PTEN is one of the most frequently inactivated tumor suppressor genes across all cancer types. The loss of PTEN activates PI3K/AKT, which inhibits GSK3?, thereby stabilizing Myc and contributing to oncogenesis. Myc recruits histone acetyltransferases to increase chromatin accessibility of target genes involved in both cell proliferation and apoptosis. Among these histone acetyltransferases, the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex preferentially acetylates histone H3 lysine 9 and histone H4 lysine 16 to activate gene expression. A pan-cancer analysis of mutually exclusive gene inactivation patterns identified a previously uncharacterized long non-coding RNA (lncRNA) as synthetic essential in the context of PTEN deficient cancer. Preliminary studies suggest that this lncRNA inhibits SAGA-mediated histone acetylation, thereby inhibiting Myc transactivation of target genes. PTEN and lncRNA double-knockout SF-763 glioma cells showed Myc pathway enrichment, impaired cell viability, and pronounced aneuploidy, which was not observed in wild-type or single knockout cell lines. We hypothesize that inhibition of SAGA-mediated acetylation by this lncRNA inhibits Myc transactivation of pro-apoptotic target genes and Myc-driven endoreduplication, thereby promoting cancer survival. This proposal will investigate the potential of targeting the poorly studied non-coding genome for cancer treatment, advance our knowledge of the role of histone acetylation on cancer genomic stability (widely targeted using genotoxic drugs and radiotherapy), and describe a novel mechanism for the regulation of Myc's dual functions in proliferation and apoptosis. We will verify lncRNA expression in cell lines from various cancer types and in clinical samples to validate the pan-cancer relevance and translational potential of this study, respectively. Annexin V and caspase 3/7 assays will be used to assess the hypothesis of Myc-driven apoptosis. Endoreduplication will also be probed using BrdU incorporation into colcemid-arrested cells. Chromatin isolation by RNA purification and chromatin immunoprecipitation sequencing will be used to demonstrate how the lncRNA inhibits histone acetylation by SAGA. To assess the role of the lncRNA in vivo, we will functionally validate its putative mouse homolog and generate a genetically engineered mouse model to characterize its effects on tumor development. The lncRNA knockout allele will be bred into a Qki;Pten;Trp53 glioblastoma mouse model to assess the effects of lncRNA suppression in the context of Pten deletion. The training plan will address gaps in the applicant's research and clinical abilities, ensuring that he can successfully complete the proposed work and preparing him for the next stage of his career. The training will be completed in Dr. Ronald DePinho's lab at MD Anderson, where the applicant will have access to the resources, facilities, and, most importantly, colleagues that will nurture his continuing development and growth into a physician-scientist.
Exploring the non-coding genome will help open up this vast frontier for the development of novel therapies that could far outreach what biomedical science has been able to accomplish up until now by only targeting the protein-coding genome. In this proposal, we will characterize a novel cancer-associated long non-coding RNA with predicted functions in regulating histone acetylation, Myc signaling, and genome stability. The successful completion of this project will highlight one example of how investigating the non-coding genome can lead to alternative strategies for targeting pathways that are widely considered to be ?undruggable,? such as Myc.
