Poster Board III-726
TNF-related apoptosis inducing ligand (TRAIL) is a death ligand with selective antitumor activity and minimal cytotoxicity toward non-malignant tissues. TRAIL is under evaluation in several clinical trials in the treatment of diverse cancers, including hematologic malignancies. Molecular mechanisms responsible for acquired TRAIL resistance (e.g. following treatment with TRAIL) are largely unknown.
TRAIL-sensitive T-lymphoblastic leukemia cell line Jurkat was cultivated for 8 weeks with TRAIL (1mg/mL). Three TRAIL-resistant Jurkat subclones (TR1-3) were derived and subjected to analysis. Acquired TRAIL-resistance was asociated with decreased sensitivity to several of the tested cytotoxic agents (including TNF-alpha, Fas-ligand, fludarabine and methotrexate), while sensitivity to other agents was unchanged (doxorubicin, cisplatin, etoposid) or even increased (cytarabine). TRAIL-resistant subclones did not show significant differences either in the cell surface expression of death ligand receptors (TRAIL-R1-4, TNF-R, Fas) or in the protein levels of key antiapoptotic regulators (e.g. Bcl2, Mcl1, XIAP, Survivin). Surprisingly, the most prominent finding was significant upregulation of apical proapoptotic caspase 10 (CASP10) in subclone TR1 on mRNA (3-fold increase) as well as on protein level. Moreover, downregulation of CASP8 was detected in subclone TR1 both by western blotting and real-time RT-PCR. siRNA-induced inhibition of protein CASP10 in subclone TR1 was associated with partial restoration of sensitivity to TRAIL-induced apoptosis. No relevant mutations were revealed by sequencing of CASP10 transcript variants A and D of TR1 subclone compared to TRAIL-sensitive Jurkat cells. Immunoprecipitation (IP) analysis of death-inducing signaling complex (DISC) using biotinylated TRAIL suggested impaired DICS formation in subclones TR2 and TR3, but not in TR1. DISC analysis also demonstrated that CASP10 was physically bound in the DISC of TR1 subclone after exposure to TRAIL. Preincubation of the TR subclones with several histone-deacetylase inhibitors (HDACi: vorinostat, sodium valproate or sodium butyrate) for 12 hours almost completely restored sensitivity to TRAIL. Immunoprecipitation analysis of DISC showed treatment with HDACi for 12h resulted in substantially increased binding of precleaved CASP8 (55kD and 43kD) and precleaved CASP10 (53kD) to the DISC. In subclone TR1 pretreatment with HDACi was associated with downregulation of overexpresssed CASP10 mRNA to the levels detected in the original Jurkat cell line. Genome-wide gene-expression profiling using Illumina chips unveiled additional changes in the transcriptome associated with acquired TRAIL resistance. Across all microarrays, the most statistically significant gene expression change was upregulation of Midkine, a heparin-binding growth factor presumably involved in the protection of cancer cells against TRAIL.
We showed that acquired TRAIL resistance of Jurkat T-lymphoblastic leukemia cells was associated with complex disruption of both extrinsic and intrinsic apoptotic pathways. While impaired formation of DISC appeared a major molecular mechanism underlying the death-ligand resistance of subclones TR2-3, deregulated expression of apical caspases 8 and 10 substantially contributed to resistance of subclone TR1. RNA interference experiments demonstrated downregulation of otherwise proapoptotic CASP10 functionally impaired extrinsic apoptotic pathway and results in increase apoptotic response to TRAIL. We speculate that displacement of CASP8 by overexpressed CASP10 from DISC might partially block downstream conveying of the proapoptotic signaling from aggregated death receptors. The microarray data implicated that additional molecular deregulations (e.g. overexpression of midkine) associated with acquired TRAIL resistance might contribute to the “multi-drug” resistant phenotype of TRAIL-resistant subclones. The ability of HDACi to restore sensitivity of TRAIL-resistant Jurkat subclones to TRAIL might influence design of novel experimental strategies in the treatment of cancer.
Financial Support: LC 06044, MSM 0021620806, MSM 0021620808, GAUK 2009/259153/86309, GAUK 259211/110709
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.