A Novel Glycogen Synthase Kinase-3 Inhibitor Optimized for Acute

Published OnlineFirst May 9, 2016; DOI: 10.1158/1535-7163.MCT-15-0566

Small Molecule Therapeutics Molecular Cancer Therapeutics A Novel Glycogen Synthase Kinase-3 Inhibitor Optimized for Acute Myeloid Leukemia Differentiation Activity Sophia Hu1, Masumi Ueda2, Lindsay Stetson1, James Ignatz-Hoover1, Stephen Moreton1, Amit Chakrabarti3, Zhiqiang Xia3, Goutam Karan3, Marcos de Lima2, Mukesh K. Agrawal3,4, and David N. Wald1,3,5

Abstract

Standard therapies used for the treatment of acute myeloid describe the discovery of a novel GSK3 inhibitor, GS87. GS87 leukemia (AML) are cytotoxic agents that target rapidly prolifer- was discovered in efforts to optimize GSK3 inhibition for AML ating cells. Unfortunately, this therapeutic approach has differentiation activity. Despite GS87's dramatic ability to induce limited efficacy and significant toxicity and the majority of AML AML differentiation, kinase profiling reveals its high specificity patients still die of their disease. In contrast to the poor prognosis in targeting GSK3 as compared with other kinases. GS87 demon- of most AML patients, most individuals with a rare subtype of strates high efficacy in a mouse AML model system and unlike AML, acute promyelocytic leukemia, can be cured by differenti- current AML therapeutics, exhibits little effect on normal bone ation therapy using regimens containing all-trans retinoic acid. marrow cells. GS87 induces potent differentiation by more GSK3 has been previously identified as a therapeutic target in effectively activating GSK3-dependent signaling components AML where its inhibition can lead to the differentiation and including MAPK signaling as compared with other GSK3 growth arrest of leukemic cells. Unfortunately, existing GSK3 inhibitors. GS87 is a novel GSK3 inhibitor with therapeutic inhibitors lead to suboptimal differentiation activity making potential as a differentiation agent for non-promyelocytic AML. them less useful as clinical AML differentiation agents. Here, we Mol Cancer Ther; 15(7); 1485–94. 2016 AACR.

Introduction In efforts to find novel strategies for inducing differentiation in non-APL leukemic cells, we and others identified that inhibiting Acute myeloid leukemia (AML), the most common form of GSK3 leads to AML differentiation (3–5). GSK3 is a serine/ acute leukemia in adults, is a heterogeneous disorder defined by threonine kinase involved in multiple signaling pathways. Orig- the arrest of proliferation of immature hematopoietic cells (1). inally identified for its role in regulating glycogen synthesis, it has Standard AML chemotherapy leads to significant toxicity and is been found to play roles in many biologic processes such as frequently ineffective. Despite the fact that AML is characterized inflammation, stem cell maintenance, cell proliferation, and by a differentiation arrest, standard AML treatment targets the regulation of normal and aberrant hematopoiesis (6). In addition, rapid proliferation of leukemic cells. In contrast to the poor GSK3 plays important roles in a variety of key signaling pathways prognosis of the majority of AML patients, most individuals with such as Wnt/b-catenin, PI3K, mTOR, and MAPK signaling (6). a rare subtype of AML, acute promyelocytic leukemia (APL), can While pharmacologic inhibition of GSK3 has historically been be cured by differentiation therapy using regimens containing all- studied in a variety of diseases (7), in recent years, GSK3 has been trans retinoic acid (ATRA; ref. 2). found to play an important role in cancer. GSK3 has been demonstrated to be overexpressed and hyperactivated in a variety 1 Department of Pathology, Case Western Reserve University, Cleve- of solid cancers. In addition, overexpression of GSK3 has been land, Ohio. 2Department of Hematology and Oncology, University Hospitals Case Medical Center and Case Western Reserve University, associated with resistance to therapy and poor prognosis (6, 8). Cleveland, Ohio. 3Invenio Therapeutics Inc., Cleveland, Ohio. 4MirX Prior studies have identified that inhibition of GSK3 results in 5 Pharmaceuticals, Cleveland, Ohio. University Hospitals Case Medical AML cell growth inhibition and monocytic differentiation (3–5, Center, Cleveland, Ohio. 9–12). Importantly, several studies have reported that targeting Note: Supplementary data for this article are available at Molecular Cancer GSK3 specifically impairs leukemic cell growth, but not normal Therapeutics Online (http://mct.aacrjournals.org/). bone marrow proliferation. Sophia Hu and Masumi Ueda contributed equally to this article. Because of the role of GSK3 in the molecular pathology of many Corresponding Authors: David N. Wald, Case Western Reserve University and diseases, development of specific inhibitors of this kinase with University Hospitals Case Medical Center, 2103 Cornell Road, Cleveland, OH therapeutic intentions has been pursued. While investigational 44106. Phone: 216-368-5668; Fax: 216-368-0495; E-mail: [email protected]; and GSK3 inhibitors exist, the potency and specificity of GSK3 inhibi- Mukesh K. Agarwal, Invenio Therapeutics, A262 Astecc Building, Lexington, KY tion is variable. Lithium (Li), the only FDA-approved GSK3 inhib- 40506. E-mail: [email protected] itor, has long been used clinically to treat bipolar disorder. Lithium þ doi: 10.1158/1535-7163.MCT-15-0566 directly binds GSK3 at an Mg2 binding site, leading to reversible 2016 American Association for Cancer Research. enzyme inhibition and inhibitory serine-9 phosphorylation

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(13–15). LY2090314 and tideglusib are more specific small-mol- Proliferation and colony assay ecule GSK3 inhibitors utilized in humans in clinical trials (16, 17). Equal numbers of THP-1, HL-60, NB4, U937, and OCI cells Other small-molecule GSK3 inhibitors have been described, such were plated and treated with Li, SB or GS87 for 72 hours. Cells as SB415285 (SB), which inhibits GSK3 by competitive binding to were analyzed for proliferation using the CellTiter 96 Aqueous the ATP-binding site (18). In general, the GSK3 inhibitors One Solution Cell Proliferation Assay (Promega) as recom- described to date also inhibit many other kinases such as mended by the manufacturer using a Molecular Q3 Devices CDK2-cyclin A (CDK2A; ref. 19). Spectramax M2 plate reader. For the colony assay, after 72 hours, Although GSK3 inhibition has been demonstrated to induce the cells were washed with PBS, an equal number of viable cells evidence of AML differentiation, existing GSK3 inhibitors lead to were suspended in 0.03% noble agar (Sigma), the cells were suboptimal levels of AML differentiation that would likely pro- incubated for one week and number of colonies was counted. hibit their use as clinically useful differentiation agents. Because of the promise of differentiation therapy for AML, we aimed to Cell-cycle analysis identify a novel GSK3 inhibitor that was optimized for AML Cells were treated with Li, SB, or GS87 for 24 hours, fixed in differentiation activity. Here, we report the identification of a 90% methanol and incubated overnight at 20C. Cells were novel and highly specific GSK3 inhibitor, GS87, which induces then washed with PBS, treated with RNase (0.5 mg/mL) and extensive AML differentiation with selective growth inhibition of propidium iodide (50 mg/mL) in the dark for 60 to 90 minutes leukemic cells and exhibits promise as an AML differentiation at room temperature and analyzed by flow cytometry. therapeutic. Western blot analysis Materials and Methods Cells treated as indicated were washed with PBS, centrifuged, Reagents and cells and lysed with a triton-containing lysis buffer for whole cell The 2,000 compound small-molecule library was custom extracts. Protein lysates (50 mg/lane) were resolved on appropriate designed based upon similarities to known chemical and struc- SDS–PAGE gel and transferred to polyvinylidene difluoride mem- tural features of previously reported GSK3 inhibitors by Enamine brane (Millipore) using Bio-Rad transfer apparatus (Bio-Rad). The (Ukraine). Additional quantities of GS87 (T5729983) were membranes were blocked, incubated with the indicated primary obtained from Enamine. SB415286 (SB) was obtained from antibodies at 4 C overnight, and incubated with the appropriate Tocris Biosciences. Lithium (Li) was obtained from Sigma. horseradish peroxidase–conjugated secondary antibodies. Immu- SB203580 (SB203), PD08959 (PD), and SP600125 (SP) were noreactive protein bands were detected by enhanced chemilumi- obtained from Selleck Chem. MTT reagent was obtained from the nescence (Pierce) using XAR-5 film. CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega). Phospho-Ser9 GSK3b (5558), GAPDH (5174), phos- RNA microarray pho-p38 (4511), phospho-ERK (4370), and phospho-JNK HL-60 cells were treated with Li, SB, or GS87 for 48 hours and (4668) was obtained from Cell Signaling Technology. b-Catenin, total RNA was isolated using TRIzol (Invitrogen). The cRNA was p21 (397), c-myc (40), c-MYB (7874), and MAFB (74521) anti- prepared using the TargetAmp-Nano Kit (Epicenter) and hybrid- bodies were obtained from Santa Cruz Biotechnology. OCI-AML3 ized to the human HT-12 Expression BeadChip (Illumina). cells were obtained from DSMZ, and HL-60, NB4, THP-1, U937, Microarray results were evaluated using Genomestudio (Illu- and HeLa cells were obtained from ATCC. All cell lines were mina) and R (version 3.1.3) software. Differential expression was obtained from ATCC or DSMZ and cultured for fewer than assessed using the lumi package for R. FDR was used to correct for 6 months after resuscitation. OCI-AML3 shb-catenin cells were multiple comparisons. Pathway analysis was performed using reported previously (3). Primary AML and normal donor bone Ingenuity Pathway Analysis software (Qiagen) for genes with marrow cells were obtained from Case Western Reserve University significantly dysregulated expression (FDR-adjusted P < 0.05) (CWRU) Hematopoietic Stem Cell Core Facility (Cleveland, OH). and an absolute log2 fold change greater than or equal to 1.5). Microarray data was submitted to Arrayexpress (accession num- Cell culture ber E-MTAB-3690). Cells were cultured in RPMI1640 media (Hyclone). All media were supplemented with 10% FBS and penicillin/streptomycin. Real-time quantitative reverse transcription PCR Cells were cultured in a humidified atmosphere of 95% (v/v) air Total RNA was isolated from cells treated with Li, SB, or GS87 and 5% (v/v) CO2 at 37 C. for 48 hours using TRIzol reagent (Invitrogen). RNA was tran- scribed into cDNA using the Enhanced Avian RT First Strand Differentiation Synthesis Kit (Sigma). Relative quantitative reverse transcription The nitrotetrazolium blue (NBT) reduction assay was per- (RT)-PCR was performed in triplicate using the FastStart SYBR formed as described previously (20). Immunophenotyping was Green Master (Roche Diagnostics) on an Applied Biosystems performed by staining cells with CD11b-PE (Becton Dickinson) 7500 Fast Real-Time PCR System (Applied Biosystems). Primers or CD14-PE (eBiosciences). Stained samples were run on a Beck- used for confirmation of microarray data are listed in Supple- man Coulter Cytomics FC 500 cytometer (Becton Coulter). For mentary Table S1 and were purchased from Sigma. morphologic assessment, cells were adhered to a slide using cytospin and then stained with Wright–Giemsa. Images were Kinase assays taken using the built in EVOS XL core camera and acquisition Kinases assays were performed by Reaction Biology Corpora- software using a 100 oil objective. Pictures were changed to gray tion using their standard 33P-ATP–based protocol. For kinase scale using Powerpoint. profiling, GS87 (1 mmol/L) was utilized for radioactive in vitro

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kinase assays on a panel of 183 kinases as shown in the Supple- differentiation. To identify novel GSK3 inhibitors optimized for mentary Data. All assays were carried out using 10 mmol/L ATP high AML differentiation activity, a focused library of approxi- and staurosporine as a positive control. For the IC50 determina- mately 2,000 novel small molecules was compiled (designed tion, a 10-dose 3-fold serial dilution assay was performed starting using structural and chemical similarities to existing GSK3 inhi- at 100 mmol/L. bitors). From screening this chemical library, we identified not only a novel and highly specific GSK3 inhibitor, but also an Mouse xenograft studies inhibitor capable of leading to dramatically higher rates of AML Six-week-old female Nod Scid IL2gR / (NSG) mice (The differentiation than previously tested GSK3 inhibitors. The screen Jackson Laboratories) were injected intravenously with 5 106 involved treating OCI-AML3 cells with the small-molecule library primary human AML cells or HL-60 cells (n ¼ 5 mice per group). and measuring differentiation using the NBT reduction assay. NBT Drug treatment was started 3 days after cell injection. GS87 reduction measures the respiratory burst activity of cells, a func- (50 mg/kg), cytarabine (50 mg/kg), or vehicle (20 mL of DMSO tion specific to differentiated cells (22, 23). From this library, and 80 mL of water) were injected as indicated intraperitoneally GS87 was as the only compound capable of inducing greater than thrice a week for 3 weeks. The mice were either assessed for 50% differentiation. The structure of GS87 which represents a survival (primary patient sample group) or sacrificed when the novel scaffold for a GSK3 inhibitor is illustrated in Fig. 1A. vehicle mice became moribund at 4 weeks after cell injection (HL- fi 60 group). The mice were sacri ced when moribund or at the end GS87 is a highly specific GSK3 inhibitor fl of the study period and analyzed by ow cytometry for human As GS87 has a novel scaffold, we confirmed that it is truly a fi leukemia cells in the bone marrow using human CD45 speci c GSK3 inhibitor using radioactive in vitro kinase assays. GS87 was antibody (BD Biosciences) as well as CD11b in the HL-60 group. found to demonstrate significant inhibition of both GSK3a and The CWRU Animal Research Committee approved the animal GSK3b (IC50 415 and 521 nmol/L, respectively) as seen in Fig. 1B. protocols used in this study. As previously reported, GSK3 inhibitors also tend to inhibit other kinases such as CDK2A, we also performed kinase profiling to Statistical analysis assess GS87's specificity in inhibiting GSK3 (19). This screening Group means were compared using two-tailed ANOVA. P < 0.05 demonstrated GS87 is among the most specific GSK3 inhibitors were considered significant. Error bars represent the root mean reported as it had little activity on a panel of 187 other kinases at square error and experiments were performed in triplicate. 1 mmol/L using in vitro kinase assays including CDK2A (Supple- mentary Table S2). Results Identification of GS87 GS87 induces AML cell differentiation GSK3 has been demonstrated to be an excellent target for AML To confirm the high level of GS87-mediated differentiation, we as inhibiting this kinase can lead to the differentiation and compared its ability to induce AML differentiation in a variety of irreversible growth arrest of AML cells yet exhibit little activity cell lines as compared with the widely used GSK3 inhibitors, or even increase the growth of normal bone marrow cells (3, 21). SB415286 (SB), and Lithium (Li). Importantly, all agents were Unfortunately, the existing GSK3 inhibitors demonstrate only used at optimal doses for inducing differentiation without leading moderate AML differentiation activity leading to suboptimal to significant cell death. Lithium was chosen as it is the only

ABN IC for GSK3a IC for GSK3b Figure 1. 50 50 120 120 GS87 is a highly speciﬁc and potent GS87 GS87 GSK3 inhibitor that induces AML cell O N 100 Staurosporine 100 Staurosporine differentiation. A, molecular structure of N 80 80 S GS87. B, GS87 is a potent GSK3 inhibitor. 60 60 In vitro kinase assay for GSK3a and O % Acvity

40 % Acvity 40 GSK3b. C, GS87 leads to extensive AML NN 20 20 differentiation as measured by respiratory burst capacity. OCI, HL-60, 0 0 -9 -8 -7 -6 -5 -4 -3 -9 -8 -7 -6 -5 -4 -3 and NB4 cells were treated with GS87 Log [Compound] (mol/L) Log [Compound] (mol/L) (30 mmol/L), Li (10 mmol/L), or SB (20 mmol/L) for 72 hours and C D differentiation capacity was measured ** ** using the NBT reduction assay. D, GS87 ** ** ** ** ** ** ** 100 ** leads to more effective AML 100 ** ** differentiation than other clinically used 80 GSK3 inhibitors. HL-60 cells were 80 UT treated with GS87 (30 mmol/L), 60 60 tideglusib (30 mmol/L), or LY-2090314 SB 40 40 (30 mmol/L) for 72 hours and Li differentiation was measured by 20 20 % NBT reducon % NBT the NBT assay. , P < 0.01. NBT, reducon % NBT GS87 0 nitrotetrazolium blue. 0 OCI HL-60 NB4 UT Tideglusib LY-2090314 GS87

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currently FDA-approved GSK3 inhibitor. OCI-AML3 (OCI), HL- studies, CD11b and CD14 were induced significantly more with 60, and NB4 cell lines showed a dramatically higher level of NBT GS87 as compared with other GSK3 inhibitors in a variety of AML reduction after treatment with GS87 (80%) as compared with cell lines. For example, CD11b was more strongly upregulated by those treated with SB (20%) or Li (10%; Fig. 1C). These levels GS87 (75%) as compared with treatment with SB (25%) and of differentiation in response to GSK3 inhibition as measured by Li (20%) in multiple AML cell lines (Fig. 2A and B and Sup- NBT reduction are similar to previous studies describing these plementary Fig. S2). As HL-60 cells can differentiate into multiple agents as well as to other GSK3 inhibitors such as TWS116, 6- lineages such as monocytes and neutrophils, we tested for CD14 bromoindirubin-30-oxime, and CHIR9902 (3). Of note, the doses induction, a specific marker of monocytic differentiation, in this used for differentiation induction due not lead to any appreciable cell line. CD14 expression in HL-60 cells was highest after GS87 cell death effects on AML cells when assessed at 72 hours after treatment (81%) as compared with treatment with SB (25%) or Li treatment (Supplementary Fig. S1). (17%; Fig. 2C). In addition, morphologic assessment of cells after In addition to Li which is used clinically, tideglusib and LY- treatment with GS87 confirmed high level of monocytic differ- 2090314 are two small-molecule GSK3 inhibitors which are in entiation as evidenced by condensation of the nucleus, change in clinical trials and were also compared with GS87 (7, 24) Treat- nuclear shape and appearance of cytoplasmic vacuoles (Fig. 2D). ment with GS87 also induced significantly more differentiation as Of note, while both cell lines treated with Li, SB, and GS87 all measured by NBT reduction than either tideglusib or LY-2090314 exhibited features of monocytic differentiation as expected, GS87- (Fig. 1D), identifying GS87 as a dramatically more effective AML treated cells demonstrated noticeably more nuclear indention. differentiation agent. We next studied the differentiation effects of GS87 on primary In addition to NBT reduction, differentiation was measured by leukemic cells derived directly from non-APL leukemia patients. immunophenotyping for the cell surface markers CD11b and Compared with untreated samples, AML cells treated with SB, Li, CD14, which are associated with general granulocytic and mono- and GS87 displayed increased differentiation characterized by cytic differentiation, respectively. Similarly to the NBT reduction CD11b expression on flow cytometry. As observed with AML cell

A ** C ** ** ** ** ** 100 ** ** ** ** ** ** ** ** ** ** ** ** 80 100 UT 60 80 SB 60 40 Li 40 20 GS87 20 % CD11b expression % CD11b expression 0

HL-60 THP OCI NB4 U937 % CD14 expression 0 NT SB Lith GS87 B D 150 UT Li

100 OCI HL-60 50 Primary AML SB GS87

% CD11b expression % CD11b expression 0 0 10 20 30 mmol/L mmol/L mmol/L mmol/L E 60 UT GS87 40 UT SB 20 Li GS87

% CD11b expression 0 Paent 1 Paent 2 Paent 3

Figure 2. GS87 promotes monocytic differentiation of AML cells. A–C, GS87 leads to extensive acute myeloid leukemia (AML) differentiation as measured by immunophenotyping. AML cell lines were treated with GS87 (30 mmol/L or as indicated), Li (10 mmol/L), or SB (20 mmol/L) for 72 hours and cell surface expression of CD11b and CD14 (HL-60 cells) were measured by ﬂow cytometry. , P < 0.01. D, treatment with GS87 induces morphologic changes consistent with monocytic differentiation in HL-60 cells compared with untreated cells. Cells were stained using Wright–Giemsa stain. Images were taken using the built-in EVOS XL core camera and acquisition software using a 100 oil objective. The arrows indicate representative cells with marked nuclear condensation or vacuoles. The boxed cells are shown expanded to better illustrate the morphology. E, GS87 leads to AML differentiation in primary patient leukemic cells. Primary patient leukemia cells treated with GS87, Li, or SB showed the highest increase in the cell surface expression of CD11b after GS87 treatment.

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lines, the greatest percentage of CD11b expression was observed increased accumulation of cells in the G0–G1 phase and a reduc- after GS87 treatment as compared with the other GSK3 inhibitors tion in the S-phase 24 hours after treatment with GS87 consistent tested (Fig. 2E). Of note, GS87 is able to exhibit potent AML with the observed growth inhibition (Fig. 3B). differentiation in a wide range of diverse primary AML samples The purpose of differentiation therapy for AML is to induce and cell lines in contrast to the clinically used differentiation agent terminal differentiation such that differentiated cells can no ATRA that is speciﬁcally potent for APL cells (Figs. 1 and 2 and longer divide and propagate disease. Thus, we evaluated the Supplementary Fig. S3). irreversibility of AML cell growth arrest by limited treatments of GS87 using colony assays. After a 72-hour treatment of OCI, HL- GS87 inhibits AML proliferation and causes terminal 60, and NB4 cells with GS87, the drug was washed away, an equal differentiation number of viable cells was plated in soft agar and subsequent We next evaluated the effects of GS87 on leukemic cell growth. colony formation was assessed. Consistent with extensive termi- Using the MTT assay, we compared cell proliferation following nal differentiation, limited treatment with GS87 was found to treatment with SB, Li, and GS87 for 72 hours using doses leading lead to a near complete inhibition of colony formation with only to maximal differentiation without signiﬁcant cytotoxic effects. 4% to 9% colony growth relative to the vehicle-treated control in GS87 treatment resulted in 35%–47% cell proliferation relative to all cell lines tested (Fig. 3C). Next, we tested the effects of GS87 on untreated controls in THP-1, HL-60, NB4, and U937 cells, whereas the colony formation of normal hematopoietic progenitor cells to SB and Li treatment showed 60%–70% and 68%–74% prolifer- investigate the potential for hematologic toxicity as this is an ation relative to vehicle control cells, respectively (Fig. 3A). extremely common side effect of most AML therapeutics. Similar Furthermore, cell-cycle analysis in HL-60 cells showed an to previous reports of inhibiting GSK3 in normal bone marrow,

A B 70 **

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to control GS87 20 20 % Expression 10 GS87

% Proliferaon % Proliferaon 0 0 HL-60 NB4 THP U937 G0−G1 SG2−M

60 80 C Untreated GS87 G −G 51.1% 60 G −G 61.3% 40 0 1 0 1 S 20.9% S 14.9% 40 100 G −M 26.5% G −M 23.0%

Count 2 Count 20 2 20 80

0 0 0 200 400 600 800 1,000 0 200 400 600 800 1,000 60 FL3 FL3

300 40 60 250 50 Li SB 20 200 G −G 55.2% relave to control

− 56.7% formaon % Colony 40 G0 G1 0 1 150 S 18.8% 30 S 20.4% 0 − G −M 22.0% Count G2 M 24.8% Count 2 100 20 OCI HL-60 NB4 Normal

50 10 bone

0 0 marrow 0 200 400 600 800 1,000 0 200 400 600 800 1,000 FL3 FL3

Figure 3. GS87 inhibits AML cell proliferation and causes irreversible growth inhibition. A, GS87 impairs AML proliferation. Equal numbers of HL-60, U937, THP-1, and NB4 cells were treated with GS87 (30 mmol/L), Li (10 mmol/L), or SB (20 mmol/L) for 72 hours and the MTT assay was performed. The MTT signal was normalized to the vehicle- treated control. B, GS87 modulates the cell cycle of AML cells. HL-60 cells were treated with GS87, Li, or SB for 24 hours and assessed for cell-cycle status. Representative ﬂow results are shown and the histogram is an average of three independent studies. , P < 0.01; NS, no signiﬁcant difference. C, GS87 inhibits colony formation of AML cells but not normal marrow mononuclear cells. OCI, HL-60, and NB4 cells and normal marrow mononuclear cells were treated with GS87 for 72 hours, after which drug was washed away and equal numbers of viable cells were cultured in semisolid soft agar medium for 7 to 10 days. Colony formation of cells was normalized to a vehicle-treated control.

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GS87 treatment did not significantly inhibit colony growth of cells three agents. We also investigated levels of c-MYB and MAFB, taken from normal human bone marrow samples (Fig. 3C). In proto-oncogenes overexpressed in immature hematopoietic cells addition, mice treated with GS87 did not show any impact on and in some hematologic malignancies that are regulated by normal hematopoiesis as measured by changes in white blood GSK3. c-MYB has previously been demonstrated to be a key target cell, red blood cell, or platelet counts (Supplementary Fig. S4). of GSK3 in mediating its cell growth effects in AML cells and MAFB is also regulated by GSK3 phosphorylation and plays a key role in GS87 modulates key GSK3 target proteins involved in cell myeloid differentiation (28–31). GSK3 inhibition has been proliferation and differentiation more effectively than lithium shown to directly downregulate c-Myb and upregulate MAFB and SB expression. GS87 more effectively modulated protein expression To explore mechanistic insights that may explain GS87's of both GSK3 target proteins c-MYB and MAFB than SB and Li enhanced differentiation activity over other GSK3 inhibitors, we further establishing the ability of GS87 to strongly impact GSK3- investigated the impact of GS87 on known GSK3-dependent dependent pathways (Fig. 4C). pathways that are important for AML differentiation and growth in leukemic cells including b-catenin, p21, c-myc, c-MYB, and GS87-mediated differentiation is dependent upon MAPK MAFB. One of the major direct target proteins of GSK3 is b-cate- signaling nin, which is phosphorylated by GSK3 and targeted for degrada- To further explore the cellular pathways involved in mediating tion. Therefore, we investigated the role of b-catenin in mediating the differentiation effects of GS87 as compared with other GSK3 the differentiation effects of GS87. GS87 treatment of OCI cells inhibitors, we performed RNA expression analysis using a gene with stable knockdown of b-catenin showed similar differentia- microarray after treatment of OCI cells with GS87, Li, and SB. The tion compared with cells transfected with empty vector (Fig. 4A), cells were treated using a short treatment (48 hours) to enable the demonstrating that the differentiation effect of GS87 is indepen- identification of mediators of differentiation instead of simply to dent of b-catenin. Although b-catenin does not appear to be appreciate the phenotype of the differentiated cells. After FDR important for GS87-mediated differentiation, GS87 did lead to correction, 1,322 genes were significantly (P < 0.05) differentially a more pronounced induction of this GSK3 target protein than the expressed with an absolute log2 fold change greater than or equal other GSK3 inhibitors tested (Fig. 4B). to 1.5 after GS87 treatment, 90 genes after treatment with SB, and p21 is a key regulator of the cell cycle and is induced during 143 genes after treatment with Li (Supplementary Fig. S5; Sup- AML differentiation (25), whereas c-myc is a promoter of cell plementary Table S3). A total of 34 genes were only significantly proliferation whose presence prevents terminal cell differentia- dysregulated by SB treatment, 47 genes were only significantly tion in cancer cells and is downregulated during AML differen- dysregulated by Li treatment, and a total of 1,206 genes were tiation (26). p21 is also known to be a direct GSK3 target protein only significantly dysregulated by treatment with GS87 (Supple- that can be degraded in a GSK3-dependent fashion (27). As mentary Table S4; Fig. 5A). shown in Fig. 4B, GS87 treatment increased p21, whereas it Next, we performed pathway analysis on genes that were reduced c-Myc protein levels more robustly than either Li or SB significantly modulated (FDR-adjusted P < 0.05, |log2 fold reinforcing the cell proliferation analysis. Importantly, the ability change| 1.5) in the GS87, Li, and SB treatment groups. This of GS87 to more effectively modulate GSK3-dependent pathways analysis identified key pathways known to be involved in AML than SB and Li occurs at doses optimized for differentiation for all differentiation and growth such as MAPK signaling as being more

A 100 ** ** 80 60 Figure 4. UT GS87 effectively modulates GSK3- 40 GS87 dependent proteins. A, GS87-induced differentiation is independent of 20 -catenin. OCI cells with stable % NBT reducon b knockdown of b-catenin showed 0 similar differentiation as compared Parental shβ-catenin with cells transfected with empty vector after treatment with GS87 for 3 B Day 2 Day 4 C days as measured by NBT reduction. , P < 0.01. B and C, GS87 modulates GSK3 target proteins important in cell UT Lith. SB GS87 UT Lith. SB GS87 UT Lith. SB GS87 proliferation and differentiation more b-Catenin effectively than Li and SB. HL-60 cells MyB were treated as indicated for 2 and 4 days (B) or 4 days (C) and assessed p21 by Western blot analysis for key AML MAFB growth and differentiation proteins that can be modulated by GSK3. NBT, c-Myc nitrotetrazolium blue. GAPDH GAPDH

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3 A SB Li 2.5

2 UT 34 6 47 1.5 SB 24 1 Li 26 66 expression GS87 0.5 Relave normalized 1,206 0 Cyclin D2 CEBPβ IRF8

Phospho-p38 Phospho-ERK2 Phospho-Jnk B GS87 0 min 30 min 1 h 1.5 h 2 h 0 min 30 min 1 h 1.5 h 2 h 0 min 30 min 1 h 1.5 h 2 h SB SB SB

Lith. Lith. Lith.

GS87 GS87 GS87

GAPDH GAPDH GAPDH

C ** **

100 100

80 80

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40 40

20 20 % NBT reducon % NBT

CD11b expression (%) 0 0 UT GS87 PD PD+GS87 UT GS87 PD PD+GS87

Figure 5. GS87-induced AML differentiation is dependent on MAPK signaling. A, comparison of GSK3 inhibitor modulation of gene expression in AML cells. HL-60 cells were treated for 48 hours and the expression of the indicated genes was assessed. Venn diagram of signiﬁcantly regulated genes (left); validation of microarray results by real-time PCR (right). B, multiple components of the MAPK signaling cascade are activated by GS87 treatment. HL-60 cells were treated with GS87, Li, or SB and the activation of p38, ERK, and JNK were analyzed by Western blot analysis at various time points using phospho-speciﬁc antibodies. C, chemical inhibition of ERK (PD08959 10 mmol/L) abrogates the differentiation capacity of GS87 in HL-60 cells. , P < 0.01.

significantly upregulated by GS87 treatment as compared with effects of GS87 (Fig. 5C and data not shown), establishing the the other treatments (Table 1 and Supplementary Table S5; role of the MEK/ERK branch of the MAPK signaling cascade as a refs. 32–35). key mediator of GS87-induced differentiation. This work also To validate the results of the microarray, genes that exhibited provided mechanistic insights into why GS87 may be a more significant changes that are also involved in myeloid differenti- effective differentiation agent. ation and growth were tested by RT-PCR. For example, CEBPb expression was upregulated after GS87 treatment, whereas IRF8 GS87 demonstrates efficacy in an AML mouse model system and cyclin D2 expression were downregulated with GS87 treat- To explore the potential of GS87 as a therapeutic, we utilized ment (Fig. 5A). CEBPb is a key regulator in myeloid differentiation two mouse models of circulating human AML. In the first model, (36, 37), whereas cyclin D2 has roles in leukemia proliferation primary AML cells derived from a patient with relapsed and (38–40). IRF8 is a regulator of myeloid differentiation and high treatment refractory disease (AML M2 subtype) were injected into expression has been linked to poor prognosis in AML (41). immunodeficient mice. GS87 demonstrated significant efficacy as Because of the known major role of MAPK signaling in AML compared with mice treated with vehicle as well as the standard differentiation, we investigated components of the MAPK signal- AML therapeutic, cytarabine. While the majority of the GS87 mice ing cascade in mediating GS87-induced differentiation. Consis- showed longer term survival, those treated with vehicle or cytar- tent with our pathway analysis, we found that p38, ERK, and JNK abine died because of leukemic cell infiltration of the bone were all activated more strongly by GS87 than Li and SB partic- marrow by approximately 45 days after cell injection (Fig. 6A). ularly at 2 hours after treatment (Fig. 5B). To investigate the Examination of the bone marrow in mice sacrificed because of importance of these pathways in GS87-mediated differentiation, moribund state or at the study endpoint of 90 days (as in the case we utilized chemical inhibitors. The ERK inhibitor (but not JNK of GS87-treated mice) showed significantly higher percentage and p38 inhibitors) significantly impaired the differentiation of blasts in the vehicle and cytarabine groups (93% 7.5% and

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65% 8.2%) compared with the surviving mice from the GS87 GS87 exhibits dramatically more differentiation activity as group (0.32% 0.53%; Fig. 6B). In the second model, the ability compared with other reported GSK3 inhibitors. GS87 induces of GS87 to induce the differentiation of the leukemic cells was differentiation and growth inhibition of AML cell lines and assessed. In this model, GS87 was found not only to significantly primary patient samples and exhibits high efficacy using aggres- reduce the burden of leukemic cells in the bone marrow at the end sive mouse models of human AML. Although GS87 exhibits of the study period but also to lead to evidence of leukemic cell extensive differentiation activity as compared with other GSK3 differentiation as measured by CD11b staining (Fig. 6C and D). inhibitors, interestingly, it leads to GSK3 inhibition at similar Finally, to confirm that GS87 can inhibit GSK3 in vivo, peripheral levels as measured by in vitro kinase assays to SB and many other blood cells were shown to induce the expression of b-catenin after reported GSK3 inhibitors. Furthermore, despite these differences drug treatment (Supplementary Fig. S6). in AML differentiation activity, GS87 was found to be a highly specific kinase inhibitor through kinase profiling. GS87, however, Discussion leads to more dramatic effects on GSK3 target proteins as compared with other GSK3 inhibitors when used at optimal differ- Here we report the identification of a novel and highly entiation doses. For example, GS87 leads to larger changes in the specific inhibitor of GSK3 that induces superior AML cell expression of c-myb, MAFB, p21, and b-catenin in AML cells as differentiation and growth arrest compared with existing GSK3 compared with SB and Li. In addition, GS87 leads to a stronger inhibitors without affecting normal hematopoietic cell prolif- activation of MAPK signaling which was found to be important for eration. The discovery of novel differentiation therapies for its differentiation activity. AML is important due to the poor efficacy and high toxicities of The reasons for the enhanced activation of GSK3-dependent existing agents. Differentiation therapy has demonstrated signaling with GS87 are not known. GSK3 activity is controlled in remarkable clinical efficacy for a small subset of AML patients a relatively complex manner by multiple posttranslational mod- with APL reinforcing its potential as an attractive therapeutic ifications, cellular localization, and interactions with other pro- strategy for this disease (2). teins (6). In addition, GSK3 can target substrates primed by Numerous groups have implicated GSK3 as a target for AML phosphorylation from other kinases or nonprimed substrates in growth inhibition and differentiation (3–5, 9–12). In addition, different manners. It is possible that GS87 impairs GSK3 activity it has been reported that GSK3 inhibition may also target in a distinct fashion as compared with other agents. In addition, leukemia initiating cells that are poorly targeted by standard despite the fact that GS87 is a specific kinase inhibitor, it is also AML chemotherapy (42). One of the major advantages of likely that it regulates other proteins that mediate pathways that targeting GSK3 as compared with the vast majority of agents can synergize with GSK3-dependent differentiation. Several path- is the selective targeting of AML cells as compared with normal ways such as retinoic acid signaling have been found to cooperate hematopoietic cells. Our studies with GS87 have further val- with GSK3-mediated differentiation (3). idated the selective effects on leukemic cells suggesting that this Further preclinical optimization of GS87 is needed to deter- approach will not lead to myelotoxicity, a major side effect of mine pharmacokinetics, pharmacodynamics, and drug formula- most leukemia therapeutics. tion prior to human testing. Whether clinically effective plasma

A B ** 100 100 90 90 Figure 6. 80 80 GS87 demonstrates efficacy in a 70 70 circulating AML mouse model system. 60 60 A, mice treated with GS87 showed 50 50 Veh longer survival compared with those 40 40 treated with vehicle or the standard GS87 30 30 AML therapeutic cytarabine in a 20 20 Cytarabine mouse model of human primary AML. Blasts in marrow (%) Percent survival (%) 10 10 B, bone marrow blast percentage in 0 0 mice with primary human AML 1 1020304050607090 Vehicle Cytarabine GS87 sacrificed because of moribund state Number of days or at study endpoint of 90 days showed significantly higher C D percentage of blasts in the vehicle and 100 100 cytarabine groups compared with the GS87 group. Note: the GS87 mice 80 80 tested were the surviving mice at the end of the study period. C and D, 60 60 mouse model of circulating HL-60 cells 40 40 showing a reduced disease burden in the bone marrow (C) and evidence of 20 20 AML differentiation (D) in the GS87- treated mice. % CD11b expression Blasts in marrow (%) 0 0 Vehicle GS87 Vehicle GS87

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Novel GSK3 Inhibitor for AML

Table 1. List of upstream regulators related to MAPK signaling identified like ATRA, requires coadministration of chemotherapy or other from pathway analysis of genes significantly modulated (>2-fold) after agents for optimal disease control (43, 44). treatment with GS87 (30 mol/L), Li (10 mmol/L), or SB (20 mol/L) m m In conclusion, GS87 is a novel GSK3 inhibitor identified in for 48 hours efforts to discover a small-molecule GSK3 inhibitor optimized for Upstream Activation fold fi regulator change P AML differentiation activity. Because of its high ef cacy in cell and animal models and its minimal apparent toxicity, GS87 is a — MAPK pathway GS87 promising new agent for clinical AML differentiation therapy. SB203580 5.84 5.43E13 PD98059 5.231 7.30E26 U0126 4.754 5.18E15 Disclosure of Potential Conflicts of Interest SP600125 3.885 1.92E09 M.K. Agarwal has ownership interest (including patents) in Invenio-MiRx. SB202190 3.024 1.36E10 D.N. Wald reports receiving a commercial research grant from Invenio Thera- MAPK10 2.197 1.37E04 peutics and has ownership interest (including patents) in Invenio Therapeutics MAPK 2.287 6.33E09 and GS87 intellectual property. No potential conflicts of interest were disclosed MAP2K1/2 2.365 6.66E09 by the other authors. MAPK13 2.404 4.15E04 MAPK12 2.418 2.17E04 MAP3K7 2.494 1.23E06 Authors' Contributions MAP2K3 2.760 1.10E05 Conception and design: M.K. Agrawal, D.N. Wald MAPK7 2.768 2.01E05 Development of methodology: M. Ueda, J. Ignatz-Hoover, S. Moreton, MAPK3 3.056 1.45E06 A. Chakrabarti, Z. Xia, M. de Lima, M.K. Agrawal, D.N. Wald MAP2K1 3.092 4.83E10 Acquisition of data (provided animals, acquired and managed patients, MAPK14 3.429 8.35E04 provided facilities, etc.): S. Hu, M. Ueda, J. Ignatz-Hoover, S. Moreton, MAP3K8 3.765 2.11E06 M.K. Agrawal, D.N. Wald ERK1/2 3.805 8.31E11 Analysis and interpretation of data (e.g., statistical analysis, biostatistics, ERK 3.961 8.06E17 computational analysis): S. Hu, M. Ueda, L. Stetson, G. Karan, M. de Lima, JNK 3.966 7.79E07 M.K. Agrawal, D.N. Wald Writing, review, and/or revision of the manuscript: S. Hu, M. Ueda, L. Stetson, MAPK pathway—SB D.N. Wald U0126 2.949 9.97E 07 Administrative, technical, or material support (i.e., reporting or organizing LY294002 2.681 6.23E 05 data, constructing databases): S. Hu, M. Ueda, S. Moreton, A. Chakrabarti, PD98059 2.662 2.13E 05 G. Karan, M.K. Agrawal, D.N. Wald SB203580 2.153 2.32E 04 Study supervision: G. Karan, M.K. Agrawal, D.N. Wald ERK 2.000 1.70E05 MAPK pathway—Li Grant Support JNK 2.221 4.88E03 The work was supported by a contract from the NCI (NIH/NCI ERK 2.592 3.47E04 HHSN261201100093C; to D.N. Wald and M.K. Agrawal) and Core Facilities of the Case Comprehensive Cancer Center (P30 CA43703). The costs of publication of this article were defrayed in part by the payment of concentrations of GS87 can be achieved in humans is unknown. page charges. This article must therefore be hereby marked advertisement in However, given its high antileukemic efficacy with limited toxicity accordance with 18 U.S.C. Section 1734 solely to indicate this fact. in our AML mouse model, GS87 has tremendous potential for drug development and first-in-human testing in persons with Received July 10, 2015; revised March 24, 2016; accepted April 6, 2016; AML. Further testing will be necessary to determine whether GS87, published OnlineFirst May 9, 2016.

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1494 Mol Cancer Ther; 15(7) July 2016 Molecular Cancer Therapeutics

A Novel Glycogen Synthase Kinase-3 Inhibitor Optimized for Acute Myeloid Leukemia Differentiation Activity

Sophia Hu, Masumi Ueda, Lindsay Stetson, et al.

Mol Cancer Ther 2016;15:1485-1494. Published OnlineFirst May 9, 2016.

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