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

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. HnRNPMmotifswere 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

Durable response to palbociclib and letrozole in ovarian cancer with CDKN2A loss.

Alterations of the Retinoblastoma (Rb) pathway are frequent in ovarian cancer, typically resulting from CDKN2A down-regulation, CCNE1 amplification, CCND1/2 amplification, and RB1 loss. However, bi-allelic CDKN2A mutation or homozygous deletion is a very rare event, concerning less than 5% of patients.Initial trials with palbociclib in serous ovarian cancer have shown very modest benefit in unselected patient populations, thus underlining the need for a biomarker predicting response. We report the case of a heavily pre-treated patient with a serous ovarian tumor harboring a homozygous deletion of the CDKN2A gene that derived significant, prolonged clinical benefit from palbociclib, a CDK4/6 oral inhibitor, with letrozole. Treatment with palbociclib and letrozole started on February 2018, with an ongoing response after 12 months.In conclusion, homozygous CDKN2A deletion is rare and could be used to predict response to CDK4/6 inhibitors in association with other genomic features. We encourage further trials in this direction

The promoter-associated noncoding RNA PNCCCND1_B assembles a protein–RNA complex to regulate cyclin D1 transcription in Ewing sarcoma

Most Ewing sarcomas are characterized by the in-frame chromosomal translocation t(11;22) generating the EWS–FLI1 oncogene. EWS–FLI1 protein interacts with the RNA helicase DHX9 and affects transcription and processing of genes involved in neoplastic transformation, including CCND1 (the cyclin D1 gene), which contributes to cell-cycle dysregulation in cancer. In this study, we found that CCND1 expression is significantly higher in patients with Ewing sarcoma compared with other sarcomas and that the pncCCND1_B RNA, a previously uncharacterized CCND1 promoter-associated noncoding (pnc) transcript, is expressed in Ewing sarcoma cells. PncCCND1_B interacted with the RNA-binding protein Sam68 and repressed CCND1 expression. Notably, knockdown of Sam68 affected pncCCND1_B subcellular localization and cyclin D1 expression. Pharmacologic impairment of DHX9/EWS–FLI1 interaction promoted RNA-dependent association of Sam68 with DHX9 and recruitment of Sam68 to the CCND1 promoter, thus repressing it. Conversely, mitogenic stimulation of Ewing sarcoma cells with IGF1 impaired Sam68/DHX9 interaction and positively regulated CCND1 expression. These studies uncover a fine-tuned modulation of the proto-oncogene CCND1 in Ewing sarcoma cells via alternative complexes formed by DHX9 with either EWS–FLI1 or pncCCND1_B-Sam68