Lung cancer is the leading cause of cancer death for veterans and in the world, for both men and women in industrialized countries, accounting for an estimated 28% of all cancer deaths in the United States. Non-small cell lung cancer (NSCLC) represents the majority of these cancers,, and NSCLC has a 5-year survival rate of ~23% with current treatment options palliative. Hence, new therapeutic strategies are needed to impact this disease, and elucidation of new mechanisms controlling the tumorigenicity of NSCLC cells will provide the foundation upon which to build. In this regard, the gene, cyclin dependent kinase inhibitor 2A (CDKN2A), that encodes the tumor suppressors, p16(INK4A) and p14(ARF), is found inactivated or deleted in NSCLC at a high percentage (e.g., ~53% deletion rate). Importantly, our laboratory linked CDKN2A inactivation to dysregulation of the RNA splicing of an uncharacterized, long non-coding RNA (lncRNA) located on Chromosome 19p13.12 (ENSG00000267053). Specifically, exon 3 inclusion/exclusion into the mature RNA transcript was dysregulated in NSCLC cell lines with CDKN2A mutated/deleted, and thus, we now term this lncRNA, Cyclin Dependent Kinase Inhibitor 2A-regulated lncRNA 1 (CyKILR-1). Initial cell signaling studies using small molecule inhibitors also identified the AKT/CDK4 axis as a regulatory pathway upstream of CyKILR-1 RNA splicing linked to CDKN2A inactivation. In human NSCLC, CyKILR-1 splice variant expression correlated with lower patient survival. These findings are congruent with studies showing the linkage of the aberrant expression of non-coding RNAs located at Chromosome 19p13.12 to cancer development. In preliminary studies, specific downregulation of CyKILR-1 splice variants in NSCLC cells induced dramatic decreases in cell survival and anchorage-independent growth. Initial mechanistic studies examining CyKILR-1 Exon 3 revealed a consensus sequence for nuclear localization (TCTAGCTCAGCCC). Fractionation experiments then demonstrated differential localization between the two splice variants with CyKILR-1B (exon 3 excluded) localized to the cytoplasm in contrast to the nuclear localization of CyKILR-1A (exon 3 included). The culmination of these data forms the hypothesis that inactivation of the tumor suppressor encoding gene, CDKN2A, in NSCLC regulates the inclusion of exon 3 producing the CyKILR-1 splice variants, which localize to the cytoplasm and imparts pro-survival phenotypes to NSCLC cells and tumors. To examine this hypothesis in depth, we propose in this application the following independent, but synergistic specific aims (SAs): SA1: To determine the biological relevance of CyKILR-1 RNA splicing in NSCLC; SA2: To determine the regulating mechanism of CyKILR-1 RNA splicing; and SA3: To determine the cell signaling pathways regulated by CyKILR-1 splice variants.
These specific aims are focused on scientific lines of investigation to address the need for new and precise therapies for treating NSCLC, which when combined with newer therapies (e.g., PD- L1/PD-1 interaction blockers; IMFINZI/durvalumab) may produce more durable results and a longer 5 yr survival. For example, the validation of our hypothesis in Aim 1 (i.e., CyKILR-1 splice variants regulate NSCLC cell survival) will show that targeting the this novel lncRNA mechanism is a promising approach to compromise lung cancer development and tumor maintenance for those tumors presenting with a CDKN2A inactivation mutation or deletion. With RNA-based therapies showing efficacy in clinical trials, these mechanisms become enticing targets for new therapeutics with the added benefit of being tailored to specific oncogenotypes.
Aims 2 and 3 synergize with Aim 1 by determining the cellular mechanisms regulating both CyKILR-1 RNA splicing and the downstream signaling pathways modulated by CyKILR-1 splice variants. These findings will then be related back to Aim 1 in regard to specific tumor biological phenotypes, which would allow clinicians to use specific targeted therapies.
Cancer is the second leading cause of death among US veterans, and lung cancer is associated with the greatest mortality in these patients. Non-small cell lung cancers (NSCLC) represent the majority of lung cancers and carry a poor prognosis with a median survival of less than 12 months. Most patients present with unresectable disease, and current treatment options of chemotherapy and radiation are palliative at best. Our laboratory has identified key mechanisms specific to a particular mutation/ inactivation of tumor suppressor genes in NSCLCs, which are plausible and highly desired targets for the development of new and targeted anti-cancer therapeutics (precision medicine). This grant application explores these cellular mechanisms in depth with the goal of developing new therapeutics to combat NSCLC, and thereby, aid US Veterans.
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