Acquired resistance

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.

KRAS mutations are the most common activating mutation in lung cancer and are identified in 25-30% of cases of lung adenocarcinoma. The clinical activity of molecularly targeted therapy in KRAS mutant lung cancer has been limited, as most prior approaches focused on downstream targets of the MAPK pathway, an approach which was largely ineffective. KRAS G12C is the most common subtype of activating KRAS mutation in NSCLC. Identification of a binding pocket of KRAS G12C enabled the development of covalent inhibitors (Ostrem et al. Nature, 2013), which selectively bind to the mutant cysteine and spare wild type or other mutant KRAS proteins (hereafter referred to as G12Ci). These drugs bind only to GDP-bound (i.e. inactive) KRAS G12C and trap the oncoprotein in its inactive state by preventing nucleotide exchange (Lito et al. Science, 2016). Of this new class of agents, two drugs (MRTX849 and AMG510) have demonstrated efficacy in early phase clinical trials, representing a remarkable breakthrough in the management of KRAS G12C mutant lung cancer. Acknowledging the limited number of patients treated thus far, response rates of 40-50% have been reported, demonstrating clinical activity far superior to prior molecularly targeted approaches for the management of KRAS mutant lung cancer. Despite this major advance it is clear that resistance to therapy, both primary and acquired, will remain a challenge and may in part be due to upstream receptor tyrosine kinase (RTK) signaling. Understanding the mechanisms of resistance to this new class of therapy is of paramount importance to improve the efficacy and durability of response, and for identifying rational combinatorial therapies for future clinical trials.

Several ALK tyrosine kinase inhibitors (TKIs) are available for the treatment of ALK-rearranged non-small cell lung cancer (NSCLC), but resistance to all ALK TKIs (including crizotinib, ceritinib, alectinib, brigatinib, and 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-positive NSCLC has demonstrated that a vaccine generated against the rearranged portion of ALK can both prevent the development of ALK-positive tumors and also more effectively treat them once they arise. In addition, we have recently found evidence of an anti-ALK immune response in patients, as a subset of them generate spontaneous ALK autoantibodies.

To more deeply characterize anti-ALK immunologic responses in patients, we propose (Aim 1) to determine if the presence of anti-ALK antibodies correlates with progression-free and overall survival among patients with ALK-positive NSCLC, and (Aim 2) to directly identify and validate antigenic ALK peptides in different HLA-haplotype backgrounds. Antigenic peptides that are identified through these studies will serve as the basis for a therapeutic ALK peptide vaccine clinical trial, for which funding has been secured independently.

Background: Osimertinib and other “third-generation” EGFR TKIs effectively overcome T790M-mediated resistance in EGFR-mutant lung cancer, but our understanding of acquired resistance in this setting remains limited. Importantly, the intrinsic heterogeneity of resistant cancers leads to clinical challenges. We hypothesize that in-depth study of 3rd-generation TKI resistance and the impact of genetic heterogeneity on treatment response will lead to improved therapeutic options and clinical outcomes. Study Design: We will analyze tumor biopsies, ctDNA and autopsy specimens using a variety of techniques such as next-generation sequencing, digital droplet PCR and whole exome sequencing to characterize heterogeneity in resistant cancers. In addition, we will run a prospective clinical trial to test a novel therapeutic combination designed to minimize heterogeneity and delay progression. On-treatment biopsies and ctDNA collection will be used to study how resistance develops. Impact: This project will leverage our broad array of clinical specimens, well-established translational research program and preexisting industry and academic collaborations to characterize the heterogeneity of resistance in EGFR-mutant lung cancer. We hope to illuminate clinical and biologic implications of heterogeneity, with the ultimate goal of identifying more effective therapies for patients.

Immune-targeted agents have generated antitumor responses that are transforming cancer therapeutics in various tumors types including lung cancer. Despite the impressive responses observed, the majority of patients eventually experience therapeutic resistance after an initial response. Considering the increased cost to patients and health systems both financially and in terms of time spent in management of immune-related adverse effects it has become clear that success of immuno-oncology approaches depends on choosing patient populations most likely to benefit. Our proposed research addresses the urgent unmet clinical need to understand the underlying biology of response and develop molecular assays of response and resistance to immune targeted agents. We propose comprehensive genome-wide analyses of tumors and cell-free circulating DNA to examine how cancer genomes change during immune checkpoint blockade. Our approach is focused on discovery of mechanisms of resistance using a multidimensional strategy that combines comprehensive genomic analyses with functional assays of autologous T-cell reactivity. The underlying premise of this proposal is that through our comprehensive molecular analyses, we will identify changes in the genomic landscape of tumors during immune checkpoint blockade that mediate emergence of primary and acquired resistance to these therapies. Unravelling the genomic mechanisms through which cancer adapts to evade anti-tumor immune responses will be critical for future development of tailored cancer immunotherapy strategies. We envision that the results of the proposed research will be used to develop patient-specific immunotherapy approaches in lung cancer and will launch investigator-initiated clinical trials at Hopkins and other institutions.

Small cell lung cancer (SCLC) is among the most aggressive and deadly human cancers. It represents approximately 15% of all lung cancers, and it is responsible for over 30,000 deaths in the US and 200,000 deaths worldwide every year. SCLC responds to treatment but recurs frequently and aggressively, with the majority of patients dying within 12 months of diagnosis. No targeted therapies are yet available for SCLC. New strategies are needed to improve outcomes for patients with SCLC. This project will expand upon preliminary data identifying at least two distinct subpopulations of cells in SCLC cell lines and patient-derived xenografts comprising a neuroendocrine (NE) and mesenchymal-like (ML) set of cell line populations. Dr. Lehman and his team will use an innovative approach using mass cytometry, using rare earth antibody conjugates to antibody label more than 30 proteins simultaneously, including phospho and signaling proteins. This technique will establish at a single-cell resolution tumor heterogeneity and signaling in small cell lung carcinoma in multiple primary human samples as well as in patient-derived xenograft samples before and after chemotherapy treatment. This proposal aims to (1) characterize the signaling of subpopulations in human primary SCLCs at a single-cell resolution, (2) identify changes in tumor heterogeneity and in these subpopulations after chemotherapy treatment and relapse, and (3) disrupt and manipulate developmental signaling/ associated subpopulations (prototypically the HH [Hedgehog] and Notch/DLL3 pathways) to reduce tumor burden and lead to targeted rational combination therapy for SCLC.

An estimated 160,000 patients die each year from non-small cell lung cancer (NSCLC), primarily due to the development of metastatic and treatment-refractory disease. Dr. Byers, Dr. Gibbons, and their group have previously demonstrated that the epithelial-mesenchymal transition (EMT) is a unifying mechanism that drives tumor progression, metastasis, resistance to standard therapies, and immune suppression. Furthermore, their group and others have shown that the receptor tyrosine kinase Axl and PD-L1 are overexpressed on tumor cells in the setting of EMT in NSCLC and that targeting either Axl or PD-L1 results in preclinical efficacy in lung cancer models.

Based on these results, they have initiated the first clinical trials of BGB324 (a first-in-class, selective Axl inhibitor) for lung cancer patients in combination with either erlotinib or chemotherapy. They hypothesize that Axl is a driver of EMT and EMT-associated immune escape. Therefore, in this project they will use pre-clinical cell line and animal models to determine the role of Axl in driving EMT, identify predictive markers of response to Axl inhibition, and investigate the efficacy of combination therapy with an Axl inhibitor and an immune checkpoint inhibitor, anti-PD-L1. The biomarkers identified in their preclinical models will be directly tested in fresh tumor biopsies from their Phase 1/2 clinical trial that combines erlotinib with BGB324.

Although NSCLC is a molecularly heterogeneous disease, EMT is a potential universal mechanism of progression and drug resistance across many molecular subtypes. It is their goal to leverage this finding to develop single agent or combination treatment strategies that can be used across the molecular spectrum.