Several transcription factors, including p53, NF-κB, and STAT3, are modified by the same enzymes that also modify histones, with important functional consequences. We have identified a previously unrecognized dimethylation of K49 of STAT3 that is crucial for the expression of many IL-6-dependent genes, catalyzed by the histone-modifying enzyme enhancer of zeste homolog 2 (EZH2). Loss of EZH2 is protumorigenic in leukemias, but its overexpression is protumorigenic in solid cancers. Connecting EZH2 to a functionally important methylation of STAT3, which is constitutively activated in many tumors, may help reveal the basis of the opposing roles of EZH2 in liquid and solid tumors and also may identify novel therapeutic opportunities.posttranslational modification | histone methyltransferase | gene expression S TAT3 is activated in 70% of all solid and hematological tumors (1, 2), where it stimulates proliferation, survival, angiogenesis, invasion, and tumor-promoting inflammation. Recently, STAT3 also was found to have an important role in maintaining cancer stem cells, both in vitro and in mouse tumor models, indicating that it is integrally involved in tumor initiation, progression, and maintenance (3). IL-6-induced constitutive activation of STAT3 was observed in neoplastic gastric tissue and is positively correlated with tumor progression (4), and the expression of tyrosine-phosphorylated STAT3 is associated with poor prognosis in colorectal cancer, independent of the mutation status of MSI, CIMP, BRAF, or KRAS (5). The STAT3 gene is rarely mutated in cancer but rather is activated by members of the IL-6 family of cytokines, receptor tyrosine kinases, mutated JAKs, or oncogenic cellular tyrosine kinases, such as SRC (6). Because STAT3 is constitutively activated during disease progression and metastasis, it is a promising therapeutic target. Even though transcription factors are difficult drug targets, accumulating evidence for the crucial roles of posttranslational modifications of transcription factors in mediating target gene expression provides an opportunity to develop drugs against the modifying enzymes rather than against the transcription factors themselves.STAT3 is activated primarily by phosphorylation of Y705 (7) and secondarily by phosphorylation of S727 (8). The acetylation of STAT3 at K685 (9, 10) has been shown recently to have little or no effect on IL-6-dependent gene expression (11). Ray et al. (12) reported that IL-6-induced gene expression requires p300-mediated acetylation of K49 and K87 and that monoubiquitination at K97 is a key mediator of BRD4-dependent gene expression (13). Like NF-κB and p53, STAT3 is reversibly methylated on lysine residues by histone-modifying enzymes, with important functional consequences (14). Furthermore, STAT3 is known to be di-or trimethylated on K140 or K180 by the histone methyltransferase SET9 (SET domain containing lysine methyltransferase 9) or EZH2 (enhancer of zeste homolog 2), respectively (15, 16). Here we show that IL-6-dependent, previously unident...

The nearly universal recurrence of glioblastoma (GBM) is driven in part by a treatment-resistant subpopulation of GBM stem cells (GSCs). To identify improved therapeutic possibilities, we combined the EGFR/HER2 inhibitor lapatinib with a novel small molecule, CBL0137, which inhibits FACT (Facilitates Chromatin Transcription), a histone chaperone complex predominantly expressed in undifferentiated cells. Lapatinib and CBL0137 synergistically inhibited the proliferation of patient-derived GBM cells. Compared to non-stem tumor cells (NSTCs) enriched from the same specimens, the GSCs were extremely sensitive to CBL0137 monotherapy or FACT knockdown. FACT expression was elevated in GSCs compared to matched NSTCs, and decreased in GSCs upon differentiation. Acute exposure of GSCs to CBL0137 increased asymmetric cell division, decreased GSC marker expression, and decreased the capacity of GSCs to form tumor spheres in vitro and to initiate tumors in vivo. Oral administration of CBL0137 to mice bearing orthotopic GBM prolonged their survival. Knockdown of FACT reduced the expression of genes encoding several core stem cell transcription factors (SOX2, OCT4, NANOG and OLIG2), and FACT occupied the promoters of these genes. FACT expression was elevated in GBM tumors compared to non-neoplastic brain tissues, portended a worse prognosis, and positively correlated with GSC markers and stem cell gene expression signatures. Preferential targeting of GSCs by CBL0137 and synergy with EGFR inhibitors support the development of clinical trials combining these two agents in GBM.

Erlotinib is a tyrosine kinase inhibitor approved for the treatment of patients with advanced non-small cell lung cancer (NSCLC). In these patients, erlotinib prolongs survival but its benefit remains modest since many tumors express wild-type (wt) epidermal growth factor receptor (EGFR) or develop a second-site EGFR mutation. To test drug combinations that could improve the efficacy of erlotinib, we combined erlotinib with quinacrine, which inhibits the FACT (facilitates chromatin transcription) complex that is required for NF-κB transcriptional activity. In A549 (wtEGFR), H1975 (EGFR-L858R/T790M) and H1993 (MET amplification) NSCLC cells, this drug combination was highly synergistic, as quantified by Chou-Talalay combination indices, and slowed xenograft tumor growth. At a sub-IC50 but more clinically attainable concentration of erlotinib, quinacrine, alone or in combination with erlotinib, significantly inhibited colony formation and induced cell cycle arrest and apoptosis. Quinacrine decreased the level of active FACT subunit SSRP1 and suppressed NF-κB-dependent luciferase activity. Knockdown of SSRP1 decreased cell growth and sensitized cells to erlotinib. Moreover, transcriptomic profiling showed that quinacrine or combination treatment significantly affected cell cycle-related genes that contain binding sites for transcription factors that regulate SSRP1 target genes. As potential biomarkers of drug combination efficacy, we identified genes that were more strongly suppressed by the combination than by either single treatment, and whose increased expression predicted poorer survival in lung adenocarcinoma patients. This preclinical study shows that quinacrine overcomes erlotinib resistance by inhibiting FACT and cell cycle progression, and supports a clinical trial testing erlotinib alone versus this combination in advanced NSCLC.

Life-sustaining responses to low oxygen, or hypoxia, depend on signal transduction by HIFs, but the underlying mechanisms by which cells sense hypoxia are not completely understood. Based on prior studies suggesting a link between the β-adrenergic receptor (β-AR) and hypoxia responses, we hypothesized that the β-AR mediates hypoxia sensing and is necessary for HIF-1α accumulation. Beta blocker treatment of mice suppressed hypoxia induction of renal HIF-1α accumulation, erythropoietin production, and erythropoiesis in vivo. Likewise, beta blocker treatment of primary human endothelial cells in vitro decreased hypoxia-mediated HIF-1α accumulation and binding to target genes and the downstream hypoxia-inducible gene expression. In mechanistic studies, cAMP-activated PKA and/or GPCR kinases (GRK), which both participate in β-AR signal transduction, were investigated. Direct activation of cAMP/PKA pathways did not induce HIF-1α accumulation, and inhibition of PKA did not blunt HIF-1α induction by hypoxia. In contrast, pharmacological inhibition of GRK, or expression of a GRK phosphorylation-deficient β-AR mutant in cells, blocked hypoxia-mediated HIF-1α accumulation. Mass spectrometry-based quantitative analyses revealed a hypoxia-mediated β-AR phosphorylation barcode that was different from the classical agonist phosphorylation barcode. These findings indicate that the β-AR is fundamental to the molecular and physiological responses to hypoxia.

Context.— The incidence and types of malignancies in effusion cytology are largely limited to studies performed in the 1970s through the 1990s. Objective.— To examine how the incidence of different types of malignancies in effusions has changed with time. Design.— A computerized search for fluid cytology from 2000 through 2016 (database included age, gender, cytologic diagnosis, and type of malignancy) was performed, and all cases were reviewed. Results.— Of 30 085 effusion specimens, 3285 (11%) were positive for malignancy (2175 pleural, 955 peritoneal, and 155 pericardial). Of those, 1023 (31%) had known primary sites (648 pleural, 267 peritoneal, and 108 pericardial). Malignancy was more common in females than males in both pleural (15% versus 9%) and peritoneal (14% versus 5%) effusions, P < .001. The most common metastatic tumors in pleural fluid were lung for males and breast for females; in peritoneal fluid, hematolymphoid for males and Müllerian tumors for females; in pericardial fluid, lung for both genders. Among invasive mammary carcinomas, lobular carcinoma tended to metastasize to peritoneal fluid, whereas ductal carcinoma tended to metastasize to pleural fluid ( P < .001). Plasma cell neoplasms metastasized to pleural and pericardial but not peritoneal fluid ( P = .002). Conclusions.— Although pulmonary and Müllerian tumors continue to be the most common origin of metastasis in pleural and peritoneal fluid for males and females, respectively, the frequencies for other malignancies have changed. Familiarity with the more common sites of metastasis in effusion cytology is important, especially in patients with unknown primary, as this will be valuable in judicious triaging of specimens for ancillary studies.

Activation of nuclear factor κB (NFκB) is a central event in the responses of normal cells to inflammatory signals, and the abnormal constitutive activation of NFκB is important for the survival of most cancer cells. In nonmalignant human cells, EGF stimulates robust activation of NFκB. The kinase activity of the EGF receptor (EGFR) is required, because the potent and specific inhibitor erlotinib blocks the response. Down-regulating EGFR expression or inhibiting EGFR with erlotinib impairs constitutive NFκB activation in several different types of cancer cells and, conversely, increased activation of NFκB leads to erlotinib resistance in these cells. We conclude that EGF is an important mediator of NFκB activation in cancer cells. To explore the mechanism, we selected an erlotinibresistant cell line in which the guanine nucleotide exchange factor Son of Sevenless 1 (SOS1), well known to be important for EGFdependent signaling to MAP kinases, is overexpressed. Increased expression of SOS1 increases NFκB activation in several different types of cancer cells, and ablation of SOS1 inhibits EGF-induced NFκB activation in these cells, indicating that SOS1 is a functional component of the pathway connecting EGFR to NFκB activation. Importantly, the guanine nucleotide exchange activity of SOS1 is not required for NFκB activation.

Background:Candida is frequently isolated from the respiratory tract and usually reflects airway colonization. True Candida pneumonia is rare. Our aim is to document a case of Candida pneumonia confirmed by cultures, molecular techniques, and surgical lung biopsy, and to highlight a previously unreported pathologic manifestation of this infection.Case summary:A 59-year-old man with a history of chronic obstructive pulmonary disease (COPD) presented with dry cough, low-grade fever, and progressive dyspnea. He was eventually diagnosed with sarcoidosis based on bilateral lung infiltrates and granulomas in a transbronchial biopsy. His condition worsened after immunosuppression, prompting surgical lung biopsy, which revealed suppurative granulomas containing Candida albicans, confirmed by cultures and polymerase chain reaction. Despite multiple episodes of respiratory failure and a prolonged course in intensive care, he recovered fully after antifungal therapy and is currently alive with COPD-related dyspnea 3 years after his initial presentation.Conclusion:Candida can rarely cause clinically significant pneumonia in adults, and should be considered in the differential diagnosis of suppurative granulomas in the lung.

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