CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

Abstract

Azathioprine and its metabolite 6-mercaptopurine (6-MP) are immunosuppressive drugs that are used in organ transplantation and autoimmune and chronic inflammatory diseases such as Crohn disease. However, their molecular mechanism of action is unknown. In the present study, we have identified a unique and unexpected role for azathioprine and its metabolites in the control of T cell apoptosis by modulation of Rac1 activation upon CD28 costimulation. We found that azathioprine and its metabolites induced apoptosis of T cells from patients with Crohn disease and control patients. Apoptosis induction required costimulation with CD28 and was mediated by specific blockade of Rac1 activation through binding of azathioprine-generated 6-thioguanine triphosphate (6-Thio-GTP) to Rac1 instead of GTP. The activation of Rac1 target genes such as mitogen-activated protein kinase kinase (MEK), NF-κB, and bcl-x L was suppressed by azathioprine, leading to a mitochondrial pathway of apoptosis. Azathioprine thus converts a costimulatory signal into an apoptotic signal by modulating Rac1 activity. These findings explain the immunosuppressive effects of azathioprine and suggest that 6-Thio-GTP derivates may be useful as potent immunosuppressive agents in autoimmune diseases and organ transplantation.

Targeting Pancreatic Cancer Metastasis by Inhibition of Vav1, a Driver of Tumor Cell Invasion

“…DanG cells were treated with 5 μM azathioprine for 48 hours, and Rac activation was analyzed using a p21-binding protein pulldown assay for active Rac/Cdc42. Indeed, azathioprine treatment caused approximately a 30–40% reduction in Rac activation, consistent with previous reports (Figure 3A, (11, 12)). However, a reduction in Rac activation was not observed in PANC1 cells, which do not express Vav1 (Figure 3B).…”

“…To determine if azathioprine was specific for Vav1, we took advantage of multiple pancreatic cancer cell lines, some of which express Vav1 (DanG, CFPAC, Panc04.03), and some of which do not (PANC1, BxPC3, L3.6) (Supplemental Figure 1, (7–9)). The cells were pre-treated with or without azathioprine for two days at 5 μM, a dose that is reported to be physiologically relevant and comparable to that in patients under azathioprine treatment (12). Azathioprine dramatically reduced transwell invasion by DanG, CFPAC, and Panc04.03 cells (Figure 1C), similar to siRNA-mediated depletion of Vav1 (9).…”

“…This study was based on the premise that azathioprine can inhibit Vav1-dependent Rac activation in primary T cells (11, 12). Therefore, we tested if azathioprine affected Rac activation in pancreatic tumor cells.…”

“…However, it was unclear how azathioprine could induce immune-specific effects. Further, levels of 6-thioguanine incorporation into the DNA did not appear to be sufficient to explain its cytotoxic effects (11, 12, 21). The recent finding that azathioprine inhibits Vav/Rac signaling was quite surprising, but is consistent with both the hematopoietic-specific effects of azathioprine and the hematopoietic expression pattern of Vav1.…”

A drug‐repositioning screen for primary pancreatic ductal adenocarcinoma cells identifies 6‐thioguanine as an effective therapeutic agent for TPMT‐low cancer cells

“…Its toxicity is mainly caused by the incorporation of 6‐TN into DNA during the S phase of the cell cycle (Lennard et al ., 1989), thereby indicating its effectiveness in cancer. Alternatively, it can inactivate a GTP‐binding protein such as Rac1 (Tiede et al ., 2003). Considering that PDAC is associated with a frequent KRas activating mutation, further research is needed to clarify whether 6‐TG can inhibit mutant KRas signaling.…”