BCR-ABL Regulates Death Receptor Expression for TNF-Related Apoptosis-Inducing Ligand (TRAIL) in Philadelphia Chromosome-Positive Leukemia

Abstract 2740

Allogeneic stem cell transplantation (allo-SCT) is potentially curative therapy for CML and Ph+ ALL patients. Graft-versus-leukemia (GVL) effect plays a central role in eradication of residual leukemia after allo-SCT, and GVL effect is mediated by cytotoxic T-lymphocytes (CTLs) and NK cells though cytotoxic ligands. TNF-related apoptosis-inducing ligand (TRAIL) is one of cytotoxic ligands expressed on CTLs and NK cells, and it has been shown that TRAIL mediates GVL effect in mice model. We and others previously demonstrated that Ph+ leukemic cells frequently express death-inducing receptors (DR4 and DR5) for TRAIL resulting in high sensitivity to pro-apoptotic activity of recombinant human soluble TRAIL (rhsTRAIL), suggesting that TRAIL-DR4/DR5 interaction mediates GVL effect against Ph+ leukemia cells at least in part. Despite the fact that TRAIL mediates tumor-specific immunity and plays a role in GVL effect, mechanisms of the DR4 and DR5 expression in leukemia cells have yet to be elucidated fully. Since we found that Ph+ leukemia cells express DR4 and/or DR5 more frequently than Ph- leukemia cells, we assumed that BCR-ABL plays a role in DR4 and DR5 expression in Ph+ leukemia cells. To verify this assumption, we analyzed the effect of imatinib on DR4 and DR5 expression in Ph+ leukemia cells. Of note, imatinib effectively downregulated gene and cell surface expression of DR4 and DR5 in all of the 12 Ph+ leukemia cell lines tested, while it did not in Ph- leukemia cell lines. Moreover, imatinib downregulated DR4 and DR5 expression on primary samples from Ph+ ALL patients. Not only imatinib but also second-generation TKIs, dasatinib and nilotinib, downregulated DR4 and DR5 expression of Ph+ leukemia cell lines. Imatinib also downregulated promoter activities of the DR4 and DR5 gene in luciferase assay. In contrast, imatinib failed to downregulate gene and cell surface expression of DR4 and DR5 in Ph+ ALL cell line having T315I mutation of BCR-ABL. These observations strongly suggested that BCR-ABL plays a role in DR4 and DR5 expression of Ph+ leukemia cells. To further verify this possibility, we transfected shRNA against bcr-abl using lentivirus vector into Ph+ leukemia cell line, and found downregulation of gene and cell surface expression of DR4 and DR5. Moreover, transfection of bcr-abl into Ph- leukemia cell line TF-1 induced gene and cell surface expression of DR4 and DR5 that was abrogated by imatinib treatment. Since BCR-ABL promotes growth and survival of Ph+ leukemia cells though activation of the MAPK, PI3K, and JAK/STAT pathways, we tested the effect of inhibitor of each pathway on the DR4 and DR5 expression of Ph+ leukemia cell lines. U0126, an inhibitor of the MAPK pathway, and LY294002, an inhibitor of the PI3K pathway, but not SD1029, an inhibitor of the JAK/STAT pathway, partially downregulated gene and cell surface expression of DR4 and DR5. These observations demonstrated a unique finding that an oncogenic fusion product derived form chromosomal translocation is implicated in DR4 and DR5 expression on leukemia cells, and provide new insight into the mechanisms of tumor-specific cytotoxic activities of TRAIL. Considering that imatinib downregulates cell surface expression of DR4 and DR5 in Ph+ leukemia cells, it is assumed that the pro-apoptotic activity of TRAIL against Ph+ leukemia cells may be functionally impaired by pretreatment with imatinib. Thus, we analyzed the effect of imatinib on the sensitivity of Ph+ leukemia cell lines to rhsTRAIL, and found that imatinib pretreatment impaired the pro-apoptotic activity of rhsTRAIL in 6 of the 7 TRAIL-sensitive Ph+ leukemia cell lines tested. Of note, imatinib pretreatment of Ph+ ALL cell line, in which DR4 and DR5 expression was not downregulated due to the T315I mutation, did not impair TRAIL sensitivity whereas imatinib pretreatment of its parental cell line with intact BCR-ABL substantially impaired TRAIL sensitivity. These observations suggested that imatinib partially attenuate the in vivo activity of CTLs and NK cells against Ph+ leukemia cells that is mediated by the TRAIL-DR4/DR5 interaction. Since imatinib is now widely used for prophylaxis of relapse and controlling hematological relapse of patients with CML and Ph+ ALL after allo-SCT, our findings are important from the clinical point of view, and suggest that careful observation is required in the clinical use of imatinib during the post-transplant period.