Published OnlineFirst July 18, 2019; DOI: 10.1158/1078-0432.CCR-19-0832 Translational Cancer Mechanisms and Therapy Clinical Cancer Research Inhibition of Bcl-2 Synergistically Enhances the Antileukemic Activity of Midostaurin and Gilteritinib in Preclinical Models of FLT3-Mutated Acute Myeloid Leukemia Jun Ma1, Shoujing Zhao1, Xinan Qiao1, Tristan Knight2,3, Holly Edwards4,5, Lisa Polin4,5, Juiwanna Kushner4,5, Sijana H. Dzinic4,5, Kathryn White4,5, Guan Wang1, Lijing Zhao6, Hai Lin7, Yue Wang8, Jeffrey W. Taub2,3, and Yubin Ge3,4,5 Abstract Purpose: To investigate the efficacy of the combination of with venetoclax. Changes of Mcl-1 transcript levels were the FLT3 inhibitors midostaurin or gilteritinib with the Bcl-2 assessed by RT-PCR. inhibitor venetoclax in FLT3-internal tandem duplication Results: The combination of midostaurin or gilteritinib (ITD) acute myeloid leukemia (AML) and the underlying with venetoclax potently and synergistically induces apo- molecular mechanism. ptosis in FLT3-ITD AML cell lines and primary patient Experimental Design: Using both FLT3-ITD cell lines and samples. The FLT3 inhibitors induced downregulation of primary patient samples, Annexin V-FITC/propidium iodide Mcl-1, enhancing venetoclax activity. Phosphorylated-ERK staining and flow cytometry analysis were used to quantify cell expression is induced by venetoclax but abolished by the death induced by midostaurin or gilteritinib, alone or in combination of venetoclax with midostaurin or gilteritinib. combination with venetoclax. Western blot analysis was per- Simultaneous downregulation of Mcl-1 by midostaurin or formed to assess changes in protein expression levels of gilteritinib and inhibition of Bcl-2 by venetoclax results in members of the JAK/STAT, MAPK/ERK, and PI3K/AKT path- "free" Bim, leading to synergistic induction of apoptosis. ways, and members of the Bcl-2 family of proteins. The In vivo results show that gilteritinib in combination with MV4-11–derived xenograft mouse model was used to assess venetoclax has therapeutic potential. in vivo efficacy of the combination of gilteritinib and veneto- Conclusions: Inhibition of Bcl-2 via venetoclax synergis- clax. Lentiviral overexpression of Mcl-1 was used to confirm tically enhances the efficacy of midostaurin and gilteritinib in its role in cell death induced by midostaurin or gilteritinib FLT3-mutated AML. Introduction remained low in both pediatric and adult patients, with 5-year survival rates approximating 61.5%–65.1% and 25%, respective- Acute myeloid leukemia (AML) is defined by the clonal pro- ly (1). FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine liferation of immature myeloid elements and survival rates have kinase and important early regulator of hematopoiesis, in which mutations are seen in approximately one third of patients with AML (2). FLT3-internal tandem duplications (ITD) mutations, 1 National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin seen in approximately 25% of patients with AML, result in University, Changchun, China. 2Division of Pediatric Hematology and Oncology, Department of Pediatrics, Children's Hospital of Michigan, Detroit, Michigan. constitutive activation of downstream pathways that promote 3Department of Pediatrics, Wayne State University School of Medicine, Detroit, cell survival and proliferation, including the MAPK/ERK, Michigan. 4Department of Oncology, Wayne State University School of Medi- PI3K/AKT, and JAK/STAT pathways; understandably, it confers cine, Detroit, Michigan. 5Molecular Therapeutics Program, Karmanos Cancer a poor prognosis (2, 3). 6 Institute, Wayne State University School of Medicine, Detroit, Michigan. Depart- Direct inhibition of FLT3 is therefore a promising therapeu- ment of Rehabilitation, School of Nursing, Jilin University, Changchun, P.R.China. 7 tic avenue, with some agents already available for immediate Department of Hematology and Oncology, The First Hospital of Jilin University, fi Changchun, P.R. China. 8Department of Pediatric Hematology and Oncology, use. Midostaurin, a rst-generation multikinase inhibitor was The First Hospital of Jilin University, Changchun, P.R. China. approved by the FDA in April 2017 for use in newly diagnosed patients with FLT3-mutated AML, in combination with stan- Note: Supplementary data for this article are available at Clinical Cancer þ Research Online (http://clincancerres.aacrjournals.org/). dard 7 3 cytarabine and daunorubicin induction and cytar- abine consolidation, on the basis of results of the RATIFY Corresponding Author: Yubin Ge, Wayne State University School of Medicine, trial (4). In patients over the age of 60, however (e.g., the 421 East Canfield Street, Detroit, MI 48201. Phone: 313-578-4285; Fax: 313-578- 4287; E-mail: [email protected] largest population of patients with AML), and those with other comorbidities, 7þ3 chemotherapy is not well tolerated because Clin Cancer Res 2019;XX:XX–XX of its extensive toxicities and severe side effects (5). Gilteritinib doi: 10.1158/1078-0432.CCR-19-0832 was approved by the FDA in November 2018 for use in adult Ó2019 American Association for Cancer Research. patients with relapsed or refractory FLT3-mutated AML, www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst July 18, 2019; DOI: 10.1158/1078-0432.CCR-19-0832 Ma et al. Translational Relevance Materials and Methods Drugs Approximately 25% of patients with acute myeloid leuke- Midostaurin (PKC-412), gilteritinib (ASP-2215), venetoclax mia (AML) have FLT3-internal tandem duplication (ITD) (ABT-199), and SCH772984 (an ERK-selective inhibitor) were mutations, which result in constitutive activation of multiple purchased from Selleck Chemicals. survival pathways. FLT3 inhibitors, midostaurin and gilteriti- nib, were recently approved by the FDA for use in patients with FLT3-mutated AML. However, efficacious but short-lived Cell cultures responses with regard to monotherapy and the extensive MOLM-13 cells were purchased from AddexBio (2012). toxicities and severe side effects of standard induction therapy, MV4-11, THP-1, and U937 cell lines were purchased from the demonstrate the need to develop new combination therapies. ATCC (2006, 2014, 2002, respectively). Cell lines were cultured – Here, we show that midostaurin and gilteritinib enhance cell using RPMI1640 media plus 10% 20% FBS (CLARK Bioscience), m death induced by venetoclax in FLT3-ITD AML cell lines and 2 mmol/L L-glutamine, and 100 U/mL penicillin and 100 g/mL streptomycin and incubated in a humidified, 5%CO2/95% air primary patient samples. Downregulation of Mcl-1 and p-ERK by these inhibitors enhance the antileukemic activity of vene- environment at 37 C. All lines were tested for the presence of Mycoplasma toclax. The combination of gilteritinib and venetoclax shows using the PCR method described by Uphoff and promising in vivo efficacy in a FLT3-ITD AML cell line–derived Drexler (15) on a monthly basis. The cell lines were authenticated xenograft mouse model. These findings support the clinical in 2017 at the Genomics Core at Karmanos Cancer Institute development of venetoclax in combination with the FLT3 (Detroit, MI) using the PowerPlex 16 System from Promega. inhibitors, midostaurin or gilteritinib, for the treatment of AML. Clinical samples Primary patient samples were obtained from the First Hospital of Jilin University (Changchun, China), following obtainment of informed consent according to the Declaration of Helsinki. The fi following the ADMIRAL trial (NCT02421939; ref. 6). As such, study itself and the obtainment of patient samples were rst treatment options for FLT3-mutated AML are beginning to approved by the Human Ethics Committee of The First Hospital exist, but carry with them certain limitations—particularly in of Jilin University (Changchun, China). All patient samples were relation to monotherapy, with efficacious but short-lived screened for FLT3-ITD, NPM1, C-kit, CEBPA, IDH1, IDH2, N-RAS, fi responses. As such, new therapies to combine with FLT3 inhi- and DNMT3A mutations by PCR ampli cation and automat- bitors are an unmet clinical need. ed DNA sequencing, cytogenetics, and fusion genes utilizing Venetoclax (ABT-199) is a selective inhibitor of Bcl-2, and RT-PCR, as described previously (16, 17). Individual patient was granted approval by the FDA in November 2018 for first- characteristics are provided in Supplementary Table S1. Patient fi line use in newly diagnosed AML in adults over 75 or those samples were puri ed using Ficoll-Hypaque density centrifugation with comorbidities precluding use of intensive induction che- and cultured in RPMI1640 media plus 20% FBS, ITS Solution motherapy, in combination with low-dose cytarabine or the (Sigma-Aldrich), and 20% supernatant of the 5637 bladder cancer b hypomethylating agents azacitidine or decitabine (7). Impor- cell line (to provide sources of GM-CSF, G-CSF, IL1 , macrophage – tantly, patients with FLT3 mutations did not fare worse than colony stimulating factor, and stem cell factor; refs. 16, 18, 19). their peers while receiving venetoclax, despite the usual poor fl prognosis associated with this mutation (8, 9). Again however, Annexin V- uorescein isothiocyanate/propidium iodide fl while encouraging, there is room to improve upon these staining and ow cytometry analyses results; these studies show median complete remission times Cells were treated with either midostaurin, gilteritinib, of 4–6 months, across