Unveiling new targets to overcome the extended chronic lymphocytic leukemia cells survival

Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature B cells in peripheral blood and lymphoid organs. The extended survival of leukemic cells is due to a combination of intrinsic alterations in the apoptotic machinery and microenvironmental factors, which both mediate evasion of immune surveillance. Among intrinsic alteration, the profound imbalance in the expression of pro- and anti-apoptotic members of the Bcl-2 family stands out. Impaired activity of the phosphatase SHP-1 in CLL cells has been related to the deficient expression of the pro-apoptotic Bcl-2 family member Bax. Since the membrane phospholipid metabolite glycerophosphoinositol (GroPIns) binds and activates SHP-1, here we asked whether GroPIns can restore Bax expression in CLL cells by reactivating SHP-1. To test this hypothesis, we cultured CLL cells in the presence of GroPIns, alone or in combination with Venetoclax, a Bcl-2 inhibitor commonly used for CLL treatment. We found that GroPIns alone increases Bax expression and apoptosis in CLL cells in a SHP-1 dependent manner. Moreover, GroPIns potentiates the pro-apoptotic activity of Venetoclax. Interestingly, among GroPIns interactors, we found Bax itself, which becomes activated when CLL cells are treated with GroPIns. These data provide evidence that GroPIns exploits two different pathways converging on Bax to promote leukemic cell apoptosis. Surface receptor/ligand inhibitory axes have recently reached attention as promoters of CLL cell survival in the tumor microenvironment (TME) by suppressing the killing activities of cytotoxic T cells (CTLs) and their ability to form the immune synapse (IS), a specialized platform which polarizes both membrane and soluble signaling mediators at the interface between T cell and antigen-presenting cell. Since leukemic cells release soluble factors which profoundly shape the TME toward a pro-survival and protective niche, here we asked whether they also contribute to suppress CTL anti-tumoral functions. We found that healthy CTLs cultured in media conditioned by leukemic cells from CLL patients or Eμ-TCL1 mice upregulate the exhaustion marker PD-1 and become unable to form functional ISs and kill target cells. These defects were more pronounced when media were conditioned by leukemic cells lacking the pro-apoptotic adaptor p66Shc, whose deficiency has been implicated in CLL aggressiveness. Multiplex ELISA assays and quantitative RT-PCR showed that interleukin (IL)-9 and IL-10 were overexpressed in leukemic cells from CLL patients, where they inversely correlated with residual p66Shc. Using neutralizing antibodies or the recombinant cytokines we show that IL-9, but not IL-10, mediates both the enhancement in PD-1 expression and the suppression of effector functions in healthy CTLs. These data demonstrate that IL-9 secreted by leukemic cells negatively modulates the anti-tumor immune abilities of CTLs. Altogether, our results highlight new intrinsic and extrinsic mechanisms underlying the defective apoptosis of CLL cells, and pave the way to new studies aimed at exploiting these molecular pathways as therapeutical targets in CLL.