Mitochondrial fusion is a therapeutic vulnerability of acute myeloid leukemia

Mitochondrial metabolism recently emerged as a critical dependency in acute myeloid leukemia (AML). The shape of mitochondria is tightly regulated by dynamin GTPase proteins, which drive opposing fusion and fission forces to consistently adapt bioenergetics to the cellular context. Here, we showed that targeting mitochondrial fusion was a new vulnerability of AML cells, when assayed in patient-derived xenograft (PDX) models. Genetic depletion of mitofusin 2 (MFN2) or optic atrophy 1 (OPA1) or pharmacological inhibition of OPA1 (MYLS22) blocked mitochondrial fusion and had significant anti-leukemic activity, while having limited impact on normal hematopoietic cells ex vivo and in vivo. Mechanistically, inhibition of mitochondrial fusion disrupted mitochondrial respiration and reactive oxygen species production, leading to cell cycle arrest at the G0/G1 transition. These results nominate the inhibition of mitochondrial fusion as a promising therapeutic approach for AML.


Lentiviral production
Briefly, we transfected HEK293T/17 cells with different plasmids together with the packaging plasmids

Human samples
Twelve-well plates were coated with 500µl retronectin (20 µg/mL in PBS) overnight at 4°C. Next, plates were blocked with 1 mL of 2% BSA PBS for 30 minutes at room temperature. Next, wells were washed once with PBS, 600 µL of concentrated lentiviral supernatant was added and plates were centrifuged (4000 rpm, 3h, 4°C) to favor virus adhesion to retronectin. After centrifugation, 0.4 mL of the viral supernatant was discarded and 1mL of the PCM culture medium containing 1-5×10 6 cells was added to each well. Plates were next centrifuged (1500 rpm, 10 minutes, RT) and incubated at 37°C for 24h.

Bioenergetic analysis assays
Oxygen consumption rate (OCR) was measured using a Seahorse XF96 extracellular flux analyzer (Seahorse after 40 min and Antimycin A/Rotenone (1µM) after 59 min.

L-CFU assays
L-CFU assays were performed as previously described 2 . Briefly, AML cells were seeded at 10 5 /mL in H4230 medium (StemCell Technologies, Vancouver, Canada) supplemented with 10% PCM. At day 7, L-CFU (colony of > 20 cells) were scored under an inverted microscope.

Normal hematopoietic progenitor clonogenic assays
Normal CD34+ hematopoietic cells were purchased from StemCell Technologies. Cells were seeded at Intracellular phosphoflow for the detection of mTORC1 activity was carried out using anti-phospho-S6R (CST). Briefly, 0.5-1x10 6 PDX AML cells were fixed 15 min in 4% paraformaldehyde (Sigma-Aldrich) and permeabilized with cold methanol for at least one hour. Next, cells were stained with anti-phospho-S6 ribosomal protein antibody for 1 hour at RT and resuspended in PBS.

Immunohistochemistry
Femurs and tibias of mice were fixed for 24h in 4% paraformaldehyde. Decalcification was carried out using 15% formic acid at 4°C for 4h, followed by a second fixation in 4% paraformaldehyde for 24h. Samples were paraffin embedded and then sliced using a ultracut E microtome (Reichert-Jung). Images were acquired and processed using the slide scanner and software Zeiss Axioscan.Z1 (Carl Zeiss AG, Oberkochen, Germany). Detection of human AML cells transduced with a mCherry-expressing lentiviral vector was done using a LSM700 confocal microscope (Zeiss, Stuttgart, Germany).

Gene chip hybridization
RNA was extracted using a RNeasy Mini Kit (Qiagen, Redwood City, CA, USA) and quality was evaluated with a Bioanalyzer 2100 (using Agilent RNA6000 nano chip kit), and 100 ng of total RNA was reverse transcribed using the GeneChip® WT Plus Reagent Kit according to the manufacturer's instructions (Affymetrix, Thermo Fischer Scientific). Briefly, cDNA were hybridized to GeneChip® Clariom S Human

Gene set enrichment analysis
We set a filter for those genes that displayed at least a ≥1,5 or ≤-1,5 fold difference in expression between groups and achieved an FDR of <0.05. Data were then interrogated for evidence of biologic pathway dysregulation using Gene set enrichment analysis (GSEA, Broad Institute). Enrichment rates were considered as significant for P-value <0.05 and FDR ≤0.1.

Quantitative PCR
Total RNA was prepared with GenElute Mammalian Total RNA miniprep (Sigma-Aldrich

Statistics
Differences between the mean values obtained for the experimental groups were analyzed using the two-