Lymphatic disorders caused by mosaic, activating KRAS variants respond to MEK inhibition

Abstract

Central conducting lymphatic anomaly (CCLA) due to congenital maldevelopment of the lymphatics can result in debilitating and life-threatening disease with limited treatment options. We identified 4 individuals with CCLA, lymphedema, and microcystic lymphatic malformation due to pathogenic, mosaic variants in KRAS . To determine the functional impact of these variants and identify a targeted therapy for these individuals, we used primary human dermal lymphatic endothelial cells (HDLECs) and zebrafish larvae to model the lymphatic dysplasia. Expression of the p.Gly12Asp and p.Gly13Asp variants in HDLECs in a 2‑dimensional (2D) model and 3D organoid model led to increased ERK phosphorylation, demonstrating these variants activate the RAS/MAPK pathway. Expression of activating KRAS variants in the venous and lymphatic endothelium in zebrafish resulted in lymphatic dysplasia and edema similar to the individuals in the study. Treatment with MEK inhibition significantly reduced the phenotypes in both the organoid and the zebrafish model systems. In conclusion, we present the molecular characterization of the observed lymphatic anomalies due to pathogenic, somatic, activating KRAS variants in humans. Our preclinical studies suggest that MEK inhibition should be studied in future clinical trials for CCLA due to activating KRAS pathogenic variants.

Targeted treatment in complex lymphatic anomaly: a case of synergistic efficacy of trametinib and sirolimus

“…Consequently, treatment with mTORC1 inhibitors may increase activation of the RAS-RAF-MEK1/2-ERK pathway [ 16 ]. Furthermore, the majority of CLAs appear to harbor a mutation that activates the RAS pathway [ 17 ]. Notably, KRAS can activate PI3K, while PI3K can also activate RAS, indicating crosstalk between these two pathways.…”

Central conducting lymphatic anomaly: from bench to bedside

“…Mosaic and germline RASopathies due to pathogenic variants that result in upregulation of the RAS/MAPK signaling pathway are the most common causes of CCLA ( Figure 1B ) ( 48 , 61 , 80 – 88 ). Specifically, somatic pathogenic variants in ARAF , BRAF , KRAS , and MAP2K1 resulting in isolated lymphatic as well as syndromic presentations have been identified ( 48 , 61 , 82 , 85 ). Germline monoallelic pathogenic variants in PTPN11 , KRAS , HRAS , BRAF , RAF1 , RIT1 , SOS1 , SOS2 , and RASA1 have been identified in individuals with CCLA and Noonan syndrome, CCLA and Costello syndrome, CCLA and cardiofaciocutaneous syndrome, or CCLA and capillary malformation-arteriovenous malformation syndrome ( 48 , 61 , 80 – 88 ).…”

“…RASopathy-associated pathogenic variants result in cellular defects as well as increased lymphangiogenesis. To evaluate this, researchers transduced mutant proteins of interest into HDLECs, followed by immunostaining for cytoskeletal proteins or conducting a spheroid sprouting assay ( 48 , 82 , 85 ). Expression of mutant ARAF protein led to increased internalization of VE-cadherin, abnormal actin cytoskeleton, and elongation of the cells, which was improved by trametinib treatment.…”

“…Similar results were seen with expression of mutant KRAS proteins. Spheroid models are also an effective three-dimensional method for evaluating the effect of a variant on lymphangiogenesis ( Figure 4A ) ( 48 , 82 , 85 ). These assays demonstrated increased sprouting behavior, including cumulative sprout length and number of sprouts per sphere in ARAF , KRAS , BRAF , and RAF1 models, which was also reduced with trametinib ( 48 , 82 , 85 ).…”

“…Spheroid models are also an effective three-dimensional method for evaluating the effect of a variant on lymphangiogenesis ( Figure 4A ) ( 48 , 82 , 85 ). These assays demonstrated increased sprouting behavior, including cumulative sprout length and number of sprouts per sphere in ARAF , KRAS , BRAF , and RAF1 models, which was also reduced with trametinib ( 48 , 82 , 85 ). Additionally, lysates from spheroid models can be used in typical biochemical assays and demonstrated increased ERK phosphorylation at Thr202/Tyr204, which was inhibited by trametinib ( 48 , 82 , 85 ).…”