More than 40 years ago, the present standard induction therapy for acute myeloid leukemia (AML) was developed. This consists of the metabolic inhibitor cytarabine (AraC) and the cytostatic topoisomerase 2 inhibitor daunorubucin (DNR). In light of the high chance for relapse, as well as the large heterogeneity, novel therapies are needed to improve patient outcome. We have tested the anti-AML activity of 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 could also have inhibitory activity against other protein kinases. Ser/Thr kinases like the Pim kinases have been identified as potential drug targets for AML therapy. The compound VS-II-173 induced AML cell death with EC50 below 5 μmol/L, and was 10 times less potent against nonmalignant cells. It perturbed Pim-kinase–mediated AML cell signaling, such as attenuation of Stat5 or MDM2 phosphorylation, and synergized with DNR to induce AML cell death. VS-II-173 induced cell death also in patients with AML blasts, including blast carrying high-risk FLT3-ITD mutations. Mutation of nucleophosmin-1 was associated with good response to VS-II-173. In conclusion new scaffolds for potential AML drugs have been explored. The selective activity toward patient AML blasts and AML cell lines of the pyrazolo-analogue VS-II-173 make it a promising drug candidate to be further tested in preclinical animal models for AML.

Acute myeloid leukemia (AML) is a highly heterogeneous disease characterized by aberrant myeloid differentiation and subsequent accumulation of immature myeloid cells in the bone marrow (BM). The standard chemotherapy regimen is based on the metabolic inhibitor cytarabin (AraC) in combination with the cytostatic topoisomerase 2 inhibitor daunorubucin (DNR), and has changed little over the past 40 years (1). Intense research efforts aim to identify drug targets that can help overcome AML chemotherapy resistance and relapse, as well as the severe toxic side effects of DNR (see ref. 2, for a recent review on potential drug targets). One of the important regulators of AML cell survival are found in the family of three serine/threonine-protein kinases, Pim1, 2, and 3 (3). They are involved in the regulation of multiple cellular processes, including cell-cycle progression and cell survival. The Pim kinases mediate signals from Abelson murine 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 constitutively active (6). Their activity is regulated by altering the intracellular levels, that is by transcription and translation, or by degradation (7). Together this makes them a likely Achilles heel for AML cells dependent on a reliable communication of cytokine signals to the nucleus in order to survive and multiply. Mice depleted of all three Pim kinase proteins have a normal life span and are 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 kinase inhibitors have been evaluated for therapeutic potential against hematologic disorders, in particular multiple myeloma and AML, but also CML, ALL, and B-cell lymphoma (9, 10). Previously, we have reported the synthesis of a series of Pim kinase inhibitors exhibiting various N-containing heteroaromatic scaffolds such as pyrrolo[2,3-a]carbazole-3-carbaldehydes, pyrazolo[3,4-c]carbazoles, pyrazolo[4,3-a]phenanthridines, and pyrrolo[2,3-g]indazoles (Fig. 1A; refs. 11–18). A broad selection of molecular scaffolds is important in order to develop the best therapeutic agents against a specific target. All the above-mentioned compound classes showed acceptable IC50 values toward Pim kinases (below 10 nmol/L in some cases, see Table 1). The ATP-binding pocket, along with the substrate-recognition residues are identified as crucial for Pim kinase inhibitors (19). These compounds were reported to have activity toward AML cells. We wanted to extend our investigation of these compounds as potential drugs for AML, and investigate the possibilities for one or two selected compounds to enter further preclinical investigations.

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).

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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Table 1.

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

IC50 (μmol/L)
Pim1Pim2Pim3
B3 (11)a 0.12 0.51 0.01 
RAG-II-45 (11) 0.0068 0.131 0.0124 
RAG54 (12) 0.57 >10 0.04 
RAG-II-115 (13) 0.075 >1 0.08 
FG587 (14) 0.009 >1 0.026 
MB032 (14) 0.042 >1 0.05 
MB071 (15) 1.4 nd 0.38 
MB069 (15) 1.56 nd 0.6 
FG644B (15) 0.39 nd 0.19 
EE057 (15) 1.1 nd 0.7 
FG610 (15) 0.74 nd 0.34 
FG773 (15) >1 nd 2.9 
LG-VII-126 (16) 0.04 49% (1 μmol/L)b 0.10 
VS-II-173 (17) 0.07 46% (1 μmol/L)b 0.02 
LG-VI-142 (18) 0.46 >10 0.033 
IC50 (μmol/L)
Pim1Pim2Pim3
B3 (11)a 0.12 0.51 0.01 
RAG-II-45 (11) 0.0068 0.131 0.0124 
RAG54 (12) 0.57 >10 0.04 
RAG-II-115 (13) 0.075 >1 0.08 
FG587 (14) 0.009 >1 0.026 
MB032 (14) 0.042 >1 0.05 
MB071 (15) 1.4 nd 0.38 
MB069 (15) 1.56 nd 0.6 
FG644B (15) 0.39 nd 0.19 
EE057 (15) 1.1 nd 0.7 
FG610 (15) 0.74 nd 0.34 
FG773 (15) >1 nd 2.9 
LG-VII-126 (16) 0.04 49% (1 μmol/L)b 0.10 
VS-II-173 (17) 0.07 46% (1 μmol/L)b 0.02 
LG-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 μmol/L.

Description of the kinase inhibitors

All compounds described in Fig. 1A are designed to bind in the ATP-recognition site of Pim kinases. Their synthesis has been described elsewhere (11–18). Compound B3 exhibited a selective profile when evaluated toward a panel of 66 protein kinases (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 by molecular modeling for analogues of this series. Compound LG-VII-126 is part of another Pim inhibitor series (Fig. 1A). 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 identified VS-II-173 as a nanomolar Pim1 and Pim3 inhibitor (17). The study of its putative binding with Pim1/Pim3 by molecular modeling demonstrated that VS-II-173 inserted deeply into the ATP site of Pim1 and Pim3 (17).

The in vitro protein kinase screening was performed at the International Centre for Kinase Profiling (ICKP; Dundee, Scotland). VS-II-173 was evaluated toward a panel of 50 protein kinases in duplicate assays at a compound concentration of 1 μmol/L as described previously (20–22). The results were expressed as the percentage of residual kinase activity. The protein kinases were of human origin except Lck (mouse) and ROCK2 (rat).

Cell culture conditions

The 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 leukemia cells (ATCC, CRL-9591), MV4-11 cells with silenced p53 (MV4-11 shp53), the rat promyeloid leukemia wild-type cell lines IPC-81 (25), IPC-81 with enforced expression of Bcl-2 (both created by Dr. Michel Lanotte, Hôpital St. Louis, Paris, France, and maintained by prof. Stein Ove Døskeland; ref. 26), and the human AML cell line OCI-AML3 (ATCC, ACC-582). The Molm-13 and MV4-11 cells with silenced p53 were created by Prof. Stein Ove Døskeland, Drs. Sjur Huseby, and Gro Gausdal by retroviral transfection for stable expression of shRNA against p53 using the pRETRO SUPER-p53 vector (27). Selection was by increasing puromycin concentration up to 400 μg/mL over 14 days. All compounds were also tested on the chronic myeloid leukemia (CML) cell line K562 (ATCC, CCL-243), NRK rat kidney epithelial (ATCC, CRL-6509), and H9c2 cardio myoblasts (ATCC, CRL-1446). Molm-13 cells were cultured in RPMI medium with 10% FBS (Invitrogen). MV4-11 and K562 were cultured in 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 supplemented with 10% horse serum. All culture media were from Sigma-Aldrich Inc., and were supplemented with 100 IU/mL penicillin and 100 mg/L streptomycin. Culturing of all cells was in a humidified atmosphere (37°C, 5% CO2). The suspension cells were kept at 10 to 80 × 104 cells/mL and diluted by adding fresh medium. 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 cell line during the time of the experiments.

Assessment of cell death

Cytotoxicity assays were performed in 96-well plates at 0.1 mL medium/well. Suspension cells were seeded at 0.5 × 106 cells/mL and used the day of seeding. The NRK and H9c2 cells were seeded at 0.05 × 106 cells/mL, and left over night to attach before experiments. The compounds were tested alone, or in combinations with the following cytostatics; DNR (Cerubidine; Sanofi Aventis Lysaker), Geldanamycin, Emetine (both purchased from Sigma-Aldrich), bortezomib (Velcade, Janssen), Etoposide (Eposin, Teva), Cisplatin (Accord Healthcare), and AraC (Cytarabin, Fresenius Kabi). The pan-Pim kinase inhibitor AZD1208 was from Tocris Bioscience, the Bcl-2 inhibitor Venetoclax / ABT-199 from Cayman Chemical, and the tyrosine-kinase inhibitor Midostaurin from Sigma-Aldrich. The viability of the treated cells was assessed using a WST-1 Cell 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 visualize the nuclei. Cell death was assessed by UV-microscopic evaluation of nuclear morphology (28), and the data from the metabolic activity were used to confirm the data from microscopy. EC50 values were estimated by nonlinear regression using the statistical software IBM SPSS statistics for Apple, ver. 24, (IMB Corp.):

formula

where Y is the response (cell death); max and min, the maximum and minimum cell death from the curve; X the concentration of the drug/compound; and h is the hill index. Determination of coefficient of drug interaction (CDI) was done by finding the ratio between the effect of treatment and control (R) and further calculates as shown in Eq. 2.

formula

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

Membrane permeability assay

The Corning Gentest pre-Coated parallel artificial membrane permeability assay PAMPA Plate System (Corning Discovery Labware) was done according to manufacturer's protocol. The compounds were tested at 50 μmol/L in PBS at 21°C. After incubation for 5 hours, 50 μL of each sample were added 15 μL acetonitrile, and injected into a reversed phase HPLC column (Kromasil 100-5 C18 150-4.6 mm; Akzo Nobel) connected to a Merck-Hitachi LaChrome HPLC-system (VWR) with a L-7455 diode array detector. Mobile phase A was 0.05% aqueous TFA, and mobile phase B was acetonitrile. The flow rate was 1.6 mL/min and compounds eluted between 1 and 4.5 minutes during a 4.5 minutes gradient from 70:30% mobile phase A:B to 0:100% mobile phase A:B. The ratios of the compound concentrations in the donor and acceptor compartments were calculated after integration of the peaks at 248 nm. Peff was calculated as described in ref. 29.

Western blot analysis

Western blotting was conducted as described previously (30). To ensure that we studied effects of kinase inhibition, we chose concentrations that gave less than 30% apoptosis. This would avoid loss of proteins due to caspase cleavage or disintegrated cells. After treatment with Pim kinase inhibitors, the cells were washed and lysed, and protein concentrations determined using the Quick Start Bradford protein assay (Bio-Rad Laboratories). SDS-polyacrylamide gels, 10% or 12% were loaded with 30 to 50 μg protein, and the prelabeled “Precision Plus Protein Standard All Blue” (Bio-Rad Laboratories). Proteins were transferred to polyvinylidene difluoride membranes (GE Healthcare Life Sciences) by electrophoresis. The following primary antibodies were used: Pim1, Pim2, and Pim3 (all from Santa Cruz Biotechnology, except for on OCI-AML3 cells, where Pim2 antibody from Sigma-Aldrich was used), Bad, pBad phospho S112, 4EBP1, p4EBP1 phospho T37/46, STAT5 and pSTAT5 phospho 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-β-actin (Sigma-Aldrich). The secondary antibodies were donkey anti-mouse and anti-rabbit antibodies conjugated to horse-radish peroxidase (Jackson Immunoresearch Laboratory). Membranes were developed using Supersignal West Pico or West Femto Chemiluminiscence Substrate from Pierce Biotechnology Inc., according to the manufacturers' protocols. The immunoblots were imaged by a LAS-4000 image reader (Fujifilm). After imaging, the membranes were stripped using Restore PLUS Western Blot Stripping Buffer (ThermoFisher Scientific) to reprobe the membranes with the antibody for the phosphorylated version of the proteins. Anti-β-actin was used for loading control.

Flow cytometric analysis of AML patient material

The collection of patient cells was approved by the regional research ethics committee (Health Region III, Bergen, Norway, REK approval numbers for biobanking: III 060.02 215.03, 231.06, and 2015/1410, and for experimental use of cells: REK Vest 2015/1759 and 2017/305) and conducted in accordance with the Declaration of Helsinki. Samples were collected after written informed consent, and stored under liquid nitrogen in biobanks approved by the Norwegian Royal Ministry of Health and the Norwegian Directorate for Health and Social Affairs. Blasts from 37 patients were used in this study. The patients were not selected after response to chemotherapy. All patients had a high count of AML blasts in peripheral blood, and blasts were isolated by density 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 and kindly provided by Professor BT Gjertsen (Health Region III; REK approval number: 2012/2247). Peripheral blood mononuclear cells (PBMC) were obtained from freshly drawn peripheral blood (Department of Immunology and Transfusion Medicine, Haukeland University Hospital, Bergen, Norway) and isolated by density gradient separation (Lymphoprep; Axis-Shield). Erythrocytes were lysed (BD Biosciences Pharm Lyse buffer) and leukocytes were suspended in Minimum Essential Medium Eagle (MEME), enriched with 10% FBS and 2 mmol/L L-glutamine, and supplemented with 100 IU/mL penicillin and 100 mg/L streptomycin (all from Sigma-Aldrich Inc.). Upon the experiment AML blasts or normal BM cells were thawed and suspended in Stem Span SFEM medium (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 was assessed by flow cytometric analysis of cells stained with PacificBlue-AnnexinV (#640918; BioLegend) and propidium iodide (PI; #421301; BioLegend). Between 10,000 and 30,000 events were collected for each sample using a BD Fortessa flow cytometer. The data were analyzed with the software Flow Jo (Tree Star, Inc.).

Statistical analysis

Response to treatments were evaluated by Student t test, correlation of data by two-tailed Pearson correlation test, and patient data by cross-table analyses for chi-square, all by using IBM SPSS statistics for Apple, ver. 24 (IMB Corp.). Significance was defined as P ≤ 0.05. Heat maps were created by MeV (31), analyses of Pim expression was done using the J-Express 2012 software (32) after normalization and log(10) transformation of data. Kinome tree was created using the Kinome Render software (33).

Characterization of anti-AML activity of kinase inhibitors

We previously found that pyrrolo[2,3-a]carbazole-3-carbaldehyde Pim kinase inhibitors (15) had promising activity against AML cells. We therefore decided to test the efficacy of an in-house selection of Pim inhibitors containing different hetero-aromatic scaffolds (Fig. 1A) against a panel of cell lines (Fig. 2A). The inhibitor VS-II-173 was found to be a potent and selective inducer of AML cell death. The cardiomyoblasts, epithelial cell lines, and the chronic myeloid leukemia cell line K562 were unaffected at VS-II-173 concentrations inducing 100% death in the AML cells. The compounds MB032, LG-VI-142, LG-VII-126, RAG54, and FG587 also showed selectivity toward AML cell lines, but were less potent than VS-II-173. The compound RAG-II-115 was a potent inducer of cell death, but did not discriminate between AML-derived and other cell lines (Fig. 2A). VS-II-173 was able to overcome the prosurvival effect of poor prognostic factors in AML such as FLT3-ITD expression (MV4-11 and Molm-13 cells), p53 silencing (Molm-13 shp53 and MV4-11 shp53) and, to some extent, Bcl-2 overexpression in IPC-81 cells (Fig. 2A). To find if the differences in efficacy was due to the compound's ability to penetrate the cellular membrane, we tested the membrane permeability using the PAMPA assay. Membrane permeability was classified 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) impermeable (LogPeff < −6.14). The compounds RAG-II-115 and MB032 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 toward cells, but low membrane permeability. There were also compounds with low efficacy toward cells, and low membrane permeability, like RAG-II-45, FG587, RAG54, and MB071. However, the compounds MB069, FG644B, EE057, and FG610 all showed high membrane permeability (Fig. 2B), but low ability to induce cell death (Fig. 2A). We also checked whether there were large differences between microscopic assessment of nuclear morphology and metabolic conversion of the WST-1 reagent (Fig. 2C and D). The WST-1 assay gave a lower EC50 value compared with microscopic evaluation, presumably due to its ability to detect cell growth inhibition. Still there was a good correlation between the two methods (Pearson's correlation test), as a decline in WST-1 signal was always accompanied with increase in apoptosis (see inset in Fig. 2D). The cell death induced by VS-II-173 had typical apoptotic morphology like cell shrinkage, nuclear condensation, and fragmentation (inset in Fig. 2C).

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 μmol/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 μmol/L VS-II-173. The bars indicate 9 μm. 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 μmol/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).

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 μmol/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 μmol/L VS-II-173. The bars indicate 9 μm. 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 μmol/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).

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We next assessed the time kinetics of the cytotoxic action by selected compounds at concentrations giving between 80% and 100% death after 24 hours. The times to obtain 50% apoptotic cells were similar (ranging from 4.4 to 6 hours) for the AML-specific compounds (Fig. 2E). RAG-II-115 induced a much more rapid 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 the initial cell experiments, VS-II-173, to be evaluated toward 50 protein kinases at 1 μmol/L concentration (Fig. 2F; Supplementary Table S1). This revealed that the compound had activity toward other kinases as well. The most notable besides Pim1 and 3 was the dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1a), AMP-activated kinase (AMPKα1), homeodomain interacting protein kinase 2 (HIPK2), protein kinase C-related protein kinase 2 (PRK2), and ribosomal protein S6 kinase A1 (RSK1), all having less than 10% residual activity after treatment with 1 μmol/L of VS-II-173.

VS-II-173 acts synergistically with the anthracycline daunorubicin, and additively with a number of other anticancer drugs

To establish whether our kinase inhibitors could have potential in combination therapy, we chose the two most potent of the AML selective compounds, VS-II-173 and MB032. We found that VS-II-173 at subtoxic concentration increased the Molm-13 AML cell death from 40% to 80% when combined with 50 nmol/L daunorubicin (DNR) for 24 hours (Fig. 3A). The compound MB032 did not synergize with DNR (Fig. 3A).

Figure 3.

Drug interactions between VS-II-173 and seven anticancer compounds. A, Molm-13 cells were treated with VS-II-173 (1 μmol/L), MB032 (100 μmol/L) in combination with DNR. After 24 hours, the cells were fixed and viability was assessed by microscopic evaluation of apoptosis using Hoechst 33342 DNA stain. The vertical dotted lines indicate EC50 values. B, Molm-13 cells were treated with 1 μmol/L VS-II-173 in combination with different anticancer compounds for 24 hours before assessing viability as in A. The CDI was calculated as described in the section Materials and Methods. The concentrations of the compounds tested were: Geldanamycin, 10 nmol/L; Etoposide, 0.6 μmol/L; Emetine, 5 nmol/L; DNR, 75 nmol/L; Cisplatine, 6 μmol/L; AraC, 6 μmol/L, Bortezomib, 10 and 3 nmol/L. In B, *** indicates P < 0.001, one-sample T test. a: For bortezomib, the CDI for the high concentration is shown. C–E, Examples of drugs which act synergistically (C; DNR), antagonistically (D; bortezomib), or additively (E; Etoposide). F and G, Synergistic activity between VS-II-173 and DNR in WT Molm-13 AML cells (left) or silenced p53 (shp53, right). The cells were incubated with increasing doses of DNR with or without 2 or 4 μmol/L VS-II-173 for 24 hours. The insets show induction of p53 by DNR expression in WT-cells (F), but not in shp53 cells (G). Viability was assessed as in A. The data in A–G are the mean and the SD from 5 to 12 experiments. H–J, Cell death inducing effect of venetoclax (H), midostaurin (I), and AZD1208 (J) on different cell lines after 24 hours incubation. Cell death was scored using the WST-1 assay and is relative to untreated cells. The data are average of two (Molm13, OCI-AML3, and MV4-11) or four (NRK) experiments and the lines are from four-parameter regression analysis as described in the section Materials and Methods.

Figure 3.

Drug interactions between VS-II-173 and seven anticancer compounds. A, Molm-13 cells were treated with VS-II-173 (1 μmol/L), MB032 (100 μmol/L) in combination with DNR. After 24 hours, the cells were fixed and viability was assessed by microscopic evaluation of apoptosis using Hoechst 33342 DNA stain. The vertical dotted lines indicate EC50 values. B, Molm-13 cells were treated with 1 μmol/L VS-II-173 in combination with different anticancer compounds for 24 hours before assessing viability as in A. The CDI was calculated as described in the section Materials and Methods. The concentrations of the compounds tested were: Geldanamycin, 10 nmol/L; Etoposide, 0.6 μmol/L; Emetine, 5 nmol/L; DNR, 75 nmol/L; Cisplatine, 6 μmol/L; AraC, 6 μmol/L, Bortezomib, 10 and 3 nmol/L. In B, *** indicates P < 0.001, one-sample T test. a: For bortezomib, the CDI for the high concentration is shown. C–E, Examples of drugs which act synergistically (C; DNR), antagonistically (D; bortezomib), or additively (E; Etoposide). F and G, Synergistic activity between VS-II-173 and DNR in WT Molm-13 AML cells (left) or silenced p53 (shp53, right). The cells were incubated with increasing doses of DNR with or without 2 or 4 μmol/L VS-II-173 for 24 hours. The insets show induction of p53 by DNR expression in WT-cells (F), but not in shp53 cells (G). Viability was assessed as in A. The data in A–G are the mean and the SD from 5 to 12 experiments. H–J, Cell death inducing effect of venetoclax (H), midostaurin (I), and AZD1208 (J) on different cell lines after 24 hours incubation. Cell death was scored using the WST-1 assay and is relative to untreated cells. The data are average of two (Molm13, OCI-AML3, and MV4-11) or four (NRK) experiments and the lines are from four-parameter regression analysis as described in the section Materials and Methods.

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We next tested VS-II-173 in combination with other anticancer drugs. The best synergy for VS-II-173 was with DNR (Fig. 3B and C), only a modest synergy being observed with cisplatin (Fig. 3B). It was also noted that high concentrations of the proteasome inhibitor bortezomib antagonized the action of VS-II-173 (Fig. 3B and D), whereas lower concentrations gave an additive effect (Fig. 3D). The other drugs tested (etoposide, emetine, and geldanamycin) 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 that cells with silenced p53 had reduced, but not abolished sensitivity to the compounds tested, including VS-II-173. We wanted to investigate if lack of p53 could affect the synergy between VS-II-173 and DNR. We found that DNR synergized well with 4 μmol/L VS-II-173, both against Molm-13 WT and Molm-13-shp53 cells, but also that a higher concentration of both drugs was needed to achieve a substantial synergistic effect (Fig. 3F and G).

Next, the anti-AML activity of three other compounds was tested. A total of 50 μmol/L of the pan-Pim kinase inhibitor AZD1208 induced around 60% cell death in MV4-11 cells after 24 hours incubation, whereas the other cell lines were mostly unaffected (Fig. 3H). The Bcl-2 inhibitor venetoclax was efficient toward Molm13 and MV4-11 in the low nanomolar range, but were nontoxic to the OCI-AML3 cells at the concentrations tested (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 substrates

Pim kinases have been shown to act downstream of FLT3 signaling (5), and we compared the efficacy of VS-II-173 on AML cell lines with different FLT3 mutation status. The VS-II-173 had higher efficacy toward cell lines harboring the FLT3-ITD mutation (Molm-13, FLT3-ITD heterozygous, and MV4-11 FLT3-ITD homozygous), with EC50 values between 2 and 3 μmol/L (Fig. 4A and B, respectively). The OCI-AML3 cells (FLT3 WT) were less sensitive to VS-II-173, with EC50 about 16 μmol/L (Fig. 4C).

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 μmol/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 μmol/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. β-Actin was used as loading control on all blots.

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 μmol/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 μmol/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. β-Actin was used as loading control on all blots.

Close modal

To study early events (the initiation phase) of the apoptosis process triggered by VS-II-173, we used concentrations that would produce apoptosis after 5 to 10 hours, in line with previous studies on for instance anthracycline and AML (34, 35). When examining the expression of Pim1, 2, and 3 in the AML cell lines treated with VS-II-173, we noted a rapid, but transient downregulation of all three kinases in Molm13 cells (Fig. 4D, see Supplementary Fig. S1 for quantification). The response was less profound in MV4-11 and OCI-AML3 cells (Fig. 4E and F). Furthermore, the Molm13 and MV4-11 cells showed a rapid dephosphorylation of Stat5 (Fig. 4G and H) upon treatment with VS-II-173. The MV4-11 cells showed a decrease of the prosurvival phospho-Bad form of the otherwise apoptogenic BH3 only protein Bad (Fig. 4H) and the OCI-AML3 cells showed a strong decrease in p-4E-BP1 and pMDM2 (Fig. 4I).

Blasts from patients with AML with poor prognostic markers are sensitive to the kinase inhibitor VS-II-173

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

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 μmol/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.

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 μmol/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.

Close modal

The results from the AML patient blast experiments showed one large group that responded well to treatment with VS-II-173, where doses between 6 and 24 μmol/L could kill more than 50% of the blasts (Responders, Fig. 5). Some of the VS-II-173 responding patient blasts also responded well to the DNR (patients 11 and 24). In some cases, even the highest concentration of DNR (100 nmol/L) did not affect the blast viability whereas VS-II-173 showed high potency against these patient blasts (patients 1 and 2). Another group responded to DNR, but not to VS-II-173, for instance patient 24 and to some extent patient 25. There were two groups that did not appear to benefit from VS-II-173. One, which appeared to be resistant to both DNR and VS-II-173, like patients 36 and 37, and one where the combination gave a reduced effect (patients 22 and 23). Despite this, the majority of the patients (24 out of 37) could be characterized as responders to VS-II-173.

Neither sex, age, nor the FAB classification seemed to influence the responsiveness of the patient blasts to VS-II-173 (Fig. 5A). The three patients with adverse cytogenetics (patients 20, 30, and 31) showed poor to intermediate response to VS-II-173, whereas the two 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 the intermediate/non responder group (3 of 13, 23%), but there was no significant difference between the occurrence of FLT3-ITD in the responder and nonresponder groups (Pearson chi-square = 1.55, P = 0.21). Furthermore, the patients with mutated NPM-1 were overrepresented in the responder group (9 of 19 patients (47%) in the responder group, and 1 of 14 patients (7%) in the intermediate/non responder group, Pearson chi-square = 6.175, P = 0.013). Three-way chi-square test of our data showed NPM1 status was important for response to VS-II-173 in patients with WT FLT3 (Pearson chi-square = 4.89, P = 0.27), but not in patients with FLT3-ITD (Pearson chi-square = 0.749, P = 0.387).

Compared with the responder group, BM aspirates or PBMC were less affected by VS-II-173 (Fig. 5A), but the AML blasts from two patients classified as nonresponders had equal or slightly lower response compared with the PBMC or BM cells.

We found no correlation between response to VS-II-173 and Pim kinase expression level (Fig. 5B), based on mRNA expression of Pim kinases related to the median expression in patients (no significance using Pearson Chi-Square statistics).

Of the 15 compounds we tested, VS-II-173 exhibited potent selective activity toward the AML cell lines. This could theoretically be explained by the compound selectively targeting rapidly growing cell lines, but the K562 CML cell line has a doubling time similar to that of Molm-13 cells, thus rapid proliferation does not seem to be the reason for the AML-selectivity observed in Fig. 2A. Importantly, VS-II-173 was active toward cells with high-risk 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 (Figs. 2A, 4A–C, and 5). We could not detect any correlation between the ability to inhibit the different Pim kinases and the ability to selectively induce AML cell death (Table 1; Fig. 2A). For instance, LG-VII-126 and VS-II-173 had similar inhibitory activity toward the three kinases, but VS-II-173 was a far more potent AML cell death inducer than LG-VII-126. This could partly be explained by differences in the ability to cross phospholipid membranes (Fig. 2B).

The kinase screen showed that VS-II-173 also inhibited other kinases than the three Pim kinases (Fig. 2F). The level of promiscuity was similar to that shown for the pan-Pim kinase inhibitor SGI-1776 (38), which is active against AML cells (39).

Many chemotherapeutic therapy regimens for AML are based on drug combinations, such as the 7+3 regimen with DNR and AraC or high-dose AraC plus a nucleoside analogue (see refs. 40, 41 for recent reviews on established and emerging therapies for AML). It became apparent that the most potent compound selective for AML cells, VS-II-173 increased the cellular response to DNR with high synergy (Fig. 3A–C). Combination with cisplatin and VS-II-173 gave more modest synergy (Fig. 3B). Thus, combinations of VS-II-173 with DNR or possibly cisplatin could be clinically relevant. This agrees with a previous study by Doshi and colleagues showing that Pim kinase inhibition sensitized AML cell lines to DNR (42). Furthermore, our results indicate that Ara-C and VS-II-173 have sub-additive action, suggesting they should not be combined simultaneously. Other Pim kinase inhibitors have acted both in a synergistic and antagonistic manner in combination with AraC in AML cell lines and primary AML cells (42, 43).

The transiently reduced level of Pim1, 2, and 3 kinases in the Molm-13 cells in response to VS-II-173 (Fig. 4D) indicates that inhibition of Pim kinases increases degradation or attenuates production of the protein. Pim kinase activity is not regulated by post-translational modifications or allosteric modulators, but by increased or attenuated expression (7). Treatment with VS-II-173 apparently perturbed the ratio between synthesis and degradation of the Pim kinases, but not irreversibly, suggesting that this was not due to cell-death associated protein degradation. The compensation mechanisms and subsequent restoration of the Pim kinase levels appears presumably too late to initiate the necessary survival mechanisms, since the cells died after eight to twelve hours (Fig. 2E).

Cells with the FLT3-ITD mutation mediate aberrant signaling through the Pim kinases (4). Thus, inhibition of Pim represents an attractive approach to target FLT3-ITD cells. Pim kinases promote survival of AML cells via phosphorylation of Bcl-2 antagonist of cell death (Bad), and also 4E-BP1 (4, 39), in accordance with reduced phosphorylation of these proteins seen upon VS-II-173 treatment, and by previous findings by Chen and colleagues (39). It has been demonstrated that Pim1 kinase directly affects the FLT3 receptor by a stabilizing serine phosphorylation (44). This positive feedback loop prompts Stat5 activation and Pim signaling (44). We found abolished phosphorylation of Stat5 in FLT3-ITD cell lines 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 signaling and MDM2 phosphorylation (45). Accordingly, our results showed reduced phosphorylation in MDM2 when using VS-II-173 in Molm-13 cells (Fig. 4G). Furthermore, we found that both VS-II-173 and the pan-Pim kinase inhibitor AZD1208 reduced the phosphorylation of Akt and p70-S6 kinase (see Supplementary Fig. S2), however VS-II-173 had a higher potency toward AML cell lines than AZD1208 in mono-treatment (Fig. 3H and Fig. 4A–C).

It must be noted that some of the perturbations in cell signaling could be caused by inhibition of other kinases. RSK1 was significantly inhibited by VS-II-173 (Fig. 2F). RSK1 is a mediator of the anti-apoptotic function of FLT3-ITD through phosphorylation of Bad at Ser112 in AML cells (46). The reduction of pBad (Fig. 4H) could be due to inhibition of RSK1 along with Pim kinase inhibition. AMPK is a stress sensor in cells, and can contribute to initiation of autophagy, and protects AML-cells from metabolic stress (47). AMPK can thus be an important target for AML therapy, particularly to prevent leukemogenesis, although another study shows that activation of AMPK initiates autophagic cell death (48). DYRK1 is a tumor suppressor, which is heavily down-regulated in AML patients (49), and mutations in the HIPK2 gene is linked to pathogenesis of AML (50). Taken together, the anti-AML activity can be inhibition of Pim kinase activity acting in concert with additional inhibition of some or all of the abovementioned factors.

The fact that VS-II-173 affects more than one target is in line with several of the so-called precision therapies, like the tyrosine kinase inhibitors. In fact, the second-generation kinase inhibitor Dasatinib has a higher level of promiscuity than its predecessor Imatinib (51). With this in mind, a possible explanation for the potency and selectivity of VS-II-173 can be its activity toward key AML survival factors like RSK1 and AMPK together with its pan-Pim kinase activity. The much more selective Pim kinase inhibitor AZD1208 (52) was less efficient 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 multityrosine kinase inhibitor midostaurin (see ref. 53; Fig. 3J). The mono-selective Bcl-2 inhibitor venetoclax (54), which emerges as therapy against AML (55), failed to induce cell death in OCI-AML3 cells (Fig. 3I) and appears to be effective against different subtypes of AML compared to VS-II-173.

A panel of nonselected AML patient blasts was included and 24 of 37 patient responded well to the treatment with VS-II-173, where doses between 6 and 12 μmol/L could kill more than 50% of the blasts (Fig. 5). Approximately 40% of patients with AML with 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 not influenced by the low-risk factor NPM1 mutation when it comes to response to VS-II-173. These findings suggest that this compound can be used irrespective of FLT3 status. There was no correlation between the response to VS-II-173 and the level of Pim kinases in the patient blasts (Fig. 5B), which could be ascribed the effect on other kinases (Fig. 2F). However, the values in Fig. 5B were compared with a pooled average, and AML cells are expected to have higher levels of Pim kinases than normal BM (3).

New strategies for AML therapy are urgently needed. Despite this, only one new drug has been developed and approved for AML during the last 15 years. Unfortunately, it appears to yield only modest improvement in survival, even in preselected patients (57). Our data point to pyrazolo[4,3-a]phenanthridine as a relevant scaffold for compounds useful in the treatment of AML. The lead compound VS-II-173 exhibited crucial properties needed to exert therapeutic effect. It is selective toward AML cells, in which it efficiently dampens prosurvival signals. It is also potent against AML patient blasts, including some that showed high tolerance to DNR, and blasts carrying mutations associated with poor prognosis. The compound and future analogs of pyrazolo[4,3-a]phenanthridines represent possible candidates for future AML treatment. Future studies will aim to reveal possible biomarkers of the patient AML cells which responded well to treatment with VS-II-173, as well as conducting preclinical studies on human AML xenografts in mice. If the abovementioned issues are resolved, we believe that pyrazolo[4,3-a]phenanthridines have a future in AML treatment.

No potential conflicts of interest were disclosed.

Conception and design: R. Bjørnstad, Ø. Bruserud, L. Herfindal

Development of methodology: R. Bjørnstad, G. Gausdal, S.O. Døskeland, L. Herfindal

Acquisition of data (acquired and managed patients, provided facilities, etc.): Ø. Bruserud, F. Giraud, T.H. Dowling, P. Moreau, F. Anizon, L. Herfindal

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R. Bjørnstad, R. Aesoy, Ø. Bruserud, A.K. Brenner, S.O. Døskeland, L. Herfindal

Writing, review, and/or revision of the manuscript: R. Bjørnstad, R. Aesoy, Ø. Bruserud, P. Moreau, S.O. Døskeland, F. Anizon, L. Herfindal

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L. Herfindal

Study supervision: L. Herfindal

The authors want to thank Ing. Nina Lied Larsen and Kaja Skålnes Knudsen, MSc for technical assistance, and Sjur Huseby, PhD for assistance with generation of the AML cell lines. Prof. Bjørn Tore Gjertsen and Misbah Sabir, MSc are thanked for supplying bone marrow samples. Kathrine Åsrud, PhD is thanked for critically reading through the manuscript. This study was supported financially by the Norwegian Western Regional Health Authorities to Lars Herfindal (Grant No. 912052), Ronja Bjørnstad (Grant No. 912028), Stein O. Døskeland (Grant No.: 303485), and Øystein Bruserud (Grant No. 911788), by the Norwegian Cancer Society to Lars Herfindal (Grant No. 112502), Stein O. Døskeland (Grant No. 705268), Øystein Bruserud (Grant No. 62370, 62371, and 100933), and by the Norwegian Research council to Lars Herfindal and Reidun Aesoy (Grant No. 254752).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Yates
JW
,
Wallace
HJ
,
Ellison
RR
,
Holland
JF
. 
Cytosine-arabinoside (Nsc-63878) and daunorubicin (Nsc-83142) therapy in acute nonlymphocytic leukemia
.
Cancer Chemoth Rep 1
1973
;
57
:
485
8
.
2.
Engen
CB
,
Hajjar
E
,
Gjertsen
BT
. 
Development of personalized molecular therapy for acute myeloid leukemia
.
Curr Pharm Biotechnol
2016
;
17
:
20
9
.
3.
Alvarado
Y
,
Giles
FJ
,
Swords
RT
. 
The PIM kinases in hematological cancers
.
Expert Rev Hematol
2012
;
5
:
81
96
.
4.
Kim
KT
,
Levis
M
,
Small
D
. 
Constitutively activated FLT3 phosphorylates BAD partially through pim-1
.
Br J Haematol
2006
;
134
:
500
9
.
5.
Adam
M
,
Pogacic
V
,
Bendit
M
,
Chappuis
R
,
Nawijn
MC
,
Duyster
J
, et al
Targeting PIM kinases impairs survival of hematopoietic cells transformed by kinase inhibitor-sensitive and kinase inhibitor-resistant forms of Fms-like tyrosine kinase 3 and BCR/ABL
.
Cancer Res
2006
;
66
:
3828
35
.
6.
Bachmann
M
,
Moroy
T
. 
The serine/threonine kinase Pim-1
.
Int J Biochem Cell Biol
2005
;
37
:
726
30
.
7.
Amaravadi
R
,
Thompson
CB
. 
The survival kinases Akt and Pim as potential pharmacological targets
.
J Clin Invest
2005
;
115
:
2618
24
.
8.
Mikkers
H
,
Nawijn
M
,
Allen
J
,
Brouwers
C
,
Verhoeven
E
,
Jonkers
J
, et al
Mice deficient for all PIM kinases display reduced body size and impaired responses to hematopoietic growth factors
.
Mol Cell Biol
2004
;
24
:
6104
15
.
9.
Garcia
PD
,
Langowski
JL
,
Wang
Y
,
Chen
M
,
Castillo
J
,
Fanton
C
, et al
Pan-PIM kinase inhibition provides a novel therapy for treating hematologic cancers
.
Clin Cancer Res
2014
;
20
:
1834
45
.
10.
Kapoor
S
,
Natarajan
K
,
Baldwin
PR
,
Doshi
KA
,
Lapidus
RG
,
Mathias
TJ
, et al
Concurrent inhibition of pim and FLT3 kinases enhances apoptosis of FLT3-ITD acute myeloid leukemia cells through increased Mcl-1 proteasomal degradation
.
Clin Cancer Res
2018
;
24
:
234
47
.
11.
Akue-Gedu
R
,
Rossignol
E
,
Azzaro
S
,
Knapp
S
,
Filippakopoulos
P
,
Bullock
AN
, et al
Synthesis, kinase inhibitory potencies, and in vitro antiproliferative evaluation of new Pim kinase inhibitors
.
J Med Chem
2009
;
52
:
6369
81
.
12.
Akue-Gedu
R
,
Nauton
L
,
Thery
V
,
Bain
J
,
Cohen
P
,
Anizon
F
, et al
Synthesis, Pim kinase inhibitory potencies and in vitro antiproliferative activities of diversely substituted pyrrolo[2,3-a]carbazoles
.
Bioorg Med Chem
2010
;
18
:
6865
73
.
13.
Akue-Gedu
R
,
Letribot
B
,
Saugues
E
,
Debiton
E
,
Anizon
F
,
Moreau
P
. 
Kinase inhibitory potencies and in vitro antiproliferative activities of N-10 substituted pyrrolo[2,3-a]carbazole derivatives
.
Bioorg Med Chem Lett
2012
;
22
:
3807
9
.
14.
Giraud
F
,
Bourhis
M
,
Nauton
L
,
Thery
V
,
Herfindal
L
,
Doskeland
SO
, et al
New N-1,N-10-bridged pyrrolo[2,3-a]carbazole-3-carbaldehydes: synthesis and biological activities
.
Bioorg Chem
2014
;
57
:
108
15
.
15.
Giraud
F
,
Bourhis
M
,
Ebrahimi
E
,
Herfindal
L
,
Choudhury
RR
,
Bjornstad
R
, et al
Synthesis and activities of new indolopyrrolobenzodiazepine derivatives toward acute myeloid leukemia cells
.
Bioorg Med Chem
2015
;
23
:
7313
23
.
16.
Suchaud
V
,
Gavara
L
,
Saugues
E
,
Nauton
L
,
Thery
V
,
Anizon
F
, et al
Identification of 1,6-dihydropyrazolo[4,3-c]carbazoles and 3,6-dihydropyrazolo[3,4-c]carbazoles as new Pim kinase inhibitors
.
Bioorg Med Chem
2013
;
21
:
4102
11
.
17.
Suchaud
V
,
Gavara
L
,
Giraud
F
,
Nauton
L
,
Thery
V
,
Anizon
F
, et al
Synthesis of pyrazolo[4,3-a]phenanthridines, a new scaffold for Pim kinase inhibition
.
Bioorg Med Chem
2014
;
22
:
4704
10
.
18.
Gavara
L
,
Suchaud
V
,
Nauton
L
,
Thery
V
,
Anizon
F
,
Moreau
P
. 
Identification of pyrrolo[2,3-g]indazoles as new Pim kinase inhibitors
.
Bioorg Med Chem Lett
2013
;
23
:
2298
301
.
19.
Ogawa
N
,
Yuki
H
,
Tanaka
A
. 
Insights from Pim1 structure for anti-cancer drug design
.
Expert Opin Drug Discov
2012
;
7
:
1177
92
.
20.
Bain
J
,
Plater
L
,
Elliott
M
,
Shpiro
N
,
Hastie
CJ
,
McLauchlan
H
, et al
The selectivity of protein kinase inhibitors: a further update
.
Biochem J
2007
;
408
:
297
315
.
21.
Bain
J
,
McLauchlan
H
,
Elliott
M
,
Cohen
P
. 
The specificities of protein kinase inhibitors: an update
.
Biochem J
2003
;
371
:
199
204
.
22.
Davies
SP
,
Reddy
H
,
Caivano
M
,
Cohen
P
. 
Specificity and mechanism of action of some commonly used protein kinase inhibitors
.
Biochem J
2000
;
351
:
95
105
.
23.
Matsuo
Y
,
MacLeod
RA
,
Uphoff
CC
,
Drexler
HG
,
Nishizaki
C
,
Katayama
Y
, et al
Two acute monocytic leukemia (AML-M5a) cell lines (MOLM-13 and MOLM-14) with interclonal phenotypic heterogeneity showing MLL-AF9 fusion resulting from an occult chromosome insertion, ins(11;9)(q23;p22p23)
.
Leukemia
1997
;
11
:
1469
77
.
24.
McCormack
E
,
Haaland
I
,
Venas
G
,
Forthun
RB
,
Huseby
S
,
Gausdal
G
, et al
Synergistic induction of p53 mediated apoptosis by valproic acid and nutlin-3 in acute myeloid leukemia
.
Leukemia
2012
;
26
:
910
7
.
25.
Lacaze
N
,
Gombaud-Saintonge
G
,
Lanotte
M
. 
Conditions controlling long-term proliferation of Brown Norway rat promyelocytic leukemia in vitro: primary growth stimulation by microenvironment and establishment of an autonomous Brown Norway ‘leukemic stem cell line’
.
Leuk Res
1983
;
7
:
145
54
.
26.
Seite
P
,
Ruchaud
S
,
Hillion
J
,
Gendron
MC
,
Bruland
O
,
Segal-Bendirdjian
E
, et al
Ectopic expression of Bcl-2 switches over nuclear signalling for cAMP- induced apoptosis to granulocytic differentiation
.
Cell Death Differ
2000
;
7
:
1081
9
.
27.
Brummelkamp
TR
,
Bernards
R
,
Agami
R
. 
Stable suppression of tumorigenicity by virus-mediated RNA interference
.
Cancer Cell
2002
;
2
:
243
7
.
28.
Bøe
R
,
Gjertsen
BT
,
Vintermyr
OK
,
Houge
G
,
Lanotte
M
,
Døskeland
SO
. 
The protein phosphatase inhibitor okadaic acid induces morphological changes typical of apoptosis in mammalian cells
.
Exp Cell Res
1991
;
195
:
237
46
.
29.
Bennion
BJ
,
Be
NA
,
McNerney
MW
,
Lao
V
,
Carlson
EM
,
Valdez
CA
, et al
Predicting a drug's membrane permeability: a computational model validated with in vitro permeability assay data
.
J Phys Chem B
2017
;
121
:
5228
37
.
30.
Wergeland
L
,
Sjoholt
G
,
Haaland
I
,
Hovland
R
,
Bruserud
O
,
Gjertsen
BT
. 
Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells
.
Mol Cancer
2007
;
6
:
33
.
31.
Saeed
AI
,
Sharov
V
,
White
J
,
Li
J
,
Liang
W
,
Bhagabati
N
, et al
TM4: a free, open-source system for microarray data management and analysis
.
Biotechniques
2003
;
34
:
374
8
.
32.
Dysvik
B
,
Jonassen
I
. 
J-Express: exploring gene expression data using Java
.
Bioinformatics
2001
;
17
:
369
70
.
33.
Chartier
M
,
Chenard
T
,
Barker
J
,
Najmanovich
R
. 
Kinome Render: a stand-alone and web-accessible tool to annotate the human protein kinome tree
.
PeerJ
2013
;
1
:
e126
.
34.
Gausdal
G
,
Gjertsen
BT
,
McCormack
E
,
Van Damme
P
,
Hovland
R
,
Krakstad
C
, et al
Abolition of stress-induced protein synthesis sensitizes leukemia cells to anthracycline-induced death
.
Blood
2008
;
111
:
2866
77
.
35.
Gausdal
G
,
Wergeland
A
,
Skavland
J
,
Nguyen
E
,
Pendino
F
,
Rouhee
N
, et al
Cyclic AMP can promote APL progression and protect myeloid leukemia cells against anthracycline-induced apoptosis
.
Cell Death Dis
2013
;
4
:
e516
.
36.
Bennett
JM
,
Catovsky
D
,
Daniel
MT
,
Flandrin
G
,
Galton
DA
,
Gralnick
HR
, et al
Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group
.
Br J Haematol
1976
;
33
:
451
8
.
37.
Grimwade
D
,
Walker
H
,
Oliver
F
,
Wheatley
K
,
Harrison
C
,
Harrison
G
, et al
The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The medical research council adult and children's leukaemia working parties
.
Blood
1998
;
92
:
2322
33
.
38.
Xu
Y
,
Brenning
BG
,
Kultgen
SG
,
Foulks
JM
,
Clifford
A
,
Lai
S
, et al
Synthesis and biological evaluation of pyrazolo[1,5-a]pyrimidine compounds as potent and selective Pim-1 Inhibitors
.
ACS Med Chem Lett
2015
;
6
:
63
7
.
39.
Chen
LS
,
Redkar
S
,
Taverna
P
,
Cortes
JE
,
Gandhi
V
. 
Mechanisms of cytotoxicity to Pim kinase inhibitor, SGI-1776, in acute myeloid leukemia
.
Blood
2011
;
118
:
693
702
.
40.
Bose
P
,
Vachhani
P
,
Cortes
JE
. 
Treatment of relapsed/refractory acute myeloid leukemia
.
Curr Treat Options Oncol
2017
;
18
:
17
.
41.
Saygin
C
,
Carraway
HE
. 
Emerging therapies for acute myeloid leukemia
.
J Hematol Oncol
2017
;
10
:
93
.
42.
Doshi
KA
,
Trotta
R
,
Natarajan
K
,
Rassool
FV
,
Tron
AE
,
Huszar
D
, et al
Pim kinase inhibition sensitizes FLT3-ITD acute myeloid leukemia cells to topoisomerase 2 inhibitors through increased DNA damage and oxidative stress
.
Oncotarget
2016
;
7
:
48280
95
.
43.
Kelly
KR
,
Espitia
CM
,
Taverna
P
,
Choy
G
,
Padmanabhan
S
,
Nawrocki
ST
, et al
Targeting PIM kinase activity significantly augments the efficacy of cytarabine
.
Br J Haematol
2012
;
156
:
129
32
.
44.
Natarajan
K
,
Xie
Y
,
Burcu
M
,
Linn
DE
,
Qiu
Y
,
Baer
MR
. 
Pim-1 kinase phosphorylates and stabilizes 130 kDa FLT3 and promotes aberrant STAT5 signaling in acute myeloid leukemia with FLT3 internal tandem duplication
.
PLoS One
2013
;
8
:
e74653
.
45.
Hogan
C
,
Hutchison
C
,
Marcar
L
,
Milne
D
,
Saville
M
,
Goodlad
J
, et al
Elevated levels of oncogenic protein kinase Pim-1 induce the p53 pathway in cultured cells and correlate with increased Mdm2 in mantle cell lymphoma
.
J Biol Chem
2008
;
283
:
18012
23
.
46.
Yang
X
,
Liu
L
,
Sternberg
D
,
Tang
L
,
Galinsky
I
,
DeAngelo
D
, et al
The FLT3 internal tandem duplication mutation prevents apoptosis in interleukin-3-deprived BaF3 cells due to protein kinase A and ribosomal S6 kinase 1-mediated BAD phosphorylation at serine 112
.
Cancer Res
2005
;
65
:
7338
47
.
47.
Saito
Y
,
Chapple
RH
,
Lin
A
,
Kitano
A
,
Nakada
D
. 
AMPK protects leukemia-initiating cells in myeloid leukemias from metabolic stress in the bone marrow
.
Cell Stem Cell
2015
;
17
:
585
96
.
48.
Sujobert
P
,
Tamburini
J
. 
Co-activation of AMPK and mTORC1 as a new therapeutic option for acute myeloid leukemia
.
Mol Cell Oncol
2016
;
3
:
e1071303
.
49.
Liu
Q
,
Liu
N
,
Zang
S
,
Liu
H
,
Wang
P
,
Ji
C
, et al
Tumor suppressor DYRK1A effects on proliferation and chemoresistance of AML cells by downregulating c-Myc
.
PLoS One
2014
;
9
:
e98853
.
50.
Li
XL
,
Arai
Y
,
Harada
H
,
Shima
Y
,
Yoshida
H
,
Rokudai
S
, et al
Mutations of the HIPK2 gene in acute myeloid leukemia and myelodysplastic syndrome impair AML1- and p53-mediated transcription
.
Oncogene
2007
;
26
:
7231
9
.
51.
Ghoreschi
K
,
Laurence
A
,
O'Shea
JJ
. 
Selectivity and therapeutic inhibition of kinases: to be or not to be?
Nat Immunol
2009
;
10
:
356
60
.
52.
Keeton
EK
,
McEachern
K
,
Dillman
KS
,
Palakurthi
S
,
Cao
Y
,
Grondine
MR
, et al
AZD1208, a potent and selective pan-Pim kinase inhibitor, demonstrates efficacy in preclinical models of acute myeloid leukemia
.
Blood
2014
;
123
:
905
13
.
53.
Schlenk
RF
,
Kayser
S
. 
Midostaurin: a multiple tyrosine kinases inhibitor in acute myeloid leukemia and systemic mastocytosis
.
Recent Results Cancer Res
2018
;
212
:
199
214
.
54.
Timucin
AC
,
Basaga
H
,
Kutuk
O
. 
Selective targeting of antiapoptotic BCL-2 proteins in cancer
.
Med Res Rev
2018
.
55.
Menghrajani
K
,
Tallman
MS
. 
New therapeutic strategies for high-risk acute myeloid leukemia
.
Curr Opin Hematol
2018
;
25
:
90
4
.
56.
Schlenk
RF
,
Dohner
K
,
Krauter
J
,
Frohling
S
,
Corbacioglu
A
,
Bullinger
L
, et al
Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia
.
N Engl J Med
2008
;
358
:
1909
18
.
57.
Levis
M
. 
Midostaurin approved for FLT3-mutated AML
.
Blood
2017
;
129
:
3403
6
.