hnRNPM guides an alternative splicing program in response to inhibition of the PI3K/AKT/mTOR pathway in Ewing sarcoma cells

Abstract

Ewing sarcomas (ES) are biologically aggressive tumors of bone and soft tissues for which no cure is currently available. Most ES patients do not respond to chemotherapeutic treatments or acquire resistance. Since the PI3K/AKT/mTOR axis is often deregulated in ES, its inhibition offers therapeutic perspective for these aggressive tumors. Herein, by using splicing sensitive arrays, we have uncovered an extensive splicing program activated upon inhibition of the PI3K/AKT/mTOR signaling pathway by BEZ235. Bioinformatics analyses identified hnRNPM as a key factor in this response. HnRNPM motifs were significantly enriched in introns flanking the regulated exons and proximity of binding represented a key determinant for hnRNPM-dependent splicing regulation. Knockdown of hnRNPM expression abolished a subset of BEZ235-induced splicing changes that contained hnRNPM binding sites, enhanced BEZ235 cytotoxicity and limited the clonogenicity of ES cells. Importantly, hnRNPM up-regulation correlates with poor outcome in sarcoma patients. These findings uncover an hnRNPM-dependent alternative splicing program set in motion by inhibition of the mTOR/AKT/PI3K pathway in ES cells that limits therapeutic efficacy of pharmacologic inhibitors, suggesting that combined inhibition of the PI3K/AKT/mTOR pathway and hnRNPM activity may represent a novel approach for ES treatment.

Poison-Exon Inclusion in DHX9 Reduces Its Expression and Sensitizes Ewing Sarcoma Cells to Chemotherapeutic Treatment

“…In breast cancer, hnRNPM contributes to metastasis by activating alternative splicing changes that promote epithelial-mesenchymal transition (EMT) [46]. In Ewing sarcoma, we previously documented that the inhibition of the mTOR/AKT/PI3K pathway sets in motion an hnRNPM-dependent alternative splicing program that limits the therapeutic efficacy of these pharmacologic inhibitors [24]. On this basis, we indicated hnRNPM as a new potential therapeutic target to counteract Ewing sarcoma malignancy [24].…”

“…In this regard, the splicing signature of human cancers is emerging as a powerful tool to distinguish tumor subtypes and stratify patients [32,33]. Notably, although alternative splicing dysregulation has also been reported in Ewing sarcoma [24,[34][35][36][37][38], limited information is available regarding the RBPs responsible for this process and their possible association with prognosis. An important regulator of RNA metabolism with strong implications in Ewing sarcoma malignancy is the DNA/RNA helicase DHX9, which interacts with the oncogene EWS-FLI1 and promotes its transcriptional activity.…”

“…In Ewing sarcoma, we previously documented that the inhibition of the mTOR/AKT/PI3K pathway sets in motion an hnRNPM-dependent alternative splicing program that limits the therapeutic efficacy of these pharmacologic inhibitors [24]. On this basis, we indicated hnRNPM as a new potential therapeutic target to counteract Ewing sarcoma malignancy [24]. Notably, our transcriptomic analysis upon inhibition of the mTOR/AKT/PI3K pathway documented the upregulation of both DHX9 and hnRNPM, and downregulation of DHX9 exon 6A [24], suggesting a direct regulation of DHX9 alternative splicing by hnRNPM.…”

“…Cross-linked and immunoprecipitation (CLIP) assays were performed as previously described [24]. In brief, TC-71 cells were irradiated once with 400 mJ/cm 2 in a Stratalinker 2400 at 254 nm.…”

The splicing factor hnRNP M is a critical regulator of innate immune gene expression in macrophages

“…One of the proteins that was significantly differentially phosphorylated in each of these datasets was hnRNP M. HnRNP M is a splicing factor and RBP that has been repeatedly implicated in cancer metastasis [22][23][24][25] and muscle differentiation 26 . It has also been identified as a component of a large splicing regulatory complex containing Rbfox which controls alternative splicing in the brain 27 .…”