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-β) 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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•WEE1 inhibition causes activation of STING and STAT1 pathways in SCLC•WEE1 inhibitor plus ICB mediates the innate and adaptive anti-immune response•Combined WEE1 inhibitor plus ICB suppresses tumor growth in SCLC mouse models
Taniguchi et al. show that WEE1 inhibition activates cGAS-STING, which increases type I interferons and pro-inflammatory chemokines, facilitating an immune response via CD8+ cytotoxic T cell infiltration in SCLC. WEE1 inhibition concomitantly activates STAT1 signaling, increasing IFN-γ and PD-L1 expression, and enhances the efficacy of PD-L1 blockade in SCLC mouse models.
Access to clinically relevant small cell lung cancer (SCLC) tissue is limited because surgical resection is rare in metastatic SCLC. Patient-derived xenografts (PDX) and circulating tumor ...cell-derived xenografts (CDX) have emerged as valuable tools to characterize SCLC. Here, we present a resource of 46 extensively annotated PDX/CDX models derived from 33 patients with SCLC. We perform multi-omic analyses, using targeted tumor next-generation sequencing, RNA-sequencing, and immunohistochemistry to deconvolute the mutational landscapes, global expression profiles, and molecular subtypes of these SCLC models. SCLC subtypes characterized by transcriptional regulators, ASCL1, NEUROD1 and POU2F3 are confirmed in this cohort. A subset of SCLC clinical specimens, including matched PDX/CDX and clinical specimen pairs, confirm that the primary features and genomic and proteomic landscapes of the tumors of origin are preserved in the derivative PDX models. This resource provides a powerful system to study SCLC biology.
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.
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.
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.
Our findings provide the first comprehensive molecular characterization of LUSC transdifferentiation, suggesting putative drivers and potential therapeutic targets to constrain or prevent lineage plasticity.
Diffuse pleural mesothelioma (DPM) is an aggressive malignancy that, despite recent treatment advances, has unacceptably poor outcomes. Therapeutic research in DPM is inhibited by a paucity of ...preclinical models that faithfully recapitulate the human disease.
We established 22 patient-derived xenografts (PDX) from 22 patients with DPM and performed multi-omic analyses to deconvolute the mutational landscapes, global expression profiles, and molecular subtypes of these PDX models and compared features to those of the matched primary patient tumors. Targeted next-generation sequencing (NGS; MSK-IMPACT), immunohistochemistry, and histologic subtyping were performed on all available samples. RNA sequencing was performed on all available PDX samples. Clinical outcomes and treatment history were annotated for all patients. Platinum-doublet progression-free survival (PFS) was determined from the start of chemotherapy until radiographic/clinical progression and grouped into < or ≥ 6 months.
PDX models were established from both treatment naïve and previously treated samples and were noted to closely resemble the histology, genomic landscape, and proteomic profiles of the parent tumor. After establishing the validity of the models, transcriptomic analyses demonstrated overexpression in WNT/β-catenin, hedgehog, and TGF-β signaling and a consistent suppression of immune-related signaling in PDXs derived from patients with worse clinical outcomes.
These data demonstrate that DPM PDX models closely resemble the genotype and phenotype of parental tumors, and identify pathways altered in DPM for future exploration in preclinical studies.
Abstract 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.
Small-cell lung cancer (SCLC) is a high-grade neuroendocrine tumor with dismal prognosis and limited treatment options. Lurbinectedin, conditionally approved as a second-line treatment for metastatic ...SCLC, drives clinical responses in about 35% of patients, and the overall survival (OS) of those who benefit from it remains very low (∼9.3 months). This finding highlights the need to develop improved mechanistic insight and predictive biomarkers of response.
We used human and patient-derived xenograft (PDX)-derived SCLC cell lines to evaluate the effect of lurbinectedin in vitro. We also demonstrate the antitumor effect of lurbinectedin in multiple de novo and transformed SCLC PDX models. Changes in gene and protein expression pre- and post-lurbinectedin treatment was assessed by RNA sequencing and Western blot analysis.
Lurbinectedin markedly reduced cell viability in the majority of SCLC models with the best response on POU2F3-driven SCLC cells. We further demonstrate that lurbinectedin, either as a single agent or in combination with osimertinib, causes an appreciable antitumor response in multiple models of EGFR-mutant lung adenocarcinoma with histologic transformation to SCLC. Transcriptomic analysis identified induction of apoptosis, repression of epithelial-mesenchymal transition, modulation of PI3K/AKT, NOTCH signaling associated with lurbinectedin response in de novo, and transformed SCLC models.
Our study provides a mechanistic insight into lurbinectedin response in SCLC and the first demonstration that lurbinectedin is a potential therapeutic target after SCLC transformation.
8042 Background: Tumor plasticity, especially histological transformation, is an emergent mechanism of therapeutic resistance. Lung adenocarcinoma (LUAD) can transform to small cell lung cancer ...(SCLC), a more aggressive tumor type of neuroendocrine (NE) histology, leading to EGFR inhibitor resistance and a more aggressive disease phenotype. TP53 and RB1 loss are enriched but insufficient for NE transformation, and it remains unknown what additional molecular changes underlie plasticity. We use single-cell RNA sequencing (scRNA-seq) to profile the intratumoral transcriptional heterogeneity of NE transformation across multiple time points. Methods: We performed 10X scRNA-seq in patient samples to create an atlas of NE plasticity (47 transformed and admixed LUAD-SCLC, 10 EGFR/RB1/TP53 mut LUAD, 17 de novo SCLC, 4 normal lung). Our pipeline performed ambient RNA removal, outlier filtering, normalization, and batch correction. Tumor cells from transformed and admixed LUAD-SCLC tumors were divided into the LUAD compartment prior to transformation (pre-transformed LUAD) and the transformed SCLC compartment based on acquisition of NE marker expression. We performed copy-number inference, pseudotime analysis, and estimation of differentiation potential to establish dynamic changes in phenotypic and subclonal architecture across longitudinal timepoints. Results: We captured 12,105 (9%) pre-transformed LUAD cells from transformed and admixed LUAD-SCLC tumors. Canonical squamous cell carcinoma genes (KRT5, KRT6A, TP63, SERPINB4) were found to be enriched in this pre-transformed LUAD compartment. By projecting a force-directed layout of cells from these samples, we modeled the transition between LUAD, LUSC, and SCLC cell types. Genetic pathways promoting angiogenesis, inflammatory signaling, epithelial-to-mesenchymal transition, and RAS/RAF signaling were highly overexpressed during LUAD-LUSC transformation. Furthermore, we show that macrophages from transforming LUAD-LUSC tumors exhibit a pro-fibrotic phenotype. CNV inference revealed distinct subclonal populations in pre-transformed LUAD tumors. Subclones with high CNV burden and high differentiation potential represented early plastic cells whose lineage could be traced to matched longitudinal post-transformation samples. Conclusions: There is remarkable intratumoral heterogeneity within transforming LUAD. Squamous transformation, mediated by early RAS/RAF signaling, may represent a precursor to SCLC transformation. Identifying early hallmarks of plasticity can help stratify patients at high risk for transformation and reveal therapeutic vulnerabilities.
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+ 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.
•CD39+ CD8+ T cells express features of exhaustion and tumor reactivity•CD39 expression enriches for CD8+ TCRs with tumor reactivity•CD39 expression on CD8+ T cells is non-redundant to tumor-based biomarkers•CD39+ CD8+ T cells are predictive of benefit from ICB
Factors predicting benefit of immune checkpoint blockade (ICB) are needed. Here, Chow et al. demonstrate that CD39 expression marks tumor-reactive CD8+ T cells. High baseline levels of CD39+ CD8+ T cells are associated with ICB efficacy in lung cancer. Thus, CD39 is a potential tumor-extrinsic biomarker for guiding cancer management.
e20105 Background: Neuroendocrine (NE) transformation occurs as a mechanism of resistance to targeted therapy in up to 14% and 30% of EGFR-mutant lung and AR-dependent prostate adenocarcinomas, ...respectively, leading to poor prognosis. Even if we know the tumor population at high risk of transformation ( TP53/RB1-mutated), no therapies to prevent NE relapse are currently available. Methods: To identify therapeutic vulnerabilities for tumors undergoing NE transformation, we performed an in vitro CRISPR screen in a NE-transformed lung tumor model, followed by functional validation in in vivo prostate and lung models of NE transformation, including pharmacologic as well as genetic (isogenic cell line with overexpression or knock of CDC7) validation. Results: Our screen identified CDC7, involved in DNA replication and DNA damage response, as a potential therapeutic target in this setting. Proteogenomic analyses revealed CDC7 upregulation in lung and prostate clinical samples undergoing NE transformation, detected already in pre-transformation adenocarcinomas. Importantly, TP53/RB1-inactivation induced sensitivity to the CDC7 inhibitor simurosertib, unraveling a therapeutic vulnerability in tumors at high risk of NE transformation. Thus, we tested the combination of simurosertib with targeted therapy in vivo in different lung and prostate patient-derived models of NE transformation. In these, simurosertib was able to suppress NE transformation and dramatically delay tumor relapse. Trajectory analysis on single-cell transcriptomic data for such models revealed a NE transformation transcriptional program occurring already in the untreated tumors before transformation. CDC7 inhibition led to increased proteasomal activity and degradation of MYC, a stemness transcription factor involved in NE transformation. Ectopic overexpression of MYC T58A , a proteasome degradation-resistant MYC isoform, rescued the NE phenotype in these transformation models, suggesting that CDC7 inhibition-induced MYC degradation is the mechanism by which NE transformation is prevented. Conclusions: In sum, CDC7 inhibition may suppress, or at least dramatically delay NE transformation in patients with lung and prostate adenocarcinomas at high risk of transformation, by inducing MYC proteasomal degradation. The clinical availability of CDC7 inhibitors, currently in phase II clinical trials after demonstrating tolerability and preliminary efficacy, will allow rapid translation of these results into the clinics.
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