Kinome screen of ferroptosis reveals a novel role of ATM in regulating iron metabolism.

Abstract

Ferroptosis is a specialized iron-dependent cell death that is associated with lethal lipid peroxidation. Modulation of ferroptosis may have therapeutic potential since it has been implicated in various human diseases as well as potential antitumor activities. However, much remains unknown about the underlying mechanisms and genetic determinants of ferroptosis. Given the critical role of kinases in most biological processes and the availability of various kinase inhibitors, we sought to systemically identify kinases essential for ferroptosis. We performed a forward genetic-based kinome screen against ferroptosis in MDA-MB-231 cells triggered by cystine deprivation. This screen identified 34 essential kinases involved in TNFα and NF-kB signaling. Unexpectedly, the DNA damage response serine/threonine kinase ATM (mutated in Ataxia-Telangiectasia) was found to be essential for ferroptosis. The pharmacological or genetic inhibition of ATM consistently rescued multiple cancer cells from ferroptosis triggered by cystine deprivation or erastin. Instead of the canonical DNA damage pathways, ATM inhibition rescued ferroptosis by increasing the expression of iron regulators involved in iron storage (ferritin heavy and light chain, FTH1 and FTL) and export (ferroportin, FPN1). The coordinated changes of these iron regulators during ATM inhibition resulted in a lowering of labile iron and prevented the iron-dependent ferroptosis. Furthermore, we found that ATM inhibition enhanced the nuclear translocation of metal-regulatory transcription factor 1 (MTF1), responsible for regulating expression of Ferritin/FPN1 and ferroptosis protection. Genetic depletion of MTF-1 abolished the regulation of iron-regulatory elements by ATM and resensitized the cells to ferroptosis. Together, we have identified an unexpected ATM-MTF1-Ferritin/FPN1 regulatory axis as novel determinants of ferroptosis through regulating labile iron levels.

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Published Version (Please cite this version)

10.1038/s41418-019-0393-7

Publication Info

Chen, Po-Han, Jianli Wu, Chien-Kuang Cornelia Ding, Chao-Chieh Lin, Samuel Pan, Nathan Bossa, Yitong Xu, Wen-Hsuan Yang, et al. (2019). Kinome screen of ferroptosis reveals a novel role of ATM in regulating iron metabolism. Cell death and differentiation. 10.1038/s41418-019-0393-7 Retrieved from https://hdl.handle.net/10161/19668.

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Scholars@Duke

Mathey-Prevot

Bernard Mathey-Prevot

Adjunct Professor in the Department of Pharmacology & Cancer Biology

The central focus of the lab is to understand how signaling pathway architecture and integration result in specific cell fates and how these properties have been hijacked in cancer cells. In particular, we are interested in assessing the extent to which cell-to-cell heterogeneity can affect the temporal dynamics and regulation of signaling pathways. We are focusing on the E2F/Rb network and have established a platform to follow in real time the activation and expression of E2F1 at the single cell level. In collaboration with the You lab (Duke Biomedical Engineering), which has developed mathematical approaches to model the behavior of the Rb/E2F network, we wish to identify functional characteristics in the signaling dynamics that may underlie disparate cell fates in response to a given external cue. Ultimately, our goal is to build an integrative understanding of the logic and structure in aberrant signaling network(s) known to drive tumor development and metastasis. 

Chi

Jen-Tsan Ashley Chi

Professor in Molecular Genetics and Microbiology

We are using functional genomic approaches to investigate the nutrient signaling and stress adaptations of cancer cells when exposed to various nutrient deprivations and microenvironmental stress conditions. Recently, we focus on two areas. First, we are elucidating the genetic determinants and disease relevance of ferroptosis, a newly recognized form of cell death. Second, we have identified the mammalian stringent response pathway which is highly similar to bacterial stringent response, but with some very interesting twists and novel mechanisms.

A. The genetic determinants and disease relevance of ferroptosis

Ferroptosis is a newly recognized form of cell death that is characterized by iron dependency and lipid peroxidation. The importance of ferroptosis is being recognized in many human diseases, including cancers, ischemia injuries, and neurodegeneration. Previously, we have identified the profound cystine addiction of renal cell carcinoma (1), breast cancer cells (2, 3), and ovarian cancer cells (4). Based on the concept that cystine deprivation triggers the ferroptosis due to the unopposed oxidative stresses, we have performed functional genomic screens to identify many novel genetic determinants of ferroptosis. For example, we have found that DNA damage response and ATM kinase regulate ferroptosis via affecting iron metabolism (5). This finding supports the potential of ionizing radiation to trigger DNA damage response and synergize with ferroptosis to treat human cancers. In addition, we found that ferroptosis is highly regulated by cell density. When cells are grown at low density, they are highly susceptible to ferroptosis. In contrast, the same cells become resistant to ferroptosis when grown at high density and confluency. we have found the Hippo pathway effectors TAZ and YAP are responsible for the cell density-dependent ferroptosis (4, 6, 7). Right now, we are pursuing several other novel determinants of ferroptosis that will reveal surprising insights into this new form of cell death.

B. A new stress pathway – mammalian stress response

All living organisms encounter a wide variety of nutrient deprivations and environmental stresses. Therefore, all organisms have developed various mechanisms to respond and promote survival under stress. In bacteria, the main strategy is “stringent response” triggered by the accumulation of the alarmone (p)ppGpp (shortened to ppGpp below) via regulation of its synthetase RelA and its hydrolase SpoT (8). The ppGpp binds to the transcription factor DksA and RNA polymerase to orchestrate extensive transcriptional changes that repress proliferation and promote stress survival (8, 9). While highly conserved among bacteria, the stringent response had not been reported in metazoans. However, a recent study identified Drosophila and human MESH1 (Metazoan SpoT Homolog 1) as the homologs of the ppGpp hydrolase domain of the bacterial SpoT (10). Both MESH1 proteins exhibit ppGpp hydrolase activity, and the deletion of Mesh1 in Drosophila led to a transcriptional response reminiscent of the bacterial stringent response (10). Recently, we have found that the genetic removal of MESH1 in tumor cells triggers extensive transcriptional changes and confers protection against oxidative stress-induced ferroptosis (11). Importantly, MESH1 removal also triggers proliferative arrest and other robust anti-tumor effects. Therefore, MESH1 knockdown leads to both stress survival and proliferation arrest, two cardinal features highly reminiscent of the bacterial stringent response. Therefore, we termed this pathway as “mammalian stringent response” (12). We have found that NADPH is the relevant MESH1 in the contexts of ferroptosis (13). Now, we are investigating how MESH1 removal leads to proliferation of arrests and anti-tumor phenotypes. Furthermore, we have found several other substrates of MESH1. We are investigating their function using culture cells, MESH1 KO mice, and other model organisms.

 

C. Genomic and single cell RNA analysis of Red Blood Cells

Red blood cells (RBC) are responsible for oxygen delivery to muscles during vigorous exercise. Therefore, many doping efforts focus on increasing RBC number and function to boost athletic performance during competition. For many decades, RBC were thought to be merely identical “sacs of hemoglobin” with no discernable differences due to factors such as age or pre-transfusion storage time. Additionally, because RBC lose their nuclei during terminal differentiation, they were not believed to retain any genetic materials.  These long-held beliefs have now been disproven and the results have significant implications for detecting autologous blood transfusion (ABT) doping in athletes.  We were among the first to discover that RBCs contain abundant and diverse species of RNAs. Using this knowledge, we subsequently optimized protocols and performed genomic analysis of the RBC transcriptome in sickle cell disease; these results revealed that heterogeneous RBCs could be divided into several subpopulations, which had implications for the mechanisms of malaria resistance. As an extension of these studies, we used high resolution Illumina RNA-Seq approaches to identify hundreds of additional known and novel microRNAs, mRNAs, and other RNA species in RBCs. This dynamic RBC transcriptome represents a significant opportunity to assess the impact that environmental factors (such as pre-transfusion refrigerate storage) on the RBC transcriptome. We have now identified a >10-fold change in miR-720 as well as several other RNA transcripts whose levels are significantly altered by RBC storage (14) which gained significant press coverage. We are pursuing the genomic and single cell analysis of RNA transcriptome in the context of blood doping, sickle cell diseases and other red cell diseases.

 

 

 

 

1.         Tang X, Wu J, Ding CK, Lu M, Keenan MM, Lin CC, et al. Cystine Deprivation Triggers Programmed Necrosis in VHL-Deficient Renal Cell Carcinomas. Cancer Res. 2016;76(7):1892-903.

2.         Tang X, Ding CK, Wu J, Sjol J, Wardell S, Spasojevic I, et al. Cystine addiction of triple-negative breast cancer associated with EMT augmented death signaling. Oncogene. 2017;36(30):4379.

3.         Lin CC, Mabe NW, Lin YT, Yang WH, Tang X, Hong L, et al. RIPK3 upregulation confers robust proliferation and collateral cystine-dependence on breast cancer recurrence. Cell Death Differ. 2020.

4.         Yang WH, Huang Z, Wu J, Ding C-KC, Murphy SK, Chi J-T. A TAZ-ANGPTL4-NOX2 axis regulates ferroptotic cell death and chemoresistance in epithelial ovarian cancer. Molecular Cancer Research. 2019: molcanres.0691.2019.

5.         Chen PH, Wu J, Ding CC, Lin CC, Pan S, Bossa N, et al. Kinome screen of ferroptosis reveals a novel role of ATM in regulating iron metabolism. Cell Death Differ. 2019.

6.         Yang W-H, Chi J-T. Hippo pathway effectors YAP/TAZ as novel determinants of ferroptosis. Molecular & Cellular Oncology. 2019:1699375.

7.         Yang WH, Ding CKC, Sun T, Hsu DS, Chi JT. The Hippo Pathway Effector TAZ Regulates Ferroptosis in Renal Cell Carcinoma Cell Reports. 2019;28(10):2501-8.e4.

8.         Potrykus K, Cashel M. (p)ppGpp: still magical? Annu Rev Microbiol. 2008;62:35-51.

9.         Kriel A, Bittner AN, Kim SH, Liu K, Tehranchi AK, Zou WY, et al. Direct regulation of GTP homeostasis by (p)ppGpp: a critical component of viability and stress resistance. Mol Cell. 2012;48(2):231-41.

10.       Sun D, Lee G, Lee JH, Kim HY, Rhee HW, Park SY, et al. A metazoan ortholog of SpoT hydrolyzes ppGpp and functions in starvation responses. Nat Struct Mol Biol. 2010;17(10):1188-94.

11.       Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060-72.

12.       Ding C-KC, Rose J, Wu J, Sun T, Chen K-Y, Chen P-H, et al. Mammalian stringent-like response mediated by the cytosolic NADPH phosphatase MESH1. bioRxiv. 2018.

13.       Ding C-KC, Rose J, Sun T, Wu J, Chen P-H, Lin C-C, et al. MESH1 is a cytosolic NADPH phosphatase that regulates ferroptosis. Nature Metabolism. 2020.

14.       Yang WH, Doss JF, Walzer KA, McNulty SM, Wu J, Roback JD, et al. Angiogenin-mediated tRNA cleavage as a novel feature of stored red blood cells. Br J Haematol. 2018.

 

 


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