Elevated NSD3 histone methylation activity drives squamous cell lung cancer.

Amplification of chromosomal region 8p11-12 is a common genetic alteration that has been implicated in the aetiology of lung squamous cell carcinoma (LUSC)1-3. The FGFR1 gene is the main candidate driver of tumorigenesis within this region4. However, clinical trials evaluating FGFR1 inhibition as a targeted therapy have been unsuccessful5. Here we identify the histone H3 lysine 36 (H3K36) methyltransferase NSD3, the gene for which is located in the 8p11-12 amplicon, as a key regulator of LUSC tumorigenesis. In contrast to other 8p11-12 candidate LUSC drivers, increased expression of NSD3 correlated strongly with its gene amplification. Ablation of NSD3, but not of FGFR1, attenuated tumour growth and extended survival in a mouse model of LUSC. We identify an LUSC-associated variant NSD3(T1232A) that shows increased catalytic activity for dimethylation of H3K36 (H3K36me2) in vitro and in vivo. Structural dynamic analyses revealed that the T1232A substitution elicited localized mobility changes throughout the catalytic domain of NSD3 to relieve auto-inhibition and to increase accessibility of the H3 substrate. Expression of NSD3(T1232A) in vivo accelerated tumorigenesis and decreased overall survival in mouse models of LUSC. Pathological generation of H3K36me2 by NSD3(T1232A) reprograms the chromatin landscape to promote oncogenic gene expression signatures. Furthermore, NSD3, in a manner dependent on its catalytic activity, promoted transformation in human tracheobronchial cells and growth of xenografted human LUSC cell lines with amplification of 8p11-12. Depletion of NSD3 in patient-derived xenografts from primary LUSCs containing NSD3 amplification or the NSD3(T1232A)-encoding variant attenuated neoplastic growth in mice. Finally, NSD3-regulated LUSC-derived xenografts were hypersensitive to bromodomain inhibition. Thus, our work identifies NSD3 as a principal 8p11-12 amplicon-associated oncogenic driver in LUSC, and suggests that NSD3-dependency renders LUSC therapeutically vulnerable to bromodomain inhibition.

Trametinib sensitizes KRAS-mutant lung adenocarcinoma tumors to PD-1/PD-L1 axis blockade via Id1 downregulation.

BACKGROUND: The identification of novel therapeutic strategies to overcome resistance to the MEK inhibitor trametinib in mutant KRAS lung adenocarcinoma (LUAD) is a challenge. This study analyzes the effects of trametinib on Id1 protein, a key factor involved in the KRAS oncogenic pathway, and investigates the role of Id1 in the acquired resistance to trametinib as well as the synergistic anticancer effect of trametinib combined with immunotherapy in KRAS-mutant LUAD. METHODS: We evaluated the effects of trametinib on KRAS-mutant LUAD by Western blot, RNA-seq and different syngeneic mouse models. Genetic modulation of Id1 expression was performed in KRAS-mutant LUAD cells by lentiviral or retroviral transductions of specific vectors. Cell viability was assessed by cell proliferation and colony formation assays. PD-L1 expression and apoptosis were measured by flow cytometry. The anti-tumor efficacy of the combined treatment with trametinib and PD-1 blockade was investigated in KRAS-mutant LUAD mouse models, and the effects on the tumor immune infiltrate were analyzed by flow cytometry and immunohistochemistry. RESULTS: We found that trametinib activates the proteasome-ubiquitin system to downregulate Id1 in KRAS-mutant LUAD tumors. Moreover, we found that Id1 plays a major role in the acquired resistance to trametinib treatment in KRAS-mutant LUAD cells. Using two preclinical syngeneic KRAS-mutant LUAD mouse models, we found that trametinib synergizes with PD-1/PD-L1 blockade to hamper lung cancer progression and increase survival. This anti-tumor activity depended on trametinib-mediated Id1 reduction and was associated with a less immunosuppressive tumor microenvironment and increased PD-L1 expression on tumor cells. CONCLUSIONS: Our data demonstrate that Id1 expression is involved in the resistance to trametinib and in the synergistic effect of trametinib with anti-PD-1 therapy in KRAS-mutant LUAD tumors. These findings suggest a potential therapeutic approach for immunotherapy-refractory KRAS-mutant lung cancers.

Experimental models to unravel the molecular pathogenesis, cell of origin and stem cell properties of cholangiocarcinoma.

Human cholangiocarcinoma (CCA) is an aggressive tumour entity arising from the biliary tree, whose molecular pathogenesis remains largely undeciphered. Over the last decade, the advent of high-throughput and cell-based techniques has significantly increased our knowledge on the molecular mechanisms underlying this disease while, at the same time, unravelling CCA complexity. In particular, it becomes clear that CCA displays pronounced inter- and intratumoural heterogeneity, which is presumably the consequence of the interplay between distinct tissues and cells of origin, the underlying diseases, and the associated molecular alterations. To better characterize these events and to design novel and more effective therapeutic strategies, a number of CCA experimental and preclinical models have been developed and are currently generated. This review summarizes the current knowledge and understanding of these models, critically underlining their translational usefulness and limitations. Furthermore, this review aims to provide a comprehensive overview on cells of origin, cancers stem cells and their dynamic interplay within CCA tissue.

Blockade of the Complement C5a/C5aR1 Axis Impairs Lung Cancer Bone Metastasis by CXCL16-mediated Effects.

RATIONALE: C5aR1 (CD88), a receptor for complement anaphylatoxin C5a, is a potent immune mediator. Its impact on malignant growth and dissemination of non-small cell lung cancer cells is poorly understood. OBJECTIVES: To investigate the contribution of the C5a/C5aR1 axis to the malignant phenotype of non-small cell lung cancer cells, particularly in skeletal colonization, a preferential lung metastasis site. METHODS: Association between C5aR1 expression and clinical outcome was assessed in silico and validated by immunohistochemistry. Functional significance was evaluated by lentiviral gene silencing and ligand l-aptamer inhibition in in vivo models of lung cancer bone metastasis. In vitro functional assays for signaling, migration, invasion, metalloprotease activity, and osteoclastogenesis were also performed. MEASUREMENTS AND MAIN RESULTS: High levels of C5aR1 in human lung tumors were significantly associated with shorter recurrence-free survival, overall survival, and bone metastasis. Silencing of C5aR1 in lung cancer cells led to a substantial reduction in skeletal metastatic burden and osteolysis in in vivo models. Furthermore, metalloproteolytic, migratory, and invasive tumor cell activities were modulated in vitro by C5aR1 stimulation or gene silencing. l-Aptamer blockade or C5aR1 silencing significantly reduced the osseous metastatic activity of lung cancer cells in vivo. This effect was associated with decreased osteoclastogenic activity in vitro and was rescued by the exogenous addition of the chemokine CXCL16. CONCLUSIONS: Disruption of C5aR1 signaling in lung cancer cells abrogates their tumor-associated osteoclastogenic activity, impairing osseous colonization. This study unveils the role played by the C5a/C5aR1 axis in lung cancer dissemination and supports its potential use as a novel therapeutic target.

KRAS oncogene in non-small cell lung cancer: clinical perspectives on the treatment of an old target.

Lung neoplasms are the leading cause of death by cancer worldwide. Non-small cell lung cancer (NSCLC) constitutes more than 80% of all lung malignancies and the majority of patients present advanced disease at onset. However, in the last decade, multiple oncogenic driver alterations have been discovered and each of them represents a potential therapeutic target. Although KRAS mutations are the most frequently oncogene aberrations in lung adenocarcinoma patients, effective therapies targeting KRAS have yet to be developed. Moreover, the role of KRAS oncogene in NSCLC remains unclear and its predictive and prognostic impact remains controversial. The study of the underlying biology of KRAS in NSCLC patients could help to determine potential candidates to evaluate novel targeted agents and combinations that may allow a tailored treatment for these patients. The aim of this review is to update the current knowledge about KRAS-mutated lung adenocarcinoma, including a historical overview, the biology of the molecular pathways involved, the clinical relevance of KRAS mutations as a prognostic and predictive marker and the potential therapeutic approaches for a personalized treatment of KRAS-mutated NSCLC patients.

Matrix-Gla protein promotes osteosarcoma lung metastasis and associates with poor prognosis.

Osteosarcoma (OS) is the most prevalent osseous tumour in children and adolescents and, within this, lung metastases remain one of the factors associated with a dismal prognosis. At present, the genetic determinants driving pulmonary metastasis are poorly understood. We adopted a novel strategy using robust filtering analysis of transcriptomic profiling in tumour osteoblastic cell populations derived from human chemo-naive primary tumours displaying extreme phenotypes (indolent versus metastatic) to uncover predictors associated with metastasis and poor survival. We identified MGP, encoding matrix-Gla protein (MGP), a non-collagenous matrix protein previously associated with the inhibition of arterial calcification. Using different orthotopic models, we found that ectopic expression of Mgp in murine and human OS cells led to a marked increase in lung metastasis. This effect was independent of the carboxylation of glutamic acid residues required for its physiological role. Abrogation of Mgp prevented lung metastatic activity, an effect that was rescued by forced expression. Mgp levels dramatically altered endothelial adhesion, trans-endothelial migration in vitro and tumour cell extravasation ability in vivo. Furthermore, Mgp modulated metalloproteinase activities and TGFß-induced Smad2/3 phosphorylation. In the clinical setting, OS patients who developed lung metastases had high serum levels of MGP at diagnosis. Thus, MGP represents a novel adverse prognostic factor and a potential therapeutic target in OS. Microarray datasets may be found at: http://bioinfow.dep.usal.es/osteosarcoma/ Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Bone metastases in lung cancer. Potential novel approaches to therapy.

The skeleton is a common site of metastases in lung cancer, an event associated with significant morbidities and poor outcomes. Current antiresorptive therapies provide limited benefit, and novel strategies of prevention and treatment are urgently needed. This review summarizes the latest advances and new perspectives on emerging experimental and clinical approaches to block this deleterious process. Progress propelled by preclinical models has led to a deeper understanding on the complex interplay of tumor cells in the osseous milieu, unveiling potential new targets for drug development. Improvements in early diagnosis through the use of sophisticated imaging techniques with bone serum biomarkers are also discussed in the context of identifying patients at risk and monitoring disease progression during the course of treatment.

Twist1 suppresses senescence programs and thereby accelerates and maintains mutant Kras-induced lung tumorigenesis.

KRAS mutant lung cancers are generally refractory to chemotherapy as well targeted agents. To date, the identification of drugs to therapeutically inhibit K-RAS have been unsuccessful, suggesting that other approaches are required. We demonstrate in both a novel transgenic mutant Kras lung cancer mouse model and in human lung tumors that the inhibition of Twist1 restores a senescence program inducing the loss of a neoplastic phenotype. The Twist1 gene encodes for a transcription factor that is essential during embryogenesis. Twist1 has been suggested to play an important role during tumor progression. However, there is no in vivo evidence that Twist1 plays a role in autochthonous tumorigenesis. Through two novel transgenic mouse models, we show that Twist1 cooperates with Kras(G12D) to markedly accelerate lung tumorigenesis by abrogating cellular senescence programs and promoting the progression from benign adenomas to adenocarcinomas. Moreover, the suppression of Twist1 to physiological levels is sufficient to cause Kras mutant lung tumors to undergo senescence and lose their neoplastic features. Finally, we analyzed more than 500 human tumors to demonstrate that TWIST1 is frequently overexpressed in primary human lung tumors. The suppression of TWIST1 in human lung cancer cells also induced cellular senescence. Hence, TWIST1 is a critical regulator of cellular senescence programs, and the suppression of TWIST1 in human tumors may be an effective example of pro-senescence therapy.

The Regulators of Peroxisomal Acyl-Carnitine Shuttle CROT and CRAT Promote Metastasis in Melanoma.

Circulating tumor cells are the key link between a primary tumor and distant metastases, but once in the bloodstream, loss of adhesion induces cell death. To identify the mechanisms relevant for melanoma circulating tumor cell survival, we performed RNA sequencing and discovered that detached melanoma cells and isolated melanoma circulating tumor cells rewire lipid metabolism by upregulating fatty acid (FA) transport and FA beta-oxidation‒related genes. In patients with melanoma, high expression of FA transporters and FA beta-oxidation enzymes significantly correlates with reduced progression-free and overall survival. Among the highest expressed regulators in melanoma circulating tumor cells were the carnitine transferases carnitine O-octanoyltransferase and carnitine acetyltransferase, which control the shuttle of peroxisome-derived medium-chain FAs toward mitochondria to fuel mitochondrial FA beta-oxidation. Knockdown of carnitine O-octanoyltransferase or carnitine acetyltransferase and short-term treatment with peroxisomal or mitochondrial FA beta-oxidation inhibitors thioridazine or ranolazine suppressed melanoma metastasis in mice. Carnitine O-octanoyltransferase and carnitine acetyltransferase depletion could be rescued by medium-chain FA supplementation, indicating that the peroxisomal supply of FAs is crucial for the survival of nonadherent melanoma cells. Our study identifies targeting the FA-based cross-talk between peroxisomes and mitochondria as a potential therapeutic opportunity to challenge melanoma progression. Moreover, the discovery of the antimetastatic activity of the Food and Drug Administration‒approved drug ranolazine carries translational potential.

TIM-3 blockade in diffuse intrinsic pontine glioma models promotes tumor regression and antitumor immune memory.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain stem tumor and the leading cause of pediatric cancer-related death. To date, these tumors remain incurable, underscoring the need for efficacious therapies. In this study, we demonstrate that the immune checkpoint TIM-3 (HAVCR2) is highly expressed in both tumor cells and microenvironmental cells, mainly microglia and macrophages, in DIPG. We show that inhibition of TIM-3 in syngeneic models of DIPG prolongs survival and produces long-term survivors free of disease that harbor immune memory. This antitumor effect is driven by the direct effect of TIM-3 inhibition in tumor cells, the coordinated action of several immune cell populations, and the secretion of chemokines/cytokines that create a proinflammatory tumor microenvironment favoring a potent antitumor immune response. This work uncovers TIM-3 as a bona fide target in DIPG and supports its clinical translation.

Preclinical models for prediction of immunotherapy outcomes and immune evasion mechanisms in genetically heterogeneous multiple myeloma.

The historical lack of preclinical models reflecting the genetic heterogeneity of multiple myeloma (MM) hampers the advance of therapeutic discoveries. To circumvent this limitation, we screened mice engineered to carry eight MM lesions (NF-κB, KRAS, MYC, TP53, BCL2, cyclin D1, MMSET/NSD2 and c-MAF) combinatorially activated in B lymphocytes following T cell-driven immunization. Fifteen genetically diverse models developed bone marrow (BM) tumors fulfilling MM pathogenesis. Integrative analyses of ∼500 mice and ∼1,000 patients revealed a common MAPK-MYC genetic pathway that accelerated time to progression from precursor states across genetically heterogeneous MM. MYC-dependent time to progression conditioned immune evasion mechanisms that remodeled the BM microenvironment differently. Rapid MYC-driven progressors exhibited a high number of activated/exhausted CD8+ T cells with reduced immunosuppressive regulatory T (Treg) cells, while late MYC acquisition in slow progressors was associated with lower CD8+ T cell infiltration and more abundant Treg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8+ T cells versus Treg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8+ T/Treg cell ratios underlie early progression in untreated smoldering MM, and correlated with early relapse in newly diagnosed patients with MM under Len/Dex therapy. In ICB-refractory MM models, increasing CD8+ T cell cytotoxicity or depleting Treg cells reversed immunotherapy resistance and yielded prolonged MM control. Our experimental models enable the correlation of MM genetic and immunological traits with preclinical therapy responses, which may inform the next-generation immunotherapy trials.

Epigenetic basis of oncogenic-Kras-mediated epithelial-cellular proliferation and plasticity.

Oncogenic Kras induces a hyper-proliferative state that permits cells to progress to neoplasms in diverse epithelial tissues. Depending on the cell of origin, this also involves lineage transformation. Although a multitude of downstream factors have been implicated in these processes, the precise chronology of molecular events controlling them remains elusive. Using mouse models, primary human tissues, and cell lines, we show that, in Kras-mutant alveolar type II cells (AEC2), FOSL1-based AP-1 factor guides the mSWI/SNF complex to increase chromatin accessibility at genomic loci controlling the expression of genes necessary for neoplastic transformation. We identified two orthogonal processes in Kras-mutant distal airway club cells. The first promoted their transdifferentiation into an AEC2-like state through NKX2.1, and the second controlled oncogenic transformation through the AP-1 complex. Our results suggest that neoplasms retain an epigenetic memory of their cell of origin through cell-type-specific transcription factors. Our analysis showed that a cross-tissue-conserved AP-1-dependent chromatin remodeling program regulates carcinogenesis.

Complement C5a induces the formation of neutrophil extracellular traps by myeloid-derived suppressor cells to promote metastasis.

Myeloid-derived suppressor cells (MDSCs) play a major role in cancer progression. In this study, we investigated the mechanisms by which complement C5a increases the capacity of polymorphonuclear MDSCs (PMN-MDSCs) to promote tumor growth and metastatic spread. Stimulation of PMN-MDSCs with C5a favored the invasion of cancer cells via a process dependent on the formation of neutrophil extracellular traps (NETs). NETosis was dependent on the production of high mobility group box 1 (HMGB1) by cancer cells. Moreover, C5a induced the surface expression of the HMGB1 receptors TLR4 and RAGE in PMN-MDSCs. In a mouse lung metastasis model, inhibition of C5a, C5a receptor-1 (C5aR1) or NETosis reduced the number of circulating-tumor cells (CTCs) and the metastatic burden. In support of the translational relevance of these findings, C5a was able to stimulate migration and NETosis in PMN-MDSCs obtained from lung cancer patients. Furthermore, myeloperoxidase (MPO)-DNA complexes, as markers of NETosis, were elevated in lung cancer patients and significantly correlated with C5a levels. In conclusion, C5a induces the formation of NETs from PMN-MDSCs in the presence of cancer cells, which may facilitate cancer cell dissemination and metastasis.

Tumor ENPP1 (CD203a)/Haptoglobin Axis Exploits Myeloid-Derived Suppressor Cells to Promote Post-Radiotherapy Local Recurrence in Breast Cancer.

ABSTRACT: Locoregional failure (LRF) in patients with breast cancer post-surgery and post-irradiation is linked to a dismal prognosis. In a refined new model, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1/CD203a (ENPP1) to be closely associated with LRF. ENPP1hi circulating tumor cells (CTC) contribute to relapse by a self-seeding mechanism. This process requires the infiltration of polymorphonuclear myeloid-derived suppressor cells and neutrophil extracellular trap (NET) formation. Genetic and pharmacologic ENPP1 inhibition or NET blockade extends relapse-free survival. Furthermore, in combination with fractionated irradiation, ENPP1 abrogation obliterates LRF. Mechanistically, ENPP1-generated adenosinergic metabolites enhance haptoglobin (HP) expression. This inflammatory mediator elicits myeloid invasiveness and promotes NET formation. Accordingly, a significant increase in ENPP1 and NET formation is detected in relapsed human breast cancer tumors. Moreover, high ENPP1 or HP levels are associated with poor prognosis. These findings unveil the ENPP1/HP axis as an unanticipated mechanism exploited by tumor cells linking inflammation to immune remodeling favoring local relapse. SIGNIFICANCE: CTC exploit the ENPP1/HP axis to promote local recurrence post-surgery and post-irradiation by subduing myeloid suppressor cells in breast tumors. Blocking this axis impairs tumor engraftment, impedes immunosuppression, and obliterates NET formation, unveiling new opportunities for therapeutic intervention to eradicate local relapse and ameliorate patient survival. This article is highlighted in the In This Issue feature, p. 1171.

The Transcription Factor SLUG Uncouples Pancreatic Cancer Progression from the RAF-MEK1/2-ERK1/2 Pathway.

Activating mutations in some isoforms of RAS or RAF are drivers of a substantial proportion of cancers. The main Raf effector, MEK1/2, can be targeted with several highly specific inhibitors. The clinical activity of these inhibitors seems to be mixed, showing efficacy against mutant BRAF-driven tumors but not KRAS-driven tumors, such as pancreatic adenocarcinomas. To improve our understanding of this context-dependent efficacy, we generated pancreatic cancer cells resistant to MEK1/2 inhibition, which were also resistant to KRAS and ERK1/2 inhibitors. Compared with parental cells, inhibitor-resistant cells showed several phenotypic changes including increased metastatic ability in vivo. The transcription factor SLUG, which is known to induce epithelial-to-mesenchymal transition, was identified as the key factor responsible for both resistance to MEK1/2 inhibition and increased metastasis. Slug, but not similar transcription factors, predicted poor prognosis of pancreatic cancer patients and induced the transition to a cellular phenotype in which cell-cycle progression becomes independent of the KRAS-RAF-MEK1/2-ERK1/2 pathway. SLUG was targeted using two independent strategies: (i) inhibition of the MEK5-ERK5 pathway, which is responsible for upregulation of SLUG upon MEK1/2 inhibition, and (ii) direct PROTAC-mediated degradation. Both strategies were efficacious in preclinical pancreatic cancer models, paving the path for the development of more effective therapies against pancreatic cancer. SIGNIFICANCE: This study demonstrates that SLUG confers resistance to MEK1/2 inhibitors in pancreatic cancer by uncoupling tumor progression from KRAS-RAF-MEK1/2-ERK1/2 signaling, providing new therapeutic opportunities. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/14/3849/F1.large.jpg.

FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy.

BACKGROUND: Fibroblast growth factor receptor (FGFR)1 and FGFR4 have been associated with tumorigenesis in a variety of tumour types. As a therapeutic approach, their inhibition has been attempted in different types of malignancies, including lung cancer, and was initially focused on FGFR1-amplified tumours, though with limited success. METHODS: In vitro and in vivo functional assessments of the oncogenic potential of downregulated/overexpressed genes in isogenic cell lines were performed, as well as inhibitor efficacy tests in vitro and in vivo in patient-derived xenografts (PDXs). mRNA was extracted from FFPE non-small cell lung cancer samples to determine the prognostic potential of the genes under study. FINDINGS: We provide in vitro and in vivo evidence showing that expression of the adhesion molecule N-cadherin is key for the oncogenic role of FGFR1/4 in non-small cell lung cancer. According to this, assessment of the expression of genes in different lung cancer patient cohorts showed that FGFR1 or FGFR4 expression alone showed no prognostic potential, and that only co-expression of FGFR1 and/or FGFR4 with N-cadherin inferred a poorer outcome. Treatment of high-FGFR1 and/or FGFR4-expressing lung cancer cell lines and patient-derived xenografts with selective FGFR inhibitors showed high efficacy, but only in models with high FGFR1/4 and N-cadherin expression. INTERPRETATION: Our data show that the determination of the expression of FGFR1 or FGFR4 alone is not sufficient to predict anti-FGFR therapy efficacy; complementary determination of N-cadherin expression may further optimise patient selection for this therapeutic strategy.

The Mir181ab1 cluster promotes KRAS-driven oncogenesis and progression in lung and pancreas.

Few therapies are currently available for patients with KRAS-driven cancers, highlighting the need to identify new molecular targets that modulate central downstream effector pathways. Here we found that the microRNA (miRNA) cluster including miR181ab1 is a key modulator of KRAS-driven oncogenesis. Ablation of Mir181ab1 in genetically engineered mouse models of Kras-driven lung and pancreatic cancer was deleterious to tumor initiation and progression. Expression of both resident miRNAs in the Mir181ab1 cluster, miR181a1 and miR181b1, was necessary to rescue the Mir181ab1-loss phenotype, underscoring their nonredundant role. In human cancer cells, depletion of miR181ab1 impaired proliferation and 3D growth, whereas overexpression provided a proliferative advantage. Lastly, we unveiled miR181ab1-regulated genes responsible for this phenotype. These studies identified what we believe to be a previously unknown role for miR181ab1 as a potential therapeutic target in 2 highly aggressive and difficult to treat KRAS-mutated cancers.

Id1 and PD-1 Combined Blockade Impairs Tumor Growth and Survival of KRAS-mutant Lung Cancer by Stimulating PD-L1 Expression and Tumor Infiltrating CD8+ T Cells.

The use of PD-1/PD-L1 checkpoint inhibitors in advanced NSCLC is associated with longer survival. However, many patients do not benefit from PD-1/PD-L1 blockade, largely because of immunosuppression. New immunotherapy-based combinations are under investigation in an attempt to improve outcomes. Id1 (inhibitor of differentiation 1) is involved in immunosuppression. In this study, we explored the potential synergistic effect of the combination of Id1 inhibition and pharmacological PD-L1 blockade in three different syngeneic murine KRAS-mutant lung adenocarcinoma models. TCGA analysis demonstrated a negative and statistically significant correlation between PD-L1 and Id1 expression levels. This observation was confirmed in vitro in human and murine KRAS-driven lung cancer cell lines. In vivo experiments in KRAS-mutant syngeneic and metastatic murine lung adenocarcinoma models showed that the combined blockade targeting Id1 and PD-1 was more effective than each treatment alone in terms of tumor growth impairment and overall survival improvement. Mechanistically, multiplex quantification of CD3+/CD4+/CD8+ T cells and flow cytometry analysis showed that combined therapy favors tumor infiltration by CD8+ T cells, whilst in vivo CD8+ T cell depletion led to tumor growth restoration. Co-culture assays using CD8+ cells and tumor cells showed that T cells present a higher antitumor effect when tumor cells lack Id1 expression. These findings highlight that Id1 blockade may contribute to a significant immune enhancement of antitumor efficacy of PD-1 inhibitors by increasing PD-L1 expression and harnessing tumor infiltration of CD8+ T lymphocytes.

Short-term starvation reduces IGF-1 levels to sensitize lung tumors to PD-1 immune checkpoint blockade.

Harnessing the immune system by blocking the programmed cell death protein 1 (PD-1) pathway has been a major breakthrough in non-small-cell lung cancer treatment. Nonetheless, many patients fail to respond to PD-1 inhibition. Using three syngeneic models, we demonstrate that short-term starvation synergizes with PD-1 blockade to inhibit lung cancer progression and metastasis. This antitumor activity was linked to a reduction in circulating insulin-like growth factor 1 (IGF-1) and a downregulation of IGF-1 receptor (IGF-1R) signaling in tumor cells. A combined inhibition of IGF-1R and PD-1 synergistically reduced tumor growth in mice. This effect required CD8 cells, boosted the intratumoral CD8/Treg ratio and led to the development of tumor-specific immunity. In patients with non-small-cell lung cancer, high plasma levels of IGF-1 or high IGF-1R expression in tumors was associated with resistance to anti-PD-1-programmed death-ligand 1 immunotherapy. In conclusion, our data strongly support the clinical evaluation of IGF-1 modulators in combination with PD-1 blockade.

Genetic Mouse Models as In Vivo Tools for Cholangiocarcinoma Research.

Cholangiocarcinoma (CCA) is a genetically and histologically complex disease with a highly dismal prognosis. A deeper understanding of the underlying cellular and molecular mechanisms of human CCA will increase our current knowledge of the disease and expedite the eventual development of novel therapeutic strategies for this fatal cancer. This endeavor is effectively supported by genetic mouse models, which serve as sophisticated tools to systematically investigate CCA pathobiology and treatment response. These in vivo models feature many of the genetic alterations found in humans, recapitulate multiple hallmarks of cholangiocarcinogenesis (encompassing cell transformation, preneoplastic lesions, established tumors and metastatic disease) and provide an ideal experimental setting to study the interplay between tumor cells and the surrounding stroma. This review is intended to serve as a compendium of CCA mouse models, including traditional transgenic models but also genetically flexible approaches based on either the direct introduction of DNA into liver cells or transplantation of pre-malignant cells, and is meant as a resource for CCA researchers to aid in the selection of the most appropriate in vivo model system.