Identification of CAR As a Novel Mediator of Erythroid Differentiation and Migration That Is Specifically Downregulated in Erythropoietic Progenitor Cells in Patients with MDS
Authors: Bauer Karin , Machherndl-Spandl Sigrid , Suessner Susanne , Danzer Martin , Pröll Johannes , Lauf Jeroen , Eisenwort Gregor , Sperr Wolfgang R. , Klein Hans-Ulrich , Hoermann Gregor , Häfner Norman , Bene Marie , Platzbecker Uwe , Weltermann Ansgar , Gabriel Christian , Germing Ulrich , Bettelheim Peter , Valent Peter 
Published: October 2019

Abstract
Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by peripheral cytopenia, an accumulation of dysplastic cells in the bone marrow (BM) and a high risk of transformation to acute myeloid leukemia. Transfusion-dependent anemia develops in most patients suffering from MDS. In patients with refractory anemias the accumulation of dysplastic erythroid BM progenitor cells (EryPC) which paradoxically accompanies peripheral anemia, is a typical finding. Although various hypotheses have been discussed, the biochemical basis of the maturation defect of erythroid cells and their abnormal accumulation in the BM in MDS remains unknown. In order to learn more about abnormally regulated and functionally relevant genes expressed in EryPC, we have recently established a screening program involving mRNA expression profiling studies of EryPC in patients with low-risk MDS (IPSS-R score<7) and control BM samples obtained from patients with other BM neoplasms as well as patients with unexplained cytopenia, normal BM (staging for lymphomas) or reactive/deficiency cytopenias. EryPC were defined as CD45low/CD105+ cells and purified from BM mononuclear cells (MNC) by multicolor flow cytometry (MFC) and cell sorting (purity>95%). mRNA expression profiles were analyzed by Affymetrix array technology (GeneChip U133 Plus 2.0 arrays) and confirmed for a panel of selected mRNA species by qPCR. In mRNA- and MFC-validation experiments, we found that the major Coxsackie-Adenovirus Receptor (CAR) is markedly and specifically down-regulated in CD45low/CD105+ EryPC in patients with MDS when compared to EryPC in normal BM or other control cohorts. In line with this observation, the immature erythroblastic cell lines HEL, K562 and KU812 all stained negative for CAR in flow cytometry analyses. Lentiviral transduction of the full-length CAR gene into these cells resulted in a significantly increased expression of various erythroid differentiation antigens, including CD36, CD71 and CD235a (Glycophorin-A) as determined by flow cytometry and qPCR. As assessed by flow cytometry, the levels of CD235a increased to 158±46% in HEL cells, to 211±23% in K562 cells and to 170±30% in KU812 cells after CAR transduction compared to the empty vector control (p<0.05). In addition, CAR transduction resulted in an increased migration of HEL cells against a serum protein-gradient in a transwell assay (empty vector control: 185±67% vs CAR-transfected cells: 356±74%, p<0.05). Transfection with truncated variants of CAR did not result in an increased expression of erythroid antigens or an increased directed migration. In conclusion, our data show that CAR is a functionally relevant antigen that promotes the expression of early erythroid differentiation antigens on myeloid progenitor cells and their migration against blood serum proteins. In patients with MDS, CAR is specifically downregulated on EryPC, which may have clinical implications both in term of the pathogenesis of the disease and the application of this novel marker in diagnostic MFC algorithms. With regard to functional consequences, we hypothesize that CAR-deficiency is pathogenetically relevant as it may not only contribute to the maturation-defect of erythroid progenitor cells in MDS EryPC but also to the related accumulation of erythroid cells in the BM that is accompanying the peripheral anemia in these patients.

Disclosures
No relevant conflicts of interest to declare.