Efficacy of SCF drug conjugate targeting c-KIT in gastrointestinal stromal tumor

Abstract

Background: Gastrointestinal stromal tumor (GIST) is a rare type of cancer that occurs in the gastrointestinal tract. The majority of GIST cases carry oncogenic forms of KIT, the receptor for stem cell factor (SCF). Small molecule kinase inhibitor imatinib is effective in prolonging the survival of GIST patients by targeting KIT. However, drug resistance often develops during the therapeutic treatment. Here, we produced a SCF-emtansine drug conjugate (SCF-DM1) with favorable drug efficacy towards GIST cells.

Methods: Recombinant human SCF (rhSCF) was expressed in E. coli cells and further purified with Ni-NTA Sepharose and Phenyl Sepharose. It was then conjugated with DM1, and the conjugated product SCF-DM1 was evaluated using in vitro cell-based assays and in vivo xenograft mouse model.

Results: SCF-DM1 was effective in inhibiting imatinib-sensitive and -resistant GIST cell lines and primary tumor cells, with IC₅₀ values of < 30 nM. It induced apoptosis and cell cycle arrest in GIST cells. In xenograft mouse model, SCF-DM1 showed favorable efficacy and safety profiles.

Conclusions: rhSCF is a convenient and effective vector for drug delivery to KIT positive GIST cells. SCF-DM1 is an effective drug candidate to treat imatinib-sensitive and -resistant GIST.

Keywords: DM1; GIST; SCF; Targeted therapy.

Fig. 1

Expression, purification, and bioactivity evaluation of rhSCF. A Immunoblotting analysis of purified rhSCF protein. Lane 1: pre-stained protein marker; lanes 2 and 4: reference rhSCF from Sf9 cells. Due to glycosylation modification, rhSCF from Sf9 cells has a higher molecule weight than rhSCF without glycosylation; lanes 3 and 5: purified rhSCF. Left panel: PVDF membrane captured by using stain free method on Bio-Rad ChemiDoc system. Right panel: western blot result of rhSCF reference and purified rhSCF. B SDS-PAGE result of rhSCF after purification. Lane 1: protein marker; lane 2: sample after Ni–NTA Sepharose purification; lane 3: sample after phenyl Sepharose purification. C Immunoblot analysis of phosphorylation status of KIT signaling proteins in THP-1 treated by rhSCF (100 ng/mL). D, E Membrane KIT expression levels in THP-1 (D) and GIST 430 (E) treated by rhSCF (100 ng/mL). The vertical dot lines in D and E were given to view the induction effect of SCF. Numbers indicated left-side or right-side cell population (%) separated by dot lines in each group

Fig. 2

Preparation and bioactivity evaluation of SCF-DM1 A. SDS-PAGE result of SCF and DM1 conjugation product. Lane 1: protein marker; lane 2: SCF and DM1 conjugation product; lane 3: rhSCF. B Membrane KIT expression levels in GIST T1 cells with treatment of drugs. Vertical dot line in B was given to view the induction effect of drugs, and numbers indicated left-side or right-side cell population (%) separated by the dot line in each group. SCF-DM1: 100 ng/mL, rhSCF: 100 ng/mL, DM1: 100 nM. C Immunoblot analysis of phosphorylation status of KIT signaling proteins in THP-1 cells with treatment of drugs. rhSCF: 100 ng/mL; SCF-DM1: 100 ng/mL. D LC–MS result of SCF and DM1 conjugation product. + X DM1 means one molecule of SCF was conjugated with X molecule(s) of DM1, + 1 SMCC means one molecule of SCF was conjugated with 1 molecule of SMCC

Fig. 3

Cytotoxicity effect of SCF-DM1 on GIST cell lines. A Cell proliferation assay of GIST cells with treatment of drugs. B FACS detection of apoptosis in GIST cells with treatment of drugs. C Immunoblot analysis of apoptosis-related proteins of GIST cells with treatment of drugs. Lane 1: control; lane 2: SCF-DM1 25 nM; lane 3: SCF-DM1 50 nM; lane 4: DM1 250 nM; lane 5: DM1 500 nM; lane 6: imatinib 500 nM. D Immunoblot analysis of p53 and p21 protein in GIST cells with treatment of SCF-DM1. E Total MDM2 expression of GIST 430 cells with treatment of drugs for 72 h. F Cell cycle analysis of GIST cells with treatment of drugs. Numbers indicated G₀/G₁ or G₂/M population (%). SCF-DM1: 50 nM, DM1: 200 nM. G Clonogenic assay of GIST cells with treatment of drugs. SCF-DM1: 50 nM; imatinib: 500 nM for GIST T1, 1000 nM for GIST 430

Fig. 4

Immunoblot analysis of phosphorylation status of KIT signaling proteins in GIST cell lines with treatment of SCF-DM1 (100 ng/mL). Cells were cultured in IMDM medium plus 10% FBS

Fig. 5

Specificity of SCF-DM1. A Membrane KIT expression level of stable HCD57-KIT/D816V cells. Vertical dot line in A was given to view the induction effect of drugs. Numbers indicated left-side or right-side cell population (%) separated by the dot line in each group. B Cell inhibition assay of HCD57 cells with treatment of drugs for 72 h. C. FACS analysis and bar chart analysis of apoptosis in GIST cells with treatment of drugs for 72 h

Fig. 6

Effect of SCF-DM1 on primary GIST cells. A FACS analysis of primary GIST cells with treatment of drugs. B Annexin V positive population of five primary GIST cells with treatment of drugs by FACS analysis. Sample 1: GIST located at stomach; samples 2–5: GIST tumor metastasized in liver. C Bar chart of increment of Annexin V + population in primary GIST patient cells with treatment of different drugs after deduction of control. Data was displayed in mean ± standard deviation

Fig. 7

Efficacy of SCF-DM1 on the mouse model. A Body weight change of mice with drug administration. B Liver and spleen of mice treated with SCF-DM1. C Tumor volume of mice with treatment of different drugs. Single injection of SCF-DM1 (50 μL, 1 mg/mL) and DM1 was intratumorally administrated on day zero. Imatinib (100 mg/kg) was given once per day until the mice were sacrificed. D Tumors dissected from mice

Fig. 8

Schematic diagram of the effects of SCF-DM1 on KIT-mutated GIST cells