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  1. Author Response:

    Reviewer #1:

    This study largely confirms prior observations, and the strength of the study is in its comprehensive nature rather than in shedding new insight into the effects of either FH or SDH loss. Nevertheless, there are some somewhat unexpected observations including a defect in proline synthesis, and changes in glutathione and NADPH metabolism that are interesting and incompletely explained. Some suggestions to strengthen the study include:

    1. Exactly how each perturbation affects cell proliferation is not clear. This should be considered, as whether some of the differences are a result in changes in growth or proliferation rate is possible, and will affect how they normalize their data.

    We thank the referee for raising a critical point and allowing us to clarify how we normalize metabolomics experiments. All the metabolomics data takes into consideration the cell number. Indeed, prior to metabolite extraction, cells are counted using a separate counting plate prepared in parallel and treated exactly like the experimental plate. In this way, differences in cell number are accounted for and the efficiency of metabolic extraction is preserved. Consumption release (CoRe) metabolomics also takes into consideration the proliferation rate during normalization since we normalise data to the final cell number. We have expanded the description in the relevant sections in the Methods.

    1. It is unclear why FH loss is different than SDH loss, and it is also somewhat surprising that the effects of acute and chronic loss of either enzyme are not that different. While explaining this is too much to ask, some additional speculation might be warranted.

    We postulate that FH loss is different to SDH loss for several reasons:

    A. FH is localized to mitochondria (specifically in the mitochondrial matrix) and the cytosol (cytFH). cytFH can translocate to the nucleus to regulate the DNA damage response (PMID: 26237645). In contrast, the SDH complex is only localized to mitochondria. As such, a loss of FH function is likely to have mitochondrial and extramitochondrial consequences.

    B. SDH is also the only TCA cycle enzyme that's physically associated with the electron transport chain (ETC) and tethered to the inner mitochondrial membrane, where it also regulates the ubiquinone pool. The different distribution of both enzymes within mitochondria is likely to influence their impact mitochondrial bioenergetics and on the overall metabolic profile.

    C. A major difference between FH and SDH loss is the accumulation of fumarate. As discussed in the manuscript, fumarate is a mildly electrophilic metabolite that can succinate GSH and protein cysteine residues to form a post-translational modification termed succination. Fumarate-mediated succination is known to impair iron-sulphur cluster metabolism and perturb aconitase and Complex I function (PMID: 29069586). This is just one example of how succination can affect cellular function. In contrast, SDH loss results in a decrease of fumarate and an accumulation of succinate.Unlike fumarate, succinate is not an electrophilic compound that can modify cystine residues, and so differences between FH and SDH loss are likely owed to succination, at least in part.

    D. While it hasn't been investigated in this study, succinate released from cells can bind to the succinate receptor SUCNR1, which is expressed in the kidney (PMID: 21803970). Autocrine and paracrine ligation of SUCNR1 by high levels succinate accumulation and release is likely to alter the metabolic and transcriptional landscape of the cells.

    Based on these observations, we also argue that the effect of acute and chronic enzyme inhibition is expectedly different regarding how the key metabolic and signalling hallmarks of FH and SDH loss develop and interact with each other over time. This hypothesis is part of work currently undergoing in our laboratory. For example, chronic SDH loss led to a significant increase in 20 metabolites however, acute SDH inhibition with TTFA and AA5 led to an increase in 60 and 50 metabolites, respectively. Chronic FH loss also led to a significant increase in 92 metabolites, whereas acute FH inhibition led to a significant increase in 49. The fact that only 2 metabolites overlap between all conditions indicates apparent differences between the loss of both enzymes on the metabolome and whether the loss is acute or chronic in nature. There are also notable differences between chronic FH loss and acute FH inhibition in relation to reductive carboxylation. Chronic FH loss triggers a higher accumulation of fumarate and succination of aconitase that impairs reductive carboxylation (PMID: 21849978); however, acute inhibition facilitates reductive carboxylation (Figure S2), likely due to lower levels of succination given the acute treatment. As such, we feel there are notable differences in the metabolite profiles and rewiring events associated with acute versus chronic enzyme inhibition. We have discussed these important points in the discussion section of the manuscript.

    1. The increase in glutathione and GSSG is interpreted as a consequence of increased oxidative stress, but that will not necessarily affect total levels.

    We agree that oxidative stress will not necessarily affect total glutathione levels and this finding is likely a time-dependent phenomenon due to persistent redox signalling. In this instance, the alterations in total glutathione levels are likely linked to transcriptional and post- transcriptional changes in GSH biosynthetic enzymes and the observed metabolic reprogramming. While ATF4 regulates the glutathione redox state and glutathione levels, it's not entirely clear if it is solely responsible for increasing total glutathione levels with TCA cycle inhibition. One possibility is that there is simultaneous activation of the transcription factor NRF2, which is a crucial regulator of glutathione synthesis and is known to be regulated by FH loss and succination (PMID: 22014567) and reactive oxygen species (ROS). ATF4 and NRF2 may cooperate to transactivate glutathione-related metabolic enzymes upon TCA cycle inhibition, as previously reported in other contexts (PMID: 23618921). Further investigation of the crosstalk between these two transcription factors is warranted in this context.

    1. The text in the Figure S1 PCA plots have legends is too small to read. This should be corrected.

    We thank you for this note and apologize for this oversight. We have now corrected the figure legends for the PCA plots.

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  2. Evaluation Summary:

    The authors of this study investigate the consequences of acute or chronic disruption of parts of the TCA cycle, and how different interventions can drive different transcriptional responses. Specifically, the authors use both pharmacological and genetic methods to disrupt succinate dehydrogenase or fumarate hydratase, and characterize the effect of each on metabolism. They also find that disruption of these enzymes elicits a transcriptional response through ATF4. This work provides insight into how metabolism is affected by TCA cycle loss, and how how this affects metabolic stress signaling.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

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  3. Reviewer #1 (Public Review):

    This study largely confirms prior observations, and the strength of the study is in its comprehensive nature rather than in shedding new insight into the effects of either FH or SDH loss. Nevertheless, there are some somewhat unexpected observations including a defect in proline synthesis, and changes in glutathione and NADPH metabolism that are interesting and incompletely explained. Some suggestions to strengthen the study include:

    1. Exactly how each perturbation affects cell proliferation is not clear. This should be considered, as whether some of the differences are a result in changes in growth or proliferation rate is possible, and will affect how they normalize their data.

    2. It is unclear why FH loss is different than SDH loss, and it is also somewhat surprising that the effects of acute and chronic loss of either enzyme are not that different. While explaining this is too much to ask, some additional speculation might be warranted.

    3. The increase in glutathione and GSSG is interpreted as a consequence of increased oxidative stress, but that will not necessarily affect total levels.

    4. The text in the Figure S1 PCA plots have legends is too small to read. This should be corrected.

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  4. Reviewer #2 (Public Review):

    In this manuscript, Ryan et al investigate how disruption of parts of the TCA cycle signals to the nucleus to drive a transcriptional response. The authors inhibited either fumarate hydratase (FH) or succinate dehydrogenase (SDH) using both pharmacological inhibitors and previously described knockdown cell lines. While there were some differences in the metabolic response between these approaches - for example in asparagine levels - the authors noted a consistent change in a number of metabolites that could be linked through their function in cellular redox homeostasis. In particular, the authors showed that a loss of TCA cycle activity through FH and SDH led to increased levels of metabolites within the glutathione synthetic pathway. This suggests that glutamate sits at a functional crossroads between the TCA cycle and glutathione synthesis, allowing the cell to rapidly compensate for mitochondrial dysfunction by increasing cellular antioxidant defences.

    Finally, the authors use transcriptional and proteomic profiling to demonstrate that in addition to the rapid metabolic response described, cells lacking SDH and FH activity respond with an ATF4-mediated transcriptional response mediated through the integrated stress response pathway.

    Strengths:

    The authors' use of both genetic models - the previously published Fh1-/- and Sdhb-/- knockout cell lines - alongside pharmacological approaches to acutely inhibit these enzymes mean that they were able to identify both rapid changes in metabolism as a result of inhibition, as well as longer-term compensatory changes. The authors also use a significant array of metabolic techniques to show changes in metabolism - for example, metabolomics, stable isotope tracers, respiration measures and compartmentalised metabolite information, which provide significant confidence in their results. The final section of the manuscript, in which the authors link TCA cycle dysfunction to the integrated stress response pathway was particularly strong - including well-described functional outcome being an ATF4-mediated transcriptional response.

    Weaknesses:

    Interestingly, the compounds the authors utilised to block SDH activity (Atpenin A5 and TTFA) have previously been described to inhibit the SQR activity (complex II) rather than specifically the proximal SDH activity of the complex. While in an enzyme complex where the ability to reduce ubiquinone is highly coupled to oxidation of succinate, it is possible that some SDH activity remains using this pharmacological approach, with enhanced ROS generation from the more reduced Fe-S centres. However, the use of the genetic model alongside these data provides additional confidence, as this has been previously shown to have no residual succinate oxidising activity. The use of mitoCDNB provides a novel means by which the authors could perturb mitochondrial thiol homeostasis specifically, it is probably that this reagent also covalently modifies other thiol-containing proteins in the mitochondrial matrix, including those exposed within the respiratory chain. These off-target effects could feasibly phenocopy the effect of TCA cycle inhibition independently of the mechanism the authors suggest. Additionally, some more detail in the methods would aid interpretation of certain experiments - for example the length of time for which some of the treatments were performed is not always clear.

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  5. Reviewer #3 (Public Review):

    The authors explore how modulation of TCA cycle impacts cell metabolism acutely, and they identify key links to non-essential amino acid metabolism. The authors employ broad omics analysis as well as targeted flux-based studies to confirm findings. They integrate genetic approaches with pharmacological inhibitors and ultimately describe a regulatory role for ATF4 stress response.

    On one hand, the dependence of NEAA synthesis on mitochondrial metabolism is well-established, and ATF4 is known to regulate amino acid metabolism. However, the authors explain the pathway regulation in some new and interesting ways while integrating recent findings pertaining to proline metabolism.

    There are limitations with respect to physiological relevance which should be outlined further, but the data are well presented and support conclusions.

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