Fig 2: PKM2 directly binds to and stabilizes VDAC3.a VDAC3 peptide sequences were observed in the 31kDa band. Flag pull down and MS analysis of PKM2 associated proteins purified from transfected HCT116 cells. b Co-immunoprecipitation analysis showed that only VDAC3 binds directly to PKM2. c Western blot analysis of the endogenous interaction of PKM2 and VDAC3. HCT116 cell lysates were subject to immunoprecipitation with control IgG and anti-VDAC3 antibodies. d, e In vitro binding assay of purified His-tagged-PKM2 and GST-tagged-VDAC3. f K433E enhanced the interaction of PKM2 and VDAC3. Stagged-WT PKM2, a succinylation mimic K433E and a succinylation null mutant K433R and flag-VDAC3 were co-transfected into HCT116 cells for Stag bead pull down that specially pulls down Stagged proteins. g Glucose starvation enhanced the interaction of PKM2 and VDAC3. HCT116 cells were transfected with HA-tagged VDAC3, cells were harvested and subjected to Stag pull down assay after 8 h glucose starvation. h Western blot analysis of VDAC3 and PKM2 in HCT116 cell lysates after RNA-interference-mediated knockdown of PKM2. i Ectopic expression of Flag-PKM2 in HCT116 cells upregulates VDAC3 in a dose-dependent manner. Treatment with the proteasome inhibitor MG132 at 10 μM for 8 h also caused VDAC3 upregulation. j Expression levels of VDAC3 and PKM2 protein analyzed with immunoblotting in PKM2 control (shControl) and PKM2 knockdown (shPKM2). Cells were treated with cycloheximide (CHX, 150 µg/ml) at indicated times. k In vivo VDAC3 ubiquitination assay. Cellular level VDAC3 ubiquitination assays were carried out in PKM2 WT or knockout MEFs. Cells were co-transfected with His-HA-ubiquitin (Ub) and GFP-Parkin, immunoprecipitated with VDAC3 antibody and immunoblotted with antibodies to HA, VDAC3, PKM2, or GFP. Cells were treated with MG132 (10 μM) for 8 h before harvest. l In vivo VDAC ubiquitination assay. Cellular level VDAC ubiquitination assays were carried out in HCT116 cells. Cells were co-transfected with His-HA-ubiquitin (Ub), Flag-VDAC3, GFP-Parkin, and cMyc-PKM2 plasmids, immunoprecipitated with Flag beads and immunoblotted with antibodies to HA and Flag. Cells were treated with MG132 (10 μM) for 8 h before harvest

Fig 3: Compound 8 blocks PKM2 mitochondrial translocation under nutritional stress and inhibits tumor growth in vivo.a Compound 8 blocks glucose starvation induced PKM2 mitochondrial translocation. Isolation of mitochondria and immunoblot of PKM2, tubulin and Tom 40 were carried out with or without compound 8 treatment. b Compound 8 disrupts interaction of PKM2 and VDAC3. HCT116 cells were transfected with HA-tagged PKM2, cells were harvested and subjected to stag pull down after 8 h 20 µM compound 8 treatment. c Compound 8 induces VDAC3 for degradation. cells were harvested and evaluated VDAC3 protein levels after 8 h 10 or 20 µM compound 8 treatment. d, e Compound 8 caused a decrease in the rate of NADH oxidation. After 8 h of treatment with 20 µM compound 8, HCT116 cells mitochondria were isolated for NADH oxidation assays. NAD/NADH ratios in HCT116 cell lysates were evaluated by LC–MS. f Compound 8 inhibited ATP production in HCT116 cells. g The tumor volume (n = 7) of HCT116 xenografts from mice with intraperitoneal injection of compound 8 and vehicle control

Fig 4: PKM2 and VDAC3 promote cell survival and tumor development under nutritional stress.a PKM2 and VDAC3 promote cell survival under nutritional stress. Survival of HCT116 cells in glucose-free media was measured with the Trypan blue exclusion method. b, c VDAC3 promotes tumor development. Tumor volume of VDAC3-kd HCT116 xenografts (n = 5). d PKM2 and VDAC3 are upregulated in human colon cancer. Fresh colon cancer and matched surrounding normal tissue from the same given patient were homogenized, and cytosolic and mitochondria fractions were separated. PKM2 was determined by western analysis. N (normal tissue), T (colon cancer). e Positive correlation of mitochondrial PKM2 and VDAC3 protein levels in human colon cancer. Statistical significance was determined with the ?2-test. R is the correlation coefficient. f, g Mitochondrial PKM2 and VDAC3 increased before solid tumor formation. f Sketch outlining the AOM/DSS-induced CRC mode. C57 mice were treated 1× with or without AOM, followed by periodic administration of 2% DSS in water. n = 12 per group. g Intestine issues were analyzed for mitochondrial PKM2 and VDAC3