Tumor microenvironment

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.

Despite high tumor response rates, patients treated with osimertinib inevitably develop disease progression. Mechanisms of both intrinsic, adaptive, and acquired drug resistance remain incompletely understood on a genomic and proteomic level. Our overall objective is to find new targeted treatments and drug combinations that can tackle cancer evolution and drug resistance.

We seek to:

1. Understand biological determinants of response prior to therapy (poor prognosis features)
2. Utilize correct combination therapies in the first line to enhance response, based on biological features of subpopulations present at the start
3. Reprogram cells toward a more drug-sensitive state through understanding network plasticity.

More specifically, our aims are:

1. To define the genomic and proteomic characteristics of drug-tolerant persister cells (DTPCs) under dynamic treatment with osimertinib. Our goal is to identify new vulnerabilities in EGFR-mutant tumors which, when therapeutically targeted, can maximize the depth and duration of benefit to first-line osimertinib therapy.
2. To decipher mechanisms of altered cell cycle progression and osimertinib-induced DNA damage that drive tumor progression.

We have assembled a collaborative, multi-disciplinary team of laboratory scientists and clinical investigators combined with cutting edge technologies to assess genetic and proteomic heterogeneity at the single cell level, access to a robust pipeline of patient samples, and strong preliminary data to support our studies. The proposed aims are expected to define the molecular determinants of tumor heterogeneity and to develop rational therapeutic strategies for delaying / overcoming resistance. These synergistic aims are highly relevant to the current landscape of EGFR-mutant lung cancer.

Several tyrosine kinase inhibitors (TKIs) are available for the treatment of ALK+ NSCLC, but resistance to all ALK TKIs (including alectinib, brigatinib, lorlatinib) has been reported and occurs through a variety of mechanisms. Once patients develop resistance to available ALK inhibitors, they are typically treated with cytotoxic chemotherapy rather than immunotherapy since response rates to PD-1 pathway inhibitors in this population are very low. However, our work in mouse models of ALK+ NSCLC has demonstrated that a vaccine generated against the rearranged portion of ALK can both prevent the development of ALK+ tumors and also more effectively treat them once they arise. We have also recently found evidence for anti-ALK immune responses in patients, as a subset of them generate spontaneous ALK autoantibodies.

We aim to translate our preclinical findings to a first-in-human clinical trial. Aim 1 will test the safety and efficacy of a novel ALK vaccine + an amphiphilic CpG toll-like receptor 9 agonist adjuvant, administered in combination with an ALK TKI or PD-1 inhibitor in patients with TKI-resistant ALK-positive NSCLC. In Aim 2, using liquid chromatography data-independent acquisition mass spectrometry, we will directly identify antigenic ALK peptides presented by MHC molecules on the surface of cancer cells from tumor biopsies obtained on trial participants. In Aim 3, we will study pre- and post-vaccination anti-ALK T-cell responses among trial participants by analyzing peripheral leukocytes and intratumoral immune cells. The overarching goal is to develop a vaccine that stimulates patients’ immune cells to recognize and eliminate ALK+ tumors.

Small cell lung cancer (SCLC) is an aggressive, neuroendocrine malignancy that accounts for approximately 15% of lung cancers. Despite recent advances, including the approval of immunotherapy combined with chemotherapy for frontline treatment, the median overall survival for patients with extensive-stage SCLC remains approximately one year. These dismal outcomes are attributable, in part, to the lack of approved biomarkers for SCLC, which, in turn, is due to the dearth of available tissue for molecular analysis. Using transcriptomic data, we have developed a gene signature that defines four distinct molecular subtypes of SCLC – each with unique biology and therapeutic vulnerabilities. These subtypes are driven by the presence (or absence) of three transcription factors (ASCL1 – SCLC-A; NEUROD1 – SCLC-N; POU2F3 – SCLC-P; and a fourth triple-negative subtype “Inflamed” – SCLC-I) that are largely mutually exclusive. Our preliminary data suggests that inter- and intra-tumoral heterogeneity among these subtypes drives response and resistance to standard and novel therapies for SCLC, including classes such as PARP inhibitors (PARPi) and immunotherapy. We propose applying bulk and single-cell molecular approaches to assign and longitudinally monitor subtype among our established cohort of patient-derived xenograft models treated with PARPi, as well as patient tumor biopsies from an investigator-initiated trial assessing a PARPi/immunotherapy combination. Based on our preliminary data, we hypothesize that SCLC-P and SCLC-I will preferentially benefit from PARP inhibitors and immunotherapy, respectively, and that subtype shifts detectable at the single-cell level along with accompanying shifts in the composition of the immune microenvironment, will govern response and resistance.

Statement of the Problem: Cancer develops in a sequenced manner. Patches of lung cells gain the ability to multiply faster than their neighboring normal cells by acquiring mutations. These patches of cells are called “premalignant lesions" (PMLs). Some of these lesions may eventually become cancer. Knowledge about how some PMLs may develop into lung cancer is central to developing tools for Cancer Interception—interrupting the cancer development process and blocking progression to advanced metastatic disease. However, we lack effective lung cancer interception approaches due to our incomplete understanding of the earliest molecular events associated with the development of lung cancer, as well as the challenge in developing personalized tools for early detection and prevention.

Proposed Solution: The Dream Team will use a holistic approach to understand the genetics, immunology, radiological (imaging) profile, and treatment response of patients who show evidence of abnormal lung tissue that puts them at high risk to develop lung cancer. The Team has access to unique groups of patient populations (adenocarcinoma—ADC and squamous cell carcinoma—SCC) with signs of lung cancer risk, and has assembled a collection of blood and tissue specimens from these individuals. The main objective of the Team is to understand how lung cancer develops and test methods that will block the development of cancer. Their three-pronged approach to solve this problem will include:

• Creation of a molecular pre-cancer genome atlas (PCGA) of the lung with the goal of identifying which types of precancerous lung tissue require aggressive treatment and which treatments will block the development of these abnormal lesions to invasive lung cancer.
• Development of two diagnostic tools that can be directly applied in the clinic for simple, yet accurate, detection of early lung cancer: 1) the use of nasal swabs, blood tests, and imaging to confirm whether lung abnormalities found on chest imaging are lung cancer or benign lung disease. 2) blood tests that can identify patients at the earliest stages of lung cancer recurrence, thus enabling their timely and effective intervention with therapies such as those that boost the immune system.
• Development of tests to identify which individuals are most likely to benefit from a number of treatment strategies to intercept lung cancer, including emerging immunotherapies.

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.