BAP1 Deficiency Inflames the Tumor Immune Microenvironment and Is a Candidate Biomarker for Immunotherapy Response in Malignant Pleural Mesothelioma.

Introduction: Malignant pleural mesothelioma (MPM) is a rare and universally lethal malignancy with limited treatment options. Immunotherapy with immune checkpoint inhibitors (ICIs) has recently been approved for unresectable MPM, but response to ICIs is heterogeneous, and reliable biomarkers for prospective selection of appropriate subpopulations likely to benefit from ICIs remain elusive. Methods: We performed multiscale integrative analyses of published primary tumor data set from The Cancer Genome Atlas (TCGA) and the French cohort E-MTAB-1719 to unravel the tumor immune microenvironment of MPM deficient in BAP1, one of the most frequently mutated tumor suppressor genes (TSGs) in the disease. The molecular profiling results were validated in independent cohorts of patients with MPM using immunohistochemistry and multiplex immunohistochemistry. Results: We revealed that BAP1 deficiency enriches immune-associated pathways in MPM, leading to increased mRNA signatures of interferon alfa/gamma response, activating dendritic cells, immune checkpoint receptors, and T-cell inflammation. This finding was confirmed in independent patient cohorts, where MPM tumors with low BAP1 levels are associated with an inflammatory tumor immune microenvironment characterized by increased exhausted precursor T-cells and macrophages but decreased myeloid-derived suppressor cells (MDSCs). In addition, BAP1low MPM cells are in close proximity to T cells and therefore can potentially be targeted with ICIs. Finally, we revealed that BAP1-proficient MPM is associated with a hyperactive mitogen-activated protein kinase (MAPK) pathway and may benefit from treatment with MEK inhibitors (MEKis). Conclusion: Our results suggest that BAP1 plays an immunomodulatory role in MPM and that BAP1-deficient MPM may benefit from immunotherapy, which merits further clinical investigation.

CRISPRing KRAS: A Winding Road with a Bright Future in Basic and Translational Cancer Research.

Once considered "undruggable" due to the strong affinity of RAS proteins for GTP and the structural lack of a hydrophobic "pocket" for drug binding, the development of proprietary therapies for KRAS-mutant tumors has long been a challenging area of research. CRISPR technology, the most successful gene-editing tool to date, is increasingly being utilized in cancer research. Here, we provide a comprehensive review of the application of the CRISPR system in basic and translational research in KRAS-mutant cancer, summarizing recent advances in the mechanistic understanding of KRAS biology and the underlying principles of drug resistance, anti-tumor immunity, epigenetic regulatory networks, and synthetic lethality co-opted by mutant KRAS.

An optimized protocol for the generation and monitoring of conditional orthotopic lung cancer in the KP mouse model using an adeno-associated virus vector compatible with biosafety level 1.

BACKGROUND: The inducible Kras/p53 lung adenocarcinoma mouse model, which faithfully recapitulates human disease, is routinely initiated by the intratracheal instillation of a virus-based Cre recombinase delivery system. Handling virus-based delivery systems requires elevated biosafety levels, e.g., biosafety level 2 (BSL-2). However, in experimental animal research facilities, following exposure to viral vectors in a BSL-2 environment, rodents may not be reclassified to BSL-1 according to standard practice, preventing access to small animal micro-computed tomography (micro-CT) scanners that are typically housed in general access areas such as BSL-1 rooms. Therefore, our goal was to adapt the protocol so that the Cre-induced KP mouse model could be handled under BSL-1 conditions during the entire procedure. RESULTS: The Kras-Lox-STOP-Lox-G12D/p53 flox/flox (KP)-based lung adenocarcinoma mouse model was activated by intratracheal instillation of either an adenoviral-based or a gutless, adeno-associated viral-based Cre delivery system. Tumor growth was monitored over time by micro-CT. We have successfully substituted the virus-based Cre delivery system with a commercially available, gutless, adeno-associated, Cre-expressing vector that allows the KP mouse model to be handled and imaged in a BSL-1 facility. By optimizing the anesthesia protocol and switching to a microscope-guided vector instillation procedure, productivity was increased and procedure-related complications were significantly reduced. In addition, repeated micro-CT analysis of individual animals allowed us to monitor tumor growth longitudinally, dramatically reducing the number of animals required per experiment. Finally, we documented the evolution of tumor volume for different doses, which revealed that individual tumor nodules induced by low-titer AAV-Cre transductions can be monitored over time by micro-CT. CONCLUSION: Modifications to the anesthesia and instillation protocols increased the productivity of the original KP protocol. In addition, the switch to a gutless, adeno-associated, Cre-expressing vector allowed longitudinal monitoring of tumor growth under BSL-1 conditions, significantly reducing the number of animals required for an experiment, in line with the 3R principles.

MEK1 drives oncogenic signaling and interacts with PARP1 for genomic and metabolic homeostasis in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a lethal malignancy etiologically caused by asbestos exposure, for which there are few effective treatment options. Although asbestos carcinogenesis is associated with reactive oxygen species (ROS), the bona fide oncogenic signaling pathways that regulate ROS homeostasis and bypass ROS-evoked apoptosis in MPM are poorly understood. In this study, we demonstrate that the mitogen-activated protein kinase (MAPK) pathway RAS-RAF-MEK-ERK is hyperactive and a molecular driver of MPM, independent of histological subtypes and genetic heterogeneity. Suppression of MAPK signaling by clinically approved MEK inhibitors (MEKi) elicits PARP1 to protect MPM cells from the cytotoxic effects of MAPK pathway blockage. Mechanistically, MEKi induces impairment of homologous recombination (HR) repair proficiency and mitochondrial metabolic activity, which is counterbalanced by pleiotropic PARP1. Consequently, the combination of MEK with PARP inhibitors enhances apoptotic cell death in vitro and in vivo that occurs through coordinated upregulation of cytotoxic ROS in MPM cells, suggesting a mechanism-based, readily translatable strategy to treat this daunting disease. Collectively, our studies uncover a previously unrecognized scenario that hyperactivation of the MAPK pathway is an essential feature of MPM and provide unprecedented evidence that MAPK signaling cooperates with PARP1 to homeostatically maintain ROS levels and escape ROS-mediated apoptosis.

Battles against aberrant KEAP1-NRF2 signaling in lung cancer: intertwined metabolic and immune networks.

The Kelch-like ECH-associated protein 1/nuclear factor erythroid-derived 2-like 2 (KEAP1/NRF2) pathway is well recognized as a key regulator of redox homeostasis, protecting cells from oxidative stress and xenobiotics under physiological circumstances. Cancer cells often hijack this pathway during initiation and progression, with aberrant KEAP1-NRF2 activity predominantly observed in non-small cell lung cancer (NSCLC), suggesting that cell/tissue-of-origin is likely to influence the genetic selection during malignant transformation. Hyperactivation of NRF2 confers a multi-faceted role, and recently, increasing evidence shows that a close interplay between metabolic reprogramming and tumor immunity remodelling contributes to its aggressiveness, treatment resistance (radio-/chemo-/immune-therapy) and susceptibility to metastases. Here, we discuss in detail the special metabolic and immune fitness enabled by KEAP1-NRF2 aberration in NSCLC. Furthermore, we summarize the similarities and differences in the dysregulated KEAP1-NRF2 pathway between two major histo-subtypes of NSCLC, provide mechanistic insights on the poor response to immunotherapy despite their high immunogenicity, and outline evolving strategies to treat this recalcitrant cancer subset. Finally, we integrate bioinformatic analysis of publicly available datasets to illustrate the new partners/effectors in NRF2-addicted cancer cells, which may provide new insights into context-directed treatment.

Non-genetic adaptive resistance to KRASG12C inhibition: EMT is not the only culprit.

Adaptions to therapeutic pressures exerted on cancer cells enable malignant progression of the tumor, culminating in escape from programmed cell death and development of resistant diseases. A common form of cancer adaptation is non-genetic alterations that exploit mechanisms already present in cancer cells and do not require genetic modifications that can also lead to resistance mechanisms. Epithelial-to-mesenchymal transition (EMT) is one of the most prevalent mechanisms of adaptive drug resistance and resulting cancer treatment failure, driven by epigenetic reprogramming and EMT-specific transcription factors. A recent breakthrough in cancer treatment is the development of KRASG12C inhibitors, which herald a new era of therapy by knocking out a unique substitution of an oncogenic driver. However, these highly selective agents targeting KRASG12C, such as FDA-approved sotorasib (AMG510) and adagrasib (MRTX849), inevitably encounter multiple mechanisms of drug resistance. In addition to EMT, cancer cells can hijack or rewire the sophisticated signaling networks that physiologically control cell proliferation, growth, and differentiation to promote malignant cancer cell phenotypes, suggesting that inhibition of multiple interconnected signaling pathways may be required to block tumor progression on KRASG12C inhibitor therapy. Furthermore, the tumor microenvironment (TME) of cancer cells, such as tumor-infiltrating lymphocytes (TILs), contribute significantly to immune escape and tumor progression, suggesting a therapeutic approach that targets not only cancer cells but also the TME. Deciphering and targeting cancer adaptions promises mechanistic insights into tumor pathobiology and improved clinical management of KRASG12C-mutant cancer. This review presents recent advances in non-genetic adaptations leading to resistance to KRASG12C inhibitors, with a focus on oncogenic pathway rewiring, TME, and EMT.

Schedule-Dependent Treatment Increases Chemotherapy Efficacy in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare but aggressive thoracic malignancy with limited treatment options. One of the standard treatments for MPM is chemotherapy, which consists of concurrent treatment with pemetrexed and cisplatin. Pemetrexed limits tumor growth by inhibiting critical metabolic enzymes involved in nucleotide synthesis. Cisplatin causes direct DNA damage, such as intra-strand and inter-strand cross-links, which are repaired by the nucleotide excision repair pathway, which depends on relatively high nucleotide levels. We hypothesized that prolonged pretreatment with pemetrexed might deplete nucleotide pools, thereby sensitizing cancer cells to subsequent cisplatin treatment. The MPM cell lines ACC-MESO-1 and NCI-H28 were treated for 72 h with pemetrexed. Three treatment schedules were evaluated by initiating 24 h of cisplatin treatment at 0 h (concomitant), 24 h, and 48 h relative to pemetrexed treatment, resulting in either concomitant administration or pemetrexed pretreatment for 24 h or 48 h, respectively. Multicolor flow cytometry was performed to detect γH2AX (phosphorylation of histone H2AX), a surrogate marker for the activation of the DNA damage response pathway. DAPI staining of DNA was used to analyze cell cycle distribution. Forward and side scatter intensity was used to distinguish subpopulations based on cellular size and granularity, respectively. Our study revealed that prolonged pemetrexed pretreatment for 48 h prior to cisplatin significantly reduced long-term cell growth. Specifically, pretreatment for 48 h with pemetrexed induced a cell cycle arrest, mainly in the G2/M phase, accumulation of persistent DNA damage, and induction of a senescence phenotype. The present study demonstrates that optimizing the treatment schedule by pretreatment with pemetrexed increases the efficacy of the pemetrexed-cisplatin combination therapy in MPM. We show that the observed benefits are associated with the persistence of treatment-induced DNA damage. Our study suggests that an adjustment of the treatment schedule could improve the efficacy of the standard chemotherapy regimen for MPM and might improve patient outcomes.

Dissecting the Immunological Profiles in NSD3-Amplified LUSC through Integrative Multi-Scale Analyses.

The histone H3 lysine 36 (H3K36) methyltransferase NSD3, a neighboring gene of FGFR1, has been identified as a critical genetic driver of lung squamous cell carcinoma (LUSC). However, the molecular characteristics, especially the immunological roles of NSD3 in driving carcinogenesis, are poorly understood. In this study, we systematically integrated multi-omics data (e.g., genome, transcriptome, proteome, and TMA array) to dissect the immunological profiles in NSD3-amplified LUSC. Next, pharmaco-transcriptomic correlation analysis was implemented to identify the molecular underpinnings and therapeutic vulnerabilities in LUSC. We revealed that NSD3-amplified LUSC presents a non-inflamed tumor immune microenvironment (TIME) state in multiple independent LUSC patient cohorts. Predictably, elevated NSD3 expression was correlated with a worse immunotherapy outcome. Further molecular characterizations revealed that the high activity of unfolded protein response (UPR) signaling might be a pivotal mediator for the non-immunogenic phenotype of NSD3-amplified LUSC. Concordantly, we showed that NSD3-amplified LUSCs exhibited a more sensitive phenotype to compounds targeting UPR branches than the wild-type group. In brief, our multi-level analyses point to a previously unappreciated immunological role for NSD3 and provide therapeutic rationales for NSD3-amplified squamous lung cancer.

Organoids as a Model for Precision Medicine in Malignant Pleural Mesothelioma: Where Are We Today?

MPM is an aggressive tumor originating from pleural mesothelial cells. A characteristic feature of the disease is the dominant prevalence of therapeutically intractable inactivating alterations in TSGs, making MPM one of the most difficult cancers to treat and the epitome of a cancer characterized by a significant lack of therapy options and an extremely poor prognosis (5-year survival rate of only 5% to 10%). Extensive interpatient heterogeneity poses another major challenge for targeted therapy of MPM, warranting stratified therapy for specific subgroups of MPM patients. Accurate preclinical models are critical for the discovery of new therapies and the development of personalized medicine. Organoids, an in vitro 'organ-like' 3D structure derived from patient tumor tissue that faithfully mimics the biology and complex architecture of cancer and largely overcomes the limitations of other existing models, are the next-generation tumor model. Although organoids have been successfully produced and used in many cancers, the development of MPM organoids is still in its infancy. Here, we provide an overview of recent advances in cancer organoids, focusing on the progress and challenges in MPM organoid development. We also elaborate the potential of MPM organoids for understanding MPM pathobiology, discovering new therapeutic targets, and developing personalized treatments for MPM patients.

Targeting lactate dehydrogenase B-dependent mitochondrial metabolism affects tumor initiating cells and inhibits tumorigenesis of non-small cell lung cancer by inducing mtDNA damage.

Once considered a waste product of anaerobic cellular metabolism, lactate has been identified as a critical regulator of tumorigenesis, maintenance, and progression. The putative primary function of lactate dehydrogenase B (LDHB) is to catalyze the conversion of lactate to pyruvate; however, its role in regulating metabolism during tumorigenesis is largely unknown. To determine whether LDHB plays a pivotal role in tumorigenesis, we performed 2D and 3D in vitro experiments, utilized a conventional xenograft tumor model, and developed a novel genetically engineered mouse model (GEMM) of non-small cell lung cancer (NSCLC), in which we combined an LDHB deletion allele with an inducible model of lung adenocarcinoma driven by the concomitant loss of p53 (also known as Trp53) and expression of oncogenic KRAS (G12D) (KP). Here, we show that epithelial-like, tumor-initiating NSCLC cells feature oxidative phosphorylation (OXPHOS) phenotype that is regulated by LDHB-mediated lactate metabolism. We show that silencing of LDHB induces persistent mitochondrial DNA damage, decreases mitochondrial respiratory complex activity and OXPHOS, resulting in reduced levels of mitochondria-dependent metabolites, e.g., TCA intermediates, amino acids, and nucleotides. Inhibition of LDHB dramatically reduced the survival of tumor-initiating cells and sphere formation in vitro, which can be partially restored by nucleotide supplementation. In addition, LDHB silencing reduced tumor initiation and growth of xenograft tumors. Furthermore, we report for the first time that homozygous deletion of LDHB significantly reduced lung tumorigenesis upon the concomitant loss of Tp53 and expression of oncogenic KRAS without considerably affecting the animal's health status, thereby identifying LDHB as a potential target for NSCLC therapy. In conclusion, our study shows for the first time that LDHB is essential for the maintenance of mitochondrial metabolism, especially nucleotide metabolism, demonstrating that LDHB is crucial for the survival and proliferation of NSCLC tumor-initiating cells and tumorigenesis.

Multi-scale integrative analyses identify THBS2+ cancer-associated fibroblasts as a key orchestrator promoting aggressiveness in early-stage lung adenocarcinoma.

Rationale: Subsets of patients with early-stage lung adenocarcinoma (LUAD) have a poor post-surgical course after curative surgery. However, biomarkers stratifying this high-risk subset and molecular underpinnings underlying the aggressive phenotype remain unclear. Methods: We integrated bulk and single-cell transcriptomics, proteomics, secretome and spatial profiling of clinical early-stage LUAD samples to identify molecular underpinnings that promote the aggressive phenotype. Results: We identified and validated THBS2, at multi-omic levels, as a tumor size-independent biomarker that robustly predicted post-surgical survival in multiple independent clinical cohorts of early-stage LUAD. Furthermore, scRNA-seq data revealed that THBS2 is exclusively derived from a specific cancer-associated fibroblast (CAF) subset that is distinct from CAFs defined by classical markers. Interestingly, our data demonstrated that THBS2 was preferentially secreted via exosomes in early-stage LUAD tumors with high aggressiveness, and its levels in the peripheral plasma associated with short recurrence-free survival. Further characterization showed that THBS2-high early-stage LUAD was characterized by suppressed antitumor immunity. Specifically, beyond tumor cells, THBS2+ CAFs mainly interact with B and CD8+ T lymphocytes as well as macrophages within tumor microenvironment of early-stage LUAD, and THBS2-high LUAD was associated with decreased immune cell infiltrates but increased immune exhaustion marker. Clinically, high THBS2 expression predicted poor response to immunotherapies and short post-treatment survival of patients. Finally, THBS2 recombinant protein suppressed ex vivo T cells proliferation and promoted in vivo LUAD tumor growth and distant micro-metastasis. Conclusions: Our multi-level analyses uncovered tumor-specific THBS2+ CAFs as a key orchestrator promoting aggressiveness in early-stage LUAD.

A Breakthrough Brought about by Targeting KRASG12C: Nonconformity Is Punished.

KRAS is the most frequently mutated oncogene in lung carcinomas, accounting for 25% of total incidence, with half of them being KRASG12C mutations. In past decades, KRAS enjoyed the notorious reputation of being untargetable-that is, until the advent of G12C inhibitors, which put an end to this legend by covalently targeting the G12C (glycine to cysteine) substitution in the switch-II pocket of the protein, inhibiting the affinity of the mutant KRAS with GTP and subsequently the downstream signaling pathways, such as Raf/MEK/ERK. KRASG12C-selective inhibitors, e.g., the FDA-approved AMG510 and MRTX849, have demonstrated potent clinical efficacy and selectivity in patients with KRASG12C-driven cancers only, which spares other driver KRAS mutations (e.g., G12D/V/S, G13D, and Q61H) and has ushered in an unprecedented breakthrough in the field in recent decades. However, accumulating evidence from preclinical and clinical studies has shown that G12C-targeted therapeutics as single agents are inevitably thwarted by drug resistance, a persistent problem associated with targeted therapies. A promising strategy to optimize G12C inhibitor therapy is combination treatments with other therapeutic agents, the identification of which is empowered by the insightful appreciation of compensatory signaling pathways or evasive mechanisms, such as those that attenuate immune responses. Here, we review recent advances in targeting KRASG12C and discuss the challenges of KRASG12C inhibitor therapy, as well as future directions.

Tumor-infiltrating lymphocytes are functionally inactivated by CD90+ stromal cells and reactivated by combined Ibrutinib and Rapamycin in human pleural mesothelioma.

Rationale: Despite evidence suggesting that the tumor microenvironment (TME) in malignant pleural mesothelioma (MPM) is linked with poor prognosis, there is a lack of studies that functionally characterize stromal cells and tumor-infiltrating lymphocytes (TILs). Here, we aim to characterize the stromal subsets within MPM, investigate their relationship to TILs, and explore the potential therapeutic targets. Methods: We curated a core set of genes defining stromal/immune signatures expressed by mesenchymal cells within the TME using molecular analysis of The Cancer Genome Atlas (TCGA) MPM cohort. Stromal and immune profiles were molecularly characterized using flow cytometry, immunohistochemistry, microarray, and functionally evaluated using T cell-activation/expansion, coculture assays and drug compounds treatment, based on samples from an independent MPM cohort. Results: We found that a high extracellular matrix (ECM)/stromal gene signature, a high ECM score, or the ratio of ECM to an immune activation gene signature are significantly associated with poor survival in the MPM cohort in TCGA. Analysis of an independent MPM cohort (n = 12) revealed that CD8+ and CD4+ TILs were characterized by PD1 overexpression and concomitant downregulation in degranulation and CD127. This coincided with an increase in CD90+ cells that overexpressed PD-L1 and were enriched for ECM/stromal genes, activated PI3K-mTOR signaling and suppressed T cells. Protein array data demonstrated that MPM samples with high PD-L1 expression were most associated with activation of the mTOR pathway. Further, to reactivate functionally indolent TILs, we reprogrammed ex vivo TILs with Ibrutinib plus Rapamycin to block interleukin-2-inducible kinase (ITK) and mTOR pathways, respectively. The combination treatment shifted effector memory (TEM) CD8+ and CD4+ TILs towards T cells that re-expressed CD45RA (TEMRA) while concomitantly downregulating exhaustion markers. Gene expression analysis confirmed that Ibrutinib plus Rapamycin downregulated coinhibitory and T cell signature pathways while upregulating pathways involved in DNA damage and repair and immune cell adhesion and migration. Conclusions: Our results suggest that targeting the TME may represent a novel strategy to redirect the fate of endogenous TILs with the goal of restoring anti-tumor immunity and control of tumor growth in MPM.

Metabolic synthetic lethality by targeting NOP56 and mTOR in KRAS-mutant lung cancer.

BACKGROUND: Oncogenic KRAS mutations are prevalent in human cancers, but effective treatment of KRAS-mutant malignancies remains a major challenge in the clinic. Increasing evidence suggests that aberrant metabolism plays a central role in KRAS-driven oncogenic transformation. The aim of this study is to identify selective metabolic dependency induced by mutant KRAS and to exploit it for the treatment of the disease. METHOD: We performed an integrated analysis of RNAi- and CRISPR-based functional genomic datasets (n = 5) to identify novel genes selectively required for KRAS-mutant cancer. We further screened a customized library of chemical inhibitors for candidates that are synthetic lethal with NOP56 depletion. Functional studies were carried out by genetic knockdown using siRNAs and shRNAs, knockout using CRISPR/Cas9, and/or pharmacological inhibition, followed by cell viability and apoptotic assays. Protein expression was determined by Western blot. Metabolic ROS was measured by flow cytometry-based quantification. RESULTS: We demonstrated that nucleolar protein 5A (NOP56), a core component of small nucleolar ribonucleoprotein complexes (snoRNPs) with an essential role in ribosome biogenesis, confers a metabolic dependency by regulating ROS homeostasis in KRAS-mutant lung cancer cells and that NOP56 depletion causes synthetic lethal susceptibility to inhibition of mTOR. Mechanistically, cancer cells with reduced NOP56 are subjected to higher levels of ROS and rely on mTOR signaling to balance oxidative stress and survive. We also discovered that IRE1α-mediated unfolded protein response (UPR) regulates this process by activating mTOR through p38 MAPK. Consequently, co-targeting of NOP56 and mTOR profoundly enhances KRAS-mutant tumor cell death in vitro and in vivo. CONCLUSIONS: Our findings reveal a previously unrecognized mechanism in which NOP56 and mTOR cooperate to play a homeostatic role in the response to oxidative stress and suggest a new rationale for the treatment of KRAS-mutant cancers.

Smoking signature is superior to programmed death-ligand 1 expression in predicting pathological response to neoadjuvant immunotherapy in lung cancer patients.

BACKGROUND: There is a paucity of biomarkers that can predict the degree of pathological response [e.g., pathological complete response (pCR) or major response (pMR)] to immunotherapy. Neoadjuvant immunotherapy provides an ideal setting for exploring responsive biomarkers because the pathological responses can be directly and accurately evaluated. METHODS: We retrospectively collected the clinicopathological characteristics and treatment outcomes of non-small cell lung cancer (NSCLC) patients who received neoadjuvant immunotherapy or chemo-immunotherapy followed by surgery between 2018 and 2020 at a large academic thoracic cancer center. Clinicopathological factors associated with pathological response were analyzed. RESULTS: A total of 39 patients (35 males and 4 females) were included. The most common histological subtype was lung squamous cell carcinoma (LUSC) (n=28, 71.8%), followed by lung adenocarcinoma (LUAD) (n=11, 28.2%). After neoadjuvant treatment, computed tomography (CT) scan-based evaluation showed poor agreement with the postoperatively pathological examination (weighted kappa =0.0225; P=0.795), suggesting the poor performance of CT scans in evaluating the response to immunotherapy. Importantly, we found that the smoking signature displayed a better performance than programmed death-ligand 1 (PD-L1) expression in predicting the pathological response (area under the curve: 0.690 vs. 0.456; P=0.0259), which might have resulted from increased tumor mutational burden (TMB) and/or microsatellite instability (MSI) relating to smoking exposure. CONCLUSIONS: These findings suggest that CT scan-based evaluation is not able to accurately reflect the pathological response to immunotherapy and that smoking signature is a superior marker to PD-L1 expression in predicting the benefit of immunotherapy in NSCLC patients.

Peritumoral CD90+CD73+ cells possess immunosuppressive features in human non-small cell lung cancer.

BACKGROUND: Although T cell abundance in solid tumours is associated with better outcomes, it also correlates with a stroma-mediated source of immune suppression driven by TGFß1 and poor overall survival. Whether this also is observed in non-small cell lung cancer (NSCLC) is unknown. METHODS: We utilized molecular analysis of The Cancer Genome Atlas (TCGA) NSLCC cohort to correlate immune activation (IA) gene expression and extracellular matrix/stromal (ECM/stromal) gene expression with patient survival. In an independent cohort of NSCLC samples, we used flow cytometry to identify mesenchymal subsets and ex vivo functional studies to characterize their immune regulatory function. FINDINGS: We observed a high enrichment in a core set of genes defining an IA gene expression signature in NSCLC across TCGA Pan-cancer cohort. High IA signature score correlates with enrichment of ECM/stromal gene signature across TCGA NSCLC datasets. Importantly, a higher ratio of ECM/stromal to IA gene signature score was associated with shorter overall survival. In tumours resected from a separate cohort of NSCLC patients, we identified CD90+CD73+ peritumoral cells that were enriched in the ECM/stromal gene signature, which was amplified by TGFß1. IFNγ and TNFα-primed peritumoral CD90+CD73+ cells upregulate immune checkpoint molecules PD-L1 and IDO1 and secrete an array of cytokines/chemokines including TGFß1. Finally, immune primed peritumoral CD90+CD73+ cells suppress T cell function, which was relieved following combined blockade of PD-L1 and TGFß1 with IDO1 inhibition but not PD-L1 or anti-CD73 alone. INTERPRETATION: Our findings suggest that targeting PD-L1 together with independent biological features of the stroma may enhance host antitumor immunity in NSCLC. FUNDING: LW and HY are supported by a 4-year China Scholarship Council award. This work was funded, in part, by a grant from the Cancer League of Bern, Switzerland to SRRH. Laser scanning microscopy imaging was funded by the R'Equip grant from the Swiss National Science Foundation Nr. 316030_145003.

Synergistic effects of FGFR1 and PLK1 inhibitors target a metabolic liability in KRAS-mutant cancer.

KRAS oncoprotein is commonly mutated in human cancer, but effective therapies specifically targeting KRAS-driven tumors remain elusive. Here, we show that combined treatment with fibroblast growth factor receptor 1 (FGFR1) and polo-like kinase 1 (PLK1) inhibitors evoke synergistic cytotoxicity in KRAS-mutant tumor models in vitro and in vivo. Pharmacological and genetic suppression of FGFR1 and PLK1 synergizes to enhance anti-proliferative effects and cell death in KRAS-mutant lung and pancreatic but not colon nor KRAS wild-type cancer cells. Mechanistically, co-targeting FGFR1 and PLK1 upregulates reactive oxygen species (ROS), leading to oxidative stress-activated c-Jun N-terminal kinase (JNK)/p38 pathway and E2F1-induced apoptosis. We further delineate that autophagy protects from PLK1/FGFR1 inhibitor cytotoxicity and that antagonizing the compensation mechanism by clinically approved chloroquine fully realizes the therapeutic potential of PLK1 and FGFR1 targeting therapy, producing potent and durable responses in KRAS-mutant patient-derived xenografts and a genetically engineered mouse model of Kras-induced lung adenocarcinoma. These results suggest a previously unappreciated role for FGFR1 and PLK1 in the surveillance of metabolic stress and demonstrate a synergistic drug combination for treating KRAS-mutant cancer.

Pharmaco-transcriptomic correlation analysis reveals novel responsive signatures to HDAC inhibitors and identifies Dasatinib as a synergistic interactor in small-cell lung cancer.

BACKGROUND: Histone acetylation/deacetylase process is one of the most studied epigenetic modifications. Histone deacetylase inhibitors (HDACis) have shown clinical benefits in haematological malignancies but failed in solid tumours due to the lack of biomarker-driven stratification. METHODS: We perform integrative pharmaco-transcriptomic analysis by correlating drug response profiles of five pan-HDACis with transcriptomes of solid cancer cell lines (n=659) to systematically identify generalizable gene signatures associated with HDACis sensitivity and resistance. The established signatures are then applied to identify cancer subtypes that are potentially sensitive or resistant to HDACis, and drugs that enhance the efficacy of HDACis. Finally, the reproductivity of the established HDACis signatures is evaluated by multiple independent drug response datasets and experimental assays. FINDINGS: We successfully delineate generalizable gene signatures predicting sensitivity (containing 46 genes) and resistance (containing 53 genes) to all five HDACis, with their reproductivity confirmed by multiple external sources and independent internal assays. Using the gene signatures, we identify low-grade glioma harbouring isocitrate dehydrogenase 1/2 (IDH1/2) mutation and non-YAP1-driven subsets of small-cell lung cancer (SCLC) that particularly benefit from HDACis monotherapy. Further, based on the resistance gene signature, we identify clinically-approved Dasatinib as a synthetic lethal drug with HDACi, synergizing in inducing apoptosis and reactive oxygen species on a panel of SCLC. Finally, Dasatinib significantly enhances the therapeutic efficacy of Vorinostat in SCLC xenografts. INTERPRETATION: Our work establishes robust gene signatures predicting HDACis sensitivity/resistance in solid cancer and uncovers combined Dasatinib/HDACi as a synthetic lethal combination therapy for SCLC. FUNDING: This work was supported by the National Natural Science Foundation of China (82072570 to F. Yao; 82002941 to B. Sun).

Epithelial-to-mesenchymal transition contributes to the downregulation of progesterone receptor expression in endometriosis lesions.

Endometriosis is a common, estrogen-dependent disease, in which endometrial tissue grows in the peritoneal cavity. These lesions often express low levels of progesterone receptors (PR), which potentially play an important role in the insufficient response to progestin treatment. Here, we uncover an interconnection between the downregulated PR expression and the epithelial-to-mesenchymal transition (EMT) in endometriotic lesions. The majority of ectopic epithelial glands (93.1 %, n = 67/72) display heterogeneous states of EMT by immunohistochemistry staining. Interestingly, low PR expression associated with high N-cadherin expression, a hallmark of EMT. In order to gain mechanistic insights, we performed in vitro functional assays with the endometriotic epithelial cell lines EM'osis and 12Z. TGF-ß-induced EMT, marked by elevations of CDH2 and SNAI1/2, led to a significant downregulation of PR gene expression in both cell lines. In contrast, silencing of SNAI1 in EM'osis and of SNAI1 plus SNAI2 in 12Z elevated PR gene expression significantly. We found that not only in vitro, but also in the epithelial component of endometriotic lesions strong expression of SNAI1/2 concurred with weak expression of PR. In summary, these results suggested the negative correlation association of the heterogeneous states of EMT and suppressed PR expression in endometriotic lesions. Our functional assays indicate that EMT contributes to the downregulation of PR expression via the upregulation of EMT-TFs, like SNAI1 and SNAI2, which may ultimately lead to progesterone resistance.