ARHGEF12 regulates erythropoiesis and is involved in erythroid regeneration after chemotherapy in acute lymphoblastic leukemia patients

Hematopoiesis is a finely regulated process in vertebrates under both homeostatic and stress conditions. By whole exome sequencing we have studied the genomics of acute lymphoid leukemia patients who needed multiple red blood cell transfusions after intensive chemotherapies. ARHGEF12, encoding a RhoA guanine nucleotide exchange factor, was found associated with chemotherapy-induced anemia by genome-wide association study analyses. A single nucleotide polymorphism (SNP) of ARHGEF12 located in an intron predicted to be a GATA1 binding site, rs10892563, is significantly associated with patients who need red blood cell transfusion (P=3.469E-03, odds ratio 5.864). A luciferase reporter assay revealed that this SNP impairs GATA1 mediated trans-regulation of ARHGEF12, and quantitative PCR studies confirmed that the homozygotes status is associated with ~61% reduction of ARHGEF12 expression (P=0.0088). Consequently, erythropoiesis was affected at the pro-erythroblast phase in the patients. A role of ARHGEF12 and its homologs in erythroid differentiation was confirmed in human K562 cells, mouse 32D cells and primary murine bone marrow cells. We further demonstrated in zebrafish by morpholino-mediated knockdown and CRISPR/Cas9-mediated knockout of arhgef12 that its reduction resulted in erythropoiesis defects. The p38 kinase pathway was affected by the ARHGEF12-RhoA signaling in K562 cells, and consistently, the arhgef12-rhoA-p38 pathway was also shown to be important for erythroid differentiation in zebrafish as active rhoA or p38 readily rescued the impaired erythropoiesis caused by arhgef12 knocking down. Finally, ARHGEF12 mediated p38 activity also appeared to be involved in phenotypes of patients of the rs10892563 homozygous genotype. Our findings present a novel SNP of ARHGEF12 that may involve ARHGEF12-RhoA-p38 signaling in erythroid regeneration in acute lymphoblastic leukemia patients after chemotherapy.