Search

Supplementary Figures S1 - S5

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 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.

Supplemental data detailing the methods and results for the synthesis and in vitro characterization of the radioimmunoconjugates, longitudinal PET imaging and ex vivo analyses in various models described within the main text.

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

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.

Supplementary Figures

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.

The repertoire of tumor-infiltrating lymphocytes (TILs) can be vast, and many of these TILs are not endowed with tumor reactivity. While a number of reports have shown that tumor-reactive TILs express CD39, few reports have demonstrated that conversely, CD39 can be leveraged to serve as a proxy of tumor-reactive CD8 T cells. Using single-cell CITE/RNA/TCRseq, we show that CD39+ CD8 T cells in human lung cancers demonstrate transcriptional and proteomic features of exhaustion, tumor reactivity, and clonal expansion. Moreover, TCR cloning revealed that CD39 enriched for tumor-reactive CD8 T cell clones. Flow cytometry of 440 lung cancer specimens revealed that CD39 level on CD8 T cells is only weakly correlated with tumoral features that currently guide lung cancer therapy, such as histology, driver mutation, PD-L1 and tumor mutation burden. PD-1 axis blockade, but not cytotoxic chemotherapy, increased intratumoral CD39+ CD8 T cells. CD39 correlated with PD-1 expression on CD8 T cells and high pre-treatment/early-on-treatment levels were associated with improved clinical outcomes, but not immune-related adverse events, from immune checkpoint blockade therapy. This comprehensive profiling of the clinical, pathological and molecular features highlights the utility of CD39 as a proxy for tumor-reactive CD8 T cells in human lung cancer.

SCLC is a highly aggressive neuroendocrine tumor that is characterized by early acquired therapeutic resistance and modest benefit from immune checkpoint blockade (ICB). Repression of the major histocompatibility complex class I (MHC-I) represents a key mechanism driving resistance to T cell-based immunotherapies.We evaluated the role of the lysine-specific demethylase 1 (LSD1) as a determinant of MHC-I expression, functional antigen presentation, and immune activation in SCLC in vitro and in vivo through evaluation of both human SCLC cell lines and immunocompetent mouse models.We found that targeted inhibition of LSD1 in SCLC restores MHC-I cell surface expression and transcriptionally activates genes encoding the antigen presentation pathway. LSD1 inhibition further activates interferon signaling, induces tumor-intrinsic immunogenicity, and sensitizes SCLC cells to MHC-I-restricted T cell cytolysis. Combination of LSD1 inhibitor with ICB augments the antitumor immune response in refractory SCLC models. Together, these data define a role for LSD1 as a potent regulator of MHC-I antigen presentation and provide rationale for combinatory use of LSD1 inhibitors with ICB to improve therapeutic response in SCLC.Epigenetic silencing of MHC-I in SCLC contributes to its poor response to ICB. Our study identifies a previously uncharacterized role for LSD1 as a regulator of MHC-I antigen presentation in SCLC. LSD1 inhibition enables MHC-I-restricted T cell cytolysis, induces immune activation, and augments the antitumor immune response to ICB in SCLC.

Improved identification of anti-tumor T cells is needed to advance cancer immunotherapies. CD39 expression is a promising surrogate of tumor-reactive CD8

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

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.

Small cell lung cancer (SCLC) is an aggressive disease characterized by early metastasis and exceptional lethality, comprising 13% of all lung cancer cases. With few treatment options, typically resulting in only transient responses, SCLC is responsible for approximately 250,000 deaths globally per year. The backbone of SCLC treatment over the past several decades has been platinum-based doublet chemotherapy, with the recent addition of immunotherapy to first-line chemotherapy showing limited benefit in a small subset of patients. Major hurdles to improving SCLC treatment include development of rapid chemoresistance and ineffective second line therapies. The identification of more durably effective therapeutic strategies is a major unmet clinical need. Here, we performed an in vitro CRISPR screen in SCLC cell lines from all major SCLC subtypes, including short-term cultured cells from patient-derived xenografts (PDXs), to identify potential therapeutic targets to enhance sensitivity to chemotherapy. Candidate hits were validated genetically and pharmacologically with in vitro synergy assays, in vivo clonal competition assays and pharmacologic assessments in PDX models. Signaling pathways were studied by RNA sequencing and western blot, and toxicity studies were performed in vivo to assess the safety of the agents at pharmacologically effective doses. We performed immunohistochemistry (IHC) to assess expression of candidate targets in tissue microarrays (TMAs). Our CRISPR screen revealed the nuclear exporter exportin 1 (encoded by the XPO1 gene) as a promising target sensitizing to chemotherapy, independently of the SCLC subtype. We found that XPO1 mRNA expression was higher in SCLC than in any other solid tumor or hematological malignancy, and demonstrated consistently high protein expression by IHC in clinical TMAs. A potent and selective exportin 1 inhibitor, selinexor, is approved for use in hematological malignancies. Combination of selinexor with cisplatin or irinotecan demonstrated synergy in vitro and efficacy in vivo in an array of chemonäive and chemoresistant SCLC PDXs, including all major SCLC subtypes. The combinations were well tolerated in mice. The chemo-sensitizing effects of selinexor were associated with suppression of chemotherapy-induced AKT activation. In conclusion, exportin 1 inhibition strongly enhances sensitivity of SCLC tumors to cisplatin and irinotecan, used in first line and second line treatment of SCLC tumors, respectively, and these effects are independent of the SCLC subtype. These results provide preclinical rationale for the combination of selinexor with cisplatin or irinotecan in naïve and relapsed SCLC. The clinical availability of selinexor will allow rapid clinical translation of these results in a disease setting with extremely limited therapeutic options. Citation Format: Alvaro Quintanal-Villalonga, Hirokazu Taniguchi, Yuan Hao, Andrew Chow, Yingqian A. Zhan, Fathema Uddin, Viola Allaj, Parvathy Manoj, Nisargbhai S. Shah, Umesh K. Bhanot, Juan Qiu, Elisa de Stanchina, Richard P. Koche, Triparna Sen, John T. Poirier, Charles M. Rudin. Exportin 1 inhibition as a therapeutic strategy for 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 3594.

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

Immune checkpoint therapies have shown tremendous promise in cancer therapy. However, tools to assess their target engagement, and hence the ability to predict their efficacy, have been lacking. Here, we show that target engagement and tumor-residence kinetics of antibody therapeutics targeting programmed death ligand-1 (PD-L1) can be quantified noninvasively. In computational docking studies, we observed that PD-L1–targeted monoclonal antibodies (atezolizumab, avelumab, and durvalumab) and a high-affinity PD-L1–binding peptide, WL12, have common interaction sites on PD-L1. Using the peptide radiotracer [((64))Cu]WL12 in vivo, we employed positron emission tomography (PET) imaging and biodistribution studies in multiple xenograft models and demonstrated that variable PD-L1 expression and its saturation by atezolizumab, avelumab, and durvalumab can be quantified independently of biophysical properties and pharmacokinetics of antibodies. Next, we used [((64))Cu]WL12 to evaluate the impact of time and dose on the unoccupied fraction of tumor PD-L1 during treatment. These quantitative measures enabled, by mathematical modeling, prediction of antibody doses needed to achieve therapeutically effective occupancy (defined as >90%). Thus, we show that peptide-based PET is a promising tool for optimizing dose and therapeutic regimens employing PD-L1 checkpoint antibodies, and can be used for improving therapeutic efficacy.

8509 Background: 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. The emerging consensus on SCLC subtypes has led to new questions, such as whether subtypes are associated with different disease stages, metastatic potential, or immune microenvironments; whether there is plasticity between subtypes; and whether novel SCLC phenotypes exist. Single cell RNA sequencing (scRNA-seq) offers a unique opportunity to address these questions by dissecting intratumoral transcriptional heterogeneity and the surrounding tumor microenvironment (TME). However, efforts to apply this technology to human SCLC tumors have been limited, as these tumors are infrequently resected. Methods: We have optimized protocols to process both surgical resections and biopsies to construct the first single-cell atlas of SCLC patient tumors (N = 21), with comparative lung adenocarcinoma (LUAD) and normal lung data. We leverage computational methods including diffusion maps and non-negative matrix factorization to perform a deep annotation of SCLC phenotypes and the surrounding immune TME. We perform validation experiments using flow cytometry, Vectra, and immunohistochemistry in independent SCLC cohorts, as well as genetic manipulation in preclinical SCLC models. Results: Our data reveals substantial transcriptional heterogeneity in SCLC both within and across tumors and confirms a pro-metastatic gene program in SCLC-N subtype characterized by epithelial-mesenchymal transformation and axonogenesis. Beyond known subtypes, we discover a PLCG2-high tumor cell population with stem-like, pro-metastatic features that recurs across subtypes and predicts significantly worse overall survival. 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. Conclusions: This atlas of SCLC illustrates how canonical subtypes and a novel PLCG2-high recurrent tumor subclone enlist diverse gene programs to create tumor heterogeneity and facilitate metastasis in a profoundly immunosuppressed TME. Our dataset provides further insight into tumor and immune biology in SCLC at single-cell resolution, with potential implications for design of novel targeted therapies and immunotherapeutic approaches.

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.

Background: Sialyl Lewis A (sLea) modification occurs on transmembrane and secreted proteins in pancreatic ductal adenocarcinoma (PDAC) and other GI cancers, leading to a high level of expression on cancer cells compared to normal tissue. sLea, also known as CA19-9, can be detected in patient serum using a CA19-9 diagnostic assay, which detects shed or secreted sLea modified proteins. MVT-5873, a fully human antibody that specifically targets the sLea antigen, is in a phase 1 clinical trial for the treatment of CA19-9 positive disease as a monotherapy (MVT-5873), as an imaging agent (MVT-2163), and as a radioimmunotherapy (MVT-1075). Methods: To understand the relevance of CA19-9 as a stratification marker, both annotated primary as well as patient-derived xenograft (PDX) tissue microarrays (TMAs) were stained with MVT-5873. To evaluate sLea as a response biomarker, serum samples from patients in the MVT-5873 phase 1 clinical trial were analyzed for CA19-9 levels using the diagnostic assay, and compared to their MVT-5873 PK profile. Available samples were also characterized by ELISA assay to determine the representation of sLea modification on MUC family proteins. Results: For the human CRC TMA, >75% of tumor cores displayed moderate to high levels of positive staining. In the PDX array, 21% of non-small cell lung cancer (NSCLC), 50% of small cell lung cancer (SCLC), and 69% of CRC cores stained positive. Overall, staining was observed with similar frequency regardless of mutational status of the tumor, suggesting that CA19-9 expression is independent of KRAS, BRAF, PIK3CA, or MMR pathway mutations. Staining was also relatively unchanged in lung PDX cancer tumor cores that were rendered chemoresistant compared to their chemosensitive precursor. The serum CA19-9 analysis of PDAC patients treated with MVT-5873 demonstrated that detectable CA19-9 is inversely proportional to MVT-5873 drug levels when CA19-9 levels are >100 U/ml. CA19-9 is not detected after MVT-5873 treatment when CA19-9 levels are Citation Format: H. Toni Jun, Christian Lohrmann, Viola Allaj, Wolfgang Scholz, G. Jonah Rainey, Wolfgang A. Weber, Jason S. Lewis, John Poirier, Paul W. Maffuid. Using CA19-9 as a translational biomarker for sLea targeted agents MVT-5873 and MVT-1075 in cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A062.

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.