Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Mediates and Sustains NF-κb Constitutive Activation in LGL Leukemia Cells

Large granular lymphocyte (LGL) leukemia results from clonal expansion of CD3⁺ cytotoxic T-lymphocytes (CTL) or CD3^-natural killer (NK) cells. Chronic antigen stimulation promotes long-term survival of LGL leukemia cells through constitutive activation of multiple survival pathways, leading to global dysregulation of apoptosis. Clinical manifestations of LGL leukemia include neutropenia, anemia and rheumatoid arthritis. Treatment for LGL leukemia patients relies on immunosuppressives such as methotrexate and is not curative. No standard therapy has been established. We reported that nuclear factor kappa B (NF-kB) is central to the leukemic LGL survival network, but the mechanisms of constitutive NF-kB activation in LGL leukemia are undefined. TNF-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis and activates NF-kB via binding to TRAIL receptor (TR) 1, 2 and decoy receptor 2 (DcR2). DcR2 is uniquely unable to transduce downstream death signals yet retains NF-kB transduction activity. The mechanisms of TRAIL expression and regulation in LGL leukemia are unknown. Thus the current study investigates these mechanisms and their potential therapeutic applications, with the use of NF-kB inhibitors ixazomib and bortezomib, in LGL leukemia.

Methods: LGL leukemia cell lines TL1 (T-LGL) and NKL (NK-LGL), peripheral blood mononuclear cells (PBMC) from LGL leukemia patients, and PBMC from normal donors were studied. NF-kB DNA binding activity was determined by EMSA. Results were confirmed using probe-based NF-kB (p50/65) transcription factor assay and immunocytochemistry (ICC). Serum TRAIL levels were detected by ELISA. Cellular TRAIL expression was determined by real-time PCR, western blot and ICC. DcR2 and Mcl-1 siRNA knock-down was performed with electroporation. Flow cytometry was used to detect TR 1-3 and DcR2 expression and apoptosis.

Results: The average serum levels of TRAIL in LGL leukemia patients were nearly 4-fold higher than normal (NL) control values (p ≤ 0.0001). Data from RT-PCR (p ≤ 0.04), western blot and ICC revealed that LGL leukemia cells were the major source of TRAIL overexpression. Identical expression levels of TR1, 2 and 3 were observed in PBMC from LGL leukemia patients and from NL controls. Like normal PBMC, LGL leukemia cells were resistant to TRAIL-induced apoptosis. In contrast, the expression frequency of DcR2 was at least 4-fold greater in LGL leukemia PBMC compared to NL control, and it correlated to the percentage of circulating LGL leukemia cells. We found that TRAIL activates NF-kB and NF-kB downstream target genes, including TRAIL and McL-1, in LGL leukemia samples. To confirm that TRAIL is responsible for constitutive NF-kB activation in LGL leukemia, T-LGL leukemia PBMC were treated with pooled sera from 3 each of either NL controls or T-LGL leukemia patients. Leukemia sera increased NF-kB activity on EMSA, and this effect was completely blocked by TRAIL neutralizing antibody. DcR2 siRNA knockdown specifically decreased RelA and NF-kB1 (p105/p50) levels in TL1 and NKL cells. Mcl-1 siRNA mediated increased apoptosis in the same cell lines. Likewise, ixazomib and bortezomib facilitated leukemia-selective apoptosis in LGL leukemia cell lines and in patient PBMC, via inhibition of NF-kB activity and of downstream targets (ixazomib, p≤0.0001; bortezomib, p≤0.03). Additionally, caspase-3 and PARP cleavage were observed in LGL leukemia cells treated with ixazomib or bortezomib. Serum TRAIL levels in LGL leukemia patients were significantly lower in methotrexate responders versus non-responders, corresponding with reduced NF-kB DNA binding activity and increased absolute neutrophil counts, indicative of treatment response.

Conclusion: These data indicate that expression of DcR2 and constitutive activation of NF-kB are responsible for TRAIL resistance in leukemic LGLs. TRAIL triggers prolonged NF-kB activation via interaction with DcR2, and activated NF-kB in turn promotes further TRAIL production in leukemic LGLs, creating a TRAIL autocrine regulatory loop. Inhibition of NF-kB activity with ixazomib and bortezomib interrupts this loop, impairs expression of Mcl-1 and induces apoptosis of leukemia cells. Our preclinical findings provide a solid framework for clinical evaluations of ixazomib and bortezomib in the treatment of LGL leukemia.