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W. Feng et al.
ing also showed similar results (Figure 4C). However, the rate of apoptosis of freshly isolated leukemia cells from the two groups was not significantly different (Figure 4D). We then assessed the response of the leukemic mice to cytarabine (Ara-C) treatment. After administration of a single dose of Ara-C to leukemic mice (with approximate- ly 5% leukemia cells in peripheral blood), a more dramatic decrease in leukemia cells was observed in MA9-P2X7 mice than in MA9 mice. Following administration of mul- tiple doses of Ara-C to leukemic mice (with approximate- ly 10% leukemia cells), there were fewer leukemia cells in the peripheral blood in MA9-P2X7 mice than in MA9 mice at 48 h. Furthermore, more apoptotic cells were detected in the bone marrow of MA9-P2X7 mice than of MA9 mice (Online Supplementary Figure S4A and B). These results demonstrate that MA9-P2X7 cells are more sensitive to Ara-C treatment. To study whether MA9-P2X7 mice have survival advantages after Ara-C treatment, leukemic mice were given Ara-C twice. Although peripheral blood leukemia cell counts were decreased to almost identical levels in MA9 and MA9-P2X7 mice on day 14, the counts in MA9-P2X7 mice increased more rapidly, so that they were significantly higher on day 19 than in MA9 mice (Figure 4E), and MA9-P2X7 mice had a shorter survival than MA9 mice (Figure 4F). Moreover, the median survival was extended by 9.5 days (35.2%) in MA9 mice but by only 3.5 days (20.0%) in MA9-P2X7 mice. These results indicate that P2X7 promotes the proliferation of AML cells and imply that there are more LSC in MA9-P2X7.
Overexpression of P2X7 increases leukemia stem cell levels in MLL-AF9-induced acute myeloid leukemia
In vitro colony-forming ability was assessed to deter- mine the self-renewal potential of leukemia cells. The MA9-P2X7 cells formed more colonies than did the MA9 cells (Figure 5A). The colonies were classified into three types:26 type A colonies, which had a compact center; type B colonies, which had a dense center surrounded by a halo of migrating cells; and type C colonies, which con- sisted of many diffuse differentiating cells (Online Supplementary Figure S5A, left). MA9-P2X7 cells formed more type A, type B and type C colonies than did MA9 cells, especially type A colonies (Online Supplementary Figure S5A, right). High levels of LSC are associated with poor prognosis in AML. Limiting dilution transplantation experiments were used to analyze LSC frequency. In the groups transplanted with 103 cells, 100% of the MA9- P2X7 mice and 40% of the MA9 mice died from AML. Furthermore, 20% of the MA9-P2X7 mice and 0% of the MA9 mice suffered from AML in the groups given 102 cells (Figure 5B). The frequency of LSC was estimated to be 1/2088 in MA9 cells and 1/288 in MA9-P2X7 cells; this represents a 7.25-fold increase in functional LSC in MA9- P2X7 cells (Figure 5C). c-Kit, an important marker for LSC, was detected. Most MA9-P2X7 cells were c-Kit+, whereas there were two populations of MA9 cells, more than half of which were c-Kit– (Figure 5D). Colonies of MA9 and MA9-P2X7 cells were collected and stained with c-Kit. The colonies showed similar results (Online Supplementary Figure S5B). Different populations were sorted and transplanted into recipient mice (Online Supplementary Figure S5C). MA9-P2X7 and MA9 c-Kit+ cells gave rise to approximately 90% and 50% c-Kit+ leukemia cells, respectively. Interestingly, MA9 c-Kit– cells also gave rise to more than 20% c-Kit+ leukemia cells in
serial transplantations (Figure 5E, Online Supplementary Figure S5C). Cell cycle analysis revealed that fewer G0/G1 but more S/G2/M phase cells were detected in c-Kit+ MA9-P2X7 cells than in the other three populations (Online Supplementary Figure S5D). To further compare the malignant potential of these cells, equal numbers of MA9, MA9 c-Kit–, MA9 c-Kit+ and MA9-P2X7 cells were trans- planted. Expectedly, MA9-P2X7 mice had the shortest survival time (Figure 5F). These results suggest that MA9- P2X7 cells are more malignant than MA9 and even MA9 c-Kit+ cells.
Taken together, the results show that MA9-P2X7 cells have both greater proliferative potential and higher LSC frequency, which are characteristics that contribute to accelerated progression of leukemia.
Identification of key molecules mediating the pro-leukemic effects of P2X7
A gene expression microarray was used to screen key molecules mediating the effects of P2X7. MA9, MA9 c- Kit+, MA9 c-Kit– and MA9-P2X7 cells were sorted and analyzed (Online Supplementary Figure S6A). The raw data were normalized to a quantile algorithm. The sum aggre- gate of DEG (n=3,329) and differentially expressed long non-coding RNA (n=1,940) were obtained. Hierarchical clustering analysis showed that MA9-P2X7 cells shared more similarities with MA9 c-Kit+ than with MA9 or MA9 c-Kit– cells (Online Supplementary Figure S6B), which was in accordance with our previous observations. The DEG of MA9-P2X7 cells versus MA9, MA9 c-Kit+ or MA9 c-Kit– cells are shown in a Venn diagram (Figure 6A). Furthermore, gene set enrichment analyses on different sets of DEG demonstrated that MA9-P2X7 cells had a more immature phenotype than that of MA9 cells and enhanced proliferative potential compared to that of MA9 c-Kit+ or MA9 c-Kit– cells (Figure 6B). Analysis of the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases for DEG between MA9- P2X7 and MA9 cells revealed that annotations related to membrane receptor signal transduction were highly enriched. Annotations related to differentiation and pro- liferation were also enriched (Online Supplementary Figure S6C). The 3,329 DEG were rearranged into 16 clusters based on their expression patterns in four samples. A cluster of genes (n=130) was highlighted since their expression was higher in MA9-P2X7 cells than in any other population. Fifty genes (Online Supplementary Table S4) were first selected since they satisfied the condition (fold-change ≥2.0) in all populations. After bloodspot analysis to examine positive correlations with P2X7 in leukemia patients, seven genes with high fold-change val- ues were selected for further verification by qRT-PCR (primers are listed in Online Supplementary Table S5). Of these seven genes, three had homeodomains (Figure 6C). Hoxa9, Meis1 and Pbx2 were also included since they are important downstream regulators of MLL-rearranged leukemia. A high fold increase in Pbx3 but not Hoxa9, Meis1 or Pbx2 was detected (Figure 6D). Furthermore, a significant positive correlation was detected between P2X7 and Pbx3 from the GSE10358, GSE19577 and GSE12417 AML datasets (Figure 6E), whereas such a cor- relation was found in the GSE10358 dataset only for HOXA9 (Online Supplementary Figure S6D) and MEIS1 (Online Supplementary Figure S6E), and no significant corre- lation was detected between P2X7 and PBX2 in the three
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