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. Author manuscript; available in PMC: 2024 Mar 27.
Published in final edited form as: Leukemia. 2023 Sep 6;37(11):2250–2260. doi: 10.1038/s41375-023-02015-7

CAR virus receptor mediates erythroid differentiation and migration and is downregulated in MDS

Karin Bauer 1,2,#, Sigrid Machherndl-Spandl 3,4,#, Lukas Kazianka 1,#, Irina Sadovnik 1,2, Sinan Gültekin 1, Susanne Suessner, Johannes Proell 4,5, Jeroen Lauf 6, Gregor Hoermann 2,7, Gregor Eisenwort 1,2, Norman Häfner 8, Mathilde Födermayr-Mayrleitner 9, Ann-Sofie Schmolke 1, Emiel van der Kouwe 1, Uwe Platzbecker 10,11, Thomas Lion 12, Ansgar Weltermann 3, Otto Zach 9, Gerald Webersinke 9, Ulrich Germing 13, Christian Gabriel 5, Wolfgang R Sperr 1,2, Marie C Béné 14, Philipp B Staber 1,2, Peter Bettelheim 6,9, Peter Valent 1,2,✉
PMCID: PMC7615770  EMSID: EMS194914  PMID: 37673973

Abstract

Myelodysplastic syndromes (MDS) are myeloid neoplasms presenting with dysplasia in the bone marrow (BM) and peripheral cytopenia. In most patients anemia develops. We screened for genes that are expressed abnormally in erythroid progenitor cells (EP) and contribute to the pathogenesis of MDS. We found that the Coxsackie-Adenovirus receptor (CAR = CXADR) is markedly downregulated in CD45low/CD105+ EP in MDS patients compared to control EP. Correspondingly, the erythroblast cell lines HEL, K562, and KU812 stained negative for CAR. Lentiviral transduction of the full-length CXADR gene into these cells resulted in an increased expression of early erythroid antigens, including CD36, CD71, and glycophorin A. In addition, CXADR-transduction resulted in an increased migration against a serum protein gradient, whereas truncated CXADR variants did not induce expression of erythroid antigens or migration. Furthermore, conditional knock-out of Cxadr in C57BL/6 mice resulted in anemia and erythroid dysplasia. Finally, decreased CAR expression on EP was found to correlate with high-risk MDS and decreased survival. Together, CAR is a functionally relevant marker that is down-regulated on EP in MDS and is of prognostic significance. Decreased CAR expression may contribute to the maturation defect and altered migration of EP and thus their pathologic accumulation in the BM in MDS.

Introduction

Myelodysplastic syndromes/neoplasms (MDS) are characterized by peripheral cytopenia, dysplasia in one or more hematopoietic lineages, and clonal instability with an increased risk to transform to secondary acute myeloid leukemia (AML) [1–3]. In most patients, anemia and transfusion dependence develop. The ‘paradoxical’ accumulation of dysplastic erythroid progenitors (EP) in the bone marrow (BM) that accompanies peripheral anemia is a pathognomonic hallmark of MDS [1–3]. However, although various hypotheses have been discussed, the biochemical basis of reduced maturation and excess accumulation of erythroid cells in the BM in MDS remains unknown.

In recent years, our knowledge about molecular players contributing to the pathogenesis of MDS increased substantially [4–9]. In fact, MDS-related genetic and chromosomal abnormalities have been identified and correlated with prognosis or a certain variant of MDS [7–12]. Furthermore, some of the abnormally expressed genes serve as diagnostic parameters. Gene array-profiling and deep-sequencing studies have intensified the research in this field and revealed a plethora of additional genes and aberration-profiles relevant to MDS [10–12].

During the past decade, neoplastic cells in MDS have also been examined extensively for abnormal expression of cell surface antigens using monoclonal antibodies (mAb) and flow cytometry. However, most studies focused on CD34+ stem- and progenitor cells or ‘granulomonocytic’ cells. By contrast, only little is known about erythroid antigens abnormally expressed in MDS [13–22].

In mRNA profiling studies and flow cytometry validation, we identified the Coxsackie-Adenovirus receptor (CAR = CXADR) as a functionally relevant EP antigen that is down-regulated in MDS. Transduction of CXADR/CAR resulted in enhanced erythroid differentiation and migration in MDS EP. Moreover, anemia and dysplasia developed in a Cxadr/Car conditional knock-out mouse model.

Materials and Methods

Reagents

Reagents and antibodies used in this study are described in the Supplement. A specification of antibodies used for flow cytometry is shown in Supplementary Table S1A and S1B and a list of PCR primers in Supplementary Table S2.

Patients and sampling of cells

BM cells were obtained during diagnostic investigations of patients with MDS and of controls. Details are described in the Supplement. MDS patients were classified according to the French-American-British (FAB) study group proposal [23] and World Health Organization (WHO) [24, 25]. Patients’ characteristics are shown in Table 1 and Supplementary Tables S3–S11. In a separate analysis, MDS patients, patients with reactive anemia, and controls without anemia were studied to define CAR expression in different stem/progenitor cell compartments (Supplementary Tables S12 and S13). All patients gave written informed consent. The study was approved by the ethics committees of the Medical University of Vienna and the Elisabethinen-Hospital Linz, Austria. Aspirated BM cells were layered over Ficoll to obtain mononuclear cells (MNC) or were directly subjected to flow cytometry. For prognostication, we applied the International Prognostic Scoring System (IPSS) [26] and revised IPSS (IPSS-R) [27]. All patients were examined for progression to AML and survival during follow-up.

Table 1. Characteristics of MDS patients (n = 117)* and WHO classification.

MDS subtype (WHO 2016-based) MDS subtype (WHO 2022-based) Number of patients Gender (m/f)
MDS-SLD MDS-LB 06 4/2
MDS-RS-SLD/MDS-RS-MLD MDS with LB and RS / MDS-SF3B1 14 7/7
MDS-MLD MDS-LB 60 43/17
MDS del(5q) MDS-5q 07 1/6
MDS-EB1 MDS-IB1 15 11/4
MDS-EB2 MDS-IB2 15 8/7
All patients All patients 117 72/45
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*

A more detailed description of our MDS patients is provided in Supplementary Table S3. MDS myelodysplastic syndromes, WHO world health organization, Hb hemoglobin, m male, f female, MDS-SLD MDS with single lineage dysplasia, MDS-RS-SLD MDS with single lineage dysplasia and ring sideroblasts (most exhibiting mutated SF3B1), MDS-RS-MLD MDS with multilineage dysplasia and ring sideroblasts (most exhibiting mutated SF3B1), MDS-MLD MDS with multilineage dysplasia, MDS del(5q) MDS with deletion of chromosome 5q, MDS-EB1 MDS with excess blasts-1, MDS-EB2 MDS with excess blasts-2, MDS-LB MDS with low blasts, MDS-IB1 MDS with increased blasts-1, MDS-IB2 MDS with increased blasts-2, LB low blasts, RS ring sideroblasts.

Flow cytometry and cell sorting

Reactivity of EP with mAb against CXADR/CAR was determined in BM samples by multi-color flow cytometry as reported and EP were defined as CD45low/CD105+ cells [28]. The gating strategy is shown in Supplementary Fig. S1. Staining results were expressed as mean fluorescence intensity (MFI) and/or absolute antibody-binding sites per cell (ABC) using Quantibrite™ PE (BD Biosciences, San Jose, CA, USA) as described [29]. In a separate set of experiments, EP were highly purified as CD45low/CD105+ cells (>95% purity) by sorting and re-sorting on an Aria III sorter (BD Biosciences). Technical details are described in the Supplement.

mRNA profiling of EP

In 24 patients with MDS, 10 with reactive anemia and 11 controls, highly purified (sorted) EP were subjected to RNA extraction, gene array profiling and qPCR. A detailed description is provided in the Supplement. Array results are available at Gene Expression Omnibus (GEO: GSE147963). To examine MDS EP for the presence of adenoviral antigens, a pan-adenovirus-specific PCR was applied on CD105+ MDS EP as described [30, 31]. Technical details are provided in the Supplement.

Lentivirus-mediated transduction of CXADR/CAR

To study the functional role of CAR in erythropoiesis, the full-length CXADR gene or empty vector were transduced into MDS MNC. In addition, we transfected the full-length CXADR gene as well as various CXADR splice-variants [32] into the CAR-negative erythroblast cell lines HEL, K562, and KU812 by lentiviral-mediated gene transfer following published protocols [33]. In select experiments, CXADR-transfected cells were cultured with or without cytokines. A detailed description is provided in the Supplement. Expression of erythroid antigens on HEL, K562, and KU812 cells was analyzed by flow cytometry using mAb shown in Supplementary Table S1. Flow cytometry results were expressed as staining index (SI). Technical details are described in the Supplement. In a separate set of experiments, CXADR-transfected and empty vector-transfected cells were cultured in various concentrations of azacytidine, decitabine, lenalidomide, or JQ1 for 48 h before CAR expression was analyzed.

Colony-formation and cell migration of normal cells and MDS cells

Normal CD34+ BM MNC were transduced with CXADR shRNA or control shRNA, and MDS patient-derived BM MNC were transduced with CAR6/7 or an empty control-vector. After transduction, CD34+ cells were purified by cell-sorting and applied in a colony assay (Stem cell technologies, Cambridge, UK). Formation of erythroid colonies (burst-forming units = BFU-E) was determined after 2 weeks. To study migration of primary cells and HEL cells against a serum-gradient (‘BM-to-blood’ transition model), chemotaxis experiments were performed using a modified Boyden-chamber assay. Technical details are provided in the Supplement.

Mouse model of Cxadr deficiency in BM cells

Mice with floxed Cxadr alleles in a C57BL/6 background (Cxadrfl/fl) and Mx1-Cre transgenic mice (Mx1-Cre) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Mx1-Cre Cxadrfl/fl mice were obtained by crossing Cxadrfl/fl and Mx1-Cre mice. Six-week old Mx1-Cre Cxadrfl/fl mice were injected intraperitoneally with 400 μg poly I:C on three consecutive days. Genotyping and confirmation of Cxadr deletion were done by PCR as described in the Supplement. Mice were kept under pathogen-free conditions. Antibodies used to detect and isolate erythroid progenitors and myeloid progenitor cells in murine BM samples are shown in Supplementary Table S16. Animal experiments were approved by the ethics committee of the Medical University of Vienna and the Austrian government (license-number: 114/2012; animal welfare committee vote: GZ 66.009/0040-II/10b/2009) and carried out in accordance with guidelines for animal care and protection and protocols approved by Austrian law. Detailed information about genotyping and mouse experiments are described in the Supplement.

Statistical analyses

Statistical analyses are described in the Supplementary appendix.

Results

EP exhibit an altered mRNA expression profile in MDS

In initial screens, we compared mRNA expression profiles of sorted EP in 10 MDS patients (#1–#10, Supplementary Table S15) and 10 with reactive anemia (#11–#20, Supplementary Table S15). A number of gene products were found to be upregulated (n = 485) or downregulated (n = 732) in MDS EP compared to EP in reactive anemia (Fig. 1). We next extended gene array studies to sorted EP in healthy controls (n = 11) and more MDS patients (n = 24), including 5 with high-risk MDS, 5 with intermediate-risk MDS, and 14 with low-risk MDS according to IPSS-R (Supplementary Table S15). Again, a number of mRNA-species were differentially expressed in EP in MDS compared to controls. In addition, several gene-products were found to be differentially expressed in EP when comparing MDS subgroups. A summary of mRNA-species differentially expressed in EP is shown in Supplementary Tables S16–S33. Next, we screened for mRNA species that generate surface molecules and are consistently up- or down-regulated in EP in MDS compared to control EP. One of these genes was found to be CXADR. In fact, CXADR was among the top 20 down-regulated mRNA-species in EP when comparing all MDS samples with normal BM (Supplementary Table S17). Moreover, CXADR was the second most downregulated gene (top 2) in a comparison between MDS and reactive (non-neoplastic) anemia (Supplementary Table S18), and the top-downregulated gene when comparing intermediate/high-risk MDS with reactive anemia (Table 2). In this latter comparison, a total number of 2324 mRNA-species were differently expressed in EP: 2303 were up-regulated and 21 (including CXADR) were down-regulated (Supplementary Table S16, Table 2). Decreased CXADR mRNA expression in MDS EP was confirmed by qPCR (Supplementary Fig. S2). We also screened for other erythroid genes that are abnormally expressed in EP in MDS. In these studies, we found a significant downregulation of CXCR4 (CD184) in MDS EP compared to control EP without anemia in our array analyses (Supplementary Fig. S3). An overview of gene-products abnormally expressed in MDS EP is provided in the Supplementary Results and in Supplementary Fig. S3.

Fig. 1. mRNA expression profiles of EP in MDS compared to EP of patients with reactive anemia and schematic presentation of the marker profile of human erythropoiesis.

Fig. 1

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A Gene expression profiles of CD105+ EP obtained from patients with MDS (n = 10) and patients with reactive anemia (n = 10). Supervised hierarchical clustering was performed on 1217 mRNA variants which were at least three-fold ‘overexpressed’ (red color) or three-fold ‘under-expressed’ (blue color) (p < 0.01): increased expression in MDS EP was seen in 485 mRNA species, whereas reduced expression in MDS EP was seen in 732 gene products. B, C Schematic illustration of the immunophenotype within the different stages of the erythroid progenitor cells in controls (B) and MDS patients (C). The black arrow represents the common leukocyte antigen CD45. CAR expression is depicted as red arrow. The red shaded area of the CAR arrow in Figure (C) means reduced CAR expression in MDS patients compared to the controls. Green arrows represent precursor-associated markers, blue arrows represent myeloid markers, orange arrows represent erythroid-associated markers and pink arrows represent non-specific. EP1 are defined as CD34+/CD105−, EP2 as CD34+/ CD105low, EP3 as CD34+/low/CD105high and EP4 as CD34−/CD105high as described by Yan et al. [49]. The erythroid stage EP1 is identical to the previously designated stage “common progenitor cells”. MDS myelodysplastic syndromes, EP erythroid progenitor cells, CD cluster of differentiation, CAR Coxsackie-Adenovirus receptor.

Table 2. Top 20 down-regulated mRNA species in EP of high and intermediate risk MDS patients vs. patients with reactive anemia.

Gene symbol Gene title p-value MDS high and intermediate risk vs. reactive anemia Fold-change MDS high and intermediate risk vs. reactive anemia
CXADR = CAR coxsackie virus and adenovirus receptor 7.57E-04 −5.05
OR2L2 olfactory receptor. family 2. subfamily L. member 2 6.66E-09 −4.45
KLRF1 killer cell lectin-like receptor subfamily F. member 1 5.10E-08 −3.72
LIFR leukemia inhibitory factor receptor alpha 4.01E-07 −3.70
ABCD2 ATP-binding cassette. subfamily D (ALD). member 2 1.06E-05 −3.68
USP25 ubiquitin specific peptidase 25 3.08E-07 −3.64
C7orf62 chromosome 7 open reading frame 62 1.08E-06 −3.39
PLCL1 phospholipase C-like 1 6.24E-06 −3.37
COPG2 coatomer protein complex. subunit gamma 2 1.68E-06 −3.25
RPS6KA6 ribosomal protein S6 kinase. 90 kDa. polypeptide 6 2.14E-09 −3.21
MYO5B myosin VB 4.71E-07 −3.17
FCGR1A /// FCGR1B
/// FCGR1C		 Fc fragment of IgG. high affinity Ia. receptor (CD64) /// Fc
fragment of IgG. high affinity Ib. receptor (CD64)/// Fc
fragment of IgG. high affinity Ic. receptor (CD64)		 2.48E-04 −3.15
GALNT3 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 3.12E-05 −3.15
DOCK1 dedicator of cytokinesis 1 4.10E-10 −3.15
LOC147791 uncharacterized LOC147791 8.67E-06 −3.15
IFNE interferon. epsilon 1.92E-07 −3.11
LOC728114 uncharacterized LOC728114 2.04E-07 −3.07
CDHR5 cadherin-related family member 5 2.87E-06 −3.02
CNTN1 contactin 1 1.57E-05 −3.02
DSG3 desmoglein 3 5.78E-07 −3.00
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Gene array studies were performed with CD105+ EP obtained from bone marrow of patients with reactive anemia (n = 10 donors) and patients with high and intermediate risk MDS (n = 10 donors). The 20 top down-regulated gene products in EP of patients with high and intermediate risk MDS compared to EP of patients with reactive anemia are shown. EP erythroid progenitor cells, MDS myelodysplastic syndromes, vs. versus.

CAR is downregulated on the surface of EP in MDS

In normal BM and MDS BM, CAR expression was found to be restricted to the erythroid lineage suggesting that CAR may play a role in erythropoiesis. When analyzing various stages of EP development, CAR was found to be expressed at an earlier stage compared to endoglin (CD105) in normal BM (Supplementary Fig. S1A+S1B) and MDS (Fig. 1B+1C, Supplementary Fig. S4). Specifically, CAR was expressed in an early CD34+ stage of erythropoiesis (EP1) as well as in EP2, EP3, and EP4 (Supplementary Figs S1 and S4). We also found significant differences in CAR expression levels when comparing MDS EP with control EP, and EP in various MDS subsets. Overall, lower CAR expression levels were found on EP in MDS patients (n = 117) compared to control EP (p < 0.05) (n = 45) (Fig. 2A). Reduced or absent CAR expression on EP was found in 100/117 MDS patients (85%), including 6/6 with single-lineage dysplasia (MDS-SLD), 10/14 with MDS-SLD and ring sideroblasts (MDS-RS-SLD), 49/60 with multi-lineage dysplasia (MDS-MLD), 7/7 with deletion of chromosome 5q (MDS del(5q)), 15/15 with excess of blasts-1 (MDS-EB1) and 13/15 with MDS-EB2. The degree of CAR deficiency on EP was found to correlate with the subtype of MDS. Median CAR expression levels on EP were lower in EB1 and EB2 patients compared to MDS-RS-SLD, MDS-MLD or del(5q) patients (p < 0.05) (Fig. 2B, Supplementary Fig. S4).

Fig. 2. Expression of CAR on EP of MDS patients.

Fig. 2

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The expression of CAR on EP was determined by multicolor flow cytometry as described in the text. The level of CAR expression on EP is shown as antibody binding per cell and is indicated by dots in each individual donor. The boxes represent the 25–75% percentile in each group, the horizontal line within boxes defines the median, and the whiskers represent the range. Figure 2(A) shows the median CAR expression in EP of all MDS patients. Asterisk (*): p < 0.05. Median CAR expression levels on EP in various MDS subtypes is shown in Fig. 2(B). Asterisk (*): p < 0.05 compared to controls. CAR Coxsackie-Adenovirus receptor, EP erythroid progenitor cells, MDS myelodysplastic syndromes, MDS-SLD MDS with single lineage dysplasia, MDS-RS-SLD MDS with single lineage dysplasia and ring sideroblasts, MDS-MLD MDS with multilineage dysplasia, MDS del(5q) MDS with deletion of chromosome 5q, MDS-EB1 MDS with excess blasts-1, MDS-EB2 MDS with excess blasts-2.

CAR surface expression on EP in other hematologic disorders

Reduced expression of CAR on EP was also seen in 38/47 patients with myeloproliferative neoplasms (MPN) (p < 0.05) and 15/18 with AML (p < 0.05) (Supplementary Fig. S5). By contrast, CAR expression on EP was normal in all non-neoplastic conditions examined (Supplementary Fig. S5). Moreover, CAR expression on BM EP was normal in most patients suffering from lymphoid neoplasms (Supplementary Fig. S5).

CXADR transduction results in an increased expression of erythroid differentiation antigens in erythroblastic cell lines

HEL, K562 and KU812 cells have only minimal erythroid differentiation-potential even when maintained in erythropoietic growth factors. As expected, all three cell lines stained negative for CAR by flow cytometry. To examine the role of CAR in erythroid differentiation, cells were transduced with the lentiviral pWPI vector containing full-length CXADR (CAR6/7), various CXADR splice-isoforms, or empty vector. CAR expression was confirmed by qPCR and flow cytometry (Supplementary Fig. S6). We found that CAR6/7-transduction promotes expression of several erythroid antigens, including CD36, CD71, CD105, and CD235a (glycophorin A) in these cells. CAR6/7-induced upregulation of erythroid antigens was demonstrable by flow cytometry and qPCR (Fig. 3A, B). Transfection of truncated CXADR variants did not result in expression of erythroid antigens (Supplementary Fig. S7). Full-length CXADR-transduction did not alter expression of CXCR4 (CD184) or E-cadherin (Supplementary Fig. S8). Next, we treated CAR6/7-transduced cell lines with erythropoietin (EPO), interleukin-3, stem cell factor or a mixture of cytokines. However, these cytokines did not promote expression of CD36, CD71, CD105 or CD235a in these cells (Supplementary Figs. S9). We also examined the effects of demethylating agents and other epigenetic drugs on CAR expression. However, neither azacytidine nor the other drugs applied (decitabine, lenalidomide, JQ1) augmented CAR expression in HEL, K562 or KU812 cells (Supplementary Fig. S10). Next, we investigated the effect of CAR expression on proliferation and apoptosis. However, CXADR transduction did not lead to enhanced or reduced proliferation or apoptosis in HEL, K562, or KU812 cells (data not shown). Finally, we asked whether abnormal CAR expression on EP is a virally triggered event. However, we were unable to detect adenoviral transcripts in patient EP (not shown).

Fig. 3. Effect of CXADR transduction on expression of erythroid differentiation antigens in HEL cells, K562 cells and KU812 cells.

Fig. 3

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A HEL, K562, and KU812 cells were transduced with pWPI plasmids containing full-length CXADR (CAR6/7) or the empty vector. Surface expression of erythroid antigens was determined by flow cytometry as described in the text. Expression levels are provided as staining index (SI) defined by the ratio of mean fluorescence intensities (MFI) obtained with specific mAb and isotype-matched control mAb (SI = MFI test mAb : MFI control mAb). Results show SI values and represent the mean ± SD of at least 3 independent experiments. Asterisk (*): p < 0.05. B After transduction, HEL, K562 and KU812 cells were subjected to RNA isolation and qPCR using primers specific for glycophorin A (CD235a), CD36, CD71 and CD105. Technical details are described in the Supplement. Results show mRNA levels as percent of ABL1 mRNA levels and represent the mean ± SD of at least 3 independent experiments. CAR Coxsackie-Adenovirus receptor, mAb monoclonal antibody.

CAR mediates colony formation in erythroid progenitor cells in normal BM and in MDS

To study the role of CAR in erythroid differentiation, we transduced CXADR shRNA into normal BM cells and CAR6/7 into MDS progenitors. shRNA-mediated depletion of CXADR in normal CD34+ BM progenitors resulted in reduced BFU-E formation compared to control shRNA (Supplementary Fig. S11). Moreover, expression of CAR in MDS cells resulted in increased BFU-E formation compared to empty vector-transduced cells or un-transduced cells (Supplementary Fig. S11). These data suggest that CAR mediates erythroid differentiation and maturation in CD34+ cells in healthy BM and MDS and might therefore serve as therapeutic target in anemic MDS patients.

Role of CAR in migration of erythroid progenitor cells against serum proteins

To explore the contribution of CAR to BM-to-blood redistribution of EP, we examined the migratory responses of non-transfected and CXADR-transfected EP and HEL cells to serum-proteins in a modified Boyden chamber. We found that MDS EP have reduced ability to migrate against a serum protein gradient compared to normal EP (Fig. 4A). Transduction of CXADR restored the migration defect of MDS EP (Fig. 4B). Moreover, CXADR shRNA was found to suppress EP migration (Fig. 4C). Knock-down and over-expression of CAR were confirmed by qPCR (Fig. 4D). Finally, HEL cells transduced with CXADR showed an increased chemotactic migration against a serum protein-gradient compared to empty vector-control or non-transduced cells (Fig. 4E). The enhanced migration was only observed with cells expressing full-length CAR6/7 gene, but not in HEL cells transduced with CXADR splice-variants (CAR2/7, CAR3/7, CAR4/7, CAR4/6) (Supplementary Fig. S12).

Fig. 4. Effects of CXADR transduction on migration of erythroid progenitor cells.

Fig. 4

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A Primary MNC of patients with MDS (n = 3; MDS #1, MDS #2, MDS #3) and MNC of normal BM (n = 3) were stained with CD34-FITC and CD105-APC antibodies. Then, MNC were resuspended in medium containing 10% human serum (control) or 0.2% human serum (0.2% serum) and were then loaded in the upper chambers in a modified Boyden chamber assay. The lower chambers were filled with medium containing 10% human serum. After five hours at 37 °C, migrated viable cells (MNC and CD34−/CD105+ MNC) were counted by flow cytometry. Results show percent of migrated cells and represent the mean ± SD of 3 experiments (3 donors). B Primary MNC of patients with MDS (n = 2; MDS #4, MDS #5) were transduced with pWPI plasmids containing CAR6/7 or empty vector control as described in the Supplement. After transduction (72 hours), MDS MNC were sorted for GFP+ cells. Then, MNC (untransfected, empty vector-transfected and CAR6/7-transfected cells) were resuspended in medium containing 10% human serum (control) or 0.2% human serum (0.2% serum) and were then loaded in the upper chambers in a modified Boyden chamber assay. The lower chambers were filled with medium containing 10% human serum. After five hours at 37 °C, migrated viable cells were counted by flow cytometry. Results show the percentage of migrated cells. C Normal BM MNC were transduced with CXADR shRNA clone 1, CXADR shRNA clone 2, or control shRNA as described in the Supplement. After transduction (72 hours), MNC were sorted for GFP+ cells. Then, MNC (untransfected, control shRNA-transfected, and CXADR shRNA-transfected cells: hairpin 1 and hairpin 2) were resuspended in medium containing 10% human serum (control) or 0.2% human serum (0.2% serum) and were then loaded in the upper chambers in a modified Boyden chamber assay. The lower chambers were filled with medium containing 10% human serum. After five hours at 37 °C, migrated viable cells were counted by flow cytometry. Results show the percentage of migrated cells. D Evaluation of efficacy of transduction of full-length CXADR. Primary MNC of a patient with MDS (MDS #4) (upper panel) were transduced with pWPI plasmids containing full-length CXADR (CAR6/7) or empty vector control as described in the method section of the Supplement. Normal BM MNC (lower panel) were transduced with CXADR shRNA clone 1, CXADR shRNA clone 2 or control shRNA as described in the method section of the Supplement. After transduction (72 hours), MDS MNC and normal BM MNC were sorted for GFP+ cells. Then, CXADR expression was determined by qPCR. CXADR mRNA levels are expressed as percent (%) of ABL1 mRNA levels. E HEL cells were transduced with pWPI plasmids containing full-length CXADR (CAR6/7) or the empty vector. For chemotactic migration, transduced and untransduced HEL cells were applied in a modified Boyden chamber assay. Cells resuspended in medium containing 10% FCS (control) or 0.2% FCS (0.2% serum) were loaded in the upper chambers. The lower chambers were filled with medium containing 10% FCS. After five hours (37 °C), the viable migrated cells were counted by flow cytometry. Results represent the mean ± SD of 6 experiments. Asterisk (*): p < 0.05. CAR Coxsackie-Adenovirus receptor, MDS myelodysplastic syndromes, MNC mononuclear cells, BM bone marrow, APC allophycocyanin, FITC fluorescein isothiocyanate, GFP green fluorescent protein, sh short hairpin, FCS fetal calf serum.

Conditional Cxadr knock out (KO) results in BM dysplasia and anemia

To further study the pathogenetic role of CXADR, we established conditional Cxadr KO mice (Mx1-Cre Cxadrfl/fl) by crossing Mx1-Cre transgenic mice with Cxadrfl/fl mice. At the age of six weeks, 400 μg poly I:C were intraperitoneally injected on three consecutive days to induce Cxadr deletion. Examples for PCR-based genotyping and confirmation of Cxadr deletion efficiency are shown in Supplementary Fig. S13. The phenotype of Mx1-Cre+Cxadrfl/fl and Mx1-Cre-Cxadrfl/fl control mice was monitored by monthly blood counts. The red blood cell (RBC) count of Mx1-Cre+Cxadrfl/fl mice showed a gradual decrease, starting at 6 months after poly I:C injection, resulting in a substantial drop at 10 months compared to control mice (p < 0.01, Fig. 5A). The decrease in RBC showed a clear dependence on the efficacy of Cxadr-deletion (Supplementary Fig. S14A) and was accompanied by a decrease in hemoglobin and hematocrit (Supplementary Fig. S14B-S14D). No decrease in leukocyte or platelet counts was observed (Fig. 5B, Supplementary Fig. S14E). After 10 months, mice were sacrificed, and EP were characterized in BM samples by flow cytometry based on expression of Ter119 and CD44 [34]. The gating strategy is shown in Supplementary Fig. S15. Although a similar distribution of erythroid cells (from erythroblast to reticulocyte stage) was found, Mx1-Cre+Cxadrfl/fl mice showed an increase in mature erythrocytes in the BM (p < 0.05, Fig. 5C). Morphological studies showed mild dysplasia in erythroid cells of Mx1-Cre+Cxadrfl/fl mice, including bi-nucleated EP and irregular nuclear shapes (Fig. 5D) [35]. Splenomegaly was only detected in a subset of animals (Supplementary Fig. S14F). Next, we applied a modified Boyden chamber assay to test the migratory capacity of murine EP. Sorted erythroid cells (combined erythroblast+reticulocyte stage of Mx1-Cre+Cxadrfl/fl mice) showed reduced migration against a serum protein-gradient compared to wild type control (p < 0.05, Fig. 5E). To further characterize the role of CAR, myeloid progenitors were sorted and subjected to RNA sequencing (RNA-seq). Efficient deletion of CAR in Mx1-Cre+Cxadrfl/fl cells was confirmed by flow cytometry (p < 0.05, Supplementary Fig. S14G) compared to control cells and CAR levels reported in hematopoietic stem cells [36]. Mx1-Cre+Cxadrfl/fl mice also displayed a general decrease in lineage−/CD117+ BM cells (p < 0.05, Fig. 5F, left panel). Within lineage−/CD117+ populations, there was a subtle, statistically not significant, trend towards megakaryocyte-erythroid progenitor (MEP) differentiation at the expense of granulocyte-monocyte progenitors (GMP) (Fig. 5F, right panel). RNA-seq and comparison of gene expression profiles revealed profound differences between Mx1-Cre+Cxadrfl/fl and Mx1-Cre−Cxadrfl/fl mice at the MEP stage, with a total of 165 differentially expressed genes (DEG) at an adjusted p-value < 0.01 (Supplementary Fig. S14H). A heat-map of DEG at the MEP stage is shown in Fig. 5G. Pathway-analysis of DEG in MEP revealed an enrichment of erythroid differentiation gene-sets in Mx1-Cre−Cxadrfl/fl control mice (Fig. 5H).

Fig. 5. Conditional knock out of Cxadr in C57BL/6 mice results in anemia and a MDS-like phenotype.

Fig. 5

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A Time course of RBC count measurements in Mx1-Cre+Cxadrfl/fl and Mx1-Cre−Cxadrfl/fl control mice shows anemia development starting at six months after poly I:C injection (n = 7–21 per time point, data presented as mean ± SEM). Asterisk (*): p < 0.05, double-asterisk (**): p < 0.01. B Leukocyte counts do not differ between Mx1-Cre+Cxadrfl/fl and Mx1-Cre−Cxadrfl/fl control mice for the entire follow-up period after conditional Cxadr deletion. C Calculated absolute numbers of BM erythroid progenitors obtained from flow cytometry based on the expression of Ter119 and CD44. Results were obtained from three independent experiments (Mx1-Cre+Cxadrfl/fl: n = 5, Mx1-Cre-Cxadrfl/fl: n = 7; data presented as mean ± SEM). Asterisk (*): p < 0.05; pro(I): proerythroblast, baso(II): basophilic erythroblast, poly(III): polychromatic erythroblast, ortho(IV): orthochromatic erythroblast, reti(V): reticulocyte, ery(VI): erythrocyte. D Representative Giemsa-stained cytospins of Mx1-Cre+Cxadrfl/fl and Mx1-Cre−Cxadrfl/fl control mice from erythrocyte-lysed whole BM (upper panel) and sorted erythroid progenitor populations (lower panel). E The modified Boyden chamber assay was performed on sorted murine progenitors (combined proerythroblast – reticulocyte stage). Cells resuspended in medium containing 10% murine serum (control) or 0.2% murine serum (0.2% serum) were loaded in the upper chambers. The lower chambers were filled with medium containing 10% murine serum. After five hours (37 °C), the viable migrated cells were counted by flow cytometry. Results were obtained from three independent experiments (p < 0.05; Mx1-Cre+Cxadrfl/fl: n = 5, Mx1-Cre-Cxadrfl/fl: n = 8; data presented as mean ± SEM). F Flow cytometry quantification of myeloid BM progenitor cells. Results are shown as percentage of parent population (triple asterisk (***): p < 0.001, Mx1-Cre+Cxadrfl/fl: n = 4, Mx1-Cre+Cxadrfl/fl: n = 4). G Heatmap showing DEG with a univariate p-value < 0.001 between Mx1-Cre+Cxadrfl/fl (D1_MEP to D4_MEP) and Mx1-Cre−Cxadrfl/fl controls (C1_MEP to C4_MEP) at the MEP stage (top 30 highlighted). After normalization of RNA-seq raw counts, differentially expressed genes were calculated using the DESeq2 package for R. H Pathway analysis of these DEG using GSEA revealed enrichment of erythroid differentiation gene sets in Mx1-Cre−Cxadrfl/fl control mice. BM bone marrow, DEG differentially expressed gene, GSEA gene set enrichment analysis, LSK Lin–/sca-1+/CD117+, GMP granulocyte-monocyte progenitor, CMP common myeloid progenitor, MEP megakaryocyte-erythroid progenitor, RBC red blood cell, SEM standard error of the mean, NES normalized enrichment score.

Prognostic impact of decreased CAR expression on EP in MDS patients

To study the prognostic impact of CAR expression on EP we first correlated CAR expression levels with IPSS-R subsets. As shown in Supplementary Fig. S16A, CAR levels on EP were significantly lower in patients with high- or very high-risk MDS by IPSS-R compared to low- or very low-risk MDS. Next we correlated CAR expression levels with overall survival (OS) and AML-free survival. Again, we found that lower CAR expression levels (below median CAR ABC of controls: <3215) correlate with reduced OS (p = 0.09) (Supplementary Fig. S16B) and reduced event-free survival (p = 0.11) (Supplementary Fig. S16C). However, these correlations were not statistically significant. Moreover, reduced CAR expression in EP did not correlate with AML-free survival (Supplementary Fig. S16D, S17). In subsequent studies, we found a significant correlation between CAR expression and platelet counts in our MDS patients (p < 0.05) (Supplementary Fig. S18). Therefore, we asked whether platelets express CAR or influence CAR expression on leukocytes. However, platelets did not stain positive for CAR and when incubated with platelets and washed, HEL cells were still found to be CAR-negative cells (not shown).

Discussion

During the past few years our knowledge about the pathogenesis of MDS increased substantially [1–3, 5–9, 37]. However, the exact mechanisms underlying the maturation defect and peripheral cytopenia remain unknown. In an attempt to identify genes responsible for altered erythroid development in MDS, we identified CAR as an erythropoiesis-associated antigen that is down-regulated on EP in MDS. Our data also show that CAR promotes erythroid differentiation as well as migration in immature erythroid cells and that a Cxadr knock-out induces a MDS-like phenotype in mice. Moreover, in a BFU-E assay, CXADR-transduction reverted the differentiation-block of MDS EP. Based on these observations, we hypothesize that CAR-deficiency contributes to the maturation defect of EP in the BM and subsequent anemia in MDS.

Recent data suggest that EP in MDS express an abnormal phenotype [14, 15]. However, so far, no comprehensive molecular studies have been performed to define abnormal gene expression profiles in MDS EP. We applied an unbiased gene array approach to compare mRNA profiles of EP in MDS patients, normal BM, and reactive anemia. In this screen, we identified a number of abnormally expressed mRNA species in MDS EP compared to controls. In subsequent analyses, we identified CAR as a most promising surface molecule that is specifically downregulated on EP in MDS.

In the validation phase, we examined a larger patient cohort and found that CAR is down-regulated on EP in most MDS patients compared to EP in controls. We also found that CAR down-regulation is more prominent in MDS-EB1/EB2 compared to patients with MDS-RS-SLD or del(5q). Especially in those with ring sideroblasts (SF3B1-mutated MDS), EP often exhibit normal CAR levels. On the other hand, decreased CAR expression was also found in other myeloid neoplasms, including AML and MPN/MDS, especially when marked dysplasia was also present.

Recent data suggest that CAR is down-regulated in neoplastic cells in a number of tumor models [38–41]. However, so far, little is known about underlying mechanisms. In some solid tumors, CAR expression can be upregulated by epigenetic drugs [42]. In our study, however, the epigenetic drugs applied, including azacytidine and decitabine, did not modulate CAR expression in erythroid cells.

Several studies have examined the functional role of CAR in normal and neoplastic cells. CAR is a member of the JAM-family of adhesion receptors that binds to type-B Coxsackie viruses and subgroup-C Adenoviruses. It has also been described that CAR regulates epithelial cell-junction stability during E-cadherin trafficking [43]. However, little is known about the biology and function of CAR in hematopoietic cells. Since CAR is a viral receptor, we first asked whether viral antigens are present in EP. However, we were unable to detect adenoviral antigens in EP in our MDS patients. Next we asked how CAR may contribute to differentiation and/or migration of EP. To address this question, we transfected full-length CXADR into MDS EP and erythroblastic cell lines. In these experiments, CXADR transduction substantially promoted erythroid burst-formation in MDS EP suggesting that CAR deficiency contributes to altered erythroid maturation of these cells in MDS. Correspondingly, CAR induced the expression of several erythroid differentiation antigens in the immature erythroblastic cell lines HEL, K562 and KU812. However, CXADR transduction did not induce full maturation in these cells, even when erythropoietic growth factors were added. This observation suggests that other differentiation signals are required to induce terminal maturation in these cells. Finally, we were able to show that CAR expression promotes the migratory response of EP and erythroid cell lines to a serum protein gradient. These observations suggest that MDS EP exhibit an altered ability to migrate into blood which may explain the paradoxical accumulation of these cells in the BM in MDS. Overall, to the best of our knowledge, this is the first report suggesting that CAR acts as key mediator of erythroid cell maturation and migration.

To further demonstrate the pathogenic role of CAR in the MDS context, we established a conditional Cxadr KO mouse model and found that Cxadr-deleted mice develop anemia and erythroid dysplasia. In addition, murine EP showed an impaired migration against serum proteins. Collectively these data suggest that CAR is an important regulator of erythropoiesis and re-distribution of EP, and that a loss of CAR in MDS may be of pathogenetic significance.

However, apart from CAR, other erythroid (surface) antigens may also be involved in the abnormal migration, distribution, and maturation of EP in MDS. For example, we found that the chemokine receptor CXCR4 that is involved in cell migration and mobilization in the BM, is downregulated on EP in MDS compared to normal EP. Other antigens downregulated in MDS EP identified in this study were CD36 and glycophorin A. Both antigens were upregulated by CAR transduction in erythroid cell lines. However, not all erythroid antigens were upregulated by CAR, which is in line with the concept that erythropoiesis is regulated by a complex network of transcription factors and other mechanisms.

During the past 10 years, flow cytometry analysis of peripheral blood and BM cells has emerged as a powerful diagnostic and prognostic approach in MDS [2, 17, 18]. Certain phenotypic abnormalities of blast cells or more mature myeloid cells are now regarded as diagnostic co-criteria in MDS [2, 44]. However, only a few studies addressed the diagnostic value of flow-abnormalities in EP. For example, it has been described that erythroid cells in MDS exhibit abnormal expression of CD36 and CD71 [14, 15]. We found that the decrease in CAR on EP in MDS is a recurrent finding. However, not all patients with MDS presented with decreased CAR expression on EP, and decreased CAR expression is not specific for MDS. Therefore, CAR expression levels on EP have to be interpreted in the context of additional flow cytometry results and on the basis of other clinical and laboratory data. A reasonable strategy might be to include CAR into diagnostic flow-panels evaluating EP in suspected MDS [17, 18].

Abnormal expression of myeloid surface antigens in MDS may be of prognostic significance, and it has been described that certain flow cytometric abnormalities can be used to create prognostic scores in these patients [45–48]. We asked whether abnormal expression of CAR on EP in MDS is of prognostic value. To address this question, MDS patients were split into two groups, one with high expression levels of CAR on EP and one with low CAR expression. The results of our study show that low expression of CAR on EP correlates with high-risk IPSS-R. Reduced CAR expression was also found to correlate with reduced survival. However, this correlation did not reach statistical significance which is best explained by the relatively low numbers of patients in whom long-term follow up data were available.

Together, our data show that CAR is a novel, specific and functionally relevant differentiation-related biomarker in MDS that is specifically down-regulated on MDS EP. Decreased CAR expression may contribute to the maturation defect of erythroid cells and their abnormal accumulation in the BM in patients with MDS.

Supplementary Material

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41375-023-02015-7.

1

Acknowledgements

We like to thank Nadine Witzeneder, Barbara Peter, Martin Danzer, Harald Herrmann, and Hans-Ulrich Klein for their skillful technical assistance.

Funding

This study was supported by The Austrian Science Fund (FWF) SFB grants F4701-B20, F4704-B20, P30625-B28, by a grant of the Upper Austrian Cancer Aid Fund and by a stem cell grant of the Medical University of Vienna, Austria.

Footnotes

Author Contributions

KB and SMS designed the study and performed key laboratory experiments. LK, SG, AS and EK performed mouse experiments. WRS provided patients and statistical analyses. HUK contributed bioinformatics. SS, MD, and JP performed molecular and gene array studies. JL performed flow cytometry experiments. GE and IS performed transduction- and cell sorting experiments. GH provided laboratory data and molecular studies. NH provided modified pBK-CMV vectors containing different CXADR splice variants. MCB, UP, OZ, and AW contributed patients and logistic support. CG and GW performed molecular studies and provided logistic support. TL performed the adenovirus peptide-binding assay. UG contributed patients and logistic support. PV, PBS and PB designed the study and wrote the manuscript. All authors contributed by writing parts of the manuscript and by critically reading the paper. All authors approved the final version of the document.

Competing Interests

The authors declare no completing interests in this study. Conflict of interest outside the study are: P.V. received research grants from Celgene, Novartis, Incyte, Pfizer, and AOP Orphan, and honoraria from Celgene, Novartis, Pfizer, Incyte, and AOP Orphan. S.M.S received honoraria from Celgene, Novartis, Pfizer, Abbvie, Amgen, Jazz, Gilead, Servier and Incyte. P.B. was supported by Alexion outside the study. T.L. received research grants from Incyte and Novartis, and honoraria from Incyte, Pfizer, Angelini, Amgen and Chimerix. O.Z. was supported by Pfizer, Takeda, Amgen, Novartis, BMS, Genzyme, Sanofi, Pierre Fabre and Roche, and he is part of advisory boards of Amgen, Pfizer and Novartis. U.G. received research support from Novartis and Celgene, and honoraria from Celgene, Jazz, Novartis and Jansen. G.W. received research grants from Abbvie, Amgen, AstraZeneca, BMS, Böhringer Ingelheim, Gilead, Incyte, Lilly, Mundipharma, Novartis, Pfizer, Roche and Tesaro. G.H. received research grants from Novartis, he received honoraria and he is part of advisory boards of Novartis, Roche, Beckman Coulter, Pfizer, Celgene, Bristol-Myers Squibb. L.K. was supported by a DOCmed Fellowship of the Austrian Academy of Sciences (OeAW) and a research grant of the Vienna Comprehensive Cancer Center (CCC). P.B.S. reports grants and personal fees from Roche, Celgene, and AbbVie; and personal fees from Amgen, Takeda, AbbVie, and Janssen Cilag outside the submitted work.The other authors declare that they have no conflicts of interest.

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Availability of Data and Materials

Results from all patients and all controls obtained in this array analysis using 54676 gene probes (raw and normalized expression data) are available at the Gene Expression Omnibus repository (GEO: GSE147963). The raw RNA-seq data of the mice reported in this paper are available at the Gene Expression Omnibus repository (GEO: GSE239578).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
EMS194914-supplement-1.pdf (7.1MB, pdf)

Data Availability Statement

Results from all patients and all controls obtained in this array analysis using 54676 gene probes (raw and normalized expression data) are available at the Gene Expression Omnibus repository (GEO: GSE147963). The raw RNA-seq data of the mice reported in this paper are available at the Gene Expression Omnibus repository (GEO: GSE239578).

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