Undrestanding the immune escape mechanisms of cancerous cells in tumor microenvironment is crucial to develop and improve new approaches for tumors targeted therapy (Teague and Kline 2013, Beatty and Gladney 2015). There are numerous mechanisms for tumor cells to escape from the host immune system, which one of the most important of them is the expression of immune checkpoint molecules (Blank et al. 2005, Drake et al. 2006, Beatty and Gladney 2015). This mechanism includes the over-expression of immune checkpoint receptors on immune cells and their ligands on tumor cells, such as PD-1/PD-L1, Tim-3/Gal-9, and TIGIT/CD155 (Blank et al. 2005, Beatty and Gladney 2015). These interactions play a pivotal role in the immune suppressing behavior of the tumor microenvironment, which may also inhibit the antitumor immunity and eventually lead to the immune escape of tumor cells (Dong et al. 2016, Jiang et al. 2019, Peng et al. 2019). Targeting these pathways is an appealing approach for cancer immunotherapy, but the clinical efficacy remains poor in some tumors. These conditions need a greater comprehension of the complex and diverse molecular mechanisms and the intracellular signaling pathways in tumor cells (Dong et al. 2016, Jiang et al. 2019). On the other hand, blockade of signaling pathways using small molecule inhibitors is a new promising strategy for cancer treatment. Various small molecule inhibitors are currently being evaluated in clinical trials for hematopoietic and non-hematopoietic malignancies. However, little is known about their possible associations with the immune evasion mechanisms. Accordingly, in this study we aimed to find any associations between the PI3K/Akt/mTOR signaling pathways and the expression of immune checkpoint ligands PD-L1, Gal-9, and CD155 on AML cells. Our results revealed that the blockade of PI3K/Akt/mTOR pathways could be a potential target for regulating the expression of PD-L1, Gal-9, and CD155 on HL-60 myeloproliferative cell line. The PI3K/Akt/mTOR pathway is of particular interest among the main abnormal intracellular signaling pathways engaged in hematological malignancies (Barrett et al. 2012, Bertacchini et al. 2015, Sandhöfer et al. 2015). Over-activation of PI3K/Akt/mTOR suggests an essential role for this axis in tumor progression of several hematological malignancies, including AML, chronic myelogenous leukemia (CML), and acute lymphoblastic leukemia (ALL), as well as in lymphoproliferative disorders (Barrett et al. 2012, Deng et al. 2017, Herschbein and Liesveld 2018). Recent studies have demonstrated that the PI3K/Akt/mTOR pathway is activated, in 50–80% of AML patients (Dos Santos et al. 2006, Martelli et al. 2010). Moreover, practically high levels of mTOR signaling is displayed in all AML patients (Park et al. 2010). Because of these discrepancies, it is assumed that dual or triple blockade of PI3K/Akt/mTOR pathway could be more effective than single blockade in AML. Previous reports suggested that the blockade of these pathways reduce cell viability and induce apoptosis of AML cells (Martelli et al. 2007, Deng et al. 2017, Hao et al. 2019). Similar to these finding, we showed that the combination blockade of PI3K/Akt/mTOR pathways by small molecule inhibitors reduces the cell viability and enhances the apoptosis on HL-60 cell line. Since the PI3K/Akt/mTOR pathway is involved in activation of some oncogenes participated in AML oncogenesis, we postulated that this pathway might regulate the immune escape mechanisms and especially the expression of immune checkpoint ligands in AML. Our findings indicated a strong correlation between the expression of PD-L1, Gal-9, and CD155 and over-activation of the PI3K/Akt/mTOR pathway in AML. Previous reports showed the associations between over-expression of immune checkpoint ligands and tumor immune escape (Lin et al. 2015, Qin et al. 2015, Taghiloo et al. 2017). Interaction of the PD-1/PD-L1, Tim-3/Gal-9, and TIGIT/CD155 pathways were able to regulate the host immune response and T-cell exhaustion in tumor microenvironment (Pardoll 2012, Allahmoradi et al. 2017, Silva et al. 2017, Taghiloo et al. 2017). Over-activation of PD-L1 and Gal-9 and CD155 is reported in a substantial number of solid and hematopoietic malignancies, but little is known about the expression of CD155 on leukemia cells (Pardoll 2012, Folgiero et al. 2015, Dong et al. 2016, Taghiloo et al. 2017, Zhao et al. 2017). Previous findings domenstrated that the activation of receptor-mediated signaling pathways, such as PI3K/Akt/mTOR, JAK/STAT, MEK/ERK, and NF-κB promote the immune escape mechanisms by regulating the expression of PD-L1 in several tumors (Ritprajak and Azuma 2015, Lastwika et al. 2016, Peng et al. 2019). In this regard, several reports indicated that the up-regulation of PD-L1 is heavily dependent on PI3K/Akt/mTOR signaling pathway in glioma, lung cancer, and pancreatic cancer (Parsa et al. 2007, Zhang et al. 2013, Zhang et al. 2017). In contrast with these reports, PD-L1 expression is also correlated with the MEK/ERK signaling pathway in myeloma cells, but not the PI3K/Akt/mTOR signaling pathways (Liu et al. 2007). It also should be noted that the expression regulation of these ligands is very complex and most probably depends on the status of underlying signaling pathways. Interestingly, most of the research in this area is related to PD-L1 expression and not much information is available about other checkpoint ligands like Gal-9 and CD155. To our knowledge, there are no evidences to investigate the association between the expression of immune checkpoint ligands with PI3K/Akt/mTOR signaling pathways in hematological malignancies. Here, our data showed that the combination blockade of PI3K/Akt/mTOR pathway by small molecule inhibitors is associated with the down-regulation of PD-L1, Gal-9, and CD155 in AML cell line. These results highlight the importance of combination blockade of PI3K/Akt/mTOR pathway as a promising target for potentiating anti-tumor immune responses by regulating the expression of immune checkpoint ligands on AML cells.
In conclusion, our findings demonstrated that the expression of PD-L1, Gal-9, and CD155 is significantly desreased after co-treatment with small molecules, including idelalisib, MK-2206, and everolimus, as PI3K/Akt/mTOR pathway inhibitors. We showed that PI3K/Akt/mTOR pathway inhibitors not only serve as cytotoxic drugs, but also regulate the expression of immune checkpoint ligands and interfere with the immune evasion mechanisms of AML leukemic cells. Combinational therapy approaches to block these pathways might be a promising and novel therapeutic strategy for AML patients via interfering in immune escape mechanisms.