Eradicating tumor dormancy that develops following epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment of EGFR-mutant non-small cell lung cancer, is an attractive ...therapeutic strategy but the mechanisms governing this process are poorly understood. Blockade of ERK1/2 reactivation following EGFR TKI treatment by combined EGFR/MEK inhibition uncovers cells that survive by entering a senescence-like dormant state characterized by high YAP/TEAD activity. YAP/TEAD engage the epithelial-to-mesenchymal transition transcription factor SLUG to directly repress pro-apoptotic BMF, limiting drug-induced apoptosis. Pharmacological co-inhibition of YAP and TEAD, or genetic deletion of YAP1, all deplete dormant cells by enhancing EGFR/MEK inhibition-induced apoptosis. Enhancing the initial efficacy of targeted therapies could ultimately lead to prolonged treatment responses in cancer patients.
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•Loss of EGFR signaling leads to senescence-like dormancy in EGFR-mutant lung cancer•YAP promotes survival and dormancy in the absence of EGFR downstream signaling•YAP/TEAD/SLUG suppress apoptosis through transcriptional repression of BMF•A TEAD inhibitor enhances EGFR inhibitor-mediated apoptosis and prevents dormancy
Kurppa et al. show that YAP activation mediates resistance to combined EGFR/MEK inhibition by inducing dormancy in non-small-cell lung cancer cells. Targeting the YAP pathway, in part by using a newly developed covalent TEAD inhibitor, promotes apoptosis of the dormant therapy-resistant cancer cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Microfluidic culture has the potential to revolutionize cancer diagnosis and therapy. Indeed, several microdevices are being developed specifically for clinical use to test novel cancer therapeutics. ...To be effective, these platforms need to replicate the continuous interactions that exist between tumor cells and non-tumor cell elements of the tumor microenvironment through direct cell-cell or cell-matrix contact or by the secretion of signaling factors such as cytokines, chemokines and growth factors. Given the challenges of personalized or precision cancer therapy, especially with the advent of novel immunotherapies, a critical need exists for more sophisticated
ex vivo
diagnostic systems that recapitulate patient-specific tumor biology with the potential to predict response to immune-based therapies in real-time. Here, we present details of a method to screen for the response of patient tumors to immune checkpoint blockade therapy, first reported in Jenkins
et al. Cancer Discovery
, 2018,
8
, 196-215, with updated evaluation of murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS), including evaluation of the requirement for 3D microfluidic culture in MDOTS, demonstration of immune-checkpoint sensitivity of PDOTS, and expanded evaluation of tumor-immune interactions using RNA-sequencing to infer changes in the tumor-immune microenvironment. We also examine some potential improvements to current systems and discuss the challenges in translating such diagnostic assays to the clinic.
Microfluidic culture has the potential to revolutionize cancer diagnosis and therapy.
Evaluating drug responses using primary patient-derived cells
represents a potentially rapid and efficient approach to screening for new treatment approaches. Here, we sought to identify neratinib ...combinations in
mutant non-small cell lung cancer (NSCLC) patient
enograft-
erived
rganotypic
pheroids (XDOTS) using a short-term
system.
We generated two
mutant NSCLC PDX models DFCI359 (
exon19 755_757LREdelinsRP) and DFCI315 (
exon20 V777_G778insGSP) and used the PDX tumors to generate XDOTS. Tumor spheroids were grown in a microfluidic device and treated
with neratinib-based drug combinations. Live/dead quantification was performed by dual-labeling deconvolution fluorescence microscopy. The most efficacious
combination was subsequently validated
using the DFCI359 and DFCI315 PDXs and a
genetically engineered mouse model.
Both neratinib and afatinib, but not gefitinib, induced cell death in DFCI359 XDOTS. The combinations of neratinib/trastuzumab and neratinib/temsirolimus enhanced the therapeutic benefit of neratinib alone in DFCI315 and DFCI359. The combination of neratinib and trastuzumab
was more effective compared with single-agent neratinib or trastuzumab and was associated with more robust inhibition of HER2 and downstream signaling.
The XDOTS platform can be used to evaluate therapies and therapeutic combinations
using PDX tumors. This approach may accelerate the identification and clinical development of therapies for targets with no or few existing models and/or therapies.
Small cell lung carcinoma (SCLC) is highly mutated, yet durable response to immune checkpoint blockade (ICB) is rare. SCLC also exhibits cellular plasticity, which could influence its immunobiology. ...Here we discover that a distinct subset of SCLC uniquely upregulates MHC I, enriching for durable ICB benefit.
modeling confirms epigenetic recovery of MHC I in SCLC following loss of neuroendocrine differentiation, which tracks with derepression of STING. Transient EZH2 inhibition expands these nonneuroendocrine cells, which display intrinsic innate immune signaling and basally restored antigen presentation. Consistent with these findings, murine nonneuroendocrine SCLC tumors are rejected in a syngeneic model, with clonal expansion of immunodominant effector CD8 T cells. Therapeutically, EZH2 inhibition followed by STING agonism enhances T-cell recognition and rejection of SCLC in mice. Together, these data identify MHC I as a novel biomarker of SCLC immune responsiveness and suggest novel immunotherapeutic approaches to co-opt SCLC's intrinsic immunogenicity. SIGNIFICANCE: SCLC is poorly immunogenic, displaying modest ICB responsiveness with rare durable activity. In profiling its plasticity, we uncover intrinsically immunogenic MHC I
subpopulations of nonneuroendocrine SCLC associated with durable ICB benefit. We also find that combined EZH2 inhibition and STING agonism uncovers this cell state, priming cells for immune rejection.
.
Single-gene missense mutations remain challenging to interpret. Here, we deploy scalable functional screening by sequencing (SEUSS), a Perturb-seq method, to generate mutations at protein interfaces ...of RUNX1 and quantify their effect on activities of downstream cellular programs. We evaluate single-cell RNA profiles of 115 mutations in myelogenous leukemia cells and categorize them into three functionally distinct groups, wild-type (WT)-like, loss-of-function (LoF)-like, and hypomorphic, that we validate in orthogonal assays. LoF-like variants dominate the DNA-binding site and are recurrent in cancer; however, recurrence alone does not predict functional impact. Hypomorphic variants share characteristics with LoF-like but favor protein interactions, promoting gene expression indicative of nerve growth factor (NGF) response and cytokine recruitment of neutrophils. Accessible DNA near differentially expressed genes frequently contains RUNX1-binding motifs. Finally, we reclassify 16 variants of uncertain significance and train a classifier to predict 103 more. Our work demonstrates the potential of targeting protein interactions to better define the landscape of phenotypes reachable by missense mutations.
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•scRNA-seq profiling of 115 variants at protein interfaces of the RUNX1 transcription factor•Clustering identifies transcriptionally distinct groups and uncovers hypomorphic variants•Hypomorphic variants alter gene programs associated with cytokine and growth factor signaling•scRNA-seq-based assay informs reclassification of variants of uncertain significance
Deciphering distinct effects of single-gene missense mutations is challenging. Ozturk et al. design an interface-guided Perturb-seq library and measure the impact of >100 RUNX1 variants on >40,000 single-cell RNA profiles. The assay identifies functionally distinct groups, demonstrating the potential of scRNA-seq to characterize cellular phenotypes reachable by individual mutations.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Treatment of cancer has been revolutionized by immune checkpoint blockade therapies. Despite the high rate of response in advanced melanoma, the majority of patients succumb to disease. To identify ...factors associated with success or failure of checkpoint therapy, we profiled transcriptomes of 16,291 individual immune cells from 48 tumor samples of melanoma patients treated with checkpoint inhibitors. Two distinct states of CD8+ T cells were defined by clustering and associated with patient tumor regression or progression. A single transcription factor, TCF7, was visualized within CD8+ T cells in fixed tumor samples and predicted positive clinical outcome in an independent cohort of checkpoint-treated patients. We delineated the epigenetic landscape and clonality of these T cell states and demonstrated enhanced antitumor immunity by targeting novel combinations of factors in exhausted cells. Our study of immune cell transcriptomes from tumors demonstrates a strategy for identifying predictors, mechanisms, and targets for enhancing checkpoint immunotherapy.
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•Single-cell RNA-seq reveals distinct CD45+ cells associated with clinical outcome•The balance between two CD8+ T cell states is linked with tumor regression•TCF7+CD8+ T cell frequency in tumor tissue predicts response and better survival•Dual blockade of CD39 with different checkpoint proteins enhances immunity
Single-cell analysis of immune cells from melanoma patients treated with immune checkpoint therapy uncovers a TCF7+ memory-like state in the cytotoxic T cell population and demonstrates its association with a positive outcome.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Resistance to oncogene-targeted therapies involves discrete drug-tolerant persister cells, originally discovered through in vitro assays. Whether a similar phenomenon limits efficacy of programmed ...cell death 1 (PD-1) blockade is poorly understood. Here, we performed dynamic single-cell RNA-Seq of murine organotypic tumor spheroids undergoing PD-1 blockade, identifying a discrete subpopulation of immunotherapy persister cells (IPCs) that resisted CD8+ T cell-mediated killing. These cells expressed Snai1 and stem cell antigen 1 (Sca-1) and exhibited hybrid epithelial-mesenchymal features characteristic of a stem cell-like state. IPCs were expanded by IL-6 but were vulnerable to TNF-α-induced cytotoxicity, relying on baculoviral IAP repeat-containing protein 2 (Birc2) and Birc3 as survival factors. Combining PD-1 blockade with Birc2/3 antagonism in mice reduced IPCs and enhanced tumor cell killing in vivo, resulting in durable responsiveness that matched TNF cytotoxicity thresholds in vitro. Together, these data demonstrate the power of high-resolution functional ex vivo profiling to uncover fundamental mechanisms of immune escape from durable anti-PD-1 responses, while identifying IPCs as a cancer cell subpopulation targetable by specific therapeutic combinations.
Abstract
Background: The complex interaction between tumor cells and the immune system has led to increased efforts focused on development of ex vivo systems that recapitulate the tumor ...microenvironment and model responses to immune checkpoint blockade. Recently, we demonstrated successful ex vivo growth of organotypic tumor spheroids and profiling of PD-1 blockade in syngeneic models and patient tumors1. Optimization of tissue processing from the operating room to the lab for patient-derived materials can further aid these efforts. Here, we identify pre-analytical variables that may impact tumor characterization in ex vivo systems.
Methods: We conducted an evaluation of pre-analytical variables for tumor tissue in a cohort of mesothelioma and non-small cell lung cancer (NSCLC) cases. Pre-analytical variables included neoadjuvant chemotherapy administration, pleurodesis status, type of surgery, presence of fibrosis, and onset of warm and cold ischemia during tissue retrieval and processing. Prior to surgery, medical records were reviewed for pathology, chemotherapy, and pleurodesis status. On surgery day, time of tissue removal or clamping of the pulmonary artery was recorded as onset of warm ischemia. Tissue was transported to pathology and allocations to research were made. Specimens were placed in culture media and ice; placement on ice was recorded as onset of cold ischemia. Arrival of tissue in lab was recorded. Chart and observational data were correlated with tissue analysis at the bench followed by 4-color immunofluorescence to characterize tumor and immune components of spheroids.
Results: A cohort of mesothelioma and NSCLC samples were followed from the operating room to the research lab. The average time between onset of warm and cold ischemia was 60 minutes (range 33-100 min). Mean time for removal of tissue in the OR to arrival in lab was 104 minutes (range 87-137 min). Two patients received neoadjuvant chemotherapy and were among 2 of 3 cases where fibrotic samples were collected. Fibrosis was often associated with more extensive surgery via extrapleural pneumonectomy and lower tumor content per microscopy and immunofluorescence.
Conclusions: Prolonged ischemic times were associated with poor tissue viability. In two cases, pre-therapeutic status was linked to tissue fibrosis, extensive surgery, low tumor yield, and failed spheroid generation. More data can help elucidate variable significance. These initial findings highlight the unique challenges associated with performing studies on live tumor tissue, and suggest that further streamlining of processes for tissue collection in the OR can optimize success of ex vivo cancer models in guiding functional precision therapies.
Citation Format: Dalia Larios, Amir Aref, Elena Ivanova, Brandon Piel, Andrew Portell, David A. Barbie, Raphael Bueno, Cloud Paweletz. From OR to bench: Identification of variables affecting success in generating patient derived organotypic spheroids (pDOTS) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5025.
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