Mitochondrial ETF insufficiency drives neoplastic growth by selectively optimizing cancer bioenergetics

Reviewer #1 (Public review):

In their manuscript, Papadopoli et al explore the role of ETFDH in transformation. They note that ETFDH protein levels are decreased in cancer, and that deletion of ETFDH in cancer cell lines results in increased tumorigenesis, elevated OXPHOS and glycolysis, and a reduction in lipid and amino acid oxidation. The authors attribute these effects to increased amino acid levels stimulating mTORC1 signaling and driving alterations in BCL6 and EIF4EBP1. They conclude that ETFDH1 is epigenetically silenced in a proportion of neoplasms, suggesting a tumor-suppressive function. Overall, the authors logically present clear data and perform appropriate experiments to support their hypotheses. I only have a few minor points related to the semantics of a few of the author's statements.

Minor Points

Authors state, "we identified ETF dehydrogenase (ETFDH) as one of the most dispensable metabolic genes in neoplasia." Surely there are thousands of genes that are dispensable for neoplasia. Perhaps the authors can revise this sentence and similar sentiments in the text.

Authors state, " These findings show that ETFDH loss elevates glutamine utilization in the CAC to support mitochondrial metabolism." While elevated glutamine to CAC flux is consistent with the statement that increased glutamine, the authors have not measured the effect of restoring glutamine utilization to baseline on mitochondrial metabolism. Thus, the causality implied by the authors can only be inferred based on the data presented. Indeed, the increased glutamine consumption may be linked to the increase in ROS, as glutamate efflux via system xCT is a major determinant of glutamine catabolism in vitro.

Authors state that the mechanism described is an example of "retrograde signaling". However, the mechanism seems to be related to a reduction in BCAA catabolism, suggesting that the observed effects may be a consequence of altered metabolic flux rather than a direct signaling pathway. The data presented do not delineate whether the observed effects stem from disrupted mitochondrial communication or from shifts in nutrient availability and metabolic regulation.

The authors should discuss which amino acids that are ETFDH substrates might affect mTORC1 activity, or consider whether other ETFDH substrates might also affect mTORC1 in their discussion. Along these lines, the authors might consider discussing why amino acids that are not ETFDH substrates are increased upon ETFDH loss.

Reviewer #2 (Public review):

Summary:

The altered metabolism of tumors enables their growth and survival. Classically, tumor metabolism often involves increased activity of a given pathway in intermediary metabolism to provide energy or substrates needed for growth. Papadopoli et al. investigate the converse - the role of mitochondrial electron transfer flavoprotein dehydrogenase (ETFDH) in cancer metabolism and growth. The authors present compelling evidence that ETFDH insufficiency, which is detrimental in non-malignant tissues, paradoxically enhances bioenergetic capacity and accelerates neoplastic growth in cancer cells in spite of the decreased metabolic fuel flexibility that this affords tumor cells. This is achieved through the retrograde activation of the mTORC1/BCL-6/4E-BP1 axis, leading to metabolic and signaling reprogramming that favors tumor progression.

Strengths:

This review focuses primarily on the cancer metabolism aspects of the manuscript.

The study provides robust evidence linking ETFDH insufficiency to enhanced cancer cell bioenergetics and tumor growth.

The use of multiple cancer cell lines and in vivo models strengthens the generalizability of the findings.

The mechanistic insights into the mTORC1/BCL-6/4E-BP1 axis and its role in metabolic reprogramming are of general interest within and outside the immediate field of tumor metabolism.

Weaknesses:

The ETFDH knockout experiments are well-controlled by the addback of sgRNA-resistant ETFDH, but do not determine if the catalytic activity of this enzyme is required for the phenotypes induced by ETFDH loss.

Although this is not critical, it would be nice to see if the increased labeled aspartate pools result in higher nucleotide pools to support tumor growth.

Conclusion:

This manuscript provides significant insights into the role of ETFDH insufficiency in cancer metabolism and growth. The findings highlight the potential of targeting the mTORC1/BCL-6/4E-BP1 axis in ETFDH-deficient cancers. The compelling data support the conclusions presented in the manuscript, which will be valuable to the cancer metabolism community.

Author response:

We are highly appreciative of your constructive criticism and that you found that our findings of interest and significance. Based on your helpful suggestions, we plan to revise the paper as following:

(1) Although ETFDH is reduced, but not mutated across neoplasia, we appreciate your point pertinent to catalytically activity of ETFDH. To this end, in the revision we are planning to compare the effects of rescues using wild type ETFDH or one of the MADD-associated mutants with compromised catalytic activity.

(2) We intend to measure steady-state nucleotide levels as a function of ETFDH status in the cell. If time and/or funding allow, we will also perform appropriate labelling experiments.

(3) We will revise the text of the manuscript to address the minor points raised by the reviewers.

Again, we would like to thank you for helpful comments, which we aim to address as outlined above and hopefully further improve our report.