The genomic analysis of erythrocyte microRNA expression in sickle cell diseases.

Dr. Telen is recognized as an expert in the biochemistry and molecular genetics of blood group antigens and the pathophysiological mechanisms of vaso-occlusion in sickle cell disease. She is the Director of the Duke Comprehensive Sickle Cell Center.

Dr. Telen's laboratory focuses on structure/function analysis of membrane proteins expressed by erythroid cells, as well as the role of these proteins in non-erythroid cells. Proteins are also studied in transfectant systems, and research focuses especially on adhesion receptors. The goals of this work are (1) to understand the mechanism and role of red cell adhesion to leukocytes and endothelium in sickle cell disease; (2) to understand the signaling mechanisms leading to activation (and inactivation) of red cell adhesion molecules; (3) to understand the molecular basis of blood group antigen expression, and (4) to understand the interactions of erythroid membrane proteins with other cells and with extracellular matrix..

Recent investigations have focused on the role of signaling pathways in the upregulation of sickle red cell adhesion. Present studies include (1) investigation of beta-adrenergic signaling pathway responsible for activation of B-CAM/LU and LW adhesion receptors; (2) understanding how nitric oxide and ATP downregulate sickle red cell adhesion; (3) studying the effect of these processes in animal models.

Dr. Telen is also involved in a large multicenter study looking for genetic polymorphisms that affect clinical outcomes in sickle cell disease, as well as a multi-center study investigating the mechanisms and treatment of pulmonary hypertension in sickle cell disease.


Key Words:

Adhesion molecules
Erythrocyte membrane
Sickle cell disease
Transfusion medicine
Immunohematology
CD44
B-CAM/LU
Genetic polymorphisms

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.