Loss of CARM1 alters the developmental programming of Glioma stem-like cells and creates a druggable NGFR/NTRK dependency

Abstract

A key driver of Glioblastoma (GBM) heterogeneity and therapy resistance is the capacity of glioma stem-like cells (GSCs) to hijack developmental signaling programs. However, it remains unclear how GSCs regulate these adapted developmental signaling pathways and how these pathways might be therapeutically exploited. The arginine methyltransferase, CARM1, has been shown to play critical roles in maintaining stem cell pluripotency, preventing differentiation, and recently was discovered to be upregulated in Glioblastoma. To date, there is little to no understanding of the role that CARM1 plays in regulating developmental processes in Glioblastoma. To address this gap in knowledge, we applied a multi-omics approach to characterize developmental processes that are specifically regulated by CARM1 in GSCs. We found that loss of CARM1 results in dysregulation of several developmental markers: ARX, GFAP, NGFR, PDGFRA and results in both a proteomic and transcriptomic shift towards the radial glia cell lineage. Moreover, CARM1 depleted cells reprogram their signaling to develop an increased survival dependency on NGFR/NTRK signaling and are hypersensitive to the FDA approved brain penetrant NTRK inhibitor-Entrectinib. Mechanistically, we find that NFIA is a CARM1 substrate and can repress NGFR signaling just as CARM1 does, and thus the CARM1/NFIA relationship is likely a key regulator of NGFR/NTRK signaling in GSCs. Altogether, we demonstrate that CARM1 regulates the cell lineage of GSCs at the transcriptomic and proteomic level, and naturally represses NGFR/NTRK signaling-likely through CARM1 dependent methylation of NFIA. Further, CARM1 depletion leads GSCs to develop a survival dependency on NGFR/NTRK signaling and creates a therapeutic vulnerability to NTRK inhibition.

Keywords: AKT; CARM1; Capivasertib; Entrectinib; Glioblastoma; MAPK; NGFR; NTRK; arginine methylation; development; epigenetics; glioma stem-like cell; histones; proteomics; radial glial cell.

Figure 1:. CARM1 predicts Glioma patient survival and regulates Glioma stem-like cell proliferation.

A) Violin plot of mRNA expression log2(normalized count+1) of CARM1 in low grade glioma (n = 344) and Glioblastoma multiforme (n = 342), and B) Kaplan Meir curve of Glioblastoma patients with high or low CARM1 mRNA expression using UCSC Xena Browser. C-D) Western blot of CARM1 following CRISPR/Cas9 depletion and negative selection in NSC11 and NSC20 GSCs. E-F) Growth curves of sgSCR and sgCARM1 ablated NSC11 and NSC20 cells measured with Cell Titer Glo 2.0, n = 3, data shows mean +/− SEM.

Figure 2:. CARM1 regulates widescale transcriptome and histone PTMs involved in development.

A) Volcano plot of NSC11 sgCARM1 (combined sgCARM1-1 and sgCARM1-2) vs sgSCR highlighting key developmental genes (adjusted p-value < 0.05, n = 3-4 per sgRNA). B) GSEA Enrichment plots of RNA-sequencing data of NSC11 sgSCR and sgCARM1 cells for ‘stem cell population maintenance’ (upper panel) and chromatin organization (lower panel) gene sets. C) Heatmap of differentially expressed genes involved in chromatin organization (adjusted p-value < 0.05) scaled by Z-score of mRNA normalized counts. D) Mass spectrometry analysis of significant histone post-translational modifications (PTMs) in NSC11 sgSCR and sgCARM1 cells, data shows mean +/− SD.

Figure 3:. CARM1 represses radial glial cell proteomic signature in Glioma stem-like cells.

A) Proteomics based heatmap of key differentially expressed proteins in NSC11 sgCARM1-1 and sgCARM1-2 (compared to sgSCR) scaled by log2 fold change (p-value < 0.05), n = 2 independent experiments. B) GSEA enrichment plot of proteomics data highlighting protein enrichment for radial glial cells in NSC11 sgCARM1 and sgSCR cells; and a C) heatmap of differentially expressed proteins (p-value < 0.05) that belong to the above mentioned GSEA pathway scaled by log 2 foldchange vs. control. D) Scatter plot of log2 fold change (averaged between sgCARM1-1 and sgCARM1-2) of RNA-seq and Proteomics data, only including genes differentially expressed in both sgCARM1-1 and sgCARM1-2 at the RNA and Protein level. E) Scatter plot of ranked genes/proteins based on their combined average log2 fold change between RNA and Protein.

Figure 4:. Loss of CARM1 drives NGFR/NTRK signaling in Glioma stem-like cells.

A) Volcano plot of differentially phosphorylated peptides between NSC11 WT and CARM1 depleted cells (sgCARM1-1) with n = 3-4 (p-value < 0.05). B) Dot plots of Qiagen IPA analysis of top upregulated pathways C) and top upstream regulators ranked by Z-score. D) Heatmap of differentially phosphorylated proteins and phospho-sites involved in NGFR/NTRK signaling scaled by Z-score of relative abundance (p-value < 0.05) and E) bar plots of phosphorylation found on NGFR/NTRK targets AKT1 and MAPK1/3 (ERK1/2). F) Western blot validation of NGFR/NTRK signaling in NSC11 sgSCR and sgCARM1 cells.

Figure 5:. CARM1 substrate, NFIA, represses NGFR expression.

A) Mass spectrometry schematic of approach to identify CARM1 asymmetric di-methyl arginine (ADMA) substrates in NSC11 GSCs WT vs CARM1-depleted (sgCARM1-1), n = 3-4. B) Pie chart of identified CARM1 substrates. C) Dot plot of Gene Ontology Molecular Functions of statistically significant (p-value < 0.05) CARM1 substrates and D) volcano plot of differentially ADMA peptides, labeling key substrates (i.e. NFIA). E) Western blots of NFIA and NGFR expression after knockdown of NFIA and F) of NGFR expression post CARM1 depletion.

Figure 6:. Loss of CARM1 sensitizes Glioma stem-like cells to NTRK inhibition.

A) Western blot of CARM1 expression after 48 hours treatment with DMSO or Entrectinib (3uM). B) Cell viability of NSC11 GSCs after 96 hours of Entrectinib measured with Cell Titer Glo 2.0, n = 3, data shows mean +/− SEM. C-D) Clonogenic assay of NSC11 GSCs treated for 72 hours with Entrectinib, n = 3, data shows mean +/− SD. E) (upper panel) Under normal conditions we propose CARM1 methylates NFIA on R389, making NFIA-ADMA. NFIA-ADMA is then able to properly form various protein-protein interactions, possibly with co-repressors, to repress NGFR transcription. Reduced NGFR transcription maintains homeostatic levels of NTRK signaling. (lower panel) Upon CARM1 depletion, NFIA is not methylated and fails to form proper protein-protein interactions. Therefore, when hypomethylated-NFIA binds chromatin, it is unable to successfully repress NGFR. NGFR expression increases which then promotes NTRK signaling which can be targeted with the NTRK inhibitor, Entrectinib.