Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.

Cycling cancer persister cells arise from lineages with distinct programs.

Non-genetic mechanisms have recently emerged as important drivers of cancer therapy failure1, where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment2. Although most cancer persisters remain arrested in the presence of the drug, a rare subset can re-enter the cell cycle under constitutive drug treatment. Little is known about the non-genetic mechanisms that enable cancer persisters to maintain proliferative capacity in the presence of drugs. To study this rare, transiently resistant, proliferative persister population, we developed Watermelon, a high-complexity expressed barcode lentiviral library for simultaneous tracing of each cell's clonal origin and proliferative and transcriptional states. Here we show that cycling and non-cycling persisters arise from different cell lineages with distinct transcriptional and metabolic programs. Upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation are associated with persister proliferative capacity across multiple cancer types. Impeding oxidative stress or metabolic reprogramming alters the fraction of cycling persisters. In human tumours, programs associated with cycling persisters are induced in minimal residual disease in response to multiple targeted therapies. The Watermelon system enabled the identification of rare persister lineages that are preferentially poised to proliferate under drug pressure, thus exposing new vulnerabilities that can be targeted to delay or even prevent disease recurrence.

An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1.

BACKGROUND: Lung cancer cells overexpress mucin 1 (MUC1) and active subunit MUC1-CT. Although a peptide blocks MUC1 signalling, metabolites targeting MUC1 are not well studied. AICAR is a purine biosynthesis intermediate. METHODS: Cell viability and apoptosis were measured in AICAR-treated EGFR-mutant and wild-type lung cells. AICAR-binding proteins were evaluated by in silico and thermal stability assays. Protein-protein interactions were visualised by dual-immunofluorescence staining and proximity ligation assay. AICAR-induced whole transcriptomic profile was determined by RNA sequencing. EGFR-TL transgenic mice-derived lung tissues were analysed for MUC1 expression. Organoids and tumours from patients and transgenic mice were treated with AICAR alone or in combination with JAK and EGFR inhibitors to evaluate treatment effects. RESULTS: AICAR reduced EGFR-mutant tumour cell growth by inducing DNA damage and apoptosis. MUC1 was one of the leading AICAR-binding and degrading proteins. AICAR negatively regulated JAK signalling and JAK1-MUC1-CT interaction. Activated EGFR upregulated MUC1-CT expression in EGFR-TL-induced lung tumour tissues. AICAR reduced EGFR-mutant cell line-derived tumour formation in vivo. Co-treating patient and transgenic mouse lung-tissue-derived tumour organoids with AICAR and JAK1 and EGFR inhibitors reduced their growth. CONCLUSIONS: AICAR represses the MUC1 activity in EGFR-mutant lung cancer, disrupting protein-protein interactions between MUC1-CT and JAK1 and EGFR.

YES1 amplification is a mechanism of acquired resistance to EGFR inhibitors identified by transposon mutagenesis and clinical genomics.

In ∼30% of patients with EGFR-mutant lung adenocarcinomas whose disease progresses on EGFR inhibitors, the basis for acquired resistance remains unclear. We have integrated transposon mutagenesis screening in an EGFR-mutant cell line and clinical genomic sequencing in cases of acquired resistance to identify mechanisms of resistance to EGFR inhibitors. The most prominent candidate genes identified by insertions in or near the genes during the screen were MET, a gene whose amplification is known to mediate resistance to EGFR inhibitors, and the gene encoding the Src family kinase YES1. Cell clones with transposon insertions that activated expression of YES1 exhibited resistance to all three generations of EGFR inhibitors and sensitivity to pharmacologic and siRNA-mediated inhibition of YES1 Analysis of clinical genomic sequencing data from cases of acquired resistance to EGFR inhibitors revealed amplification of YES1 in five cases, four of which lacked any other known mechanisms of resistance. Preinhibitor samples, available for two of the five patients, lacked YES1 amplification. None of 136 postinhibitor samples had detectable amplification of other Src family kinases (SRC and FYN). YES1 amplification was also found in 2 of 17 samples from ALK fusion-positive lung cancer patients who had progressed on ALK TKIs. Taken together, our findings identify acquired amplification of YES1 as a recurrent and targetable mechanism of resistance to EGFR inhibition in EGFR-mutant lung cancers and demonstrate the utility of transposon mutagenesis in discovering clinically relevant mechanisms of drug resistance.

Mutational landscape of EGFR-, MYC-, and Kras-driven genetically engineered mouse models of lung adenocarcinoma.

Genetically engineered mouse models (GEMMs) of cancer are increasingly being used to assess putative driver mutations identified by large-scale sequencing of human cancer genomes. To accurately interpret experiments that introduce additional mutations, an understanding of the somatic genetic profile and evolution of GEMM tumors is necessary. Here, we performed whole-exome sequencing of tumors from three GEMMs of lung adenocarcinoma driven by mutant epidermal growth factor receptor (EGFR), mutant Kirsten rat sarcoma viral oncogene homolog (Kras), or overexpression of MYC proto-oncogene. Tumors from EGFR- and Kras-driven models exhibited, respectively, 0.02 and 0.07 nonsynonymous mutations per megabase, a dramatically lower average mutational frequency than observed in human lung adenocarcinomas. Tumors from models driven by strong cancer drivers (mutant EGFR and Kras) harbored few mutations in known cancer genes, whereas tumors driven by MYC, a weaker initiating oncogene in the murine lung, acquired recurrent clonal oncogenic Kras mutations. In addition, although EGFR- and Kras-driven models both exhibited recurrent whole-chromosome DNA copy number alterations, the specific chromosomes altered by gain or loss were different in each model. These data demonstrate that GEMM tumors exhibit relatively simple somatic genotypes compared with human cancers of a similar type, making these autochthonous model systems useful for additive engineering approaches to assess the potential of novel mutations on tumorigenesis, cancer progression, and drug sensitivity.

De novo selection of oncogenes.

All cellular proteins are derived from preexisting ones by natural selection. Because of the random nature of this process, many potentially useful protein structures never arose or were discarded during evolution. Here, we used a single round of genetic selection in mouse cells to isolate chemically simple, biologically active transmembrane proteins that do not contain any amino acid sequences from preexisting proteins. We screened a retroviral library expressing hundreds of thousands of proteins consisting of hydrophobic amino acids in random order to isolate four 29-aa proteins that induced focus formation in mouse and human fibroblasts and tumors in mice. These proteins share no amino acid sequences with known cellular or viral proteins, and the simplest of them contains only seven different amino acids. They transformed cells by forming a stable complex with the platelet-derived growth factor ß receptor transmembrane domain and causing ligand-independent receptor activation. We term this approach de novo selection and suggest that it can be used to generate structures and activities not observed in nature, create prototypes for novel research reagents and therapeutics, and provide insight into cell biology, transmembrane protein-protein interactions, and possibly virus evolution and the origin of life.

Next-generation sequencing of paired tyrosine kinase inhibitor-sensitive and -resistant EGFR mutant lung cancer cell lines identifies spectrum of DNA changes associated with drug resistance.

Somatic mutations in kinase genes are associated with sensitivity of solid tumors to kinase inhibitors, but patients with metastatic cancer eventually develop disease progression. In EGFR mutant lung cancer, modeling of acquired resistance (AR) with drug-sensitive cell lines has identified clinically relevant EGFR tyrosine kinase inhibitor (TKI) resistance mechanisms such as the second-site mutation, EGFR T790M, amplification of the gene encoding an alternative kinase, MET, and epithelial-mesenchymal transition (EMT). The full spectrum of DNA changes associated with AR remains unknown. We used next-generation sequencing to characterize mutational changes associated with four populations of EGFR mutant drug-sensitive and five matched drug-resistant cell lines. Comparing resistant cells with parental counterparts, 18-91 coding SNVs/indels were predicted to be acquired and 1-27 were lost; few SNVs/indels were shared across resistant lines. Comparison of two related parental lines revealed no unique coding SNVs/indels, suggesting that changes in the resistant lines were due to drug selection. Surprisingly, we observed more CNV changes across all resistant lines, and the line with EMT displayed significantly higher levels of CNV changes than the other lines with AR. These results demonstrate a framework for studying the evolution of AR and provide the first genome-wide spectrum of mutations associated with the development of cellular drug resistance in an oncogene-addicted cancer. Collectively, the data suggest that CNV changes may play a larger role than previously appreciated in the acquisition of drug resistance and highlight that resistance may be heterogeneous in the context of different tumor cell backgrounds.

Isolation of epithelial, endothelial, and immune cells from lungs of transgenic mice with oncogene-induced lung adenocarcinomas.

Genetically engineered mouse models of lung adenocarcinoma have proven invaluable for understanding mechanisms of tumorigenesis, therapy response, and drug resistance. However, mechanistic studies focused on studying these processes in tumor-bearing mouse lungs are confounded by the fact that, in most cases, relevant signaling pathways are analyzed in whole-lung preparations, which are composed of a heterogeneous mixture of cells. Given our increasing knowledge about the roles played by different subpopulations of cells in the development of lung adenocarcinoma, separating the major cellular compartments of the tumor microenvironment is recommended to allow for a precise analysis of relevant pathways in each isolated cell type. In this study, we optimized magnetic- and fluorescence-based isolation protocols to segregate lung epithelial (CD326/epithelial cell adhesion molecule-positive), endothelial (CD31-positive), and immune (CD45-positive) cells, with high purity, from the lungs of transgenic mice with mutant epidermal growth factor receptor-induced lung adenocarcinomas. This approach, which can potentially be extended to additional lung adenocarcinoma models, enables delineation of the molecular features of individual cell types that can be used to gain insight into their roles in lung adenocarcinoma initiation, progression, and response to therapy.

The Evolution of Mouse Models of Cancer: Past, Present, and Future.

In the nearly 50 years since the original models of cancer first hit the stage, mouse models have become a major contributor to virtually all aspects of cancer research, and these have evolved well beyond simple transgenic or xenograft models to encompass a wide range of more complex models. As the sophistication of mouse models has increased, an explosion of new technologies has expanded the potential to both further develop and apply these models to address major challenges in cancer research. In the current era, cancer modeling has expanded to include nongermline genetically engineered mouse models (GEMMs), patient-derived models, organoids, and adaptations of the models better suited for cancer immunology research. New technologies that have transformed the field include the application of CRISPR-Cas9-mediated genome editing, in vivo imaging, and single-cell analysis to cancer modeling. Here, we provide a historical perspective on the evolution of mouse models of cancer, focusing on how far we have come in a relatively short time and how new technologies will shape the future development of mouse models of cancer.

Co-Occurring Alterations in Multiple Tumor Suppressor Genes Are Associated With Worse Outcomes in Patients With EGFR-Mutant Lung Cancer.

INTRODUCTION: Patients with metastatic EGFR-mutant NSCLC inevitably have disease progression while on tyrosine kinase inhibitor (TKI) therapy. Co-occurring tumor suppressor gene (TSG) alterations have been associated with poor outcomes, however, detailed analyses of their impact on patient outcomes are limited. METHODS: Patients with EGFR-mutant NSCLC treated with EGFR TKIs who had tumor genomic profiling were included. Alterations in TP53 and five additional TSGs (RB1, NF1, ARID1A, BRCA1, and PTEN) were used to stratify the cohort into the following three subgroups: patients with tumors harboring a TP53 mutation plus a mutation in at least one additional TSG (TP53mut/TSGmut), those having a TP53 mutation without additional TSG mutations (TP53mut/TSGwt), and those with TP53wt. Patient characteristics and clinical outcomes were assessed in two independent cohorts. RESULTS: A total of 101 patients from the Yale Cancer Center and 182 patients from the American Association for Cancer Research Project GENIE database were included. In the Yale cohort, TP53 mutations were identified in 65 cases (64%), of which 23 were TP53mut/TSGmut and 42 were TP53mut/TSGwt. Although the presence of a TP53 mutation was associated with worse outcomes, the additional TSG alteration in TP53mut tumors identified a subset of patients associated with particularly aggressive disease and inferior clinical outcome in both the Yale and the GENIE cohorts. Specifically, in the Yale cohort for patients receiving first-line TKIs, those with TP53mut/TSGmut tumors had shorter progression-free survival (PFS) and overall survival (OS) than TP53mut/TSGwt (PFS: hazard ratio [HR] = 2.03, confidence interval [CI]: 1.12-3.69, p < 0.01, OS: HR = 1.58, CI: 0.82-3.04, p = 0.12) or TP53wt cases (PFS: HR 2.4, CI: 1.28-4.47, p < 0.001, OS: HR = 2.54, CI: 1.21-5.34, p < 0.005). Inferior outcomes in patients with TP53mut/TSGmut tumors were also found in those receiving osimertinib as second-line therapy. Similar findings were seen in patients in the GENIE cohort. CONCLUSIONS: Patients with TP53mut/TSGmut tumors represent a patient subgroup characterized by an aggressive disease phenotype and inferior outcomes on EGFR TKIs. This information is important for understanding the biological underpinnings of differential outcomes with TKI treatment and has implications for identifying patients who may benefit from additional therapeutic interventions beyond osimertinib monotherapy.

Overexpression of Malat1 drives metastasis through inflammatory reprogramming of the tumor microenvironment.

Expression of the long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) correlates with tumor progression and metastasis in many tumor types. However, the impact and mechanism of action by which MALAT1 promotes metastatic disease remain elusive. Here, we used CRISPR activation (CRISPRa) to overexpress MALAT1/Malat1 in patient-derived lung adenocarcinoma (LUAD) cell lines and in the autochthonous K-ras/p53 LUAD mouse model. Malat1 overexpression was sufficient to promote the progression of LUAD to metastatic disease in mice. Overexpression of MALAT1/Malat1 enhanced cell mobility and promoted the recruitment of protumorigenic macrophages to the tumor microenvironment through paracrine secretion of CCL2/Ccl2. Ccl2 up-regulation was the result of increased global chromatin accessibility upon Malat1 overexpression. Macrophage depletion and Ccl2 blockade counteracted the effects of Malat1 overexpression. These data demonstrate that a single lncRNA can drive LUAD metastasis through reprogramming of the tumor microenvironment.

Chitinase 3-like-1 (CHI3L1) in the pathogenesis of epidermal growth factor receptor mutant non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) accounts for 85 % of all lung cancers. In NSCLC, 10-20 % of Caucasian patients and 30-50 % of Asian patients have tumors with activating mutations in the Epidermal Growth Factor Receptor (EGFR). A high percentage of these patients exhibit favorable responses to treatment with tyrosine kinase inhibitors (TKI). Unfortunately, a majority of these patients develop therapeutic resistance with progression free survival lasting 9-18 months. The mechanisms that underlie the tumorigenic effects of EGFR and the ability of NSCLC to develop resistance to TKI therapies, however, are poorly understood. Here we demonstrate that CHI3L1 is produced by EGFR activation of normal epithelial cells, transformed epithelial cells with wild type EGFR and cells with cancer-associated, activating EGFR mutations. We also demonstrate that CHI3L1 auto-induces itself and feeds back to stimulate EGFR and its ligands via a STAT3-dependent mechanism(s). Highly specific antibodies against CHI3L1 (anti-CHI3L1/FRG) and TKI, individually and in combination, abrogated the effects of EGFR activation on CHI3L1 and the ability of CHI3L1 to stimulate the EGFR axis. Anti-CHI3L1 also interacted with osimertinib to reverse TKI therapeutic resistance and induce tumor cell death and inhibit pulmonary metastasis while stimulating tumor suppressor genes including KEAP1. CHI3L1 is a downstream target of EGFR that feeds back to stimulate and activate the EGFR axis. Anti-CHI3L1 is an exciting potential therapeutic for EGFR mutant NSCLC, alone and in combination with osimertinib or other TKIs.

EGFR-driven lung adenocarcinomas coopt alveolar macrophage metabolism and function to support EGFR signaling and growth.

The limited efficacy of currently approved immunotherapies in EGFR-driven lung adenocarcinoma (LUAD) underscores the need to better understand alternative mechanisms governing local immunosuppression to fuel novel therapies. Elevated surfactant and GM-CSF secretion from the transformed epithelium induces tumor-associated alveolar macrophage (TA-AM) proliferation which supports tumor growth by rewiring inflammatory functions and lipid metabolism. TA-AM properties are driven by increased GM-CSF-PPARγ signaling and inhibition of airway GM-CSF or PPARγ in TA-AMs suppresses cholesterol efflux to tumor cells, which impairs EGFR phosphorylation and restrains LUAD progression. In the absence of TA-AM metabolic support, LUAD cells compensate by increasing cholesterol synthesis, and blocking PPARγ in TA-AMs simultaneous with statin therapy further suppresses tumor progression and increases proinflammatory immune responses. These results reveal new therapeutic combinations for immunotherapy resistant EGFR-mutant LUADs and demonstrate how cancer cells can metabolically co-opt TA-AMs through GM-CSF-PPARγ signaling to provide nutrients that promote oncogenic signaling and growth.

ASCL1 Drives Tolerance to Osimertinib in EGFR Mutant Lung Cancer in Permissive Cellular Contexts.

The majority of EGFR mutant lung adenocarcinomas respond well to EGFR tyrosine kinase inhibitors (TKI). However, most of these responses are partial, with drug-tolerant residual disease remaining even at the time of maximal response. This residual disease can ultimately lead to relapses, which eventually develop in most patients. To investigate the cellular and molecular properties of residual tumor cells in vivo, we leveraged patient-derived xenograft (PDX) models of EGFR mutant lung cancer. Subcutaneous EGFR mutant PDXs were treated with the third-generation TKI osimertinib until maximal tumor regression. Residual tissue inevitably harbored tumor cells that were transcriptionally distinct from bulk pretreatment tumor. Single-cell transcriptional profiling provided evidence of cells matching the profiles of drug-tolerant cells present in the pretreatment tumor. In one of the PDXs analyzed, osimertinib treatment caused dramatic transcriptomic changes that featured upregulation of the neuroendocrine lineage transcription factor ASCL1. Mechanistically, ASCL1 conferred drug tolerance by initiating an epithelial-to-mesenchymal gene-expression program in permissive cellular contexts. This study reveals fundamental insights into the biology of drug tolerance, the plasticity of cells through TKI treatment, and why specific phenotypes are observed only in certain tumors. SIGNIFICANCE: Analysis of residual disease following tyrosine kinase inhibitor treatment identified heterogeneous and context-specific mechanisms of drug tolerance in lung cancer that could lead to the development of strategies to forestall drug resistance. See related commentary by Rumde and Burns, p. 1188.

Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy in pancreatic ductal adenocarcinoma.

Acquired resistance to immunotherapy remains a critical yet incompletely understood biological mechanism. Here, using a mouse model of pancreatic ductal adenocarcinoma (PDAC) to study tumor relapse following immunotherapy-induced responses, we find that resistance is reproducibly associated with an epithelial-to-mesenchymal transition (EMT), with EMT-transcription factors ZEB1 and SNAIL functioning as master genetic and epigenetic regulators of this effect. Acquired resistance in this model is not due to immunosuppression in the tumor immune microenvironment, disruptions in the antigen presentation machinery, or altered expression of immune checkpoints. Rather, resistance is due to a tumor cell-intrinsic defect in T-cell killing. Molecularly, EMT leads to the epigenetic and transcriptional silencing of interferon regulatory factor 6 (Irf6), rendering tumor cells less sensitive to the pro-apoptotic effects of TNF-α. These findings indicate that acquired resistance to immunotherapy may be mediated by programs distinct from those governing primary resistance, including plasticity programs that render tumor cells impervious to T-cell killing.

EGFR-Driven Lung Adenocarcinomas Co-opt Alveolar Macrophage Metabolism and Function to Support EGFR Signaling and Growth.

The limited efficacy of currently approved immunotherapies in EGFR-driven lung adenocarcinoma (LUAD) underscores the need to better understand alternative mechanisms governing local immunosuppression to fuel novel therapies. Elevated surfactant and GM-CSF secretion from the transformed epithelium induces tumor-associated alveolar macrophage (TA-AM) proliferation, which supports tumor growth by rewiring inflammatory functions and lipid metabolism. TA-AM properties are driven by increased GM-CSF-PPARγ signaling and inhibition of airway GM-CSF or PPARγ in TA-AMs suppresses cholesterol efflux to tumor cells, which impairs EGFR phosphorylation and restrains LUAD progression. In the absence of TA-AM metabolic support, LUAD cells compensate by increasing cholesterol synthesis, and blocking PPARγ in TA-AMs simultaneous with statin therapy further suppresses tumor progression and increases proinflammatory immune responses. These results reveal new therapeutic combinations for immunotherapy-resistant EGFR-mutant LUADs and demonstrate how cancer cells can metabolically co-opt TA-AMs through GM-CSF-PPARγ signaling to provide nutrients that promote oncogenic signaling and growth. SIGNIFICANCE: Alternate strategies harnessing anticancer innate immunity are required for lung cancers with poor response rates to T cell-based immunotherapies. This study identifies a targetable, mutually supportive, metabolic relationship between macrophages and transformed epithelium, which is exploited by tumors to obtain metabolic and immunologic support to sustain proliferation and oncogenic signaling.

Patient-Derived Models of Cancer in the NCI PDMC Consortium: Selection, Pitfalls, and Practical Recommendations.

For over a century, early researchers sought to study biological organisms in a laboratory setting, leading to the generation of both in vitro and in vivo model systems. Patient-derived models of cancer (PDMCs) have more recently come to the forefront of preclinical cancer models and are even finding their way into clinical practice as part of functional precision medicine programs. The PDMC Consortium, supported by the Division of Cancer Biology in the National Cancer Institute of the National Institutes of Health, seeks to understand the biological principles that govern the various PDMC behaviors, particularly in response to perturbagens, such as cancer therapeutics. Based on collective experience from the consortium groups, we provide insight regarding PDMCs established both in vitro and in vivo, with a focus on practical matters related to developing and maintaining key cancer models through a series of vignettes. Although every model has the potential to offer valuable insights, the choice of the right model should be guided by the research question. However, recognizing the inherent constraints in each model is crucial. Our objective here is to delineate the strengths and limitations of each model as established by individual vignettes. Further advances in PDMCs and the development of novel model systems will enable us to better understand human biology and improve the study of human pathology in the lab.

Effect of Dietary Supplementation with a Mixture of Natural Antioxidants on Milk Yield, Composition, Oxidation Stability and Udder Health in Dairy Ewes.

Due to the limitations in the use of antibiotic agents, researchers are constantly seeking natural bioactive compounds that could benefit udder health status but also milk quality characteristics in dairy animals. The aim of the current study was therefore to examine the effects of a standardized mixture of plant bioactive components (MPBC) originated from thyme, anise and olive on milk yield, composition, oxidative stability and somatic cell count in dairy ewes. Thirty-six ewes approximately 75 days after parturition were randomly allocated into three experimental treatments, which were provided with three diets: control (C); without the addition of the mixture, B1; supplemented with MPBC at 0.05% and B2; supplemented with rumen protected MPBC at 0.025%. The duration of the experiment was 11 weeks, and milk production was weekly recorded, while individual milk samples for the determination of composition, oxidative stability, somatic cell count (SCC), pH and electric conductivity were collected. Every two weeks, macrophage, lymphocyte, and polymorphonuclear leukocyte counts were also determined in individual milk samples. It was observed that milk yield was the greatest in the B2 group, with significant differences within the seventh and ninth week (p < 0.05), whereas no significant differences were found for milk composition, with the exception of the seventh week, when protein, lactose and non-fat solid levels were lower in MPBC groups (p < 0.05). Oxidative stability was improved in the groups that received the MPBC, with significant differences at the third, seventh, tenth and eleventh week (p < 0.05). SCC was also significantly lower at the second, eighth and ninth week in B2 compared to the other groups (p < 0.05), while no significant effects on the macrophage, lymphocyte, and polymorphonuclear leukocyte counts were observed. In conclusion, the MPBC addition had a positive effect on sheep milk yield, oxidative stability and somatic cell count, without any negative effect on its composition.

Overexpressed Malat1 Drives Metastasis through Inflammatory Reprogramming of Lung Adenocarcinoma Microenvironment.

Metastasis is the main cause of cancer deaths but the molecular events leading to metastatic dissemination remain incompletely understood. Despite reports linking aberrant expression of long noncoding RNAs (lncRNAs) with increased metastatic incidence , in vivo evidence establishing driver roles for lncRNAs in metastatic progression is lacking. Here, we report that overexpression of the metastasis-associated lncRNA Malat1 (metastasis-associated lung adenocarcinoma transcript 1) in the autochthonous K-ras/p53 mouse model of lung adenocarcinoma (LUAD) is sufficient to drive cancer progression and metastatic dissemination. We show that increased expression of endogenous Malat1 RNA cooperates with p53 loss to promote widespread LUAD progression to a poorly differentiated, invasive, and metastatic disease. Mechanistically, we observe that Malat1 overexpression leads to the inappropriate transcription and paracrine secretion of the inflammatory cytokine, Ccl2, to augment the mobility of tumor and stromal cells in vitro and to trigger inflammatory responses in the tumor microenvironment in vivo . Notably, Ccl2 blockade fully reverses cellular and organismal phenotypes of Malat1 overexpression. We propose that Malat1 overexpression in advanced tumors activates Ccl2 signaling to reprogram the tumor microenvironment to an inflammatory and pro-metastatic state.

Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy.

Acquired resistance to immune checkpoint immunotherapy remains a critical yet incompletely understood biological mechanism. Here, using a mouse model of pancreatic ductal adenocarcinoma (PDAC) to study tumor relapse following immunotherapy-induced responses, we found that tumors underwent an epithelial-to-mesenchymal transition (EMT) that resulted in reduced sensitivity to T cell-mediated killing. EMT-transcription factors (EMT-TFs) ZEB1 and SNAIL function as master genetic and epigenetic regulators of this tumor-intrinsic effect. Acquired resistance was not due to immunosuppression in the tumor immune microenvironment, disruptions in the antigen presentation machinery, or altered expression of immune checkpoints. Rather, EMT was associated with epigenetic and transcriptional silencing of interferon regulatory factor 6 (Irf6), which renders tumor cells less sensitive to the pro-apoptotic effects of TNF-α. These findings show how resistance to immunotherapy in PDAC can be acquired through plasticity programs that render tumor cells impervious to T cell killing.