Targeting the AAA ATPase p97 as an Approach to Treat Cancer through Disruption of Protein Homeostasis

Anderson, Daniel J.; Moigne, Ronan Le; Djakovic, Stevan; Kumar, Brajesh; Rice, Julie; Wong, Steve; Wang, Jinhai; Yao, Bing; Valle, Eduardo; Soly, Szerenke Kiss von; Madriaga, Antonett; Soriano, Ferdie; Menon, Mary‐Kamala; Wu, Zhi Yong; Kampmann, Martin; Chen, Yu-Wen; Weissman, Jonathan S.; Aftab, Blake T.; Yakes, F. Michael; Shawver, Laura K.; Zhou, Han-Jie; Wustrow, David; Rolfe, Mark

doi:10.1016/j.ccell.2015.10.002

Cited by 347 publications

(444 citation statements)

References 55 publications

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“…To distinguish between the role of Sec61 in import and export, we assessed the ability of the preformed ER pool of ddVenus to be retrotranslocated to the cytosol. Cells were pulsed with cycloheximide for 15 min to prevent protein synthesis, and then chased with MG-132 ± mycolactone or CB-5083, a potent p97 inhibitor (23), in the continued presence of cycloheximide (Fig. 3A).…”

Section: Resultsmentioning

confidence: 99%

Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export

Grotzke

Kozik

Morel

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Although antigen cross-presentation in dendritic cells (DCs) is critical to the initiation of most cytotoxic immune responses, the intracellular mechanisms and traffic pathways involved are still unclear. One of the most critical steps in this process, the export of internalized antigen to the cytosol, has been suggested to be mediated by Sec61. Sec61 is the channel that translocates signal peptide-bearing nascent polypeptides into the endoplasmic reticulum (ER), and it was also proposed to mediate protein retrotranslocation during ER-associated degradation (a process called ERAD). Here, we used a newly identified Sec61 blocker, mycolactone, to analyze Sec61's contribution to antigen cross-presentation, ERAD, and transport of internalized antigens into the cytosol. As shown previously in other cell types, mycolactone prevented protein import into the ER of DCs. Mycolactone-mediated Sec61 blockade also potently suppressed both antigen cross-presentation and direct presentation of synthetic peptides to CD8 + T cells. In contrast, it did not affect protein export from the ER lumen or from endosomes into the cytosol, suggesting that the inhibition of cross-presentation was not related to either of these trafficking pathways. Proteomic profiling of mycolactoneexposed DCs showed that expression of mediators of antigen presentation, including MHC class I and β2 microglobulin, were highly susceptible to mycolactone treatment, indicating that Sec61 blockade affects antigen cross-presentation indirectly. Together, our data suggest that the defective translocation and subsequent degradation of Sec61 substrates is the cause of altered antigen cross-presentation in Sec61-blocked DCs.Sec61 | cross-presentation | ERAD | mycolactone

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Section: Resultsmentioning

confidence: 99%

Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export

Grotzke

Kozik

Morel

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Subsequent optimization of ML240 resulted in the identification of CB-5083 (43), which is currently being tested in phase I clinical trials. CB-5083 exhibits potent antitumor activity in both multiple myeloma and solid tumor xenograft models (44). However, the precise mechanisms by which p97 regulates substrates under physiological conditions remain poorly understood, and only a limited number of substrates have been studied in great detail.…”

Section: Significancementioning

confidence: 99%

p97/VCP promotes degradation of CRBN substrate glutamine synthetase and neosubstrates

Nguyen

Lu³

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Glutamine synthetase (GS) plays an essential role in metabolism by catalyzing the synthesis of glutamine from glutamate and ammonia. Our recent study showed that CRBN, a direct protein target for the teratogenic and antitumor activities of immunomodulatory drugs such as thalidomide, lenalidomide, and pomalidomide, recognizes an acetyl degron of GS, resulting in ubiquitylation and degradation of GS in response to glutamine. Here, we report that valosin-containing protein (VCP)/p97 promotes the degradation of ubiquitylated GS, resulting in its accumulation in cells with compromised p97 function. Notably, p97 is also required for the degradation of all four known CRBN neo-substrates [Ikaros family zinc finger proteins 1 (IKZF1) and 3 (IKZF3), casein kinase 1α (CK1α), and the translation termination factor GSPT1] whose ubiquitylation is induced by immunomodulatory drugs. Together, these data point to an unexpectedly intimate relationship between the E3 ubiquitin ligase CRL4 CRBN and p97 pathways.lutamine plays important roles in many cellular processes, including oxidative metabolism and ATP generation, biosynthesis of proteins, lipids, and nucleic acids, and cell growth and proliferation through the regulation of the mTOR signaling pathway, translation, and autophagy (1, 2). In mammals, it is the most abundant amino acid in plasma with a concentration of 0.5-0.9 mM (3), accounting for ∼20% of its free amino acid pool. Glutamine synthetase (GS) is the only enzyme that is capable of de novo synthesis of glutamine and also functions to detoxify glutamate and ammonia, depending on tissue localization. Skeletal muscles and lungs are major sites of glutamine synthesis, whereas cells of the gut and the immune system, such as lymphocytes and macrophages, consume large amounts of glutamine in plasma (4). GS protects neurons against excitotoxicity by converting glutamate into glutamine in brain, detoxifies ammonia in liver, and maintains physiologic pH in kidney (5). In an attempt to investigate the role of GS in development, He et al. generated GS-knockout mice and reported that GS is essential in early embryogenesis, because deletion of the murine GLUL gene causes lethality at the blastocyst stage (embryonic day 3.5) (6). Interestingly, mouse ES cells maintain pluripotency and proliferate when grown in the absence of exogenous glutamine (7). However, inhibition of GS with the small molecule methionine sulfoximine (MSO) is sufficient to block the proliferation of ES cells in glutamine-free medium (7). In humans, congenital systemic glutamine deficiency caused by homozygous GS mutations results in multiorgan failure and neonatal death (8).Recent studies highlight the importance of glutamine metabolism in metabolic reprogramming, because many tumor cells display "glutamine addiction" (9). Activation of oncogenes such as MYC, KRAS, and HIF1α and/or loss of tumor suppressor genes including p53 can directly mediate the reprogramming of glutamine metabolism by selectively activating their downstream signaling or metabolic pathway...

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“…The ability of VCP to facilitate the retro-translocation of ER-associated degradation (ERAD) substrates prior to proteasome-mediated degradation is one of the more well-characterized roles for VCP (50). Several VCP inhibitors have been developed with more recent VCP ATPase inhibitors showing antitumor activity in xenograft tumor models (39,51,52). Motivated by our results demonstrating that the ub-modified proteome can be used to monitor protein homeostasis dysfunction upon proteasome inhibition or distinct challenge with an ER stressor, we employed our quantitative ub-proteomics strategy to interrogate the impact of pharmacological VCP inhibition on the ub-modified proteome.…”

Section: Vcp Inhibition Induces Distinct Alterations To the Ub-modifimentioning

confidence: 99%

“…These site-specific regulatory events mark distinct functional outputs and combining ub-proteomics with subcellular fractionation can effectively separate degradative and regulatory ubiquitylation events on distinct protein populations. To establish if different protein homeostasis stressors induce general or specific alterations within the ub-modified proteome we used a recently developed potent inhibitor of the critical protein homeostasis factor, p97/VCP (hereafter referred to as VCP) (39). We demonstrate that VCP inhibition, despite similarly impacting global ubiquitin homeostasis, results in largely distinct alterations of the ub-modified proteome compared with proteasome inhibition.…”

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confidence: 95%

Using the Ubiquitin-modified Proteome to Monitor Distinct and Spatially Restricted Protein Homeostasis Dysfunction

Gendron

Webb

Yang

et al. 2016

Molecular & Cellular Proteomics

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Protein homeostasis dysfunction has been implicated in the development and progression of aging related human pathologies. There is a need for the establishment of quantitative methods to evaluate global protein homoeostasis function. As the ubiquitin (ub) proteasome system plays a key role in regulating protein homeostasis, we applied quantitative proteomic methods to evaluate the sensitivity of site-specific ubiquitylation events as markers for protein homeostasis dysfunction. Here, we demonstrate that the ub-modified proteome can exceed the sensitivity of engineered fluorescent reporters as a marker for proteasome dysfunction and can provide unique signatures for distinct proteome challenges which is not possible with engineered reporters. We demonstrate that combining ub-proteomics with subcellular fractionation can effectively separate degradative and regulatory ubiquitylation events on distinct protein populations. Using a recently developed potent inhibitor of the critical protein homeostasis factor p97/VCP, we demonstrate that distinct insults to protein homeostasis function can elicit robust and largely unique alterations to the ub-modified proteome. Taken together, we demonstrate that proteomic approaches to monitor the ubmodified proteome can be used to evaluate global protein homeostasis and can be used to monitor distinct functional outcomes for spatially separated protein populations. Molecular & Cellular

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Targeting the AAA ATPase p97 as an Approach to Treat Cancer through Disruption of Protein Homeostasis

Cited by 347 publications

References 55 publications

Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export

Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export

p97/VCP promotes degradation of CRBN substrate glutamine synthetase and neosubstrates

Using the Ubiquitin-modified Proteome to Monitor Distinct and Spatially Restricted Protein Homeostasis Dysfunction

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