Dengyang Zhang1, Yao Guo1, Yuming Zhao1, Liuting Yu1, Zhiguang Chang1, Hanzhong Pei1, Junbin Huang2, Chun Chen2, Hongman Xue2, Xiaojun Xu3, Yihang Pan1, Ningning Li1, Chengming Zhu1, Zhizhuang Joe Zhao4, Jian Yu5, Yun Chen6. 1. Edmond H. Fischer Translational Medical Research Laboratory, Scientific Research Center, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, 518107, Guangdong, China. 2. Department of Pediatrics, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, 518107, Guangdong, China. 3. Department of Hematology, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, 518107, Guangdong, China. 4. Department of Pathology, University of Oklahoma Health Sciences Center, 1100 N. Lindsay, Oklahoma City, OK, 73104, USA. 5. Beijing Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing, 100083, China. yulab@buaa.edu.cn. 6. Edmond H. Fischer Translational Medical Research Laboratory, Scientific Research Center, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, 518107, Guangdong, China. cheny653@mail.sysu.edu.cn.
Abstract
BACKGROUND: Most patients with acute myeloid leukemia (AML) remain uncurable and require novel therapeutic methods. Gain-of-function FMS-like tyrosine kinase 3 (FLT3) mutations are present in 30-40% of AML patients and serve as an attractive therapeutic target. In addition, FLT3 is aberrantly expressed on blasts in > 90% of patients with AML, making the FLT3 ligand-based drug conjugate a promising therapeutic strategy for the treatment of patients with AML. Here, E. coli was used as a host to express recombinant human FLT3 ligand (rhFL), which was used as a specific vehicle to deliver cytotoxic drugs to FLT3 + AML cells. METHODS: Recombinant hFL was expressed and purified from induced recombinant BL21 (DE3) E. coli. Purified rhFL and emtansine (DM1) were conjugated by an N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) linker. We evaluated the potency of the conjugation product FL-DM1 against FLT3-expressing AML cells by examining viability, apoptosis and the cell cycle. The activation of proteins related to the activation of FLT3 signaling and apoptosis pathways was detected by immunoblotting. The selectivity of FL-DM1 was assessed in our unique HCD-57 cell line, which was transformed with the FLT3 internal tandem duplication mutant (FLT3-ITD). RESULTS: Soluble rhFL was successfully expressed in the periplasm of recombinant E. coli. The purified rhFL was bioactive in stimulating FLT3 signaling in AML cells, and the drug conjugate FL-DM1 showed activity in cell signaling and internalization. FL-DM1 was effective in inhibiting the survival of FLT3-expressing THP-1 and MV-4-11 AML cells, with half maximal inhibitory concentration (IC50) of 12.9 nM and 1.1 nM. Additionally, FL-DM1 induced caspase-3-dependent apoptosis and arrested the cell cycle at the G2/M phase. Moreover, FL-DM1 selectively targeted HCD-57 cells transformed by FLT3-ITD but not parental HCD-57 cells without FLT3 expression. FL-DM1 can also induce obvious apoptosis in primary FLT3-positive AML cells ex vivo. CONCLUSIONS: Our data demonstrated that soluble rhFL can be produced in a bioactive form in the periplasm of recombinant E. coli. FL can be used as a specific vehicle to deliver DM1 into FLT3-expressing AML cells. FL-DM1 exhibited cytotoxicity in FLT3-expressing AML cell lines and primary AML cells. FL-DM1 may have potential clinical applications in treating patients with FLT3-positive AML.
BACKGROUND: Most patients with acute myeloid leukemia (AML) remain uncurable and require novel therapeutic methods. Gain-of-function FMS-like tyrosine kinase 3 (FLT3) mutations are present in 30-40% of AMLpatients and serve as an attractive therapeutic target. In addition, FLT3 is aberrantly expressed on blasts in > 90% of patients with AML, making the FLT3 ligand-based drug conjugate a promising therapeutic strategy for the treatment of patients with AML. Here, E. coli was used as a host to express recombinant humanFLT3 ligand (rhFL), which was used as a specific vehicle to deliver cytotoxic drugs to FLT3 + AML cells. METHODS: Recombinant hFL was expressed and purified from induced recombinant BL21 (DE3) E. coli. Purified rhFL and emtansine (DM1) were conjugated by an N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) linker. We evaluated the potency of the conjugation product FL-DM1 against FLT3-expressing AML cells by examining viability, apoptosis and the cell cycle. The activation of proteins related to the activation of FLT3 signaling and apoptosis pathways was detected by immunoblotting. The selectivity of FL-DM1 was assessed in our unique HCD-57 cell line, which was transformed with the FLT3 internal tandem duplication mutant (FLT3-ITD). RESULTS: Soluble rhFL was successfully expressed in the periplasm of recombinant E. coli. The purified rhFL was bioactive in stimulating FLT3 signaling in AML cells, and the drug conjugateFL-DM1 showed activity in cell signaling and internalization. FL-DM1 was effective in inhibiting the survival of FLT3-expressing THP-1 and MV-4-11AML cells, with half maximal inhibitory concentration (IC50) of 12.9 nM and 1.1 nM. Additionally, FL-DM1 induced caspase-3-dependent apoptosis and arrested the cell cycle at the G2/M phase. Moreover, FL-DM1 selectively targeted HCD-57 cells transformed by FLT3-ITD but not parental HCD-57 cells without FLT3 expression. FL-DM1 can also induce obvious apoptosis in primary FLT3-positive AML cells ex vivo. CONCLUSIONS: Our data demonstrated that soluble rhFL can be produced in a bioactive form in the periplasm of recombinant E. coli. FL can be used as a specific vehicle to deliver DM1 into FLT3-expressing AML cells. FL-DM1 exhibited cytotoxicity in FLT3-expressing AML cell lines and primary AML cells. FL-DM1 may have potential clinical applications in treating patients with FLT3-positive AML.
Authors: Philip R Hamann; Lois M Hinman; Irwin Hollander; Carl F Beyer; Delores Lindh; Ryan Holcomb; William Hallett; Hwei-Ru Tsou; Janis Upeslacis; Dan Shochat; Andrew Mountain; David A Flowers; Irwin Bernstein Journal: Bioconjug Chem Date: 2002 Jan-Feb Impact factor: 4.774
Authors: M Nakao; S Yokota; T Iwai; H Kaneko; S Horiike; K Kashima; Y Sonoda; T Fujimoto; S Misawa Journal: Leukemia Date: 1996-12 Impact factor: 11.528
Authors: Richard M Stone; Sumithra J Mandrekar; Ben L Sanford; Kristina Laumann; Susan Geyer; Clara D Bloomfield; Christian Thiede; Thomas W Prior; Konstanze Döhner; Guido Marcucci; Francesco Lo-Coco; Rebecca B Klisovic; Andrew Wei; Jorge Sierra; Miguel A Sanz; Joseph M Brandwein; Theo de Witte; Dietger Niederwieser; Frederick R Appelbaum; Bruno C Medeiros; Martin S Tallman; Jürgen Krauter; Richard F Schlenk; Arnold Ganser; Hubert Serve; Gerhard Ehninger; Sergio Amadori; Richard A Larson; Hartmut Döhner Journal: N Engl J Med Date: 2017-06-23 Impact factor: 91.245
Authors: Hagop M Kantarjian; Daniel J DeAngelo; Matthias Stelljes; Giovanni Martinelli; Michaela Liedtke; Wendy Stock; Nicola Gökbuget; Susan O'Brien; Kongming Wang; Tao Wang; M Luisa Paccagnella; Barbara Sleight; Erik Vandendries; Anjali S Advani Journal: N Engl J Med Date: 2016-06-12 Impact factor: 91.245
Authors: C E Carow; M Levenstein; S H Kaufmann; J Chen; S Amin; P Rockwell; L Witte; M J Borowitz; C I Civin; D Small Journal: Blood Date: 1996-02-01 Impact factor: 22.113