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Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

Background Lineage plasticity, the ability to transdifferentiate among distinct phenotypic identities, facilitates therapeutic resistance in cancer. In lung adenocarcinomas (LUADs), this phenomenon includes small cell and squamous cell (LUSC) histologic transformation in the context of acquired resistance to targeted inhibition of driver mutations. LUAD-to-LUSC transdifferentiation, occurring in up to 9% of EGFR-mutant patients relapsed on osimertinib, is associated with notably poor prognosis. We hypothesized that multi-parameter profiling of the components of mixed histology (LUAD/LUSC) tumors could provide insight into factors licensing lineage plasticity between these histologies. Methods We performed genomic, epigenomics, transcriptomics and protein analyses of microdissected LUAD and LUSC components from mixed histology tumors, pre-/post-transformation tumors and reference non-transformed LUAD and LUSC samples. We validated our findings through genetic manipulation of preclinical models in vitro and in vivo and performed patient-derived xenograft (PDX) treatments to validate potential therapeutic targets in a LUAD PDX model acquiring LUSC features after osimertinib treatment. Results Our data suggest that LUSC transdifferentiation is primarily driven by transcriptional reprogramming rather than mutational events. We observed consistent relative upregulation of PI3K/AKT, MYC and PRC2 pathway genes. Concurrent activation of PI3K/AKT and MYC induced squamous features in EGFR-mutant LUAD preclinical models. Pharmacologic inhibition of EZH1/2 in combination with osimertinib prevented relapse with squamous-features in an EGFR-mutant patient-derived xenograft model, and inhibition of EZH1/2 or PI3K/AKT signaling re-sensitized resistant squamous-like tumors to osimertinib. Conclusions Our findings provide the first comprehensive molecular characterization of LUSC transdifferentiation, suggesting putative drivers and potential therapeutic targets to constrain or prevent lineage plasticity.

Summary Small cell lung cancer (SCLC) is an aggressive malignancy that includes subtypes defined by differential expression of ASCL1, NEUROD1, and POU2F3 (SCLC-A, -N, and -P, respectively). To define the heterogeneity of tumors and their associated microenvironments across subtypes, we sequenced 155,098 transcriptomes from 21 human biospecimens, including 54,523 SCLC transcriptomes. We observe greater tumor diversity in SCLC than lung adenocarcinoma, driven by canonical, intermediate, and admixed subtypes. We discover a PLCG2-high SCLC phenotype with stem-like, pro-metastatic features that recurs across subtypes and predicts worse overall survival. SCLC exhibits greater immune sequestration and less immune infiltration than lung adenocarcinoma, and SCLC-N shows less immune infiltrate and greater T cell dysfunction than SCLC-A. We identify a profibrotic, immunosuppressive monocyte/macrophage population in SCLC tumors that is particularly associated with the recurrent, PLCG2-high subpopulation.

Paired T cell receptor and RNA single cell sequencing (scTCR/RNA-seq) has allowed for enhanced resolution of clonal T cell dynamics in cancer. Here, we report a scTCR/RNA-seq dataset of 162,062 single T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients who underwent resections for progressing lung cancers after immune checkpoint blockade (ICB). We found marked regional heterogeneity in tumor persistence that was associated with heterogeneity in CD4 and CD8 T cell phenotypes; regions with persistent cancer cells were enriched for follicular helper CD4 T cells (TFH), regulatory T cells (Treg), and exhausted CD8 T cells. Clonal analysis demonstrated that highly-expanded T cell clones were predominantly of the CD8 subtype, were ubiquitously present across all sampled regions, found in the peripheral circulation, and expressed gene signatures of ‘large’ and ‘dual-expanded’ clones that have been predictive of response to ICB. Longitudinal tracking of CD8 T cell clones in the peripheral blood revealed that the persistence of ubiquitous CD8 T cell clones, as well as phenotypically distinct clones with tumor-reactive features, correlated with systemic tumor control. Finally, tracking CD8 T cell clones across tissues revealed the presence of TCF-1+precursor exhausted CD8 T cells in tumor draining LNs that were clonally linked to expanded exhausted CD8 T cells in tumors. Altogether, this comprehensive scTCR/RNA-seq dataset with regional, longitudinal, and clonal resolution provides fundamental insights into the tissue distribution, persistence, and differentiation trajectories of ICB-responsive T cells that underlie clinical responses to ICB.

Small cell lung cancer (SCLC) has limited therapeutic options and an exceptionally poor prognosis. Understanding the oncogenic drivers of SCLC may help define novel therapeutic targets. Recurrent genomic rearrangements have been identified in SCLC, most notably an in-frame gene fusion between RLF and MYCL found in up to 7% of the predominant ASCL1-expressing subtype. To explore the role of this fusion in oncogenesis and tumor progression, we used CRISPR/Cas9 somatic editing to generate a Rlf–Mycl-driven mouse model of SCLC. RLF–MYCL fusion accelerated transformation and proliferation of murine SCLC and increased metastatic dissemination and the diversity of metastatic sites. Tumors from the RLF–MYCL genetically engineered mouse model displayed gene expression similarities with human RLF–MYCL SCLC. Together, our studies support RLF–MYCL as the first demonstrated fusion oncogenic driver in SCLC and provide a new preclinical mouse model for the study of this subtype of SCLC. Significance: The biological and therapeutic implications of gene fusions in SCLC, an aggressive metastatic lung cancer, are unknown. Our study investigates the functional significance of the in-frame RLF–MYCL gene fusion by developing a Rlf–Mycl-driven genetically engineered mouse model and defining the impact on tumor growth and metastasis. This article is highlighted in the In This Issue feature, p. 2945

Small cell lung cancer (SCLC) is an aggressive malignancy that includes subtypes defined by differential expression of ASCL1, NEUROD1, and POU2F3 (SCLC-A, -N, and -P, respectively), which are associated with distinct therapeutic vulnerabilities. To define the heterogeneity of tumors and their associated microenvironments across subtypes, we sequenced 54,523 cellular transcriptomes from 21 human biospecimens. Our single-cell SCLC atlas reveals tumor diversity exceeding lung adenocarcinoma, driven by canonical, intermediate, and admixed subtypes. We discovered a PLCG2-high tumor cell population with stem-like, pro-metastatic features that recurs across subtypes and predicts worse overall survival, and manipulation of PLCG2 expression in cells confirms correlation with key metastatic markers. Treatment and subtype are associated with substantial phenotypic changes in the SCLC immune microenvironment, with greater T-cell dysfunction in SCLC-N than SCLC-A. Moreover, the recurrent, PLCG2-high subclone is associated with exhausted CD8+ T-cells and a pro-fibrotic, immunosuppressive monocyte/macrophage population, suggesting possible tumor-immune coordination to promote metastasis.

Human clinical samples pose a challenge for single-cell RNA-seq (scRNA-seq) and single-nucleus ATAC-seq (snATAC-seq) analysis, due to their heterogeneity in cell type composition and degree of degradation or necrosis. Moreover, although some tissues can be sampled by surgical resection, most biospecimens are derived from core needle biopsies or fine needle aspirates, and must be allocated to pressing needs such as pathological analysis, thereby severely limiting the number of cells available for analysis. We have developed a protocol for obtaining robust, high-quality single-cell sequencing data from human biospecimens from a variety of biopsy types, enabling greater access to clinical cohorts. A critical factor is to limit ischemic time and transport to the lab to under an hour, and to restrict subsequent processing time to under 3 hours. This protocol uses a commercial tissue disruptor for dissociation. It was optimized on diverse human lung tumor samples, including pleural effusions, but is widely applicable to normal and diseased human clinical tissues from different organ sites.

Treating KRAS-mutant lung adenocarcinoma (LUAD) remains a major challenge in cancer treatment given the difficulties associated with directly inhibiting the KRAS oncoprotein. One approach to addressing this challenge is to define mutations that frequently co-occur with those in KRAS, which themselves may lead to therapeutic vulnerabilities in tumors. Approximately 20% of KRAS-mutant LUAD tumors carry loss-of-function mutations in the KEAP1 gene encoding Kelch-like ECH-associated protein 1 (refs. 2, 3, 4), a negative regulator of nuclear factor erythroid 2-like 2 (NFE2L2; hereafter NRF2), which is the master transcriptional regulator of the endogenous antioxidant response. The high frequency of mutations in KEAP1 suggests an important role for the oxidative stress response in lung tumorigenesis. Using a CRISPR-Cas9-based approach in a mouse model of KRAS-driven LUAD, we examined the effects of Keap1 loss in lung cancer progression. We show that loss of Keap1 hyperactivates NRF2 and promotes KRAS-driven LUAD in mice. Through a combination of CRISPR-Cas9-based genetic screening and metabolomic analyses, we show that Keap1- or Nrf2-mutant cancers are dependent on increased glutaminolysis, and this property can be therapeutically exploited through the pharmacological inhibition of glutaminase. Finally, we provide a rationale for stratification of human patients with lung cancer harboring KRAS/KEAP1- or KRAS/NRF2-mutant lung tumors as likely to respond to glutaminase inhibition.

The Notch ligand DLL3 has emerged as a novel therapeutic target expressed in small cell lung cancer (SCLC) and high-grade neuroendocrine carcinomas. Rovalpituzumab teserine (Rova-T; SC16LD6.5) is a first-in-class DLL3-targeted antibody–drug conjugate with encouraging initial safety and efficacy profiles in SCLC in the clinic. Here we demonstrate that tumor expression of DLL3, although orders of magnitude lower in surface protein expression than typical oncology targets of immunoPET, can serve as an imaging biomarker for SCLC. We developed 89Zr-labeled SC16 antibody as a companion diagnostic agent to facilitate selection of patients for treatment with Rova-T based on a noninvasive interrogation of the in vivo status of DLL3 expression using PET imaging. Despite low cell-surface abundance of DLL3, immunoPET imaging with 89Zr-labeled SC16 antibody enabled delineation of subcutaneous and orthotopic SCLC tumor xenografts as well as distant organ metastases with high sensitivity. Uptake of the radiotracer in tumors was concordant with levels of DLL3 expression and, most notably, DLL3 immunoPET yielded rank-order correlation for response to SC16LD6.5 therapy in SCLC patient–derived xenograft models. Cancer Res; 77(14); 3931–41. ©2017 AACR.

Developmental processes underlying normal tissue regeneration have been implicated in cancer, but the degree of their enactment during tumor progression and under the selective pressures of immune surveillance, remain unknown. Here we show that human primary lung adenocarcinomas are characterized by the emergence of regenerative cell types, typically seen in response to lung injury, and by striking infidelity among transcription factors specifying most alveolar and bronchial epithelial lineages. In contrast, metastases are enriched for key endoderm and lung-specifying transcription factors, SOX2 and SOX9, and recapitulate more primitive transcriptional programs spanning stem-like to regenerative pulmonary epithelial progenitor states. This developmental continuum mirrors the progressive stages of spontaneous outbreak from metastatic dormancy in a mouse model and exhibits SOX9-dependent resistance to natural killer cells. Loss of developmental stage-specific constraint in macrometastases triggered by natural killer cell depletion suggests a dynamic interplay between developmental plasticity and immune-mediated pruning during metastasis. Single-cell analysis of lung cancer progression uncovers developmental and regenerative programs co-opted by cancer cells and immune-mediated pruning during metastatic outbreak

Background: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, accounting for about 15% of all lung cancer cases and approximately 200,000 deaths per year. SCLC genetic alterations are characterized by near-universal loss of function of the tumor suppressor genes TP53 and RB1. Several in-frame gene fusions have been identified in SCLC tumors and cell lines, with recurrent fusion events noted in up to 5% of cases. However, the biological and therapeutic implications of these fusions, including the recurrent in-frame RLF-MYCL1 gene fusion, are unknown. MYCL1 is frequently expressed in SCLC and differential expression of MYC family members has been associated with different SCLC subtypes. Here we investigate the functional significance of the RLF-MYCL1 gene fusion by developing a novel Rlf-Mycl1-driven genetically engineered mouse model (GEMM) of SCLC using CRISPR-Cas9 genome engineering. Methods: SgRNAs targeting intronic sequences of the Rlf and Mycl1 genes were cloned into a Cas9-expressing plasmid and co-transfected in NIH/3T3 cells. The presence of the Rlf-Mycl1 fusion was confirmed at the DNA and protein level. To test whether the RLF-MYCL1 gene fusion is important for the oncogenic switch during early SCLC development, we transduced Rb1Δ/Δ;Trp53Δ/Δ;Rbl2+/Δ precancerous neuroendocrine cells (preSCs), which were isolated from an early stage in tumorigenesis, with a lentiviral vector encoding Cas9 and with the dual sgRNAs targeting Rlf and Mycl1 genes (P-RM). Results: The P-RM transduced preSCs formed significantly more colonies in soft agar than those infected with non-targeting sgRNAs (P-NT) (p Summary: A major hurdle to SCLC clinical advancement is the absence of targetable driver mutations. Our findings indicate that the Rlf-Mycl1 gene fusion accelerates SCLC tumor growth. Defining mechanisms by which Rlf-Mycl1 accelerates tumorigenesis may identify therapeutic strategies to target SCLC harboring this fusion oncogene. Our study further supports a careful analysis of the involvement of other gene-fusions in SCLC. Citation Format: Metamia Ciampricotti, Triantafyllia Karakousi, Alvaro Quintanal Villalonga, Viola Allaj, Andrea Ventura, Triparna Sen, JT Poirier, Thales Papagiannakopoulos, Charles M. Rudin. Rlf-Mycl1 gene fusion as a novel oncogenic driver in a mouse model of small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 420.

Patient-derived xenograft (PDX) mouse models are increasingly used for preclinical therapeutic testing of human cancer. A limitation in molecular and genetic characterization of PDX tumors is the presence of integral murine stroma. This is particularly problematic for genomic sequencing of PDX models. Rapid and dependable approaches for quantitating stromal content and purifying the malignant human component of these tumors are needed. We used a recently developed technique exploiting species-specific polymerase chain reaction (PCR) amplicon length (ssPAL) differences to define the fractional composition of murine and human DNA, which was proportional to the fractional composition of cells in a series of lung cancer PDX lines. We compared four methods of human cancer cell isolation: fluorescence-activated cell sorting (FACS), an immunomagnetic mouse cell depletion (MCD) approach, and two distinct EpCAM-based immunomagnetic positive selection methods. We further analyzed DNA extracted from the resulting enriched human cancer cells by targeted sequencing using a clinically validated multi-gene panel. Stromal content varied widely among tumors of similar histology, but appeared stable over multiple serial tumor passages of an individual model. FACS and MCD were superior to either positive selection approach, especially in cases of high stromal content, and consistently allowed high quality human-specific genomic profiling. ssPAL is a dependable approach to quantitation of murine stromal content, and MCD is a simple, efficient, and high yield approach to human cancer cell isolation for genomic analysis of PDX tumors.

Treating KRAS mutant lung adenocarcinoma (LUAD) remains a major challenge in cancer treatment given the difficulties associated with directly inhibiting the KRAS oncoprotein1. One approach to addressing this challenge is to define frequently co-occurring mutations with KRAS, which themselves may lead to therapeutic vulnerabilities in tumors. Approximately 20% of KRAS mutant LUAD tumors carry loss-of-function (LOF) mutations in Kelch-like ECH-associated protein 1 (KEAP1)2-4, a negative regulator of nuclear factor erythroid 2-like 2 (NFE2L2; hereafter NRF2), which is the master transcriptional regulator of the endogenous antioxidant response5-10. The high frequency of mutations in KEAP1, which would be predicted to result in activation of the NRF2 pathway, suggests an important role for the oxidative stress response in lung tumorigenesis. To test this directly, we used a CRISPR/Cas9-based approach in a mouse model of Kras-driven LUAD to examine the effects of loss of KEAP1 function in lung cancer progression. We show that loss of KEAP1 hyper-activates Nrf2 and promotes Kras-driven LUAD. Combining CRISPR/Cas9-based genetic screening and metabolomic analyses, we also show that KEAP1/NRF2 mutant cancers are dependent on increased glutaminolysis, and this property can be therapeutically exploited through the pharmacologic inhibition of glutaminase. Finally, we provide a rationale for sub-stratification of human lung cancer patients with KRAS-KEAP1 or -NRF2 mutant tumors as likely to respond to glutaminase inhibition. Citation Format: Rodrigo Romero, Volkan I. Sayin, Davidson M. Shawn, Matthew Bauer, Simranjit X. Singh, Sarah LeBoeuf, Triantafyllia R. Karakousi, Donald C. Ellis, Arjun Bhutkar, Francisco Sanchez-Rivera, Lakshmipriya Subbaraj, Britney Martinez, Roderick T. Bronson, Justin R. Prigge, Edward E. Schmidt, Craig J. Thomas, Angela Davies, Igor Dolgalev, Adriana Heguy, Viola Allaj, John T. Piorier, Andre L. Moreira, Charles M. Rudin, Harvey I. Pass, Matthew G. Vander Heiden, Tyler Jacks, Thales Papagiannakopoulos. Loss of Keap1 promotes KRAS-driven lung cancer and results in genotype-specific vulnerabilities [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A43.

Fatty acids are involved in multiple pathways and play a pivotal role in health. Eicosanoids, derived from arachidonic acid, have received extensive attention in the field of cancer research. Following release from the phospholipid membrane, arachidonic acid can be metabolized into different classes of eicosanoids through cyclooxygenases, lipoxygenases, or p450 epoxygenase pathways. Non-steroid anti-inflammatory drugs (NSAIDs) are widely consumed as analgesics to relieve minor aches and pains, as antipyretics to reduce fever, and as anti-inflammatory medications. Most NSAIDs are nonselective inhibitors of cyclooxygenases, the rate limiting enzymes in the formation of prostaglandins. Long term use of some NSAIDs has been linked with reduced incidence and mortality in many cancers. In this review, we appraise the biological activities of prostanoids and their cognate receptors in the context of cancer biology. The existing literature supports that these lipid mediators are involved to a great extent in the occurrence and progression of cancer.