Autophagy degrades and is thought to recycle proteins, other macromolecules, and organelles. In genetically engineered mouse models (GEMMs) for Kras-driven lung cancer, autophagy prevents the accumulation of defective mitochondria and promotes malignancy. Autophagy-deficient tumor-derived cell lines are respiration-impaired and starvation-sensitive. However, to what extent their sensitivity to starvation arises from defective mitochondria or an impaired supply of metabolic substrates remains unclear. Here, we sequenced the mitochondrial genomes of wild-type or autophagy-deficient (Atg7) Kras-driven lung tumors. Although Atg7 deletion resulted in increased mitochondrial mutations, there were too few nonsynonymous mutations to cause generalized mitochondrial dysfunction. In contrast, pulse-chase studies with isotope-labeled nutrients revealed impaired mitochondrial substrate supply during starvation of the autophagy-deficient cells. This was associated with increased reactive oxygen species (ROS), lower energy charge, and a dramatic drop in total nucleotide pools. While starvation survival of the autophagy-deficient cells was not rescued by the general antioxidant N-acetyl-cysteine, it was fully rescued by glutamine or glutamate (both amino acids that feed the TCA cycle and nucleotide synthesis) or nucleosides. Thus, maintenance of nucleotide pools is a critical challenge for starving Kras-driven tumor cells. By providing bioenergetic and biosynthetic substrates, autophagy supports nucleotide pools and thereby starvation survival.

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Autophagy captures intracellular components and delivers them to lysosomes for degradation and recycling. Conditional autophagy deficiency in adult mice causes liver damage, shortens life span to 3 mo due to neurodegeneration, and is lethal upon fasting. As autophagy deficiency causes p53 induction and cell death in neurons, we sought to test whether p53 mediates the lethal consequences of autophagy deficiency. Here, we conditionally deleted Trp53 (p53 hereafter) and/or the essential autophagy gene Atg7 throughout adult mice. Compared with Atg7 Δ/Δ mice, the life span of Atg7 Δ/Δ p53 Δ/Δ mice was extended due to delayed neurodegeneration and resistance to death upon fasting. Atg7 also suppressed apoptosis induced by p53 activator Nutlin-3, suggesting that autophagy inhibited p53 activation. To test whether increased oxidative stress in Atg7 Δ/Δ mice was responsible for p53 activation, Atg7 was deleted in the presence or absence of the master regulator of antioxidant defense nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 −/− Atg7 Δ/Δ mice died rapidly due to small intestine damage, which was not rescued by p53 codeletion. Thus, Atg7 limits p53 activation and p53-mediated neurodegeneration. In turn, NRF2 mitigates lethal intestine degeneration upon autophagy loss. These findings illustrate the tissue-specific roles for autophagy and functional dependencies on the p53 and NRF2 stress response mechanisms.

The mechanisms underlying metabolic adaptation of pancreatic ductal adenocarcinoma (PDA) cells to pharmacological inhibition of RAS-MAPK signaling are largely unknown. Using transcriptome and chromatin immunoprecipitation profiling of PDA cells treated with the MEK inhibitor, Trametinib (MEKi), we identify transcriptional antagonism between c-MYC and the master transcription factors for lysosome gene expression, the MiT/TFE proteins. Under baseline conditions, c-MYC and MiT/TFE factors compete for binding to lysosome gene promoters to fine-tune gene expression. Treatment of PDA cells or patient organoids with MEKi leads to c-MYC downregulation and increased MiT/TFE-dependent lysosome biogenesis. Quantitative proteomics of immunopurified lysosomes uncovered reliance on ferritinophagy, the selective degradation of the iron storage complex ferritin, in MEKi treated cells. Ferritinophagy promotes mitochondrial iron-sulfur cluster protein synthesis and enhanced mitochondrial respiration. Accordingly, suppressing iron utilization sensitizes PDA cells to MEKi, highlighting a critical and targetable reliance on lysosome-dependent iron supply during adaptation to KRAS-MAPK inhibition.

Macroautophagy (hereafter autophagy) plays an important role in maintaining cellular homeostasis under stress conditions. We previously demonstrated that conditional autophagy deficiency in adult mice causes selective tissue damage, is lethal upon fasting, and shortens lifespan to less than three months primarily due to neurodegeneration, but not all the mechanisms are known. We conditionally deleted Trp53/p53 and/or the essential autophagy gene Atg7 throughout adult mice to test whether TRP53 is responsible for any of these phenotypes. atg7 Δ/Δ trp53 Δ/Δ mice have extended lifespan due to delayed tissue damage and neurodegeneration, and are resistant to death upon fasting compared to atg7 Δ/Δ mice. Atg7 also suppresses apoptosis induced by the TRP53 activator Nutlin-3 in liver and brain. We then deleted Atg7 in the presence or absence of the master regulator of antioxidant defense NFE2L2/NRF2 (nuclear factor, erythroid derived 2, like 2) to test if increased oxidative stress causes TRP53 activation in atg7 Δ/Δ mice. nfe2l2 −/-atg7 Δ/Δ mice die rapidly due to intestinal damage, which is not rescued by trp53 co-deletion. Therefore, these data demonstrate the tissue specificities and functional dependencies between autophagy, TRP53 and NFE2L2 stress response mechanisms.

Significance Autophagy defects are a risk factor for inflammatory bowel diseases (IBDs), but the mechanism remains unknown. We show here that conditional whole-body deletion of Atg5 or Fip200 , but not Atg7 , is lethal due to loss of ileum stem cells and barrier function likely caused by different kinetics of autophagy loss, which was rescued by slow deletion. Specific autophagy loss in PDGFRα+ mesenchymal cells (PMCs) resulted in loss of Wnt signaling responsible for failed stem cell renewal. We also observed depletion of aspartate and nucleotides throughout the ileum. Our results illustrate that autophagy is required for PMC metabolism and survival necessary to sustain intestinal stem cells and mouse survival, and failure to maintain PMCs through autophagy contributes to IBD.

<div>Abstract<p>The mechanisms underlying metabolic adaptation of pancreatic ductal adenocarcinoma (PDA) cells to pharmacologic inhibition of RAS–MAPK signaling are largely unknown. Using transcriptome and chromatin immunoprecipitation profiling of PDA cells treated with the MEK inhibitor (MEKi) trametinib, we identify transcriptional antagonism between c-MYC and the master transcription factors for lysosome gene expression, the MiT/TFE proteins. Under baseline conditions, c-MYC and MiT/TFE factors compete for binding to lysosome gene promoters to fine-tune gene expression. Treatment of PDA cells or patient organoids with MEKi leads to c-MYC downregulation and increased MiT/TFE-dependent lysosome biogenesis. Quantitative proteomics of immunopurified lysosomes uncovered reliance on ferritinophagy, the selective degradation of the iron storage complex ferritin, in MEKi-treated cells. Ferritinophagy promotes mitochondrial iron–sulfur cluster protein synthesis and enhanced mitochondrial respiration. Accordingly, suppressing iron utilization sensitizes PDA cells to MEKi, highlighting a critical and targetable reliance on lysosome-dependent iron supply during adaptation to KRAS–MAPK inhibition.</p>Significance:<p>Reduced c-MYC levels following MAPK pathway suppression facilitate the upregulation of autophagy and lysosome biogenesis. Increased autophagy–lysosome activity is required for increased ferritinophagy-mediated iron supply, which supports mitochondrial respiration under therapy stress. Disruption of ferritinophagy synergizes with KRAS–MAPK inhibition and blocks PDA growth, thus highlighting a key targetable metabolic dependency.</p><p><i><a href="https://aacrjournals.org/cancerdiscovery/article/doi/10.1158/2159-8290.CD-22-0734" target="_blank">See related commentary by Jain and Amaravadi, p. 2023</a>.</i></p><p><i><a href="https://aacrjournals.org/cancerdiscovery/article/doi/10.1158/2159-8290.CD-22-0043" target="_blank">See related article by Santana-Codina et al., p. 2180</a>.</i></p><p><i><a href="https://aacrjournals.org/cancerdiscovery/article/doi/10.1158/2159-8290.CD-12-9-ITI" target="_blank">This article is highlighted in the In This Issue feature, p. 2007</a></i></p><p><i></i></p></div>

Supplementary Figure from Coordinated Transcriptional and Catabolic Programs Support Iron-Dependent Adaptation to RAS–MAPK Pathway Inhibition in Pancreatic Cancer