A novel class of anti-tumoral compounds, pyrrolonaphtoxazepines (PNOXs), targets the p66Shc/STAT4 axis in CLL prymary cells and reduces tumor burden in Eμ-TCL1 mice

Chronic lymphocytic leukemia is an haematological malignancy characterized by the accumulation of CD5+CD19+ mature B cell clones in peripheral blood, spleen, lymph nodes and bone marrow (B-CLL). Leukemic cells accumulation is both dependent upon enhanced B cell receptor (BCR)-driven proliferation and defects in the apoptotic machinery. Additionally, alterations of surface chemokine receptors have been accounted for the enhanced accumulation and survival of CLL cells within the secondary lymphoid organs (SLOs). At present, treatment options for CLL patients include drugs targeting proteins participating in the BCR signaling pathway (i.e Ibrutinib, Idelalisib) and in the apoptotic process (i.e Venetoclax). However, relapsed or refractory CLL is still a clinical problem, and prolonged treatment with the above mentioned drugs leads to resistance occurrence as well as severe side effects, indicating that novel therapeutic options are needed. The adaptor protein p66Shc is a negative regulator of BCR-signaling and a pro-apoptotic protein whose loss in CLL B cells accounts for the extended survival of tumoral cells and poor prognosis. p66Shc has been also shown to control B cells trafficking by altering CXCR4 function, CCR7 and S1PR1 expression. The pathogenetic role of p66Shc in CLL was recently confirmed in the Eμ-TCL1 mouse model of CLL. Eμ-TCL1 mice lacking the p66Shc gene (Eμ-TCL1p66Shc-/- mice), showed indeed a more severe disease characterized by earlier onset and higher nodal and extranodal accumulation of leukemic cells compared with the Eμ-TCL1 mice. Hence, identification of compounds that up-regulate p66Shc expression and function in malignant B cells are likely to target cell proliferation, migration and apoptosis and may represent an appealing therapeutic option for CLL. In this study, we decided to investigate the molecular mechanism underlying the anti tumoral efficacy of a recently developed class of compounds, the pyrrolonaphthoxazepines (PNOXs), which have been demonstrated to be effective in vitro in several tumoral cells, including primary human CLL cells and to test, for the first time, these novel compounds in the murine model of human CLL, the Eμ-TCL1 mouse. Our results show that PNOXs restore p66Shc expression and activate transcription factor STAT4 in primary human CLL cells without affecting normal B cells. Accordingly, our data show that the recovery of p66Shc expression in human CLL cells directly correlates with the apoptosis rate induced by PNOXs. In agreement with human CLL, we found that PNOXs efficiently upregulate p66Shc and promote apoptosis in murine CLL cells. Moreover, we demonstrated that PNOXs were able to activate pro-apoptotic activity of p66Shc through a JNK dependent phosphorylation of Ser36 residue on p66Shc, suggesting that the anti-tumor effect of PNOXs in human CLL cells relies at least in part on its ability to first promote p66Shc expression and subsequently to foster its apoptotic function. PNOXs treatment of Eμ-TCL1 mice resulted in a significantly longer overall survival and in the reduction in tumor burden in the spleen and in the peritoneum. The powerful antitumor effect of PNOXs in vivo correlates with upregulation of S1PR1 and in the mobilization of leukemic cells from the spleen into the blood. Interestingly, treatment with PNOXs compounds on Eμ-TCL1p66Shc-/- mice highlighted that only one compound, PNOX-3, exerts a p66Shc-independent pro-apoptotic activity towards circulating CLL cells, while absence of p66Shc impairs the ability of PNOXs to mobilize CLL cells from the spleen to peripheral blood. Since accumulation of leukemic cells within secondary lymphoid organs is responsible for the enhanced survival of leukemic cells and drug resistance, our data indicate that PNOXs may represent a novel effective treatment for CLL by favouring the mobilization of leukemic cells from the protective tumor microenvironment of the SLOs into the blood and by promoting apoptosis through the upregulation of p66Shc.