Ezatiostat Hydrochloride

Glutathione S‐transferase pi (GSTP1), a phase II detoxification enzyme, is known to be overexpressed and mediates chemotherapeutic resistance in lung cancer. However, whether GSTP1 supports cancer stem cells (CSCs) and the underlying mechanisms in lung adenocarcinoma (LUAD) remain largely unknown. This study unveiled that GSTP1 is upregulated in lung CSCs and supports tumor self‐renewal, metastasis, and resistance to targeted tyrosine kinase inhibitors of LUAD both in vitro and in vivo. Mechanistically, CaMK2A (calcium/calmodulin‐dependent protein kinase 2 isoform A)/NRF2 (nuclear factor erythroid 2‐related factor 2)/GSTP1 is uncovered as a regulatory axis under hypoxia. CaMK2A increased GSTP1 expression through phosphorylating the Sersine558 residue of NRF2 and promoting its nuclear translocation, a novel mechanism for NRF2 activation apart from conventional oxidization‐dependent activation. Upregulation of GSTP1 in turn suppressed reactive oxygen species levels and supported CSC phenotypes. Clinically, GSTP1 analyzed by immunohistochemistry is upregulated in a proportion of LUAD and serves as a prognostic marker for survival. Using patient‐derived organoids from an ALK‐translocated LUAD, the therapeutic potential of a specific GSTP1 inhibitor ezatiostat in combination treatment with the ALK inhibitor crizotinib is demonstrated. This study demonstrates GSTP1 to be a promising therapeutic target for long‐term control of LUAD through targeting CSCs.

To test this hypothesis, we first preformed mouse aortic endothelial cell (MAEC) outgrowth assays on aortic segments dissected from mice homozygous (KO) and heterozygous (Het) for GCLm knockout as well as wildtype controls (WT). VEGF stimulated chemotaxis was determined using the under‐agarose migration assay. Primary human aortic endothelial cells (HAEC) were purchased from Lonza and cultured following suppliers’ recomendations. Phospho‐Y1175 VEGFR2/total VEGFR2 and phospho‐Y416 proto‐oncogene c‐Src (Src)/total Src were determined by western blotting.

Outgrowth area in the Het group was significantly larger than WT and KO at day 7 (2.15 fold, n=4, P<0.001). Het MAECs were 1.25 fold faster (n=4 p<0.001) while KO MAECs were 0.90 fold slower than WT (n=3, p<0.05) migrating towards VEGF‐A. Concurrently, we inhibited glutathione reductase in HAECs using 2‐acetylamino‐3‐[4‐(2‐acetylamino‐2‐carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2‐AAPA). 2‐AAPA (75 µM) alone didn’t significantly activate VEGFR2. However, 2‐AAPA (75 µM) in combination with H2O2 (100 µM) induced a 5.5 fold (p<0.01, n=4) increase in the relative amount of active VEGFR2 compared to vehicle treated HAECs. 2‐AAPA both with and without H2O2 induced a significant increase in active Src, 5.3 and 9.0 fold (p<0.05, p<0.01, n=6) respectively. This effect was attenuated by the addition of Dithiothreitol (1mM). Furthermore, Src kinase inhibitor 1 (SKI) (200 nM) attenuated the effect of 2‐AAPA + H2O2 on VEGFR2. While the VEGFR2 kinase inhibitor SU1948 inhibited activation of VEGFR2, it had no significant effect on the 2‐AAPA + H2O2 mediated activation of Src. Glutathione S‐transferase P1 (GSTP1) catalyzes the enzymatic glutathionylation of proteins, The GSTP1 inhibitor TLK199 (50 µM) abrogated the effects of 2‐AAPA + H2O2 on VEGFR2.

Combined these data suggest Src mediates the 2‐AAPA + H2O2 activation of VEGFR2. And that glutathionylation is necessary for this effect, co‐immunoprecipitation experiments are underway to determine the glutathionylation status of Src in our system and its effect on the interaction of Src and VEGFR2.