The Role of RNA Splicing in Non-Small Cell Lung Cancer

About This Grant

Significance to VA: Lung cancer (LC) is the leading cause of cancer death for US veterans (USvets). Non-small cell LC (NSCLC) represents the majority of LCs with a poor 5-yr survival rate (~23%). NSCLC patients in the VA Health Care System are increasing as USvets often acquire tobacco addiction during military service fostering a large percentage of high-risk current & former smokers. The lung cancer incidence is also higher in USvets with lower survival rates, and LC is linked to service-connected exposure to carcinogens. Current treatment options for NSCLC are palliative, but have recently evolved with the use of immune checkpoint inhibitors (ICIs). Unfortunately, the effectiveness of ICIs in NSCLC remains modest with underlying resistance mechanisms elusive. Our research will define these resistance mechanisms and identify potential molecular targets & strategies to target both the NSCLC tumor & enhance ICI efficacy to produce a more durable outcome for USvets. Innovation & Impact: STK11 mutations (mtSTK11) are common in NSCLC and associated with resistance to immune checkpoint inhibitors (ICIs). Our data connected mtSTK11 to a novel dysregulation of caspase 9 (C9) alternative RNA splicing (ARS). Specifically, mtSTK11 NSCLC preferentially expressed C9b, which induces tumorigenesis, and a tumor immunosuppressive microenvironment (TIME) that supports ICI resistance. Genetic removal of C9b sensitized mtSTK11 NSCLC to ICI therapy in a NSCLC mouse model, thus highlighting the potential utility of modulating dysregulated ARS as a therapeutic. Human mtSTK11 NSCLC tumors also presented with the dysregulation of additional ARS events, which our data also support roles in NSCLC tumorigenesis & TIMEs. These data support the hypothesis that dysregulated ARS in mtSTK11 NSCLC modulates tumorigenesis and induces a TIME that promotes ICI resistance. Specific Aims (SAs): To interrogate our innovative hypothesis, we are proposing three specific aims: SA1: Determine the role of ARS events linked to the mtSTK11 oncogenotype in NSCLC tumorigenesis; SA2: Determine the role of specific STK11-regulated ARS events in ICI responses; & SA3: Determine the signaling mechanisms driving dysregulated ARS in mtSTK11 NSCLC. Methodologies: Unbiased “splicomic” analysis in human NSCLC will identify ARS events dysregulated in the mtSTK11 oncogenotype. Once validated for STK11/LKB1-regulation (e.g., by qRT-PCR), their roles in cancer biology will be determined using cells models (e.g., clonogenic potential) and mtSTK11 NSCLC mouse models (e.g., tumorigenesis & ICI resistance). The mechanism of action for specific ARS events will be determined by interrogation of the cell composition of the TIME using multiplex immunofluorescent histology & Aurora flow cytometry. State-of-the-art molecular manipulations (e.g., CRISPR), novel molecular “tools”, and complementary biophysical studies will be employed to modulate specific ARS events in our cellular & in vivo models and determine mechanistic function. Important ARS events and mechanisms will be interrogated in human NSCLC tumors for translational outcomes (e.g., survival, ICI resistance). Path to translation/implementation: Validation of our hypothesis would lead to the identification of new chemical entities (NCEs) that specifically block cancer-related ARS to foster a new generation of therapeutics for NSCLC. These NCEs would have limited toxic side effects: the “Achilles Heel” for some global ARS inhibitors. Additionally, our laboratory has shown that ceramide induction in NSCLC cells will reverse the dysregulated C9 ARS and sensitize cells to standard of care NSCLC treatments. A new ceramide-induction therapy, ceramide nanoliposomes, recently completed a phase I clinical trial (NCT02834611) for solid tumors (e.g., NSCLC) with an excellent safety profile. Thus, our proposed studies will build the molecular, mechanistic, & pre-clinical foundation for the development of new targeted ARS therapeutics and the clinical implementation of combination therapies using both ARS inhibitors & ceramide-induction therapy with ICIs in NSCLC to improve outcomes.