Esistance to AZD1480 therapy in HL patients. The fact that AZD1480 synergized AS-252424  characterized by AZD1480 induced ERK hyperactivation strongly supports the hypothesis that ERK hyperactivation may be an important mechanism of resistance to the JAK inhibition in HL. In this study, we show that higher concentrations of AZD1480 inhibited Aurora kinases and induced G2/M cell cycle arrest and cell death in all HL cell lines, irrespective of JAK/STAT pathway status. Aurora A has a crucial role in mitotic bipolar spindle formation and localizes to centrosomes at the proximal mitotic spindle, whereas Aurora B localizes to kinetochores, phosphorylates histone H3 at Ser10, and has a role in chromosome alignment and cytokinesis.
35 Inhibition of Aurora kinases promotes G2/M arrest and apoptosis in multiple human cancers including lymphoid malignancies,36 38 but no data are available, which suggest a role of Aurora kinases in Hodgkin lymphomagenesis. We found that Aurora A and B kinases were overexpressed in the four HL cell lines compared with PBMCs from healthy donors. AZD1480 promoted Bafetinib a dose dependent inhibition of Aurora A autophosphorylation at Thr288 in all the four HL cell lines. We observed a tight correlation between the dose dependent inhibition of Aurora A and the changes observed in cell cycle fraction and apoptosis in HL cells. A dose dependent inhibition of histone H3 phosphorylation at ser 10 was detected in two cell lines, suggesting that high doses of AZD1480 may also inhibit Aurora B in these cell lines.
Due to the fact that Reed Sternberg cells account for less than 5% of the entire tumor mass, being very rare in the affected lymph nodes, we were not able to microdissect viable primary HRS cells from patients, lymph nodes to perform in vitro viability and functional assays. Figure 6 Model for AZD1480 activity in HL cells. A model showing the dual, dose dependent mechanism of action of AZD1480 in HL cells. At low doses, AZD1480 inhibits the JAK/STAT pathway, showing predominantly immunoregulatory effects. At high doses, AZD1480 also inhibits the Aurora kinases, promoting G2/M arrest and apoptosis in HL cells. Activity of AZD1480 in Hodgkin lymphoma E Derenzini et al However, our data clearly demonstrate that AZD1480 inhibits JAK/STAT activation in cultured HL cells at submicromolar concentrations, by blocking the function of JAKs and determining immunomodulatory effects.
Moreover, the two cell lines, which showed MAP kinase hyperactivation following treatment with AZD1480, were resistant to the drug at concentrations clearly able to inhibit STATs phosphorylation. The fact that different MEK inhibitors synergized with AZD1480 in these two resistant cell lines, suggest that this negative feedback loop activating MAP kinases could be an important mechanism of resistance to AZD1480. In summary, our results provide preclinical rationale for further clinical investigation of AZD1480 in HL and provide molecular rationale for incorporating biomarker studies according to the primary target inhibition. Furthermore, our data demonstrate the importance of evaluating the in vivo effect of small molecule inhibitors on secondary signaling pathways that may mediate resistance to therapy and pr