Ameen A. Salahudeen

An E3 ligase possessing an iron-responsive hemerythrin domain is a regulator of iron homeostasis.

Iron Sensor Intracellular iron is an essential cofactor for many proteins, but can also damage macromolecules, so its levels are carefully controlled. Cellular iron homeostasis is mediated by iron regulatory proteins that regulate the expression of genes involved in iron uptake and storage. However, it is not clear how cells sense iron bioavailability (see the Perspective by Rouault). Using different approaches, Salahudeen et al. (p. 722, published online 17 September) and Vashisht et al. (p. 718, published online 17 September) have identified the F-box protein FBXL5 as a human iron sensor. FBXL5 is part of an E3 ubiquitin ligase complex that regulates the degradation of iron regulatory proteins and thereby cellular iron levels. It contains a hemerythrin domain that binds iron and acts as an iron-dependent regulatory switch, causing the degradation of FBXL5 under low iron conditions. This alternative pathway for the regulation of iron homeostasis has implications for both normal cellular physiology and disease. A vertebrate hemerythrin domain in an E3 ubiquitin ligase complex senses and regulates cellular iron levels. Cellular iron homeostasis is maintained by the coordinate posttranscriptional regulation of genes responsible for iron uptake, release, use, and storage through the actions of the iron regulatory proteins IRP1 and IRP2. However, the manner in which iron levels are sensed to affect IRP2 activity is poorly understood. We found that an E3 ubiquitin ligase complex containing the FBXL5 protein targets IRP2 for proteasomal degradation. The stability of FBXL5 itself was regulated, accumulating under iron- and oxygen-replete conditions and degraded upon iron depletion. FBXL5 contains an iron- and oxygen-binding hemerythrin domain that acted as a ligand-dependent regulatory switch mediating FBXL5’s differential stability. These observations suggest a mechanistic link between iron sensing via the FBXL5 hemerythrin domain, IRP2 regulation, and cellular responses to maintain mammalian iron homeostasis.

Structural and Molecular Characterization of Iron-sensing Hemerythrin-like Domain within F-box and Leucine-rich Repeat Protein 5 (FBXL5)

Background: FBXL5 is required for proper regulation of cellular iron homeostasis. Results: FBXL5 contains a structurally characterized hemerythrin-like domain. Conclusion: The atypical features of the hemerythrin-like domain facilitate its role as a metabolic sensor. Significance: FBXL5 is the only identified mammalian protein containing a hemerythrin-like domain. Resolution of the structure of the domain provides mechanistic insights into its iron and oxygen responsiveness. Mammalian cells maintain iron homeostasis by sensing changes in bioavailable iron levels and promoting adaptive responses. FBXL5 is a subunit of an E3 ubiquitin ligase complex that mediates the stability of iron regulatory protein 2, an important posttranscriptional regulator of several genes involved in iron metabolism. The stability of FBXL5 is regulated in an iron- and oxygen-responsive manner, contingent upon the presence of its N-terminal domain. Here we present the atomic structure of the FBXL5 N terminus, a hemerythrin-like α-helical bundle fold not previously observed in mammalian proteins. The core of this domain employs an unusual assortment of amino acids necessary for the assembly and sensing properties of its diiron center. These regulatory features govern the accessibility of a mapped sequence required for proteasomal degradation of FBXL5. Detailed molecular and structural characterization of the ligand-responsive hemerythrin domain provides insights into the mechanisms by which FBXL5 serves as a unique mammalian metabolic sensor.

Overexpression of heme oxygenase protects renal tubular cells against cold storage injury: studies using hemin induction and HO-1 gene transfer.

Heme oxygenase-1 (HO-1), a 32-kd microsomal enzyme, is induced as an adaptive response to a wide variety of injurious stimuli. We examined the possible role of HO-1 in cold storage of renal proximal tubular epithelial (RPTE) cells. Hemin, a potent HO-1-inducer, caused a time-dependent increase in HO-1 mRNA and protein expression. Hemin pretreatment of human RPTE cells before cold storage conferred cytoprotection. Increased HO-1 protein was associated with a brisk and early increase in catalytically active iron and a robust increase in cellular ferritin. Deferoxamine, an iron sequestrating antioxidant, prevented hemin-induced iron release and the increase in ferritin, suggesting iron release as an antecedent mechanism for ferritin induction. To verify that the proximate cause of hemin cytoprotection was due to HO-1 induction, we transiently transfected LL-CPK1 porcine kidney cells with a HO-1 expression vector before cold storage. HO-1 transfection resulted in increased expression of HO-1 protein and reduced cell injury during cold storage. The novel observation that prior induction of HO-1 prevents cold storage–induced cell injury suggests that a similar strategy may prove efficacious in preventing cold storage–induced organ damage during transplantation.

Maintaining Mammalian Iron and Oxygen Homeostasis: Sensors, Regulation, and Cross-Talk

Though iron and oxygen are required to sustain essential biological processes, an excess of either can result in oxidative stress. Therefore, mammals tightly regulate cellular and systemic iron and oxygen homeostasis. At the cellular level, the hypoxia‐inducible transcription factors (HIFs) are key mediators of oxygen homeostasis through their regulation of genes involved in anaerobic metabolism and oxygen delivery, among others. Iron regulatory proteins (IRPs) largely govern cellular iron homeostasis through their effects on the translation and stability of mRNAs involved in iron uptake, utilization, export, and storage. Here, we describe regulatory factors for each pathway that sense both iron and oxygen availability and coordinate the maintenance of mammalian iron and oxygen homeostasis at both the cellular and systemic levels.

Zinc protoporphyrin regulates cyclin D1 expression independent of heme oxygenase inhibition.

Zinc protoporphyrin IX (ZnPP), an endogenous heme analogue that inhibits heme oxygenase (HO) activity, represses tumor growth. It can also translocate into the nucleus and up-regulate heme oxygenase 1 (HMOX1) gene expression. Here, we demonstrate that tumor cell proliferation was inhibited by ZnPP, whereas tin protoporphyrin (SnPP), another equally potent HO-1 inhibitor, had no effect. Microarray analysis on 128 tumorigenesis related genes showed that ZnPP suppressed genes involved in cell proliferation and angiogenesis. Among these genes, CYCLIN D1 (CCND1) was specifically inhibited as were its mRNA and protein levels. Additionally, ZnPP inhibited CCND1 promoter activity through an Sp1 and Egr1 overlapping binding site (S/E). We confirmed that ZnPP modulated the S/E site, at least partially by associating with Sp1 and Egr1 proteins rather than direct binding to DNA targets. Furthermore, administration of ZnPP significantly inhibited cyclin D1 expression and progression of a B-cell leukemia/lymphoma 1 tumor in mice by preferentially targeting tumor cells. These observations show HO independent effects of ZnPP on cyclin D1 expression and tumorigenesis.

Rociletinib, a third generation EGFR tyrosine kinase inhibitor: current data and future directions

ABSTRACT Introduction: Major advances have been made since the discovery of driver mutations and their targeted therapies, especially in the treatment of patients with epidermal growth factor receptor (EGFR) mutations. Despite their initial efficacy in the majority of the patients with such driver mutations, all targeted therapies are limited by the eventual development of resistance mechanisms. Areas Covered: EGFR T790M mutation is a common resistance mechanism after treatment with first or second generation EGFR tyrosine kinase inhibitors (TKI). Rociletinib is one of the third generation EGFR TKIs with activity against T790M and activating EGFR mutations while sparing the wild-type EGFR. In this review, we discuss the current understanding and available data on rociletinib, including the side effects associated with the medication. We will also review the BEAMing plasma test to detect T790M mutation without the need for repeat biopsy. Lastly, we review the potential resistance mechanisms after progression on rociletinib, and future directions. Expert Opinion: It is important to note that there are other 3rd generation EGFR TKIs with activity against T790M already approved by the US FDA (osimertinib) and many others in development. Future research will focus on figuring out which patients can benefit the most from a particular medication with minimal side effects, and further resistance mechanisms after rociletinib.

Toward recreating colon cancer in human organoids.

Experimental modeling of cancer typically uses in vitro culture of transformed cell lines or in vivo animal models. A new study using CRISPR-Cas9 to engineer oncogenic mutations into three-dimensional human colon organoid cultures yields insights into colorectal cancer tumorigenesis.

Overview of Thoracic Oncology Trials in Cooperative Groups Around the Globe

Abstract Survival rates of patients with either early and advanced stage non–small‐cell lung cancer (NSCLC) have improved with newer systemic therapy and radiation techniques, including combination regimens, targeted therapies, and immunotherapies. The cancer cooperative groups have historically played a critical role in the advancement of NSCLC therapy. Annually, representatives from cooperative groups worldwide convene at the International Lung Cancer Congress (ILCC). In summer 2015, the ILCC reached its 16th anniversary. This article highlights the NSCLC studies presented by participating groups in 2015.

Update on International Cooperative Groups Studies in Thoracic Malignancies: The Emergence of Immunotherapy

Abstract Cancer cooperative groups have historically played a critical role in the advancement of non–small‐cell lung cancer therapy. Representatives from cooperative groups worldwide convene at the International Lung Cancer Congress annually. The International Lung Cancer Congress had its 17th anniversary in the summer of 2016. The present review highlights the thoracic malignancy studies discussed by presenters. The included studies are merely a sample of the trials of thoracic malignancies ongoing globally.

Abstract B30: Three-dimensional organoid model for acquired drug resistance in non-small cell lung cancer

Background: Targeted therapies against specific driver mutations of cancer progression have been used to improve survival of lung adenocarcinoma patients. In KRAS mutant NSCLC specifically, however, after some initial improvement in lung cancer patients, targeted therapies often fail due to acquired drug resistance. To uncover mechanisms of resistance and to discover new drivers, genome-scale sequencing of lung cancers has identified candidate genes, but these data have not rapidly translated in preclinical validation. A major obstacle in lung cancer research has been the deficiencies of standard in vitro models. Methods: To address the deficiencies within standard models we have developed an in vitro 3-dimensional, KRAS-mutated “organoid” model of lung adenocarcinoma that surpasses both in vitro and in vivo models by possessing the tractability of cell lines and the 3-dimensional architecture and morphology of animal models. We have engineered a p53 knockout and KRAS mutation on top of normal wild-type lung epithelium to achieve oncogenicity. Result: Through an optimized growth period in the presence of drug, an organoid model of resistance has been developed through which de novo genetic events underlying acquired resistance can be studied. Conclusion: The highly defined genetic background of the KRAS-mutated 3-D organoid model serves as a tabula rasa upon which stochastic secondary genetic and epigenetic changes can be identified and mechanistically studied by forward and reverse genetics approaches in order to rapidly identify mechanisms of acquired drug resistance and validate therapeutic options. Citation Format: Navika D. Shukla, Ameen A. Salahudeen, Sukhmani K. Padda, Joel W. Neal, Heather A. Wakelee, Calvin J. Kuo. Three-dimensional organoid model for acquired drug resistance in non-small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B30.