AbstractHematopoiesis is a finely regulated process in vertebrates under both homeostatic and stress conditions. By whole exome sequencing, we studied the genomics of acute lymphoblastic leukemia (ALL) patients who needed multiple red blood cell (RBC) transfusions after intensive chemotherapy treatment. ARHGEF12, encoding a RhoA guanine nucleotide exchange factor, was found to be 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 RBC 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 polymerase chain reaction studies confirmed that the homozygotes status is associated with an approximately 61% reduction in ARHGEF12 expression (P=0.0088). Consequently, erythropoiesis was affected at the pro-erythroblast phases. The 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 knockdown. 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 ALL patients after chemotherapy.
Chemotherapy for hematologic malignancies such as acute lymphoblastic leukemia (ALL) often causes anemia. To alleviate chemotherapy-induced anemia, red blood cell (RBC) transfusion has become standard care. The need for RBC transfusion varies significantly among patients who have undergone similar treatment protocols at similar intensities. Sensitivity of erythrocytes to the cytotoxicity of chemotherapy and the recovery rate of erythropoiesis are contributing factors related to the severity and duration of the anemia. Genetic diversity in genes regulating these response processes can be a cause for the variations between patients. Uncovering the genetic basis for the variable response is important for understanding the molecular mechanisms underlying erythropoiesis and its relationship to chemotherapy-induced anemia.
In this study, we performed genome-wide association study (GWAS) analyses of samples from individuals who had undergone multiple RBC transfusions (MRT) and from those who received no RBC transfusion (NRT) when a remission was achieved. By counting the cell line, primary bone marrow (BM) cells, and considering the results of animal model and human genetic studies, we suggest a novel molecular pathway involved in erythroid regeneration in ALL patients after chemotherapy.
From January 1 2001 to December 31 2014, a total of 452 patients diagnosed with childhood ALL were recruited in this study. The patients included were enrolled on Shanghai Children’s Medical Center -Acute Lymphoblastic Leukemia-2005 (SCMC-ALL-2005) protocol. Standard induction and consolidation chemotherapy were used. Blood transfusion records were collected from the transfusion department of SCMC and clinical data were reviewed to exclude the events needed for additional transfusions of RBC, such as transplantation, gastrointestinal bleeding, surgery, etc. Patients who abandoned treatment or who died were not included in this study. Only total RBC transfusion units after achieving complete remission was included in the count and this number was normalized by body surface area (Unit/m) to exclude the influence of patient’s age, mass, and the disease itself on blood transfusion units. Patients who received more than eight RBC units/m were defined as MRT. This study has been approved by Shanghai Children’s Medical Center Ethics Committee (n. SCMCIRB-K2018052).
Dual Luciferase reporter assays
A total of 933 bp DNA fragments surrounding rs10892563 CC and TT genotype were cloned into the firefly Luciferase vector pGl4.27 (Promega); 293T cells were transfected with 5 g PRL-TK vector and 300 ng PCDNA3-Flag expression GATA1 via lipofectemin. Luciferase activity was measured in a Varioskan Flash spectral scanning multimode reader (Thermo) using the Dual-Luciferase Reporter Assay system kit (Promega).
Targeted single nucleotide polymorphism genotyping by polymerase chain reaction
The candidate SNP rs10892563 on ARHGEF12 at position 119,729,754 bp was analyzed by polymerase chain reaction (PCR) on 381 ALL patients enrolled in the SCMC-ALL-2005 protocols for which genomic DNA samples were still available. The DNA segment containing the candidate mutation was amplified by PCR using the following primers:
- 5′-00ATAGGGATACCTGGCCCCTA-3′ and 5′-ndATAGGGATACCTGGCCCCTA-3′
These PCR products were subsequently Sanger sequenced.
Whole-mount in situ hybridization
The antisense probes of arhgef12a and arhgef12b were obtained by PCR with the primers
- (arhgef12a forward primer, 5′-GCGGAATTCCCACCTCAAGGAGATGGAAA-3′;
- reverse primer, 5′-GCGGGTACCCCAAAAGCATGCAAGAAACA-3′;
- arhgef12b forward primer, 5′-GCCGAATTCTCCAGCATGAGTGGTTGGTA-3′;
reverse primer, 5′-ATTGGTACCCTCAACAGAAAGCCGA-GACC3′), and added with EcoR1/Kpn1 restriction enzyme sites for cloning into pCS2+ vector. Antisense digoxigenin (DIG)– labeled RNA probes were generated by in vitro transcription and whole-mount in situ hybridization (WISH) was performed as described previously.1 The results were imaged using a stereomicroscope Nikon SMZ1500 with a 1 x HR Plan Apo objective and ACT-1 vision software.
One-cell-stage embryos were injected with 2 nL of morpholino (MO) or mismatch morpholino (MIS) mixes (arhgef12a and arhgef12b) purchased from Gene-Tools. The MO sequences and concentrations are listed in Table 1.
The arhgef12a gRNA (5′-GGACGTGGGTCTCGAGTCAC-3′) and arhgef12b gRNA (5'-GGAATCTGAGGCAGGCCCGG-3') were synthesized. The zebrafish optimized Cas9 mRNA was syn-thesized in vitro from the pCS2-nCas9n plasmid (addgene, #47929) as described.2 The Cas9 mRNA was synthesized in vitro by SP6 mMessage mMachine Transcription Kit (Ambion). arhgef12a gRNA (50pg), arhgef12b gRNA (50pg), and Cas9 mRNA (150pg) were co-injected into one-cell stage embryos.
Results are expressed as mean±standard deviation considering the number of experiments. Statistical comparisons between groups were performed by two-tailed t-test or one-sided t-test using Graphpad Prism version 6.0.
Whole exome sequencing, GWAS, cell sorting, quantitative real-time (qRT)-PCR, plasmid construction, in vitro RNA synthesis, micro-injection and anisomycin treatment were performed as described in the Online Supplementary Appendix.
An ARHGEF12 polymorphism in acute lymphoblastic leukemia patients is associated with susceptibility to chemotherapy-induced anemia
We performed whole exome sequencing in 31 individuals who had undergone at least eight RBC transfusions (MRT) and 31 patients with no RBC transfusion (NRT), all from the SCMC-ALL-2005 cohort (Figure 1A). Considering the variations in patient age and body weight, the RBC transfusion amount was normalized by patient body surface area taking into account only post-remission transfusions in order to minimize the effect of ALL itself. By a GWAS analysis, 1,708 SNPs of 281 genes passed the criterion of the primary cut: a call rate of >95% and P<0.01. Of interest, most of the SNPs were located in introns adjacent to exons, suggesting that these polymorphisms are relevant to chemotherapy-induced anemia by regulating gene expression. These genes could be highly expressed in hematopoietic cells and involved in erythroid differentiation from hematopoietic stem/progenitor cells. To address this possibility, we sorted the primary gene list with expression patterns in primitive CD34 cells before erythroid differentiation and in erythrocytes based on the Differentiation Map Portal (DMAP) database.3 A total of 35 genes were enriched by this analysis. Among them, 12 genes were highly expressed in hematopoietic stem/progenitor cells (HSPC) and 23 genes were expressed in erythrocytes with over two times the average expressions. At the top of this enriched list were GUCY1A3,4 NUCB2, TFDP2,5 CHPT1,6 PLCB1,7 LPIN2,8 TNS1,109 BSG,1211 COL5A1,13 ANXA7,14 EPB42,1615 RAP1GAP,17 ARHGEF12,1810 ABCC419 and FARP120 (Tables 2 and 3 and Figure 1B). Interestingly, most of these genes are well-known in association with erythropoiesis or cytotoxicity 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 possibly 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 binding 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-positive 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 associated with an approximately 61% reduction in ARHGEF12 expression (P=0.0088) (Figure 2C and D).
Further verifying an involvement of rs10892563, additional 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/m in patients with CC genotype, 2.353 and 2.335 in patients with CT and TT genotypes, 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 lineage (Online Supplementary Figure S2A) based on the analyses of several public databases3 suggest that ARHGEF12 is involved in erythropoiesis. As an initial test, we performed ARHGEF12 knockdown in the human erythroleukemia cell line K562 using lentiviral shRNA constructs (Online Supplementary Figure S2B). GPA expression and the benzidine cytochemical test showed that erythroid differentiation of the cells, under hemin induction, decreased significantly compared to the non-targeted (NT) cells (Online Supplementary Figure S2C).
ARHGEEF12 or its orthologs is involved in erythroid differentiation in murine progenitor cells and in a zebrafish model
To rule out potential effects by the neoplastic background of K562 cells, we knocked down Arhgef12 expression in mouse hematopoietic cell line 32D cells and in primary mouse BM cells by lentiviral shRNA transduction. Erythropoietin-induced erythroid differentiation was significantly blocked by the interference of Arhgef12 expression as by observed erythroid immunophenotyping by flow cytometry and in the burst forming units-erythroid and colony forming unit-erythroid colony forming assays (Online Supplementary Figure S3).
Zebrafish genome harbors two orthologs of ARHGEF12: arhgef12a on chromosome 15 and arhgef12b on chromosome 5. By comparing their sequences (arhgef12a, ENS-DARG00000030532; arhgef12b, ENSDARG00000067634) with human ARHGEF12, we found that the similarities were 54% and 55%, respectively. Synteny analysis also showed the relatively conserved positions for both arhgef12a and arhgef12b (Online Supplementary Figure S4A). Arhgef12a is selectively enriched in early erythroid progenitors (Online Supplementary Figure S4B) whereas arhgef12b is expressed in early erythroid progenitors (Online Supplementary Figure S4C).
To study the role of arhgef12 in erythropoiesis, we performed microinjections of arhgef12a and arhgef12b morpholino both in combination (arhgef12 MO) and separately (arhgef12a MO and arhgef12b MO). Firstly, we performed WISH at 22 hours post fertilization (hpf) to analyze the primitive wave24 of hematopoiesis. Expressions of the erythroid progenitor marker gata1, the mature erythrocyte marker αe1-globin, the hematopoietic lineage marker scl, and the myeloid markers pu.1 and lysozyme C remained unchanged in arhgef12 MO-injected embryos (Online Supplementary Figure S5). At 36 hpf, the definitive hematopoiesis stage of zebrafish, the expression of αe1-globin was dramatically decreased in arhgef12a- and arhgef12b-deficient embryos, whereas the hematopoietic stem cells (HSC) markers runx1 and c-myb and the vascular morphology and marker flk1 were unchanged (Figure 3A). At four days post-fertilization (dpf), the markers representing mature RBC including αe1-globin, βe1-globin, βe2-globin, band3, and alas2 were severely reduced (Figure 3B). Of interest, gata1 was associated with an obvious increase in caudal hematopoietic tissue (CHT) (Figure 3B), indicating that the erythroid defect may be caused by an impaired differentiation. Of interest, gata1 was associated with an obvious CHT (Figure 3B), indicating that the erythroid defect may be caused by an impaired differentiation. Consistent with this possibility, o-Dianisidine stained hemoglobin showed that erythrocytes from arhgef12 MO-injected embryos were more immature than those from the control group at 36 hpf and 4 dpf (Figure 3C). Subsequent examinations of arhgef12a and arhgef12b double knockout mutants by using the CRISPR/Cas9 method followed by o-Dianisidine staining found that mature erythrocytes were significantly decreased at 4 dpf in the mutant CHT and heart (Figure 3D and E). These results indicate that arhgef12a and arhgef12b are required for erythroid differentiation and maturation in zebrafish.
ARHGEF12 regulates erythroid differentiation through a RhoA-p38 pathway
RhoA is a well-defined substrate of ARHGEF12, which activates the exchange of RhoA bound GDP in the inactivated form for GTP to yield the active RhoA-GTP.26 We hypothesized that RhoA is the key target of ARHGEF12 to mediate its function in erythrocyte maturation. Because zebrafish harbors 5 rhoa genes27 and they all have an amino acid sequence which is quite similar (identity >90%) to human RhoA, we inferred that human RhoA mutant mRNA dominant-negative (DN) mutant RhoA T19N28 and constitutively active mutant RhoA Q6329 would also function in zebrafish. In fact, injection of dominant-negative RhoA mRNA led to anemia, which mimicked the arhgef12 deficiency phenotype (Figure 4A), but the anemia seemed to be less severe than with MO injection, possibly due to mRNA instability. On the other hand, based on o-Dianisidine staining and WISH analysis of αe1-globin, a co-injection of constitutively active mutant RhoA Q63L mRNA was able to restore the erythropoiesis defect caused by arhgef12 MO (Figure 4A). These results indicate that Arhgef12 activates RhoA to control erythroid differentiation.
To further understand the molecular events downstream of RhoA in erythropoiesis, the K562 cell line in which ARHGEF12 is important for its erythroid differentiation was examined. An antibody microarray screen found that phosphorylation of molecules in the p38 MAPK pathway was significantly decreased in the ARHGEF12 knockdown K562 cells (Online Supplementary Figure S6A). Western blotting confirmed this effect on p38 phosphorylation (Online Supplementary Figure S6B), suggesting that the p38 MAPK signaling pathway may contribute to the ARHGEF12-regulated erythropoiesis. Further confirmation using the p38 inhibitor SB202190 in zebrafish found that p38 inhibition resulted in a similar block of erythropoiesis as in the arhgef12 morphants at 4 dpf (Figure 4B). Application of anisomycin, a p38 MAPK activator, was able to restore the erythrocyte maturation in the arhgef12 morphants (Figure 4C). Thus, an ARHGEF12-RhoA-p38 pathway is likely to be involved in erythroid differentiation.
STAT1 expression can rescue the erythroid phenotype caused by arhgef12 knockdown in zebrafish
Human STAT1 produces two splicing variants that differ at their carboxy terminus. Zebrafish has two orthologous genes related to human STAT1: stat1a and stat1b. It has been shown that p38 MAPK-STAT1 pathways can regulate neutrophil development. Meanwhile, our antibody microarray screen showed that the phosphorylation of STAT1 at serine (S) 727 was decreased approximately 2-fold in ARHGEF12 knockdown K562 cells (Online Supplementary Figure S6A). We thus further examined whether STAT1 may be downstream of p38 MAPK in regulating erythropoiesis. We co-injected the HA-stat1a construct together with arhgef12 MO in zebrafish, and observed that the phenotype of erythropenia was restored (Figure 5A a-c, a′-c′) and αe1-globin expression recovered (Figure 5A e-g, e′-g′), compared with control embryos. A cytology assay by Wright-Giemsa staining showed that the co-injection of stat1a mRNA with arhgef12 MO appeared to promote the immature erythrocyte differentiation (Figure 5B). In addition, stat1 MO injection increased gata1 expression (Figure 5C a, a′, b, b′) but reduced ae1-globin expression (Figure 5C c, c′, d, d′), similar to that by arhgef12 MO injection. It is thus likely that STAT1 is involved in the ARHGEF12-p38 MAPK signaling function in erythroid differentiation.
The ARHGEF12-p38 pathway is associated with erythroid regeneration in acute lymphoblastic leukemia patients after chemotherapy
To examine whether ARHGEF12 polymorphism-associated anemia after chemotherapy in ALL patients may engage the p38 pathway, we measured p38 phosphorylation in erythroid cells in seven remission-related BM from ALL patients during maintenance therapy by phospho-flow.30 All seven patient samples with the rs10892563 CC genotype showed consistently reduced phosphorylated p38 in pro-erythroblasts (Figure 6C and D). Similar to the case of arhgef12 MO, where the p38 activity is inhibited in the zebrafish, these rs10892563 CC genotype patients showed an erythroid differentiation block at the erythroblast stage (Figure 6A and B), suggesting a strong association with the ARHGEF12-p38 pathway.
For leukemia patients, hematologic toxicity is the most common side effect of chemotherapy as the hematopoietic cells are among the tissues most vulnerable to therapy-related damage, in part due to their active cell cycle status. Anemia is one of the most frequently recorded manifestations of the hematopoietic toxic effects during the course of chemotherapy. Chemotherapy-induced anemia can be caused by cytotoxic inhibition of normal hematopoiesis similar to chemotherapy-induced neutropenia and thrombocytopenia. Chemotherapy agent-related autoimmune hemolysis3231 and chemotherapy-induced eryptosis can also cause anemia.33 Our current GWAS studies have found that chemotherapy-induced anemia is associated with SNP in CHPT1,6 BSG,1211 ANAX7,14 EPB42,1615 and ABCC417 that may be related to increased erythrocyte loss. There is evidence to suggest that: (i) ANAX7 is related to erythrocyte death;34 (ii) EPB421615 is related to hereditary spherocytosis in which eryptosis is an important cause of anemia; (iii) CHPT16 and BSG1211 may be related to the vulnerability of RBC to chemotherapy; and (iv) ABCC4,17 a membrane transporter, is related to accumulation of cytotoxic agents in cells and chemo-sensitivity of hematopoietic cells. Other direct or indirect evidence based on the known functions of the gene products suggests that the remaining genes in the top ten list of GWAS in our study, TFDP2, LPIN2, TNS1, RAP1GAP, ARHGEF12, could also be related to impaired hematopoiesis in response to chemotherapy.
Erythropoiesis is a tightly regulated process in which the hemoglobin level is maintained in a narrow window between 135g/L and 155g/L under normal conditions or in response to stress such as chemotherapy. A complex lineage-specific transcription factor network underlies the homeostatic hematopoiesis and erythropoiesis mechanisms. In such a transcription network, the GATA transcription 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 expression patterns in vertebrates, and GATA-1, GATA-2 and GATA-3 are classified into the hematopoietic GATA subfamily 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 erythroid 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 cellular 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 erythroblasts 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 activator 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 number of studies have reported that p38 MAPK is involved in erythroid differentiation,4139 yet the role of p38 MAPK in stress erythropoiesis is still poorly understood. P38a regulates erythroblast enucleation in a cell-autonomous manner in vivo during fetal and anemic stress erythropoiesis.42 Remarkably, loss of p38a leads to downregulation of p21Cip1, and decreased activation of the p21Cip1 inactivates Rb, both of which are critical regulators of erythroblast enucleation. Hu et al. suggested P38a could act as a molecular brake to limit over-active erythropoiesis in response to stress-relief of this molecular brake by inhibiting P38-enhanced stress erythropoiesis and accelerated recovery from anemia.43 Our observed association of a down-regulated erythroid p38 phosphorylation in patients with the ARHGEF12 polymorphism who need multiple RBC transfusions to overcome chemotherapy-induced anemia also supports the involvement of such a pathway. Pharmacological activation of wild-type p53 is a logical therapeutic strategy for leukemia where the p53 pathway could be down-regulated by abnormalities in p53-regulatory proteins.44 It has been reported that p38 kinase can positively regulate p53, and activation of p38 not only promotes erythropoiesis, but also potentially maintains a higher level of p53 in cancer cells, which can be a dual benefit for cancer patients who carry wild-type p53 alleles.
Several reported GWAS studies related to hematologic traits failed to find a correlation between ARHGEF12 and erythroid phenotypes4645 in normal populations, which may suggest there are functional redundancies to the ARHGEF12-RhoA-p38 pathway in homeostatic erythropoiesis. Suboptimal level of guanine nucleotide exchange activity may be compensated by down-regulated RhoA negative regulator, GTPase-activating proteins, or by other guanine nucleotide exchange factors, such as ARHGEF3, which was shown to be important for erythropoiesis through RhoA in a zebrafish model.47 Our GWAS results draw a clear association between ARHGEF12 at rs10892563 with erythrocyte regeneration under chemotherapy stress, suggesting that this gene/SNP status may be considered a biomarker to predict severity of chemotherapy-induced anemia among the patients.
In addition to the erythropoiesis differentiation mechanism, genes expressed in HSPC can also be associated with chemotherapy-associated anemia. To this end, we analyzed the primary gene list with expression patterns in the CD34 cell population before erythroid differentiation with the same database as we did for the erythrocyte-specific genes. Among the genes highly expressed in HSPC, four of the top five have known functional connection with erythropoiesis (Figure 1C). While a correlation analysis between RBC transfusion and severity of neutropenia reveals that RBC transfusions had no significant correlation with neutropenia, there was a significance in correlation with thrombocytopenia (Online Supplymentary Figure S7). This suggests that it is possible that effects on HSPC such as megakaryocyte-erythroid progenitors could be a contributing factor to chemotherapy-induced anemia.
Combining our patient SNPs and phenotype observations, biochemical analyses of patient samples, and human and murine cells, together with the zebrafish genetic model characterizations, our studies unveil a novel SNP related to chemotherapy-induced anemia in ARHGEF12 and the associated signaling pathway. These findings will be useful for future consideration of strategies to overcome the chemotherapy-induced anemia in some ALL patients.
The authors would like to thank the staff of Shanghai Institute of Hematology for their assistance with zebrafish husbandry, particularly Yi Chen. We thank Yongjuan Zhang for the SNP linkage disequilibrium analysis and Professor Xiaojian Sun for helpful discussions.
- ↵* YX and LG contributed equally to this work.
- Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/105/4/925
- FundingThis research was supported by grants from the National Natural Science Foundation of China (n. 81270623), The National Key R&D Program of China, Stem Cell and Translation Research (n. 2016YFA0102000) and The fourth round of Three-Year Public Health Action Plan (2015-2017) (GWIV-25). This research was also supported by the National Natural Science Foundation of China (No. 81900114).
- Received October 30, 2018.
- Accepted August 22, 2019.
- Sun XJ, Xu PF, Zhou T. Genome-Wide Survey and Developmental Expression Mapping of Zebrafish SET Domain-Containing Genes. PLoS One. 2008; 3(1):e1499. PubMedhttps://doi.org/10.1371/journal.pone.0001499Google Scholar
- Jao LE, Wente SR, Chen W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci U S A. 2013; 110(34):13904-13909. PubMedhttps://doi.org/10.1073/pnas.1308335110Google Scholar
- Novershtern N, Subramanian A, Lawton LN. Densely interconnected transcriptional circuits control cell states in human hematopoiesis. Cell. 2011; 144(2):296-309. PubMedhttps://doi.org/10.1016/j.cell.2011.01.004Google Scholar
- Ikuta T, Sellak H, Odo N, Adekile AD, Gaensler KML. Nitric Oxide-cGMP Signaling Stimulates Erythropoiesis through Multiple Lineage- Specific Transcription Factors : Clinical Implications and a Novel Target for Erythropoiesis. Plos One. 2016; 11(1):e0144561. Google Scholar
- Chen C, Lodish HF. Global analysis of induced transcription factors and cofactors identifies Tfdp2 as an essential coregulator during terminal erythropoiesis. Exp Hematol. 2014; 42(6):464-476. PubMedhttps://doi.org/10.1016/j.exphem.2014.03.001Google Scholar
- Henneberry AL, Wistow G, Mcmaster CR. Cloning, genomic organization, and characterization of a human cholinephosphotransferase. J Biol Chem. 2000; 8(4):29808-29815. Google Scholar
- Cocco L, Manzoli L, Faenza I. Advances in Biological Regulation Modulation of nuclear PI-PLCbeta1 during cell differentiation. Adv Biol Regul. 2016; 60:1-5. Google Scholar
- Ferguson PJ, Chen S, Tayeh MK. Homozygous mutations in LPIN2 are responsible for the syndrome of chronic recurrent multifocal osteomyelitis and congenital dyserythropoietic anaemia (Majeed syndrome). J Med Genet. 2005; 42(7):551-557. PubMedhttps://doi.org/10.1136/jmg.2005.030759Google Scholar
- Shih YP, Sun P, Wang A, Lo SH. Tensin1 positively regulates RhoA activity through its interaction with DLC1. Biochim Biophys Acta - Mol Cell Res. 2015; 1853(12):3258-3265. Google Scholar
- Konstantinidis DG, Giger KM, Risinger M. Cytokinesis failure in RhoA-deficient mouse erythroblasts involves actomyosin and midbody dysregulation and triggers p53 activation. Blood. 2015; 126(12):1473-1482. PubMedhttps://doi.org/10.1182/blood-2014-12-616169Google Scholar
- Coste I, Gauchat JF, Wilson A. Unavailability of CD147 leads to selective erythrocyte trapping in the spleen. Blood. 2001; 97(12):3984-3988. PubMedhttps://doi.org/10.1182/blood.V97.12.3984Google Scholar
- Ma C, Wang J, Fan L, Guo Y. Inhibition of CD147 expression promotes chemosensitivity in HNSCC cells by deactivating MAPK/ERK signaling pathway. Exp Mol Pathol. 2017; 102(1):59-64. Google Scholar
- Dorn I, Meyer G, Lindner U, Driller B, Schlenke P. The influence of extracellular matrix proteins and mesenchymal stem cells on erythropoietic cell maturation. Vox Sang. 2011; 101(1):65-76. PubMedGoogle Scholar
- Abed M, Balasaheb S, Towhid ST, Daniel C, Amann K, Lang F. Adhesion of Annexin 7 Deficient Erythrocytes to Endothelial Cells. PLoS One. 2013; 8(2):1-10. PubMedhttps://doi.org/10.1371/journal.pone.0054583Google Scholar
- Hayette S, Dhermy D, dos Santos ME. A deletional frameshift mutation in protein 4.2 gene (allele 4.2 Lisboa) associated with hereditary hemolytic anemia. Blood. 1995; 85(1):250-256. PubMedGoogle Scholar
- Yawata Y, Kanzaki A, Yawata A, Doerfler W, Ozcan R, Eber SW. Characteristic features of the genotype and phenotype of hereditary spherocytosis in the Japanese population. Int J Hematol. 2000; 71(2):118-135. PubMedGoogle Scholar
- Satyanarayana A, Gudmundsson KO, Chen X. RapGEF2 is essential for embryonic hematopoiesis but dispensable for adult hematopoiesis. Blood. 2010; 116(16):2921-2931. PubMedhttps://doi.org/10.1182/blood-2010-01-262964Google Scholar
- Kourlas PJ, Strout MP, Becknell B. Identification of a gene at 11q23 encoding a guanine nucleotide exchange factor: evidence for its fusion with MLL in acute myeloid leukemia. Proc Natl Acad Sci U S A. 2000; 97(5):2145-2150. PubMedhttps://doi.org/10.1073/pnas.040569197Google Scholar
- Drenberg C, Hu S, Li L. ABCC4 Is a Determinant of Cytarabine-Induced Cytotoxicity and Myelosuppression. Clin Transl Sci. 2016; 9(1):51-59. Google Scholar
- Yang L, Wang L, Kalfa TA. Cdc42 critically regulates the balance between myelopoiesis and erythropoiesis. Blood. 2007; 110(12):3853-3861. PubMedhttps://doi.org/10.1182/blood-2007-03-079582Google Scholar
- Ghiaur G, Lee A, Bailey J, JA C, Zheng Y, DA W. Inhibition of RhoA GTPase activity enhances hematopoietic stem and progenitor cell proliferation and engraftment. Blood. 2006; 108(6):2087-2094. PubMedhttps://doi.org/10.1182/blood-2006-02-001560Google Scholar
- Sankaran VG, Ghazvinian R, Do R. Exome sequencing identifies GATA1 mutations resulting in Diamond-Blackfan anemia. J Clin Invest. 2012; 122(7):2439-2443. PubMedhttps://doi.org/10.1172/JCI63597Google Scholar
- van Lochem EG, van der Velden VHJ, Wind HK, te Marvelde JG, Westerdaal NAC, van Dongen JJM. Immunophenotypic differentiation patterns of normal hematopoiesis in human bone marrow: Reference patterns for age-related changes and disease-induced shifts. Cytometry. 2004; 60B(1):1-13. Google Scholar
- Orkin SH, Zon LI. Hematopoiesis: an evolving paradigm for stem cell biology. Cell. 2008; 132(4):631-644. PubMedhttps://doi.org/10.1016/j.cell.2008.01.025Google Scholar
- Paffett-Lugassy NN, Zon LI. Analysis of hematopoietic development in the zebrafish. Methods Mol Med. 2005; 105:171-198. PubMedGoogle Scholar
- Reuther GW, Lambert QT, Booden MA. Leukemia-associated Rho Guanine Nucleotide Exchange Factor, a Dbl Family Protein Found Mutated in Leukemia, Causes Transformation by Activation of RhoA. J Biol Chem. 2001; 276(29):27145-27151. PubMedhttps://doi.org/10.1074/jbc.M103565200Google Scholar
- Salasvidal E, Meijer AH, Cheng X, Spaink HP. Genomic annotation and expression analysis of the zebrafish Rho small GTPase family during development and bacterial infection. Genomics. 2005; 86(1):25-37. PubMedhttps://doi.org/10.1016/j.ygeno.2005.03.010Google Scholar
- Qiu RG, Chen J, Mccormick F, Symons M. A Role for Rho in Ras Transformation. Proc Natl Acad Sci U S A. 1995; 92(25):11781-11785. PubMedhttps://doi.org/10.1073/pnas.92.25.11781Google Scholar
- Dvorsky R, Ahmadian MR. Always look on the bright site of Rho: structural implications for a conserved intermolecular interface. EMBO Rep. 2004; 5(12):1130-1136. PubMedhttps://doi.org/10.1038/sj.embor.7400293Google Scholar
- Perez OD, Mitchell D, Campos R, Gao GJ, Li L, Nolan GP. Multiparameter analysis of intracellular phosphoepitopes in immunophenotyped cell populations by flow cytometry. Curr Protoc Cytom. 2005. Google Scholar
- Ahlgren J, Patel N, Simmens S. O-MAX chemotherapy: high activity in metastatic esophagogastric adenocarcinoma and possible relation to subclinical hemolysis. Oncology. 2014; 87(6):371-380. Google Scholar
- Betensky M, Witmer C, Fisher MJ, Nance S, Weiss MJ, Sesok-Pizzini DA. Immune hemolytic anemia with drug-induced antibodies to carboplatin and vincristine in a pediatric patient with an optic pathway glioma. Transfusion. 2015; 54(11):2901-2905. Google Scholar
- Lang E, Bissinger R, Qadri SM, Lang F. Suicidal death of erythrocytes in cancer and its chemotherapy: A potential target in the treatment of tumor-associated anemia. Int J Cancer. 2017; 141(8):1522-1528. Google Scholar
- Lang E, Lang PA, Shumilina E. Enhanced eryptosis of erythrocytes from gene-targeted mice lacking annexin A7. Pflugers Arch. 2010; 460(3):667-676. PubMedhttps://doi.org/10.1007/s00424-010-0829-xGoogle Scholar
- Abiko H, Fujiwara S, Ohashi K. Rho guanine nucleotide exchange factors involved in cyclic-stretch-induced reorientation of vascular endothelial cells. J Cell Sci. 2015; 128(9):1683-1695. PubMedhttps://doi.org/10.1242/jcs.157503Google Scholar
- Thompson WR, Yen SS, Uzer G. LARG GEF and ARHGAP18 orchestrate RhoA activity to control mesenchymal stem cell lineage. Bone. 2018; 107:172-180. Google Scholar
- Shi GX, Yang WS, Jin L, Matter ML, Ramos JW. RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases. Proc Natl Acad Sci U S A. 2018; 115(2):E190-E199. PubMedhttps://doi.org/10.1073/pnas.1708584115Google Scholar
- Williams CM, Harper MT, Goggs R, Walsh TG, Offermanns S, Poole AW. Leukemia-associated Rho guanine-nucleotide exchange factor is not critical for RhoA regulation, yet is important for platelet activation and thrombosis in mice. J Thromb Haemost. 2015; 13(11):2102-2107. Google Scholar
- Hua WK, Chang YI, Yao CL, Hwang SM, Chang CY, Lin WJ. Protein Arginine Methyltransferase 1 Interacts with and Activates p38α to Facilitate Erythroid Differentiation. PLoS One. 2013; 8(3):e56715. PubMedhttps://doi.org/10.1371/journal.pone.0056715Google Scholar
- Shahab U, Jeong AK, Jodie U, Dolores M, Amittha W. Differentiation stage-specific activation of p38 mitogen-activated protein kinase isoforms in primary human erythroid cells. Proc Natl Acad Sci U S A. 2004; 101(1):147-152. PubMedhttps://doi.org/10.1073/pnas.0307075101Google Scholar
- Pace BS, Qian XH, Sangerman J. p38 MAP kinase activation mediates γ-globin gene induction in erythroid progenitors. Exp Hematol. 2003; 31(11):1089-1096. PubMedhttps://doi.org/10.1016/S0301-472X(03)00235-2Google Scholar
- Schultze SM, Mairhofer A, Li D. p38α controls erythroblast enucleation and Rb sig-naling in stress erythropoiesis. Cell Res. 2012; 22(3):539-550. PubMedhttps://doi.org/10.1038/cr.2011.159Google Scholar
- Hanenberg H, Filippi M-D, Ivan M. P38α/JNK signaling restrains erythropoiesis by suppressing Ezh2-mediated epigenetic silencing of Bim. Nat Commun. 2018; 9(1):3518. Google Scholar
- Bulavin DV, Saito S, Hollander MC. Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation. Embo J. 1999; 18(23):6845-6854. PubMedhttps://doi.org/10.1093/emboj/18.23.6845Google Scholar
- Kuhnel B, Aspelund T, Yang Q. Multiple loci influence erythrocyte phenotypes in the CHARGE Consortium. Nat Genet. 2009; 41(11):1191-1198. PubMedhttps://doi.org/10.1038/ng.466Google Scholar
- Kamatani Y, Matsuda K, Okada Y. Genome-wide association study of hematological and biochemical traits in a Japanese population. Nat Publ Gr. 2010; 42(3):210-215. Google Scholar
- Serbanovic-Canic J, Cvejic A, Soranzo N, Stemple DL, Ouwehand WH, Freson K. Silencing of RhoA nucleotide exchange factor , ARHGEF3, reveals its unexpected role in iron uptake. Blood. 2011; 118(18):4967-4977. PubMedhttps://doi.org/10.1182/blood-2011-02-337295Google Scholar