P2X7 Variants in Pathophysiology.

P2X7 receptor activation by extracellular adenosine triphosphate (eATP) modulates different intracellular pathways, including pro-inflammatory and tumor-promoting cascades. ATP is released by cells and necrotic tissues during stressful conditions and accumulates mainly in the inflammatory and tumoral microenvironments. As a consequence, both the P2X7 blockade and agonism have been proposed as therapeutic strategies in phlogosis and cancer. Nevertheless, most studies have been carried out on the WT fully functional receptor variant. In recent years, the discovery of P2X7 variants derived by alternative splicing mechanisms or single-nucleotide substitutions gave rise to the investigation of these new P2X7 variants' roles in different processes and diseases. Here, we provide an overview of the literature covering the function of human P2X7 splice variants and polymorphisms in diverse pathophysiological contexts, paying particular attention to their role in oncological and neuroinflammatory conditions.

The purinergic receptor P2X7 as a modulator of viral vector-mediated antigen cross-presentation.

Introduction: Modified Vaccinia Virus Ankara (MVA) is a safe vaccine vector inducing long- lasting and potent immune responses. MVA-mediated CD8+T cell responses are optimally induced, if both, direct- and cross-presentation of viral or recombinant antigens by dendritic cells are contributing. Methods: To improve the adaptive immune responses, we investigated the role of the purinergic receptor P2X7 (P2RX7) in MVA-infected feeder cells as a modulator of cross-presentation by non-infected dendritic cells. The infected feeder cells serve as source of antigen and provide signals that help to attract dendritic cells for antigen take up and to license these cells for cross-presentation. Results: We demonstrate that presence of an active P2RX7 in major histocompatibility complex (MHC) class I (MHCI) mismatched feeder cells significantly enhanced MVA-mediated antigen cross-presentation. This was partly regulated by P2RX7-specific processes, such as the increased availability of extracellular particles as well as the altered cellular energy metabolism by mitochondria in the feeder cells. Furthermore, functional P2RX7 in feeder cells resulted in a delayed but also prolonged antigen expression after infection. Discussion: We conclude that a combination of the above mentioned P2RX7-depending processes leads to significantly increased T cell activation via cross- presentation of MVA-derived antigens. To this day, P2RX7 has been mostly investigated in regards to neuroinflammatory diseases and cancer progression. However, we report for the first time the crucial role of P2RX7 for antigen- specific T cell immunity in a viral infection model.

Growth Charts for Shwachman-Diamond Syndrome at Ages 0 to 18 Years.

Shwachman-Diamond syndrome (SDS) is one of the most common inherited bone marrow failure syndromes. SDS is characterized by hypocellular bone marrow, with a severe impairment of the myeloid lineage, resulting in neutropenia, thrombocytopenia, and, more rarely, anemia. Almost 15% of patients with SDS develop myelodysplastic syndrome or acute myeloid leukemia as early as childhood or young adulthood. Exocrine pancreatic insufficiency is another common feature of SDS. Almost all patients with SDS show failure to thrive, which is associated with skeletal abnormalities due to defective ossification. Considering these observations, it remains unfeasible to use the common growth charts already available for the general population. To address this issue, we report how we drew up growth charts of patients with SDS aged 0 to 18 years. We analyzed height, weight, and body max index (BMI) in 121 Italian patients with SDS. Results indicated that the 50th and 3rd percentiles of weight and height of the pediatric general population correspond to the 97th and 50th percentiles of patients with SDS aged 0-18 years, respectively. In addition, the percentage increment in weight of subjects aged 14-18 years was higher in patients with SDS than in the general population. SDS-specific growth charts, such as those described here, afford a new tool, which is potentially useful for both clinical and research purposes in SDS.

Ataluren improves myelopoiesis and neutrophil chemotaxis by restoring ribosome biogenesis and reducing p53 levels in Shwachman-Diamond syndrome cells.

Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, exocrine pancreatic insufficiency and skeletal abnormalities. SDS bone marrow haematopoietic progenitors show increased apoptosis and impairment in granulocytic differentiation. Loss of Shwachman-Bodian-Diamond syndrome (SBDS) expression results in reduced eukaryotic 80S ribosome maturation. Biallelic mutations in the SBDS gene are found in ~90% of SDS patients, ~55% of whom carry the c.183-184TA>CT nonsense mutation. Several translational readthrough-inducing drugs aimed at suppressing nonsense mutations have been developed. One of these, ataluren, has received approval in Europe for the treatment of Duchenne muscular dystrophy. We previously showed that ataluren can restore full-length SBDS protein synthesis in SDS-derived bone marrow cells. Here, we extend our preclinical study to assess the functional restoration of SBDS capabilities in vitro and ex vivo. Ataluren improved 80S ribosome assembly and total protein synthesis in SDS-derived cells, restored myelopoiesis in myeloid progenitors, improved neutrophil chemotaxis in vitro and reduced neutrophil dysplastic markers ex vivo. Ataluren also restored full-length SBDS synthesis in primary osteoblasts, suggesting that its beneficial role may go beyond the myeloid compartment. Altogether, our results strengthened the rationale for a Phase I/II clinical trial of ataluren in SDS patients who harbour the nonsense mutation.

The P2X7 Receptor in Oncogenesis and Metastatic Dissemination: New Insights on Vesicular Release and Adenosinergic Crosstalk.

The tumor niche is an environment rich in extracellular ATP (eATP) where purinergic receptors have essential roles in different cell subtypes, including cancer, immune, and stromal cells. Here, we give an overview of recent discoveries regarding the role of probably the best-characterized purinergic receptor in the tumor microenvironment: P2X7. We cover the activities of the P2X7 receptor and its human splice variants in solid and liquid cancer proliferation, dissemination, and crosstalk with immune and endothelial cells. Particular attention is paid to the P2X7-dependent release of microvesicles and exosomes, their content, including ATP and miRNAs, and, in general, P2X7-activated mechanisms favoring metastatic spread and niche conditioning. Moreover, the emerging role of P2X7 in influencing the adenosinergic axis, formed by the ectonucleotidases CD39 and CD73 and the adenosine receptor A2A in cancer, is analyzed. Finally, we cover how antitumor therapy responses can be influenced by or can change P2X7 expression and function. This converging evidence suggests that P2X7 is an attractive therapeutic target for oncological conditions.

Emerging roles of purinergic signaling in anti-cancer therapy resistance.

Cancer is a complex disease with a rapid growing incidence and often characterized by a poor prognosis. Although impressive advances have been made in cancer treatments, resistance to therapy remains a critical obstacle for the improvement of patients outcome. Current treatment approaches as chemo-, radio-, and immuno-therapy deeply affect the tumor microenvironment (TME), inducing an extensive selective pressure on cancer cells through the activation of the immune system, the induction of cell death and the release of inflammatory and damage-associated molecular patterns (DAMPS), including nucleosides (adenosine) and nucleotides (ATP and ADP). To survive in this hostile environment, resistant cells engage a variety of mitigation pathways related to metabolism, DNA repair, stemness, inflammation and resistance to apoptosis. In this context, purinergic signaling exerts a pivotal role being involved in mitochondrial function, stemness, inflammation and cancer development. The activity of ATP and adenosine released in the TME depend upon the repertoire of purinergic P2 and adenosine receptors engaged, as well as, by the expression of ectonucleotidases (CD39 and CD73) on tumor, immune and stromal cells. Besides its well established role in the pathogenesis of several tumors and in host-tumor interaction, purinergic signaling has been recently shown to be profoundly involved in the development of therapy resistance. In this review we summarize the current advances on the role of purinergic signaling in response and resistance to anti-cancer therapies, also describing the translational applications of combining conventional anticancer interventions with therapies targeting purinergic signaling.

Administration of P2X7 Receptor Blockers in Oncological Experimental Models.

The tumor microenvironment is rich in components that strongly influence cancer cell survival. One of the pivotal molecules present at the tumor bed is ATP, which has an essential role in promoting cancer proliferation and metastasis and immune responses via its receptor P2X7. Several studies have proved the efficacy of P2X7 pharmacological blockade in inhibiting primary and metastatic tumor growth in preclinical models. Here we describe the experimental procedures that we optimized to test P2X7 roles in carcinogenesis by antagonist administration. Special attention is paid to their concentrations and routes of administration. The depicted in vitro models include cell count and viability assays, which are useful to test P2X7 roles in cell proliferation and vitality, and the soft agar colony formation test that allows investigation of the transforming and invading abilities of tumor cells. We also describe systemic and intramass administration of P2X7 blockers in murine models of melanoma and leukemia. Both xenotransplant and syngeneic experimental tumor models are detailed.

A2A Receptor Contributes to Tumor Progression in P2X7 Null Mice.

ATP and adenosine are key constituents of the tumor niche where they exert opposite and complementary roles. ATP can be released in response to cell damage or actively released by tumor cells and subsequently degraded into adenosine, which accumulates within the tumor microenvironment. Notably, while ATP promotes immune eradicating responses mainly via the P2X7 receptor (P2X7R), extracellular adenosine acts as a potent immune suppressor and facilitates neovascularization thanks to the A2A receptor (A2AR). To date, studies exploring the interplay between P2X7R and A2AR in the tumor microenvironment are as yet missing. Here, we show that, in C57/bl6 P2X7 null mice inoculated with B16-F10 melanoma cells, several pro-inflammatory cytokines, including interleukin 1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), interleukin 12 (IL-12), interleukin 17 (IL-17), interferon gamma (IFN-γ) were significantly decreased, while the immune suppressant transforming growth factor beta (TGF-ß) was almost three-fold increased. Interestingly, tumors growing in P2X7-null mice upregulated tumor-associated and splenic A2AR, suggesting that immunosuppression linked to lack of the P2X7R might depend upon A2AR overexpression. Immunohistochemical analysis showed that tumor cells' A2AR expression was increased, especially around necrotic areas, and that vascular endothelial growth factor (VEGF) and the endothelial marker CD31 were upregulated. A2AR antagonist SCH58261 treatment reduced tumor growth similarly in the P2X7 wild type or null mice strain. However, SCH58261 reduced VEGF only in the P2X7 knock out mice, thus supporting the hypothesis of an A2AR-mediated increase in vascularization observed in the P2X7-null host. SCH58261 administration also significantly reduced intratumor TGF-ß levels, thus supporting a key immune suppressive role of A2AR in our model. Altogether, these results indicate that in the absence of host P2X7R, the A2AR favors tumor growth via immune suppression and neovascularization. This study shows a novel direct correlation between P2X7R and A2AR in oncogenesis and paves the way for new combined therapies promoting anti-cancer immune responses and reducing tumor vascularization.

Novel Translational Read-through-Inducing Drugs as a Therapeutic Option for Shwachman-Diamond Syndrome.

Shwachman-Diamond syndrome (SDS) is one of the most commonly inherited bone marrow failure syndromes (IBMFS). In SDS, bone marrow is hypocellular, with marked neutropenia. Moreover, SDS patients have a high risk of developing myelodysplastic syndrome (MDS), which in turn increases the risk of acute myeloid leukemia (AML) from an early age. Most SDS patients are heterozygous for the c.183-184TA>CT (K62X) SBDS nonsense mutation. Fortunately, a plethora of translational read-through inducing drugs (TRIDs) have been developed and tested for several rare inherited diseases due to nonsense mutations so far. The authors previously demonstrated that ataluren (PTC124) can restore full-length SBDS protein expression in bone marrow stem cells isolated from SDS patients carrying the nonsense mutation K62X. In this study, the authors evaluated the effect of a panel of ataluren analogues in restoring SBDS protein resynthesis and function both in hematological and non-hematological SDS cells. Besides confirming that ataluren can efficiently induce SBDS protein re-expression in SDS cells, the authors found that another analogue, namely NV848, can restore full-length SBDS protein synthesis as well, showing very low toxicity in zebrafish. Furthermore, NV848 can improve myeloid differentiation in bone marrow hematopoietic progenitors, enhancing neutrophil maturation and reducing the number of dysplastic granulocytes in vitro. Therefore, these findings broaden the possibilities of developing novel therapeutic options in terms of nonsense mutation suppression for SDS. Eventually, this study may act as a proof of concept for the development of similar approaches for other IBMFS caused by nonsense mutations.