Funded Projects

Small cell lung cancer (SCLC) is a common, aggressive, and exceptionally lethal malignancy that is usually metastatic at the time of diagnosis. Newly diagnosed SCLC is remarkably sensitive to chemotherapy, with response rates of over 60% to cisplatin and etoposide even in patients with extensive-stage disease. These chemotherapy responses are often both rapid and deep, but are never curative: extensive-stage disease recurs universally, in most cases within a few months after completion of first-line therapy. Recurrent SCLC is remarkably refractory to treatment. The median overall survival of patients with extensive-stage SCLC is about nine months from the time of initial diagnosis, and the 5-year survival is less than 1%. Defining the basis of acquired chemotherapy resistance in SCLC could have clear and important ramifications for clinical management of this disease. Targeting the key determinants of acquired chemoresistance could be integrated into first-line treatment, and/or could drive rational targeted therapeutic studies for recurrent disease. We now have in hand multiple technical approaches that could comprehensively define the genomic and epigenetic changes that drive acquired resistance in SCLC. In this award application, we propose two parallel approaches to analyzing acquired resistance in SCLC: analysis of primary samples and patient-derived xenografts before and after acquired resistance.

Prognosis for non-small cell lung cancer (NSCLC) is substantially better in early-stage as opposed to late-stage disease. If high-risk individuals could be effectively identified, they could significantly benefit from intensive surveillance, early detection, and prevention strategies. While smoking is a primary risk factor, only about 15% of smokers develop the disease. Consistent with a genetic predisposition, some patients have a family history of the disease and are affected at a young age, although they have never smoked. Unfortunately, NSCLC predisposing gene mutations are poorly characterized. We aim to identify novel NSCLC germline risk gene mutations. Our team members have previously identified novel risk gene mutations for several familial cancers. We have assembled a large clinically and molecularly well-characterized NSCLC case-control cohort of never-smokers, performed whole-exome sequencing (WES) on familial and early onset NSCLC probands, identified promising candidate risk genes, and externally validated selected samples. We follow a resource-conscious approach by focusing on a founder population, Ashkenazi Jews (AJ), to decrease genetic heterogeneity and to increase the allele frequencies of candidate recurrent risk mutations. We will perform WES on additional familial and early onset NSCLC probands to identify recurrently mutated NSCLC genes and cross-validate the genes using whole-genome sequencing on matching tumors. The gene candidates identified will be validated using a targeted sequencing approach in a larger non-AJ cohort. These are anticipated to provide new biological insights, serve as future chemoprevention targets, and be used in the development of a genetic diagnostic test to identify high-risk individuals.

The release of small amounts of cell-free DNA into the bloodstream from dying tumor cells has been well documented in patients with various malignancies. Driven by improvements in assay technologies, such circulating tumor DNA (ctDNA) is beginning to show excellent promise as a non-invasive cancer biomarker. Because unique somatic mutations can serve as telltale marks to distinguish tumor-derived DNA in plasma, the cancer specificity of ctDNA is expected to be very high, making it an attractive candidate as a biomarker for cancer screening. Because many somatic mutations in non-small cell lung cancer (NSCLC) are found clustered with high frequency within a small set of genes, detection of ctDNA in such patients is technically more tractable. We have created a highly multiplexed, ultrasensitive assay that uses next-generation sequencing techniques to quantify low-abundance mutant DNA fragments in plasma against a large excess of background wild-type DNA. Our assay can simultaneously evaluate mutations in 40 different hotspots in up to 96 patient samples, using novel error suppression methods to optimize detection sensitivity. We have shown that the assay is capable of measuring very small amounts of mutant ctDNA in a limited set of patients with early-stage disease or with small, localized recurrences. We now aim to test the clinical performance of the assay on a larger scale. We propose to perform a case-control study comparing patients with early-stage NSCLC to individuals with a heavy smoking history or individuals having benign pulmonary nodules.

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.

Advanced (stage IV) lung adenocarcinoma is an incurable disease. Treatment mostly consists of chemotherapy, which provides a consistent but small survival benefit. Standard therapy includes a platinum-doublet (pemetrexed or paclitaxel) often in combination with bevacizumab, followed by maintenance bevacizumab or pemetrexed. Programmed death 1 (PD-1) is a T-cell surface receptor that when activated, delivers a negative signal to T cells. This feedback mechanism dampens immune responses when no longer needed, preventing damaging autoimmune reactions. Tumors can co-opt this T-cell control mechanism to escape surveillance and/or rejection by the immune system. Thus, reversing this tumor action can result in antitumor activity worthy of clinical evaluation. Recently it has been demonstrated that inhibition of the PD-1 receptor with an anti-PD-1 monoclonal antibody (nivolumab) has significant clinical activity in lung cancer with an ORR of 26%. As remarkable as this may be, given the heavily pretreated population of lung cancer patients studied, this also indicates that 74% of patients did not respond and remained resistant to this approach. Therefore, going forward it is important to develop combination therapies to improve the efficacy of this approach, and to discover resistance mechanisms that may allow for better patient (and tumor) selection. One potential class of agents to be used in combination with anti-PD-1 is the adenosine A2AR antagonists. Given that A2AR on T cells within the tumor microenvironment contributes to immune escape and therapeutic resistance, the addition of PBF-509 is expected to improve the efficacy of nivolumab.

Melanoma has the highest propensity to metastasize to the brain once the disease has spread, while non-small cell lung cancer (NSCLC) has the highest incidence of brain metastases among all cancers. Approximately 50,000 patients develop brain metastases from these diseases every year. Patients with brain metastases have exceedingly limited therapeutic options since they are typically excluded from clinical trials. Recently a number of new systemic immune therapies, such as nivolumab (BMS-936558) and lambrolizumab (MK-3475), two inhibitors of PD-1 (an immune inhibitory molecule), have led to dramatic responses in metastatic melanoma (MM) and NSCLC. However, these therapies have not been studied in patients with untreated brain metastases.  Little is known about immune cells in the brain, and whether the immune system can be stimulated to reject cancer cells. Moreover, little is known about what tumor characteristics are associated with response to MK-3475 in brain metastases. This proposal includes innovative correlative biomarker studies incorporated into a clinical trial uniquely designed to study the activity of MK-3475 in patients with limited, small brain metastases in MM and NSCLC. This drug carries the hope to provide prolonged responses similar to responses seen in other metastatic sites, and might enable patients to avoid radiation therapy, and improve their outcome. As part of this clinical trial, we will collect brain and other metastatic specimens from patients enrolled and study the expression of immune inhibitory molecules utilizing a novel quantitative assay and algorithms that we have developed in preliminary work. These studies will facilitate selection of patients who are more likely to respond to this class of drugs.

Small cell lung carcinoma (SCLC) remains one of the most lethal human cancers. More than 25,000 patients are diagnosed with SCLC every year in the United States. Unfortunately, most patients usually survive only 6-12 months after diagnosis. Except for the combination of radiation therapy and chemotherapy, there has been no novel therapeutic approach implemented in the past 30 years. Despite a large number of clinical trials, no targeted therapy has been approved for SCLC patients, whose survival rate hovers around 5%. Previous attempts to identify therapeutic targets in SCLC patients have mostly focused on signaling networks within the tumor cells. Here we propose to test a novel immunotherapeutic approach to promote the anti-cancer activity of macrophages. Specifically, we found that SCLC cells express high levels of the CD47 antigen, a marker of self that inhibits the phagocytic activity of macrophages. Based on previous studies from the laboratories of Dr. Weissman and Dr. Garcia at Stanford, we propose to block the interaction between CD47 and SIRPa, an inhibitory receptor expressed on macrophages, to stimulate the engulfment of SCLC cells by macrophages and their eradication. Furthermore, we will identify SCLC-specific antigens and combine CD47-blocking therapies with these antibodies to enhance the recruitment and the activity of macrophages against SCLC cells. Clinical trials testing the CD47-blocking strategies are being initiated in patients with blood cancer and some solid tumors. If successful, our pre-clinical experiments may lead to the rapid implementation of this novel anti-cancer immunotherapy strategy in SCLC patients.

Cancer-associated fibroblasts (CAFs) support the growth of lung cancer cells in vivo by secretion of soluble factors that stimulate the growth of tumor cells. One such soluble factor is CLCF1. Functional studies in our laboratory identified an important role for CLCF1–CNTFR signaling in promoting growth of NSCLCs. NSCLC cell lines as well as patient-derived xenografts were found to express CNTFR, and knock-down of CNTFR leads to decreased proliferation. To test the use of CNTFR blockade as an anti-cancer therapy, we developed a soluble version of CNTFR (i.e., a CNTFR “receptor decoy”) that can be expressed from an adenoviral vector (Ad-sCNTFR-Fc). Preliminary experiments indicate that Ad-sCNTFR-Fc can block proliferation of a NSCLC cell line in vitro. Our proposed studies build on an established translational research program that includes intensive efforts to harvest and analyze primary tumor samples and CAFs directly from patients. In Aim 1, we will test the therapeutic efficacy of Ad-sCNTFR-Fc in a panel of patient-derived xenografts (PDX) as well as established cell lines. In Aim 2, to increase therapeutic efficacy, we will use protein engineering to develop a high-affinity sCNTFR that can more effectively block CLCF1-CNTFR interactions, and will evaluate this therapeutic candidate in PDX models. In Aim 3, we will establish the utility of using plasma CLCF1 as a biomarker for CLCF1 production in CAFs isolated directly from patient tumors. Parallel analysis of CAF and plasma CLCF1 will establish if CLCF1 in plasma correlates with CLCF1 in tumors and is thus a useful biomarker.

A significant fraction of NSCLC have mutant KRAS which predicts poor response to cytotoxic therapy and portends a poor prognosis. Despite the discovery of the first mutation in the KRAS oncogene in NSCLC over two decades ago, there are no current therapies targeting this critical oncogene. In this proposal we explore a novel and exciting therapeutic strategy to target KRAS-mutant NSCLC through the combination of Hsp90 and mTOR inhibition. We have preclinical evidence suggesting that the combination of ganetespib and an mTOR inhibitor may be an effective therapeutic strategy for patients with KRAS- mutant NSCLC. In Aim 1, we will define resistance mechanisms to single-agent ganetespib in KRAS- mutant NSCLC. The identification of resistance mechanisms to single-agent ganetespib will reveal novel pathways that we can then target in combination with an Hsp90 inhibitor. In Aim 2, we will test the hypothesis that activation of the mTOR pathway through alterations in STK11 or PI3KCA mutations will predict response to the combination. Finally, in Aim 3 we will translate our studies to the clinic in a phase I/II trial of the combination of ganetespib and an mTOR inhibitor in patients with advanced KRAS-mutant lung adenocarcinoma. Importantly, we will have correlative studies looking at response in subpopulations of KRAS-mutant tumors that are hyperactive for the mTOR pathway (STK11, PIK3CA mutations). This translational approach will allow us to identify biomarkers that we can use to effectively target this combination to the patients with greatest chance to benefit from this therapy.

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.

Lung cancer remains the leading cause of cancer death in the world for both men and women. Prevention studies have largely been ineffective. A chemoprevention trial using the prostacyclin analogue iloprost demonstrated improvement in endobronchial histology in former smokers. An ongoing trial is investigating lung cancer chemoprevention with pioglitazone, which decreases lung cancer risk in diabetics. The effects of iloprost and pioglitazone are mediated by PPARγ and its downstream targets. We hypothesize that response to iloprost and pioglitazone depends on expression of PPARγ and that measures of prostacyclin and PPARγ activity will correlate with histology. We will measure a PPARγ expression in biopsy tissues to determine if expression can predict response to iloprost. We will assess methylation and acetylation in tissues with loss of PPARγ expression, which could identify a potential role for demethylation or deacetylation agents in creating a response to chemoprevention. We will measure activity of iloprost and pioglitazone through QPCR of downstream targets. Previously generated tissues from mouse models of lung cancer chemoprevention will be used to correlate markers with human samples. miRNA have the potential to be important markers but there is little information on miRNA associated with lung cancer chemoprevention. We identify potential miRNA markers of iloprost and pioglitazone activity in dysplastic cell lines treated with iloprost or pioglitazone and in tissues from mouse models of chemoprevention. Data from this project will inform future clinical trials using iloprost and pioglitazone, improve targeted lung cancer chemoprevention, and identify relevant miRNA markers for future chemoprevention studies.

Screening smokers with CT can detect lung cancer at relatively early stages, and thus reduce mortality. However, CT screening for lung cancer has a poor specificity, often resulting in unnecessary treatments to smokers who have benign diseases. The objective of this proposal is to develop genomic- and non-coding RNA (ncRNA)-based biomarkers in sputum that can complement CT for lung cancer early detection in smokers.  Proposed aim 1 is to define sputum ncRNA signatures of early-stage lung cancer using next-generation sequencing (NGS). Proposed aim 2 is to optimize a panel of sputum ncRNA biomarkers with CT for the early detection of lung cancer. Proposed aim 3 is to integrate our previously identified genomic biomarkers and the newly optimized panel of ncRNA biomarkers with CT for the early detection of lung cancer in independent case-control series. Future use of the biomarkers in clinical practice for screening smokers for lung cancer will increase CT’s specificity, and hence reduce harmful and unnecessary treatments to many smokers who have benign diseases. Integrating the biomarkers with CT could assist making decisions for the management of abnormal findings in the lungs and enabling effective treatments to be immediately initiated for lung cancer. The study will translate the strengths of novel ncRNA profiling data developed from NGS into clinical settings by using a simple and cost-effective approach, and thus bridge the gap between basic research and patient care.

Small cell lung cancer (SCLC) exhibits aggressive clinical behavior and poor outcome and response to therapy. Moreover, there are no effective strategies for early detection of SCLC. Therefore, there is an urgent need to identify new strategies and markers for SCLC detection. We recently characterized the molecular airway field cancerization (FC) in non-small cell lung carcinoma (NSCLC) patients and identified airway profiles that varied spatially (n=1165) and with time (n=1395) following surgery. Moreover, we characterized the adjacent airway FC in NSCLC (n=1606 genes), a subset of which (n=299) significantly distinguished airways of smokers with lung cancer from those of healthy smokers and another (n=208) separated airways of different NSCLC subtypes. Although we previously showed that SCLCs and adjacent airways are characterized by higher level of allelic losses compared to those of NSCLC cases, the global airway FC in SCLC and its relevance to detection has not been explored. We hypothesize that the molecular airway FC in SCLC is different compared to that in NSCLC or benign lung disease and comprises biomarkers specific for early detection of SCLC. We will 1) perform global expression profiling of the airway FC and tumors or nodules in patients with SCLC, NSCLC, and benign disease for identification of airway markers specific to SCLC and 2) test identified airway mRNA markers for SCLC detection in bronchial and nasal compartments in patients with suspect lung cancer. We anticipate that our proposal, due to its unique resources and approaches, will identify novel markers for SCLC detection.

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.

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.