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  1. Evaluation Summary:

    The authors demonstrate a molecular mechanism responsible for the rewiring of stroma cells that makes them supportive of acute myeloid leukaemia (AML) cells. Understanding metabolic interactions between leukaemia cells and the bone marrow microenvironment may provide new targets for leukaemia treatments. It has already been shown that the inhibition of gap junctions reduces AML growth in vivo, and here the authors provide evidence for a potential mechanism explaining that finding. The work will be very interesting for those working in the fields of hematopoiesis, leukemia (especially AML), cancer metabolism and cancer microenvironment.

    (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 #3 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    The authors demonstrate a molecular mechanism responsible for the rewiring of stroma cells that makes them supportive of acute myeloid leukaemia cells. That metabolic interactions takes place between leukaemia cells and the bone marrow microenvironment is an emerging topic, which hopes to provide new targets for the development of less toxic and more effective leukaemia treatments. It has already been shown that inhibiting gap junctions reduces AML growth in vivo (reference 10 in the manuscript), and here the authors provide evidence for a potential mechanism explaining that finding. Here, the authors demonstrate using co-culture models that AML cells use gap junctions to offload ROS to stroma cells, which in turn provide acetate that fuels AML cells' metabolism. This is linked to a transcriptional rewiring of stroma cells leading to overexpression of gap junctions themselves and of genes encoding enzymes involved in acetate production. The work is very interesting, and it should be feasible for the authors to fill a few gaps that would allow this to become a complete demonstration of the phenomenon uncovered.

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  3. Reviewer #2 (Public Review):

    The manuscript by Vilaplana-Lopera et al. highlights a novel interesting metabolic cross-talk between the stroma and AML cells through production of acetate. The main conclusion of the paper, ie that acetate production is increased in co-culture most likely because of stroma production and is taken up by AML cells, is supported by the data. Some of the other conclusions re the utilisation of acetate in AML cells and the exact mechanism leading to acetate production might require further experimental work to be fully supported. The authors make an effort to validate their findings in primary AML cells model and in vivo although the mechanistic insight is mostly done in AML cell lines grown on a MS-5 stromal layer. As a result, whether the described interaction is also present in other stroma/AML combination remains to be demonstrated.

    Technically the paper appears rigorous although I would not be able to comment on the technical aspects of the NMR analysis and I would hope some of the other reviewers can comment on that.

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  4. Reviewer #3 (Public Review):

    Vilaplana-Lopera et al., present a study which predominantly focusses on the in vitro interaction of three different human AML cell lines with the murine bone marrow stromal cell line MS-5. The authors describe a novel observation where the co-culture of AML and MS-5 cell lines results in elevated production and secretion of acetate via a mechanism which requires direct contact between these two cell types. Importantly, increased extracellular acetate could also be detected in both an in vitro setting where primary patient AML samples were co-cultured with MS-5 cells; and in an in vivo mouse model of MLL-AF9-driven AML. In the context of in vitro co-culture, the authors demonstrate that AML cell lines are capable of taking up extracellular labelled acetate and incorporating this into various TCA-related metabolites. Underlying this observation, the authors found evidence of metabolic changes in stromal cells under co-culture conditions, namely relating to elevated glucose metabolism and production of acetate via upregulated glycolysis. Following on from this observation, evidence is presented to suggest that AML cells transfer reactive oxygen species (ROS) to stromal cells via gap junctions upon co-culture, where the ROS can then mediate a non-enzymatic decarboxylation of pyruvate into acetate. The authors speculate that this metabolic reprogramming of stromal cells by AML cells may help support the increased energy demands of AML cells by producing acetate which can then be metabolised in the TCA cycle.

    Strengths of the study

    The authors have succeeded in documenting a novel phenomenon where leukemia cells are able to metabolically reprogram stromal cells, resulting in a very interesting model where acetate is shuttled from niche cells to AML cells, apparently to the benefit of the AML cells.

    The authors have validated some key aspects of their cell line data using both patient samples and a mouse model of AML.

    NMR-based tracing of metabolites unequivocally demonstrates that AML cells are capable of taking up and subsequently metabolising extracellular acetate in the context of co-culture with stromal cells.

    The concept that the intercellular shuttling of ROS is responsible for the catabolic conversion of pyruvate to acetate in stromal cells is highly novel.

    Weaknesses of the study

    The biological relevance of this mechanism is not clear, since coculture does not alter the growth of AML cell lines. Therefore, the resulting transfer of acetate appears to have no obvious impact on leukemia cell biology.

    The authors have not directly demonstrated that acetate generated by stromal cells is metabolised by AML cell lines.

    The use of NAC as a method to reduce ROS confounds interpretation of the data in this particular experimental setting, since NAC is precursor for several substrates in the metabolic pathways that are examined in this study. That is, NAC is the pro-drug for the generation of intracellular glutathione and can also be deacetylated to generate intracellular acetate.

    The experiments which explore the mechanistic role of gap junctions in this phenomenon do not show any direct impact on acetate production nor secretion by stromal cells.

    The culture conditions that have been used for growth of primary AML and control samples appeared to have been altered between different experimental repeats, which makes interpretation of data difficult. In addition, it is not entirely clear what cell type has been used as a control cell population for these experiments.

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