Selective killing of human T-ALL cells: an integrated approach targeting redox homeostasis and the OMA1/OPA1 axis

Approximately 20% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients are currently incurable due to primary or secondary resistance to glucocorticoid-based therapies. Here we employed an integrated approach to selectively kill T-ALL cells by increasing mitochondrial reactive oxygen species (ROS) using NS1619, a benzimidazolone that activates the K+ (BK) channel, and dehydroepiandrosterone (DHEA), which blunts ROS scavenging through inhibition of the pentose phosphate pathway. These compounds selectively killed T-ALL cell lines, patient-derived xenografts and primary cells from patients with refractory T-ALL, but did not kill normal human thymocytes. T-ALL cells treated with NS1619 and DHEA showed activation of the ROS-responsive transcription factor NRF2, indicating engagement of antioxidant 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.

Cell death was calculated using the formula [(% dead cells in the treated sample -% dead cells in the control sample) / (100 -% dead cells in control)] x 100.
Immunoblot analysis. Protein lysates were separated by SDS/PAGE, transferred to a nitrocellulose membrane and probed with the following antibodies: mouse anti-OPA1 (BD Biosciences), rabbit anti-Caspase 3 (Cell Signaling), rabbit anti-PARP1 (Cell Signaling), rabbit anti-βactin (Sigma-Aldrich), mouse anti Hsp70 (BD Biosciences) and rabbit anti-vinculin (Cell Signaling), followed by incubation with an HRP-conjugated anti-mouse or anti-rabbit antibody (Pierce) and detection reagents (Super Signal West Femto Maximum sensitivity substrate, Thermo Fisher Scientific or LiteaBlot turbo substrate, Euroclone). Chemiluminescent signals were detected using a Cambridge UVITEC imaging system. The cleaved OPA1 ratio was calculated by dividing the signals of bands c, d, and e by the signals of all bands (see Fig. 4).
Electron microscopy. TALL-1 cells were seeded at 700,000 cells/ml and treated for 24 h with the indicated compounds. Cells were then washed in PBS and fixed with 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer, pH 7.4 for 1 hour at 4°C, postfixed with a mixture of 1% osmium tetroxide and 1% potassium ferrocyanide in 0.1M sodium cacodylate buffer for 1 hour at 4°C and incubated overnight in 0.25% uranyl acetate at 4°C. After three water washes, samples were dehydrated in a graded ethanol series and embedded in an epoxy resin (Sigma-Aldrich). Ultrathin sections (60-70 nm) were obtained with an Ultrotome V (LKB) ultramicrotome, counterstained with uranyl acetate and lead citrate and viewed with a Tecnai G 2 (FEI) transmission electron microscope operating at 100 kV.
Images were captured with a Veleta (Olympus Soft Imaging System) digital camera at 37000x magnification. At least 130 mitochondria for each sample were analyzed using ImageJ (IJ 1.46, NIH).
Regions of Interest (ROIs) were manually traced and shape descriptors were obtained to analyze mean mitochondrial area and circularity (4 x Area/Perimeter 2 ), which is a value between 0 (tubular) and 1 (circular). Computed data were analyzed using SigmaPlot to generate graphs and evaluate statistical significance (Mann-Whitney Rank Sum Test).
RNA samples were treated with DNase I (Invitrogen) for 15 minutes at 37°C followed by addition of EDTA and incubation at 70°C for 10 minutes to inactivate the enzyme. The RNA was reversetranscribed using SuperScript II reverse transcriptase (Invitrogen) and random hexamers. Aliquots of the resulting cDNA were PCR-amplified by using SYBR Green (Roche) and the following primers: β2microglobulin-s: (TGACTTTGTCACAGCCCAAG); β2-microglobulin-as: (TTCAAACCTCCATGATGCTG); TRAILR2-s: (CCAGGTGTGATTCAGGTGAA); TRAILR2-as: (CCCCACTGTGCTTTGTACCT); OMA1-s (CATTGTAGGCAGGGGCATAA); OMA1-as (CACCACAAAGAGCAATCCAAAA). The OPA1 cDNA was amplified using primers OPA1-s (TCAAGAAAAACTTGATGCTTTCA), OPA1-as (GCAGAGCTGATTATGAGTACGATT) and probe #2 from the Roche Universal Probe Library. NRF2 target genes were analyzed using TaqMan Gene Expression Assays (ThermoFisher Scientific). The PCR reactions were performed in a LightCycler 480 thermal cycler (Roche) according the manufacturer's protocol. β2-microglobulin was used as a housekeeper mRNA for the calculation of relative expression values.
RNA silencing experiments. Five million TALL-1 cells were mixed with 312.5 pMol of siRNA (Oma1#S41777 or Silencer Negative Control 1 #4390843, Thermo Scientific) and electroporated with a Neon transfection system (Thermo Scientific) using a single 1410V-30 msec pulse. Following overnight culture in complete RPMI with 30% FCS, the electroporated cells were seeded at 7X10 5 /ml in complete RPMI-10% FCS and treated as indicated in the figure legends.  Age indicates years at diagnosis, except where marked by*, which indicates age at relapse.