KLF4K409Q-mutated meningiomas show enhanced hypoxia signaling and respond to mTORC1 inhibitor treatment

Abstract

Meningioma represents the most common primary brain tumor in adults. Recently several non-NF2 mutations in meningioma have been identified and correlated with certain pathological subtypes, locations and clinical observations. Alterations of cellular pathways due to these mutations, however, have largely remained elusive. Here we report that the Krueppel like factor 4 (KLF4)-K409Q mutation in skull base meningiomas triggers a distinct tumor phenotype. Transcriptomic analysis of 17 meningioma samples revealed that KLF4^K409Q mutated tumors harbor an upregulation of hypoxia dependent pathways. Detailed in vitro investigation further showed that the KLF4^K409Q mutation induces HIF-1α through the reduction of prolyl hydroxylase activity and causes an upregulation of downstream HIF-1α targets. Finally, we demonstrate that KLF4^K409Q mutated tumors are susceptible to mTOR inhibition by Temsirolimus. Taken together, our data link the KLF4^K409Q mediated upregulation of HIF pathways to the clinical and biological characteristics of these skull base meningiomas possibly opening new therapeutic avenues for this distinct meningioma subtype.

Keywords: Edema; HIF; Hypoxia; K409Q; KLF4; Meningioma; Mutation.

Fig. 1

KLF4^K409Q Mutation results in a significant shift of gene expression and upregulates hypoxia driven pathways. a Transcriptomic analysis of 7 KLF4^K409Q and 10 KLF4^wt tumors. Nineteen thousand seven hundred ninety-nine protein-coding transcripts were considered, Trimmed Mean of M-value-normalization of all data. False discovery rate (FDR) < 0.1. Heatmap indicates the NES values derived for indicated hallmark gene sets by GSEA. Only gene sets with an FDR < 0.05 were considered. b Selective heatmap of genes relevant to hypoxia/glycolysis pathways. Red indicates significantly (FDR < 0.05) upregulated genes in KLF4^K409Q tumors, bold is upregulated but not significant. c Boxplots of KLF4, VEGF-A, SLC2A3 and HK2 expression within the analyzed samples. d, e Exemplary Western blot, its quantification of HK2 and TMA-IHC (e) of patient derived tumor-tissue confirming the increased expression of hypoxia dependent genes as well as KLF4 on the protein level in KLF^K409Q Meningiomas. (Western blot: n = 8, 4 KLF4^wt and 4 KLF4K409Q, TMA: n = 27, 17 KLF4^wt and 10 KLF4^K409Q)

Fig. 2

Effects of the KLF4^K409Q mutation on cell viability, growth rate and hypoxia response. a Western blot analysis of KLF4 overexpression in transfected IOMM cells. b Colony formation assay (n = 60), cell viability assay after 4, 24 and 48 h (n = 5 / timepoint) and cell proliferation assay (n = 15) of KLF4^wt- and KLF4^K409Q -cell lines. c, d Western Blot and RT-qPCR (n = 3) analyses of cell lines under normoxic and hypoxic conditions. e Correlation of KLF4-mRNA and mRNA levels of hypoxia dependent genes in patient derived tumor samples. f Quantification of relative KLF4^wt/^K409Q protein stability with GFP-KLF4 constructs and blocking of protein translation with emetin at specified timepoints. (n = 3). g Quantification of hydroxylation dependent HIF1-α degradation in IOMM-KLF4^wt/^K409Q cell lines after transient transfection of HIF1-ODD-Luc (n = 3). h Western Blot analysis of hydroxylated HIF1-α after proteasome inhibition with MG132

Fig. 3

Effect of Temsirolimus treatment in vitro. a TMA staining of human tumor samples for HIF1-α and Pho-p70S6k. (n = 27, 17 KLF4^wt, 10 KLF4^K409Q). b Colony formation assay (CFA) and size analysis after Temsirolimus treatment. (n = 60). c Confocal imaging of actin stained IOMM cells. d RT-qPCR analysis of HIF1-α dependent genes after treatment with Temsirolimus under normoxic conditions. (n = 3). e Western Blot analysis of HIF1-α and (Ph)-p70S6K protein levels after treatment with Temsirolimus under hypoxia

Fig. 4

Survival studies of orthotopic xenograft models in mice. a Kaplan-Meier-Survival curve after establishing tumors through intracranial injection of IOMM-KLF4^wt or -KLF4^K409Q cells in the convexity (IOMM-KLF4^wt vs IOMM-KLF4^K409Q) and exemplary MRI. No difference in OS was observed (p = .4305). b Exemplary MRI of KLF4^wt (I) and KLF4^K409Q (II) tumors and histological cross section of the KLF4^K409Q skull base tumor (III and IV). c Analysis of tumor size on MRI (n = 3). d Kaplan-Meier-Survival curve of mice bearing skull base tumors with and without treatement. e Combined Kaplan-Meier-Survival curve of both cell lines (KLF4^wt and KLF4^K409Q) with and without treatment