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. Ó2016 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 Ó2016 American Association for Cancer Research. enzyme inhibition and inhibitory serine-9 phosphorylation www.aacrjournals.org 1485 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst May 9, 2016; DOI: 10.1158/1535-7163.MCT-15-0566 Hu et al. (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 À20C. 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