Radiotherapy

Whole genome pooled retroviral shRNA screens were performed in NSCLC cell lines A549 and NCI-H460. Out of nearly 18,000 genes, the top hit whose knockdown most reproducibly showed cytoxicity was PSMA1, a core subunit of the 20S proteasome. The combination of radiotherapy and PSMA1 knockdown significantly enhanced tumor control in a mouse xenograft model. We observed that proteasome inhibition results in increases in nuclear 1κBa, which in turn decreases NF-κB binding to the promoters of Fanconi Anemia/Homologous Recombination (HR) genes including FANCD2 and BRCA1. This reduces the expression and subsequent availability of these DNA repair proteins for recruitment to double strand breaks (DSBs) induced by radiation. These mechanistic studies provided the basis for the proposed hypothesis-driven translational research.

The central hypothesis is that high levels of HR-mediated repair of radiation-induced DNA DSBs are associated with poor tumor control, which may be improved with proteasome inhibitors. First, CT-guided fractionated radiotherapy ± bortezomib will be tested in diverse genetically engineered mouse models of NSCLC to identify mutations and other biomarkers associated with responses to radiation and/or proteasome inhibitors. Immunofluorescence and immunohistochemistry against proteins, identified in prior mechanistic studies, as well as gene expression profiling (GEP), will be performed. Then, the biomarkers will be examined in archived specimens from patients treated with radiotherapy, to assess associations with outcomes. DASL will be used to perform GEP studies using formalin-fixed, paraffin-embedded specimens. The goal will be to identify pathway-based expression signatures that predict poor outcomes with radiotherapy and improved outcomes with addition of proteasome inhibitors.

Platinum-based concurrent chemoradiation (CRT) remains the standard treatment for patients with locally advanced, stage III non-small cell lung cancer (NSCLC). Outcomes for this large subgroup remain dismal, and no progress has been made in this area in the last three decades. Novel treatment options and predictive biomarkers to improve upon the outcome and reduce the toxicity of our current treatment paradigms are sorely needed. Our overall hypothesis is that the identification of acquired resistance pathways and synergistic targets for platinum-radiation therapy will lead to improved treatments and patient selection. We will pursue in vitro and in vivo xenograft studies to assess the synergistic role of PARP inhibition and ERCC1 modulation in the setting of CRT and will correlate these findings with a biomarker study using human tumor specimens. We will further validate YAP as a promising actionable target and will assess the interactions of concurrent chemoradiation with YAP blockade based on our preliminary data of synergy. Then, we will pursue an unbiased, functional shRNA genetic screen to identify further essential treatment resistance pathways, and carefully selected targets will be validated through a series of biochemical and functional assessment steps. The predictive value of biomarkers will be tested in retrospective cohorts of patients treated with cisplatin-radiation for locally advanced non-small cell lung cancer enrolled in our lung cancer database. Last, our unique tissue bank of pre- and post-neoadjuvant treatment specimens will be used for the identification of novel resistance markers based on modulation of expression upon neoadjuvant therapy, and functional validation of resistance biomarkers and novel treatment candidates will be pursued. These studies should provide the foundation for biomarker-driven translational and clinical studies in this area of major unmet clinical need.