Fig. 1: Mitochondrial fusion is an AML dependency.
From: Mitochondrial fusion is a therapeutic vulnerability of acute myeloid leukemia
A–E MOLM-14 and OCI-AML2 cells were transduced with doxycycline (dox) inducible shRNA against MFN1, MFN2, OPA1, MFF or DRP1, or control (CTL) shRNA (n = 3). A Western blots performed 72 h after dox using anti-MFN1, -MFN2, -OPA1, -MFF, -DRP1 and -β-actin (ACTB) antibodies. B Cell death measured by DAPI staining 72 h after dox. C Cell proliferation measured by daily counting using trypan blue exclusion assay for the indicated time after dox. D Electron microscopy imaging at a 7100x magnification five days after dox. Scale bars = 5 μm. E Quantification of mitochondria (Mt) length (left panel) and area (right panel) in control, or MFN2- or OPA1-depleted MOLM-14 cells. Each dot represents Mt length in μm or area in μ2 (n = 30 cells, 5–50 Mt were measured in each cell). F–K PDX AML cells or normal immature CD34+ hematopoietic cells were transduced with mCherry-tagged shRNA targeting MFN2 or OPA1, or CTL shRNAs. In these mCherry+ transduced cells (input), we measured mitochondrial length (readout), performed ex vivo methylcellulose cultures to measure leukemia colony-forming units (L-CFUs), and evaluated the proportion of mCherry+ compared to mCherry- cells (output). G Quantification of MFN2 or OPA1 transcripts relative to cells transduced with CTL shRNA in PDX AML cells (n = 6 different PDXs). H L-CFU assays with PDX AML samples scored after 7–10 days (shMFN2, n = 8 different PDXs; shOPA1, n = 6 different PDXs). I, J mCherry staining in human AML cells transduced by mCherry+ lentiviral vectors before or after methylcellulose culture. I Representative contour plots in CTL or MFN2 shRNA conditions. J Ratio of mCherry+ cells between the input (before methylcellulose) and the output (after methylcellulose) (n = 6). K Quantification of erythroid (BFU-E, left panel) and myeloid (CFU-GM, right panel) colonies from CD34+ cells after MFN2 or OPA1 depletion under an inverted microscope after 10 days of methylcellulose culture (n = 4). Vertical bars indicate standard deviations. ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
