Biochemical and cellular properties of insulin receptor signalling.

The mechanism of insulin action is a central theme in biology and medicine. In addition to the rather rare condition of insulin deficiency caused by autoimmune destruction of pancreatic ß-cells, genetic and acquired abnormalities of insulin action underlie the far more common conditions of type 2 diabetes, obesity and insulin resistance. The latter predisposes to diseases ranging from hypertension to Alzheimer disease and cancer. Hence, understanding the biochemical and cellular properties of insulin receptor signalling is arguably a priority in biomedical research. In the past decade, major progress has led to the delineation of mechanisms of glucose transport, lipid synthesis, storage and mobilization. In addition to direct effects of insulin on signalling kinases and metabolic enzymes, the discovery of mechanisms of insulin-regulated gene transcription has led to a reassessment of the general principles of insulin action. These advances will accelerate the discovery of new treatment modalities for diabetes.

Discovery of a class of endogenous mammalian lipids with anti-diabetic and anti-inflammatory effects.

Increased adipose tissue lipogenesis is associated with enhanced insulin sensitivity. Mice overexpressing the Glut4 glucose transporter in adipocytes have elevated lipogenesis and increased glucose tolerance despite being obese with elevated circulating fatty acids. Lipidomic analysis of adipose tissue revealed the existence of branched fatty acid esters of hydroxy fatty acids (FAHFAs) that were elevated 16- to 18-fold in these mice. FAHFA isomers differ by the branched ester position on the hydroxy fatty acid (e.g., palmitic-acid-9-hydroxy-stearic-acid, 9-PAHSA). PAHSAs are synthesized in vivo and regulated by fasting and high-fat feeding. PAHSA levels correlate highly with insulin sensitivity and are reduced in adipose tissue and serum of insulin-resistant humans. PAHSA administration in mice lowers ambient glycemia and improves glucose tolerance while stimulating GLP-1 and insulin secretion. PAHSAs also reduce adipose tissue inflammation. In adipocytes, PAHSAs signal through GPR120 to enhance insulin-stimulated glucose uptake. Thus, FAHFAs are endogenous lipids with the potential to treat type 2 diabetes.

Regulated dynamic subcellular GLUT4 localization revealed by proximal proteome mapping in human muscle cells.

Regulation of glucose transport, which is central for control of whole-body metabolism, is determined by the amount of GLUT4 glucose transporter (also known as SLC2A4) in the plasma membrane (PM) of fat and muscle cells. Physiologic signals [such as activated insulin receptor or AMP-activated protein kinase (AMPK)] increase PM GLUT4. Here, we show that the distribution of GLUT4 between the PM and interior of human muscle cells is dynamically maintained, and that AMPK promotes PM redistribution of GLUT4 by regulating exocytosis and endocytosis. Stimulation of exocytosis by AMPK is mediated by Rab10 and the Rab GTPase-activating protein TBC1D4. APEX2 proximity mapping reveals that GLUT4 traverses both PM-proximal and PM-distal compartments in unstimulated muscle cells, further supporting retention of GLUT4 by a constitutive retrieval mechanism. AMPK-stimulated translocation involves GLUT4 redistribution among the same compartments traversed in unstimulated cells, with a significant recruitment of GLUT4 from the Golgi and trans-Golgi network compartments. Our comprehensive proximal protein mapping provides an integrated, high-density, whole-cell accounting of the localization of GLUT4 at a resolution of ∼20 nm that serves as a structural framework for understanding the molecular mechanisms regulating GLUT4 trafficking downstream of different signaling inputs in a physiologically relevant cell type.

Proximity proteome mapping reveals PD-L1-dependent pathways disrupted by anti-PD-L1 antibody specifically in EGFR-mutant lung cancer cells.

BACKGROUND: PD-L1, a transmembrane ligand for immune checkpoint receptor PD1, has been successfully targeted to activate an anti-tumor immune response in a variety of solid tumors, including non-small cell lung cancer (NSCLC). Despite the success of targeting PD-L1, only about 20% of patients achieve a durable response. The reasons for the heterogeneity in response are not understood, although some molecular subtypes (e.g., mutant EGF receptor tumors) are generally poor responders. Although PD-L1 is best characterized as a transmembrane PD1 ligand, the emerging view is that PD-L1 has functions independent of activating PD1 signaling. It is not known whether these cell-intrinsic functions of PD-L1 are shared among non-transformed and transformed cells, if they vary among cancer molecular subtypes, or if they are impacted by anti-PD-L1 therapy. METHODS: Here we use quantitative microscopy techniques and APEX2 proximity mapping to describe the behavior of PD-L1 and to identify PD-L1's proximal proteome in human lung epithelial cells. RESULTS: Our data reveal growth factor control of PD-L1 recycling as a mechanism for acute and reversible regulation of PD-L1 density on the plasma membrane. In addition, we describe novel PD-L1 biology restricted to mutant EGFR cells. Anti-PD-L1 antibody treatment of mutant EGFR cells perturbs cell intrinsic PD-L1 functions, leading to reduced cell migration, increased half-life of EGFR and increased extracellular vesicle biogenesis, whereas anti-PD-L1 antibody does not induce these changes in wild type EGFR cells. CONCLUSIONS: Growth factor acute regulation of PD-L1 trafficking, by contributing to the control of plasma membrane density, might contribute to the regulation of PD-L1's immune checkpoint activity, whereas the specific effects of anti-PD-L1 on mutant EGFR cells might contribute to the poor anti-PD-L1 response of mutant EGFR tumors. Video Abstract.

Neoadjuvant durvalumab with or without stereotactic body radiotherapy in patients with early-stage non-small-cell lung cancer: a single-centre, randomised phase 2 trial.

BACKGROUND: Previous phase 2 trials of neoadjuvant anti-PD-1 or anti-PD-L1 monotherapy in patients with early-stage non-small-cell lung cancer have reported major pathological response rates in the range of 15-45%. Evidence suggests that stereotactic body radiotherapy might be a potent immunomodulator in advanced non-small-cell lung cancer (NSCLC). In this trial, we aimed to evaluate the use of stereotactic body radiotherapy in patients with early-stage NSCLC as an immunomodulator to enhance the anti-tumour immune response associated with the anti-PD-L1 antibody durvalumab. METHODS: We did a single-centre, open-label, randomised, controlled, phase 2 trial, comparing neoadjuvant durvalumab alone with neoadjuvant durvalumab plus stereotactic radiotherapy in patients with early-stage NSCLC, at NewYork-Presbyterian and Weill Cornell Medical Center (New York, NY, USA). We enrolled patients with potentially resectable early-stage NSCLC (clinical stages I-IIIA as per the 7th edition of the American Joint Committee on Cancer) who were aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 0 or 1. Eligible patients were randomly assigned (1:1) to either neoadjuvant durvalumab monotherapy or neoadjuvant durvalumab plus stereotactic body radiotherapy (8 Gy × 3 fractions), using permuted blocks with varied sizes and no stratification for clinical or molecular variables. Patients, treating physicians, and all study personnel were unmasked to treatment assignment after all patients were randomly assigned. All patients received two cycles of durvalumab 3 weeks apart at a dose of 1·12 g by intravenous infusion over 60 min. Those in the durvalumab plus radiotherapy group also received three consecutive daily fractions of 8 Gy stereotactic body radiotherapy delivered to the primary tumour immediately before the first cycle of durvalumab. Patients without systemic disease progression proceeded to surgical resection. The primary endpoint was major pathological response in the primary tumour. All analyses were done on an intention-to-treat basis. This trial is registered with ClinicalTrial.gov, NCT02904954, and is ongoing but closed to accrual. FINDINGS: Between Jan 25, 2017, and Sept 15, 2020, 96 patients were screened and 60 were enrolled and randomly assigned to either the durvalumab monotherapy group (n=30) or the durvalumab plus radiotherapy group (n=30). 26 (87%) of 30 patients in each group had their tumours surgically resected. Major pathological response was observed in two (6·7% [95% CI 0·8-22·1]) of 30 patients in the durvalumab monotherapy group and 16 (53·3% [34·3-71·7]) of 30 patients in the durvalumab plus radiotherapy group. The difference in the major pathological response rates between both groups was significant (crude odds ratio 16·0 [95% CI 3·2-79·6]; p<0·0001). In the 16 patients in the dual therapy group with a major pathological response, eight (50%) had a complete pathological response. The second cycle of durvalumab was withheld in three (10%) of 30 patients in the dual therapy group due to immune-related adverse events (grade 3 hepatitis, grade 2 pancreatitis, and grade 3 fatigue and thrombocytopaenia). Grade 3-4 adverse events occurred in five (17%) of 30 patients in the durvalumab monotherapy group and six (20%) of 30 patients in the durvalumab plus radiotherapy group. The most frequent grade 3-4 events were hyponatraemia (three [10%] patients in the durvalumab monotherapy group) and hyperlipasaemia (three [10%] patients in the durvalumab plus radiotherapy group). Two patients in each group had serious adverse events (pulmonary embolism [n=1] and stroke [n=1] in the durvalumab monotherapy group, and pancreatitis [n=1] and fatigue [n=1] in the durvalumab plus radiotherapy group). No treatment-related deaths or deaths within 30 days of surgery were reported. INTERPRETATION: Neoadjuvant durvalumab combined with stereotactic body radiotherapy is well tolerated, safe, and associated with a high major pathological response rate. This neoadjuvant strategy should be validated in a larger trial. FUNDING: AstraZeneca.

AMPK-dependent degradation of TXNIP upon energy stress leads to enhanced glucose uptake via GLUT1.

Thioredoxin-interacting protein (TXNIP) is an α-arrestin family protein that is induced in response to glucose elevation. It has been shown to provide a negative feedback loop to regulate glucose uptake into cells, though the biochemical mechanism of action has been obscure. Here, we report that TXNIP suppresses glucose uptake directly, by binding to the glucose transporter GLUT1 and inducing GLUT1 internalization through clathrin-coated pits, as well as indirectly, by reducing the level of GLUT1 messenger RNA (mRNA). In addition, we show that energy stress results in the phosphorylation of TXNIP by AMP-dependent protein kinase (AMPK), leading to its rapid degradation. This suppression of TXNIP results in an acute increase in GLUT1 function and an increase in GLUT1 mRNA (hence the total protein levels) for long-term adaptation. The glucose influx through GLUT1 restores ATP-to-ADP ratios in the short run and ultimately induces TXNIP protein production to suppress glucose uptake once energy homeostasis is reestablished.

Thirty sweet years of GLUT4.

A pivotal metabolic function of insulin is the stimulation of glucose uptake into muscle and adipose tissues. The discovery of the insulin-responsive glucose transporter type 4 (GLUT4) protein in 1988 inspired its molecular cloning in the following year. It also spurred numerous cellular mechanistic studies laying the foundations for how insulin regulates glucose uptake by muscle and fat cells. Here, we reflect on the importance of the GLUT4 discovery and chronicle additional key findings made in the past 30 years. That exocytosis of a multispanning membrane protein regulates cellular glucose transport illuminated a novel adaptation of the secretory pathway, which is to transiently modulate the protein composition of the cellular plasma membrane. GLUT4 controls glucose transport into fat and muscle tissues in response to insulin and also into muscle during exercise. Thus, investigation of regulated GLUT4 trafficking provides a major means by which to map the essential signaling components that transmit the effects of insulin and exercise. Manipulation of the expression of GLUT4 or GLUT4-regulating molecules in mice has revealed the impact of glucose uptake on whole-body metabolism. Remaining gaps in our understanding of GLUT4 function and regulation are highlighted here, along with opportunities for future discoveries and for the development of therapeutic approaches to manage metabolic disease.

Altered Plasma Profile of Antioxidant Proteins as an Early Correlate of Pancreatic ß Cell Dysfunction.

Insulin resistance and ß cell dysfunction contribute to the pathogenesis of type 2 diabetes. Unlike insulin resistance, ß cell dysfunction remains difficult to predict and monitor, because of the inaccessibility of the endocrine pancreas, the integrated relationship with insulin sensitivity, and the paracrine effects of incretins. The goal of our study was to survey the plasma response to a metabolic challenge in order to identify factors predictive of ß cell dysfunction. To this end, we combined (i) the power of unbiased iTRAQ (isobaric tag for relative and absolute quantification) mass spectrometry with (ii) direct sampling of the portal vein following an intravenous glucose/arginine challenge (IVGATT) in (iii) mice with a genetic ß cell defect. By so doing, we excluded the effects of peripheral insulin sensitivity as well as those of incretins on ß cells, and focused on the first phase of insulin secretion to capture the early pathophysiology of ß cell dysfunction. We compared plasma protein profiles with ex vivo islet secretome and transcriptome analyses. We detected changes to 418 plasma proteins in vivo, and detected changes to 262 proteins ex vivo The impairment of insulin secretion was associated with greater overall changes in the plasma response to IVGATT, possibly reflecting metabolic instability. Reduced levels of proteins regulating redox state and neuronal stress markers, as well as increased levels of coagulation factors, antedated the loss of insulin secretion in diabetic mice. These results suggest that a reduced complement of antioxidants in response to a mixed secretagogue challenge is an early correlate of future ß cell failure.

Development of a new model system to dissect isoform specific Akt signalling in adipocytes.

Protein kinase B (Akt) kinases are critical signal transducers mediating insulin action. Genetic studies revealed that Akt1 and Akt2 signalling differentially contribute to sustain lipid and glucose homoeostasis; however Akt isoform-specific effectors remain elusive due to the lack of a suitable model system to mechanistically interrogate Akt isoform-specific signalling. To overcome those technical limitations we developed a novel model system that provides acute and specific control of signalling by Akt isoforms. We generated mutants of Akt1 and Akt2 resistant to the allosteric Akt inhibitor MK-2206. We then developed adipocyte cell lines, in which endogenous Akt1 or Akt2 has been replaced by their corresponding drug-resistant Akt mutant. Treatment of those cells with MK-2206 allowed for acute and specific control of either Akt1 or Akt2 function. Our data showed that Akt1(W80A) and Akt2(W80A) mutants are resistant to MK-2206, dynamically regulated by insulin and able to signal to Akt downstream effectors. Analyses of insulin action in this cellular system showed that Akt1 and Akt2 are both able to mediate insulin regulation of the transcription factor forkhead box O1 (FoxO1) and the glucose transporter 4 (GLUT4), revealing a redundant role for these Akt kinases in the control of glucose transport into fat cells. In contrast, Akt1 signalling is uniquely required for adipogenesis, by controlling the mitotic clonal expansion (MCE) of pre-adipocytes that precedes white adipose cell differentiation. Our data provide new insights into the role of Akt kinases in glucose transport and adipogenesis and support our model system as a valuable tool for the biochemical characterization of signalling by specific Akt isoforms.

The Microenvironment of Lung Cancer and Therapeutic Implications.

The tumor microenvironment (TME) represents a milieu that enables tumor cells to acquire the hallmarks of cancer. The TME is heterogeneous in composition and consists of cellular components, growth factors, proteases, and extracellular matrix. Concerted interactions between genetically altered tumor cells and genetically stable intratumoral stromal cells result in an "activated/reprogramed" stroma that promotes carcinogenesis by contributing to inflammation, immune suppression, therapeutic resistance, and generating premetastatic niches that support the initiation and establishment of distant metastasis. The lungs present a unique milieu in which tumors progress in collusion with the TME, as evidenced by regions of aberrant angiogenesis, acidosis and hypoxia. Inflammation plays an important role in the pathogenesis of lung cancer, and pulmonary disorders in lung cancer patients such as chronic obstructive pulmonary disease (COPD) and emphysema, constitute comorbid conditions and are independent risk factors for lung cancer. The TME also contributes to immune suppression, induces epithelial-to-mesenchymal transition (EMT) and diminishes efficacy of chemotherapies. Thus, the TME has begun to emerge as the "Achilles heel" of the disease, and constitutes an attractive target for anti-cancer therapy. Drugs targeting the components of the TME are making their way into clinical trials. Here, we will focus on recent advances and emerging concepts regarding the intriguing role of the TME in lung cancer progression, and discuss future directions in the context of novel diagnostic and therapeutic opportunities.

Hyperinsulinemia leads to uncoupled insulin regulation of the GLUT4 glucose transporter and the FoxO1 transcription factor.

Insulin resistance is a component of the metabolic syndrome and Type 2 diabetes. It has been recently shown that in liver insulin resistance is not complete. This so-called selective insulin resistance is characterized by defective insulin inhibition of hepatic glucose output while insulin-induced lipogenesis is maintained. How this occurs and whether uncoupled insulin action develops in other tissues is unknown. Here we show in a model of chronic hyperinsulinemia that adipocytes develop selective insulin resistance in which translocation of the GLUT4 glucose transporter to the cell surface is blunted yet nuclear exclusion of the FoxO1 transcription factor is preserved, rendering uncoupled insulin-controlled carbohydrate and lipid metabolisms. We found that in adipocytes FoxO1 nuclear exclusion has a lower half-maximal insulin dose than GLUT4 translocation, and it is because of this inherent greater sensitivity that control of FoxO1 by physiological insulin concentrations is maintained in adipocytes with compromised insulin signaling. Pharmacological and genetic interventions revealed that insulin regulates GLUT4 and FoxO1 through the PI3-kinase isoform p110α, although FoxO1 showed higher sensitivity to p110α activity than GLUT4. Transient down-regulation and overexpression of Akt isoforms in adipocytes demonstrated that insulin-activated PI3-kinase signals to GLUT4 primarily through Akt2 kinase, whereas Akt1 and Akt2 signal to FoxO1. We propose that the lower threshold of insulin activity for FoxO1's nuclear exclusion is in part due to its regulation by both Akt isoforms. Identification of uncoupled insulin action in adipocytes suggests this condition might be a general phenomenon of insulin target tissues contributing to insulin resistance's pathophysiology.

A signature of enhanced proliferation associated with response and survival to anti-PD-L1 therapy in early-stage non-small cell lung cancer.

In early-stage non-small cell lung cancer, the combination of neoadjuvant anti-PD-L1 and subablative stereotactic body radiation therapy (SBRT) is associated with higher rates of major pathologic response compared to anti-PD-L1 alone. Here, we identify a 140-gene set, enriched in genes characteristic of highly proliferating cells, associated with response to the dual therapy. Analysis of on-treatment transcriptome data indicate roles for T and B cells in response. The 140-gene set is associated with disease-free survival when applied to the combined trial arms. This 140-gene set identifies a subclass of tumors in all 7 of The Cancer Genome Atlas tumor types examined. Worse survival is associated with the 140-gene signature in 5 of these tumor types. Collectively, our data support that this 140-gene set, discovered in association with response to combined anti-PD-L1 and SBRT, identifies a clinically aggressive subclass of solid tumors that may be more likely to respond to immunotherapies.

Transferrin receptor-based circulating tumor cell enrichment provides a snapshot of the molecular landscape of solid tumors and correlates with clinical outcomes.

Circulating tumor cells (CTCs) captured from the bloodstream of patients with solid tumors have the potential to accelerate precision oncology by providing insight into tumor biology, disease progression and response to treatment. However, their potential is hampered by the lack of standardized CTC enrichment platforms across tumor types. EpCAM-based CTC enrichment, the most commonly used platform, is limited by EpCAM downregulation during metastasis and the low EpCAM expression in certain tumor types, including the highly prevalent and lethal NSCLC. In this study we demonstrate that Transferrin Receptor (TfR) is a selective, efficient biomarker for CTC identification and capture in patients with prostate, pancreatic and NSCLC. TfR identifies significantly higher CTC counts than EpCAM, and TfR + -CTC enumeration correlates with disease progression in metastatic prostate and pancreatic cancers, and overall survival and osimetrinib-resistance in non-small cell lung cancer (NSCLC). Profiling of TfR + -CTCs provides a snapshot of the molecular landscape of each respective tumor type and identifies potential mechanisms underlying treatment response to EGFR TKi and immune checkpoint inhibitors in NSCLC. One sentence summary: Transferrin Receptor identifies circulating tumor cells in solid tumors.

Regulation of PD-L1 Trafficking from Synthesis to Degradation.

Programmed death-ligand 1 (PD-L1) is a transmembrane ligand for the programmed cell death protein 1 (PD-1), a receptor that inhibits T-cell activity. The PD-L1/PD-1 immune checkpoint axis has been successfully targeted to enhance antitumor immune responses. Tethering PD-L1 to the membrane spatially restricts its ability to inhibit immune responses, and it provides for the acute and reversible modulation of PD-L1 plasma membrane density by regulation of its trafficking. PD-L1 has functions that are independent of its role as a ligand for PD-1, and control of PD-L1 residence in different intracellular compartments might contribute to the regulation of those activities. Thus, control of PD-L1 trafficking is emerging as a key feature of its biology. Herein, we focus on current understating of PD-L1 trafficking and review current attempts to therapeutically target this process in cancer cells to enhance antitumor immunity.

Spatiotemporal regulation of GIPR signaling impacts glucose homeostasis as revealed in studies of a common GIPR variant.

OBJECTIVE: Glucose-dependent insulinotropic polypeptide (GIP) has a role in controlling postprandial metabolic tone. In humans, a GIP receptor (GIPR) variant (Q354, rs1800437) is associated with a lower body mass index (BMI) and increased risk for Type 2 Diabetes. To better understand the impacts of GIPR-Q354 on metabolism, it is necessary to study it in an isogeneic background to the predominant GIPR isoform, E354. To accomplish this objective, we used CRISPR-CAS9 editing to generate mouse models of GIPR-Q354 and GIPR-E354. Here we characterize the metabolic effects of GIPR-Q354 variant in a mouse model (GIPR-Q350). METHODS: We generated the GIPR-Q350 mice for in vivo studies of metabolic impact of the variant. We isolated pancreatic islets from GIPR-Q350 mice to study insulin secretion ex vivo. We used a ß-cell cell line to understand the impact of the GIPR-Q354 variant on the receptor traffic. RESULTS: We found that female GIPR-Q350 mice are leaner than littermate controls, and male GIPR-Q350 mice are resistant to diet-induced obesity, in line with the association of the variant with reduced BMI in humans. GIPR-Q350 mice of both sexes are more glucose tolerant and exhibit an increased sensitivity to GIP. Postprandial GIP levels are reduced in GIPR-Q350 mice, revealing feedback regulation that balances the increased sensitivity of GIP target tissues to secretion of GIP from intestinal endocrine cells. The increased GIP sensitivity is recapitulated ex vivo during glucose stimulated insulin secretion assays in islets. Generation of cAMP in islets downstream of GIPR activation is not affected by the Q354 substitution. However, post-activation traffic of GIPR-Q354 variant in ß-cells is altered, characterized by enhanced intracellular dwell time and increased localization to the Trans-Golgi Network (TGN). CONCLUSIONS: Our data link altered intracellular traffic of the GIPR-Q354 variant with GIP control of metabolism. We propose that this change in spatiotemporal signaling underlies the physiologic effects of GIPR-Q350/4 and GIPR-E350/4 in mice and humans. These findings contribute to a more complete understanding of the impact of GIPR-Q354 variant on glucose homeostasis that could perhaps be leveraged to enhance pharmacologic targeting of GIPR for the treatment of metabolic disease.

Tumor-intrinsic IRE1α signaling controls protective immunity in lung cancer.

IRE1α-XBP1 signaling is emerging as a central orchestrator of malignant progression and immunosuppression in various cancer types. Employing a computational XBP1s detection method applied to TCGA datasets, we demonstrate that expression of the XBP1s mRNA isoform predicts poor survival in non-small cell lung cancer (NSCLC) patients. Ablation of IRE1α in malignant cells delays tumor progression and extends survival in mouse models of NSCLC. This protective effect is accompanied by alterations in intratumoral immune cell subsets eliciting durable adaptive anti-cancer immunity. Mechanistically, cancer cell-intrinsic IRE1α activation sustains mPGES-1 expression, enabling production of the immunosuppressive lipid mediator prostaglandin E2. Accordingly, restoring mPGES-1 expression in IRE1αKO cancer cells rescues normal tumor progression. We have developed an IRE1α gene signature that predicts immune cell infiltration and overall survival in human NSCLC. Our study unveils an immunoregulatory role for cancer cell-intrinsic IRE1α activation and suggests that targeting this pathway may help enhance anti-tumor immunity in NSCLC.

Deficiency of metabolic regulator PKM2 activates the pentose phosphate pathway and generates TCF1+ progenitor CD8+ T cells to improve checkpoint blockade.

TCF1high progenitor CD8+ T cells mediate the efficacy of PD-1 blockade, however the mechanisms that govern their generation and maintenance are poorly understood. Here, we show that targeting glycolysis through deletion of pyruvate kinase muscle 2 (PKM2) results in elevated pentose phosphate pathway (PPP) activity, leading to enrichment of a TCF1high central memory-like phenotype and increased responsiveness to PD-1 blockade in vivo. PKM2KO CD8+ T cells showed reduced glycolytic flux, accumulation of glycolytic intermediates and PPP metabolites, and increased PPP cycling as determined by 1,2 13C glucose carbon tracing. Small molecule agonism of the PPP without acute glycolytic impairment skewed CD8+ T cells towards a TCF1high population, generated a unique transcriptional landscape, enhanced tumor control in mice in combination with PD-1 blockade, and promoted tumor killing in patient-derived tumor organoids. Our study demonstrates a new metabolic reprogramming that contributes to a progenitor-like T cell state amenable to checkpoint blockade.

TIMP1 is an early biomarker for detection and prognosis of lung cancer.

BACKGROUND: Lung cancer remains the major cause of cancer-related deaths worldwide. Early stages of lung cancer are characterized by long asymptomatic periods that are ineffectively identified with the current screening programs. This deficiency represents a lost opportunity to improve the overall survival of patients. Serum biomarkers are among the most effective strategies for cancer screening and follow up. METHODS: Using bead-based multiplexing assays we screened plasma and tumours of the KrasG12D/+; Lkb1f/f (KL) mouse model of lung cancer for cytokines that could be used as biomarkers. We identified tissue inhibitor of metalloproteinase 1 (TIMP1) as an early biomarker and validated this finding in the plasma of lung cancer patients. We used immunohistochemistry (IHC), previously published single-cell RNA-seq and bulk RNA-seq data to assess the source and expression of TIMP1in the tumour. The prognostic value of TIMP1 was assessed using publicly available human proteomic and transcriptomic databases. RESULTS: We found that TIMP1 is a tumour-secreted protein with high sensitivity and specificity for aggressive cancer, even at early stages in mice. We showed that TIMP1 levels in the tumour and serum correlate with tumour burden and worse survival in mice. We validated this finding using clinical samples from our institution and publicly available human proteomic and transcriptomic databases. These data support the finding that high tumour expression of TIMP1 correlates with an unfavorable prognosis in lung cancer patients. CONCLUSION: TIMP1 is a suitable biomarker for lung cancer detection.

Neoadjuvant durvalumab plus radiation versus durvalumab alone in stages I-III non-small cell lung cancer: survival outcomes and molecular correlates of a randomized phase II trial.

We previously reported the results of a randomized phase II trial (NCT02904954) in patients with early-stage non-small cell lung cancer (NSCLC) who were treated with either two preoperative cycles of the anti-PD-L1 antibody durvalumab alone or combined with immunomodulatory doses of stereotactic radiation (DRT). The trial met its primary endpoint of major pathological response, which was significantly higher following DRT with no new safety signals. Here, we report on the prespecified secondary endpoint of disease-free survival (DFS) regardless of treatment assignment and the prespecified exploratory analysis of DFS in each arm of the trial. DFS at 2 and 3 years across patients in both arms of the trial were 73% (95% CI: 62.1-84.5) and 65% (95% CI: 52.5-76.9) respectively. For the exploratory endpoint of DFS in each arm of the trial, three-year DFS was 63% (95% CI: 46.0-80.4) in the durvalumab monotherapy arm compared to 67% (95% CI: 49.6-83.4) in the dual therapy arm. In addition, we report post hoc exploratory analysis of progression-free survival as well as molecular correlates of response and recurrence through high-plex immunophenotyping of sequentially collected peripheral blood and gene expression profiles from resected tumors in both treatment arms. Together, our results contribute to the evolving landscape of neoadjuvant treatment regimens for NSCLC and identify easily measurable potential biomarkers of response and recurrence.

Rab10, a target of the AS160 Rab GAP, is required for insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane.

GLUT4 trafficking to the plasma membrane of muscle and fat cells is regulated by insulin. An important component of insulin-regulated GLUT4 distribution is the Akt substrate AS160 rab GTPase-activating protein. Here we show that Rab10 functions as a downstream target of AS160 in the insulin-signaling pathway that regulates GLUT4 translocation in adipocytes. Overexpression of a mutant of Rab10 defective for GTP hydrolysis increased GLUT4 on the surface of basal adipocytes. Rab10 knockdown resulted in an attenuation of insulin-induced GLUT4 redistribution to the plasma membrane and a concomitant 2-fold decrease in GLUT4 exocytosis rate. Re-expression of a wild-type Rab10 restored normal GLUT4 translocation. The basal increase in plasma-membrane GLUT4 due to AS160 knockdown was partially blocked by knocking down Rab10 in the same cells, further indicating that Rab10 is a target of AS160 and a positive regulator of GLUT4 trafficking to the cell surface upon insulin stimulation.