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MEK Inhibition Reduces Vascular Malformations and Gene Dysregulation in NRASQ61R Human Endothelial Cells
Sara Alharbi1,2,3, Andrew Wagner3, Svatava Merkle2,3,4
11Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
Background:
An activating NRAS p.Q61R (NRASQ61R) somatic mutation occurs in the lesions of patients with kaposiform lymphangiomatosis (KLA). Therapies for KLA patients are limited and since the mitogen-activated protein kinase pathway is hyperactivated by NRASQ61R, we evaluated the potency of Mitogen-activated protein kinase kinase (MEK) inhibitors (trametinib, selumetinib, and cobimetinib) on human NRASQ61R endothelial cells (ECs). RNA sequencing analysis was performed to identify dysregulated genes and those restored by trametinib. Trametinib was tested in a mouse xenograft model of NRASQ61R ECs.
Procedure:
Doxycycline (Dox)-inducible NRASQ61R ECs were treated with trametinib, selumetinib, cobimetinib, or vehicle (control). Cell signaling pathways, proliferation, migration, morphology, and angiopoietin-2 (ANG-2) levels were assessed. RNA-sequencing analysis was performed on Dox-induced NRAS wild-type (NRASWT), NRASQ61R, and trametinib-treated NRASQ61R ECs. Selected proteins were verified by immunoblotting. NRASQ61R ECs were injected into nude mice on Dox-containing diet to generate xenografts and treated with trametinib or vehicle.
Results:
Trametinib reduced NRASQ61R-activated ERK phosphorylation, cell proliferation, migration, spindling, and ANG-2 at lower doses than selumetinib or cobimetinib. RNA-sequencing detected 1315 upregulated and 1773 downregulated genes in NRASQ61R ECs compared with NRASWT. Trametinib corrected 19% upregulated and 8% downregulated genes, including elevated Notch ligands (DLL4, JAG1), activated Notch 1, and HES1. Trametinib reduced NRASQ61R ECs xenograft weights by 46%, vascular area by 63%, and phosphorylated ERK staining.
Conclusion:
Trametinib was the most effective MEK inhibitor, correcting some dysregulated genes in NRASQ61R ECs, including some in the Notch pathway. Trametinib reduced vessel overgrowth in xenografts of NRASQ61R ECs. These studies support using trametinib treatment for KLA patients with an NRASQ61R mutation.
Insights
Trametinib effectively targets NRASQ61R mutations in kaposiform lymphangiomatosis (KLA) by inhibiting MEK signaling. This MEK inhibitor reduced cell proliferation and vessel overgrowth in preclinical models, supporting its use in KLA patients.
Area of Science:
- Oncology
- Vascular Biology
- Molecular Biology
Background:
- Kaposiform lymphangiomatosis (KLA) is associated with activating NRAS p.Q61R (NRASQ61R) somatic mutations.
- Hyperactivation of the mitogen-activated protein kinase pathway by NRASQ61R drives KLA pathogenesis.
- Limited therapeutic options exist for KLA patients.
Purpose of the Study:
- To evaluate the efficacy of Mitogen-activated protein kinase kinase (MEK) inhibitors (trametinib, selumetinib, cobimetinib) against NRASQ61R-driven endothelial cell (EC) dysfunction.
- To identify molecular targets and pathways affected by NRASQ61R and modulated by trametinib using RNA sequencing.
- To assess the therapeutic potential of trametinib in a preclinical KLA xenograft model.
Main Methods:
- Human NRASQ61R endothelial cells (ECs) were treated with MEK inhibitors (trametinib, selumetinib, cobimetinib) or vehicle.
- Cell signaling, proliferation, migration, morphology, and angiopoietin-2 (ANG-2) levels were assessed.
- RNA sequencing was performed on NRAS wild-type (NRASWT), NRASQ61R ECs, and trametinib-treated NRASQ61R ECs. A mouse xenograft model was utilized.
Main Results:
- Trametinib demonstrated superior potency in inhibiting NRASQ61R-induced ERK phosphorylation, cell proliferation, migration, and ANG-2 secretion compared to selumetinib and cobimetinib.
- RNA sequencing revealed significant gene expression dysregulation in NRASQ61R ECs, with trametinib partially restoring expression of genes, including Notch pathway components (DLL4, JAG1, Notch 1, HES1).
- In vivo, trametinib treatment significantly reduced xenograft weight (46%) and vascular area (63%) in NRASQ61R EC xenografts.
Conclusions:
- Trametinib is the most effective MEK inhibitor evaluated, demonstrating ability to correct NRASQ61R-driven gene dysregulation, including in the Notch pathway.
- Trametinib effectively reduced pathological vessel overgrowth in a preclinical model of KLA.
- These findings support the clinical investigation of trametinib for KLA patients harboring NRASQ61R mutations.
