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The mitochondrial translation machinery as a therapeutic target in Myc-driven lymphomas

The oncogenic transcription factor Myc is required for the progression and maintenance of diverse tumors. This has led to the concept that Myc itself, Myc-activated gene products, or associated biological processes might constitute prime targets for cancer therapy. Here, we present an in vivo reverse-genetic screen targeting a set of 241 Myc-activated mRNAs in mouse B-cell lymphomas, unraveling a critical role for the mitochondrial ribosomal protein (MRP) Ptcd3 in tumor maintenance. Other MRP-coding genes were also up regulated in Myc-induced lymphoma, pointing to a coordinate activation of the mitochondrial translation machinery. Inhibition of mitochondrial translation with the antibiotic Tigecycline was synthetic-lethal with Myc activation, impaired respiratory activity and tumor cell survival in vitro, and significantly extended lifespan in lymphoma-bearing mice. We have thus identified a novel Myc-induced metabolic dependency that can be targeted by common antibiotics, opening new therapeutic perspectives in Myc-overexpressing tumors.

1x10 6 cells were transplanted by tail-vein injection into syngeneic C57BL/6 females for in vivo expansion. Genomic DNA was used to determined shRNA distributions in the tumor populations by Illumina sequencing and depleted shRNAs (see below) selected for the in vitro secondary validation screen. In the secondary screen viral preparations were titrated in order to achieve a transduction efficiency of ~10 % to ~60%, and the fraction of GFP + cells was measured 2 d after infection (day 0) and again after 10 day of culture.

-Tumor sequencing and Analysis
For evaluating the shRNA distributions in tumor cell populations before and after passage in vivo, we designed a PCR-based barcoding strategy. Briefly, three different lymphomas were collected and processed from each mouse. DNA was extracted from lymphoma cell pellets using the Gentra Puregene kit (Qiagen). shRNA sequences integrated into genomic DNA were recovered by PCR amplification with primers annealing to the shRNA loop (forward primer) and the miR30 region (reverse primer).
The primers also contained the P5 and P7 adaptor sequences required for sequencing on the Illumina Hi-Seq 2000 platform. Furthermore, the forward primer carried a sixnucleotide barcode sequence between the loop-and the P7-sequence (modified from Scuoppo et al., 2012). Approximately 10x10 6 50-base-pair reads were acquired for each sample. shRNAs were identified by sequence alignment using the BWA algorithm (Li & Durbin, 2009). To determine shRNA distributions among different samples, raw read counts were normalized based on the size of each sequencing library by dividing each value to the number of total aligned reads for that sample and multiplying to 10^6. shRNA abundance ratios were calculated, for each shRNA, as the number of normalized reads in each tumor divided by the number of normalized reads at T0 (before injection).
Normalized Fold Changes from three lymphomas/mouse were then averaged and log2 transformed. See Supplemental supplemental table S7 for the full list of primers.

Cell cycle kinetics
For BrdU pulse-chase experiment Eµ-myc lymphoma cells transduced with either empty vector (EV) or shPTCD3 #4 were grown in the presence of 33 µM BrdU for 15 minutes. At the end of BrdU incubation the medium was removed, cells were washed twice with PBS, supplemented with fresh medium and maintained in culture over the time-course. At 0, 2, 4, 6, 8, 10, 12, 14 and 24 hours, 3x10 6 cells were collected, fixed in 70% Ethanol and kept at +4 • C before staining. Staining was performed as described previously with minor modifications (Erba et al., 1999). For detection of BrdU incorporation into DNA, the fixed cells were washed with cold PBS and the DNA was denatured with 1 ml of 3N hydrochloric acid for 20 min at room temperature. DNA denaturation was stopped by adding 3 ml of 0.1M sodium tetraborate pH 8.5. After centrifugation, the pellet was washed once with 1 ml of PBS+1% BSA and incubated with a mouse monoclonal anti-BrdU antibody (Becton Dickinson, cat. #340649) diluted 1:5 in PBS+1% BSA and kept for 1 h at room temperature in the dark. The pellet was washed once with 1 ml of PBS+1% BSA before incubation with an allophycocyanin (APC)conjugated donkey anti-mouse secondary antibody (Invitrogen, cat. #A-31571) diluted 1:100 in PBS+1%BSA for 1 h at room temperature in the dark. The cells were finally resuspended in 1 ml of a solution containing 2.5 µg/ml of propidium iodide (PI) in PBS and 40µg/ml RNAse A, and stained overnight at 4°C in the dark.
For DNA content analysis cells were fixed with 70% EtOH, resuspended in a solution containing 50 µg/ml of propidium iodide (PI) in PBS and 40µg/ml RNAse A and stained overnight at 4°C in the dark. Samples were acquired on a FACSCalibur flow cytometer (Becton Dickinson).

Electron Microscopy
After treatment of cells in suspension with 1 or 2.5 µM Tigecycline for 12, 24 and 48 hrs, cells were fixed with 1% glutaraldehyde for 1 hour and then with reduced OsO4 for 2 hours and thiocarbohydrazide as described in (Polishchuk et al., 1999) and (Cutrona et al., 2013) and embedded into Epon. Standard 60 nm sections with the thicknes fo 60 nm were examined udner the Tecnai 20 electron microscope and the severity of mitochondrial damages was evaluated blindly by two experts.

Assessment of Tigecycline Activity in Mouse lymphomas models
For in vivo treatment with tigecycline, 2x10 5 Eµ-myc lymphoma cells (LY36 or LY35) were transplanted by tail-vein injection into 6-8 weeks old healthy syngeneic C57Bl/6 recipient mice. Mice were monitored daily for lymphoma development by peripheral lymph node palpation. Treatment with tigecycline was started when lymphnodes became palpable and mice showed evident signs of disease. For the xenograft model of human Burkitt's lymphoma, 10 7 Raji cells were transplanted subcutaneously in previously irradiated (3 Gray) nude mice (Hudson et al., 1998). Treatment with tigecycline started upon the appearance of measurable tumors. Tumor volumes was assessed from the start of the treatment every two days with a digital caliper. In both cases we used the following treatment scheme: Two rounds of treatment twice a day for 5 days, with two days off at each round, for a total of 14 days and 20 treatments. Time interval between the two injections was ~6 hours. Tigecycline was dissolved in saline solution (0.8% NaCl) at 100mg/kg and was freshly prepared from the dry powder at each injection.
For Blood analysis, 50µl of blood was collected by tail-vein bleeding and 10µl 0.5M EDTA was immediately added to prevent coagulation. Whole blood was analysed using the Hematological Analyzer (Beckman Dickinson). White Blood Cells (WBC) were isolated from spleen by mechanical disruption and filtration thruogh a 70µM nylon cell strainer. Following lysis of erythrocytes peripheral or splenic WBC were stained with an anti-B220 PE-cy7 antibody (#12-0452-82, eBiosciences) and analyzed by flow-cytometry on a FACSCalibur (BD biosciences). Treatment with 2.5µM led to a more consistent reduction in number and lenght of cristae