EPCO-11. IN VIVO FUNCTIONAL GENOMIC SCREEN IDENTIFIES WISP1 AS AN OVEREXPRESSED DRIVER OF GLIOBLASTOMA

There is a tremendous need to identify new genetic drivers of glioblastoma which can serve as potential therapeutic targets. In order to find new drivers, we leveraged genomic datasets to conduct a context specific in vivo functional genomic screen of overexpressed and/or amplified genes in GBM. We identified WISP1, a secreted extracellular matrix protein, to be an overexpressed driver in GBM. Overexpression of WISP1 was able to drive tumor growth in various in vivo models. Knockdown of WISP1 with shRNAs resulted in reduced colony formation in vitro and reduced tumor growth in vivo. Rescue experiments validated that the shRNAs were on target. Functional characterization of the protein revealed that the TSP module is necessary for the phenotype. Intriguingly, overexpression of WISP1 lacking the signal peptide module for secretion resulted in a strong phenotype. Co-culture and conditioned medium experiments further supported a secretion independent intracellular role of WISP1 in GBM. Though WISP1 is a secreted protein we have found some basal localization in the cytosol. Overall, we have revealed WISP1 to be a driver of GBM with possible therapeutic potential as a target. This study has expanded our understanding of WISP1 by supporting a new role as a driver in GBM which can function in a non-canonical manner in the cytosol. Overall, we have revealed WISP1 to be a driver of GBM with possible therapeutic potential as a target.

N6-methyladenosine mRNA marking promotes selective translation of regulons required for human erythropoiesis

Many of the regulatory features governing erythrocyte specification, maturation, and associated disorders remain enigmatic. To identify new regulators of erythropoiesis, we utilize a functional genomic screen for genes affecting expression of the erythroid marker CD235a/GYPA. Among validating hits are genes coding for the N6-methyladenosine (m6A) mRNA methyltransferase (MTase) complex, including, METTL14, METTL3, and WTAP. We demonstrate that m6A MTase activity promotes erythroid gene expression programs through selective translation of ~300 m6A marked mRNAs, including those coding for SETD histone methyltransferases, ribosomal components, and polyA RNA binding proteins. Remarkably, loss of m6A marks results in dramatic loss of H3K4me3 marks across key erythroid-specific KLF1 transcriptional targets (e.g., Heme biosynthesis genes). Further, each m6A MTase subunit and a subset of their mRNAs targets are required for human erythroid specification in primary bone-marrow derived progenitors. Thus, m6A mRNA marks promote the translation of a network of genes required for human erythropoiesis.

N6-methyladenosine mRNA marking promotes selective translation of regulons required for human erythropoiesis

Many of the regulatory features governing erythrocyte specification, maturation, and associated disorders remain enigmatic. To identify new regulators of erythropoiesis, we performed a functional genomic screen for genes affecting expression of the erythroid marker CD235a/GYPA. Among validating hits were genes coding for the N6-methyladenosine (m6A) mRNA methyltransferase (MTase) complex, including,METTL14,METTL3, andWTAP. We found that m6A MTase activity promotes erythroid gene expression programs and lineage specification through selective translation of >200 m6A marked mRNAs, including those coding for SETD methyltransferase, ribosome, and polyA RNA binding proteins. Remarkably, loss of m6A marks resulted in dramatic loss of H3K4me3 across key erythroid-specific KLF1 transcriptional targets (e.g., Heme biosynthesis genes). Further, each m6A MTase subunit and a subset of their mRNAs targets, includingBRD7,CXXC1,PABPC1,PABPC4,STK40, andTADA2B, were required for erythroid specification. Thus, m6A mRNA marks promote the translation of a network of genes required for human erythropoiesis.