Abstract NG05: Multi-omic approaches to study the role of plasticity in therapy resistance and metastasis in lung cancer
Authors: Quintanal-Villalonga Alvaro , Chan Joseph M. , Gao Vianne R. , Xie Yubin , Pe’er Dana , Rudin Charles M. 
Published: April 2023

Abstract
Lineage plasticity, the ability of cells to transdifferentiate between committed developmental pathways, has been proposed as a source of intratumoral heterogeneity and as a mechanism for tumor adaptation to stringent environmental conditions. Lineage plasticity is increasingly recognized as a contributor to both drug resistance and metastasis, as recently highlighted by our team (Quintanal-Villalonga et al., Nat Rev Clin Oncol 2020), thus representing a biological phenomenon with high clinical relevance. Leveraging bulk and single-cell multi-omic approaches in clinical specimens, we aimed to perform a comprehensive molecular characterization of lung cancer cellular plasticity in the settings of (1) histological transdifferentiation as a mechanism of resistance to targeted therapy, and (2) disease progression. In lung adenocarcinomas (LUADs), lineage plasticity drives small cell (SCLC) and squamous cell (LUSC) transdifferentiation in the context of acquired resistance to targeted inhibitors. In the EGFR -mutant cancers, transformation to SCLC and to LUSC as a mechanism of acquired TKI resistance has been reported in 14% and 9% of cases, respectively. Transdifferentiated tumors portend poorer prognosis than non-transformed tumors. To date, no preventative therapies for transformation are available, although tumor subsets at high risk of transformation (concomitant TP53/RB1 inactivation in the case of SCLC transdifferentiation) have been identified. Defining molecular mechanisms of histological transformation in lung cancer has been challenging due in part to a paucity of well-annotated pre- and post-transformation clinical samples. We hypothesized that mixed histology tumors (LUAD/SCLC and LUAD/LUSC), containing different histological components and sharing common driver mutations, may represent an intermediate step of transdifferentiation, and an ideal substrate to study lineage plasticity, with both histological components sharing location, time, and treatment influence. By selecting mixed histology specimens amenable for clean microdissection of each histological component (10 LUAD/LUSC and 11 LUAD/SCLC), as well as pre- and post-transdifferentiated samples (N=17), we performed comprehensive genomic, epigenomic, transcriptomic, and protein characterization. Our data supports that histological transdifferentiation from LUAD to LUSC or SCLC tumors is driven by epigenetic remodeling rather than by mutational events, and indicates that transdifferentiated tumors retain epigenomic features of their previous LUAD state. Integrative epigenomic, transcriptomic, and protein analysis revealed divergent biological pathways dysregulated for each histological outcome, such as upregulation of genes involved in Hedgehog and Notch signaling and MYC targets in LUSC-transdifferentiated tumors. Most interestingly, these analyses identified commonly dysregulated pathways in both SCLC- and LUSC-transdifferentiating tumors, including remarkable downregulation of a variety of immune-related pathways and upregulation of genes involved in the PRC2 epigenetic remodeling complex and AKT signaling. To validate drivers of transdifferentiation from our multi-omic data, preclinical in vivo experiments indicated that concurrent activation of AKT and MYC overexpression induced a LUSC phenotype with increased P40 and CK5/6 expression in EGFR -mutant LUAD patient-derived models. LUSC features in these models were further accentuated by the EGFR inhibitor osimertinib, which enriched for transdifferentiated LUSC cells. With the aim to validate potential therapeutic targets to prevent transdifferentiation from our multi-omic data, we tested the efficacy of EZH2 (the catalytic subunit of PRC2) or AKT pharmacological inhibitors in combination with osimertinib in an EGFR -mutant patient-derived xenograft model of LUAD-to-LUSC transdifferentiation and observed that inhibition of either pathway dramatically delayed relapse and prevented emergence of LUSC phenotypic markers. Additionally, pharmacological targeting of AKT and EGFR delayed relapse in a mixed LUAD/SCLC patient-derived xenograft model representing an intermediate step of LUAD-to-SCLC transdifferentiation. Interestingly, AKT inhibition selectively targeted the SCLC compartment of the tumor and prevented full SCLC transformation.
This work was published in two manuscripts, of which I am the first and co-corresponding author (Quintanal-Villalonga et al., Cancer Discov 2021; Quintanal-Villalonga et al., J Hematol Oncol 2021) and defines a novel landscape of potential drivers and therapeutic vulnerabilities of histological transdifferentiation in lung cancer. Both transformed and de novo SCLCs are aggressive and rapidly metastatic lung tumors. Limited treatment options and transient responses translate to poor prognosis for patients with SCLC; 5-year survival rates are &lt;1% for extensive disease, and SCLC accounts for &gt;200,000 annual deaths worldwide. Metastasis is the main cause of mortality among patients with SCLC. To characterize SCLC metastasis, we combined single-cell RNA sequencing (scRNA-seq) and multiplexed ion beam imaging (MIBI) technologies to study intratumoral heterogeneity and the surrounding tumor microenvironment (TME). Efforts to apply these technologies to human SCLC tumors have been limited, as surgical resections of primary tumors are performed in &lt;5% of patients with SCLC, and scRNA-seq processing of biopsied samples is extremely challenging. Additionally, since resection is only clinically indicated for exceptionally early stage de novo disease, these samples fail to capture the spectrum of disease progression. Through the optimization of protocols allowing single cell profiling of difficult samples such as small tissue biopsies, pleural effusions, and fine needle aspirations, along with larger volume resections, we constructed a single-cell atlas of SCLC patient tumors, with 155,098 transcriptomes, including 54,523 transcriptomes from 21 SCLC clinical specimens. Despite substantial heterogeneity among SCLC tumors, we detected a minor cell subpopulation that was shared among tumors across subtypes, treatments, and tissue locations, pointing to a potentially universal characteristic of this malignancy. This subpopulation demonstrated a pro-metastatic, highly plastic (stem-like) phenotype and exhibited profound PLCG2 overexpression. Direct genetic manipulation validated that PLCG2 expression promotes metastatic features and induced plasticity in vitro and in vivo. Consistently, we found that higher representation of this subpopulation in clinical samples, as well as PLCG2 expression itself, are strong predictors of shorter overall survival in patients with SCLC. Additionally, we found that SCLC is enriched for a profibrotic, immunosuppressive monocyte/macrophage population associated with the recurrent pro-metastatic PLCG2-high subpopulation, with potential implications in the metastatic process. I am the co-first author of this work published in Cancer Cell (Chan JM*, Quintanal-Villalonga* et al., Cancer Cell 2021) defining a novel mechanism of plasticity-mediated metastasis in SCLC. These works highlight the key role of plasticity in disease progression and therapy resistance in lung cancer, and describe molecular events occurring during fate reprogramming, thus nominating potential drivers and therapeutic vulnerabilities in plasticity-driven clinically relevant biological processes.
Citation Format: Alvaro Quintanal-Villalonga, Joseph M. Chan, Vianne R. Gao, Yubin Xie, Dana Pe’er, Charles M. Rudin. Multi-omic approaches to study the role of plasticity in therapy resistance and metastasis in lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr NG05.