Difluoromethylornithine rebalances aberrant polyamine ratios in Snyder–Robinson syndrome

Abstract Snyder–Robinson syndrome (SRS) results from mutations in spermine synthase (SMS), which converts the polyamine spermidine into spermine. Affecting primarily males, common manifestations of SRS include intellectual disability, osteoporosis, hypotonia, and seizures. Symptom management is the only treatment. Reduced SMS activity causes spermidine accumulation while spermine levels are reduced. The resulting exaggerated spermidine:spermine ratio is a biochemical hallmark of SRS that tends to correlate with symptom severity. Our studies aim to pharmacologically manipulate polyamine metabolism to correct this imbalance as a therapeutic strategy for SRS. Here we report the repurposing of 2‐difluoromethylornithine (DFMO), an FDA‐approved inhibitor of polyamine biosynthesis, in rebalancing spermidine:spermine ratios in SRS patient cells. Mechanistic in vitro studies demonstrate that, while reducing spermidine biosynthesis, DFMO also stimulates the conversion of spermidine into spermine in hypomorphic SMS cells and induces uptake of exogenous spermine, altogether reducing the aberrant ratios. In a Drosophila SRS model characterized by reduced lifespan, DFMO improves longevity. As nearly all SRS patient mutations are hypomorphic, these studies form a strong foundation for translational studies with significant therapeutic potential.

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General comments
The manuscript by Murray Stewart et al reports a study that is an outstanding example of translational biomedical research.The paper addresses an unmet medical need in children with Snyder Robinson Syndrome (SRS), which is the lack of any currently effective pharmacotherapies.The authors build on previous studies implicating a loss of activity of the polyamine biosynthetic enzyme spermine synthase (SMS) in SRS, and others indicating that an imbalance in the relative amounts of the polyamines spermidine and spermine is associated with disease severity in SRS patients.The authors hypothesized that rebalancing the relative amounts of these polyamines could have a therapeutic benefit in these patients.Murray Stewart and co-workers used novel model systems to test their hypothesis, including human cells derived from SRS patients and a novel Drosophila SRS model characterized by reduced lifespan.
The authors recognized that most SRS patients are hypomorphic for germline SMS alterations.They found an expected decrease in pool sizes of putrescine, the diamine precursor of spermidine, and spermidine in SRS cells treated with difluoromethylornithine (DFMO), an enzyme-activated inhibitor of ornithine decarboxylase, but also found evidence of increased spermine synthesis in SRS cells with some residual SMS activity.This finding is consistent with other studies indicating that polyamine biosynthesis is itself highly regulated by a series of feedback mechanisms.They show that SRS cells retain the normal mechanism of regulating the production of the propyl amine precursor (dcSAM) by AMD1, but demonstrate that this mechanism only leads to spermine formation in SRS cells retaining some SMS activity.
The authors recognize that tissue polyamines can be derived from several processes including uptake from dietary or intestinal luminal sources.They use an interesting spermine mimetic, 1,12-Me2-spermine, to augment mechanistic studies indicating that cellular transport mechanisms involving uptake from outside to inside of cells is functional in SRS cells.The spermine mimetic is interesting in that it is resistant to catabolism by serum amine oxidases that might degrade it in patients.In cell culture experiments employing an inhibitor of amine oxidases, both spermine and the spermine mimetic (with or without the amine oxidase inhibitor) restored growth defects and corrected the relative amounts of spermidine and spermine in SRS cells.
Finally, the authors demonstrate that DFMO increases the lifespan of flies in the Drosophila model system, from 21 to 35 days, providing evidence of a therapeutic benefit for DFMO, and potentially the spermine mimetic, in a novel model of this disease.

Specific comments
1. See last line of abstract -The authors should consider the potential inclusion of a genetic marker capable of distinguishing hypomorphic genotypes from total loss of spermine synthase as a potential companion diagnostic for therapies like DFMO and/or the spermine mimetic.They note that "nearly all SRS patient mutations are hypomorphic..." Given the report by Pegg and co-workers that Gy mice, which lack SMS activity, are extremely sensitive to DFMO treatment, such a companion diagnostic could provide important patient safety data in any future clinical trials of this strategy.
2. Last para Intro -The fact that DFMO has been approved by regulatory agencies for other indications may be of limited relevance in this case.Here, the authors are considering chronic administration at low doses (see page 8 line 19 in manuscript) of both DFMO and/or the mimetic.Prior approvals have been for acute IV delivery of high doses of DFMO (sleeping sickness) or topical delivery (Vaniqa).Both these prior approvals are quite different from those contemplated for patients with SRS.Of more relevance will be ongoing studies involving chronic administration of lower doses, like those used to treat children with neuroblastoma.

Referee #3 (Remarks for Author):
This well-designed and well-written manuscript describes the potential use of DFMO in the treatment of patients with Snyder Robinson syndrome (SRS).Since most SRS patients have hypomorphic mutations of the SMS gene causing reduced SMS activity and reduced spermine levels, DFMO can rebalance the spermidine:spermine ratios in these patients by increasing production of dcSAM to facilitate spermine synthesis and by increasing cellular import of polyamines.There are a few points that should be addressed before publication: 1. Fig. 2A: What are the concentrations of DFMO used to treat the SRS patient-derived lymphoblastoid cells?Also, correct the spelling of lymphoblastoid in the figure title.2. Since the effect of DFMO on the spermidine:spermine ratio is variable depending on the extent to which the mutation affects SMS activity, is there a possibility that lowering the ratio to where there is so little spermidine accumulated (as shown in Fig. S1 with c329+5g>a cells treated with 100 µM DFMO) would cause pathology too?Since spermidine is required for hypusination of eIF5A, how may DFMO affect activation of eIF5A? 3. Fig. 4A: in SMS-WT cells, what causes the decrease in the spermidine:spermine ratio -DFMO?This should be indicated.4. Since DFMO cannot rebalance the spermidine:spermine ratio in cells with a complete loss of SMS activity, it is not clear how the Drosophila model with SMS knocked out is a good model for hypomorphic mutations of SMS in humans, nor the mechanism by which DFMO extends their lifespan.Please discuss.A mouse model with a hypomorphic mutation of SMS would be ideal for future preclinical studies.
Referee #4 (Remarks for Author): In Snyder-Robinson syndrome, an incurable disease for which no fundamental treatment exists, the SPD/SPM ratio is increased as a result of the accumulation of SPD, the precursor of SPM, in addition to a decrease in the amount of SPM due to partial dysfunction of spermine synthase.The SPD/SPM ratio tends to be higher the more severe the symptoms of Snyder-Robinson syndrome.The authors carried out this study with the idea that the disease can be treated by reducing the elevated SPD/SPM ratio in patients with Snyder-Robinson syndrome.In a further in vivo study, using a fly model of Snyder-Robinson syndrome, it was shown that oral administration of DMFO increased the life span shortened by the disease.The study is of great interest, but there are some major problems with the organization of the manuscript and some additional experiments are needed to integrate the results of the in intro and in vivo studies.6.In the Discussion, please specifically cite Figs for the results of this study.It should be cited in at least the following three places, but also in other areas where it is possible to cite it.(a) While the extent of induction was similar between WT cells and the G56S variant, induction in the c.329+5 variant significantly exceeded either of these.(b) Consequently, cells with this variant have measurable SMS activity that maintains a SPD/SPM ratio approximately double that of WT controls (our study and (5)).(c) resulting in a ratio approximately 6-fold greater than controls (our study and (10)) 7. Please explain the results more carefully, by mentioning data marked with Asterisk in the figures (What you have shown to be statistically significant differences) in the text.
8. Please provide statistical treatment for the bottom panel of Fig. 2A and indicate significant differences for data described as different in the text.9. Two results for the SMSWT group for the same conditions are shown in Fig. 2A and Fig. 4A respectively.Please explain the reason, or delete one of them.

Minor points Abstract
"Mechanistic studies" Change to "In vitro studies".

P. 4
Consistent with DFMO-mediated inhibition of biosynthesis, SPD was reduced in a dose-dependent manner;.Insufficient data to describe "in a dose-dependent manner", as there is no statistically significant difference between DFMO concentrations of 0.1 mM and 0.5 mM (Fig. 2E).P. 5 "These results indicate that the most common SRS-associated mutations, all of which are hypomorphic, increase resistance to the growth inhibitory effects of DFMO.Additionally, as extracellular sources of SPM include the diet and microbiota, growth inhibition by DFMO is not anticipated to be a treatment limitation in SRS." Please move this sentence to Discussion.Also cite the appropriate literature on the provision of polyamines derived from the diet and microbiota.Furthermore, in the introduction the author described that food-derived spermine does not alter the Spd/Spm ratio, which seems contradictory at first glance.Please explain this more carefully.Murray Stewart and co-workers used novel model systems to test their hypothesis, including human cells derived from SRS pa ents and a novel Drosophila SRS model characterized by reduced lifespan.

General comments
The manuscript by Murray Stewart et al reports a study that is an outstanding example of transla onal biomedical research.The paper addresses an unmet medical need in children with Snyder Robinson Syndrome (SRS), which is the lack of any currently effec ve pharmacotherapies.The authors build on previous studies implica ng a loss of ac vity of the polyamine biosynthe c enzyme spermine synthase (SMS) in SRS, and others indica ng that an imbalance in the rela ve amounts of the polyamines spermidine and spermine is associated with disease severity in SRS pa ents.The authors hypothesized that rebalancing the rela ve amounts of these polyamines could have a therapeu c benefit in these pa ents.Murray Stewart and co-workers used novel model systems to test their hypothesis, including human cells derived from SRS pa ents and a novel Drosophila SRS model characterized by reduced lifespan.
The authors recognized that most SRS pa ents are hypomorphic for germline SMS altera ons.They found an expected decrease in pool sizes of putrescine, the diamine precursor of spermidine, and spermidine in SRS cells treated with difluoromethylornithine (DFMO), an enzyme-ac vated inhibitor of ornithine decarboxylase, but also found evidence of increased spermine synthesis in SRS cells with some residual SMS ac vity.This finding is consistent with other studies indica ng that polyamine biosynthesis is itself highly regulated by a series of feedback mechanisms.They show that SRS cells retain the normal mechanism of regula ng the produc on of the propyl amine precursor (dcSAM) by AMD1, but demonstrate that this mechanism only leads to spermine forma on in SRS cells retaining some SMS ac vity.
The authors recognize that ssue polyamines can be derived from several processes including uptake from dietary or intes nal luminal sources.They use an interes ng spermine mime c, 1,12-Me2spermine, to augment mechanis c studies indica ng that cellular transport mechanisms involving uptake from outside to inside of cells is func onal in SRS cells.The spermine mime c is interes ng in that it is resistant to catabolism by serum amine oxidases that might degrade it in pa ents.In cell culture experiments employing an inhibitor of amine oxidases, both spermine and the spermine mime c (with or without the amine oxidase inhibitor) restored growth defects and corrected the rela ve amounts of spermidine and spermine in SRS cells.
Finally, the authors demonstrate that DFMO increases the lifespan of flies in the Drosophila model system, from 21 to 35 days, providing evidence of a therapeu c benefit for DFMO, and poten ally the spermine mime c, in a novel model of this disease.

Specific comments
1. See last line of abstract -The authors should consider the poten al inclusion of a gene c marker 5th Jul 2023 1st Authors' Response to Reviewers capable of dis nguishing hypomorphic genotypes from total loss of spermine synthase as a poten al companion diagnos c for therapies like DFMO and/or the spermine mime c.They note that "nearly all SRS pa ent muta ons are hypomorphic..." Given the report by Pegg and co-workers that Gy mice, which lack SMS ac vity, are extremely sensi ve to DFMO treatment, such a companion diagnos c could provide important pa ent safety data in any future clinical trials of this strategy.Thank you for this sugges on.We have included the following in the Discussion: "These results also suggest that clinical use of DFMO to treat SRS patients may need to be tailored to the individual depending on the amount of residual SMS activity or SPD/SPM ratio.Indeed, in cell lines with the c.329+5 g>a variant (Fig 2A , S1), exposure to DFMO ultimately depleted SPD pools, which may also be detrimental.However, we anticipate the in vivo situation to be less responsive in this regard due to compensatory uptake of extracellular PUT and/or SPD.Future studies in relevant mammalian animal models should clarify this potential.Regardless, the results of these studies indicate that a functional readout of SMS activity, such as SPD/SPM ratio, should be an important consideration in DFMO clinical trial design." "However, an early mouse model that completely lacked the chromosomal region including the Sms gene was extremely sensitive to DFMO, which had detrimental effects (16).Whether this resulted from the lack of Sms activity or an alternative function is unclear, but caution is warranted when considering patient inclusion criteria for clinical trial design." 2. Last para Intro -The fact that DFMO has been approved by regulatory agencies for other indica ons may be of limited relevance in this case.Here, the authors are considering chronic administra on at low doses (see page 8 line 19 in manuscript) of both DFMO and/or the mime c. Prior approvals have been for acute IV delivery of high doses of DFMO (sleeping sickness) or topical delivery (Vaniqa).Both these prior approvals are quite different from those contemplated for pa ents with SRS.Of more relevance will be ongoing studies involving chronic administra on of lower doses, like those used to treat children with neuroblastoma.We agree and have now emphasized the chronic administra on of DFMO, such as that in NB, by adding the following sentence to the last paragraph of the Introduc on: "Notably, both BABS and neuroblastoma patients receive multiple oral doses of DFMO daily on a continuing basis, representing patient populations and dosing schedules similar to those anticipated in an SRS clinical trial."

Referee #3 (Remarks for Author):
This well-designed and well-wri en manuscript describes the poten al use of DFMO in the treatment of pa ents with Snyder Robinson syndrome (SRS).Since most SRS pa ents have hypomorphic muta ons of the SMS gene causing reduced SMS ac vity and reduced spermine levels, DFMO can rebalance the spermidine:spermine ra os in these pa ents by increasing produc on of dcSAM to facilitate spermine synthesis and by increasing cellular import of polyamines.There are a few points that should be addressed before publica on: 1. Fig. 2A: What are the concentra ons of DFMO used to treat the SRS pa ent-derived lymphoblastoid cells?The DFMO concentra ons are noted below the bo om panel.This has now been clarified in the figure legend as follows: "Cells were treated with increasing concentrations of DFMO as indicated below the bottom panel."Also, correct the spelling of lymphoblastoid in the figure tle.-done 2. Since the effect of DFMO on the spermidine:spermine ra o is variable depending on the extent to which the muta on affects SMS ac vity, is there a possibility that lowering the ra o to where there is so li le spermidine accumulated (as shown in Fig. S1 with c329+5g>a cells treated with 100 µM DFMO) would cause pathology too?Since spermidine is required for hypusina on of eIF5A, how may DFMO affect ac va on of eIF5A?Thank you for the reminder to include a discussion of this differen al response and what it might mean for pa ents.Between the reduc on in SMS ac vity and the compensatory uptake of polyamines that DFMO causes, it is unlikely that SPD will be depleted in the organismal se ng in the drama c manner observed in our cell lines in culture (in the absence of exogenous polyamines).We an cipate that cells will import enough PUT/SPD to maintain these polyamines at homeosta c levels.Future studies in animal models will be needed to fully address this ques on.We have included the following in the Discussion: "These results also suggest that clinical use of DFMO to treat SRS patients may need to be tailored to the individual depending on the amount of residual SMS activity or SPD/SPM ratio.Indeed, in cell lines with the c.329+5 g>a variant (Fig 2A , S1), exposure to DFMO ultimately depleted SPD pools, which may also be detrimental.However, we anticipate the in vivo situation to be less responsive in this regard due to compensatory uptake of extracellular PUT and/or SPD.Future studies in a relevant mammalian animal model should clarify this potential.Regardless, the results of these studies will be important considerations for future clinical trial design.""Untreated lymphoblast lines from 2 different SMS WT healthy donors are included for comparison of polyamine levels and SPD/SPM ratio." 4. Since DFMO cannot rebalance the spermidine:spermine ra o in cells with a complete loss of SMS ac vity, it is not clear how the Drosophila model with SMS knocked out is a good model for hypomorphic muta ons of SMS in humans, nor the mechanism by which DFMO extends their lifespan.Please discuss.We agree completely that the fly model is not an ideal model, but it is currently the only system readily available with a defined phenotype amenable to a enua on.As our primary goal with this study was to define the mechanism-of-ac on of DFMO in SRS pa ent cells, we chose to use the fly model to expand the study to the organismal level and demonstrate an effect beyond that at the biochemical/molecular level.We have now included the following sentence in the Discussion to clarify that SPM is a component of the fly medium, sugges ng that DFMO may improve SPM availability in support of lifespan extension: "Additionally, the standard media used for Drosophila studies includes measurable SPM (approximately 4 nmol/mg), suggesting its enhanced uptake by the dSms -/-flies may be responsible for the extended lifespan in response to DFMO." A mouse model with a hypomorphic muta on of SMS would be ideal for future preclinical studies.We agree, and further studies are planned in a newly available mouse SRS model, once baseline phenotyping studies have been completed and a phenotype relevant to the human popula on has been iden fied.We have included the following sentence in the Discussion to indicate the status of the mouse studies: "A mouse model of SRS, which harbors the clinically relevant Sms G56S mutation, recently became available (The Jackson Laboratory, strain #033707).Our plans for future studies include determining the effects of DFMO, SPM, and Me 2 SPM on these mice; however, a patient-relevant phenotype must first be identified."

Referee #4 (Remarks for Author):
In Snyder-Robinson syndrome, an incurable disease for which no fundamental treatment exists, the SPD/SPM ra o is increased as a result of the accumula on of SPD, the precursor of SPM, in addi on to a decrease in the amount of SPM due to par al dysfunc on of spermine synthase.The SPD/SPM ra o tends to be higher the more severe the symptoms of Snyder-Robinson syndrome.The authors carried out this study with the idea that the disease can be treated by reducing the elevated SPD/SPM ra o in pa ents with Snyder-Robinson syndrome.In a further in vivo study, using a fly model of Snyder-Robinson syndrome, it was shown that oral administra on of DMFO increased the life span shortened by the disease.
The study is of great interest, but there are some major problems with the organiza on of the manuscript and some addi onal experiments are needed to integrate the results of the in intro and in vivo studies.1.To integrate the fly experiments with your in vitro experiments up to that point, the following addi onal experiments should be added.(a) Test the respec ve polyamine levels in the body of the flies.This is a good sugges on and was in our original plan.Unfortunately, technical issues prohibited the collec on of this data and our system has since been out of commission.However, we would like to emphasize that the choice to include the fly model was made with the goal of demonstra ng an effect at the organismal level.As the biochemical MOA has been deciphered in human pa ent cells, we do not believe that the lack of Drosophila biochemistry diminishes the study.The SRS fly model has some major limita ons (it is a complete knockout vs. hypomorph, dSms is autosomal vs. X-linked, and the fly polyamine pathway may differ from that in mammals), but it is the only readily available model with a defined, reversible phenotype.Thus, we used it to show a phenotypic benefit beyond the change in biochemistry observed in pa ent cells, but respec ully do not believe that further molecular studies in the fly model will add value to the manuscript.(b) Analyse the changes in expression of polyamine-related enzyme genes (odc, srm, sms, amd1) in the fly body following DFMO administra on.Due to the limita ons described in 1a, we do not believe these experiments will add value to our study.Addi onally, most polyamine-related genes, including those men oned, are majorly regulated at the post-transcrip onal/transla onal level, so measuring gene expression provides li le indica on of true metabolic ac vity.(c) Analyse the effects of Spm alone and simultaneous administra on of Spm and DFMO on the lifespan of flies.This is an important point we did not clarify.We did not supplement the fly growth medium with SPM, as we had determined it was already present in the ingredients.So, the condi ons suggested are the same as those performed.This has been clarified in the Discussion as follows: "Notably, the standard media used for Drosophila studies includes measurable SPM (approximately 4 nmol/mg), suggesting its enhanced uptake by the dSms -/-flies may be responsible for the extended lifespan in response to DFMO." 2. Please prepare a Table showing genotype and characteris cs, spermidine synthase ac vity and first published literature for the cells isolated from Snyder-Robinson syndrome pa ents and used in this study, and introduce them in the introduc on.
The following text with corresponding Table has been added in the Introduc on.Note that although we like the idea of including the pa ent characteris cs, this requires much space and was recently provided in another publica on, which is now emphasized for the reader.Also, we chose to include the ra o as a func onal readout of SMS ac vity, as an actual ac vity assay is not currently in use.
"A summary of patient cell lines with ratios included in this study can be found in 3. The Legend for Fig. 2 and Fig. 4 describes the experimental results.The legend should contain detailed informa on about the experimental condi ons and the axes of the graphs, while the experimental results should be integrated in the text.Please revise.
We have revised the figure legends according to the EMBO Molecular Medicine Author Guidelines.
4. There is a cita on to Fig. 3D in the text, but Fig. 3D does not exist in this manuscript.Either delete the cita on in the text or add Fig. 3D.(p. 5, "included during treatment (Fig. 3B-D).")Thank you for catching this.The "D" has been changed to "C".(b) Consequently, cells with this variant have measurable SMS ac vity that maintains a SPD/SPM ra o approximately double that of WT controls (our study and ( 5)).
(c) resul ng in a ra o approximately 6-fold greater than controls (our study and ( 10)) References to figures have now been added throughout the Discussion.
7. Please explain the results more carefully, by men oning data marked with Asterisk in the figures (What you have shown to be sta s cally significant differences) in the text.We have carefully reviewed the Results and added text in cases of ambiguity.
8. Please provide sta s cal treatment for the bo om panel of Fig. 2A and indicate significant differences for data described as different in the text.Sta s cally significant differences are indicated in figures in accordance with EMBO's guidelines, which advise against providing sta s cs for studies with n = 2. 9. Two results for the SMSWT group for the same condi ons are shown in Fig. 2A and Fig. 4A respec vely.Please explain the reason, or delete one of them.Thank you for no ng this.The WT results are from 2 different donors.We have included the following in the figure legend: "Untreated lymphoblast lines from 2 different SMS WT healthy donors are included for comparison of polyamine levels and SPD/SPM ratio." Minor points Abstract "Mechanis c studies" Change to "In vitro studies".We have added "in vitro"

P. 4
Consistent with DFMO-mediated inhibi on of biosynthesis, SPD was reduced in a dose-dependent manner;.Insufficient data to describe "in a dose-dependent manner", as there is no sta s cally significant difference between DFMO concentra ons of 0.1 mM and 0.5 mM (Fig. 2E).
"in a dose-dependent manner" has been removed P. 5 "These results indicate that the most common SRS-associated muta ons, all of which are hypomorphic, increase resistance to the growth inhibitory effects of DFMO.Addi onally, as extracellular sources of SPM include the diet and microbiota, growth inhibi on by DFMO is not an cipated to be a treatment limita on in SRS." Please move this sentence to Discussion.Also cite the appropriate literature on the provision of polyamines derived from the diet and microbiota.Done Furthermore, in the introduc on the author described that food-derived spermine does not alter the Spd/Spm ra o, which seems contradictory at first glance.Please explain this more carefully.Food-derived spermine seems to have limited effect on its own -this is the problem we hope to negate with DFMO.One possible explana on is that the cell type necessary for uptake and entry into systemic circula on has downregulated polyamine transport, unlike the fibroblasts and lymphoblasts we can easily study in culture.These types of studies will benefit from an appropriate mouse model.DFMO should s mulate uptake in all cell types, as well as s mulate the conversion of SPD to SPM.We have added the following in the Introduc on: "However, the fact remains that SPM is a common dietary component that fails to rescue the SRS phenotype in vivo, suggesting that improvements to SPM bioavailability may enhance efficacy."Thank you for the submission of your revised manuscript to EMBO Molecular Medicine, and please accept my apologies for the delay in getting back to you in this busy time of the year.We have now received the reports from the 2 referees who re-reviewed your manuscript.As mentioned in a previous correspondence and as you will see below, while referee #3 is supportive of publication, referee #4 regrets that concerns raised during the initial review have not been addressed.Based on the explanations you provided and on the technical limitations mentioned, and after discussion with my colleagues, we agree that it would not be productive to wait for these limitations to be overcome, and I am therefore pleased to inform you that I will be able to accept your manuscript once the following editorial points will be addressed: 1/ Please provide a detailed response to referee #4's remaining concerns.

2/ Main manuscript text:
-Please address the queries from our data editors in the related manuscript file.Please accept previous changes and only keep in track changes mode any new modification.
-Please move the Data Availability section to the end of the Materials and Methods.
-Acknowledgements: the information provided in this section should be the same as provided in the submission system.Currently, Commonwealth Foundation for Cancer Research, Chan-Zuckerberg Initiative have not been entered in the submission system.

3/ Figures:
-please make sure all figures/figure panels are referenced in the text.A callout is currently missing for Fig. 5A.
-please remove error bars in figure with n=2.4/ Thank you for providing a nice synopsis image.Could you please send it as a jpg/png/tiff version as 550 pixels wide x 200-400 high?5/ As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329),EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts.This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript.Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication.Please note that the Authors checklist will be published at the end of the RPF.I look forward to receiving your revised manuscript.New materials and reagents need to be available; do any restrictions apply?Not Applicable

Antibodies
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(Reagents and Tools Cell lines: Provide species information, strain.Provide accession number in repository OR supplier name, catalog number, clone number, and/OR RRID.

Yes
Experimental procedures Cell lines, culture conditions, and compounds and Table 1 Primary cultures: Provide species, strain, sex of origin, genetic modification status.

Yes
Experimental procedures Cell lines, culture conditions, and compounds and Table 1 Report if the cell lines were recently authenticated (e.g., by STR profiling) and tested for mycoplasma contamination.

Yes
Experimental procedures Cell lines, culture conditions, and compounds

Experimental animals
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(Reagents and Tools

Plants and microbes
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(Reagents and Tools the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner.

Study protocol
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(Reagents and Tools

Reporting
Adherence to community standards Information included in the manuscript?
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(Reagents and Tools Have primary datasets been deposited according to the journal's guidelines (see 'Data Deposition' section) and the respective accession numbers provided in the Data Availability Section?

Not Applicable
Were human clinical and genomic datasets deposited in a public accesscontrolled repository in accordance to ethical obligations to the patients and to the applicable consent agreement?

Not Applicable
Are computational models that are central and integral to a study available without restrictions in a machine-readable form?Were the relevant accession numbers or links provided?

Not Applicable
If publicly available data were reused, provide the respective data citations in the reference list.

Not Applicable
The MDAR framework recommends adoption of discipline-specific guidelines, established and endorsed through community initiatives.Journals have their own policy about requiring specific guidelines and recommendations to complement MDAR.

9)
We replaced Supplementary Information with Expanded View (EV) Figures and Tables that are collapsible/expandable online.A maximum of 5 EV Figures can be typeset.EV Figures should be citedas 'Figure EV1, Figure EV2" etc... in the text and their respective legends should be included in the main text after the legends of regular figures.-For the figures that you do NOT wish to display as Expanded View figures, they should be bundled together with their legends in a single PDF file called *Appendix*, which should start with a short Table of Content.Appendix figures should be referred to in the main text as: "Appendix Figure S1, Appendix Figure S2" etc.
** Reviewer's comments ***** Referee #2 (Comments on Novelty/Model System for Author): Murray Stewart and co-workers used novel model systems to test their hypothesis, including human cells derived from SRS patients and a novel Drosophila SRS model characterized by reduced lifespan.Referee #2 (Remarks for Author): 1. To integrate the fly experiments with your in vitro experiments up to that point, the following additional experiments should be added.(a) Test the respective polyamine levels in the body of the flies.(b) Analyse the changes in expression of polyamine-related enzyme genes (odc, srm, sms, amd1) in the fly body following DFMO administration.(c) Analyse the effects of Spm alone and simultaneous administration of Spm and DFMO on the lifespan of flies.2. Please prepare a Table showing genotype and characteristics, spermidine synthase activity and first published literature for the cells isolated from Snyder-Robinson syndrome patients and used in this study, and introduce them in the introduction.3. The Legend for Fig. 2 and Fig. 4 describes the experimental results.The legend should contain detailed information about the experimental conditions and the axes of the graphs, while the experimental results should be integrated in the text.Please revise.4.There is a citation to Fig. 3D in the text, but Fig. 3D does not exist in this manuscript.Either delete the citation in the text or add Fig. 3D.(p. 5, "included during treatment (Fig. 3B-D).") 5. Fig. 4C is present in Figures, but a citation to fig.4C is missing in the text of this manuscript; please explain the results while citing fig.4C, or delete fig.4C.

Figure
Figure legend for Fig. 2 Please specify in the legend what the abbreviations NT and WT refer to.
3.Fig.4A: in SMS-WT cells, what causes the decrease in the spermidine:spermine ra o -DFMO?This should be indicated.Thank you for no ng this.The WT results are from 2 different donors.We have included the following in the figure legend: 5.Fig.4C is present in Figures, but a cita on to fig.4C is missing in the text of this manuscript; please explain the results while ci ng fig.4C, or delete fig.4C.Thank you.Fig 4C has now been cited.6.In the Discussion, please specifically cite Figs for the results of this study.It should be cited in at least the following three places, but also in other areas where it is possible to cite it.(a) While the extent of induc on was similar between WT cells and the G56S variant, induc on in the c.329+5 variant significantly exceeded either of these.

Figure
Figure legend for Fig. 2 Please specify in the legend what the abbrevia ons NT and WT refer to.-done

Table 1 .
Additionally, a detailed collection of the clinical features of the individual patients was recently published by Larcher and colleagues (14)."

Table 1 -Variants and ratios of SRS patient-derived cell lines in the current study.
1 Ratio of N/A is due to complete lack of SPM; WT, wildtype; (L), lymphoblast; (f), fibroblast

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(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)

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Table, Materials and Methods, Figures, Data Availability Section) (Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)

Short novel DNA or RNA including primers, probes: provide the sequences. Not Applicable Cell materials Information included in the manuscript? In which section is the information available?
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Provide species, strain, sex, age, genetic modification status.Provide accession number in repository OR supplier name, catalog number, clone number, OR RRID.

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Table, Materials and Methods, Figures, Data Availability Section) (Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) If collected and within the bounds of privacy constraints report on age, sex and gender or ethnicity for all study participants.

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(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section), such as t-test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section;

Please complete ALL of the questions below. Select "Not Applicable" only when the requested information is not relevant for your study. if
n<5, the individual data points from each experiment should be plotted.Any statistical test employed should be justified.Source Data should be included to report the data underlying figures according to the guidelines set out in the authorship guidelines on Data Each figure caption should contain the following information, for each panel where they are relevant: a specification of the experimental system investigated (eg cell line, species name).theassay(s)and method(s) used to carry out the reported observations and measurements.anexplicitmention of the biological and chemical entity(ies) that are being measured.anexplicitmention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.ideally,figurepanels should include only measurements that are directly comparable to each other and obtained with the same assay.plotsinclude clearly labeled error bars for independent experiments and sample sizes.Unless justified, error bars should not be shown for technical the exact sample size (n) for each experimental group/condition, given as a number, not a range; a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).a statement of how many times the experiment shown was independently replicated in the laboratory.This checklist is adapted from Materials Design Analysis Reporting (MDAR) Checklist for Authors.MDAR establishes a minimum set of requirements in transparent reporting in the life sciences (see Statement of Task: 10.31222/osf.io/9sm4x).Please follow the journal's guidelines in preparing your manuscript.

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Table, Materials and Methods, Figures, Data Availability Section) If study protocol has been pre-registered, provide DOI in the manuscript.For clinical trials, provide the trial registration number OR cite DOI.(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)If sample or data points were omitted from analysis, report if this was due to attrition or intentional exclusion and provide justification.

definition and in-laboratory replication Information included in the manuscript? In which section is the information available?
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)In the figure legends: state number of times the experiment was replicated in laboratory.

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Include a statement confirming that informed consent was obtained from all subjects and that the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.For publication of patient photos, include a statement confirming that consent to publish was obtained.Not Applicable Studies involving experimental animals: State details of authority granting ethics approval (IRB or equivalent committee(s), provide reference number for approval.Include a statement of compliance with ethical regulations.
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)Studies involving human participants: State details of authority granting ethics approval (IRB or equivalent committee(s), provide reference number for approval.Not ApplicableStudies involving human participants:

Use Research of Concern (DURC) Information included in the manuscript? In which section is the information available?
(Reagents and ToolsTable, Materials and Methods, Figures, Data Availability Section) Could your study fall under dual use research restrictions?Please check biosecurity documents and list of select agents and toxins (CDC): https://www.selectagents.gov/sat/list.htmNot Applicable If you used a select agent, is the security level of the lab appropriate and reported in the manuscript?Not Applicable If a study is subject to dual use research of concern regulations, is the name of the

authority granting approval and reference number for
the regulatory approval provided in the manuscript?

and III randomized controlled trials
Table, Materials and Methods, Figures, Data Availability Section) State if relevant guidelines or checklists (e.g., ICMJE, MIBBI, ARRIVE, PRISMA) have been followed or provided.Yes ICMJE guidelines for authorship -during submission For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at top right).See author guidelines, under 'Reporting Guidelines'.Please confirm you have followed these guidelines., please refer to the CONSORT flow diagram (see link list at top right) and submit the CONSORT checklist (see link list at top right) with your submission.See author guidelines, under 'Reporting Guidelines'.Please confirm you have submitted this list.Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) (