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Immune checkpoint inhibitors have significantly improved the clinical outcomes of many cancer types, but they induce a range of immune-related adverse events (irAEs). Although adrenal insufficiency (AI) is a rare irAE, it can lead to serious consequences. This study aimed to determine the clinical features of patients with advanced non-small-cell lung cancer (NSCLC) who developed AI following pembrolizumab treatment.We retrospectively reviewed and analyzed the clinical data of all patients with NSCLC treated with pembrolizumab at Juntendo University Hospital from February 2017 to December 2020. The diagnosis of AI was established based on the Endocrine Emergency Guidance for the acute management of endocrine complications of checkpoint inhibitor therapy in the UK and the clinical practice guidelines of the Japan Endocrine Society.AI was clinically suspected in 59 out of 186 patients treated with pembrolizumab, and 10 (5.4%) cases were confirmed. The symptoms included hyponatremia (n = 9), fatigue (n = 8), and loss of appetite (n = 6). All patients had low adrenocorticotropic hormone (ACTH) levels, and five patients were diagnosed with isolated ACTH deficiency. All patients completely recovered with corticosteroid replacement. The median time to onset of AI was 8.0 (range 3.8-15.2) months. The median progression-free survival in these patients was 22.4 (95% confidence interval 11.2-not reached) months.The incidence of AI among patients treated with pembrolizumab is more frequent than previously reported. In addition, secondary AI, especially isolated ACTH deficiency, is a major form of AI induced by pembrolizumab.

Supplementary Figure S5 shows role of MFGE8 in MPM.

Supplementary Figure Legends

Table S3 shows list of KRAS WT; LKB1 WT and KRAS WT; LKB1 Mut lung adenocarcinoma cell lines related with supplementary figure S1.

Table S1: KLF5 E419Q-gained super-enhancers and the nearest genes

Figure S1. Super-enhancers near the KLF5 gene are focally amplified in cancer cell lines;Figure S2. The regulatory potential of the KLF5/KLF12 intervening region;Figure S3. Expression level of KLF12, DIS3 and PIBF1 after repression of KLF5 superenhancers;Figure S4. Genomic analysis of KLF5 binding sites in BICR31 cells;Figure S5. Target genes of KLF5 in head and neck squamous cell carcinomas;Figure S6. The effect of KLF5 silencing on H3K27ac profile;Figure S7. Mutation hotspots in KLF5;Figure S8. Mutation profile in FBXW7 in colorectal cancers; Figure S9. Ectopic expression of KLF5 mutants in HEK293T cells; Figure S10. Ectopic expression of KLF5 E419Q in HCC95 cells

Supplementary Table S1 shows the selected genes with statistically significant changes in transcriptome analysis using two different shRNA constructs.

Table S2: Oligos used in the study

The supplementary file shows Supplementary figure S1-S4 and their legends, and the legends for Supplementary table S1-S4.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that has a low overall survival; however, no significant treatment advances have been made in the past 15 years. Large-scale molecular studies have identified a poor prognostic subset of MPM linked to the epithelial–mesenchymal transition (EMT) that may contribute toward resistance to chemotherapy, suggesting that EMT could be targeted to treat patients with MPM. Previously, we reported that histone modifiers regulating EMT could be therapeutic targets; therefore, in this study, we investigated whether targeting lysine-specific demethylase 1 (LSD1/KDM1), a histone-modifying enzyme responsible for demethylating histone H3 lysine 4 and lysine 9, could represent a novel therapeutic strategy for MPM. We suppressed LSD1 and investigated the EMT phenotype using EMT marker expression and wound-healing assay; and chemosensitivity using apoptosis assay. We found that suppressing LSD1 induces an epithelial phenotype in sarcomatoid MPM cells, while attenuating the mesenchymal phenotype sensitized MPM cells to cisplatin-induced apoptosis. Subsequent genome-wide identification, comprehensive microarray analysis, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to assess genome-wide changes in chromatin accessibility suggested that LSD1 directly regulates milk fat globulin protein E8 (MFGE8), an integrin ligand that is involved in the FAK pathway. Furthermore, we found that LSD1 regulates the mesenchymal phenotype and apoptosis by activating the FAK–AKT–GSK3β pathway via a positive feedback loop involving MFGE8 and Snail expression, thereby leading to cisplatin resistance.Implications:This study suggests that LSD1 regulates the mesenchymal phenotype and apoptosis, and that LSD1 inhibitors could be combined with the cisplatin as a novel therapy for patients with MPM.

The Krüppel-like family of transcription factors plays critical roles in human development and is associated with cancer pathogenesis. Krüppel-like factor 5 gene (KLF5) has been shown to promote cancer cell proliferation and tumorigenesis and to be genomically amplified in cancer cells. We recently reported that the KLF5 gene is also subject to other types of somatic coding and noncoding genomic alterations in diverse cancer types. Here, we show that these alterations activate KLF5 by three distinct mechanisms: (i) Focal amplification of superenhancers activates KLF5 expression in squamous cell carcinomas; (ii) Missense mutations disrupt KLF5–FBXW7 interactions to increase KLF5 protein stability in colorectal cancer; (iii) Cancer type–specific hotspot mutations within a zinc-finger DNA binding domain of KLF5 change its DNA binding specificity and reshape cellular transcription. Utilizing data from CRISPR/Cas9 gene knockout screening, we reveal that cancer cells with KLF5 overexpression are dependent on KLF5 for their proliferation, suggesting KLF5 as a putative therapeutic target.Significance: Our observations, together with previous studies that identified oncogenic properties of KLF5, establish the importance of KLF5 activation in human cancers, delineate the varied genomic mechanisms underlying this occurrence, and nominate KLF5 as a putative target for therapeutic intervention in cancer. Cancer Discov; 8(1); 108–25. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1

Inherited lung cancer risk, particularly in nonsmokers, is poorly understood. Genomic and ancestry analysis of 1,153 lung cancers from Latin America revealed striking associations between Native American ancestry and their somatic landscape, including tumor mutational burden, and specific driver mutations in EGFR, KRAS, and STK11. A local Native American ancestry risk score was more strongly correlated with EGFR mutation frequency compared with global ancestry correlation, suggesting that germline genetics (rather than environmental exposure) underlie these disparities.Significance:The frequency of somatic EGFR and KRAS mutations in lung cancer varies by ethnicity, but we do not understand why. Our study suggests that the variation in EGFR and KRAS mutation frequency is associated with genetic ancestry and suggests further studies to identify germline alleles that underpin this association.See related commentary by Gomez et al., p. 534.This article is highlighted in the In This Issue feature, p. 521

Supplementary Figures

Introduction: Cisplatin-based chemotherapy was established in the 1980s, and it has been improved by the development of a short hydration protocol in lung cancer therapy. However, cisplatin-based chemotherapy is still associated with renal toxicity. Because 5-aminolevulinic acid (5-ALA) with sodium ferrous citrate (SFC) is known to be a mitochondrial activator and a heme oxygenase-1 (HO-1) inducer, 5-ALA with SFC is speculated to mitigate cisplatin-induced renal inflammation. Methods: We investigated the effects of oral administration of 5-ALA with SFC for preventing cisplatin-based nephrotoxicity in patients with lung cancer and evaluated its benefits for patients who received cisplatin-based chemotherapy. The primary endpoint was the significance of the difference between the serum creatinine (sCr) levels of the patients administered 5-ALA with SFC and those given placebo after course 1 of chemotherapy. The difference in the estimated glomerular filtration rate (eGFR) between the two groups was also evaluated as the secondary endpoint. Results: The double-blind, randomized two-arm studies were conducted at 15 medical facilities in Japan; 54 male and 20 female patients with lung cancer who received cisplatin-based chemotherapy between the ages of 42 and 75 years were included in the study. The compliance rate was greater than 94% in the primary assessment and subsequent drug administration periods. All enrolled patients completed the four cycles of cisplatin-based chemotherapy with short hydration. The average level of sCr on day 22 of course 1 was 0.707 mg/dL in the group treated with 5-ALA and SFC and 0.735 mg/dL in the placebo group, respectively, and the sCr in the test group was significantly lower than that in the placebo group (p = 0.038). In addition, the eGFR was significantly higher in the SPP-003 group than in the placebo group up to day 1 of course 3 (84.66 and 75.68 mL/min/1.73 m2, respectively, p = 0.02) and kept better even after the last administration of the study drug (82.37 and 73.49 mL/min/1.73 m2, respectively, p = 0.013). Conclusions: The oral administration of 5-ALA with SFC is beneficial to patients undergoing cisplatin-based chemotherapy for lung cancer with short hydration.

Background Zinc‐finger E‐box‐binding homeobox 1 (ZEB1) is an important regulator of epithelial‐mesenchymal transition (EMT) and is involved in the maintenance of cancer stem cells (CSCs) via miR‐200c and BMI1 pathway. Recent studies revealed that ZEB1 contributes to the EMT‐mediated acquired resistance to gefitinib in EGFR‐mutant non‐small cell lung cancer (NSCLC). However, the precise role of ZEB1 in the maintenance of lung CSCs that lead to acquired resistance to gefitinib remains unclear. Methods PC9 and HCC827 NSCLC cell lines were treated with high concentrations of gefitinib, and surviving cells were referred to as “gefitinib‐resistant persisters” (GRPs). ZEB1 knockdown or overexpression was performed to determine the biological significance of ZEB1 in the CSC features of GRPs, and animal models were studied for in vivo validation. Expression of ZEB1, BMI1, and ALDH1A1 was analyzed by immunohistochemistry in tumor specimens from NSCLC patients with acquired resistance to gefitinib. Results GRPs had characteristic features of mesenchymal and CSC phenotypes with high expression of ZEB1 and BMI1, and decreased miR‐200c, in vitro and in vivo. ZEB1 silencing attenuated the suppression of miR‐200c, resulting in the reduction in BMI1 and reversed the mesenchymal and CSC features of GRPs. Furthermore, ZEB1 overexpression induced EMT and increased the levels of CD133‐ and BMI1‐positive GRPs in vitro and gefitinib resistance in vivo. Finally, ZEB1, BMI1, and ALDH1A1 were highly expressed in tumor specimens from EGFR‐mutant NSCLC patients with gefitinib resistance. Conclusions ZEB1 plays an important role in gefitinib‐resistant lung CSCs with EMT features via regulation of miR‐200c and BMI1., Gefitinib‐resistant persisters (GRPs) of EGFR‐mutant NSCLC cells have characteristic features of mesenchymal and CSC phenotypes. ZEB1 is highly expressed in GRPs and involved in the maintenance of lung CSCs resistant to gefitinib via miR‐200c‐BMI1 pathway.

The development of molecular targeted therapy has improved clinical outcomes in patients with life-threatening advanced lung cancers with driver oncogenes. However, selective treatment for KRAS-mutant lung cancer remains underdeveloped. We have successfully characterised specific molecular and pathological features of KRAS-mutant lung cancer utilising newly developed cell line models that can elucidate the differences in driver oncogenes among tissues with identical genetic backgrounds. Among these KRAS-mutation-associated specific features, we focused on the IGF2-IGF1R pathway, which has been implicated in the drug resistance mechanisms to AMG 510, a recently developed selective inhibitor of KRAS G12C lung cancer. Experimental data derived from our cell line model can be used as a tool for clinical treatment strategy development through understanding of the biology of lung cancer. The model developed in this paper may help understand the mechanism of anticancer drug resistance in KRAS-mutated lung cancer and help develop new targeted therapies to treat patients with this disease.

Purpose Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix (ECM) protein in the lungs. Transforming growth factor (TGF) β-induced ECM protein synthesis contributes to the development of IPF. Tranilast, an anti-allergy drug, suppresses TGFβ expression and inhibits interstitial renal fibrosis in animal models. However, the beneficial effects of tranilast or its mechanism as a therapy for pulmonary fibrosis have not been clarified. Methods We investigated the in vitro effect of tranilast on ECM production and TGFβ/SMAD2 pathway in TGFβ2-stimulated A549 human alveolar epithelial cells, using quantitative polymerase chain reaction, Western blotting, and immunofluorescence. In vitro observations were validated in the lungs of a murine pulmonary fibrosis model, which we developed by intravenous injection of bleomycin. Results Treatment with tranilast suppressed the expression of ECM proteins, such as fibronectin and type IV collagen, and attenuated SMAD2 phosphorylation in TGFβ2-stimulated A549 cells. In addition, based on a wound healing assay in these cells, tranilast significantly inhibited cell motility, with foci formation that comprised of ECM proteins. Histological analyses revealed that the administration of tranilast significantly attenuated lung fibrosis in mice. Furthermore, tranilast treatment significantly reduced levels of TGFβ, collagen, fibronectin, and phosphorylated SMAD2 in pulmonary fibrotic tissues in mice. Conclusion These findings suggest that tranilast inhibits pulmonary fibrosis by suppressing TGFβ/SMAD2-mediated ECM protein production, presenting tranilast as a promising and novel anti-fibrotic agent for the treatment of IPF.

Genomic analysis of lung adenocarcinomas has revealed that the MGA gene, which encodes a heterodimeric partner of the MYC-interacting protein MAX, is significantly mutated or deleted in lung adenocarcinomas. Most of the mutations are loss of function for MGA, suggesting that MGA may act as a tumor suppressor. Here, we characterize both the molecular and cellular role of MGA in lung adenocarcinomas and illustrate its functional relevance in the MYC pathway. Although MGA and MYC interact with the same binding partner, MAX, and recognize the same E-box DNA motif, we show that the molecular function of MGA appears to be antagonistic to that of MYC. Using mass spectrometry–based affinity proteomics, we demonstrate that MGA interacts with a noncanonical PCGF6-PRC1 complex containing MAX and E2F6 that is involved in gene repression, while MYC is not part of this MGA complex, in agreement with previous studies describing the interactomes of E2F6 and PCGF6. Chromatin immunoprecipitation-sequencing and RNA sequencing assays show that MGA binds to and represses genes that are bound and activated by MYC. In addition, we show that, as opposed to the MYC oncoprotein, MGA acts as a negative regulator for cancer cell proliferation. Our study defines a novel MYC/MAX/MGA pathway, in which MYC and MGA play opposite roles in protein interaction, transcriptional regulation, and cellular proliferation. Implications: This study expands the range of key cancer-associated genes whose dysregulation is functionally equivalent to MYC activation and places MYC within a linear pathway analogous to cell-cycle or receptor tyrosine kinase/RAS/RAF pathways in lung adenocarcinomas.

Background Osimertinib (AZD9291) is a third-generation EGFR-tyrosine kinase inhibitor (TKI) that selectively inhibits the activating EGFR mutation and T790M mutation, and is currently used globally to treat EGFR-mutant non-small cell lung cancer (NSCLC). However, acquired resistance to osimertinib is inevitable. Methods We established osimertinib-resistant cells (PC9/T790M/AZDR and H1975/AZDR) derived from EGFR-mutant NSCLC cells harboring T790M mutation, and investigated the mechanism of acquired resistance to osimertinib by whole-exome sequencing and multiple phospho-receptor tyrosine kinase (RTK) array. A tumor specimen from an EGFR-mutant NSCLC patient with acquired resistance to osimertinib was also subjected to immunohistochemical analysis. Results Whole-exome sequencing analysis demonstrated that genetic alterations, such as acquisition of EGFR C797S, loss of T790M mutation, MET amplification, or mutated KRAS, MEK, BRAF, PIK3CA, were not detected. Analysis of phospho-RTK array revealed that insulin-like growth factor-1 receptor (IGF1R) was activated in PC9/T790M/AZDR and H1975/AZDR cells. Knockdown of IGF1R by siRNA as well as inhibition of IGF1R activation by linstinib (IGF1R inhibitor) significantly restored the sensitivity to osimertinib. Immunohistochemical analysis revealed that the expression level of phosphorylated IGF1R was higher in the tumor specimen from the EGFR-mutant NSCLC patient with acquired resistance to osimertinib than in the specimen collected prior to the treatment. Conclusions IGF1R activation could occur following treatment with osimertinib in EGFR-mutant NSCLC with T790M mutation, and might be one of the mechanisms underlying osimertinib resistance. Combined treatment of osimertinib and IGF1R inhibitor might be effective in overcoming the acquired resistance to osimertinib induced by IGF1R activation. Key points Significant findings of the study: Using osimertinib-resistant cells, we found that IGF1R activation induced by osimertinib treatment in EGFR-mutant NSCLC with T790M mutation is involved in resistance. Increased phosphorylation of IGF1R was observed in the tumor specimen from an EGFR-mutant NSCLC patient with acquired osimertinib resistance. What this study adds IGF1R activation might be one of the mechanisms of osimertinib resistance. A combination therapy with osimertinib and an IGF1R inhibitor might be an optimal approach for overcoming the acquired resistance to osimertinib induced by IGF1R activation.

Cancer cachexia is associated with poor immunotherapeutic outcomes. This prospective observational study longitudinally evaluated the role of cachexia-related circulating cytokines in predicting the risk and benefit of PD-1/PD-L1 blockade in advanced lung cancer. Forty-one circulating cytokines at baseline and after one cycle of PD-1/PD-L1 blockade treatment were measured in patients with advanced lung cancer between 2019 and 2020. The cachexia-related cytokines were identified by comparing the levels of circulating cytokines between cachectic and non-cachectic patients. Among 55 patients, 49.1% were diagnosed with cachexia at the beginning of PD-1/PD-L1 blockade therapy. Baseline levels of the circulating cytokines IL-6, IL-8, IL-10, IL-15, and IP-10 were significantly higher in cachectic patients. In contrast, the level of eotaxin-1 was lower in cachectic patients than in those without cachexia. Higher IL-6 at baseline and during treatment was associated with a greater risk of immune-related adverse events, while higher IL-10 at baseline was linked to worse overall survival. More importantly, increased eotaxin-1 after one cycle of PD-1/PD-L1 blockade treatment was associated with higher objective response and better overall survival. A blood-based, cachexia-related cytokine assay may yield potential biomarkers for the early prediction of clinical response to PD-1/PD-L1 blockade and provide clues for improving the outcomes of cachectic patients.

Background The prognosis of patients with NSCLC harboring oncogenic driver gene alterations, such as EGFR gene mutations or ALK fusion, has improved dramatically with the advent of corresponding molecularly targeted drugs. As patients were followed up for about five years in most clinical trials, the long-term outcomes beyond 5 years are unclear. The objectives of this study are to explore the clinical course beyond five years of chemotherapy initiation and to investigate factors that lead to long-term survival. Methods One hundred and seventy-seven patients with advanced, EGFR-mutated or ALK-rearranged NSCLC who received their first chemotherapy between December 2008 and September 2015 were included. Kaplan Meier curves were drawn for the total cohort and according to subgroups of patients’ characteristics. Results Median OS in the total cohort was 40.6 months, the one-year survival rate was 89%, the three-year survival rate was 54%, and the five-year survival rate was 28%. Median OS was 36.9 months in EGFR-mutated patients and 55.4 months in ALK-rearranged patients. The OS curve seemed to plateau after 72 months, and most of the patients who were still alive after more than five years are on treatment. Female sex, age under 75 years, an ECOG PS of 0 to 1, ALK rearrangement, postoperative recurrence, and presence of brain metastasis were significantly associated with longer OS. Conclusions A tail plateau was found in the survival curves of patients with advanced, EGFR-mutated and ALK-rearranged NSCLC, but most were on treatment, especially with EGFR-mutated NSCLC.

Small cell lung cancer (SCLC) is a type of high-grade neuroendocrine carcinoma. It initially responds to chemotherapy but rapidly becomes chemoresistant and it is highly proliferative. The prognosis in SCLC is poor. We have established a novel SCLC cell line, SCLC-J1, from a malignant pleural effusion in a patient with advanced SCLC. SCLC-J1 cells express ganglioside GD2, CD276, and Delta-like protein 3. RB1 is lost. These features of the new SCLC cell line may be useful in understanding the cellular and molecular biology of SCLC and in designing better treatment., Highlight • A novel small lung cancer cell line, SCLC-J1, was successfully established. • SCLC-J1 cells express the tumor-specific antigens ganglioside GD2, CD276, and Delta-like protein 3. RB1 is lost. • SCLC-J1 will provide insights into SCLC biology that may permit better therapeutic targeting.

Background Immunodeficient patients are recommended to receive pneumococcal vaccination. However, there is limited evidence showing effectiveness of the polysaccharide vaccine. Polysaccharide vaccination has shown an association with cardiovascular event risk reduction. We assessed the efficacy of the 23-valent pneumococcal polysaccharide vaccine (PPSV23) in relation to the risk of hospitalization and death due to pneumonia and acute cardiac events. Methods The medical records of all dialysis patients attending our 8 study centers in 2010 were studied, and we selected 1038 consecutive patients. One-to-one propensity score matching was used to correct for potential selection bias in a PPSV23-vaccinated group versus a non-vaccinated group, and a total of 510 patients were identified for outcome analysis. Time to first admission, or deaths due to all-cause pneumonia or cardiac events until 2015 were compared between both groups. Results The all-cause death rate was significantly decreased in the PPSV23-vaccinated group, (hazard ratio [HR] 0.62, 95% confidence interval [CI]; 0.46–0.83, P = 0.002). All-cause death was considered to be a competing risk for the other outcomes. Further outcomes were evaluated by competing risk analysis adjusting for mortality. There was no statistically significant difference in the hospitalization rate for pneumonia; however, the hospitalization rate due to cardiac events was significantly lower in the PPSV23-vaccinated group than in the non-vaccinated group (HR 0.44, 95% CI; 0.20–0.96, P = 0.040). There was no statistically significant difference in the death rate due to pneumonia; however, the rate of cardiac death was significantly lower in the PPSV23-vaccinated group than in the non-vaccinated group (HR 0.36, 95% CI; 0.18–0.71, P = 0.003). Conclusions The PPSV23 vaccination is associated with a good prognosis and a low-risk of cardiac events in dialysis patients; however, there was no evidence indicating enhanced protective efficacy against pneumonia, suggesting the PPSV23 vaccination might improve the prognosis by directly preventing cardiovascular events.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that has a low overall survival; however, no significant treatment advances have been made in the past 15 years. Large-scale molecular studies have identified a poor prognostic subset of MPM linked to the epithelial–mesenchymal transition (EMT) that may contribute toward resistance to chemotherapy, suggesting that EMT could be targeted to treat patients with MPM. Previously, we reported that histone modifiers regulating EMT could be therapeutic targets; therefore, in this study, we investigated whether targeting lysine-specific demethylase 1 (LSD1/KDM1), a histone-modifying enzyme responsible for demethylating histone H3 lysine 4 and lysine 9, could represent a novel therapeutic strategy for MPM. We suppressed LSD1 and investigated the EMT phenotype using EMT marker expression and wound-healing assay; and chemosensitivity using apoptosis assay. We found that suppressing LSD1 induces an epithelial phenotype in sarcomatoid MPM cells, while attenuating the mesenchymal phenotype sensitized MPM cells to cisplatin-induced apoptosis. Subsequent genome-wide identification, comprehensive microarray analysis, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to assess genome-wide changes in chromatin accessibility suggested that LSD1 directly regulates milk fat globulin protein E8 (MFGE8), an integrin ligand that is involved in the FAK pathway. Furthermore, we found that LSD1 regulates the mesenchymal phenotype and apoptosis by activating the FAK–AKT–GSK3β pathway via a positive feedback loop involving MFGE8 and Snail expression, thereby leading to cisplatin resistance. Implications: This study suggests that LSD1 regulates the mesenchymal phenotype and apoptosis, and that LSD1 inhibitors could be combined with the cisplatin as a novel therapy for patients with MPM.

Inherited lung cancer risk, particularly in nonsmokers, is poorly understood. Genomic and ancestry analysis of 1,153 lung cancers from Latin America revealed striking associations between Native American ancestry and their somatic landscape, including tumor mutational burden, and specific driver mutations in EGFR, KRAS, and STK11. A local Native American ancestry risk score was more strongly correlated with EGFR mutation frequency compared with global ancestry correlation, suggesting that germline genetics (rather than environmental exposure) underlie these disparities. Significance: The frequency of somatic EGFR and KRAS mutations in lung cancer varies by ethnicity, but we do not understand why. Our study suggests that the variation in EGFR and KRAS mutation frequency is associated with genetic ancestry and suggests further studies to identify germline alleles that underpin this association. See related commentary by Gomez et al., p. 534. This article is highlighted in the In This Issue feature, p. 521

Background Hermansky-Pudlak syndrome (HPS) is an extremely rare disease with pulmonary fibrosis (PF), oculocutaneous albinism, induced platelet dysfunction, and granulomatous colitis. Although patients with HPS-associated PF (HPS-PF) often receive treatment with anti-fibrotic agents, including pirfenidone, many HPS-PF cases are progressive. The development of pneumothorax is known to be rare in HPS-PF. Pneumothorax development is generally important for prognosis in patients with interstitial pneumonia. However, there are few reports regarding the development of pneumothorax in patients with HPS-PF. Case presentation A 50-year-old Japanese man with chestnut hair, white skin, and light brown squint eyes visited our hospital for interstitial pneumonia examination. Chest high-resolution computed tomography (HRCT) demonstrated diffuse bilateral reticular opacities along the bronchovascular bundles and traction bronchiectasis predominantly in the upper lung fields. He was definitively diagnosed with HPS because genetic analysis showed that he had a homozygous mutation, c.398 + 5G > A, in the HPS-1 gene. After diagnosis with HPS-PF, he initiated home oxygen therapy due to gradually progressive hypoxemia. Three months after the HPS-PF diagnosis, the patient suddenly developed severe chest pain and dyspnea and was admitted to our hospital on emergency. He was diagnosed with pneumothorax by chest radiological findings. He immediately received chest drainage; however, his pneumothorax did not improve. Therefore, he underwent video-assisted surgery by thoracic surgeons. The leak point was not detected, but multiple bullae were found, mainly in the upper lung lobes. Thus, the surgeons did not perform bullectomy and only covered the apical areas. Fifteen days after the surgery, the patient developed high fever and dyspnea with a new diffuse reticular shadow found through HRCT. We first initiated the patient on broad-spectrum antibiotics; however, the symptoms and radiological findings worsened. Therefore, we started treatment with pirfenidone for inhibition of PF progression. The patient re-developed pneumothorax with severe respiratory failure. Although he re-underwent chest drainage, he died of progressive respiratory failure. Conclusions We herein report the case of a rare HPS patient who developed pneumothorax with progressive PF. Pneumothorax may cause rapid progressive respiratory failure and may be associated with PF progression in HPS-PF.

Background There are few studies that directly compare the effects of osimertinib on patients with non‐small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) T790M mutation between the generation of prior EGFR tyrosine kinase inhibitors (TKIs). Methods We retrospectively reviewed clinical data from the medical records of consecutive patients with advanced NSCLC who had developed resistance to first‐ or second‐generation EGFR‐TKIs due to EGFR T790M mutation and were subsequently treated with osimertinib at Juntendo University Hospital. In patients with available tumor samples, target amplicon sequencing analyses were performed to explore the genetic biomarkers. Results A total of 38 patients with NSCLC harboring EGFR T790M mutation were treated with osimertinib. Eight patients were classified into group A (afatinib followed by osimertinib) and 30 patients were classified into group B (first‐generation EGFR‐TKI followed by osimertinib). Progression‐free survival (PFS) was significantly longer in group A than in group B (median PFS; not reached vs. 11.0 months, P = 0.018). Fourteen patients had available tissue samples collected before osimertinib treatment for target sequencing. In group A we found no additional mutations, other than EGFR T790M mutation. On the other hand, there were three samples in which other mutations emerged, in addition to EGFR T790M mutation, in group B. Conclusions PFS of osimertinib was significantly longer in patients with NSCLC harboring EGFR T790M mutation after treatment with afatinib than in patients after treatment with first generation EGFR‐TKIs. Additional mutations other than EGFR T790M may affect the efficacy of osimertinib treatment. Key points Significant findings of the study: PFS of osimertinib was significantly longer in patients with NSCLC harboring EGFR T790M mutation after treatment with afatinib than in patients after treatment with first generation EGFR‐TKIs. What this study adds: Additional mutations other than EGFR T790M may affect the efficacy of osimertinib treatment., There are few studies that directly compare the difference between the generation of prior EGFR tyrosine kinase inhibitors (TKIs) before osimertinib treatment in patients with non‐small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) T790M mutation. In our retrospective study, progression‐free survival of osimertinib was significantly longer in patients with NSCLC harboring EGFR T790M mutation after treatment with afatinib than in patients after treatment with first generation EGFR‐TKIs.

Purpose Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix (ECM) protein in the lungs. Transforming growth factor (TGF) β-induced ECM protein synthesis contributes to the development of IPF. Tranilast, an anti-allergy drug, suppresses TGFβ expression and inhibits interstitial renal fibrosis in animal models. However, the beneficial effects of tranilast or its mechanism as a therapy for pulmonary fibrosis have not been clarified. Methods We investigated the in vitro effect of tranilast on ECM production and TGFβ/SMAD2 pathway in TGFβ2-stimulated A549 human alveolar epithelial cells, using quantitative polymerase chain reaction, Western blotting, and immunofluorescence. In vitro observations were validated in the lungs of a murine pulmonary fibrosis model, which we developed by intravenous injection of bleomycin. Results Treatment with tranilast suppressed the expression of ECM proteins, such as fibronectin and type IV collagen, and attenuated SMAD2 phosphorylation in TGFβ2-stimulated A549 cells. In addition, based on a wound healing assay in these cells, tranilast significantly inhibited cell motility, with foci formation that comprised of ECM proteins. Histological analyses revealed that the administration of tranilast significantly attenuated lung fibrosis in mice. Furthermore, tranilast treatment significantly reduced levels of TGFβ, collagen, fibronectin, and phosphorylated SMAD2 in pulmonary fibrotic tissues in mice. Conclusion These findings suggest that tranilast inhibits pulmonary fibrosis by suppressing TGFβ/SMAD2-mediated ECM protein production, presenting tranilast as a promising and novel anti-fibrotic agent for the treatment of IPF.

Inherited lung cancer risk, particularly in nonsmokers, is poorly understood. In particular, the frequency of somatic EGFR and KRAS mutations in lung cancer varies by ethnicity. Somatic EGFR mutation rates are higher in lung cancers from patients with East Asian ancestry and lower in patients with European or African ancestry. Somatic KRAS mutation rates show the opposite pattern. In patients from Latin America, somatic lung cancer EGFR mutation rates vary by country, highest in countries with more Native American ancestry. To ask whether the somatic genome in lung cancer is affected by ethnicity related germline risk, we studied genomes from 1,153 lung cancers from Mexico and Colombia. This study revealed striking associations between ancestry and somatic lung cancer alterations, including tumor mutational burden, and specific driver mutations in EGFR, KRAS, and STK11. A local ancestry score was more strongly correlated with EGFR mutation frequency compared with global ancestry correlation, suggesting that germline genetics (rather than environmental exposure) underlie these disparities. Our study suggests that the variation in EGFR and KRAS mutation frequency in lung cancer is associated with genetic ancestry in patients from Latin America, and suggests further studies to identify germline alleles that underpin this association. If we find a germline locus, this might help in improving lung cancer prevention and screening for populations of Latin American origin and others. Furthermore, multiple studies now highlight the special importance of EGFR mutation screening and EGFR-directed targeted therapy for lung cancer patients in Latin America and with origins in Latin America. Citation Format: Jian Carrot-Zhang, Giovanny Soca-Chafre, Nick Patterson, Aaron Thorner, Anwesha Nag, Jacqueline Watson, Giulio Genovese, July Rodriguez, Maya Gelbard, Luis Corrales-Rodriguez, Yoichiro Mitsuishi, Gavin Ha, Joshua Campbell, Geoffrey Oxnard, Oscar Arrieta, Andres Cardona, Alexander Gusev, Matthew Meyerson. Genetic ancestry and lung cancer somatic mutations in patients of Latin American descent: Etiology and treatment implications [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr IA-07.

Autocrine and paracrine factors, including glutamate and epidermal growth factor (EGF), are potent inducers of brain tumor cell invasion, a pathological hallmark of malignant gliomas. System xc(-) consists of xCT and CD98hc subunits and functions as a plasma membrane antiporter for the uptake of extracellular cystine in exchange for intracellular glutamate. We previously showed that the EGF receptor (EGFR) interacts with xCT and thereby promotes the activity of system xc(-) in a kinase-independent manner, resulting in enhanced glutamate release in glioma cells. However, the molecular mechanism underlying EGFR-mediated glioma progression in a glutamate-rich microenvironment has remained unclear. Here we show that the GluN2B subunit of the N-methyl-d-aspartate-sensitive glutamate receptor (NMDAR) is a substrate of EGFR in glioma cells. In response to EGF stimulation, EGFR phosphorylated the COOH-terminal domain of GluN2B and thereby enhanced glutamate-NMDAR signaling and consequent cell migration in EGFR-overexpressing glioma cells. Treatment with the NMDAR inhibitor MK-801 or the system xc(-) inhibitor sulfasalazine suppressed EGF-elicited glioma cell migration. The administration of sulfasalazine and MK-801 also synergistically suppressed the growth of subcutaneous tumors formed by EGFR-overexpressing glioma cells. Furthermore, shRNA-mediated knockdown of xCT and GluN2B cooperatively prolonged the survival of mice injected intracerebrally with such glioma cells. Our findings thus establish a central role for EGFR in the signaling crosstalk between xCT and GluN2B-containing NMDAR in glioma cells.

Background Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disorder. Recent studies have suggested that epithelial-mesenchymal transition (EMT) of alveolar epithelial cells influences development of pulmonary fibrosis, which is mediated by transforming growth factor β (TGF-β). Tumor necrosis factor α (TNF-α), an important proinflammatory cytokine in IPF, has been shown to enhance TGF-β-induced EMT. Nintedanib, a multiple tyrosine kinase inhibitor that is currently used to treat IPF, has been shown to suppress EMT in various cancer cell lines. However, the mechanism of EMT inhibition by nintedanib and its effect on TGF-β and TNF-α signaling pathways in alveolar epithelial cells have not been fully elucidated. Methods A549 alveolar epithelial cells were stimulated with TGF-β2 and TNF-α, and the effects of nintedanib on global gene expression were evaluated using microarray analysis. Furthermore, Smad2/3 phosphorylation was assessed using western blotting. Results We found that in A549 cells, TGF-β2 and TNF-α treatment induces EMT, which was inhibited by nintedanib. Gene ontology analysis showed that nintedanib significantly attenuates the gene expression of EMT-related cellular pathways and the TGF-β signaling pathway, but not in the TNF-α-mediated signaling pathway. Furthermore, hierarchical cluster analysis revealed that EMT-related genes were attenuated in nintedanib-treated cells. Additionally, nintedanib was found to markedly suppress phosphorylation of Smad2/3. Conclusion Nintedanib inhibits EMT by mediating EMT-related gene expression and the TGF-β/Smad pathway in A549 alveolar epithelial cells.

Several recent studies suggest that cancer stem cells (CSCs) are involved in intrinsic resistance to cancer treatment. Maintenance of quiescence is crucial for establishing resistance of CSCs to cancer therapeutics. F-box/WD repeat-containing protein 7 (FBXW7) is a ubiquitin ligase that regulates quiescence by targeting the c-MYC protein for ubiquitination. We previously reported that gefitinib-resistant persisters (GRPs) in EGFR-mutant non-small cell lung cancer (NSCLC) cells highly expressed octamer-binding transcription factor 4 (Oct-4) as well as the lung CSC marker CD133, and they exhibited distinctive features of the CSC phenotype. However, the role of FBXW7 in lung CSCs and their resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors in NSCLC is not fully understood. In this study, we developed GRPs from the two NSCLC cell lines PC9 and HCC827, which express an EGFR exon 19 deletion mutation, by treatment with a high concentration of gefitinib. The GRPs from both PC9 and HCC827 cells expressed high levels of CD133 and FBXW7, but low levels of c-MYC. Cell cycle analysis demonstrated that the majority of GRPs existed in the G0/G1 phase. Knockdown of the FBXW7 gene significantly reduced the cell number of CD133-positive GRPs and reversed the cell population in the G0/G1-phase. We also found that FBXW7 expression in CD133-positive cells was increased and c-MYC expression was decreased in gefitinib-resistant tumors of PC9 cells in mice and in 9 out of 14 tumor specimens from EGFR-mutant NSCLC patients with acquired resistance to gefitinib. These findings suggest that FBXW7 plays a pivotal role in the maintenance of quiescence in gefitinib-resistant lung CSCs in EGFR mutation-positive NSCLC.

KRAS-driven lung cancers frequently inactivate TP53 and/or STK11/LKB1, defining tumor subclasses with emerging clinical relevance. Specifically, KRAS-LKB1 (KL)–mutant lung cancers are particularly aggressive, lack PD-L1, and respond poorly to immune checkpoint blockade (ICB). The mechanistic basis for this impaired immunogenicity, despite the overall high mutational load of KRAS-mutant lung cancers, remains obscure. Here, we report that LKB1 loss results in marked silencing of stimulator of interferon genes (STING) expression and insensitivity to cytoplasmic double-strand DNA (dsDNA) sensing. This effect is mediated at least in part by hyperactivation of DNMT1 and EZH2 activity related to elevated S-adenylmethionine levels and reinforced by DNMT1 upregulation. Ectopic expression of STING in KL cells engages IRF3 and STAT1 signaling downstream of TBK1 and impairs cellular fitness, due to the pathologic accumulation of cytoplasmic mitochondrial dsDNA associated with mitochondrial dysfunction. Thus, silencing of STING avoids these negative consequences of LKB1 inactivation, while facilitating immune escape. Significance: Oncogenic KRAS-mutant lung cancers remain treatment-refractory and are resistant to ICB in the setting of LKB1 loss. These results begin to uncover the key underlying mechanism and identify strategies to restore STING expression, with important therapeutic implications because mitochondrial dysfunction is an obligate component of this tumor subtype. See related commentary by Corte and Byers, p. 16. This article is highlighted in the In This Issue feature, p. 1

Transforming growth factor β (TGFβ)-mediated epithelial–mesenchymal transition (EMT) of alveolar epithelial cells contributes to pulmonary fibrosis. Dasatinib (DAS), a potent and broad-spectrum tyrosine kinase inhibitor, has been widely studied as an anti-cancer agent. However, the therapeutic application of DAS for pulmonary fibrosis has not been clarified. Our purpose here is to investigate the effect of DAS on TGFβ1-induced EMT in human alveolar and bronchial epithelial cells in vitro and to evaluate the efficacy of DAS on lung fibrosis in vivo. TGFβ1-stimulated human alveolar epithelial (A549) and bronchial epithelial (BEAS-2B) cells were treated with or without DAS in vitro. Murine pulmonary fibrosis model was generated by injection of bleomycin (BLM). A549 and BEAS-2B cells exposed to TGFβ1 underwent EMT, as indicated by downregulation of epithelial protein E-cadherin and induction of the mesenchymal proteins, fibronectin and type I and type IV collagen. These effects were dramatically suppressed by DAS treatment, which also prevented Smad2 and Smad3 phosphorylation. DAS inhibited TGFβ1-induced cell motility and migration. Furthermore, DAS administration significantly attenuated lung fibrosis in mice by histological analysis. Treatment with DAS also significantly reduced the levels of collagen and fibronectin and phosphorylation of Smad2 in the lung tissues of the murine model. These findings suggest that DAS inhibited TGFβ-mediated EMT of alveolar and bronchial epithelial cells and attenuated BLM-induced lung fibrosis in mice by suppressing the TGFβ/Smad pathway. DAS may be a promising and novel anti-fibrotic agent for preventing lung fibrosis.

The Krüppel-like family of transcription factors plays critical roles in human development and is associated with cancer pathogenesis. Krüppel-like factor 5 gene (KLF5) has been shown to promote cancer cell proliferation and tumorigenesis and to be genomically amplified in cancer cells. We recently reported that the KLF5 gene is also subject to other types of somatic coding and noncoding genomic alterations in diverse cancer types. Here, we show that these alterations activate KLF5 by three distinct mechanisms: (i) Focal amplification of superenhancers activates KLF5 expression in squamous cell carcinomas; (ii) Missense mutations disrupt KLF5–FBXW7 interactions to increase KLF5 protein stability in colorectal cancer; (iii) Cancer type–specific hotspot mutations within a zinc-finger DNA binding domain of KLF5 change its DNA binding specificity and reshape cellular transcription. Utilizing data from CRISPR/Cas9 gene knockout screening, we reveal that cancer cells with KLF5 overexpression are dependent on KLF5 for their proliferation, suggesting KLF5 as a putative therapeutic target. Significance: Our observations, together with previous studies that identified oncogenic properties of KLF5, establish the importance of KLF5 activation in human cancers, delineate the varied genomic mechanisms underlying this occurrence, and nominate KLF5 as a putative target for therapeutic intervention in cancer. Cancer Discov; 8(1); 108–25. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1