A Kinase Inhibitor with Anti-Pim Kinase Activity Is a Potent And

Published OnlineFirst January 24, 2019; DOI: 10.1158/1535-7163.MCT-17-1234

Small Molecule Therapeutics Molecular Cancer Therapeutics A Kinase Inhibitor with Anti-Pim Kinase Activity is a Potent and Selective Cytotoxic Agent Toward Acute Myeloid Leukemia Ronja 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 induction VS-II-173 induced AML cell death with EC50 below therapy for acute myeloid leukemia (AML) was developed. 5 mmol/L, and was 10 times less potent against nonma- This consists of the metabolic inhibitor cytarabine (AraC) and lignant cells. It perturbed Pim-kinase–mediated AML cell the cytostatic topoisomerase 2 inhibitor daunorubucin signaling, such as attenuation of Stat5 or MDM2 phos- (DNR). In light of the high chance for relapse, as well as phorylation, and synergized with DNR to induce AML cell the large heterogeneity, novel therapies are needed to improve death. VS-II-173 induced cell death also in patients with patient outcome. We have tested the anti-AML activity AML blasts, including blast carrying high-risk FLT3-ITD of 15 novel compounds based on the scaffolds pyrrolo mutations. Mutation of nucleophosmin-1 was associated [2,3-a]carbazole-3-carbaldehyde, pyrazolo[3,4-c]carbazole, with good response to VS-II-173. In conclusion new pyrazolo[4,3-a]phenanthridine, or pyrrolo[2,3-g]indazole. scaffolds for potential AML drugs have been explored. The compounds were inhibitors of Pim kinases, but could The selective activity toward patient AML blasts and AML also have inhibitory activity against other protein kinases. cell lines of the pyrazolo-analogue VS-II-173 make it a Ser/Thr kinases like the Pim kinases have been identiﬁed promising drug candidate to be further tested in preclin- as potential drug targets for AML therapy. The compound ical animal models for AML.

Introduction Pim1, 2, and 3 (3). They are involved in the regulation of Acute myeloid leukemia (AML) is a highly heterogeneous multiple cellular processes, including cell-cycle progression and disease characterized by aberrant myeloid differentiation and cell survival. The Pim kinases mediate signals from Abelson subsequent accumulation of immature myeloid cells in the bone murine leukemia viral oncogene (ABL), Janus kinase 2 (JAK2), marrow (BM). The standard chemotherapy regimen is based on and fms like tyrosine kinase 3 (FLT3; refs. 4, 5), all oncogenes. the metabolic inhibitor cytarabin (AraC) in combination with the Pim kinases lack regulatory domains, and are constitutively cytostatic topoisomerase 2 inhibitor daunorubucin (DNR), and active (6). Their activity is regulated by altering the intracellular has changed little over the past 40 years (1). Intense research levels, that is by transcription and translation, or by degrada- efforts aim to identify drug targets that can help overcome AML tion (7). Together this makes them a likely Achilles heel for AML chemotherapy resistance and relapse, as well as the severe toxic cells dependent on a reliable communication of cytokine signals side effects of DNR (see ref. 2, for a recent review on potential drug to the nucleus in order to survive and multiply. Mice depleted of targets). One of the important regulators of AML cell survival are all three Pim kinase proteins have a normal life span and are found in the family of three serine/threonine-protein kinases, fertile, only exhibiting a slight deficient growth response with a reduced body size (8). Consequently, no severe side effects from inhibition of selective Pim kinases are expected. Several Pim 1Department of Clinical Science, Centre for Pharmacy, University of Bergen, kinase inhibitors have been evaluated for therapeutic potential Bergen, Norway. 2Hospital Pharmacy in western Norway, Bergen. 3Department against hematologic disorders, in particular multiple myeloma of Clinical Science, University of Bergen, Bergen, Norway. 4Universite Clermont and AML, but also CML, ALL, and B-cell lymphoma (9, 10). Auvergne, CNRS, Sigma Clermont, ICCF, F-63000 Clermont-Ferrand, France. Previously, we have reported the synthesis of a series of Pim 5Centre for Cancer Biomarkers, Department of Clinical Science, University of 6 7 kinase inhibitors exhibiting various N-containing heteroaro- Bergen, Bergen, Norway. BerGenBio AS, Bergen, Norway. Department of matic scaffolds such as pyrrolo[2,3-a]carbazole-3-carbaldehydes, Biomedicine, University of Bergen, Bergen, Norway. pyrazolo[3,4-c]carbazoles, pyrazolo[4,3-a]phenanthridines, and Note: Supplementary data for this article are available at Molecular Cancer pyrrolo[2,3-g]indazoles (Fig. 1A; refs. 11–18). A broad selection Therapeutics Online (http://mct.aacrjournals.org/). of molecular scaffolds is important in order to develop the best Corresponding Author: Lars Herfindal, University of Bergen, Laboratoriebyg- therapeutic agents against a specific target. All the above-men- get, Jonas Lies vei 87, Bergen N-5021, Norway. Phone: 4755974628; E-mail: tioned compound classes showed acceptable IC values toward lars.herfi[email protected] 50 Pim kinases (below 10 nmol/L in some cases, see Table 1). The doi: 10.1158/1535-7163.MCT-17-1234 ATP-binding pocket, along with the substrate-recognition resi- 2019 American Association for Cancer Research. dues are identified as crucial for Pim kinase inhibitors (19).

www.aacrjournals.org OF1

Bjørnstad et al.

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).

These compounds were reported to have activity toward AML pocket in a manner typical for non-ATP mimetic Pim inhibitors cells. We wanted to extend our investigation of these compounds (Fig. 1B; ref. 11). A similar binding mode was observed by as potential drugs for AML, and investigate the possibilities for molecular modeling for analogues of this series. Compound one or two selected compounds to enter further preclinical LG-VII-126 is part of another Pim inhibitor series (Fig. 1A). investigations. Molecular modeling showed that the indazole ring inserted into the ATP-binding pocket with high-affinity interactions in Pim1 and Pim3 (16). The synthesis of a homolog series Materials and Methods identified VS-II-173 as a nanomolar Pim1 and Pim3 inhibi- Description of the kinase inhibitors tor (17). The study of its putative binding with Pim1/Pim3 by All compounds described in Fig. 1A are designed to bind in molecular modeling demonstrated that VS-II-173 inserted the ATP-recognition site of Pim kinases. Their synthesis has deeply into the ATP site of Pim1 and Pim3 (17). been described elsewhere (11–18). Compound B3 exhibited a The in vitro protein kinase screening was performed at the selective profile when evaluated toward a panel of 66 protein International Centre for Kinase Profiling (ICKP; Dundee, Scot- kinases (11). X-ray co-crystal structure with Pim1 (PDB code: land). VS-II-173 was evaluated toward a panel of 50 protein 3JPV) demonstrated that it inserted into the ATP-binding kinases in duplicate assays at a compound concentration of

OF2 Mol Cancer Ther; 18(3) March 2019 Molecular Cancer Therapeutics

New Pim Kinase Inhibitor for AML Therapy

Table 1. Ability of the novel compounds to inhibit activity of Pim-1, 2, and 3 every second month. No positive tests were obtained in any cell kinases line during the time of the experiments. IC50 (mmol/L) Pim1 Pim2 Pim3 Assessment of cell death B3 (11)a 0.12 0.51 0.01 RAG-II-45 (11) 0.0068 0.131 0.0124 Cytotoxicity assays were performed in 96-well plates at 0.1 mL 6 RAG54 (12) 0.57 >10 0.04 medium/well. Suspension cells were seeded at 0.5 10 cells/ RAG-II-115 (13) 0.075 >1 0.08 mL and used the day of seeding. The NRK and H9c2 cells were FG587 (14) 0.009 >1 0.026 seeded at 0.05 106 cells/mL, and left over night to attach before MB032 (14) 0.042 >1 0.05 experiments. The compounds were tested alone, or in combina- MB071 (15) 1.4 nd 0.38 tions with the following cytostatics; DNR (Cerubidine; MB069 (15) 1.56 nd 0.6 fi FG644B (15) 0.39 nd 0.19 Sano Aventis Lysaker), Geldanamycin, Emetine (both pur- EE057 (15) 1.1 nd 0.7 chased from Sigma-Aldrich), bortezomib (Velcade, Janssen), FG610 (15) 0.74 nd 0.34 Etoposide (Eposin, Teva), Cisplatin (Accord Healthcare), FG773 (15) >1nd 2.9and AraC (Cytarabin, Fresenius Kabi). The pan-Pim kinase b LG-VII-126 (16) 0.04 49% (1 mmol/L) 0.10 inhibitor AZD1208 was from Tocris Bioscience, the Bcl-2 inhib- m b VS-II-173 (17) 0.07 46% (1 mol/L) 0.02 itor Venetoclax / ABT-199 from Cayman Chemical, and the LG-VI-142 (18) 0.46 >10 0.033 tyrosine-kinase inhibitor Midostaurin from Sigma-Aldrich. The nd, not determined. viability of the treated cells was assessed using a WST-1 Cell aThe numbers in parentheses refer to the citation in the reference list. b% residual kinase activity at 1 mmol/L. Proliferation Assay (Roche Applied Sciences). After recording metabolic 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- 1 mmol/L as described previously (20–22). The results were alize the nuclei. Cell death was assessed by UV-microscopic expressed as the percentage of residual kinase activity. The evaluation of nuclear morphology (28), and the data from the protein kinases were of human origin except Lck (mouse) and metabolic activity were used to confirm the data from micros- ROCK2 (rat). copy. EC50 values were estimated by nonlinear regression using the statistical software IBM SPSS statistics for Apple, ver. 24, Cell culture conditions (IMB Corp.): The following AML cell lines were utilized in this study; the ðÞmax min ¼ þ ÀÁ; ð Þ human acute monocytic leukemia cells Molm-13 (ACC, 554; Y min h 1 þ EC50 ref. 23), Molm-13 cells with shRNA mediated silencing of p53 1 X (Molm-13 shp53; ref. 24), MV4-11 human monocytic leukemia where Y is the response (cell death); max and min, the maximum cells (ATCC, CRL-9591), MV4-11 cells with silenced p53 and minimum cell death from the curve; X the concentration of (MV4-11 shp53), the rat promyeloid leukemia wild-type cell the drug/compound; and h is the hill index. Determination of lines IPC-81 (25), IPC-81 with enforced expression of Bcl-2 coefficient of drug interaction (CDI) was done by finding the ratio (both created by Dr. Michel Lanotte, Hopital^ St. Louis, Paris, between the effect of treatment and control (R) and further France, and maintained by prof. Stein Ove Døskeland; ref. 26), calculates as shown in Eq. 2. and the human AML cell line OCI-AML3 (ATCC, ACC-582). The Molm-13 and MV4-11 cells with silenced p53 were created by ¼ RCombination ð Þ CDI 2 Prof. Stein Ove Døskeland, Drs. Sjur Huseby, and Gro Gausdal RDrug1 RDrug2 by retroviral transfection for stable expression of shRNA against A value around one indicates additive effect, values below 0.7 p53 using the pRETRO SUPER-p53 vector (27). Selection was by demonstrate clear synergy, and higher than one antagonistic m increasing puromycin concentration up to 400 g/mL over 14 effect. days. All compounds were also tested on the chronic myeloid leukemia (CML) cell line K562 (ATCC, CCL-243), NRK rat Membrane permeability assay kidney epithelial (ATCC, CRL-6509), and H9c2 cardio myoblasts The Corning Gentest pre-Coated parallel artificial membrane (ATCC, CRL-1446). Molm-13 cells were cultured in RPMI medi- permeability assay PAMPA Plate System (Corning Discovery um with 10% FBS (Invitrogen). MV4-11 and K562 were cultured Labware) was done according to manufacturer's protocol. The in Iscove medium added 8 mmol/L L-glutamine and 10% FBS. compounds were tested at 50 mmol/L in PBS at 21 C. After OCI-AML3, NRK, and H9c2 cells were cultured in DMEM, incubation for 5 hours, 50 mL of each sample were added 15 mL enriched with 10% FBS and the IPC cells were supplemented acetonitrile, and injected into a reversed phase HPLC column with 10% horse serum. All culture media were from Sigma- (Kromasil 100-5 C18 150-4.6 mm; Akzo Nobel) connected to a Aldrich Inc., and were supplemented with 100 IU/mL penicillin Merck-Hitachi LaChrome HPLC-system (VWR) with a L-7455 and 100 mg/L streptomycin. Culturing of all cells was in a diode array detector. Mobile phase A was 0.05% aqueous humidified atmosphere (37 C, 5% CO2). The suspension cells TFA, and mobile phase B was acetonitrile. The flow rate was were kept at 10 to 80 104 cells/mL and diluted by adding fresh 1.6 mL/min and compounds eluted between 1 and 4.5 minutes medium. The adherent cells were cultured until reaching 90% during a 4.5 minutes gradient from 70:30% mobile phase A:B confluence. They were then detached by mild trypsination, to 0:100% mobile phase A:B. The ratios of the compound con- centrifuged, and reseeded in fresh medium at 40% confluence. centrations in the donor and acceptor compartments were calcu- Adherent cells that had more than 12 passages were not used. lated after integration of the peaks at 248 nm. Peff was calculated The cells were tested for mycoplasma infection using MycoAlert as described in ref. 29.

www.aacrjournals.org Mol Cancer Ther; 18(3) March 2019 OF3

Bjørnstad et al.

Western blot analysis fresh, and suspended in MEME medium. Treatment with VS-II- Western blotting was conducted as described previously (30). 173 and DNR were for 24 hours. Drug-induced cell death was To ensure that we studied effects of kinase inhibition, we chose assessed by flow cytometric analysis of cells stained with Pacific- concentrations that gave less than 30% apoptosis. This would Blue-AnnexinV (#640918; BioLegend) and propidium iodide (PI; avoid loss of proteins due to caspase cleavage or disintegrated #421301; BioLegend). Between 10,000 and 30,000 events were cells. After treatment with Pim kinase inhibitors, the cells were collected for each sample using a BD Fortessa flow cytometer. The washed and lysed, and protein concentrations determined using data were analyzed with the software Flow Jo (Tree Star, Inc.). the Quick Start Bradford protein assay (Bio-Rad Laboratories). SDS-polyacrylamide gels, 10% or 12% were loaded with 30 Statistical analysis to 50 mg protein, and the prelabeled "Precision Plus Protein Response to treatments were evaluated by Student t test, cor- Standard All Blue" (Bio-Rad Laboratories). Proteins were trans- relation of data by two-tailed Pearson correlation test, and patient ferred to polyvinylidene difluoride membranes (GE Healthcare data by cross-table analyses for chi-square, all by using IBM SPSS Life Sciences) by electrophoresis. The following primary antibo- statistics for Apple, ver. 24 (IMB Corp.). Significance was defined dies were used: Pim1, Pim2, and Pim3 (all from Santa Cruz as P 0.05. Heat maps were created by MeV (31), analyses of Pim Biotechnology, except for on OCI-AML3 cells, where Pim2 anti- expression was done using the J-Express 2012 software (32) after body from Sigma-Aldrich was used), Bad, pBad phospho S112, normalization and log(10) transformation of data. Kinome tree 4EBP1, p4EBP1 phospho T37/46, STAT5 and pSTAT5 phospho was created using the Kinome Render software (33). Y694, Akt and Akt phospho S473, p70 S6 kinase and p70 S6 kinase phospho S371 (from Cell Signaling Technology), MDM2 and pMDM2 phospho S166 antibody (Abcam plc), anti-b-actin Results (Sigma-Aldrich). The secondary antibodies were donkey anti- Characterization of anti-AML activity of kinase inhibitors mouse and anti-rabbit antibodies conjugated to horse-radish We previously found that pyrrolo[2,3-a]carbazole-3-carbalde- peroxidase (Jackson Immunoresearch Laboratory). Membranes hyde Pim kinase inhibitors (15) had promising activity against were developed using Supersignal West Pico or West Femto AML cells. We therefore decided to test the efficacy of an in-house Chemiluminiscence Substrate from Pierce Biotechnology Inc., selection of Pim inhibitors containing different hetero-aromatic according to the manufacturers' protocols. The immunoblots scaffolds (Fig. 1A) against a panel of cell lines (Fig. 2A). The were imaged by a LAS-4000 image reader (Fujifilm). After imag- inhibitor VS-II-173 was found to be a potent and selective inducer ing, the membranes were stripped using Restore PLUS Western of AML cell death. The cardiomyoblasts, epithelial cell lines, and Blot Stripping Buffer (ThermoFisher Scientific) to reprobe the the chronic myeloid leukemia cell line K562 were unaffected at membranes with the antibody for the phosphorylated version of VS-II-173 concentrations inducing 100% death in the AML cells. the proteins. Anti-b-actin was used for loading control. The compounds MB032, LG-VI-142, LG-VII-126, RAG54, and FG587 also showed selectivity toward AML cell lines, but were Flow cytometric analysis of AML patient material less potent than VS-II-173. The compound RAG-II-115 was a The collection of patient cells was approved by the regional potent inducer of cell death, but did not discriminate between research ethics committee (Health Region III, Bergen, Norway, AML-derived and other cell lines (Fig. 2A). VS-II-173 was able to REK approval numbers for biobanking: III 060.02 215.03, 231.06, overcome the prosurvival effect of poor prognostic factors in AML and 2015/1410, and for experimental use of cells: REK Vest 2015/ such as FLT3-ITD expression (MV4-11 and Molm-13 cells), p53 1759 and 2017/305) and conducted in accordance with the silencing (Molm-13 shp53 and MV4-11 shp53) and, to some Declaration of Helsinki. Samples were collected after written extent, Bcl-2 overexpression in IPC-81 cells (Fig. 2A). To find if the informed consent, and stored under liquid nitrogen in biobanks differences in efficacy was due to the compound's ability to approved by the Norwegian Royal Ministry of Health and the penetrate the cellular membrane, we tested the membrane per- Norwegian Directorate for Health and Social Affairs. Blasts from meability using the PAMPA assay. Membrane permeability was 37 patients were used in this study. The patients were not selected classified by the range defined by Bennion and colleagues (29), after response to chemotherapy. All patients had a high count of which divides compounds into four groups: high permeability > > < AML blasts in peripheral blood, and blasts were isolated by (LogPeff 5.33), intermediate permeability (LogPeff 5.66 to density gradient separation of peripheral blood (Lymphoprep; 5.33), low permeability (LogPeff > 6.14 to < 5.66) imper- < Axis-Shield). After isolation, the samples contained at least 95% meable (LogPeff 6.14). The compounds RAG-II-115 and leukemic blasts. The BM aspirate was from a healthy donor and MB032 showed intermediate to high efficacy toward cells kindly provided by Professor BT Gjertsen (Health Region III; REK (Fig. 2A) and intermediate to high membrane permeability approval number: 2012/2247). Peripheral blood mononuclear (Fig. 2B), whereas compound VS-II-173 had high efficacy toward cells (PBMC) were obtained from freshly drawn peripheral blood cells, but low membrane permeability. There were also com- (Department of Immunology and Transfusion Medicine, Hauke- pounds with low efficacy toward cells, and low membrane per- land University Hospital, Bergen, Norway) and isolated by den- meability, like RAG-II-45, FG587, RAG54, and MB071. However, sity gradient separation (Lymphoprep; Axis-Shield). Erythrocytes the compounds MB069, FG644B, EE057, and FG610 all showed were lysed (BD Biosciences Pharm Lyse buffer) and leukocytes high membrane permeability (Fig. 2B), but low ability to induce were suspended in Minimum Essential Medium Eagle (MEME), cell death (Fig. 2A). We also checked whether there were large enriched with 10% FBS and 2 mmol/L L-glutamine, and supple- differences between microscopic assessment of nuclear morphol- mented with 100 IU/mL penicillin and 100 mg/L streptomycin ogy and metabolic conversion of the WST-1 reagent (Fig. 2C and (all from Sigma-Aldrich Inc.). Upon the experiment AML blasts or D). The WST-1 assay gave a lower EC50 value compared with normal BM cells were thawed and suspended in Stem Span SFEM microscopic evaluation, presumably due to its ability to detect cell medium (Stem Span Technologies), whereas PBMCs were used growth inhibition. Still there was a good correlation between the

OF4 Mol Cancer Ther; 18(3) March 2019 Molecular Cancer Therapeutics

New Pim Kinase Inhibitor for AML Therapy

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) before assessment of metabolic activity using the WST-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-13 cells with VS-II-173 over 24 hours. The same experiments were assessed by both microscopy (C) and metabolic activity by WST-1 assay (D) and EC50 values determined by nonlinear regression. The inset in C shows surface and nuclear morphology of Molm-13 cells after 24 hours of treatment with either solvent (upper images) or 10 mmol/L VS-II-173. The bars indicate 9 mm. The inset in D shows correlation between the WST-1 signal and apoptotic morphology, using Pearson Correlation 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 in A–E are from 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. The size of the circles is inversely proportional to the residual activity of the kinases. The tree was generated using the Kinome Render software, and the illustration reproduced courtesy of Cell Signaling Technology (www.cellsignal.com). A table with the values from F is available as supplementary information (Supplementary Table S1). www.aacrjournals.org Mol Cancer Ther; 18(3) March 2019 OF5

Bjørnstad et al.

two methods (Pearson's correlation test), as a decline in WST-1 and MV4-11 cells, but to a small degree toward OCI-AML3 signal was always accompanied with increase in apoptosis (see (Fig. 3J). inset in Fig. 2D). The cell death induced by VS-II-173 had typical apoptotic morphology like cell shrinkage, nuclear condensation, VS-II-173 attenuates phosphorylation of Pim kinase substrates and fragmentation (inset in Fig. 2C). Pim kinases have been shown to act downstream of FLT3 We next assessed the time kinetics of the cytotoxic action by signaling (5), and we compared the efficacy of VS-II-173 on AML selected compounds at concentrations giving between 80% and cell lines with different FLT3 mutation status. The VS-II-173 had 100% death after 24 hours. The times to obtain 50% apoptotic higher efficacy toward cell lines harboring the FLT3-ITD mutation cells were similar (ranging from 4.4 to 6 hours) for the AML- (Molm-13, FLT3-ITD heterozygous, and MV4-11 FLT3-ITD specific compounds (Fig. 2E). RAG-II-115 induced a much more homozygous), with EC50 values between 2 and 3 mmol/L rapid cell death (about 2 hours; Fig. 2E). (Fig. 4A and B, respectively). The OCI-AML3 cells (FLT3 WT) All compounds showed high activity toward the Pim kinases were less sensitive to VS-II-173, with EC50 about 16 mmol/L (Table 1), and we chose the most promising compound from the (Fig. 4C). initial cell experiments, VS-II-173, to be evaluated toward 50 To study early events (the initiation phase) of the apoptosis protein kinases at 1 mmol/L concentration (Fig. 2F; Supplemen- process triggered by VS-II-173, we used concentrations that would tary Table S1). This revealed that the compound had activity produce apoptosis after 5 to 10 hours, in line with previous toward other kinases as well. The most notable besides Pim1 and studies on for instance anthracycline and AML (34, 35). When 3 was the dual specificity tyrosine phosphorylation regulated examining the expression of Pim1, 2, and 3 in the AML cell lines kinase 1A (DYRK1a), AMP-activated kinase (AMPKa1), home- treated with VS-II-173, we noted a rapid, but transient down- odomain interacting protein kinase 2 (HIPK2), protein kinase regulation of all three kinases in Molm13 cells (Fig. 4D, see C-related protein kinase 2 (PRK2), and ribosomal protein S6 Supplementary Fig. S1 for quantification). The response was less kinase A1 (RSK1), all having less than 10% residual activity after profound in MV4-11 and OCI-AML3 cells (Fig. 4E and F). Fur- treatment with 1 mmol/L of VS-II-173. thermore, the Molm13 and MV4-11 cells showed a rapid dephos- phorylation of Stat5 (Fig. 4G and H) upon treatment with VS-II- VS-II-173 acts synergistically with the anthracycline 173. The MV4-11 cells showed a decrease of the prosurvival daunorubicin, and additively with a number of other anticancer phospho-Bad form of the otherwise apoptogenic BH3 only pro- drugs tein Bad (Fig. 4H) and the OCI-AML3 cells showed a strong To establish whether our kinase inhibitors could have potential decrease in p-4E-BP1 and pMDM2 (Fig. 4I). in combination therapy, we chose the two most potent of the AML selective compounds, VS-II-173 and MB032. We found that VS-II- Blasts from patients with AML with poor prognostic markers are 173 at subtoxic concentration increased the Molm-13 AML cell sensitive to the kinase inhibitor VS-II-173 death from 40% to 80% when combined with 50 nmol/L dau- To better assess the potential of VS-II-173 as a lead com- norubicin (DNR) for 24 hours (Fig. 3A). The compound MB032 pound for the treatment of AML, it was tested on a panel did not synergize with DNR (Fig. 3A). of nonselected AML patient blasts. The blasts were treated with We next tested VS-II-173 in combination with other anticancer VS-II-173 alone, or in combination with DNR. Our experience drugs. The best synergy for VS-II-173 was with DNR (Fig. 3B and with AML patient blasts is that they are generally more resistant C), only a modest synergy being observed with cisplatin (Fig. 3B). to cytostatics compared with most AML cell lines, and in order It was also noted that high concentrations of the proteasome to ensure an effect, we increased the doses of both VS-II-173 inhibitor bortezomib antagonized the action of VS-II-173 (Fig. 3B and DNR. The age of the patients were from 19 to 86 years, with and D), whereas lower concentrations gave an additive effect a median of 69 years. They were classified using the French– (Fig. 3D). The other drugs tested (etoposide, emetine, and gelda- American–British (FAB) classification (36), and cytogenetics namycin) exhibited additive effects in combination with VS-II- analyzed based on the MRC classification (37). Eleven 173 (Fig. 3B and E). patients had FLT3-ITD (high-risk characteristic), 21 had WT The firsttestonthepanelofAMLcells(Fig.2A)revealedthat FLT3, and 3 were not tested. Ten patients had insertion muta- cells with silenced p53 had reduced, but not abolished sensi- tion in NPM-1 (favorable prognostic factor), and 22 had wild- tivity to the compounds tested, including VS-II-173. We type NPM-1 (three not tested). See Fig. 5A for further patient wanted to investigate if lack of p53 could affect the synergy information. between VS-II-173 and DNR. We found that DNR synergized The results from the AML patient blast experiments showed one well with 4 mmol/L VS-II-173, both against Molm-13 WT and large group that responded well to treatment with VS-II-173, Molm-13-shp53 cells, but also that a higher concentration of where doses between 6 and 24 mmol/L could kill more than both drugs was needed to achieve a substantial synergistic 50% of the blasts (Responders, Fig. 5). Some of the VS-II-173 effect (Fig. 3F and G). responding patient blasts also responded well to the DNR Next, the anti-AML activity of three other compounds was (patients 11 and 24). In some cases, even the highest concentra- tested. A total of 50 mmol/L of the pan-Pim kinase inhibitor tion of DNR (100 nmol/L) did not affect the blast viability AZD1208 induced around 60% cell death in MV4-11 cells after whereas VS-II-173 showed high potency against these patient 24 hours incubation, whereas the other cell lines were mostly blasts (patients 1 and 2). Another group responded to DNR, but unaffected (Fig. 3H). The Bcl-2 inhibitor venetoclax was effi- not to VS-II-173, for instance patient 24 and to some extent cient toward Molm13 and MV4-11 in the low nanomolar range, patient 25. There were two groups that did not appear to benefit but were nontoxic to the OCI-AML3 cells at the concentrations from VS-II-173. One, which appeared to be resistant to both DNR tested (Fig. 3I). Finally, the AML drug midostaurin was tested. and VS-II-173, like patients 36 and 37, and one where the This multitarget kinase inhibitor was efficient toward Molm13 combination gave a reduced effect (patients 22 and 23). Despite

OF6 Mol Cancer Ther; 18(3) March 2019 Molecular Cancer Therapeutics

New Pim Kinase Inhibitor for AML Therapy

C A 100 100 VS-II-173 80 80 DNR

60 Control (DNR + vehicle) 60 EC50 = 53 ± 0.8 nmol/L 40 Figure 3. 40 DNR + MB032 Dead cells (%) Dead cells Drug interactions between VS-II-173 and (%) Dead cells EC50 = 49 ± 2.3 nmol/L 20 20 DNR + VS-II-173 seven anticancer compounds. A, Molm-13 0 EC50 = 27 ± 1.7 nmol/L cells were treated with VS-II-173 (1 mmol/L), 0 0642 m VS-II-173 (μmol/L) MB032 (100 mol/L) in combination with 0 20 40 60 80 100 120 ﬁ DNR. After 24 hours, the cells were xed and B Concentration of DNR (nmol/L) D 100 VS-II-173 viability was assessed by microscopic 80 Bortezomib evaluation of apoptosis using Hoechst 10 nmol/L ***a 60 33342 DNA stain. The vertical dotted lines Bortezomiba Bortezomib (3 nmol/L) 40 indicate EC50 values. B, Molm-13 cells were m Geldanamycin 20

treated with 1 mol/L VS-II-173 in (%) Dead cells combination with different anticancer 0 Etoposide compounds for 24 hours before assessing 062 4 viability as in A. The CDI was calculated as VS-II-173 (μmol/L) Emetine described in the section Materials and E 100 VS-II-173 Methods. The concentrations of the Cisplatin 80 Etoposide compounds tested were: Geldanamycin, m 60 10 nmol/L; Etoposide, 0.6 mol/L; Emetine, AraC 5 nmol/L; DNR, 75 nmol/L; Cisplatine, 40 m m *** 6 mol/L; AraC, 6 mol/L, Bortezomib, DNR (%) Dead cells 20 10 and 3 nmol/L. In B, indicates 0 P < 0.001, one-sample T test. a: For 0 642 bortezomib, the CDI for the high 0.80.60.4 321.21.0 4 VS-II-173 (μmol/L) concentration is shown. C–E, Examples of Coefficient of drug interaction drugs which act synergistically (C; DNR), FH100 Molm13 antagonistically (D; bortezomib), or 100 Molm-13 WT 80 MV4-11 additively (E; Etoposide). F and G, OCI-AML3

Synergistic activity between VS-II-173 and 80 Ctr DNR 60 NRK DNR in WT Molm-13 AML cells (left) or p53 40 silenced p53 (shp53, right). The cells were 60 Actin death % cell 20 incubated with increasing doses of DNR 0 40 0.1 1 10 100 with or without 2 or 4 mmol/L VS-II-173 for

Dead cells (%) Dead cells AZD1208 (μmol/L) 24 hours. The insets show induction of p53 20 by DNR expression in WT-cells (F), but not I 100 Molm13 in shp53 cells (G). Viability was assessed as 0 10 20 40 100 80 MV4-11 in A. The data in A–G are the mean and the OCI-AML3 60 SD from 5 to 12 experiments. H–J, Cell death G NRK 100 Molm-13 shp53 40 inducing effect of venetoclax (H),

% Cell death % Cell 20 DNR midostaurin (I), and AZD1208 (J)on 80 Ctr different cell lines after 24 hours incubation. p53 0 60 0 110010 Cell death was scored using the WST-1 assay Actin Venetoclax (nmol/L) and is relative to untreated cells. The data 40 are average of two (Molm13, OCI-AML3, and (%) Dead cells J 100 Molm13 MV4-11) or four (NRK) experiments and the 20 MV4-11 80 OCI-AML3 lines are from four-parameter regression 60 NRK analysis as described in the section Materials 0 10 20 40 100 40 and Methods. Concentration of DNR (nmol/L) death % Cell 20 0 Without VS-II-173 10100 1000 With 2 μmol/L VS-II-173 Midostaurin (nmol/L) With 4 μmol/L VS-II-173 this, the majority of the patients (24 out of 37) could be char- the responder and nonresponder groups (Pearson chi-square ¼ acterized as responders to VS-II-173. 1.55, P ¼ 0.21). Furthermore, the patients with mutated NPM-1 Neither sex, age, nor the FAB classification seemed to influence were overrepresented in the responder group (9 of 19 patients the responsiveness of the patient blasts to VS-II-173 (Fig. 5A). The (47%) in the responder group, and 1 of 14 patients (7%) in the three patients with adverse cytogenetics (patients 20, 30, and 31) intermediate/non responder group, Pearson chi-square ¼ 6.175, showed poor to intermediate response to VS-II-173, whereas the P ¼ 0.013). Three-way chi-square test of our data showed NPM1 two patients with good risk cytogenetics showed high response. status was important for response to VS-II-173 in patients with WT Patients with FLT3-ITD were represented in the responder group FLT3 (Pearson chi-square ¼ 4.89, P ¼ 0.27), but not in patients (8 of 20, 40%) of the patients with known status, and in the with FLT3-ITD (Pearson chi-square ¼ 0.749, P ¼ 0.387). intermediate/non responder group (3 of 13, 23%), but there was Compared with the responder group, BM aspirates or PBMC no significant difference between the occurrence of FLT3-ITD in were less affected by VS-II-173 (Fig. 5A), but the AML blasts from

www.aacrjournals.org Mol Cancer Ther; 18(3) March 2019 OF7

Bjørnstad et al.

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 formaldehyde containing the DNA stain Hoechst 33342. Apoptotic cells were identified by microscopic evaluation of nuclear morphology. See section Materials and Methods for 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), and OCI-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 the expression 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 pMDM2 in Molm-13 (G), MV4-11 (H), and OCI-AML3 (I) cells. b-Actin was used as loading control on all blots.

two patients classiﬁed as nonresponders had equal or slightly cally be explained by the compound selectively targeting rapidly lower response compared with the PBMC or BM cells. growing cell lines, but the K562 CML cell line has a doubling time We found no correlation between response to VS-II-173 and similar to that of Molm-13 cells, thus rapid proliferation does not Pim kinase expression level (Fig. 5B), based on mRNA expression seem to be the reason for the AML-selectivity observed in Fig. 2A. of Pim kinases related to the median expression in patients (no Importantly, VS-II-173 was active toward cells with high-risk signiﬁcance using Pearson Chi-Square statistics). characteristics like decreased expression of p53 (Molm-13 shp53 and MV4-11 shp53; Figs. 2A and 3G), and cells overexpressing the survival protein Bcl-2 (IPC-81-Bcl2; Fig. 2A) as well as FLT3-ITD Discussion (Figs. 2A, 4A–C, and 5). We could not detect any correlation Of the 15 compounds we tested, VS-II-173 exhibited potent between the ability to inhibit the different Pim kinases and the selective activity toward the AML cell lines. This could theoreti- ability to selectively induce AML cell death (Table 1; Fig. 2A). For

OF8 Mol Cancer Ther; 18(3) March 2019 Molecular Cancer Therapeutics

New Pim Kinase Inhibitor for AML Therapy

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 PBMC were suspended in MEME medium, 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 in combination with DNR, for 24 hours. Apoptosis was assessed by flow cytometric analysis of cells stained with PacificBlue-AnnexinV and PI. Between 10,000 and 30,000 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 their response to VS-II-173. Abbreviations: F, female; M, male; N, normal; I, intermediate; A, adverse; ITD, FLT3 internal tandem duplications; INS, insertion mutation in NPM-1; nt, not tested. instance, LG-VII-126 and VS-II-173 had similar inhibitory activity Thus, combinations of VS-II-173 with DNR or possibly cisplat- toward the three kinases, but VS-II-173 was a far more potent AML in could be clinically relevant. This agrees with a previous study cell death inducer than LG-VII-126. This could partly be explained by Doshi and colleagues showing that Pim kinase inhibition by differences in the ability to cross phospholipid membranes sensitized AML cell lines to DNR (42). Furthermore, our results (Fig. 2B). indicate that Ara-C and VS-II-173 have sub-additive action, The kinase screen showed that VS-II-173 also inhibited other suggesting they should not be combined simultaneously. Other kinases than the three Pim kinases (Fig. 2F). The level of Pim kinase inhibitors have actedbothinasynergisticand promiscuity was similar to that shown for the pan-Pim kinase antagonistic manner in combination with AraC in AML cell inhibitor SGI-1776 (38), which is active against AML lines and primary AML cells (42, 43). cells (39). The transiently reduced level of Pim1, 2, and 3 kinases in the Many chemotherapeutic therapy regimens for AML are based Molm-13 cells in response to VS-II-173 (Fig. 4D) indicates that on drug combinations, such as the 7þ3 regimen with DNR and inhibition of Pim kinases increases degradation or attenuates AraC or high-dose AraC plus a nucleoside analogue (see refs. 40, production of the protein. Pim kinase activity is not regulated 41 for recent reviews on established and emerging therapies by post-translational modifications or allosteric modulators, but for AML). It became apparent that the most potent compound by increased or attenuated expression (7). Treatment with VS-II- selective for AML cells, VS-II-173 increased the cellular response 173 apparently perturbed the ratio between synthesis and deg- to DNR with high synergy (Fig. 3A–C). Combination with radation of the Pim kinases, but not irreversibly, suggesting that cisplatin and VS-II-173 gave more modest synergy (Fig. 3B). this was not due to cell-death associated protein degradation. The

www.aacrjournals.org Mol Cancer Ther; 18(3) March 2019 OF9

Bjørnstad et al.

compensation mechanisms and subsequent restoration of the A panel of nonselected AML patient blasts was included and 24 Pim kinase levels appears presumably too late to initiate the of 37 patient responded well to the treatment with VS-II-173, necessary survival mechanisms, since the cells died after eight to where doses between 6 and 12 mmol/L could kill more than 50% twelve hours (Fig. 2E). of the blasts (Fig. 5). Approximately 40% of patients with AML Cells with the FLT3-ITD mutation mediate aberrant signal- with FLT3-ITD mutation also harbor the NPM1 mutation (56). ing through the Pim kinases (4). Thus, inhibition of Pim Our data showed NPM1 status was important for response to VS- represents an attractive approach to target FLT3-ITD cells. Pim II-173 in patients with FLT3-WT, but not in patients with FLT3- kinases promote survival of AML cells via phosphorylation of ITD. Thus, the presence of the high-risk factor FLT3-ITD is not Bcl-2 antagonist of cell death (Bad), and also 4E-BP1 (4, 39), influenced by the low-risk factor NPM1 mutation when it comes in accordance with reduced phosphorylation of these proteins to response to VS-II-173. These findings suggest that this com- seen upon VS-II-173 treatment, and by previous findings by pound can be used irrespective of FLT3 status. There was no Chen and colleagues (39). It has been demonstrated that correlation between the response to VS-II-173 and the level of Pim1 kinase directly affects the FLT3 receptor by a stabilizing Pim kinases in the patient blasts (Fig. 5B), which could be ascribed serine phosphorylation (44). This positive feedback loop the effect on other kinases (Fig. 2F). However, the values in Fig. 5B prompts Stat5 activation and Pim signaling (44). We found were compared with a pooled average, and AML cells are expected abolished phosphorylation of Stat5 in FLT3-ITD cell lines to have higher levels of Pim kinases than normal BM (3). Molm-13 and MV4-11 (Fig. 4G and H). We did not detect Y694 phosphorylated Stat5 in OCI-AML3 (Fig. 4I), which has wild-type FLT3. Elevated levels of Pim1 kinase induce p53 Conclusion signaling and MDM2 phosphorylation (45). Accordingly, our New strategies for AML therapy are urgently needed. Despite results showed reduced phosphorylation in MDM2 when using this, only one new drug has been developed and approved for VS-II-173 in Molm-13 cells (Fig. 4G). Furthermore, we found AML during the last 15 years. Unfortunately, it appears to yield that both VS-II-173 and the pan-Pim kinase inhibitor AZD1208 only modest improvement in survival, even in preselected reduced the phosphorylation of Akt and p70-S6 kinase (see patients (57). Our data point to pyrazolo[4,3-a]phenanthridine Supplementary Fig. S2), however VS-II-173 had a higher poten- as a relevant scaffold for compounds useful in the treatment of cy toward AML cell lines than AZD1208 in mono-treatment AML. The lead compound VS-II-173 exhibited crucial properties (Fig. 3H and Fig. 4A–C). needed to exert therapeutic effect. It is selective toward AML It must be noted that some of the perturbations in cell signaling cells, in which it efficiently dampens prosurvival signals. It is also could be caused by inhibition of other kinases. RSK1 was signif- potent against AML patient blasts, including some that showed icantly inhibited by VS-II-173 (Fig. 2F). RSK1 is a mediator of the high tolerance to DNR, and blasts carrying mutations associat- anti-apoptotic function of FLT3-ITD through phosphorylation of ed with poor prognosis. The compound and future analogs of Bad at Ser112 in AML cells (46). The reduction of pBad (Fig. 4H) pyrazolo[4,3-a]phenanthridines represent possible candidates for could be due to inhibition of RSK1 along with Pim kinase future AML treatment. Future studies will aim to reveal possible inhibition. AMPK is a stress sensor in cells, and can contribute biomarkers of the patient AML cells which responded well to to initiation of autophagy, and protects AML-cells from metabolic treatment with VS-II-173, as well as conducting preclinical studies stress (47). AMPK can thus be an important target for AML on human AML xenografts in mice. If the abovementioned issues therapy, particularly to prevent leukemogenesis, although anoth- are resolved, we believe that pyrazolo[4,3-a]phenanthridines er study shows that activation of AMPK initiates autophagic cell have a future in AML treatment. death (48). DYRK1 is a tumor suppressor, which is heavily down- regulated in AML patients (49), and mutations in the HIPK2 gene Disclosure of Potential Conflicts of Interest is linked to pathogenesis of AML (50). Taken together, the anti- No potential conflicts of interest were disclosed. AML activity can be inhibition of Pim kinase activity acting in concert with additional inhibition of some or all of the above- Authors' Contributions mentioned factors. Conception and design: R. Bjørnstad, . Bruserud, L. Herfindal The fact that VS-II-173 affects more than one target is in line Development of methodology: R. Bjørnstad, G. Gausdal, S.O. Døskeland, with several of the so-called precision therapies, like the tyro- L. Herfindal sine kinase inhibitors. In fact, the second-generation kinase Acquisition of data (acquired and managed patients, provided facilities, etc.): fi inhibitor Dasatinib has a higher level of promiscuity than its . Bruserud, F. Giraud, T.H. Dowling, P. Moreau, F. Anizon, L. Her ndal Analysis and interpretation of data (e.g., statistical analysis, biostatistics, predecessor Imatinib (51). With this in mind, a possible computational analysis): R. Bjørnstad, R. Aesoy, . Bruserud, A.K. Brenner, explanation for the potency and selectivity of VS-II-173 can S.O. Døskeland, L. Herfindal be its activity toward key AML survival factors like RSK1 and Writing, review, and/or revision of the manuscript: R. Bjørnstad, R. Aesoy, AMPK together with its pan-Pim kinase activity. The much . Bruserud, P. Moreau, S.O. Døskeland, F. Anizon, L. Herfindal more selective Pim kinase inhibitor AZD1208 (52) was less Administrative, technical, or material support (i.e., reporting or organizing fi efficient toward our AML cell lines than VS-II-173 (Fig. 3H), data, constructing databases): L. Her ndal Study supervision: L. Herfindal perhaps due to the broader range of substrates of the latter. Pan- kinase inhibition is believed to be favorable to the multityr- Acknowledgments osine kinase inhibitor midostaurin (see ref. 53; Fig. 3J). The mono-selective Bcl-2 inhibitor venetoclax (54), which emerges The authors want to thank Ing. Nina Lied Larsen and Kaja Skalnes Knudsen, MSc for technical assistance, and Sjur Huseby, PhD for assistance with gener- as therapy against AML (55), failed to induce cell death in OCI- ation of the AML cell lines. Prof. Bjørn Tore Gjertsen and Misbah Sabir, MSc AML3 cells (Fig. 3I) and appears to be effective against different are thanked for supplying bone marrow samples. Kathrine Åsrud, PhD is subtypes of AML compared to VS-II-173. thanked for critically reading through the manuscript. This study was supported

OF10 Mol Cancer Ther; 18(3) March 2019 Molecular Cancer Therapeutics

New Pim Kinase Inhibitor for AML Therapy

financially by the Norwegian Western Regional Health Authorities to Lars The costs of publication of this article were defrayed in part by the Herfindal (Grant No. 912052), Ronja Bjørnstad (Grant No. 912028), Stein payment of page charges. This article must therefore be hereby marked O. Døskeland (Grant No.: 303485), and ystein Bruserud (Grant No. 911788), advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate by the Norwegian Cancer Society to Lars Herfindal (Grant No. 112502), this fact. Stein O. Døskeland (Grant No. 705268), ystein Bruserud (Grant No. 62370, 62371, and 100933), and by the Norwegian Research council to Lars Received December 13, 2017; revised May 5, 2018; accepted January 14, 2019; Herfindal and Reidun Aesoy (Grant No. 254752). published first January 24, 2019.

References 1. Yates JW, Wallace HJ, Ellison RR, Holland JF. Cytosine-arabinoside (Nsc- 19. Ogawa N, Yuki H, Tanaka A. Insights from Pim1 structure for anti-cancer 63878) and daunorubicin (Nsc-83142) therapy in acute nonlymphocytic drug design. Expert Opin Drug Discov 2012;7:1177–92. leukemia. Cancer Chemoth Rep 1 1973;57:485–8. 20. Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, et al. The 2. Engen CB, Hajjar E, Gjertsen BT. Development of personalized molecular selectivity of protein kinase inhibitors: a further update. Biochem J 2007; therapy for acute myeloid leukemia. Curr Pharm Biotechnol 2016;17: 408:297–315. 20–9. 21. Bain J, McLauchlan H, Elliott M, Cohen P. The specificities of protein kinase 3. Alvarado Y, Giles FJ, Swords RT. The PIM kinases in hematological cancers. inhibitors: an update. Biochem J 2003;371:199–204. Expert Rev Hematol 2012;5:81–96. 22. Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of 4. Kim KT, Levis M, Small D. Constitutively activated FLT3 phosphorylates action of some commonly used protein kinase inhibitors. Biochem J 2000; BAD partially through pim-1. Br J Haematol 2006;134:500–9. 351:95–105. 5. Adam M, Pogacic V, Bendit M, Chappuis R, Nawijn MC, Duyster J, et al. 23. Matsuo Y, MacLeod RA, Uphoff CC, Drexler HG, Nishizaki C, Katayama Y, Targeting PIM kinases impairs survival of hematopoietic cells trans- et al. Two acute monocytic leukemia (AML-M5a) cell lines (MOLM-13 and formed by kinase inhibitor-sensitive and kinase inhibitor-resistant MOLM-14) with interclonal phenotypic heterogeneity showing MLL-AF9 forms of Fms-like tyrosine kinase 3 and BCR/ABL. Cancer Res 2006; fusion resulting from an occult chromosome insertion, ins(11;9)(q23; 66:3828–35. p22p23). Leukemia 1997;11:1469–77. 6. Bachmann M, Moroy T. The serine/threonine kinase Pim-1. Int J Biochem 24. McCormack E, Haaland I, Venas G, Forthun RB, Huseby S, Gausdal G, Cell Biol 2005;37:726–30. et al. Synergistic induction of p53 mediated apoptosis by valproic 7. Amaravadi R, Thompson CB. The survival kinases Akt and Pim as potential acid and nutlin-3 in acute myeloid leukemia. Leukemia 2012;26: pharmacological targets. J Clin Invest 2005;115:2618–24. 910–7. 8. Mikkers H, Nawijn M, Allen J, Brouwers C, Verhoeven E, Jonkers J, et al. 25. Lacaze N, Gombaud-Saintonge G, Lanotte M. Conditions controlling long- Mice deficient for all PIM kinases display reduced body size and impaired term proliferation of Brown Norway rat promyelocytic leukemia in vitro: responses to hematopoietic growth factors. Mol Cell Biol 2004;24: primary growth stimulation by microenvironment and establishment of 6104–15. an autonomous Brown Norway `leukemic stem cell line'. Leuk Res 1983;7: 9. Garcia PD, Langowski JL, Wang Y, Chen M, Castillo J, Fanton C, et al. Pan- 145–54. PIM kinase inhibition provides a novel therapy for treating hematologic 26. Seite P, Ruchaud S, Hillion J, Gendron MC, Bruland O, Segal-Bendirdjian E, cancers. Clin Cancer Res 2014;20:1834–45. et al. Ectopic expression of Bcl-2 switches over nuclear signalling for cAMP- 10. Kapoor S, Natarajan K, Baldwin PR, Doshi KA, Lapidus RG, Mathias TJ, et al. induced apoptosis to granulocytic differentiation. Cell Death Differ 2000; Concurrent inhibition of pim and FLT3 kinases enhances apoptosis of 7:1081–9. FLT3-ITD acute myeloid leukemia cells through increased Mcl-1 protea- 27. Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumor- somal degradation. Clin Cancer Res 2018;24:234–47. igenicity by virus-mediated RNA interference. Cancer Cell 2002;2: 11. Akue-Gedu R, Rossignol E, Azzaro S, Knapp S, Filippakopoulos P, Bullock 243–7. AN, et al. Synthesis, kinase inhibitory potencies, and in vitro antiproli- 28. Bøe R, Gjertsen BT, Vintermyr OK, Houge G, Lanotte M, Døskeland SO. The ferative evaluation of new Pim kinase inhibitors. J Med Chem 2009;52: protein phosphatase inhibitor okadaic acid induces morphological 6369–81. changes typical of apoptosis in mammalian cells. Exp Cell Res 1991; 12. Akue-Gedu R, Nauton L, Thery V, Bain J, Cohen P, Anizon F, et al. Synthesis, 195:237–46. Pim kinase inhibitory potencies and in vitro antiproliferative activities of 29. Bennion BJ, Be NA, McNerney MW, Lao V, Carlson EM, Valdez CA, et al. diversely substituted pyrrolo[2,3-a]carbazoles. Bioorg Med Chem 2010; Predicting a drug's membrane permeability: a computational model 18:6865–73. validated with in vitro permeability assay data. J Phys Chem B 2017; 13. Akue-Gedu R, Letribot B, Saugues E, Debiton E, Anizon F, Moreau P. Kinase 121:5228–37. inhibitory potencies and in vitro antiproliferative activities of N-10 substi- 30. Wergeland L, Sjoholt G, Haaland I, Hovland R, Bruserud O, Gjertsen BT. tuted pyrrolo[2,3-a]carbazole derivatives. Bioorg Med Chem Lett 2012;22: Pre-apoptotic response to therapeutic DNA damage involves protein 3807–9. modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells. 14. Giraud F, Bourhis M, Nauton L, Thery V, Herfindal L, Doskeland SO, et al. Mol Cancer 2007;6:33. New N-1,N-10-bridged pyrrolo[2,3-a]carbazole-3-carbaldehydes: synthe- 31. Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, et al. TM4: a free, sis and biological activities. Bioorg Chem 2014;57:108–15. open-source system for microarray data management and analysis. 15. Giraud F, Bourhis M, Ebrahimi E, Herfindal L, Choudhury RR, Bjornstad R, Biotechniques 2003;34:374–8. et al. Synthesis and activities of new indolopyrrolobenzodiazepine deri- 32. Dysvik B, Jonassen I. J-Express: exploring gene expression data using Java. vatives toward acute myeloid leukemia cells. Bioorg Med Chem 2015;23: Bioinformatics 2001;17:369–70. 7313–23. 33. Chartier M, Chenard T, Barker J, Najmanovich R. Kinome Render: a stand- 16. Suchaud V, Gavara L, Saugues E, Nauton L, Thery V, Anizon F, et al. alone and web-accessible tool to annotate the human protein kinome tree. Identification of 1,6-dihydropyrazolo[4,3-c]carbazoles and 3,6- PeerJ 2013;1:e126. dihydropyrazolo[3,4-c]carbazoles as new Pim kinase inhibitors. Bioorg 34.GausdalG,GjertsenBT,McCormackE,VanDammeP,HovlandR, Med Chem 2013;21:4102–11. Krakstad C, et al. Abolition of stress-induced protein synthesis sensitizes 17. Suchaud V, Gavara L, Giraud F, Nauton L, Thery V, Anizon F, et al. Synthesis leukemia cells to anthracycline-induced death. Blood 2008;111: of pyrazolo[4,3-a]phenanthridines, a new scaffold for Pim kinase inhibi- 2866–77. tion. Bioorg Med Chem 2014;22:4704–10. 35. Gausdal G, Wergeland A, Skavland J, Nguyen E, Pendino F, Rouhee N, 18. Gavara L, Suchaud V, Nauton L, Thery V, Anizon F, Moreau P. Identification et al. Cyclic AMP can promote APL progression and protect myeloid of pyrrolo[2,3-g]indazoles as new Pim kinase inhibitors. Bioorg Med Chem leukemia cells against anthracycline-induced apoptosis. Cell Death Dis Lett 2013;23:2298–301. 2013;4:e516.

www.aacrjournals.org Mol Cancer Ther; 18(3) March 2019 OF11

Bjørnstad et al.

36. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, deprived BaF3 cells due to protein kinase A and ribosomal S6 kinase et al. Proposals for the classification of the acute leukaemias. French- 1-mediated BAD phosphorylation at serine 112. Cancer Res 2005;65: American-British (FAB) co-operative group. Br J Haematol 1976;33:451–8. 7338–47. 37. Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G, et al. 47. Saito Y, Chapple RH, Lin A, Kitano A, Nakada D. AMPK protects leukemia- The importance of diagnostic cytogenetics on outcome in AML: analysis of initiating cells in myeloid leukemias from metabolic stress in the bone 1,612 patients entered into the MRC AML 10 trial. The medical research marrow. Cell Stem Cell 2015;17:585–96. council adult and children's leukaemia working parties. Blood 1998;92: 48. Sujobert P, Tamburini J. Co-activation of AMPK and mTORC1 as a new 2322–33. therapeutic option for acute myeloid leukemia. Mol Cell Oncol 2016;3: 38. Xu Y, Brenning BG, Kultgen SG, Foulks JM, Clifford A, Lai S, et al. e1071303. Synthesis and biological evaluation of pyrazolo[1,5-a]pyrimidine com- 49. Liu Q, Liu N, Zang S, Liu H, Wang P, Ji C, et al. Tumor suppressor DYRK1A pounds as potent and selective Pim-1 Inhibitors. ACS Med Chem Lett effects on proliferation and chemoresistance of AML cells by downregulat- 2015;6:63–7. ing c-Myc. PLoS One 2014;9:e98853. 39. Chen LS, Redkar S, Taverna P, Cortes JE, Gandhi V. Mechanisms of 50. Li XL, Arai Y, Harada H, Shima Y, Yoshida H, Rokudai S, et al. Mutations of cytotoxicity to Pim kinase inhibitor, SGI-1776, in acute myeloid leukemia. the HIPK2 gene in acute myeloid leukemia and myelodysplastic syndrome Blood 2011;118:693–702. impair AML1- and p53-mediated transcription. Oncogene 2007;26: 40. Bose P, Vachhani P, Cortes JE. Treatment of relapsed/refractory acute 7231–9. myeloid leukemia. Curr Treat Options Oncol 2017;18:17. 51. Ghoreschi K, Laurence A, O'Shea JJ. Selectivity and therapeutic 41. Saygin C, Carraway HE. Emerging therapies for acute myeloid leukemia. inhibition of kinases: to be or not to be? Nat Immunol 2009; J Hematol Oncol 2017;10:93. 10:356–60. 42. Doshi KA, Trotta R, Natarajan K, Rassool FV, Tron AE, Huszar D, et al. Pim 52. Keeton EK, McEachern K, Dillman KS, Palakurthi S, Cao Y, Grondine MR, kinase inhibition sensitizes FLT3-ITD acute myeloid leukemia cells to et al. AZD1208, a potent and selective pan-Pim kinase inhibitor, demon- topoisomerase 2 inhibitors through increased DNA damage and oxidative strates efficacy in preclinical models of acute myeloid leukemia. Blood stress. Oncotarget 2016;7:48280–95. 2014;123:905–13. 43. Kelly KR, Espitia CM, Taverna P, Choy G, Padmanabhan S, Nawrocki ST, 53. Schlenk RF, Kayser S. Midostaurin: a multiple tyrosine kinases inhibitor in et al. Targeting PIM kinase activity significantly augments the efficacy of acute myeloid leukemia and systemic mastocytosis. Recent Results Cancer cytarabine. Br J Haematol 2012;156:129–32. Res 2018;212:199–214. 44. Natarajan K, Xie Y, Burcu M, Linn DE, Qiu Y, Baer MR. Pim-1 kinase 54. Timucin AC, Basaga H, Kutuk O. Selective targeting of antiapoptotic BCL-2 phosphorylates and stabilizes 130 kDa FLT3 and promotes aberrant STAT5 proteins in cancer. Med Res Rev 2018. signaling in acute myeloid leukemia with FLT3 internal tandem duplica- 55. Menghrajani K, Tallman MS. New therapeutic strategies for high-risk acute tion. PLoS One 2013;8:e74653. myeloid leukemia. Curr Opin Hematol 2018;25:90–4. 45. Hogan C, Hutchison C, Marcar L, Milne D, Saville M, Goodlad J, et al. 56. Schlenk RF, Dohner K, Krauter J, Frohling S, Corbacioglu A, Elevated levels of oncogenic protein kinase Pim-1 induce the p53 pathway Bullinger L, et al. Mutations and treatment outcome in cytogeneti- in cultured cells and correlate with increased Mdm2 in mantle cell lym- cally normal acute myeloid leukemia. N Engl J Med 2008;358: phoma. J Biol Chem 2008;283:18012–23. 1909–18. 46. Yang X, Liu L, Sternberg D, Tang L, Galinsky I, DeAngelo D, et al. The FLT3 57. Levis M. Midostaurin approved for FLT3-mutated AML. Blood 2017;129: internal tandem duplication mutation prevents apoptosis in interleukin-3- 3403–6.

OF12 Mol Cancer Ther; 18(3) March 2019 Molecular Cancer Therapeutics

A Kinase Inhibitor with Anti-Pim Kinase Activity is a Potent and Selective Cytotoxic Agent Toward Acute Myeloid Leukemia

Ronja Bjørnstad, Reidun Aesoy, Øystein Bruserud, et al.

Mol Cancer Ther Published OnlineFirst January 24, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-17-1234

Supplementary Access the most recent supplemental material at: Material http://mct.aacrjournals.org/content/suppl/2019/01/24/1535-7163.MCT-17-1234.DC1

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

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/early/2019/02/19/1535-7163.MCT-17-1234. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.