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Targeted therapies such as erlotinib or gefitinib for EGFR-mutated lung cancer and crizotinib for ALK-translocated lung cancer can yield remarkable responses and significant clinical benefit. Other mutations in genes such as K-RAS, BRAF, and PI3K, among others, are also being actively investigated as targets of new drugs in clinical trials. However, a substantial number of patients (~40%) have no mutations when tested with a panel of known oncogenic mutations. The aim of this research proposal is to identify novel somatic genomic alterations in lung cancer, using an existing tumor bank of lung cancer cases with known clinical and tumor genetic information. We will focus on those lung cancers that are known to be wildtype on SNaPshot and ALK FISH (fluorescence in situ hybridization) (i.e., negative for over 50 tested oncogenic mutations), and perform both deep sequencing of exomes and a comprehensive search for translocations involving kinases. We believe that this effort will identify novel tumor genomic changes that can be targets for therapy.

Lung cancer remains the leading cause of cancer-related death due to the limited ability to detect the disease at an early and potentially curable stage. Low-dose computerized tomography (LDCT) screening of high-risk patients improves mortality. However, the high proportion of false positive tests, the majority of which are pulmonary nodules, has limited widespread adoption of this life-saving screening modality. Since screen-detected pulmonary nodules contribute to the overall cost of screening and potential morbidity due to the need for further diagnostic testing, there is an urgent need for accurate tools to stratify patients with screen-detected nodules for routine follow-up or additional intervention. Our data suggests that, similar to the smoking-associated field effect, the lung cancer-associated airway field of injury can be measured less invasively using gene-expression profiling of the nasal epithelium. Because early-stage lung cancers detected by CT screening may have distinct biologic characteristics compared to those diagnosed in patients evaluated for clinical symptoms, we propose to develop an accurate biomarker integrating expression profiling of genes and microRNA, clinical variables, and nodule characteristics in order to distinguish early lung malignancies from benign pulmonary nodules. The immediate clinical application of this test will be in the evaluation of patients with LDCT-detected lung nodules, and will allow clinicians to more accurately stratify patients to or from further diagnostic intervention. Ultimately, this test may also have utility for further stratifying lung cancer risk in patients eligible for screening prior to undergoing LDCT.

Recently, the National Lung Screening Trial reported a 20% reduction in lung cancer mortality and lung cancer chemoprevention trials targeting the arachidonic acid pathway have demonstrated decreases in lung cancer associate markers .These studies highlight possibilities for future reductions in lung cancer mortality through early detection and chemoprevention. Our group has previously identified smoking- and lung cancer-specific gene expression alterations in cytologically normal airway epithelial cells that can serve s a clinically relevant biomarker for the early detection of lung cancer. We propose to extend our studies of the field of injury by profiling  these cells from high-risk smokers with preinvasive lesions (dysplasia) and matched controls without lesions by RNA-Seq. We will characterize the molecular alterations (coding, noncoding, and novel RNA expression and alterative splicing) associated with the field of injury in premalignancy and identify whether these differences are similar in both current and former smokers. Next, we will develop molecular signatures that predict subsequent progression or regression of premalignant lesions as well as changes that occur as a result of progression or regression. Collectively, these signatures will aid in the stratification of high-risk smokers for chemoprevention trials, serve as intermediate markers of efficacy in these trials, and identify new targets for chemoprevention. Finally, we will explore whether these molecular signatures re associated with occult lung cancer or future lung cancer development in independent cohorts as common alterations may indicate early events in lung carcinogenesis and have additional utility as biomarkers in the early detection of lung cancer.

Cancer therapy needs to be better personalized to individual patients, each of whom carries a unique spectrum of mutations, inherited and/or acquired. One of the best cases for this is lung cancer, where molecular subgrouping patients and targeting their mutations has shown promise. While many groups have searched fervently for additional tumor-acquired mutations that are associated with drug sensitivity and resistance, few have emerged since the discovery of the initial mutations, such as EGFR. In contrast, we have applied our expertise in microRNAs (miRNAs), their biologic function and interaction with oncogenes, and as a result have discovered a new class of inherited, germ-line mutations that appear to have strong potential as companion diagnostics. These mutations are in a relatively unexplored region of the genome, the 3’ untranslated regions (UTRs), once thought to be junk DNA. The first such discovered biomarker, the “KRAS-variant,” is an inherited 3’UTR mutation in KRAS, which affects microRNA (miRNA) binding and KRAS pathway expression, as well as tumor biology. The KRAS-variant was first shown to be a genetic marker for an increased risk of developing non-small cell lung cancer (NSCLC). In addition, our data indicate that this variant represents an inherited form of an activated KRAS allele, one of the most important human oncogenes. Several groups have found that the KRAS-variant acts as a biomarker of poor outcomes across multiple cancers, including head and neck cancer, ovarian cancer, and colon cancer. We as well as others have shown that this biomarker is predictive of response to specific cancer treatments, and is not just prognostic of aggressive tumor biology. For example, the KRAS-variant predicts resistance to cisplatin in ovarian and head and neck cancer, resistance to cetuximab in combination with irinotecan chemotherapy, but sensitivity to cetuximab when delivered alone. Furthermore, our preliminary data in this proposal indicate that the KRAS-variant is a powerful companion diagnostic predicting poor response to specific agents, and excellent response to others. These findings, combined with evidence that this allele of KRAS can be targeted using siRNA and miRNA strategies that we have pioneered, leads us to believe that this KRAS-3’UTR mutation is a powerful companion diagnostic in lung cancer, and ultimately may be a critical new target for cancer therapy.

The genotype-directed use of EGFR TKIs in EGFR-mutant lung cancer patients has fundamentally changed the face of the disease with improved response to therapy, preserved quality of life, and prolonged progression-free survival. However, tumors that initially respond become resistant after ~ 1 year (i.e., acquired resistance, AR). The biology of AR is incompletely understood and is largely inferred from pre-clinical models. Our group at Mass General implemented a wide-scale program to perform repeat biopsies on EGFR-mutant patients with AR to first-generation EGFR inhibitors (gefitinib, erlotinib). This translational research paradigm has been instrumental in gaining insight into AR and has led to novel observations such as PIK3CA and BRAF mutations as mechanisms of AR and the abundant frequency of phenotypic changes at the time of AR like small cell lung cancer and epithelial-to-mesenchymal transitions. The repeat biopsy results have directly helped our patients as well, by informing choices about the most relevant clinical trials or alternative treatments. Currently, a new cohort of EGFR TKIs is entering the clinic: 2^nd-generation irreversible pan-HER inhibitors and 3^rd-generation T790M-specific inhibitors. Little is known about how AR develops to these newer therapies or how the biology of EGFR-mutant cancers evolves over time after exposure to sequential EGFR blockers.

Our proposal aims to discover the mechanisms of AR to next-generation EGFR TKIs and to understand the evolution of EGFR-mutant cancers as they face the selective pressure of sequential types of EGFR-directed therapies. To accomplish this we will collaborate across institutions to amass a large number of pre-treatment and post-treatment paired patient biopsies which we will interrogate via allele-specific genotyping as well as whole genome sequencing to define mechanisms of AR. In addition we will utilize an innovative technique of taking tumor tissue directly from a biopsy and deriving patient-specific in vitro and in vivo models in the laboratory. Expansion of these models will yield a larger supply of AR cancer material to aid descriptive and functional research about the biology of AR. In addition, establishment of pre-treatment patient-specific models will allow us to verify whether the mechanism of AR derived in the lab through ex-vivo therapy mirrors the eventual clinical mode of AR.

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.