Combined Inhibition of Janus and Aurora Kinases by the Novel Small Molecule Inhibitor AZD1480 Induces Cell Death and Downregulates PD-L1 and PD-L2 Expression In Hodgkin Lymphoma

Abstract 2848

Hodgkin Lymphoma (HL) cell proliferation and survival is sustained by a complex network of cytokine signaling, involving the Hodgkin and Reed-Sternberg cells and tumor microenvironment. Following cytokine stimulation, JAK-STAT activation promotes the transcription of target genes involved in proliferation, survival, and immune escape. Programmed Death-ligands 1 and 2 (PD-L1 and PD-L2) and the Th2 chemokine TARC are immune-modulators involved in immune evasion, respectively through inhibition of effector T cell function (PD-L1, PD-L2) and attraction and homing of Th2 cells (TARC). Aurora kinases are frequently overexpressed in human cancers and play essential functions in chromosome alignment and cytokinesis. The role of Aurora kinases in Hodgkin lymphomagenesis is not defined yet. In this study we report the activity profile of the JAK2 inhibitor AZD1480 in HL cell lines (HD-LM2, L-428, KM-H2, L-540).

To assess the effect of AZD1480 on cell proliferation, cells were incubated with increasing concentrations of AZD1480 (from 0.1 to 10 μM) for 24, 48 and 72 hours (hrs). A significant growth inhibition was evident after 72 hrs of incubation, specially using the high doses of AZD1480 (5μM). The L-540 cell line showed the highest sensitivity, with a decrease in cell viability close to 50% following incubation with AZD1480 1μM. Inhibition of STAT3, STAT5 and STAT6 phosphorylation in the L-540, L-428 and HD-LM2 cell lines was observed with concentrations equal to 0.1 μM or higher. Using Annexin V- propidium iodide staining, we found that AZD1480 induced cell death by apoptosis in a dose dependent manner after 72 hrs of incubation when a high concentration (5μM) of the drug was used. Lower concentrations of AZD1480 (1μM) promoted a statistically significant increase in cell death only in the L-540 and to a lesser extent in the L-428 cell line. Consistent with this data, also caspase 9, 3 and PARP cleavage was observed in all the cell lines exposed to AZD1480 5 μM. AZD1480 5μM promoted a marked increase in the G2/M fraction in all the cell lines as soon as 24 hrs after incubation, especially in the HD-LM2 and L-428 cell lines. Treatment with lower doses (1μM) did not affect significantly the cell cycle. Since AZD1480 was also reported to inhibit Aurora A kinase at nanomolar concentrations in enzymatic assays, we assessed if the significant increase in the G2/M fraction was related to the inhibition of the Aurora A kinase. We evaluated the levels of autophosphorylation on Thr-288 by western blotting. Cells were pretreated with Nocodazole 400 ng/ml for 18 hrs in order to achieve a mitotic block, and then exposed to AZD1480 (1-5μM) and/or the proteasome inhibitor MG132 (20μM) (in order to prevent the potential overriding of the Nocodazole induced mitotic block), for 3 hours. A dose-dependent inhibition of Aurora A was detected in all the cell lines, with a complete abrogation when higher doses of AZD1480 were used (5μM). These findings are consistent with the analysis of the cell cycle fractions, showing dose-dependent changes of the cell cycle at 24 hrs following incubation with AZD1480. AZD1480 also decreased the secretion of key cytokines involved autocrine and paracrine survival loops and immune escape. Following incubation with AZD1480 1μM for 72 hrs cell culture supernatants were analyzed by ELISA: decreased levels of IL-6, IL-13, TARC, and IL-21 were observed in HD-LM2, L-428 and L-540 cells. Moreover we assessed the expression of PD-L1 and PD-L2 by flow cytometry and observed significant downregulation in the PD-L1/PD-L2 overexpressing cell lines (L-540 and HD-LM2). These data suggest that AZD1480 has a pleiotropic mechanism of action in HL by targeting the JAK-STAT and the Aurora kinase pathway, and by altering the pattern of cytokine and chemokine secretion and the expression of factors involved in immune escape. Our study provides the rationale for further clinical investigation of AZD1480 in HL.