Adenocarcinoma

Lung cancer is the leading cause of cancer related death among women and men and the second most diagnosed cancer in the world, causing 1.8 million deaths annually. Surgical resection of the primary tumor is the standard of care whenever possible. However, 90% of lung cancer deaths are caused by metastases months-to-years after surgery. Current screening tools such as high-resolution Computed Tomography (CT) and Positron Emission Tomography (PET) can only identify tumors that have reached a detectable size, that often correlates with advanced disease. However, this technology carries side problems, including the false positive rate that led to follow-up tests and invasive procedures that confers additional risks to patients.  

Therapeutic strategies targeting specific driver mutations have emerged in recent years for the treatment of metastatic non-small cell lung cancer and have improved survival, yet the initial identification of these molecular alterations depends on biopsies of metastatic lesions after the resection of the primary tumor. For biopsy, the metastatic lesion must be large enough to be detectable by clinical imaging tools, and this large lesion indicates a relatively advanced stage of disease. Biopsy of these lesions is often difficult from both location and patient-safety perspective. An emerging option is a blood draw or liquid biopsy to obtain circulation tumor DNA (ctDNA) for mutation analyses, yet the ctDNA is not abundant in circulation until relatively large metastases have already formed.  

Therefore, novel, efficient, and affordable approaches are needed for disease surveillance. We propose a high sensitive, minimally-invasive and efficient cell capture device for easy isolation of tumor cells at early stages of disease, whose analysis can identify targeted therapeutics that can be applied while the disease burden and heterogeneity are low. Furthermore, this technology would increase the coverage of the eligible population and reduce the clinical costs by using histopathology analysis of the sentinel implant. This device is a porous biomaterial scaffold that, when implanted, is vascularized, and infiltrated by immune cells and subsequently cancer cells, acting as a synthetic sentinel niche that provides information about the disease development and progression. Our previous studies in breast and pancreatic cancer models have demonstrated that tumor cells are detectable early after tumor initiation and during the progression of the disease. A comparative analysis of scaffolds and ctDNA showed that the implant captured and therefore detected cancer cells before positive detection of ctDNA.  

We will determine the genomic and transcriptomic prolife of inoculated lung cancer cells in PDX’s murine model at early time points. We anticipate that the tumor cells captured by the scaffolds will recapitulate the genotype of the parental PDX. The scaffold safety will be investigated in a Phase I clinical study of metastatic lung cancer patients implanted with our scaffold, with biopsy and subsequent removal to analyze captured cells. Sequencing analysis will be applied to captured cancer cells to assess their genomic and transcriptomic profile. We foresee the use of these bioengineered implants for surveillance in high-risk lung cancer patients, with analysis of the collected cells employed to identify personalized therapeutic strategies to improve patient outcome.

EGFR mutations render lung cancer tumors resistant to immunotherapy via checkpoint blockade. Fortunately, the development of EGFR tyrosine kinase inhibitors (EGFRi) such as Osimertinib has provided impressive clinical responses in a majority of patients. However, many of these patients eventually recur. Studies by our group and others have identified some of the mechanisms associated with resistance to EGFRi. Although EGFRi eliminate the overwhelming proportion of tumor cells, a limited number of cells persist and have thus been termed DTPCs (drug-tolerant persister cells). Importantly, it is these DTPCs which eventually become resistant and are to blame for patient recurrence, highlighting the importance of eradicating these cells before they become fully resistant. Resistance to EGFR TKIs can emerge via two main mechanisms: oncogene-dependent and independent. While oncogene-dependent mechanisms include accumulation of additional mutations in EGFR such as T790M and C797S, oncogene-independent mechanisms include epithelial to mesenchymal transition (EMT) and small cell transformation. Despite their lack of responsiveness to EGFRi at this juncture, each of these mechanisms offers a unique opportunity to leverage cellular immunotherapy to eradicate EGFR mutant DTPCs before they become resistant, thereby improving clinical outcomes. Over the last few years, we have built a library of T cell receptors (TCRs) which confer the ability to recognize epitopes derived from common EGFR mutations including L858R, T790M, and C797S, as well as from the tumor-associated antigen (TAA) FOXM1 which is overexpressed in tumors, and upregulated upon resistance to EGFRi through EMT and small cell transformation. Therefore, we hypothesize that engineering T cells with receptors targeting these EGFR- and FOXM1-derived antigens allows the destruction of DTPCs following treatment with EGFRi, thereby preventing the emergence of resistance. We propose the following specific aims: Aim 1. Assess the impact of drug-tolerant persister cells on CD8 T cell phenotype and function. In this aim, we will identify immune suppressive mechanisms deployed by DTPCs to inhibit T cell response and function. Aim 2. Evaluate the potential of T cell engineering to eliminate DTPCs. In this aim, we will test the ability of TCR-engineered T cells recognizing epitopes derived from EGFR(L858R) and EGFR(T790M) and from the FOXM1 TAA to eradicate DTPCs in vitro and in vivo. Overall, our proposal aims to demonstrate for the first time that TCR engineering can be used in combination with EGFRi to eradicate EGFR-mutant DTPCs. As a result, this work lays the foundation for use of TCR-engineered T cells in this treatment setting. Most importantly, MD Anderson is currently running a clinical trial employing TCR engineering for the treatment of lung cancer with Alaunos Therapeutics which could provide a clear pathway towards the clinic.

The development of tyrosine kinase inhibitors (TKIs) revolutionized treatment of advanced/metastatic non-small-cell lung cancer with EGFR mutations. Osimertinib is one of several third-generation EGFR TKIs and shows improvement in progression-free survival and overall survival compared to first- and second-generation EGFR TKIs. However, intrinsic or acquired resistance to osimertinib emerges in essentially all patients, which prevents a cure for EGFR-mutated lung cancer. Therefore, it is necessary to develop better strategies to improve clinical outcomes. Recent evidence suggests that drug-tolerant persister (DTP) populations of cancer cells, which survive and adapt to TKIs during early phases of therapy, play an important role in the emergence of resistance to targeted therapies. Such DTP cells give rise to resistant cells via diverse mechanisms and contribute to spatial and temporal heterogeneity of tumors. Therefore, targeting DTP cells may be an efficient strategy to prevent resistance to EGFR TKIs. Using lung cancer cell line models and clinical specimens, we recently identified CD74 as a novel gene that plays a critical role in the drug-tolerant state. In vitro and in vivo experiments demonstrate that CD74 upregulation signals osimertinib resistance and blocks apoptosis, enabling tumor regrowth. Based on preliminary evidence, we hypothesize that CD74 increases tumor cell survival in the presence of TKIs and that elimination of CD74 positive cells by CD74-antibody-drug conjugates (ADCs) may suppress the emergence of DTP cells, which underlie relapse. In this proposal, we will test this hypothesis by: 1) confirming that CD74-positive cells are tolerant to EGFR TKIs; 2) investigating the mechanisms by which osimertinib induces CD74 expression; and 3) evaluating a combination of CD74-ADCs and osimertinib to prevent the emergence of acquired resistance. We will address these questions using cell culture systems and animal models. These experiments should unveil the mechanisms underlying resistance to EGFR-TKIs and the emergence of DTP cells, and provide preclinical evidence that a combination of CD74-ADC and EGFR TKIs is a rational and efficacious treatment strategy for EGFR-mutated lung cancer.

Lung cancer is the leading cause of cancer in the U.S., with over 238,000 new cases and more than 125,000 deaths expected in  2023. Early detection is crucial for improving prognosis, and low-dose computed tomography (LDCT) scans are recommended for high-risk populations. However, LDCT screening has limitations, including a significant false positive rate and low compliance. New approaches are therefore needed to enhance early detection in high-risk individuals. Liquid biopsy assays that can detect circulating tumor DNA (ctDNA) represent a promising alternative early detection approach. Our group has developed three state-of-the-art liquid biopsy techniques that each have the potential to detect lung cancer noninvasively, including a somatic mutation approach (Lung-CLiP), a methylation-based approach (meCAPP-Seq), and a fragmentomic-based approach (EPIC-Seq). We hypothesize that combining mutation-, methylation-, and fragmentomic-based ctDNA analysis will maximize sensitivity for detecting early stage lung cancers. Our proposal includes three specific aims. In the first aim, we will compare the clinical sensitivity and specificity of meCAPP- Seq, EPIC-Seq, and Lung-CLiP for early lung cancer detection. In the second aim, we will determine the analytical limits of detection for the three methods, which is important for future regulatory considerations. In the third aim, we will develop an integrated liquid biopsy assay that combines meCAPP-Seq, EPIC-Seq, and/or Lung-CLiP. We will test if combining two or all three assays can improve performance over the best- performing single assay. The deliverable of this project will be the identification of the combination of liquid biopsy assays that achieves the best performance for early lung cancer detection. If successful, our work has the potential to significantly improve early lung cancer detection which would lead to improved outcomes for lung cancer patients.

Among mRNA modifications, N6- Methyladenosine (m6A) is the most abundant, catalyzed by a complex that includes methyltransferase-like 3 (METTL3). Although the function of m6A in cancer remains understudied, Gregory’s lab provided functional evidence of METTL3 as an oncogenic protein, promoting translation of oncogenes to promote lung cancer cell growth, survival, and invasion in human cancer cell lines. Lung adenocarcinoma (LUAD) is the most common form of lung cancer, with a survival rate of about 18%, highlighting the need for more effective treatments and understanding LUAD biology.  

Our hypothesis: METTL3 is a novel oncogenic factor in lung cancer and plays a key role in tumor initiation and progression. I will test the role of METTL3 in a LUAD mouse model, and in tumor organoid culture system.

Aim 1: Determine the effects of METTL3 manipulation on tumor progression in a LUAD mouse model. Lentivirus will be used to knockdown or overexpress METTL3 simultaneously with activation of oncogenic Kras in the LUAD mouse model for comparison of tumor number, size and histopathological features.  

Aim 2: Test the effects of METTL3 on early-stage lung cancer by manipulating METTL3 expression in a tumor organoid model of cancer initiation. Alveolar epithelial (AT2) cells from Kras mice will be infected with Cre and shMETTL3 or METTL3 cDNA to initiate early-stage lung cancer in organoid cultures, to assess organoid size, proliferation and differentiation features.  

This proposal could establish METTL3 as a therapeutic target for lung cancer and pave the way for understanding links between epitranscriptome and cancer biology.

While immunotherapies in cancer have allowed for major advances in the field, most patients with advanced or metastatic solid tumors do not achieve durable responses. While CAR-T cell therapy and bispecific antibody therapies which utilize T-cells as their effector have shown promise and durable responses in hematologic malignancies such as DLBCL and multiple myeloma, the promise of similar responses in solid tumors remains an unmet goal. TROP2, a cell surface protein normally expressed during fetal development and at low levels in some normal adult tissue, is over expressed on a variety of solid tumors including NSCLC, and has emerged as an ideal target. TROP2 is the target of the recently FDA approved ADC sacitizumab-govitecan for breast cancer as well as for patients with locally advanced or metastatic urothelial cancer and is currently being tested in phase II/III trials in NSCLC. However, response in patients and in animal models are not durable, even when targeting minimal residual disease (MRD; defined as the residual disease after effective genotype directed systemic treatment). We propose utilizing TROP2 directed CAR-T to target EGFRm NSCLC at MRD, which we hypothesize when compared to TROP2 directed ADC will demonstrate a more durable response. Our preliminary data support the high activity of TROP2 directed CAR-T against EGFRm NSCLC including in the MRD state after osimertinib treatment. We expect these findings to translate to approaches utilizing CAR-T cellular therapy to target the MRD state after targeted therapy.

Checkpoint immunotherapy has advanced the treatment paradigm for non-small cell lung cancer (NSCLC), but only some patients respond to treatment and many patients experience immune related adverse events (irAE). Understanding the distinct and overlapping mechanisms regulating anti-tumor immunity and autoimmunity due to checkpoint immunotherapy is critical for developing more effective and less toxic therapies. A subset of CD8+ T cells expressing killer cell immunoglobulin-like receptors (KIR) have recently been shown to suppress autoimmunity without impairing anti-viral immunity by eliminating autoreactive T cells. We reason that KIR+ CD8+ T cells may also play an important role in regulating autoimmunity arising from checkpoint immunotherapy. Furthermore, we reason that KIR+ CD8+ T cells may also regulate the activity of tumor-reactive T cells to varying degrees, since tumor-reactive T cells are a specialized type of autoreactive T cells. This study examines the hypothesis that KIR+ CD8+ T cells dampen autoimmunity and anti-tumor immunity during checkpoint immunotherapy treatment. To test this hypothesis, we will first interrogate the activity of KIR+ CD8+ T cells during the development of irAE after immune checkpoint blockade (specific aim 1). Next, we will evaluate the impact of KIR+ CD8+ T cells on anti-tumor immunity in NSCLC (specific aim 2). This study may shed light on the biological mechanisms regulating autoimmunity and anti-tumor immunity in lung cancer patients undergoing checkpoint immunotherapy. Insights gained from this study may pave the way toward clinical strategies for improving immunotherapy efficacy while minimizing irAEs.

Lung cancer is the most common cause of cancer-related mortality in North America. Although the average age at diagnosis is 70, thousands of new lung cancers are diagnosed each year in patients under 45 years over 45 years old (median age of diagnosis = 67). The work proposed here will further define this difference in a larger cohort, and address whether the identified variants or genes affect tumor evolution and treatment outcomes. The aims of our study are to identify a population of young lung cancer patients with elevated germline risk, and to investigate the mechanism of the germline-somatic interaction in young lung cancer. The young lung cancer population also provides a lens through which to better understand oncogene-driven lung cancer that occurs in non-smokers – increasingly important as global smoking rates decline.old, most of whom have never smoked. Given that younger patients develop lung cancer decades earlier and without known environmental insults, we hypothesized that underlying germline genetic variation predisposes to developing lung cancer at a young age. Here, we assembled a cohort of over 350 patients diagnosed with lung cancer at age 45 or younger and performed germline whole-genome sequencing (WGS). Preliminary data from 243 patients with median diagnosis age of 37 showed at least 30% of patients carry clinically actionable pathogenic germline alterations in cancer-associated genes, at rates exceeding those seen in many other cancer types – with key age-specific differences when compared to ancestry-matched lung cancer patients from The Cancer Genome Atlas diagnosed over 45 years old (median age of diagnosis = 67). The work proposed here will further define this difference in a larger cohort, and address whether the identified variants or genes affect tumor evolution and treatment outcomes. The aims of our study are to identify a population of young lung cancer patients with elevated germline risk, and to investigate the mechanism of the germline-somatic interaction in young lung cancer. The young lung cancer population also provides a lens through which to better understand oncogene-driven lung cancer that occurs in non-smokers – increasingly important as global smoking rates decline.

Lorlatinib is currently the only approved ALK TKI (tyrosine kinase inhibitor) for patients with ALK NSCLC that have progressed on prior lines of therapy. The clinical benefit of lorlatinib is modest in previously-treated patients with a median PFS of 7 months (1). Mechanisms of lorlatinib resistance are diverse and include novel ALK mutations, compound ALK mutations, and activation of bypass pathways including AXL. Gilteritinib is a dual inhibitor of FLT3/AXL that is approved for relapsed/refractory FLT3+ acute myeloid leukemia (AML). Pre-clinical work using ALK fusion positive cell lines (including those containing compound mutations), patient-derived tumor cells, and in vivo mouse studies all demonstrate the anti-tumor activity of gilteritinib specifically against lorlatinib-resistant ALK NSCLC.

We will recruit patients with ALK NSCLC with progression on lorlatinib and collect patient-derived tumor samples. In addition to surgical, biopsy, or effusion fluid samples, plasma will be collected for isolation of circulating tumor DNA and circulating tumor cells. Whole exome sequencing (WES) will be used to identify molecular alterations that define resistance mechanisms. Ex vivo drug testing will be conducted using our existing platform and will include ALK inhibitors and inhibitors of pathways implicated in resistance such as AXL, MET, mTOR, and MAPK. Results of ex vivo drug testing will be correlated with findings from WES.

Simultaneously, we will open a phase I clinical trial to assess the safety and preliminary efficacy of gilteritinib in patients with lorlatinib-resistant ALK NSCLC. Crucially, clinical endpoints will be correlated with sequencing data and ex vivo drug testing results.

About 5-7% of non-small cell lung cancers (NSCLCs) have a rearrangement of the anaplastic lymphoma kinase (ALK) gene. ALK-rearranged NSCLC is typically treated with ALK tyrosine kinase inhibitors (TKIs), but no effective immunotherapies are available for refractory or relapsed tumors. In previous work, we have shown that ALK is an immunogenic oncoprotein that induces specific T cell responses with potent killing activity against ALK+ tumor cells in the lung and in distant metastatic sites, such as the brain. Thus, the lack of efficacy of standard immunotherapy in ALK+ NSCLC can be bypassed by the development of tools to induce ALK-specific T cell responses. Our central hypothesis is that the generation of T cells engineered to express TCRs (TCR-T cells) that selectively target ALK+ NSCLC will be a powerful tool to completely eradicate ALK+ lung cancer cells.

In preliminary experiments, we have shown the feasibility of the cloning of ALK-specific TCRs against an ALK peptide expressed by a mouse model of ALK+ lung cancer. In Aim 1, we will test the potency, the safety and the in vivo efficacy of ALK-specific TCR-T cells obtained by T cells engineered to express TCRs against ALK in a mouse model of ALK+ lung cancer. TCR-T cells will be tested alone or in combination with ALK TKI. Their efficacy will be compared to natural anti-ALK T cells induced by vaccination in mice. In Aim 2, we will discover and study the activity of ALK-specific TCRs against human ALK peptides. We will leverage ALK peptides we previously identified to be expressed by human ALK+ NSCLC. The killing activity of these human specific TCR-T cells will be tested against human ALK+ NSCLC in vitro and in vivo, alone or in combination with ALK TKIs. We expect to identify potent and specific TCRs as leading candidates for a TCR-T cell-based clinical trial for patients with TKI-resistant ALK+ NSCLC.