The pyrrolo-1,5-benzoxazepine, PBOX-15, enhances TRAIL-induced apoptosis by upregulation of DR5 and downregulation of core cell survival proteins in acute lymphoblastic leukaemia cells

Abstract

Apoptotic defects are frequently associated with poor outcome in pediatric acute lymphoblastic leukaemia (ALL) hence there is an ongoing demand for novel strategies that counteract apoptotic resistance. The death ligand TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) and its selective tumour receptor system has attracted exceptional clinical interest. However, many malignancies including ALL are resistant to TRAIL monotherapy. Tumour resistance can be overcome by drug combination therapy. TRAIL and its agonist antibodies are currently undergoing phase II clinical trials with established chemotherapeutics. Herein, we present promising therapeutic benefits in combining TRAIL with the selective anti-leukaemic agents, the pyrrolo-1,5-benzoxazepines (PBOXs) for the treatment of ALL. PBOX-15 synergistically enhanced apoptosis induced by TRAIL and a DR5-selective TRAIL variant in ALL-derived cells. PBOX-15 enhanced TRAIL-induced apoptosis by dual activation of extrinsic and intrinsic apoptotic pathways. The specific caspase-8 inhibitor, Z-IETD-FMK, identified the extrinsic pathway as the principal mode of apoptosis. We demonstrate that PBOX-15 can enhance TRAIL-induced apoptosis by upregulation of DR5, reduction of cellular mitochondrial potential, activation of the caspase cascade and downregulation of PI3K/Akt, c-FLIP, Mcl-1 and IAP survival pathways. Of note, the PI3K pathway inhibitor LY-294002 significantly enhanced the apoptotic potential of TRAIL and PBOX-15 validating the importance of Akt downregulation in the TRAIL/PBOX-15 synergistic combination. Considering the lack of cytotoxicity to normal cells and ability to downregulate several survival pathways, PBOX-15 may represent an effective agent for use in combination with TRAIL for the treatment of ALL.

Pre-clinical evaluation of a novel class of anti-cancer agents, the Pyrrolo-1, 5-benzoxazepines.

“…Group one (PBOX-1, -2, and -21) do not induce apoptosis in cancer cells 3, 5, 8, 9. Group two (PBOX-3, -4, -5, -6, -15 and -16) induce apoptosis in cancer cells 3-6, 10-28. The non-apoptotic subset of the PBOXs (PBOX-1,-2,-21) exert their anti-proliferative effects by inducing a G1 cell cycle arrest 2, 8, 9.…”

“…It is noteworthy that PBOX-induced apoptosis can also occur independent of change to Bcl-2 32, 21. To date, PBOX-6- and PBOX-15-induced apoptosis associates with a decrease in Mcl-1 expression 21, 23, 24, 27, 28. SiRNA-mediated downregulation of Mcl-1 augmented PBOX-6-induced apoptosis, suggesting that downregulation of Mcl-1 enhances or facilitates PBOX-6-induced apoptosis, rather than being the effective target 16.…”

“…Extracellular death ligands such as TNF-related apoptosis-inducing ligand (TRAIL) interact with its corresponding death receptor/s (DR4, DR5) leading to the formation of the death-inducing signalling complex and subsequent activation of caspase-8 50. PBOX-15 can also trigger apoptosis, albeit to a lesser extent, by the extrinsic pathway 21, 28. PBOX-15 increased DR5 expression in malignant myeloma and T-cell acute lymphoblastic leukemia (T-ALL) cells facilitating enhanced activation of the extrinsic pathway 21, 28.…”

“…PBOX-15 can also trigger apoptosis, albeit to a lesser extent, by the extrinsic pathway 21, 28. PBOX-15 increased DR5 expression in malignant myeloma and T-cell acute lymphoblastic leukemia (T-ALL) cells facilitating enhanced activation of the extrinsic pathway 21, 28. Accordingly, activation of the extrinsic pathway initiator caspase, caspase-8 by PBOX-15 was identified in CLL, malignant myeloma and T-ALL cells 21, 28, 32.…”

“…PBOX-15 increased DR5 expression in malignant myeloma and T-cell acute lymphoblastic leukemia (T-ALL) cells facilitating enhanced activation of the extrinsic pathway 21, 28. Accordingly, activation of the extrinsic pathway initiator caspase, caspase-8 by PBOX-15 was identified in CLL, malignant myeloma and T-ALL cells 21, 28, 32. The addition of the DR5 ligand TRAIL to cells further enhanced PBOX-15-induced activation of the extrinsic pathway 21, 28.…”