Pyruvate Kinase Activity Regulates Cystine Starvation Induced Ferroptosis through Malic Enzyme 1 in Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with high mortality and limited efficacious therapeutic options. PDAC cells undergo metabolic alterations to survive within a nutrient-depleted tumor microenvironment. One critical metabolic shift in PDAC cells occurs through altered isoform expression of the glycolytic enzyme, pyruvate kinase (PK). Pancreatic cancer cells preferentially upregulate pyruvate kinase muscle isoform 2 isoform (PKM2). PKM2 expression reprograms many metabolic pathways, but little is known about its impact on cystine metabolism. Cystine metabolism is critical for supporting survival through its role in defense against ferroptosis, a non-apoptotic iron-dependent form of cell death characterized by unchecked lipid peroxidation. To improve our understanding of the role of PKM2 in cystine metabolism and ferroptosis in PDAC, we generated PKM2 knockout (KO) human PDAC cells. Fascinatingly, PKM2KO cells demonstrate a remarkable resistance to cystine starvation mediated ferroptosis. This resistance to ferroptosis is caused by decreased PK activity, rather than an isoform-specific effect. We further utilized stable isotope tracing to evaluate the impact of glucose and glutamine reprogramming in PKM2KO cells. PKM2KO cells depend on glutamine metabolism to support antioxidant defenses against lipid peroxidation, primarily by increased glutamine flux through the malate aspartate shuttle and utilization of ME1 to produce NADPH. Ferroptosis can be synergistically induced by the combination of PKM2 activation and inhibition of the cystine/glutamate antiporter in vitro. Proof-of-concept in vivo experiments demonstrate the efficacy of this mechanism as a novel treatment strategy for PDAC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/557984v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@fa23c2org.highwire.dtl.DTLVardef@41c01dorg.highwire.dtl.DTLVardef@abc283org.highwire.dtl.DTLVardef@6f53ef_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPKM2KO in pancreatic ductal adenocarcinoma (PDAC) cells produces enhanced defense against cystine starvation induced ferroptosis. C_LIO_LIPharmacologic activation of pyruvate kinase (PK) activity promotes ferroptosis under cystine starvation, while inhibition promotes ferroptosis survival in PDAC cells. C_LIO_LIDecrease in PK activity reprograms glutamine metabolism to increase use of malic enzyme 1 and promote survival under cystine starvation in PDAC cells. C_LIO_LICystine starvation and activation of pyruvate kinase synergistically decreases progression of pancreatic cancer in vivo. C_LI