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Pro-leukemic effects of P2X7
been identified in MLL-rearranged leukemia, the most common translocation partners are AF4, AF9, ENL, AF10 and ELL.17 Among these, MLL-AF9 accounts for approxi- mately 30% of cases of MLL-rearranged acute myeloid leukemia (AML).18 Given the broad range of partners, dif- ferent mechanisms by which MLL fusion proteins cause leukemia have been suggested, with abnormalities in the epigenetic regulatory network being considered the most important events.19 Nevertheless, constitutive activation of homeobox (HOX) genes by fusion proteins is suggested as the central mechanism causing aberrant self-renewal in many MLL-rearranged cases of leukemia.20 HOX proteins form cooperative DNA binding complexes with other atypical homeodomain proteins, such as PBX and MEIS, which belong to the three-amino-acid loop extension (TALE) family.21 HOXA7, HOXA9 and cofactor MEIS1 are suggested to be key mediators in the transformation caused by MLL rearrangements.22 Notably, increased expression of HOX proteins and their cofactors has also been detected in other leukemia subtypes.23 Overexpression of the Hox gene with Meis induces leuke- mogenesis.24 Furthermore, coexpression of PBX3 and MEIS1 is sufficient to transform normal hematopoietic stem/progenitor cells and induce AML in mice.25 Hence, dysfunction of these factors plays important roles in differ- ent subtypes of leukemia.
Although P2X7 was suggested to have adverse effects in leukemia, the molecular mechanism has not been eluci- dated. In this study, we analyzed the correlation between P2X7 expression and clinical outcome and explored the mechanism of action of P2X7 in leukemia progression using mouse AML, nude mouse xenograft and patient- derived xenograft models. High levels of expression of P2X7 were correlated with worse survival in AML. Furthermore, P2X7 accelerated AML progression by pro- moting proliferation and increasing leukemia stem cells (LSC). Moreover, Pbx3 mediated the effects of P2X7.
Methods
Patients’ samples
Bone marrow samples were collected from 20 newly diagnosed AML patients (Online Supplementary Table S1) and 12 normal donors at the Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College with informed consent (ethical review approval number KT2018045- EC-2).
Mouse acute myeloid leukemia models
All animal experiments were approved by the Animal Care and Use Committee at the institution.
To establish a mouse AML model overexpressing P2X7, GFP+ cells were sorted from mice with MLL-AF9-induced AML15 and infected with blank or pMSCV-P2X7 retrovirus. GFP+BFP+ cells were sorted and transplanted into C57BL/6J mice by intravenous tail injection. These cells were named MA9 and MA9-P2X7 cells, and the corresponding AML mice were named MA9 and MA9- P2X7 mice, respectively.
To establish mouse Pbx3 knockdown (KD) AML models, MA9- P2X7 cells were infected with pLV-SC or pLV-mPbx3sh1 lentiviruses. GFP+BFP+RFP+ cells were sorted and transplanted into C57BL/6J mice. These cells were named MA9-P2X7-SC and MA9- P2X7-mPbx3sh1, and the corresponding AML mice were named MA9-P2X7-SC and MA9-P2X7-mPbx3sh1 mice, respectively.
Characterization of MLL-AF9-induced acute myeloid leukemia mouse models overexpressing P2X7
In most experiments, 3×105 MA9 or MA9-P2X7 cells were transplanted. In limiting-dilution transplantation experiments, 1×102, 1×103 and 1×104 MA9 or MA9-P2X7 cells were transplanted (n≥7). In Pbx3 KD experiments, 1×103 GFP+BFP+RFP+ leukemia cells were transplanted (n=12). Leukemia cells in the peripheral blood, bone marrow and spleen were monitored, and the survival of mice was recorded. The cell cycle stage and apoptosis rate of freshly sorted leukemia cells were studied. In homing experi- ments, 9×106 leukemia cells were transplanted, and mice were sac- rificed 16 h later (n=4). Total bone marrow and spleen cells were counted, and the proportion of leukemia cells was determined by flow cytometry.
Microarray studies
MA9, MA9 c-Kit+, MA9 c-Kit- and MA9-P2X7 cells were sorted by FACS. The microarrays were carried out by Oebiotech using Agilent Mouse Gene Expression (Format: 8*60K, Design ID: 028005). Feature Extraction software (version 10.7.1.1, Agilent Technologies) was used to obtain raw data, and Genespring soft- ware (version 12.5; Agilent Technologies) was used for the basic analysis. The raw data were normalized with a quantile algo- rithm. Fold-change ≥2.0 was used as the cutoff for screening for differentially expressed genes (DEG). The K-Mean cluster was analyzed by MeV 4.8.1, and the Venn diagram was analyzed online (http://bioinfogp.cnb.csic.es/tools/venny/). Gene set enrichment analysis was used to determine the biological functions or path- ways primarily affected by the DEG. The datasets are available in the NCBI Gene Expression Omnibus (GEO) database (GSE92969).
Statistical analysis
The results are represented as means ± standard error of mean. GraphPad Prism 6.0 (San Diego, CA, USA) and SPSS 16.0 (SPSS, Chicago, IL, USA) were used. An unpaired Student t test was used for comparisons between two groups, whereas one-way analysis of variance was used for comparisons among multiple groups. Survival curves were produced using Kaplan-Meier estimates. The results for the limiting-dilution transplantation assays were com- pared using extreme limiting dilution analysis (ELDA). P<0.05 was considered statistically significant.
Additional experimental procedures are described in the Online Supplementary Appendix.
Results
High levels of P2X7 expression correlate with worse prognosis in patients with acute myeloid leukemia
In this study we used several public free databases and datasets to analyze the expression of P2X7 and the corre- lation between P2X7 and other genes. The detailed char- acteristics of patients (in some cases including the type of treatment received) can be accessed from the websites summarized in Online Supplementary Table S2. To deter- mine the overall expression pattern of P2X7 in hematopoi- etic malignancies, a large-cohort leukemia microarray dataset (GSE13204, n=1,962) was analyzed. P2X7 expres- sion was significantly higher in AML and CLL but lower in acute B-cell lymphoblastic leukemia (B-ALL) and chron- ic myelogenous leukemia than in normal controls (Figure 1A). We detected its expression in 20 newly diagnosed AML patients (Online Supplementary Table S1) showing that its expression was higher in AML than in normal bone marrow (Figure 1B). To further assess its clinical sig-
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