Author Manuscript Published OnlineFirst on July 18, 2019; DOI: 10.1158/1078-0432.CCR-19-0832 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Inhibition of Bcl-2 Synergistically Enhances the Antileukemic Activity of Midostaurin and 2 Gilteritinib in Preclinical Models of FLT3-mutated Acute Myeloid Leukemia 3 4 Jun Ma1, Shoujing Zhao1, Xinan Qiao1, Tristan Knight2,3, Holly Edwards4,5, Lisa Polin4,5, 5 Juiwanna Kushner4,5, Sijana H. Dzinic4,5, Kathryn White4,5, Guan Wang1, Lijing Zhao6, Hai Lin7, 6 Yue Wang8, Jeffrey W. Taub2,3, and Yubin Ge3,4,5* 7 8 1National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, 9 Changchun, China 10 2Division of Pediatric Hematology and Oncology, Department of Pediatrics, Children's Hospital 11 of Michigan, Detroit, Michigan, USA 12 3Department of Pediatrics, Wayne State University School of Medicine, Detroit, Michigan, USA 13 4Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan, USA 14 5Molecular Therapeutics Program, Karmanos Cancer Institute, Wayne State University School of 15 Medicine, Detroit, Michigan, USA 16 6Department of Rehabilitation, School of Nursing, Jilin University, Changchun, P.R.China 17 7Department of Hematology and Oncology, The First Hospital of Jilin University, Changchun, 18 P.R. China 19 8Department of Pediatric Hematology and Oncology, The First Hospital of Jilin University, 20 Changchun, P.R. China 21 22 *Corresponding author 23 24 Running title: Joint FLT3 and BCL-2 Inhibition in FLT3-mutated AML 25 Keywords: acute myeloid leukemia; FLT3; venetoclax 26 27 GRANT SUPPORT 28 This study was supported by Jilin University, Changchun, China, the Barbara Ann 29 Karmanos Cancer Institute, Wayne State University School of Medicine, and by grants from the 30 National Natural Science Foundation of China, NSFC 31671438 and NSFC 31471295, Graduate 31 Innovation Fund of Jilin University, Hyundai Hope on Wheels, LaFontaine Family/U Can-Cer 32 Vive Foundation, Kids Without Cancer, Children’s Hospital of Michigan Foundation, 33 Decerchio/Guisewite Family, Justin’s Gift, Elana Fund, Ginopolis/Karmanos Endowment and 34 the Ring Screw Textron Endowed Chair for Pediatric Cancer Research. The Animal Model and 35 Therapeutics Evaluation Core and Genomics Core are supported, in part, by NIH Center Grant 36 P30 CA022453 to the Karmanos Cancer Institute at Wayne State University. The funders had no 37 role in study design, data collection, analysis and interpretation of data, decision to publish, or 38 preparation of the manuscript. 39 40 Correspondence: 41 Yubin Ge, PhD 42 Department of Oncology 43 Wayne State University School of Medicine 44 421 East Canfield 45 Detroit, MI, 48201 46 Email: [email protected] 1 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 18, 2019; DOI: 10.1158/1078-0432.CCR-19-0832 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 47 Tel: 313 578-4285 48 49 Conflict of interest disclosure: The authors declare no potential conflicts of interest. 50 Abstract word count: 249 51 Manuscript Word Count: 4,158 52 Figures: 6 53 Tables: 0 54 References: 35 55 2 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 18, 2019; DOI: 10.1158/1078-0432.CCR-19-0832 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 56 TRANSLATIONAL RELEVANCE 57 Approximately 25% of patients with acute myeloid leukemia (AML) have FLT3-internal tandem 58 duplication (ITD) mutations, which results in constitutive activation of multiple survival 59 pathways. FLT3 inhibitors midostaurin and gilteritinib were recently approved by the US FDA 60 for use in FLT3-mutated AML patients. However, efficacious but short-lived responses with 61 regard to monotherapy and the extensive toxicities and severe side effects of standard induction 62 therapy, demonstrate the need to develop new combination therapies. Here, we show that 63 midostaurin and gilteritinib enhance cell death induced by venetoclax in FLT3-ITD AML cell 64 lines and primary patient samples. Downregulation of Mcl-1 and p-ERK by these inhibitors 65 enhance the antileukemic activity of venetoclax. The combination of gilteritinib and venetoclax 66 shows promising in vivo efficacy in a FLT3-ITD AML cell line-derived xenograft mouse model. 67 These findings support the clinical development of venetoclax in combination with the FLT3 68 inhibitors midostaurin or gilteritinib for the treatment of AML. 69 3 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 18, 2019; DOI: 10.1158/1078-0432.CCR-19-0832 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 70 ABSTRACT 71 Purpose: To investigate the efficacy of the combination of the FLT3 inhibitors midostaurin or 72 gilteritinib with the Bcl-2 inhibitor venetoclax in FLT3-ITD acute myeloid leukemia (AML) and 73 the underlying molecular mechanism. 74 Experimental Design: Using both FLT3-ITD cell lines and primary patient samples, annexin V- 75 FITC/propidium iodide staining and flow cytometry analysis were used to quantify cell death 76 induced by midostaurin or gilteritinib, alone or in combination with venetoclax. Western blot 77 analysis was performed to assess changes in protein expression levels of members of the 78 JAK/STAT, MAPK/ERK, and PI3K/AKT pathways, and members of the Bcl-2 family of 79 proteins. The MV4-11-derived xenograft mouse model was used to assess in vivo efficacy of the 80 combination of gilteritinib and venetoclax. Lentiviral overexpression of Mcl-1 was used to 81 confirm its role in cell death induced by midostaurin or gilteritinib with venetoclax. Changes of 82 Mcl-1 transcript levels were assessed by real-time RT-PCR. 83 Results: The combination of midostaurin or gilteritinib with venetoclax potently and 84 synergistically induces apoptosis in FLT3-ITD AML cell lines and primary patient samples. The 85 FLT3 inhibitors induced downregulation of Mcl-1, enhancing venetoclax activity. 86 Phosphorylated-ERK expression is induced by venetoclax but abolished by the combination of 87 venetoclax with midostaurin or gilteritinib. Simultaneous downregulation of Mcl-1 by 88 midostaurin or gilteritinib and inhibition of Bcl-2 by venetoclax results in “free” Bim, leading to 89 synergistic induction of apoptosis. In vivo results show that gilteritinib in combination with 90 venetoclax has therapeutic potential. 91 Conclusion: Inhibition of Bcl-2 via venetoclax synergistically enhances the efficacy of 92 midostaurin and gilteritinib in FLT3-mutated AML. 4 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 18, 2019; DOI: 10.1158/1078-0432.CCR-19-0832 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 93 INTRODUCTION 94 Acute myeloid leukemia (AML) is defined by the clonal proliferation of immature 95 myeloid elements and survival rates have remained low in both pediatric and adult patients, with 96 5-year survival rates approximating 61.5-65.1% and 25%, respectively (1). FMS-like tyrosine 97 kinase 3 (FLT3) is a receptor tyrosine kinase and important early regulator of hematopoiesis, in 98 which mutations are seen in approximately one third of AML patients (2). FLT3-internal tandem 99 duplications (ITD) mutations, seen in approximately 25% of patients with AML, result in 100 constitutive activation of downstream pathways which promote cell survival and proliferation, 101 including the MAPK/ERK, PI3K/AKT, and JAK/STAT pathways; understandably, it confers a 102 poor prognosis (2, 3). 103 Direct inhibition of FLT3 is therefore a promising therapeutic avenue, with some agents 104 already available for immediate use. Midostaurin, a first generation multi-kinase inhibitor was 105 approved by the US Food and Drug Administration (FDA) in April 2017 for use in newly 106 diagnosed FLT3-mutated AML patients, in combination with standard 7+3 cytarabine and 107 daunorubicin induction and cytarabine consolidation, on the basis of results of the RATIFY trial 108 (4). In patients over the age of 60 however (e.g. the largest population of AML patients), and 109 those with other comorbidities, 7+3 chemotherapy is not well tolerated due to its extensive 110 toxicities and severe side effects (5). Gilteritinib was approved by the US FDA in November 111 2018 for use in adult patients with relapsed or refractory FLT3-mutated AML, following the 112 ADMIRAL trial (NCT02421939) (6). As such, treatment options for FLT3-mutated AML are 113 beginning to exist, but carry with them certain limitations – particularly in relation to 114 monotherapy, with efficacious but short-lived responses. As such, new therapies to combine with 115 FLT3 inhibitors are an unmet clinical need. 5 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 18, 2019; DOI: 10.1158/1078-0432.CCR-19-0832 Author manuscripts have been peer reviewed and accepted for publication