Supplementary Materialssupplemental material 41419_2018_870_MOESM1_ESM. pathways, as well as increased cleavage of OPA1, a mitochondrial protein that promotes mitochondrial fusion and regulates apoptosis. Consistent with these observations, transmission electron microscopy analysis indicated that NS1619 and DHEA increased mitochondrial fission. OPA1 cleavage and cell death were inhibited by ROS scavengers and by siRNA-mediated knockdown of the mitochondrial protease OMA1, indicating the engagement of a ROS-OMA1-OPA1 axis in T-ALL cells. Furthermore, NS1619 and DHEA sensitized T-ALL cells to TRAIL-induced apoptosis. In vivo, the combination of dexamethasone and NS1619 significantly reduced the growth of a glucocorticoid-resistant patient-derived T-ALL xenograft. Taken together, our findings provide proof-of-principle for an integrated ROS-based pharmacological approach to target refractory T-ALL. Introduction Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm of precursor T-cells1. Despite significant improvements in treatment, approximately one out of five patients exhibit main or secondary resistance to current therapies2,3, which include glucocorticoids as a key component; indeed, the overall clinical outcome depends on the initial response to glucocorticoids4,5. Investigations of the genetics of T-ALL cells have identified a wide variety of mutations affecting several oncogenic pathways6C8. As more than 60% of T-ALL patients harbor activating mutations of (observe Materials and Methods). After 24?h of treatment, NS1619 and DHEA alone or in combination induced a relative increase in the cleaved OPA1 ratio. This effect was confirmed in the other T-ALL cell lines (Fig?S6A-C) and in PDX (Fig.?S6D). NS1619?+?DHEA also reduced the overall expression of OPA1 mRNA measured by qRT-PCR (Fig.?S6E), suggesting a ROS-mediated control Natamycin reversible enzyme inhibition of OPA1 expression. Open in a separate window Fig. 4 Effects of NS1619 and DHEA on OPA1.A Immunoblot of a representative experiment showing the five major OPA1 isoforms (ACE) in TALL-1 cells after 24?h of the indicated treatments. (see Materials and Methods) are shown below the blots. NAC (observe Materials and Methods) are shown below the blots. D Specific cell death of TALL-1 cells after electroporation with control siRNA (continuous lines) or OMA1-specific siRNA (dashed lines) followed by treatment with NS1619 (red), DHEA (green) or NS1619?+?DHEA (blue). Mean values of specific cell death and SE bars from three impartial experiments are shown The effects of NS1619 and DHEA on OPA1 cleavage were less obvious in the presence of NAC (Fig.?4A), indicating their ROS dependence and suggesting the involvement of OMA124,25. To test this hypothesis, we analyzed the effects of NS1619 and DHEA in TALL-1 cells following small interfering RNA (siRNA)-mediated knockdown of OMA1, which resulted in an 80% reduction of its mRNA (Fig.?4B). Interestingly, both OPA1 cleavage (Fig.?4C) and cell death (Fig.?4D) induced by NS1619 and DHEA were reduced in OMA1-silenced cells. Consistent with these findings, the cleavage of OPA1 and induction of Rabbit polyclonal to AEBP2 apoptosis (measured as cleaved Caspase 3) in response to NS1619?+?DHEA was abrogated in fibroblasts obtained from OMA1?/? mice24,33 (Fig.?S7). OPA1 controls mitochondrial function and dynamics in part by promoting mitochondrial fusion23C25,31. We therefore tested whether the increased OPA1 cleavage induced by NS1619 and DHEA was accompanied by a switch in mitochondrial morphology. Results of transmission Natamycin reversible enzyme inhibition electron microscopy analysis (Fig.?5) showed that 24?h of treatment of TALL-1 cells with DHEA alone or in combination with NS1619 significantly reduced the mean mitochondrial area, whereas circularity was unchanged, indicating a relative increase in mitochondrial fission, a finding that is consistent with a decrease in OPA1 function following its processing by OMA1. Open in a separate windows Fig. 5 Effects of NS1619?+?DHEA on mitochondrial morphology.A Representative images of electron microscopy analysis showing mitochondria of TALL-1 cells after 24?h of treatment with NS1619 and DHEA. B, C Quantification of mitochondrial area (B) and circularity (C) (observe Materials and Methods) in TALL-1 cells subjected to the indicated treatments for 24?h. The graph shows mean values and SE bars from analysis of at least 130 mitochondria per treatment NS1619 and DHEA sensitize T-ALL cells to TRAIL-induced death We next investigated whether NS1619 and DHEA sensitize T-ALL cells to killing by TRAIL, which induces apoptosis through tBid-mediated opening of the Bax/Bak pore26,34C37. As shown in Fig.?6A, TALL-1 cells exhibited a modest response to 24?h of treatment with TRAIL alone, but showed more substantial death when TRAIL was combined with NS1619?+?DHEA. Comparable results were obtained in Molt-3 and Jurkat cells, whereas CEM cells were refractory to TRAIL (Fig.?S8A-C, upper panels). qRT-PCR analysis showed that NS1619?+?DHEA induced a significant upregulation of TRAIL-receptor-2 (R2) mRNA Natamycin reversible enzyme inhibition in TALL-1 cells (Fig.?S9A). Interestingly, TRAIL-R2 mRNA levels were very low in CEM cells (Fig.?S9B), which might explain their resistance to TRAIL. Consistent Natamycin reversible enzyme inhibition with cell death results, treatment of TALL-1 cells with NS1619, DHEA, and TRAIL induced mitochondrial depolarization, release of cytochrome from mitochondria, and cleavage of Caspase 3 (Fig.?S10), which are key events Natamycin reversible enzyme inhibition in the intrinsic apoptotic.