Page 92 - Haematologica April 2020

Y. Xie et al.
response to stress such as chemotherapy. A complex line- age-specific transcription factor network underlies the homeostatic hematopoiesis and erythropoiesis mecha- nisms. In such a transcription network, the GATA tran- scription factor family plays a central role in the proper differentiation of erythroid cells together with Friend of GATA (FOG-1). The GATA family is composed of six members in mammals that are highly conserved in expres- sion patterns in vertebrates, and GATA-1, GATA-2 and GATA-3 are classified into the hematopoietic GATA sub- family based on their expression profiles and domain structures. GATA-1 is important in adult hematopoiesis especially for erythropoiesis and regulates multiple target genes during the development and differentiation of ery- throid and megakaryocytic lineages by binding to the GATA motif (A/T)GATA(A/G). In this study, we identified a novel SNP of ARHGEF12 gene, rs10892563, located in a regulatory GATA motif and found that the erythroid expression of AEHGEF12 is significantly down-regulated in rs10892563 homogeneous ALL patients who have undergone chemotherapy.
ARHGEF12 encodes a RhoA specific guanine nucleotide exchange factor which positively regulates the RhoA GDP/GTP exchange reaction. ARHGEF12 plays crucial roles in the cyclic-stretch-induced cell and stress fiber reorientation responses,35 mesenchymal stem cell fate,36 and cell migration and invasion,37 by regulating RhoA activity. ARHGEF12 is important for platelet activation
and thrombosis in mice,38 but its role in erythropoiesis has not been defined. As a founding member of the Rho GTPase family, RhoA is involved in many important cel- lular functions, including gene transcription, survival, adhesion, and cytoskeleton reorganization. RhoA is important for hematopoiesis, regulating HSPC trafficking, interaction with the BM microenvironment, proliferation, survival, and self-renewal, and for fetal erythropoiesis in mitosis and cytokinesis.10 Interestingly, among the top ten in our GWAS list, there are three genes related to small GTPase functions: TNS1, RAP1GAP, and ARHGEF12. We focused our attention on ARHGEF12 because RhoA knockout in mice causes cytokinesis failure in erythrob- lasts through actomyosin and midbody dysregulation and p53 activation.10
To define the functional and mechanistic role of arhgef12 in erythropoiesis, we have used a zebrafish model to knockdown or knockout arhgef12 isoforms. We show a causal role of the ARHGEF12-RhoA signaling in this model in mediating the p38 MAPK and Stat1 pathway in erythropoiesis. In zebrafish, erythroid defects caused by arhgef12 knockdown can be rescued by p38 MAPK activa- tor and stat1 expression. Conversely, a p38 inhibitor can induce erythropoiesis defects mimicking that of the arhgef12 knockout or knockdown. This signaling effect seems to be conserved in mammals, as the ARHGEF12- RhoA-p38 function appears to also regulate the erythroid differentiation of erythroleukemia cell line K562. A num-
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Figure 6. The ARHGEF12-RhoA-p38 pathway is associated with erythroid regeneration from chemotherapy-induced anemia in acute lymphoblastic leukemia (ALL) patients. (A) Erythroid differentiation evaluation by flow cytometry of cryopreserved bone marrow (BM) samples from patients in remission with different rs10892563 genotypes. Three populations of different development stages, i.e. CD71+CD235a- pro-erythroblasts (Pro-E), CD71+CD235a+ erythroblasts and CD71-CD235a+ ery- throcytes can be discriminated. Representative flow cytometry dot plots showing the percentage of erythroblasts in patients with CC genotype was higher than TT genotype, whereas the pro-erythroblasts (Pro-E) and erythrocytes were reduced in CC genotype. (B) The TT group and the CC group each had nine samples. Proportion statistics of each stage of erythroid differentiation are shown. (C) Statistics of mean fluorescence intensity of phospho-p38. We compared the base-2 log of phos- pho-p38 levels between the CC and TT genotypes, phospho-p38 levels are lower in the CC genotype patients (P=0.0471, one-tailed t-test). (D) Phospho-flow his- tograms of phospho-p38 on pro-erythroblasts with different rs10892563 genotypes. Phosphorylated p38 was stained with anti-pT180/pY182 antibody on cryopre- served BM samples from patients in remission with different rs10892563 genotypes as well as CD71 and CD235a monoclonal antibody. Histograms showing phos- pho-p38 peaks shifted left in CC genotype (black) comparing TT genotype (gray).
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haematologica | 2020; 105(4)