MED12 mutation induces RTK inhibitor resistance in NSCLC via MEK/ERK pathway activation by inflammatory cytokines

Abstract

Non-small cell lung cancer (NSCLC) is frequently associated with mutations in receptor tyrosine kinases (RTKs), such as EGFR and ALK. While RTK inhibitors (RTKIs) have proven effective in treating patients with specific RTK mutations, the emergence of resistance to these therapies remains a significant clinical obstacle. As such, there is still an unmet need for the identification of new biomarkers that can predict resistance to RTK inhibitors in clinical use. In the present study, we demonstrate that MED12 mutations are a key driver of RTKi resistance in NSCLC cells. This resistance is mediated through the release of inflammatory cytokines triggered by MED12 degradation. Notably, we observed that of the two major downstream signaling pathways activated by inflammatory cytokines, only the MEK/ERK pathway was upregulated, while the PI3K/AKT pathway was unaffected in MED12 knock-out (KO) cells. The degradation of MED12 results in the dissociation of the MED12 complex, which subsequently leads to YAP phosphorylation. This phosphorylated YAP increases PTEN expression by inhibiting miR-29, thereby suppressing the PI3K/AKT signaling pathway. Importantly, treatment with trametinib, a MEK inhibitor, effectively suppressed tumor growth in MED12KO NSCLC cells and in xenograft models derived from these cells. These findings suggest that targeting the MEK/ERK signaling pathway, such as with trametinib, may provide a viable strategy to overcome RTKi resistance in MED12-mutant NSCLC. Furthermore, MED12 is identified as a crucial biomarker and potential therapeutic target for overcoming RTKi resistance.

Keywords: Drug resistance; MED12 mutation; MEK inhibitor; NSCLC; Receptor tyrosine kinase inhibitor.

Fig. 1

MED12 mutation induces resistance to RTK inhibitors in NSCLC via the suppression of its expression and predicts poor survival to RTK inhibitors in NSCLC patients A Cell viability of the ceritinib-resistant H3122 cell line (H3122CR) was assessed using the MTS assay. B Targeted sequencing analysis of H3122CR identified the presence of the L1283P (3848T > C) mutation in the MED12 gene, represented as a proportion (%) among various gene mutations. C Western blotting analysis confirmed decreased MED12 expression in both ceritinib-resistant and mutant MED12 overexpression cell line compared to the control group. D Western blot analysis validated the efficient knockout of MED12 in the generated knockout (KO) cell lines, exhibiting a notable reduction in MED12 protein expression compared to the control group. E, F MTS assay was performed to evaluate the sensitivity of MED12 KO cell lines to RTK inhibitors (ceritinib, alectinib, lorlatinib, and osimertinib). Cell viability of MED12 KO cell lines was compared to the respective control cell lines. Re-expression of wild-type MED12 in KO cells restored RTK inhibitor sensitivity to levels comparable to the parental cells. G Kaplan-Meier survival analysis of progression-free survival (PFS) for EGFR-TKI or ALKi treated NSCLC patients with MED12 mutations compared to those without. The hazard ratio (HR) was 1.979 (p-value = 0.0246), indicating an unfavorable prognosis for patients with MED12 mutations following RTKi treatment

Fig. 2

MED12 mutation, L1283P, induces its proteasomal degradation, which lead to break of MED12 complex. A Western blot analysis showing the protein expression levels of MED12 complex components (MED12, CDK8, MED13, CCNC) in the parental cell line (H3122) and MED12 knockout (KO) cell line with mutant MED12 overexpression (OE) before and after treatment with 2 µM MG132 (proteasome inhibitor) for 24 h. B Co-immunoprecipitation was performed to assess the direct interaction between the mutant MED12 and ubiquitin. Protein lysates from the MED12 KO/mutant MED12 OE cell line were immunoprecipitated with anti-MED12 antibody, followed by immunoblotting with anti-ubiquitin antibody. C Fluorescence imaging of GFP-tagged MED12 in the MED12 KO/mutant MED12 OE cell line before and after treatment with MG132, demonstrating the blockade of ubiquitin-mediated proteasomal degradation

Fig. 3

Inflammatory cytokines release by MED12 mutation induce RTK inhibitor resistance through only the MEK/ERK pathway activation, not the PI3K/AKT pathway. A Gene Set Enrichment Analysis (GSEA) of RNA-seq data from NSCLC patients with MED12 mutations, as well as MED12 knock-out H3122 and PC9 cell lines, showing significant enrichment in the cytokine-cytokine receptor interaction gene set of the KEGG_LEGACY subset. B Western blot analysis confirming elevated chromatin-bound MED1 levels in MED12 knock-out H3122 and PC9 cell lines compared to parental cell lines. C Olink proteomics analysis of culture media from MED12 knock-out cell lines, identifying increased levels of inflammatory cytokines. D Cell image of the trans-well insert co-culture system used to expose parental NSCLC cell lines to cytokines released from MED12 knock-out cell lines, resulting in increased RTKi resistance, assessed by clonogenic assay. Schematic of the trans-well co-culture system was created with BioRender.com. E Western blot analysis showing the activation of both AKT and ERK1/2 pathways in parental cell lines exposed to cytokines from MED12 knock-out cell lines. F In H3122/MED12 KO cell lines, inhibition of the AKT pathway and activation of the ERK1/2 pathway were observed by Western blotting analysis. G Western blot analysis demonstrating the reactivation of the AKT pathway and suppression of the ERK1/2 pathway in H3122/MED12 KO cell lines after overexpression of wild type MED12

Fig. 4

MEK inhibitor, trametinib, alone overcomes RTK-induced resistance by repression of MED12 expression by its mutation. A MTS assay was performed to assess the cell viability of MED12 KO cell lines and control cell lines treated with ceritinib/osimertinib and trametinib. B Caspase 3/7 assay was performed to evaluate apoptosis in MED12 KO cell lines and control cell lines treated with ceritinib, osimertinib, or trametinib, with or without co-treatment of the pan-caspase inhibitor Z-VAD-FMK. C Western blot analysis showing the expression of the apoptosis marker cleaved PARP in MED12 KO cell lines treated with trametinib compared to control groups. Ceritinib and trametinib were each administered at a concentration of 200 nM for 72 h. D Tumor volume measurements of xenograft mouse models implanted with parental cell line (H3122, n = 3) and MED12 knockout (KO) NSCLC cells (H3122/MED12 KO, n = 3) and treated with daily administration of vehicle, ceritinib, or trametinib for three weeks. E Representative images of tumors obtained from the xenograft mouse model showing the differences in tumor growth among the treatment groups. F Immunohistochemistry (IHC) analysis performed on tumor tissues derived from the xenograft mouse model to evaluate the expression levels of Ki-67, a proliferation marker, and MED12

Fig. 5

Inhibiting physical interaction between MED12 and YAP leads to increased PTEN expression and subsequent inhibition of the AKT pathway. A Co-immunoprecipitation was performed to confirm the direct interaction between MED12 and YAP. Protein lysates from the cell lines were immunoprecipitated with anti-MED12 antibody, followed by immunoblotting with anti-YAP antibody. B Western blot analysis showing increased levels of phospho-YAP (ser127) and PTEN in the MED12 KO cell line compared to the control, indicating the inhibition of the PI3K/AKT pathway. C Restoration of wild type MED12 expression in the MED12 KO cell line resulted in decreased levels of phospho-YAP (ser127) and PTEN, indicating the reactivation of the PI3K/AKT pathway. D The expression levels of miR-29, a mediator of PTEN suppression by YAP, were downregulated in the MED12 KO cell line and restored after wild-type MED12 recovery, as determined by qPCR analysis. E Western blot analysis showing the effects of YAP overexpression (YAP, YAP-5SA, YAP-S94A) in both the parental (H3122) and MED12 KO (H3122/MED12 KO) cell lines. Changes in PTEN and phospho-AKT (p-AKT) levels were evaluated. F The expression levels of miR-29 in the different YAP overexpressed cell lines were evaluated by qRT-PCR. G Resistance to ceritinib and trametinib in the various YAP overexpressed cell lines was assessed using MTS assay

Fig. 6

MEK inhibitor could be the most suitable treatment option for MED12 mutation-induced RTK inhibitor-resistant NSCLC. Created with BioRender.com Schemas illustrating the identified mechanisms of MED12 mutation-mediated resistance to RTK inhibitions. A Targeting EGFR or ALK in EGFR-mutated or EML4-ALK/wild-type MED12 cells. B Although MED12 mutation-induced inflammatory cytokine induces both PI3K/AKT activation and MEK/ERK activation, MED12 mutation blocks PI3K/AKT activation by PTEN induction via YAP regulation. Therefore, MEK inhibitor alone provides sufficient benefit for MED12-mutated RTKi-resistant NSCLC. Solid lines indicate the effects