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Omic analysis of clinical specimens undergoing histological transformation defines targetable drivers to prevent plasticity and treatment resistance.
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Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/patologíaRESUMEN
INTRODUCTION: Small Cell Lung Cancer (SCLC) can be classified into transcriptional subtypes with distinct degrees of neuroendocrine (NE) differentiation. Recent evidence supports plasticity among subtypes with a bias toward adoption of low-NE states during disease progression or upon acquired chemotherapy resistance. Here, we identify a role for SMARCA4, the catalytic subunit of the SWI/SNF complex, as a regulator of subtype shift in SCLC. METHODS: ATACseq and RNAseq experiments were performed in SCLC cells after pharmacological inhibition of SMARCA4. DNA binding of SMARCA4 was characterized by ChIPseq in high-NE SCLC patient derived xenografts (PDXs). Enrichment analyses were applied to transcriptomic data. Combination of FHD-286 and afatinib was tested in vitro and in a set of chemo-resistant SCLC PDXs in vivo. RESULTS: SMARCA4 expression positively correlates with that of NE genes in both SCLC cell lines and patient tumors. Pharmacological inhibition of SMARCA4 with FHD-286 induces the loss of NE features and downregulates neuroendocrine and neuronal signaling pathways while activating non-NE factors. SMARCA4 binds to gene loci encoding NE-lineage transcription factors ASCL1 and NEUROD1 and alters chromatin accessibility, enhancing NE programs. Enrichment analysis applied to high-confidence SMARCA4 targets confirmed neuron related pathways as the top GO Biological processes regulated by SMARCA4 in SCLC. In parallel, SMARCA4 also controls REST, a known suppressor of the NE phenotype, by regulating SRRM4-dependent REST transcript splicing. Furthermore, SMARCA4 inhibition drives ERBB pathway activation in SCLC, rendering SCLC tumors sensitive to afatinib. CONCLUSIONS: This study nominates SMARCA4 as a key regulator of the NE state plasticity and defines a novel therapeutic strategy for SCLC.
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ADN Helicasas , Neoplasias Pulmonares , Proteínas Nucleares , Carcinoma Pulmonar de Células Pequeñas , Factores de Transcripción , Humanos , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patología , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/tratamiento farmacológico , ADN Helicasas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Línea Celular Tumoral , Animales , Ratones , Regulación Neoplásica de la Expresión Génica , Proteínas RepresorasRESUMEN
PURPOSE: The impact of the intratumoral microbiome on immune checkpoint inhibitor (ICI) efficacy in patients with non-small-cell lung cancer (NSCLC) is unknown. Preclinically, intratumoral Escherichia is associated with a proinflammatory tumor microenvironment and decreased metastases. We sought to determine whether intratumoral Escherichia is associated with outcome to ICI in patients with NSCLC. PATIENTS AND METHODS: We examined the intratumoral microbiome in 958 patients with advanced NSCLC treated with ICI by querying unmapped next-generation sequencing reads against a bacterial genome database. Putative environmental contaminants were filtered using no-template controls (n = 2,378). The impact of intratumoral Escherichia detection on overall survival (OS) was assessed using univariable and multivariable analyses. The findings were further validated in an external independent cohort of 772 patients. Escherichia fluorescence in situ hybridization (FISH) and transcriptomic profiling were performed. RESULTS: In the discovery cohort, read mapping to intratumoral Escherichia was associated with significantly longer OS (16 v 11 months; hazard ratio, 0.73 [95% CI, 0.59 to 0.92]; P = .0065) in patients treated with single-agent ICI, but not combination chemoimmunotherapy. The association with OS in the single-agent ICI cohort remained statistically significant in multivariable analysis adjusting for prognostic features including PD-L1 expression (P = .023). Analysis of an external validation cohort confirmed the association with improved OS in univariable and multivariable analyses of patients treated with single-agent ICI, and not in patients treated with chemoimmunotherapy. Escherichia localization within tumor cells was supported by coregistration of FISH staining and serial hematoxylin and eosin sections. Transcriptomic analysis correlated Escherichia-positive samples with expression signatures of immune cell infiltration. CONCLUSION: Read mapping to potential intratumoral Escherichia was associated with survival to single-agent ICI in two independent cohorts of patients with NSCLC.
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Neuroendocrine (NE) transformation is a mechanism of resistance to targeted therapy in lung and prostate adenocarcinomas leading to poor prognosis. Up to date, even if patients at high risk of transformation can be identified by the occurrence of Tumor Protein P53 (TP53) and Retinoblastoma Transcriptional Corepressor 1 (RB1) mutations in their tumors, no therapeutic strategies are available to prevent or delay histological transformation. Upregulation of the cell cycle kinase Cell Division Cycle 7 (CDC7) occurred in tumors during the initial steps of NE transformation, already after TP53/RB1 co-inactivation, leading to induced sensitivity to the CDC7 inhibitor simurosertib. CDC7 inhibition suppressed NE transdifferentiation and extended response to targeted therapy in in vivo models of NE transformation by inducing the proteasome-mediated degradation of the MYC Proto-Oncogen (MYC), implicated in stemness and histological transformation. Ectopic overexpression of a degradation-resistant MYC isoform reestablished the NE transformation phenotype observed on targeted therapy, even in the presence of simurosertib. CDC7 inhibition also markedly extended response to standard cytotoxics (cisplatin, irinotecan) in lung and prostate small cell carcinoma models. These results nominate CDC7 inhibition as a therapeutic strategy to constrain lineage plasticity, as well as to effectively treat NE tumors de novo or after transformation. As simurosertib clinical efficacy trials are ongoing, this concept could be readily translated for patients at risk of transformation.
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Proteínas de Ciclo Celular , Neoplasias Pulmonares , Neoplasias de la Próstata , Proteínas Proto-Oncogénicas c-myc , Humanos , Masculino , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Línea Celular Tumoral , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ratones , Animales , Tumores Neuroendocrinos/genética , Tumores Neuroendocrinos/patología , Tumores Neuroendocrinos/metabolismo , Tumores Neuroendocrinos/tratamiento farmacológico , Proteolisis/efectos de los fármacos , Proteínas de Unión a Retinoblastoma/genética , Proteínas de Unión a Retinoblastoma/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
We report an approach for cancer phenotyping based on targeted sequencing of cell-free DNA (cfDNA) for small cell lung cancer (SCLC). In SCLC, differential activation of transcription factors (TFs), such as ASCL1, NEUROD1, POU2F3, and REST defines molecular subtypes. We designed a targeted capture panel that identifies chromatin organization signatures at 1535 TF binding sites and 13,240 gene transcription start sites and detects exonic mutations in 842 genes. Sequencing of cfDNA from SCLC patient-derived xenograft models captured TF activity and gene expression and revealed individual highly informative loci. Prediction models of ASCL1 and NEUROD1 activity using informative loci achieved areas under the receiver operating characteristic curve (AUCs) from 0.84 to 0.88 in patients with SCLC. As non-SCLC (NSCLC) often transforms to SCLC following targeted therapy, we applied our framework to distinguish NSCLC from SCLC and achieved an AUC of 0.99. Our approach shows promising utility for SCLC subtyping and transformation monitoring, with potential applicability to diverse tumor types.
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Carcinoma de Pulmón de Células no Pequeñas , Ácidos Nucleicos Libres de Células , Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Humanos , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Secuencias Reguladoras de Ácidos Nucleicos , Regulación Neoplásica de la Expresión GénicaRESUMEN
The use of 90 kDa heat shock protein (HSP90) inhibition as a therapy in lung adenocarcinoma remains limited due to moderate drug efficacy, the emergence of drug resistance, and early tumor recurrence. The main objective of this research is to maximize treatment efficacy in lung adenocarcinoma by identifying key proteins underlying HSP90 inhibition according to molecular background, and to search for potential biomarkers of response to this therapeutic strategy. Inhibition of the HSP90 chaperone was evaluated in different lung adenocarcinoma cell lines representing the most relevant molecular alterations (EGFR mutations, KRAS mutations, or EML4-ALK translocation) and wild-type genes found in each tumor subtype. The proteomic technique iTRAQ was used to identify proteomic profiles and determine which biological pathways are involved in the response to HSP90 inhibition in lung adenocarcinoma. We corroborated the greater efficacy of HSP90 inhibition in EGFR mutated or EML4-ALK translocated cell lines. We identified proteins specifically and significantly deregulated after HSP90 inhibition for each molecular alteration. Two proteins, ADI1 and RRP1, showed independently deregulated molecular patterns. Functional annotation of the altered proteins suggested that apoptosis was the only pathway affected by HSP90 inhibition across all molecular subgroups. The expression of ADI1 and RRP1 could be used to monitor the correct inhibition of HSP90 in lung adenocarcinoma. In addition, proteins such as ASS1, ITCH, or UBE2L3 involved in pathways related to the inhibition of a particular molecular background could be used as potential response biomarkers, thereby improving the efficacy of this therapeutic approach to combat lung adenocarcinoma.
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Adenocarcinoma del Pulmón , Adenocarcinoma , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Proteómica , Receptores ErbB/genética , Receptores ErbB/metabolismo , Recurrencia Local de Neoplasia/genética , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/genética , Proteínas Tirosina Quinasas Receptoras/genética , Oncogenes , Mutación , Línea Celular Tumoral , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismoRESUMEN
In lung and prostate adenocarcinomas, neuroendocrine (NE) transformation to an aggressive derivative resembling small cell lung cancer (SCLC) is associated with poor prognosis. We previously described dependency of SCLC on the nuclear transporter exportin 1. Here, we explored the role of exportin 1 in NE transformation. We observed up-regulated exportin 1 in lung and prostate pretransformation adenocarcinomas. Exportin 1 was up-regulated after genetic inactivation of TP53 and RB1 in lung and prostate adenocarcinoma cell lines, accompanied by increased sensitivity to the exportin 1 inhibitor selinexor in vitro. Exportin 1 inhibition prevented NE transformation in different TP53/RB1-inactivated prostate adenocarcinoma xenograft models that acquire NE features upon treatment with the aromatase inhibitor enzalutamide and extended response to the EGFR inhibitor osimertinib in a lung cancer transformation patient-derived xenograft (PDX) model exhibiting combined adenocarcinoma/SCLC histology. Ectopic SOX2 expression restored the enzalutamide-promoted NE phenotype on adenocarcinoma-to-NE transformation xenograft models despite selinexor treatment. Selinexor sensitized NE-transformed lung and prostate small cell carcinoma PDXs to standard cytotoxics. Together, these data nominate exportin 1 inhibition as a potential therapeutic target to constrain lineage plasticity and prevent or treat NE transformation in lung and prostate adenocarcinoma.
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Adenocarcinoma , Neoplasias Pulmonares , Neoplasias de la Próstata , Factores de Transcripción SOXB1 , Carcinoma Pulmonar de Células Pequeñas , Humanos , Masculino , Adenocarcinoma/patología , Regulación hacia Abajo , Neoplasias Pulmonares/patología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Carcinoma Pulmonar de Células Pequeñas/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Animales , Proteína Exportina 1RESUMEN
INTRODUCTION: Although targeted therapies have revolutionized the therapeutic landscape of lung adenocarcinomas (LUADs), disease progression on single-agent targeted therapy against known oncogenic drivers is common, and therapeutic options after disease progression are limited. In patients with MDM2 amplification (MDM2amp) and a concurrent oncogenic driver alteration, we hypothesized that targeting of the tumor-suppressor pathway (by means of restoration of p53 using MDM2 inhibition) and simultaneous targeting of co-occurring MAPK oncogenic pathway might represent a more durably effective therapeutic strategy. METHODS: We evaluated genomic next-generation sequencing data using the Memorial Sloan Kettering Cancer Center-Integrated Mutation Profiling of Actionable Cancer Targets platform to nominate potential targets for combination therapy in LUAD. We investigated the small molecule MDM2 inhibitor milademetan in cell lines and patient-derived xenografts of LUAD with a known driver alteration and MDM2amp. RESULTS: Of 10,587 patient samples from 7121 patients with LUAD profiled by next-generation sequencing, 6% (410 of 7121) harbored MDM2amp. MDM2amp was significantly enriched among tumors with driver alterations in METex14 (36%, p < 0.001), EGFR (8%, p < 0.001), RET (12%, p < 0.01), and ALK (10%, p < 0.01). The combination of milademetan and the MEK inhibitor trametinib was synergistic in growth inhibition of ECLC5-GLx (TRIM33-RET/MDM2amp), LUAD12c (METex14/KRASG12S/MDM2amp), SW1573 (KRASG12C, TP53 wild type), and A549 (KRASG12S) cells and in increasing expression of proapoptotic proteins PUMA and BIM. Treatment of ECLC5-GLx and LUAD12c with single-agent milademetan increased ERK phosphorylation, consistent with previous data on ERK activation with MDM2 inhibition. This ERK activation was effectively suppressed by concomitant administration of trametinib. In contrast, ERK phosphorylation induced by milademetan was not suppressed by concurrent RET inhibition using selpercatinib (in ECLC5-GLx) or MET inhibition using capmatinib (in LUAD12c). In vivo, combination milademetan and trametinib was more effective than either agent alone in ECLC5-GLx, LX-285 (EGFRex19del/MDM2amp), L13BS1 (METex14/MDM2amp), and A549 (KRASG12S, TP53 wild type). CONCLUSIONS: Combined MDM2/MEK inhibition was found to have efficacy across multiple patient-derived LUAD models harboring MDM2amp and concurrent oncogenic drivers. This combination, potentially applicable to LUADs with a wide variety of oncogenic driver mutations and kinase fusions activating the MAPK pathway, has evident clinical implications and will be investigated as part of a planned phase 1/2 clinical trial.
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Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Mutación , Progresión de la Enfermedad , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/uso terapéutico , Proteínas Proto-Oncogénicas c-mdm2/genética , Factores de Transcripción/genéticaRESUMEN
While regulatory T (Treg) cells are traditionally viewed as professional suppressors of antigen presenting cells and effector T cells in both autoimmunity and cancer, recent findings of distinct Treg cell functions in tissue maintenance suggest that their regulatory purview extends to a wider range of cells and is broader than previously assumed. To elucidate tumoral Treg cell 'connectivity' to diverse tumor-supporting accessory cell types, we explored immediate early changes in their single-cell transcriptomes upon punctual Treg cell depletion in experimental lung cancer and injury-induced inflammation. Before any notable T cell activation and inflammation, fibroblasts, endothelial and myeloid cells exhibited pronounced changes in their gene expression in both cancer and injury settings. Factor analysis revealed shared Treg cell-dependent gene programs, foremost, prominent upregulation of VEGF and CCR2 signaling-related genes upon Treg cell deprivation in either setting, as well as in Treg cell-poor versus Treg cell-rich human lung adenocarcinomas. Accordingly, punctual Treg cell depletion combined with short-term VEGF blockade showed markedly improved control of PD-1 blockade-resistant lung adenocarcinoma progression in mice compared to the corresponding monotherapies, highlighting a promising factor-based querying approach to elucidating new rational combination treatments of solid organ cancers.
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Neoplasias , Linfocitos T Reguladores , Animales , Ratones , Humanos , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Microambiente Tumoral , Neoplasias/metabolismoRESUMEN
Paired single-cell RNA and T cell receptor sequencing (scRNA/TCR-seq) has allowed for enhanced resolution of clonal T cell dynamics in cancer. Here, we report a scRNA/TCR-seq analysis of 187,650 T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients with non-small cell lung cancer after immune checkpoint blockade (ICB). Regions with viable cancer cells are enriched for exhausted CD8+ T cells, regulatory CD4+ T cells (Treg), and follicular helper CD4+ T cells (TFH). Tracking T cell clonotypes across tissues, combined with neoantigen specificity assays, reveals that TFH and tumor-specific exhausted CD8+ T cells are clonally linked to TCF7+SELL+ progenitors in tumor draining LNs, and progressive exhaustion trajectories of CD8+ T, Treg, and TFH cells with proximity to the tumor microenvironment. Finally, longitudinal tracking of tumor-specific CD8+ and CD4+ T cell clones reveals persistence in the peripheral blood for years after ICB therapy.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Linfocitos T CD8-positivos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Receptores de Antígenos de Linfocitos T , Células Clonales , Microambiente TumoralRESUMEN
Improved identification of anti-tumor T cells is needed to advance cancer immunotherapies. CD39 expression is a promising surrogate of tumor-reactive CD8+ T cells. Here, we comprehensively profiled CD39 expression in human lung cancer. CD39 expression enriched for CD8+ T cells with features of exhaustion, tumor reactivity, and clonal expansion. Flow cytometry of 440 lung cancer biospecimens revealed weak association between CD39+ CD8+ T cells and tumoral features, such as programmed death-ligand 1 (PD-L1), tumor mutation burden, and driver mutations. Immune checkpoint blockade (ICB), but not cytotoxic chemotherapy, increased intratumoral CD39+ CD8+ T cells. Higher baseline frequency of CD39+ CD8+ T cells conferred improved clinical outcomes from ICB therapy. Furthermore, a gene signature of CD39+ CD8+ T cells predicted benefit from ICB, but not chemotherapy, in a phase III clinical trial of non-small cell lung cancer. These findings highlight CD39 as a proxy of tumor-reactive CD8+ T cells in human lung cancer.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Linfocitos T CD8-positivos , InmunoterapiaRESUMEN
Neuroendocrine neoplasms (NENs) can develop in almost any organ and span a spectrum from well-differentiated and indolent neuroendocrine tumours (NETs) to poorly differentiated and highly aggressive neuroendocrine carcinomas (NECs), including small-cell lung cancer (SCLC). These neoplasms are thought to primarily derive from neuroendocrine precursor cells located throughout the body and can also arise through neuroendocrine transdifferentiation of organ-specific epithelial cell types. Hence, NENs constitute a group of tumour types that share key genomic and phenotypic characteristics irrespective of their site of origin, albeit with some organ-specific differences. The establishment of representative preclinical models for several of these disease entities together with analyses of human tumour specimens has provided important insights into crucial aspects of their biology with therapeutic implications. In this Review, we provide a comprehensive overview of the current understanding of NENs of the gastrointestinal system and lung from clinical and biological perspectives. Research on NENs has typically been siloed by the tumour site of origin, and a cross-cutting view might enable advances in one area to accelerate research in others. Therefore, we aim to emphasize that a better understanding of the commonalities and differences of NENs arising in different organs might more effectively inform clinical research to define therapeutic targets and ultimately optimize patient care.
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Carcinoma Neuroendocrino , Neoplasias Gastrointestinales , Neoplasias Pulmonares , Tumores Neuroendocrinos , Neoplasias Pancreáticas , Carcinoma Pulmonar de Células Pequeñas , Humanos , Tumores Neuroendocrinos/patología , Neoplasias Pulmonares/patología , Pulmón/metabolismo , Pulmón/patología , Biología , Neoplasias Pancreáticas/patologíaRESUMEN
We report a protocol for obtaining high-quality single-cell transcriptomics data from human lung biospecimens acquired from core needle biopsies, fine-needle aspirates, surgical resection, and pleural effusions. The protocol relies upon the brief mechanical and enzymatic disruption of tissue, enrichment of live cells by fluorescence-activated cell sorting (FACS), and droplet-based single-cell RNA sequencing (scRNA-seq). The protocol also details a procedure for analyzing the scRNA-seq data. For complete details on the use and execution of this protocol, please refer to Chan et al. (2021).
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Perfilación de la Expresión Génica , Pulmón , Humanos , Análisis de Secuencia de ARN/métodos , RNA-Seq , Perfilación de la Expresión Génica/métodos , Biopsia con Aguja Fina/métodosRESUMEN
PURPOSE: RB1 mutations and loss of retinoblastoma (Rb) expression represent consistent but not entirely invariable hallmarks of small cell lung cancer (SCLC). The prevalence and characteristics of SCLC retaining wild-type Rb are not well-established. Furthermore, the performance of targeted next-generation sequencing (NGS) versus immunohistochemistry for Rb assessment is not well-defined. EXPERIMENTAL DESIGN: A total of 208 clinical SCLC samples were analyzed by comprehensive targeted NGS, covering all exons of RB1, and Rb IHC. On the basis of established coordination of Rb/p16/cyclinD1 expression, p16-high/cyclinD1-low profile was used as a marker of constitutive Rb deficiency. RESULTS: Fourteen of 208 (6%) SCLC expressed wild-type Rb, accompanied by a unique p16-low/cyclinD1-high profile supporting Rb proficiency. Rb-proficient SCLC was associated with neuroendocrine-low phenotype, combined SCLC with non-SCLC (NSCLC) histology and aggressive behavior. These tumors exclusively harbored CCND1 amplification (29%), and were markedly enriched in CDKN2A mutations (50%) and NSCLC-type alterations (KEAP1, STK11, FGFR1). The remaining 194 of 208 SCLC were Rb-deficient (p16-high/cyclinD1-low), including 184 cases with Rb loss (of which 29% lacked detectable RB1 alterations by clinical NGS pipeline), and 10 cases with mutated but expressed Rb. CONCLUSIONS: This is the largest study to date to concurrently analyze Rb by NGS and IHC in SCLC, identifying a 6% rate of Rb proficiency. Pathologic-genomic data implicate NSCLC-related progenitors as a putative source of Rb-proficient SCLC. Consistent upstream Rb inactivation via CDKN2A/p16↓ and CCND1/cyclinD1↑ suggests the potential utility of CDK4/6 inhibitors in this aggressive SCLC subset. The study also clarifies technical aspects of Rb status determination in clinical practice, highlighting the limitations of exon-only sequencing for RB1 interrogation. See related commentary by Mahadevan and Sholl, p. 4603.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Neoplasias de la Retina , Retinoblastoma , Carcinoma Pulmonar de Células Pequeñas , Humanos , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/patología , Inmunohistoquímica , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Genómica , Neoplasias Pulmonares/patologíaRESUMEN
INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
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Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I , Histona Demetilasas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Animales , Antígenos de Neoplasias , Antígeno B7-H1 , Genes MHC Clase I , Antígenos de Histocompatibilidad Clase I/genética , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Humanos , Neoplasias Pulmonares/patología , Ratones , Carcinoma Pulmonar de Células Pequeñas/patologíaRESUMEN
Non-small-cell lung cancer (NSCLC) is the leading cause of cancer death worldwide, generating an enormous economic and social impact that has not stopped growing in recent years. Cancer treatment for this neoplasm usually includes surgery, chemotherapy, molecular targeted treatments, and ionizing radiation. The prognosis in terms of overall survival (OS) and the disparate therapeutic responses among patients can be explained, to a great extent, by the existence of widely heterogeneous molecular profiles. The main objective of this study was to identify prognostic and predictive gene signatures of response to cancer treatment involving radiotherapy, which could help in making therapeutic decisions in patients with NSCLC. To achieve this, we took as a reference the differential gene expression pattern among commercial cell lines, differentiated by their response profile to ionizing radiation (radiosensitive versus radioresistant lines), and extrapolated these results to a cohort of 107 patients with NSCLC who had received radiotherapy (among other therapies). We obtained a six-gene signature (APOBEC3B, GOLM1, FAM117A, KCNQ1OT1, PCDHB2, and USP43) with the ability to predict overall survival and progression-free survival (PFS), which could translate into a prediction of the response to the cancer treatment received. Patients who had an unfavorable prognostic signature had a median OS of 24.13 months versus 71.47 months for those with a favorable signature, and the median PFS was 12.65 months versus 47.11 months, respectively. We also carried out a univariate analysis of multiple clinical and pathological variables and a bivariate analysis by Cox regression without any factors that substantially modified the HR value of the proposed gene signature.
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Small cell lung cancers (SCLCs) have high mutational burden but are relatively unresponsive to immune checkpoint blockade (ICB). Using SCLC models, we demonstrate that inhibition of WEE1, a G2/M checkpoint regulator induced by DNA damage, activates the STING-TBK1-IRF3 pathway, which increases type I interferons (IFN-α and IFN-ß) and pro-inflammatory chemokines (CXCL10 and CCL5), facilitating an immune response via CD8+ cytotoxic T cell infiltration. We further show that WEE1 inhibition concomitantly activates the STAT1 pathway, increasing IFN-γ and PD-L1 expression. Consistent with these findings, combined WEE1 inhibition (AZD1775) and PD-L1 blockade causes remarkable tumor regression, activation of type I and II interferon pathways, and infiltration of cytotoxic T cells in multiple immunocompetent SCLC genetically engineered mouse models, including an aggressive model with stabilized MYC. Our study demonstrates cell-autonomous and immune-stimulating activity of WEE1 inhibition in SCLC models. Combined inhibition of WEE1 plus PD-L1 blockade represents a promising immunotherapeutic approach in SCLC.
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Protocolos de Quimioterapia Combinada Antineoplásica , Antígeno B7-H1 , Proteínas de Ciclo Celular , Neoplasias Pulmonares , Proteínas de la Membrana , Proteínas Tirosina Quinasas , Factor de Transcripción STAT1 , Carcinoma Pulmonar de Células Pequeñas , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Sinergismo Farmacológico , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas de la Membrana/metabolismo , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT1/metabolismo , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/inmunología , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patologíaRESUMEN
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.
Asunto(s)
Carioferinas/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/metabolismo , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Animales , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/patología , Ratones , Carcinoma Pulmonar de Células Pequeñas/patología , Proteína Exportina 1RESUMEN
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.
Asunto(s)
Adenocarcinoma del Pulmón/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Transdiferenciación Celular , Humanos , Ratones Endogámicos NOD , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-myc/genética , Transducción de Señal , TranscriptomaRESUMEN
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.