Mutation profile

EGFR-mutant lung cancers (LC) serve as the prototype oncogene-driven cancer and mechanisms of resistance to EGFR therapy are well-established. Selective pressure of EGFR inhibitors can induce lineage plasticity to escape oncogene-dependence. While we have shown EGFR/TP53/RB1-mutations are necessary but insufficient to induce small cell transformation, no other genomic signatures and transcriptomic/epigenomic effectors that induce transformation have been identified.

Using parallel exome sequencing, bulk RNA sequencing, and bisulfite sequencing, we will characterize the biomarkers of transformation in EGFR/TP53/RB1-mutant LC and mixed adenocarcinoma/squamous cell tumors. We will also utilize single-cell RNA sequencing to identify the transcriptional changes associated with transformation and to identify distinct cell types within a heterogenous tumor. We will reconstruct a phenotypic landscape to identify subclones in a transitional state between histologies. The information obtained from these studies will nominate novel biomarkers of lineage plasticity that will be validated and proposed for study in clinical trials.

The current standard of care for ALK rearranged non-small cell lung cancer (NSCLC) is targeted therapy with ALK tyrosine kinase inhibitors (TKIs) which bind within the kinase domain of ALK. Due to the development of ALK resistance mutations or bypass activation of parallel or downstream signal pathways, patients inevitably relapse. Even with the introduction of second or third generation TKIs, median overall survival has been estimated to be 6.8 years . Recent advances in immunotherapy have demonstrated progress in lung cancer treatment. However, a majority of ALK positive NSCLC patients have yet to realize the benefits from checkpoint inhibitors or CAR-T cell therapies due to primary resistance or toxicity. Therefore, the development of new treatments for ALK-rearranged NSCLC is an area of critical need. We believe that an effective assault on ALK-driven tumors requires the implementation of creative strategies that allow us to expand our arsenal of drugs to target ALK beyond its kinase domain. To meet this challenge, we have developed and employed cysteine druggability mapping (CDM), a method to define the landscape of inhibitor-protein interactions in a cancer cell. CDM identifies cysteines that can bind to covalent drug-like molecules which serve as early prototypes on which to base cancer drug development programs.
Cysteine-reactive covalent inhibitors have already emerged as an effective approach to targeting protein kinases. Osimertinib, a third-generation epidermal growth factor receptor (EGFR) TKI covalent inhibitor, overcomes EGFR T790M resistance mutations following treatment with EGFR TKI, demonstrating significant improvements over standard EGFR TKIs when used as first line therapy. Importantly, targeting cysteines with covalent inhibitors massively expands the number of proteins which are targetable with small molecule inhibitors, including many targets previously considered undruggable.

We will use ALK fusion positive cancer cell lines, together with tumor tissues from patients who developed resistance mechanisms, to generate druggable cysteine profiles by CDM. Druggable proteins specific to ALK fusion positive samples, including EML4-ALK itself, will be prioritized in our screening for protein degraders from a library of over 2,500 advanced cysteine reactive compounds. After screening, we will assess the specificity of the compound hits and validate their anti-tumor activity using ALK fusion positive cell lines and patient derived ALK TKI-resistant cell lines. We consider the use of the novel, cutting-edge cysteine targeting technology to screen for potent therapeutic compounds as a valuable proposition to improve management of patients with ALK positive lung cancer, with the goal of transforming this cancer into a chronic condition.

With the development of increasingly potent and selective ALK tyrosine kinase inhibitors (TKIs), resistance is commonly mediated by ALK-independent mechanisms, such as bypass pathway activation. As bypass signaling dependencies vary across ALK-rearranged (ALK+) lung cancers, a multipronged approach is needed to broadly suppress potential bypass signals. We have identified MET activation in 20% of ALK+ tumors that are resistant to next-generation ALK TKIs. In preclinical studies, dual ALK/MET blockade re-sensitized MET-amplified ALK+ tumors to lorlatinib. MEK and SHP2 have recently been identified as important effector proteins downstream of several bypass signaling pathways. In ALK+ models harboring diverse bypass signals, pairing an ALK TKI with either a MEK or SHP2 inhibitor demonstrated broad anti-proliferative activity. Based on these findings, we will conduct a clinical trial to evaluate lorlatinib in combination with a MET, MEK, or SHP2 inhibitor for patients with ALK+ lung cancers with ALK-independent resistance. Serial tumor biopsies will be analyzed to characterize molecular determinants of response to combination therapy and identify expression changes induced by treatment. This innovative multi-arm trial will also assess whether dynamics of genetic alterations in plasma can be used to guide intensification of treatment from monotherapy to early combination therapy for patients who have not yet developed resistance to ALK-directed therapy. Collectively, the studies in this grant strive to develop diverse strategies for overcoming ALK-independent resistance, a heterogeneous molecular entity that undermines efficacy of current ALK therapeutics.

Metastatic non-small cell lung cancer (mNSCLC) is the leading cause of cancer-related death in the United States. Most patients with mNSCLC are treated with systemic agents, which prolong survival and improve symptoms, but are typically not curative. Although recent advancements in immune checkpoint inhibitors have revolutionized the management of mNSCLC lacking targetable driver mutations, only a fraction of mNSCLC patients benefit from immunotherapy. Emerging evidence suggests that ablative radiotherapy can augment immunotherapy responses in metastatic disease; however, strategies to optimize the synergy between local radiation and the immune system are not well defined. We are conducting a randomized phase I/II clinical trial designed to evaluate the safety and efficacy of combination immune checkpoint blockade (ipilimumab + nivolumab) plus multisite ablative radiotherapy as a first-line treatment for patients with mNSCLC. As a secondary analysis of the phase I trial, we propose to investigate biomarkers associated with treatment response. In Aim 1, we propose to compare the diagnostic accuracy of established clinical biomarkers, including PD-L1 immunohistochemistry (IHC), tumor mutational burden, gene expression profiles, and multiplex IHC in predicting response to combination ablative radiotherapy and immunotherapy. In Aim 2, we propose to characterize global transcriptional and genomic changes induced by radiation when given prior to or concurrently with immunotherapy and evaluate whether these changes are associated with treatment response. In addition, in Aim 3, we propose to validate a novel biomarker derived from alternative splicing as a predictor of radio-immunotherapy response. By elucidating the immunomodulatory properties of radiotherapy, we may be able to improve treatment responses and long-term survival in patients with mNSCLC.

The majority of lung cancer patients present with advanced disease that is largely incurable. Efforts to improve the early detection of this disease are critical to decrease lung cancer-related mortality. Low-dose computed tomography (CT) screening in high risk patients has demonstrated reduction in lung cancer related mortality through diagnosis of earlier stage disease at the cost of high false-positive rates and the frequent need for invasive diagnostic testing when indeterminate lung nodules are identified. Detecting tumor specific somatic mutations and/or epigenetic signatures in circulating tumor DNA (ctDNA) represents an attractive non-invasive method for identifying early stage lung cancer. However, to realize this promise for lung cancer screening, where ctDNA from small tumors is miniscule, the development of sensitive, specific, and economical ctDNA based assays is critical. 
The bacterial innate immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and their associated endonucleases (CRISPR-Cas) are specifically adapted to recognize foreign nucleic acids. The recent identification and diagnostic adaptation of certain classes of CRISPR-Cas systems, such as CRISPR-Cas12a and -Cas13a, offer unique platforms on which to develop sensitive, specific, and economical assays for the detection of ctDNA. The goal of the current project is to develop CRISPR-Cas12a and -Cas13a based ctDNA assays that can sensitively detect somatic cancer mutations and aberrant promoter hypermethylation often found in lung cancer patients and to validate these assays in plasma samples of patients with early stage NSCLC and in discriminating between benign and malignant nodules identified by low-dose CT screening studies. 

One of the challenges for early detection and prevention of lung squamous cell carcinoma is the lack of understanding of the molecular and cellular changes that cause initiation and progression of this disease. Airway premalignant lesions are the precursors of lung squamous cell carcinoma and can be detected and monitored by autofluorescence bronchoscopy. However, many of these lesions will regress without clinical intervention, and only a subset will progress to invasive carcinoma. The goal of this study is to characterize the landscape of somatic mutations in airway premalignant lesions and to develop an early detection biomarker using targeted DNA sequencing that can predict risk of lesion progression. Ultra-deep whole-exome sequencing will be performed on a previously collected cohort of airway premalignant lesions from high-risk smokers that have been sampled over time, characterized histologically, and classified into those that are stable, regress, or progress. Significantly mutated genes, recurrent copy number changes, and mutational signatures will be identified, and a determination will be made whether they are associated with lesion histology or progression or regression status. Finally, the findings from this study will be combined with the driver genes from The Cancer Genome Atlas (TCGA) to build a targeted DNA sequencing panel. This panel will serve as a rapid and relatively inexpensive method for monitoring premalignant lesions from patients over time and can be used in a clinical trial to assess the ability of autofluorescence bronchoscopy in combination with molecular profiling to stratify patients and serve as a companion diagnostic in chemoprevention trials.

Circulating tumor cells (CTCs) are cancer cells that break away from either the primary tumor or metastatic sites. Increasingly, CTCs have been used as a readily accessible source of tumor cells (“liquid biopsy”). However, shortcomings in current CTC technologies limit downstream analyses to assessment of a selected set of nucleic acid markers. Genome and transcriptome-wide analyses of CTCs have been hampered by the low purity of CTC isolates. The complex and protracted nature of CTC capture protocols further impedes analysis.

Dr. Kulkarni and his group have developed a CTC capture technology known as Vortex Chip, which allows for rapid isolation of highly purified CTCs. In addition to standard immunofluorescence, they have generated proof-of-principle evidence that their capture technology allows for analysis of genetic material. Moreover, because the Vortex Chip isolates CTCs based on their size, their chip does not require antibody-based capture that can potentially bias isolation of CTC subpopulations. Serial CTC analyses performed in the context of treatment changes can characterize the mutational landscape of tumors as they evolve in response to therapy and thereby identify molecular predictors and drivers of treatment resistance and sensitivity to novel anti-tumor therapies.

Their project has two goals: 1) validate use of CTCs as biomarker in lung cancer and 2) explore use of CTC analysis to obtain genetic information about the tumor. They hypothesize that the unique microfluidic characteristics of lung CTCs will drive rapid isolation of pure CTC populations for analysis to characterize heterogeneity and profile therapeutic responsiveness.