Small Cell Lung Cancer Plasticity Enables NFIB-Independent Metastasis.

Metastasis is a major cause of morbidity and mortality in patients with cancer, highlighting the need to identify improved treatment and prevention strategies. Previous observations in preclinical models and tumors from patients with small cell lung cancer (SCLC), a fatal form of lung cancer with high metastatic potential, identified the transcription factor NFIB as a driver of tumor growth and metastasis. However, investigation into the requirement for NFIB activity for tumor growth and metastasis in relevant in vivo models is needed to establish NFIB as a therapeutic target. Here, using conditional gene knockout strategies in genetically engineered mouse models of SCLC, we found that upregulation of NFIB contributes to tumor progression, but NFIB is not required for metastasis. Molecular studies in NFIB wild-type and knockout tumors identified the pioneer transcription factors FOXA1/2 as candidate drivers of metastatic progression. Thus, while NFIB upregulation is a frequent event in SCLC during tumor progression, SCLC tumors can employ NFIB-independent mechanisms for metastasis, further highlighting the plasticity of these tumors. SIGNIFICANCE: Small cell lung cancer cells overcome deficiency of the prometastatic oncogene NFIB to gain metastatic potential through various molecular mechanisms, which may represent targets to block progression of this fatal cancer type.

Integrative Analysis of a Large Real-World Cohort of Small Cell Lung Cancer Identifies Distinct Genetic Subtypes and Insights into Histologic Transformation.

Small cell lung cancer (SCLC) is a recalcitrant neuroendocrine carcinoma with dismal survival outcomes. A major barrier in the field has been the relative paucity of human tumors studied. Here we provide an integrated analysis of 3,600 "real-world" SCLC cases. This large cohort allowed us to identify new recurrent alterations and genetic subtypes, including STK11-mutant tumors (1.7%) and TP53/RB1 wild-type tumors (5.5%), as well as rare cases that were human papillomavirus-positive. In our cohort, gene amplifications on 4q12 are associated with increased overall survival, whereas CCNE1 amplification is associated with decreased overall survival. We also identify more frequent alterations in the PTEN pathway in brain metastases. Finally, profiling cases of SCLC containing oncogenic drivers typically associated with NSCLC demonstrates that SCLC transformation may occur across multiple distinct molecular cohorts of NSCLC. These novel and unsuspected genetic features of SCLC may help personalize treatment approaches for this fatal form of cancer. SIGNIFICANCE: Minimal changes in therapy and survival outcomes have occurred in SCLC for the past four decades. The identification of new genetic subtypes and novel recurrent mutations as well as an improved understanding of the mechanisms of transformation to SCLC from NSCLC may guide the development of personalized therapies for subsets of patients with SCLC. This article is highlighted in the In This Issue feature, p. 1501.

Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH.

Extrachromosomal DNA (ecDNA) is a common mode of oncogene amplification but is challenging to analyze. Here, we adapt CRISPR-CATCH, in vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA, previously developed for bacterial chromosome segments, to isolate megabase-sized human ecDNAs. We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells and NRAS ecDNA from human metastatic melanoma with acquired therapeutic resistance. Targeted enrichment of ecDNA versus chromosomal DNA enabled phasing of genetic variants, identified the presence of an EGFRvIII mutation exclusively on ecDNAs and supported an excision model of ecDNA genesis in a glioblastoma model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNAs. We distinguished heterogeneous ecDNA species within the same sample by size and sequence with base-pair resolution and discovered functionally specialized ecDNAs that amplify select enhancers or oncogene-coding sequences.

Combinatorial Inactivation of Tumor Suppressors Efficiently Initiates Lung Adenocarcinoma with Therapeutic Vulnerabilities.

Lung cancer is the leading cause of cancer death worldwide, with lung adenocarcinoma being the most common subtype. Many oncogenes and tumor suppressor genes are altered in this cancer type, and the discovery of oncogene mutations has led to the development of targeted therapies that have improved clinical outcomes. However, a large fraction of lung adenocarcinomas lacks mutations in known oncogenes, and the genesis and treatment of these oncogene-negative tumors remain enigmatic. Here, we perform iterative in vivo functional screens using quantitative autochthonous mouse model systems to uncover the genetic and biochemical changes that enable efficient lung tumor initiation in the absence of oncogene alterations. Generation of hundreds of diverse combinations of tumor suppressor alterations demonstrates that inactivation of suppressors of the RAS and PI3K pathways drives the development of oncogene-negative lung adenocarcinoma. Human genomic data and histology identified RAS/MAPK and PI3K pathway activation as a common feature of an event in oncogene-negative human lung adenocarcinomas. These Onc-negativeRAS/PI3K tumors and related cell lines are vulnerable to pharmacologic inhibition of these signaling axes. These results transform our understanding of this prevalent yet understudied subtype of lung adenocarcinoma. SIGNIFICANCE: To address the large fraction of lung adenocarcinomas lacking mutations in proto-oncogenes for which targeted therapies are unavailable, this work uncovers driver pathways of oncogene-negative lung adenocarcinomas and demonstrates their therapeutic vulnerabilities.

Keap1 mutation renders lung adenocarcinomas dependent on Slc33a1.

Approximately 20-30% of human lung adenocarcinomas (LUAD) harbor loss-of-function (LOF) mutations in Kelch-like ECH Associated-Protein 1 (KEAP1), which lead to hyperactivation of the nuclear factor, erythroid 2-like 2 (NRF2) antioxidant pathway and correlate with poor prognosis1-3. We previously showed that Keap1 mutation accelerates KRAS-driven LUAD and produces a marked dependency on glutaminolysis4. To extend the investigation of genetic dependencies in the context of Keap1 mutation, we performed a druggable genome CRISPR-Cas9 screen in Keap1-mutant cells. This analysis uncovered a profound Keap1 mutant-specific dependency on solute carrier family 33 member 1 (Slc33a1), an endomembrane-associated protein with roles in autophagy regulation5, as well as a series of functionally-related genes implicated in the unfolded protein response. Targeted genetic and biochemical experiments using mouse and human Keap1-mutant tumor lines, as well as preclinical genetically-engineered mouse models (GEMMs) of LUAD, validate Slc33a1 as a robust Keap1-mutant-specific dependency. Furthermore, unbiased genome-wide CRISPR screening identified additional genes related to Slc33a1 dependency. Overall, our study provides a strong rationale for stratification of patients harboring KEAP1-mutant or NRF2-hyperactivated tumors as likely responders to targeted SLC33A1 inhibition and underscores the value of integrating functional genetic approaches with GEMMs to identify and validate genotype-specific therapeutic targets.

Identification of DHODH as a therapeutic target in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive lung cancer subtype with extremely poor prognosis. No targetable genetic driver events have been identified, and the treatment landscape for this disease has remained nearly unchanged for over 30 years. Here, we have taken a CRISPR-based screening approach to identify genetic vulnerabilities in SCLC that may serve as potential therapeutic targets. We used a single-guide RNA (sgRNA) library targeting ~5000 genes deemed to encode "druggable" proteins to perform loss-of-function genetic screens in a panel of cell lines derived from autochthonous genetically engineered mouse models (GEMMs) of SCLC, lung adenocarcinoma (LUAD), and pancreatic ductal adenocarcinoma (PDAC). Cross-cancer analyses allowed us to identify SCLC-selective vulnerabilities. In particular, we observed enhanced sensitivity of SCLC cells toward disruption of the pyrimidine biosynthesis pathway. Pharmacological inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in this pathway, reduced the viability of SCLC cells in vitro and strongly suppressed SCLC tumor growth in human patient-derived xenograft (PDX) models and in an autochthonous mouse model. These results indicate that DHODH inhibition may be an approach to treat SCLC.