Page 86 - Haematologica April 2020

Y. Xie et al.
susceptibility to chemotherapy.21 Of note, four of these genes, i.e. RAP1GAP, ARHGEF12, TNS1 and FARP1, are related to small GTPase regulation (Table 2 and 3). ARHGEF12, a RhoA-specific guanine-exchange factor (GEF), can specifically activate RhoA18 which is essential for embryonic erythropoiesis.10 ARHGEF12 is thus possi- bly one of the associated genes involved in the regulatory mechanism of erythroid regeneration from anemia induced by chemotherapy. Among the SNP found in ARHGEF12, the most significant association Was rs76693355 (P=3.469E-03, odds ratio 5.864). All SNP were screened with linkage disequilibrium 0.2<r2<1 related to rs76693355 in the 5-kb flanking regions of ARHGEF12. We found that rs10892563 is located at a predicted bind- ing site of the erythroid-specific transcription factor GATA1.22 To test if such a variant could disrupt this GATA1 binding site function, we employed a dual luciferase assay in 293T cells with an expression vector containing this intron motif of rs10892563 in the promoter region. The expression assay showed that the minor allele change of rs10892563 was able to down-regulate ARHGEF12 transcriptional regulation by GATA1 (Figure 2A and B). To examine if the rs10892563 SNP is actually associated with ALL patient RBC function, the CD71-pos- itive erythroid cells from the ALL patient BM samples were isolated by flow cytometry23 for sequencing and gene expression verifications. qRT-PCR analysis found that rs10892563 homozygosity in the patients is associat- ed with an approximately 61% reduction in ARHGEF12 expression (P=0.0088) (Figure 2C and D).
Further verifying an involvement of rs10892563, addi-
tional targeted SNP genotyping of 452 ALL patients enrolled in the SCMC-ALL-2005 protocol showed that the genotype frequencies were CC in 7.52%, CT in 41.37%, and TT in 51.11% patients. The average normalized RBC transfusion was 4.533 units/m2 in patients with CC geno- type, 2.353 and 2.335 in patients with CT and TT geno- types, respectively (Figure 2E and Online Supplementary Figure S1). All patients who were homozygous needed RBC transfusion to maintain hemoglobin >65 g/L during the course of chemotherapy, whereas among those who were heterozygous or wild-type, the frequencies were 61.497% and 70.996%, respectively. Patients who were homozygous or heterozygous had a significantly higher probability of requiring MRT than patients carrying wild- type alleles (Figure 2F). Collectively, these results suggest that the ARHGEF12 polymorphism rs10892563 is involved in the susceptibility to chemotherapy-induced anemia
ARHGEF12 reduction blocks erythroid differentiation of K562 cells
The findings that a polymorphism of ARHGEF12 is associated with chemotherapy-induced anemia and that this gene is heavily transcribed in the human erythroid lin- eage (Online Supplementary Figure S2A) based on the analy- ses of several public databases3 suggest that ARHGEF12 is involved in erythropoiesis. As an initial test, we per- formed ARHGEF12 knockdown in the human ery- throleukemia cell line K562 using lentiviral shRNA con- structs (Online Supplementary Figure S2B). GPA expression and the benzidine cytochemical test showed that ery-
Table 2. Gene function of top 10 genes highly expressed in hematopoietic cells along erythroid differentiation from hematopoietic stem cells.
928
haematologica | 2020; 105(4)