Abstract
BackgroundMost 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.MethodsRecombinant 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).ResultsSoluble 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.ConclusionsOur 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.
Highlights
Most patients with acute myeloid leukemia (AML) remain uncurable and require novel therapeutic methods
Our data demonstrated that soluble recombinant human FLT3 ligand (rhFL) can be produced in a bioactive form in the periplasm of recombinant E. coli
FMS-like tyrosine kinase 3 (FLT3) ligand (FL) can be used as a specific vehicle to deliver DM1 into FLT3-expressing AML cells
Summary
Most patients with acute myeloid leukemia (AML) remain uncurable and require novel therapeutic methods. The phosphorylated tyrosine domains can recruit Src homology domain− (SH2−) or phosphotyrosine-binding domain− (PTB−) containing adaptors and phosphorylate downstream signaling proteins, such as AKT, MAPK, STAT5, and SFK family members. Such signaling can result in anti-apoptosis and cell survival and proliferation [6]. The total FLT3 expression level in the bone marrow of AML patients is 5–6 times higher than that in healthy donors [7] These properties can reduce the undesirable side effects of FLT3-targeted drugs while maximizing the cytotoxic effect [10, 11]
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