and 24 mmol/L could kill more than50% of the blasts (Responders, Fig. 5). Some of the VS-II-173responding patient blasts also responded well to the DNR(patients 11 and 24). In some cases, even the highest concentra-tion of DNR (100 nmol/L) did not affect the blast viabilitywhereas VS-II-173 showed high potency against these patientblasts (patients 1 and 2). Another group responded to DNR, butnot to VS-II-173, for instance patient 24 and to some extentpatient 25. There were two groups that did not appear to benefitfrom VS-II-173. One, which appeared to be resistant to bothDNRand VS-II-173, like patients 36 and 37, and one where thecombination gave a reduced effect (patients 22 and 23). Despite

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this, the majority of the patients (24 out of 37) could be char-acterized as responders to VS-II-173.

Neither sex, age, nor the FAB classification seemed to influencethe responsiveness of the patient blasts to VS-II-173 (Fig. 5A). Thethree patients with adverse cytogenetics (patients 20, 30, and 31)showed poor to intermediate response to VS-II-173, whereas thetwo patients with good risk cytogenetics showed high response.Patients with FLT3-ITD were represented in the responder group(8 of 20, 40%) of the patients with known status, and in theintermediate/non responder group (3 of 13, 23%), but there wasno significant difference between the occurrence of FLT3-ITD in

the responder and nonresponder groups (Pearson chi-square ¼1.55, P ¼ 0.21). Furthermore, the patients with mutated NPM-1were overrepresented in the responder group (9 of 19 patients(47%) in the responder group, and 1 of 14 patients (7%) in theintermediate/non responder group, Pearson chi-square ¼ 6.175,P ¼ 0.013). Three-way chi-square test of our data showed NPM1statuswas important for response toVS-II-173 inpatientswithWTFLT3 (Pearson chi-square ¼ 4.89, P ¼ 0.27), but not in patientswith FLT3-ITD (Pearson chi-square ¼ 0.749, P ¼ 0.387).

Compared with the responder group, BM aspirates or PBMCwere less affected by VS-II-173 (Fig. 5A), but the AML blasts from

321.21.0

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Drug interactions between VS-II-173 andseven anticancer compounds. A,Molm-13cells were treated with VS-II-173 (1 mmol/L),MB032 (100 mmol/L) in combination withDNR. After 24 hours, the cells were fixed andviability was assessed by microscopicevaluation of apoptosis using Hoechst33342 DNA stain. The vertical dotted linesindicate EC50 values. B,Molm-13 cells weretreated with 1 mmol/L VS-II-173 incombination with different anticancercompounds for 24 hours before assessingviability as inA. The CDI was calculated asdescribed in the section Materials andMethods. The concentrations of thecompounds tested were: Geldanamycin,10 nmol/L; Etoposide, 0.6 mmol/L; Emetine,5 nmol/L; DNR, 75 nmol/L; Cisplatine,6 mmol/L; AraC, 6 mmol/L, Bortezomib,10 and 3 nmol/L. In B, ��� indicatesP < 0.001, one-sample T test. a: Forbortezomib, the CDI for the highconcentration is shown. C–E, Examples ofdrugs which act synergistically (C; DNR),antagonistically (D; bortezomib), oradditively (E; Etoposide). F and G,Synergistic activity between VS-II-173 andDNR inWTMolm-13 AML cells (left) orsilenced p53 (shp53, right). The cells wereincubated with increasing doses of DNRwith or without 2 or 4 mmol/L VS-II-173 for24 hours. The insets show induction of p53by DNR expression inWT-cells (F), but notin shp53 cells (G). Viability was assessed asinA. The data inA–G are the mean and theSD from 5 to 12 experiments. H–J, Cell deathinducing effect of venetoclax (H),midostaurin (I), and AZD1208 (J) ondifferent cell lines after 24 hours incubation.Cell death was scored using theWST-1 assayand is relative to untreated cells. The dataare average of two (Molm13, OCI-AML3, andMV4-11) or four (NRK) experiments and thelines are from four-parameter regressionanalysis as described in the section Materialsand Methods.

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two patients classified as nonresponders had equal or slightlylower response compared with the PBMC or BM cells.

We found no correlation between response to VS-II-173 andPim kinase expression level (Fig. 5B), based onmRNA expressionof Pim kinases related to the median expression in patients (nosignificance using Pearson Chi-Square statistics).

DiscussionOf the 15 compounds we tested, VS-II-173 exhibited potent

selective activity toward the AML cell lines. This could theoreti-

cally be explained by the compound selectively targeting rapidlygrowing cell lines, but the K562 CML cell line has a doubling timesimilar to that of Molm-13 cells, thus rapid proliferation does notseem to be the reason for the AML-selectivity observed in Fig. 2A.Importantly, VS-II-173 was active toward cells with high-riskcharacteristics like decreased expression of p53 (Molm-13 shp53andMV4-11 shp53; Figs. 2A and 3G), and cells overexpressing thesurvival protein Bcl-2 (IPC-81-Bcl2; Fig. 2A) as well as FLT3-ITD(Figs. 2A, 4A–C, and 5). We could not detect any correlationbetween the ability to inhibit the different Pim kinases and theability to selectively induce AML cell death (Table 1; Fig. 2A). For

Figure 4.

Modulation of cell signaling in AML cell lines Molm-13, MV4-11, and OCI-AML3 effectuated by VS-II-173. A–C, Dose–response curve of VS-II-173 on Molm-13 (A),MV4-11 (B), and OCI-AML3 (C). The cells were incubated with various concentrations of VS-II-173 for 24 hours and then fixed in 2% buffered formaldehydecontaining the DNA stain Hoechst 33342. Apoptotic cells were identified by microscopic evaluation of nuclear morphology. See section Materials and Methodsfor description of cell lines. Data are presented as mean of two to five separate experiments and SD. D–F,Western blot analysis of Molm-13 (D), MV4-11 (E), andOCI-AML3 (F) cells treated with 3, 6, or 12 mmol/L VS-II-173 for 1, 3, or 6 hours. Total cell lysates were prepared and immunoblot analyses were conducted for theexpression levels of Pim1, 2, and 3. G–I, Western blot analysis of Molm-13, MV4-11, and OCI-AML3 cells treated with 3, 6, or 12 mmol/L VS-II-173 for 1, 3, or 6 hours.Cell lysates were prepared and immunoblot analyses were conducted for the expression levels of Bad, pBad, 4E-BP1, p4E-BP1, Stat5, pStat5, MDM2, and pMDM2in Molm-13 (G), MV4-11 (H), and OCI-AML3 (I) cells. b-Actin was used as loading control on all blots.

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instance, LG-VII-126 and VS-II-173 had similar inhibitory activitytoward the three kinases, but VS-II-173was a farmore potent AMLcell death inducer than LG-VII-126. This could partly be explainedby differences in the ability to cross phospholipid membranes(Fig. 2B).

The kinase screen showed that VS-II-173 also inhibited otherkinases than the three Pim kinases (Fig. 2F). The level ofpromiscuity was similar to that shown for the pan-Pim kinaseinhibitor SGI-1776 (38), which is active against AMLcells (39).

Many chemotherapeutic therapy regimens for AML are basedon drug combinations, such as the 7þ3 regimen with DNR andAraC or high-dose AraC plus a nucleoside analogue (see refs. 40,41 for recent reviews on established and emerging therapiesfor AML). It became apparent that the most potent compoundselective for AML cells, VS-II-173 increased the cellular responseto DNR with high synergy (Fig. 3A–C). Combination withcisplatin and VS-II-173 gave more modest synergy (Fig. 3B).

Thus, combinations of VS-II-173 with DNR or possibly cisplat-in could be clinically relevant. This agrees with a previous studyby Doshi and colleagues showing that Pim kinase inhibitionsensitized AML cell lines to DNR (42). Furthermore, our resultsindicate that Ara-C and VS-II-173 have sub-additive action,suggesting they should not be combined simultaneously. OtherPim kinase inhibitors have acted both in a synergistic andantagonistic manner in combination with AraC in AML celllines and primary AML cells (42, 43).

The transiently reduced level of Pim1, 2, and 3 kinases in theMolm-13 cells in response to VS-II-173 (Fig. 4D) indicates thatinhibition of Pim kinases increases degradation or attenuatesproduction of the protein. Pim kinase activity is not regulatedby post-translational modifications or allosteric modulators, butby increased or attenuated expression (7). Treatment with VS-II-173 apparently perturbed the ratio between synthesis and deg-radation of the Pim kinases, but not irreversibly, suggesting thatthis was not due to cell-death associated protein degradation. The

Figure 5.

VS-II-173 induces cell death in blasts from patients with AML. A, Patient blasts and BM cells were suspended at 2� 106 cells/mL in StemSpan culture medium,whereas PBMCwere suspended in MEMEmedium, and plated at 100,000 cells/well in 96-well plates before adding VS-II-173 at 6, 12, and 24 mmol/L alone or incombination with DNR, for 24 hours. Apoptosis was assessed by flow cytometric analysis of cells stained with PacificBlue-AnnexinV and PI. Between 10,000 and30,000 events were collected for each sample on a BD Fortessa flow cytometer. B,Median levels of Pim1 to 3 mRNA in selected patients compared with theirresponse to VS-II-173. Abbreviations: F, female; M, male; N, normal; I, intermediate; A, adverse; ITD, FLT3 internal tandem duplications; INS, insertion mutation inNPM-1; nt, not tested.

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compensation mechanisms and subsequent restoration of thePim kinase levels appears presumably too late to initiate thenecessary survival mechanisms, since the cells died after eight totwelve hours (Fig. 2E).

Cells with the FLT3-ITD mutation mediate aberrant signal-ing through the Pim kinases (4). Thus, inhibition of Pimrepresents an attractive approach to target FLT3-ITD cells. Pimkinases promote survival of AML cells via phosphorylation ofBcl-2 antagonist of cell death (Bad), and also 4E-BP1 (4, 39),in accordance with reduced phosphorylation of these proteinsseen upon VS-II-173 treatment, and by previous findings byChen and colleagues (39). It has been demonstrated thatPim1 kinase directly affects the FLT3 receptor by a stabilizingserine phosphorylation (44). This positive feedback loopprompts Stat5 activation and Pim signaling (44). We foundabolished phosphorylation of Stat5 in FLT3-ITD cell linesMolm-13 and MV4-11 (Fig. 4G and H). We did not detectY694 phosphorylated Stat5 in OCI-AML3 (Fig. 4I), which haswild-type FLT3. Elevated levels of Pim1 kinase induce p53signaling and MDM2 phosphorylation (45). Accordingly, ourresults showed reduced phosphorylation in MDM2 when usingVS-II-173 in Molm-13 cells (Fig. 4G). Furthermore, we foundthat both VS-II-173 and the pan-Pim kinase inhibitor AZD1208reduced the phosphorylation of Akt and p70-S6 kinase (seeSupplementary Fig. S2), however VS-II-173 had a higher poten-cy toward AML cell lines than AZD1208 in mono-treatment(Fig. 3H and Fig. 4A–C).

Itmust be noted that some of the perturbations in cell signalingcould be caused by inhibition of other kinases. RSK1 was signif-icantly inhibited by VS-II-173 (Fig. 2F). RSK1 is a mediator of theanti-apoptotic function of FLT3-ITD through phosphorylation ofBad at Ser112 in AML cells (46). The reduction of pBad (Fig. 4H)could be due to inhibition of RSK1 along with Pim kinaseinhibition. AMPK is a stress sensor in cells, and can contributeto initiation of autophagy, and protects AML-cells frommetabolicstress (47). AMPK can thus be an important target for AMLtherapy, particularly to prevent leukemogenesis, although anoth-er study shows that activation of AMPK initiates autophagic celldeath (48). DYRK1 is a tumor suppressor, which is heavily down-regulated in AML patients (49), andmutations in the HIPK2 geneis linked to pathogenesis of AML (50). Taken together, the anti-AML activity can be inhibition of Pim kinase activity acting inconcert with additional inhibition of some or all of the above-mentioned factors.

The fact that VS-II-173 affects more than one target is in linewith several of the so-called precision therapies, like the tyro-sine kinase inhibitors. In fact, the second-generation kinaseinhibitor Dasatinib has a higher level of promiscuity than itspredecessor Imatinib (51). With this in mind, a possibleexplanation for the potency and selectivity of VS-II-173 canbe its activity toward key AML survival factors like RSK1 andAMPK together with its pan-Pim kinase activity. The muchmore selective Pim kinase inhibitor AZD1208 (52) was lessefficient toward our AML cell lines than VS-II-173 (Fig. 3H),perhaps due to the broader range of substrates of the latter. Pan-kinase inhibition is believed to be favorable to the multityr-osine kinase inhibitor midostaurin (see ref. 53; Fig. 3J). Themono-selective Bcl-2 inhibitor venetoclax (54), which emergesas therapy against AML (55), failed to induce cell death in OCI-AML3 cells (Fig. 3I) and appears to be effective against differentsubtypes of AML compared to VS-II-173.

A panel of nonselected AML patient blasts was included and 24of 37 patient responded well to the treatment with VS-II-173,where doses between 6 and 12 mmol/L could kill more than 50%of the blasts (Fig. 5). Approximately 40% of patients with AMLwith FLT3-ITD mutation also harbor the NPM1 mutation (56).Our data showed NPM1 status was important for response to VS-II-173 in patients with FLT3-WT, but not in patients with FLT3-ITD. Thus, the presence of the high-risk factor FLT3-ITD is notinfluenced by the low-risk factor NPM1 mutation when it comesto response to VS-II-173. These findings suggest that this com-pound can be used irrespective of FLT3 status. There was nocorrelation between the response to VS-II-173 and the level ofPimkinases in thepatient blasts (Fig. 5B),which could be ascribedthe effect on other kinases (Fig. 2F). However, the values in Fig. 5Bwere compared with a pooled average, and AML cells are expectedto have higher levels of Pim kinases than normal BM (3).

ConclusionNew strategies for AML therapy are urgently needed. Despite

this, only one new drug has been developed and approved forAML during the last 15 years. Unfortunately, it appears to yieldonly modest improvement in survival, even in preselectedpatients (57). Our data point to pyrazolo[4,3-a]phenanthridineas a relevant scaffold for compounds useful in the treatment ofAML. The lead compound VS-II-173 exhibited crucial propertiesneeded to exert therapeutic effect. It is selective toward AMLcells, in which it efficiently dampens prosurvival signals. It is alsopotent against AML patient blasts, including some that showedhigh tolerance to DNR, and blasts carrying mutations associat-ed with poor prognosis. The compound and future analogs ofpyrazolo[4,3-a]phenanthridines represent possible candidates forfuture AML treatment. Future studies will aim to reveal possiblebiomarkers of the patient AML cells which responded well totreatment with VS-II-173, as well as conducting preclinical studieson human AML xenografts in mice. If the abovementioned issuesare resolved, we believe that pyrazolo[4,3-a]phenanthridineshave a future in AML treatment.

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

Authors' ContributionsConception and design: R. Bjørnstad, �. Bruserud, L. HerfindalDevelopment of methodology: R. Bjørnstad, G. Gausdal, S.O. Døskeland,L. HerfindalAcquisition of data (acquired andmanagedpatients, provided facilities, etc.):�. Bruserud, F. Giraud, T.H. Dowling, P. Moreau, F. Anizon, L. HerfindalAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Bjørnstad, R. Aesoy, �. Bruserud, A.K. Brenner,S.O. Døskeland, L. HerfindalWriting, review, and/or revision of the manuscript: R. Bjørnstad, R. Aesoy,�. Bruserud, P. Moreau, S.O. Døskeland, F. Anizon, L. HerfindalAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. HerfindalStudy supervision: L. Herfindal

AcknowledgmentsThe authors want to thank Ing. Nina Lied Larsen and Kaja Ska

�lnes Knudsen,

MSc for technical assistance, and Sjur Huseby, PhD for assistance with gener-ation of the AML cell lines. Prof. Bjørn Tore Gjertsen and Misbah Sabir, MScare thanked for supplying bone marrow samples. Kathrine Åsrud, PhD isthanked for critically reading through themanuscript. This study was supported

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financially by the Norwegian Western Regional Health Authorities to LarsHerfindal (Grant No. 912052), Ronja Bjørnstad (Grant No. 912028), SteinO.Døskeland (Grant No.: 303485), and�ystein Bruserud (Grant No. 911788),by the Norwegian Cancer Society to Lars Herfindal (Grant No. 112502),Stein O. Døskeland (Grant No. 705268), �ystein Bruserud (Grant No.62370, 62371, and 100933), and by the Norwegian Research council to LarsHerfindal and Reidun Aesoy (Grant No. 254752).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedDecember 13, 2017; revisedMay5, 2018; accepted January14, 2019;published first January 24, 2019.

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Mol Cancer Ther; 18(3) March 2019 Molecular Cancer Therapeutics578

Bjørnstad et al.

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2019;18:567-578. Published OnlineFirst January 24, 2019.Mol Cancer Ther   Ronja Bjørnstad, Reidun Aesoy, Øystein Bruserud, et al.   Selective Cytotoxic Agent Toward Acute Myeloid LeukemiaA Kinase Inhibitor with Anti-Pim Kinase Activity is a Potent and

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