lucasbsullivan.bsky.social
Associate Professor @ Fred Hutch
Studying metabolic constraints of cancer cell proliferation
https://research.fredhutch.org/sullivan/en.html
On a mote of dust suspended in a sunbeam
32 posts
650 followers
458 following
Getting Started
Active Commenter
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Thanks Jakub!
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Thank you!
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Thanks to Madeleine for wanting to stick around post-graduation to figure out “the boomerang” and the rest of the team for support, esp Kristian for quantitative efforts. Thanks to NIGMS and NCI for funding. Also, watch out for Madeleine's next act with @saranowinski.bsky.social!
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TLDR – we find that, upon SDH loss, the combined effects of ASP depletion and succinate accumulation profoundly inhibit ATCase. Pyrimidine loss impairs ASP consumption for biosynthesis, causing ASP to accumulate until it outcompetes succinate to restart pyrimidines/biosynthesis once again.
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The full paper has plenty more (ASP flux measurements, succinate sufficiency, replication stress), so I invite you to give it a full read. There are also fun phenotypes that epitomize some of the logical pitfalls that we metabolism nerds often warn about (e.g. levels =/= flux).
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We found SDH impaired cells can reach >10 mM!
Conclusion: succinate competes with diminished ASP at ATCase, blocking pyrimidine synthesis. To our knowledge the first description of this relationship in cells + revealing another potential regulatory function of succinate.
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A literature search found a goldmine: E Coli ATCase was a workhorse of biochemistry for decades (e.g. ⬇️) One compound used to impair its function in vitro? Succinate! While the concentrations needed to impair ATCase in vitro seemed unrealistic at ~1+ mM...
pubmed.ncbi.nlm.nih.gov/3894357/
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SDH inhibition is distinct from other mitochondrial impairments due to the accumulation of succinate, which we hypothesized to play a role. First, we decreased succinate production by cotreating with the CI inhibitor rotenone. Result: restoration of pyrimidine synthesis, even with comparable ASP.
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So, what happens if we restore pyrimidines to these cells?
A complete loss of the ASP rebound and a return to the (very reasonable) phenotype of monotonic ASP depletion.
So, SDH loss causes a disproportionate effect on pyrimidine synthesis by impairing ATCase, but why?
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We next investigated the biosynthetic fates of aspartate and found:
Aspartyl tRNA charge = OK
Purine nucleotides = OK
ASN = OK - Rebounds with ASP
Pyrimidine nucleotides = Depleted at the first step (ATCase, which makes Carb-ASP) - and stays depleted even with rebounding ASP:
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When we did the same experiment with the SDH inhibitor AA5, we get a strange result:
ASP declines for ~24 hours until proliferation slows, then..ASP rebounds???
Even stranger:
Proliferation doesn't recover with ASP even though we know ASP supplementation would solve the proliferation defect!
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Deploying our ASP biosensor, jAspSnFR3, Madeleine & co used live cell imaging to continuously measure ASP levels (GFP) and cell number. In two cases – CI inhibition and GOT1/2 DKO cells, ASP levels crash over 24 hours then cell proliferation decays, leading to a new ~steady state.
Very reasonable.
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Thanks Alex!
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Re: ITS - the product number is in the methods. There are many "ITS" products available with different nutrient iterations, so check out our note about it (in the Methods) for more info. I would also be happy to discuss the details via DM/Zoom if you interested!
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but it would be interesting to find out! If they don't activate, then investigating the salient metabolic/signaling differences for why not could reveal new, manipulable biology. Or at least, that is the hope!
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Thanks Dylan!
Almost certainly many effects in many systems, which (to me) is exactly the point. Neither FBS nor defined media = physiological, but comparisons across them (+other environments) could be illuminating. Would macrophages proliferate or activate in defined media? We don't know...
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Update to tag the author (and new arrival to bsky) - @olivernewsom.bsky.social!
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Thanks Greg!
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Many thanks to Oliver for his dedication to a project that had many unexpected twists and turns, and to the NIGMS for flexible funding that enabled us to pursue this work even when it went well off script.
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This work also supports a crucial role for lipid scavenging, which we believe is an underappreciated requirement for cancer cell metabolism. We also think that the description of FA uptake as primarily concentration driven helps contextualize well described effects of specific FAs on lipotoxicity.
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Recent research has clearly shown that the metabolic environment is a major determinant of cell function. Our hope is that this work will provide opportunities to compare serum-free conditions with physiological contexts to better understand the nutritive functions of the physiological milieu.
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Oliver went on to use this defined system, free from FBS confounding the lipid environment, to characterize albumin-associated lipid uptake kinetics (mass action driven) and to determine that lipid scavenging is a necessary lipid acquisition route for many cancer cells.
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Quantitative lipidomics from media gave a clear answer – albumin-associated lipids (fatty acids, LPCs, LPEs). Importantly, supplementing cells with an ITS additive (containing missing metals) and any of these albumin-associated lipids equivalently supported serum-free cancer cell proliferation.
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Indeed, testing various additives, he found that this “depletion phenotype” was largely driven by the need for metals and lipids, both absent from most basal media formulations. However - serum provides a multitude of potential lipid species to uptake – which lipids are actually consumed by cells?
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Using live cell imaging of cancer cell proliferation in reduced serum conditions, Oliver found that cell proliferation doesn’t immediately slow, it decays over time, suggesting the consumption and depletion of essential nutrients.
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In cancer cell culture, the growth factor component of FBS seems particularly unnecessary, given that cancer is a post-natal disease and cancer cells are defined by cell autonomous growth factor signaling. Yet, FBS is required for rapid cancer cell proliferation. Why? Oliver went to find out.
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Adding FBS is…weird. It’s expensive, undefined, morally dubious, and variable across lots. It introduces unknown factors that can obfuscate the effects of biological perturbations by complementation of metabolic and signaling processes…
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Tissue culture is critical for biomedical research by providing a simple and controllable system to understand complex cellular processes. However, a pervasive issue is that nearly all cell culture medias require the addition of animal products, typically FBS, to support cell proliferation.
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We are finalizing a paper about exactly this - should be on bioRxiv in the next month. I’ll email you a draft!
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Thanks for including us on this illustrious list. 🍻