Hematopoietic stem progenitor cells with malignancy‐related gene mutations in patients with acquired aplastic anemia are characterized by the increased expression of CXCR4

Abstract The phenotypic changes in hematopoietic stem progenitor cells (HSPCs) with somatic mutations of malignancy‐related genes in patients with acquired aplastic anemia (AA) are poorly understood. As our initial study showed increased CXCR4 expression on HLA allele‐lacking (HLA[−]) HSPCs that solely support hematopoiesis in comparison to redundant HLA(+) HSPCs in AA patients, we screened the HSPCs of patients with various types of bone marrow (BM) failure to investigate their CXCR4 expression. In comparison to healthy individuals (n = 15, 12.3%–49.9%, median 43.2%), the median CXCR4+ cell percentages in the HSPCs of patients without somatic mutations were low: 29.3% (14.3%–37.3%) in the eight patients without HLA(−) granulocytes, 8.8% (4.1%–9.8%) in the five patients with HLA(−) cells accounting for >90% of granulocytes, and 7.8 (2.1%–8.7%) in the six patients with paroxysmal nocturnal hemoglobinuria. In contrast, the median percentage was much higher (78% [61.4%–88.7%]) in the five AA patients without HLA(−) granulocytes possessing somatic mutations (c‐kit, t[8;21], monosomy 7 [one for each], ASXL1 [n = 2]), findings that were comparable to those (66.5%, 63.1%–88.9%) in the four patients with advanced myelodysplastic syndromes. The increased expression of CXCR4 may therefore reflect intrinsic abnormalities of HSPCs caused by somatic mutations that allow them to evade restriction by BM stromal cells.


INTRODUCTION
Acquired aplastic anemia (AA) is a syndrome characterized by pancytopenia and bone marrow (BM) hypoplasia without apparent dysplasia in the BM cells and an increase in the number of blasts [1]. As the main pathophysiology of AA is a reduction of hematopoietic stem progenitor cells (HSPCs) due to T-cell attacks against HSPCs, HSPCs themselves are thought to be healthy and give rise to polyclonal hematopoiesis [2][3][4][5]. However, recent studies using next-generation sequencing have revealed that approximately one third of newly diagnosed AA patients have clonal hematopoiesis by HSPCs with somatic mutations of myeloid malignancy-related genes [6,7]. Some of these somatic mutations indeed predispose secondary myelodysplastic syndromes (MDS) [1,8,9], whereas others do not directly contribute to the development of secondary MDS [6]. The prognostic significance of such somatic mutations therefore remains unclear. Phenotypic changes in the HSPCs associated with somatic mutations are unknown either.
Hematopoiesis in humans is thought to be supported by a limited number of HSPCs selected from a large number of HSPCs that remain dormant in the BM [10,11]. Different from murine HSPCs that can be marked by transduced genes, it is generally impossible to know which HSPCs do or do not contribute to hematopoiesis due to a lack of useful markers on healthy HSPCs, which are responsible for active hematopoiesis. Rare exceptions include HLA class I allelelacking (HLA[−]) HSPCs, which are detected in approximately 30% of AA patients [4,5,12]. HLA(−) HSPCs are thought to be healthy HSPCs that escape T-cell attack due to the lack of particular HLA-class I alleles that present autoantigens of HSPCs to T cells in AA [13,14]. As HLA(−) HSPCs often produce clonal hematopoiesis with their HLA(−) progenies, studying HLA(−) HSPCs may be useful for clarifying the phenotype unique to healthy HSPCs, which substantially contribute to hematopoiesis and could be different from the phenotype of HSPCs with myeloid malignancy-related gene mutations.
To address this issue, we investigated the expression of the HLA alleles in the HSPCs of AA patients whose granulocytes are completely replaced with HLA(−) cells. In the process of studying the gene expression profiles in HLA(−) HSPCs and HLA(+) HSPCs of an AA patient, we found that the gene expression of a chemokine CXCL12 was diminished in HLA(−) HSPCs, and that the expression of its receptor CXCR4 on HSPCs was also decreased in comparison to HLA(+) HSPCs. As the CXCR4 expression on HSPCs has been shown to increase in patients with MDS, we hypothesized that the CXCR4 expression level may be useful for discriminating clonal hematopoiesis by healthy HSPCs from premalignant clonal hematopoiesis by HSPCs with somatic mutations.
We then screened HSPCs from the peripheral blood (PB) of patients with various types of BM failure with or without somatic mutations of myeloid malignancy-related genes for the expression of CXCR4.

Patients
Patient information is provided in

Flow cytometry analysis and cell sorting
Details of methods are provided in Table S2 and Figure S1.

Generation of iPS cells (iPSCs)
Heparinized PB was drawn from an AA patient (UPN 2) in convalescence with HLA(−) leukocytes of which hematopoietic function depended on cyclosporine for 14 years after anti-thymocyte globulin therapy and induced pluripotent stem cells (iPSCs) were generated from the circulating monocytes at the Center for iPS Cell Research and Application (Kyoto University, Kyoto, Japan) as described previously [14]. Several clones with either HLA(−) or HLA(+) phenotype were obtained and verified as previously reported [14].
Maintenance and expansion of iPSCs were achieved by coculturing them with mitomycin C-treated SNL feeder cells in Dulbecco'smodified Eagle medium/F12 supplemented with 20% knockout serum replacement medium, fibroblast growth factor (10 ng/ml), 2-mM L-glutamine, nonessential amino acids, and 1% penicillin and streptomycin.

Induction of HSPCs from iPSCs
CD34 + HSPCs derived from iPSCs were generated by culture in the conditioned medium (CM) derived from OP9 cells and WEHI cells and were resuspended in phosphate-buffered saline (PBS) contain-ing 1% of bovine serum albumin and stained with various monoclonal antibodies (mAbs) directed against iPSCs and hematopoietic cell markers (Table S2) [14]. In some experiments, CD34 + HSPCs derived from iPSCs were purified by cell sorting using a FACSAria Fusion instrument and were then subjected to colony-forming assays using methylcellulose medium (MethoCult GF H4034; STEMCELL Technologies, Vancouver, BC, Canada). Enriched CD34 + HSPCs derived from iPSCs were resuspended at the density of 50,000 cells per 1 mL. An amount of 200 µl of the cell suspension was mixed with 1-ml methylcellulose medium, and then plated into a 35-mm dish and incubated at 37 • C with 5% CO 2 for 2 weeks. Lineage classification was identified by the morphologic analysis of Giemsa staining with an inverted microscope.

Transplantation of human CD34 + iPSC-HSPCs cells into immunodeficient mice
The immunodeficient mouse strain C57BL/6.Rag2 null Il2rg null NODSirpa (BRGS), which lacks T, B, and NK cells, was used as a humanized model of human hematopoiesis. Sublethally irradiated (250 cGy) mice of 4 weeks of age received a total of 1 × 10 5 CD34 + HSPCs derived from iPSCs by direct injection into the BM of femur bones. At 12 weeks after transplantation, animals were euthanized and spleen was harvested from recipient mice and subjected to flow cytometry analysis as described earlier [14].

Analysis of somatic mutations in PB leukocytes
PB samples from AA and MDS patients were subjected to DNA extraction using a DNA extraction kit (Qiagen, Hilden, Germany).
The DNA samples were subjected to targeted-capture sequencing as previously described [15]. A custom RNA bait was designed for the detection of oncogenic variants in 390 known driver genes implicated in myeloid malignancies (SureSelect; Agilent Technology). This bait included additional 1158 single-nucleotide polymorphisms to calculate genome-wide copy numbers. These probes were deliberately selected so that they cover the human genome uniformly to allow for prospective detection of copy-number change and loss of heterozygosity (LOH) on the next-generation sequencing platform.

Statistical analyses
Data were analyzed using the Spearman's correlation using the EZR software package [16] and the GraphPad Prism software package, ver-

The expression of CXCR4 by HLA(−) cells in CMPs of AA patients was lower than that of HLA(+) cells in CMPs
The CXCR4 expression on MEPs and GMPs, which consisted of almost entirely HLA(−) cells, was also lower than in HIs (data not shown).

The CXCR4 expression on CD34 + HSPCs induced from iPSC clones with different genotypes
To ascertain the diminished CXCR4 expression on HLA(−) HSPCs in AA patients at the clonal level, we examined HSPCs derived from iPSCs with different genotypes, including wild-type (WT), LOH of chromosome 6p (6pLOH), and a loss-of-function mutation in HLA-B genes from one of the three patients (AA2) [14], and from another patient (AA4) who possessed HLA-B5401-lacking leukocytes [19]. Incubation of iPSCs in CM-induced CD34 + cells capable of generating different types of colony-forming cells (Figure 2A) as well as engrafting in immunodeficient mice (C57BL/6.Rag2 null Il2rg null NODSirpa [BRGS]), as previously described ( Figure 2B) [14]. Both 6pLOH(+) CD45 + CD34 + cells and HLA-B (B4002 in AA2 and B5401 in AA4)-lacking CD45 + CD34 + cells expressed much less CXCR4 than WT cells ( Figure 2C). Figure 2D summarizes the percentages of CXCR4 + cells in CD34 + cells induced from all iPSC clones with different genotypes, showing that the CXCR4 expression was consistently lower in HLA-B(−) iPSC-derived CD34 + cells than in WT cells in both patients. In contrast, B lymphocytes identified in the spleen of iPSC-derived HSPC-engrafted mice highly expressed CXCR4, regardless of the HLA expression ( Figure 2E), suggesting that the CXCR4 expression reduction by iPSC-HSPCs occurred by some epigenetic mechanism.  Figure 4A,B). In contrast, B cells of the same PNH patients highly expressed CXCR4, regardless of the GPI expression ( Figure 4C).

F I G U R E 2 A schematic diagram for the induction of CD34 + cells from induced pluripotent stem cells (iPSCs) and intrafemoral injection of the iPSC-hematopoietic stem progenitor cells (HSPCs) into BRGS mice and the CXCR4 expression on CD34
Although we confirmed that the CXCR4 expression on GPI(−) HSPCs of PNH patients was much lower than that on GPI(+) HSPCs of HIs, the failed lipid raft formation cannot explain the comparable expression levels of CXCR4 between GPI(−) B and GPI(+) B cells of the same patients. It is more reasonable to interpret that the decrease in the CXCR4 expression level, which was seen in both HLA(−) and GPI(−) HSPCs, is a common feature of HSPCs that actively support hematopoiesis in humans.
We observed the increased expression of CXCR4 by PB CD34 + cells not only in MDS patients but also in AA patients who exhibited clonal hematopoiesis associated with somatic mutations of myeloid

CONFLICT OF INTEREST
All authors declare no conflicts of interest in association with the present study.

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DATA AVAILABILITY STATEMENT
All data generated or analyzed during this study are included in this article and Supporting Information section. The data described in this article are openly available.