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Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor
Published on: May 2, 2022
Activated tyrosine kinases in gastrointestinal stromal tumor with loss of KIT oncoprotein expression
1a Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Sciences , Zhejiang Sci-Tech University , Hangzhou , China.
Abstract:
Oncogenic KIT or PDGFRA receptor tyrosine kinase (TK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GISTs), and the KIT/PDGFRA kinase inhibitor, imatinib, is the standard of care for patients with metastatic GIST. However, approximately 10% of KIT-positive GIST metastases lose KIT expression at the time of clinical progression during imatinib therapy. In the present report, we performed TK-activation screens, using phosphotyrosine-TK double immunoaffinity purification and mass spectrometry, in GIST in vitro models lacking KIT expression. These studies demonstrated tyrosine-phosphorylated EGFR, AXL, and EPHA2 in four of six KIT-negative GIST lines (GIST62, GIST522, GIST54, GIST226, GIST48B, and GIST430B), and tyrosine-phosphorylated focal adhesion kinase (FAK) in each of the six KIT-negative lines. AXL expression was strong in KIT-negative or -weak clinical GIST samples that were obtained from progressing metastases during imatinib therapy. AXL knockdown inhibited viability in three KIT-negative GIST cell lines (GIST62, GIST54, and GIST522), but not in an AXL-negative, KIT-positive GIST control cell line (GIST430). AXL inhibition by R428, a specific AXL kinase inhibitor, reduced viability in AXL-activated GIST54. AXL knockdown in GIST62, GIST522, and GIST54 was accompanied by an increase in p21, p27, and p53 expression. By contrast, gefitinib-mediated EGFR inhibition, PF562271-mediated FAK inactivation, and shRNA-mediated knockdowns of EPHA2 and FAK had no effect on viability or colony formation of the KIT-negative GISTs. These findings highlight the potential relevance of AXL/p53 signaling as a therapeutic target in a subset of GISTs that have lost KIT oncoprotein expression.
Insights
Gastrointestinal stromal tumors (GISTs) can lose KIT expression during imatinib therapy. Research identified AXL receptor tyrosine kinase as a potential therapeutic target in KIT-negative GISTs, showing AXL inhibition impacts cancer cell viability.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Gastrointestinal stromal tumors (GISTs) are often driven by mutations in KIT or PDGFRA receptor tyrosine kinases (TKs).
- Imatinib is the standard therapy for metastatic GIST, but resistance develops in about 10% of cases due to loss of KIT expression.
- Identifying alternative therapeutic targets in KIT-negative GIST is crucial for improving patient outcomes.
Purpose of the Study:
- To identify novel tyrosine kinase (TK) targets in GIST models that have lost KIT expression.
- To investigate the role of identified TKs in the viability and progression of KIT-negative GIST cells.
- To evaluate the therapeutic potential of targeting these novel pathways in GIST.
Main Methods:
- Utilized TK-activation screens involving phosphotyrosine-TK double immunoaffinity purification and mass spectrometry in KIT-negative GIST cell lines.
- Performed AXL knockdown and inhibition studies using specific inhibitors (R428) to assess effects on cell viability.
- Analyzed the expression of cell cycle regulators (p21, p27, p53) following AXL modulation.
Main Results:
- Identified tyrosine-phosphorylated EGFR, AXL, and EPHA2, and focal adhesion kinase (FAK) in KIT-negative GIST cell lines.
- Observed strong AXL expression in clinical GIST samples from patients progressing on imatinib.
- AXL knockdown or inhibition significantly reduced viability in KIT-negative GIST cell lines and increased p53 pathway activation.
Conclusions:
- AXL receptor tyrosine kinase is activated in a subset of KIT-negative GISTs that develop resistance to imatinib.
- AXL signaling, potentially through p53 pathway modulation, represents a promising therapeutic target in imatinib-resistant GIST.
- Targeting AXL may offer a new treatment strategy for patients with advanced GIST who have lost KIT expression.
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