Search

Supplemental tables S1-S15

Lineage plasticity is implicated in treatment resistance in multiple cancers. In lung adenocarcinomas (LUAD) amenable to targeted therapy, transformation to small cell lung cancer (SCLC) is a recognized resistance mechanism. Defining molecular mechanisms of neuroendocrine (NE) transformation in lung cancer has been limited by a paucity of pre/posttransformation clinical samples. Detailed genomic, epigenomic, transcriptomic, and protein characterization of combined LUAD/SCLC tumors, as well as pre/posttransformation samples, supports that NE transformation is primarily driven by transcriptional reprogramming rather than mutational events. We identify genomic contexts in which NE transformation is favored, including frequent loss of the 3p chromosome arm. We observed enhanced expression of genes involved in the PRC2 complex and PI3K/AKT and NOTCH pathways. Pharmacologic inhibition of the PI3K/AKT pathway delayed tumor growth and NE transformation in an EGFR-mutant patient-derived xenograft model. Our findings define a novel landscape of potential drivers and therapeutic vulnerabilities of NE transformation in lung cancer.Significance:The difficulty in collection of transformation samples has precluded the performance of molecular analyses, and thus little is known about the lineage plasticity mechanisms leading to LUAD-to-SCLC transformation. Here, we describe biological pathways dysregulated upon transformation and identify potential predictors and potential therapeutic vulnerabilities of NE transformation in the lung.See related commentary by Meador and Lovly, p. 2962.This article is highlighted in the In This Issue feature, p. 2945

Supplemental figures and figure legends.

Supplementary Legend

Small cell lung cancer (SCLC) is an aggressive malignancy characterized by early metastasis and extreme lethality. The backbone of SCLC treatment over the past several decades has been platinum-based doublet chemotherapy, with the recent addition of immunotherapy providing modest benefits in a subset of patients. However, nearly all patients treated with systemic therapy quickly develop resistant disease, and there is an absence of effective therapies for recurrent and progressive disease. Here we conducted CRISPR-Cas9 screens using a druggable genome library in multiple SCLC cell lines representing distinct molecular subtypes. This screen nominated exportin-1, encoded by XPO1, as a therapeutic target. XPO1 was highly and ubiquitously expressed in SCLC relative to other lung cancer histologies and other tumor types. XPO1 knockout enhanced chemosensitivity, and exportin-1 inhibition demonstrated synergy with both first- and second-line chemotherapy. The small molecule exportin-1 inhibitor selinexor in combination with cisplatin or irinotecan dramatically inhibited tumor growth in chemonaïve and chemorelapsed SCLC patient-derived xenografts, respectively. Together these data identify exportin-1 as a promising therapeutic target in SCLC, with the potential to markedly augment the efficacy of cytotoxic agents commonly used in treating this disease.Significance:CRISPR-Cas9 screening nominates exportin-1 as a therapeutic target in SCLC, and exportin-1 inhibition enhances chemotherapy efficacy in patient-derived xenografts, providing a novel therapeutic opportunity in this disease.

Subtype information

Overall survival by combination of STK11, KEAP1, and SMARCA4 alteration status.

Purpose:SMARCA4 mutations are among the most common recurrent alterations in non–small cell lung cancer (NSCLC), but the relationship to other genomic abnormalities and clinical impact has not been established.Experimental Design:To characterize SMARCA4 alterations in NSCLC, we analyzed the genomic, protein expression, and clinical outcome data of patients with SMARCA4 alterations treated at Memorial Sloan Kettering.Results:In 4,813 tumors from patients with NSCLC, we identified 8% (n = 407) of patients with SMARCA4-mutant lung cancer. We describe two categories of SMARCA4 mutations: class 1 mutations (truncating mutations, fusions, and homozygous deletion) and class 2 mutations (missense mutations). Protein expression loss was associated with class 1 mutation (81% vs. 0%, P < 0.001). Both classes of mutation co-occurred more frequently with KRAS, STK11, and KEAP1 mutations compared with SMARCA4 wild-type tumors (P < 0.001). In patients with metastatic NSCLC, SMARCA4 alterations were associated with shorter overall survival, with class 1 alterations associated with shortest survival times (P < 0.001). Conversely, we found that treatment with immune checkpoint inhibitors (ICI) was associated with improved outcomes in patients with SMARCA4-mutant tumors (P = 0.01), with class 1 mutations having the best response to ICIs (P = 0.027).Conclusions:SMARCA4 alterations can be divided into two clinically relevant genomic classes associated with differential protein expression as well as distinct prognostic and treatment implications. Both classes co-occur with KEAP1, STK11, and KRAS mutations, but individually represent independent predictors of poor prognosis. Despite association with poor outcomes, SMARCA4-mutant lung cancers may be more sensitive to immunotherapy.

Frequency of altered individual genes within SMARCA4 Class 1 vs SMARCA4 Class 2 subgroups.

Supplementary Figures

Populations analyzed in genomic and clinical analyses.

Lineage plasticity is the ability for a cell to change its phenotypic state under evolutionary pressure. This process mediates acquired resistance to EGFR-targeted therapies in lung adenocarcinoma (LUAD), through transformation to small cell lung cancer (SCLC), an aggressive cancer type of neuroendocrine (NE) histology. This transformed SCLC has worse clinical outcomes than either its LUAD or de novo SCLC counterparts, with less than one-year overall survival following transformation. Classical SCLC itself can display plasticity through interconversion between classical, variant, and non-NE transcriptional subtypes, with non-classical subtypes displaying increased metastasis and chemoresistance. It is poorly understood what factors drive SCLC transformation or subtype switching. Prior studies poorly model intratumoral heterogeneity because they profile tumors in bulk, which only estimates the average phenotype. However, SCLC transformation often results in admixed LUAD/SCLC tumors. We sought to capture the full intratumoral heterogeneity of SCLC transformation and identify potential molecular determinants of plasticity by applying single-cell RNA sequencing (scRNA-seq) to 41 tumors and patient-derived xenografts from 22 patients with transformed or combined LUAD/NE histology, with matched targeted DNA sequencing and 17 de novo SCLC tumors, 10 LUAD tumors with concurrent EGFR/RB1/TP53 mutations, and 4 tumor-adjacent normal lung samples for comparison. Of 234,322 single transcriptomes assessed, we found 89,113 NE cancer cells and 18,197 LUAD cancer cells. We confirmed that NE and LUAD components within the same tumor shared clonal mutations and common ancestry. Compared to de novo, transformed SCLC harbored greater phenotypic diversity across patients (p < 1 × 10−10), driven largely by enrichment of variant and non-NE subtypes (p < 0.02), including NEUROD1, POU2F3, and YAP1-high subtypes, the latter of which was completely absent in de novo. Within each tumor, transformed SCLC displayed higher intratumoral subtype diversity than de novo SCLC (likelihood ratio p < 0.025). After adjusting for SCLC subtype, differential expression and pathway analysis demonstrated that transformed SCLC is a distinct phenotype from de novo and shares features of the ancestral clone that include residual EGFR and NSCLC gene signatures, as well as pathways in neuronal stemness, MYC targets, AKT/MTOR signaling, JAK/STAT inflammation, and chromatin remodeling. In sum, we find increased intratumoral phenotypic diversity in transformed SCLC, including variant and non-NE subtypes, that may explain worse clinical outcomes. We show that transformed SCLC is a distinct phenotype from de novo, marked by pathways that may offer new potential drug targets to constrain plasticity, with the goal of restoring original sensitivity to targeted therapies. Citation Format: Joseph Minhow Chan, Alvaro Quintanal-Villalonga, Amin Sabet, Parvathy Manoj, Ojasvi Chaudhary, Tianhao Xu, Ignas Masilionis, Jacklynn Egger, Noor Sohail, Jaeyoung Chun, Tal Nawy, Linas Mazutis, Triparna Sen, Ronan Chaligne, Helena Yu, Dana Pe'er, Charles Rudin. Single-cell transcriptomic profiling of SCLC transformation reveals increased intratumoral diversity of variant and non-neuroendocrine subtypes [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 1167.

Response to immune checkpoint blockade (ICB) in non-small cell lung cancers (NSCLC) is associated with recurring mutations in tumor suppressor genes STK11 and TP53. Whereas STK11-mutated patients are mostly insensitive, TP53-mutated patients commonly respond to ICB. Previous studies have linked mutational status in these genes to differences in cell type composition of the tumor microenvironment (TME). However, it remains unclear if differences in cell type compositions as well as cell-cell interactions in turn could account for the observed differences in treatment response and overall survival in NSCLC. Here, we perform spatial profiling of immune and stromal phenotypes in the TME of 119 NSCLC patients using imaging mass cytometry (IMC) on tissue microarrays (TMA). Matching data from MSK IMPACT (clinical sequencing) was used to establish mutation profiles and therapeutic response was included in the analysis. We find that STK11-mutated NSCLC is characterized by decreased CD4 T cell and increased neutrophil abundance, while TP53-mutated NSCLC is associated with increased CD8 T cell and decreased endothelial cell abundance. Accordingly, we stratified the patient population into TME classes by cell type composition. We found that while mutational status does not inform overall survival in our cohort, stratification by cell type abundance strongly associates with patient outcome (p-value: 0.000146, logRank test). Patients with neutrophil-rich TMEs show worse overall survival (25% 5-year survival), while patients with increased endothelial cell and macrophage abundance have a tendency to live longer (80% and 75% 5-year survival respectively). Furthermore, we interrogate pairwise proximity of immune cell types and states to construct cellular networks in NSCLC. Together, our findings suggest that TME cell type composition and cellular networks are potential molecular determinants for ICB therapeutic response in NSCLC patients. Citation Format: Florian Uhlitz, Douglas Linn, Elsa Beyer Krall, Jacklynn Egger, Hira Rizvi, Jason Chang, Benjamin Nicholson, Rami Vanguri, Andrew Chow, Matthew Hellmann, Sohrab Shah. Spatial immune determinants of ICB response in STK11-mutated non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB049.

Lineage plasticity contributes to therapeutic resistance in cancer. In lung adenocarcinomas (LUADs), this phenomenon drives neuroendocrine (NE) and squamous cell (LUSC) histologic transdifferentiation in the context of acquired resistance to targeted inhibition of driver mutations, with up to 14% and 9% incidences in EGFR-mutant tumors relapsed on EGFR inhibitors, respectively. Notably, survival of patients with NE- or LUSC-transdifferentiated tumors is lower than that of either LUAD or de novo LUSC patients. To date, little is known about the molecular effectors enhancing lineage plasticity and driving histological transdifferentiation due to the paucity of well annotated pre- and post-transdifferentiation clinical samples amenable for molecular analyses. Currently no specific therapies for LUSC or NE transdifferentiation prevention are available for patients at high risk of transformation. We performed multi-omic profiling of transdifferentiating clinical samples, as well as control never-transformed LUAD and de novo LUSC and small cell carcinomas, including comprehensive and integrative genomic (whole exome sequencing), epigenomic (bisulfite sequencing), transcriptomic (RNAseq) and protein (antibody arrays) characterization. Findings were validated in preclinical models including cell lines as well as LUSC- and NE-transdifferentiation patient-derived xenograft models. Our data suggest that histological transdifferentiation is driven by epigenetic -rather than mutational- events, and indicate that transdifferentiated tumors retain molecular features of their previous LUAD state. Integrative analysis revealed biological pathways dysregulated specifically for distinct histological outcomes, including downregulation of RTK signaling and Notch-related genes in NE-transformed tumors, and upregulation of genes involved in Hedgehog and Notch signaling and MYC targets in LUSC-transdifferentiated tumors. Most interestingly, these analyses revealed commonly dysregulated pathways for transdifferentiated tumors, including marked downregulation of a variety of immune-related pathways and upregulation of genes involved in AKT signaling and in the PRC2 epigenetic remodeling complex. Concurrent activation of AKT and MYC overexpression induced a squamous phenotype in EGFR-mutant LUAD preclinical models, further accentuated by EGFR inhibition. Pharmacological targeting of AKT in combination with osimertinib delayed both squamous and NE transformation in EGFR-mutant patient-derived xenograft transdifferentiation models. These results identify common and histology-specific drivers and dysregulated pathways in NE and LUSC transdifferentiation, and nominate AKT as a therapeutic target to constrain lineage plasticity and prevent the acquisition of resistance to EGFR-targeted therapies through histological transdifferentiation. Citation Format: Alvaro Quintanal-Villalonga, Hirokazu Taniguchi, Yingqian A. Zhan, Fathema Uddin, Viola Allaj, Parvathy Manoj, Nisargbhai S. Shah, Umesh K. Bhanot, Jacklynn Egger, Juan Qiu, Elisa de Stanchina, Natasha Rekhtman, Brian Houck-Loomis, Richard P. Koche, Helena A. Yu, Triparna Sen, Charles M. Rudin. AKT pathway as a therapeutic target to constrain lineage plasticity leading to histological transdifferentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 658.

Introduction: Small-cell lung cancer (SCLC) is a high-grade neuroendocrine carcinoma characterised by high proliferation rate and early, rapid metastatic spread. Although SCLC is treated as a homogenous disease, recent studies revealed morphologic and transcriptomic heterogeneity with several molecular subtypes described based on predominant transcription factor expression (ASCL1, NEUROD1, ATOH1, POU2F3, YAP1) (Rudin et al., 2019; Simpson et al., 2020) which in preclinical studies exhibit differing vulnerabilities raising the potential of stratified therapy. DNA methylation is also thought be an important regulator of SCLC biology (Gazdar et al., 2017) and epigenetically distinct subtypes derived from SCLC primary tumour samples reported (Poirier et al., 2015). Here, we developed a robust workflow for genome-wide DNA methylation profiling to examine the potential use of cfDNA methylation profiling for detection and subtyping of SCLC. Results: To evaluate SCLC genome-wide DNA methylation patterns we employed a bisulfite-free enrichment-based approach (T7-MBD-seq). We tested this approach on tissue samples from preclinical models and from normal lung (n=110) and on cfDNA samples from both patients with SCLC and from non-cancer controls (n=157). Methylation profiles from preclinical models (patient-derived xenografts (PDX) and CTC derived explant (CDX) models) were comparable to previously described methylation patterns from SCLC primary tumours and were recapitulated in patients’ cfDNA samples. A tumour/normal classifier, based on 4,061 genomic regions detected as being hypermethylated in SCLC preclinical models, correctly assigned 93% and 100% cfDNA samples from patients with limited and extensive stage SCLC respectively, with a statistically significant correlation of prediction scores with disease stage (P=0.0076). Finally, to determine whether cfDNA methylation profiling could subtype SCLC patients, we built a subtype classifier, based on methylation signatures derived from 59 established SCLC cell lines. We applied the classifier to cfDNA samples from 56 patients and 10/11 with known subtypes (identified from a donor matched CDX model) were correctly classified. Overall, 73% of cfDNA samples were classified as ASCL1, 13% were classified as NEUROD1 and 14% were classified as being double negative with the distribution of the subtypes correlating closely to previously published IHC data from SCLC tissue samples (Baine et al., 2020). Conclusions: Our data reveal two potential clinical utilities of cfDNA methylation profiling; a universally applicable liquid biopsy approach for more sensitive detection and monitoring of SCLC and molecular subtyping to ease the path to future clinical trials of subtype stratified treatments for patients with SCLC. Citation Format: Dominic G. Rothwell, Francesca Chemi, Simon Pearce, Alex Clipson, Steven Hill, Alicia Marie Conway, Sophie Richardson, Katarzyna Murat, Rebecca Caeser, Jacklynn Egger, John T. Poirier, Alastair Kerr, Fiona Blackhall, Charles M. Rudin, Caroline Dive. Profiling of the circulating cell-free DNA methylome for detection and subtyping of small cell lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6238.

Introduction: Mutations in the RAS family of proto-oncogenes are frequently found in NSCLC, with KRAS being the most prevalent mutated isoform. Of KRAS mutations, the most common is KRAS G12C, representing ~40% of KRAS mutations and occurring in ~13% of all lung adenocarcinoma cases. The objective of this study is to describe clinicopathological and molecular characteristics, treatment patterns, and outcomes in patients with KRAS G12C-mutated metastatic NSCLC using the AACR Project GENIE database. Methods: An observational retrospective study of adult patients (>18 years of age) with KRAS G12C-mutated metastatic NSCLC from three US academic comprehensive cancer centers in the AACR Project GENIE database was undertaken using de-identified data. Patients were eligible if their tumor tested positive for KRAS G12C mutation by next-generation sequencing at the participating institutions. Event-time distributions were estimated using the Kaplan-Meier method, and multivariable models were fitted to estimate adjusted hazard ratios (aHRs) by Cox proportional models. Patients with large panel sequencing performed up to Dec 31, 2018 were included. All available data until Aug 1, 2019 were collected. Results: Of the 416 eligible patients included in this analysis, 91.6% were white, 64.4% were female, median age at metastatic diagnosis was 68.2 years (range: 37.6-86.2), and 97.1% were former or current smokers. 65.1% of patients had stage 4 NSCLC at diagnosis, and most tumors were adenocarcinomas (85.8%). The KRAS G12C mutation was nearly mutually exclusive (≤ 1.0%) with other actionable driver mutations in EGFR, ALK, ROS1, and BRAF but was co-occurred with STK11 (23.6%) and KEAP1 (9.9%) mutations. Among patients who received PD-(L)1 monotherapy (n=110), median progression-free (mPFS) and overall survival (mOS) were 4.6 and 11.9 months, respectively, while among those who received chemotherapy only (n=133) mPFS and mOS were 4.1 and 12.1 months, respectively. When clinical outcomes were analyzed according to co-occurring genomic alterations, we found that KRAS/STK11 co-mutations were associated with significantly worse outcomes with immunotherapy (PFS: aHR 0.44, P=0.002; OS: aHR 0.42, P=0.003) but not with chemotherapy (PFS: aHR 0.82, P=0.44; OS: aHR 0.81, P=0.43). There was no significant impact of co-occurring KRAS mutations with KEAP1, SMARCA4, ATM, RBM10 and TP53 on clinical outcomes to either PD-(L)1 inhibition or chemotherapy. Conclusions: KRAS G12C mutant NSCLC represents a heterogeneous group of lung cancers and its genomic diversity is reflected in differential responses to therapy. These findings highlight the urgent clinical need for molecular biomarker-driven therapies in this subset of patients. Citation Format: Biagio Ricciuti, Chenguang Wang, Hira Rizvi, Jacklynn Egger, Michele LeNoue-Newton, Marilyn Holt, Shivani Aggarwal, Huakang Tu, Hil Hsu, Xuena Wang, Gataree Ngarmchamnanrith, Victoria Chia, Jocelyn Lee, Robert Scharpf, Valsamo Anagnostou, Christine Lovly, Greg Riely, Mark Awad. Clinicopathological and molecular characteristics, treatment patterns, and outcomes in patients with KRAS p.G12C-mutated metastatic non-small cell lung cancer (NSCLC) in the AACR Project GENIE database [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 102.