The human genome produces many thousands of long noncoding RNAs (lncRNAs) ? transcripts >200 nucleotides long with little evidence of protein coding potential. It is now clear that lncRNAs can have critical biological functions and roles in human disease including cancer. Because lncRNAs are particularly cell and disease specific, they are attractive as therapeutic targets. However, our understanding of lncRNAs in primary brain tumors is still very limited. Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor. Despite surgery, chemotherapy and radiation treatment, the median survival after diagnosis of GBM is only 14-16 months. Our long-term goal is to develop highly specific and effective new therapies for the treatment of primary brain tumors including GBM. In pursuit of this goal, our immediate objective is to identify and pursue specific lncRNAs as therapeutic targets in human gliomas. CRISPR interference (CRISPRi) is a readily scalable and highly specific technology for transcriptional regulation, and we have recently implemented this as a method for lncRNA knockdown in human GBM cells. In Preliminary Studies, we have developed CRISPRi for large-scale, genome-wide screening of lncRNA function in human brain tumor cells. In our pilot screen of ~1300 GBM-expressed lncRNAs, we identified 27 that regulate the propagation of GBM cells in culture. CRISPRi screen ?hits? could be individually validated with both CRISPRi and antisense oligonucleotide (ASO) mediated knockdown. Knockdown of one particular lncRNA ? LINC00909 ? strongly reduced glioma cell propagation in culture and in a xenograft mouse model. LINC00909 overexpression is very specific to GBM tumors, and higher LINC00909 expression predicts shorter survival of patients with mesenchymal GBM. Interestingly, in the developing human brain, LINC00909 was enriched in normal neural stem cells, and expression analysis suggests LINC00909 expression in GBM cancer stem cells (CSCs). Given these Preliminary Studies, our central hypothesis is that specific lncRNAs such as LINC00909 are uniquely required for GBM tumor growth but not the viability of normal adult human brain cells. In this proposal, our first Aim is to determine the role of LINC00909 in models of human GBM both in vitro and in vivo. Whether LINC00909 has essential function in normal glia and neurons will also be tested. For our second Aim, we will use CRISPRi to more comprehensively identify lncRNAs that regulate GBM tumor growth. In addition to further testing our central hypothesis, this work will provide an important data resource (lncRNA hit identification) and novel tools (large-scale CRISPRi screening libraries and methods) ? both of which we will make available for distribution. In addition to our expertise with lncRNAs and Preliminary Studies, our ongoing local collaborations with Dr. Jonathan Weissman (CRISPRi), Dr. Aaron Diaz (bioinformatics) and Dr. Nalin Gupta (primary human brain tumor models) support the feasibility of this work. By accomplishing these studies, we will lay novel, important groundwork for the development of lncRNAs as targets for glioma therapy.
Glioblastoma multiforme (GBM) is the most common primary brain tumor. Despite surgery, chemotherapy and radiation, the median survival of patients with GBM is only approximately 14 months. The human genome encodes tens of thousands of long noncoding RNA (lncRNA) molecules, and the abnormal production of specific lncRNAs may underlie some of the most devastating human diseases including cancer. This work will identify specific lncRNAs that may be targeted for the development of novel GBM therapies.
