AZD1480 effectively inhibited the function of the target JAK2 CHIR-124 protein, as evident by dephosphorylation of the downstream STAT proteins, these concentrations had no significant antiproliferative effect in the HD LM2 and L 428 cell lines. Submicromolar concentrations of AZD1480 inhibited the phosphorylation of STAT1, STAT3, STAT5 and STAT6, with no apparent differential effect. This is in contrast with what was recently reported with selective silencing of STAT6 gene expression experiments, as it resulted in activation of STAT1 in the same cell line, which may have contributed to induction of cell death. 32 At low concentrations, AZD1480 displayed predominantly immunomodulatory effects, downregulating the expression of Th2 cytokines and chemokines, and factors involved in mechanisms of immune escape.
Collectively, these data suggest that AZD1480 may enhance anti tumor immunity by increasing the activity of cytotoxic T cells. Regarding the mechanism involved in the resistance of the HD LM2 and L 428 cell lines to low doses of AZD1480, this may be related to a negative feedback loop involving hyperphosphorylation of JAK2 and activation of secondary ERK and p38 signaling pathways AZD8931 that promote HL survival. In fact, even though the function of JAK2 was effectively inhibited as demonstrated by the abrogation of downstream STATs phosphorylation, we observed a paradoxical increase in the JAK2 and TYK2 phosphorylation status after incubation with AZD1480. Although the mechanism of AZD1480 induced JAK2 phosphorylation is currently unclear, it may be related to the conformational changes and/or induction of negative feedback loops involving activating cytokines.
Similar results were previously reported by Okuzumi et al. 33, who described paradoxical hyperphosphorylation of AKT after treatment with an ATP competitive kinase inhibitor. On the other hand, our data suggest that AZD1480 induced ERK and p38 phosphorylation may involve two major regulators of the JAK and MAPK pathways: SOCS 3 21 and SHP 2. 24 According to other reports showing sustained SHP 2 and ERK activation after SOCS 3 deletion in various in vitro and in vivo models,22,23,25 we observed a similar regulation of MAPK signaling, at least in the HD LM2 and L 428 cells that were resistant to AZD1480. These observations are consistent with a model in which SOCS 3, JAK2 and SHP 2 are reciprocally regulated as previously reported.
22 It is important to note that these molecular negative feedback loop events were associated with an increase in the level of IL 8, IP 10 and RANTES cytokines in HL cell lines supernatants. As RANTES may activate and phosphorylate JAK2 and TYK2, it is possible that the increased baseline levels of pJAK2 and pTYK2 in the HDLM2 and L428 cells, but not L 540 cells, may be related to the differential production of RANTES and other cytokines among these cell lines. Similarly, an increased secretion of these cytokines, such as RANTES, in response to AZD1480 treatment, may have also contributed to the observed JAK2 hyperphosphorylation, following incubation with AZD1480. 34 This observation should be further investigated in the clinical setting to determine whether changes in the levels of certain cytokines in serial plasma specimens may serve as a surrogate biomarker for r