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W. Feng et al.
However, the significance of P2X7 seems obscure since opposite effects have been observed.29 Early studies focused on apoptosis caused by its cytolytic effects.30 Exposure to high levels of ATP induces cell death in colon cancer due to disruption of the balance between cell growth and autophagy, which depends on the activation of the PI3K/AKT axis and AMPK-PRAS40-mTOR.31 In contrast, P2X7 promotes cell growth in neuroblastoma, non-melanoma skin cancer, prostate cancer and thyroid papillary cancer by different mechanisms.32 Typically, P2X7-dependent calcium influx results in the activation of downstream signaling pathways and the subsequent release of cytokines, inflammatory factors, microparti- cles, etc., which finally promote the proliferation and metastasis of tumor cells.10
Abnormal expression and function of P2X7 have been detected In various hematopoietic malignancies.6-8 Overexpression of P2X7 failed to induce leukemic transfor- mation,11 but the role of P2X7 in leukemia progression remains largely unknown. Although an apoptosis-related mechanism was proposed in an early study,30 leukemia is heterogeneous and it is thought that different mechanisms are involved in the initiation and progression of leukemia subtypes. We found that P2X7 was highly expressed in CLL and AML (Figure 1A) and that the levels of P2X7 in MLL-rearranged AML and CLL were equivalent (Online Supplementary Figure S1C). Adinolfi et al. reported that the expression of P2X7 was higher in evolutive B-cell CLL patients and suggested that high-level expression of P2X7 had a proliferative advantage and was associated with a poor prognosis in B-cell CLL.8 However, the molecular mechanism in CLL has not been elucidated, partly because of the lack of appropriate cell lines and mouse models, although a mouse model based on B-cell-restricted expres- sion of Sf3b1 mutation and Atm deletion has just been reported.33 In contrast, there are suitable cell lines and mouse models to study the molecular mechanisms in AML, typically MLL-rearranged AML. Hence, we focused on AML. We found that overexpression of P2X7 accelerat- ed the progression of MLL-rearranged AML by promoting proliferation and increasing LSC levels through the activa- tion of the HOXA9/PBX3/MEIS1 axis by upregulation of Pbx3. Since more LSC and rapid proliferation are frequent- ly related to worse clinical outcomes, our results suggest that P2X7 plays unfavorable roles in the progression of MLL-rearranged AML.
As a cofactor of Hoxa9, Pbx3 was previously thought to make a redundant contribution to cell transformation.34 However, emerging evidence indicates that it has inde- pendent roles in leukemogenesis.35 We demonstrated that the expression of Pbx3, but not of HOXA9 or MEIS1, is positively correlated with P2X7. Knockdown of Pbx3 in leukemia cells overexpressing P2X7 inhibited cell prolifera- tion in different models, decreased LSC (forming type A colonies) and eventually prolonged the survival of AML mice. These effects of Pbx3 seem to be contradictory since LSC are considered a quiescent population.36 However, leukemia cells comprise heterogeneous populations. Pbx3 not only plays vital roles in the maintenance of LSC37 and tumor-initiating cells,38 but also promotes the proliferation of non-LSC, which is also observed in solid tumors.39 It is worth noting that there is still controversy about whether quiescence is necessary for LSC since there is evidence of a non-quiescent AML LSC population, which is required for the development of MLL-rearranged AML.40 Hence, Pbx3
mediates the adverse effects of P2X7 in the progression of MLL-rearrangedAML.
Although they have mainly been observed in models of MLL-rearranged AML, the effects and mechanism of P2X7 should also be present in other types of leukemia. P2X7high AML had higher Pbx3 expression and worse prognosis than P2X7low AML (Figures 6E and Figures 6F and 1C and D). Overexpression of P2X7 in leukemia cells without MLL rearrangements results in the upregulation of Pbx3. In fact, HOX proteins, which are also regulated by MEN1, CDX, etc.,41 are widely involved in normal and malignant hematopoiesis.42 Dysregulation of HOX gene expression is also found in other types of leukemia without MLL rearrangements, such as those with PML-RARA, FLT3-ITD and NUP98-related fusion genes.43 Specifically, Pbx3 should play a role in the progression of other types of leukemia since upregulation of Pbx3 is detected not only in patients with MLL translocations44 but also in those with FLT3-ITD and NPM1 mutations as well as NUP98-related fusion genes.43 This mechanism may also be involved in non-hematopoietic malignancies since Pbx3 is a key factor promoting metastasis, proliferation and poor prognosis in various solid tumors.45 Pbx3 induces an infiltrative tumor cell phenotype in gastric cancer46 and plays determinant roles in hepatocellular carcinoma tumor-initiating cells by regulating their self-renewal and tumorigenicity potential.38 Hence, P2X7 may promote the progression of certain malignancies through the upregulation of Pbx3.
Although there is a positive correlation between P2X7 and Pbx3, the mechanism remains unclear. Abnormal acti- vation of P2X7 is probably involved because high levels of ATP are detected in the microenvironment of various malignancies.10 Ca2+ influx and K+/Na+ efflux, which may be involved in the P2X7-related upregulation of Pbx3, are early events upon P2X7 activation.47 A rapid rise in intracel- lular Ca2+ concentration is an important event in the activa- tion of hormone signals,48 which regulates the expression of HOX genes.49 Moreover, ion signaling is also required for post-translational modification of homeoproteins. For example, calcium binding to histone tails induces confor- mational changes essential for their functions.50 Further work is required to elucidate the detailed interactions between P2X7 and Pbx3.
In summary, we provide compelling evidence that P2X7 accelerates the progression of MLL-rearranged AML by promoting proliferation and increasing LSC through the upregulation of Pbx3. This novel pathway links dysfunc- tion in P2X7 signaling and poor clinical outcome in MLL- rearranged AML, and probably in other types of malignan- cies with high levels of P2X7 expression. Our study pro- vides new insights into malignant progression caused by abnormal purinergic signaling.
Disclosures
No conflicts of interest to disclose.
Contributions
WF and XY designed the experiments, performed research and drafted the paper; they contributed equally to this work. RW, FY, HW, XL and RQ performed research and critically revised the paper. LW, YZ and XZ analyzed the data and crit- ically revised the paper. GZ obtained funding, designed the experiments, acquired and analyzed the data and critically revised the paper. All authors are responsible for the accuracy and integrity of all aspects of the manuscript.
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haematologica | 2021; 106(5)