Succinate receptor 1 restricts hematopoiesis and prevents acute myeloid leukemia progression

Domain: neuroscience
Published: 2026-02-05
Editorial outcome: major_revision

Abstract
Despite intriguing roles for the Succinate receptor (Sucnr1) in inflammation, few studies have explored its role in hematopoiesis. Here, we show that low SUCNR1 represents a marker for reduced overall and progression-free survival in acute myeloid leukemia (AML) patients. Succinic acid, which displays Sucnr1-dependent and independent effects, promotes disease in mouse models of pre-leukemic myelopoiesis, AML and AML xenografts, expressing low SUCNR1. In vivo global or hematopoietic deletion of Sucnr1 induces expansion of hematopoietic stem and progenitor cells (HSPC) and hematopoiesis, whilst Sucnr1-tomato+ HSPC display restricted engraftment potential. Mechanistically, activation of Sucnr1 counterbalances the stimulatory effect of intracellular succinate in HSPC and preserves HSPC transcriptional programs via control of S100a8/S100a9. Blocking S100a9 with tasquinimod rescues the defects of Sucnr1 knock-out mice, and combined with a potent Sucnr1 agonist shows therapeutic value in AML mice. In AML xenografts, single-cell RNA-sequencing reanalyses confirm SUCNR1 as a therapeutic vulnerability in patients. Together, Sucnr1 signaling restricts hematopoiesis at least partially through HSPC and via control of S100a8/S100a9. Its dysregulation emerges as contributor to malignancy that opens therapeutic avenues for AML patients.

Introduction
Hematopoietic stem cell (HSC) ability to self-renew and retain its identity depends on intrinsic features and on the microenvironment provided by non-HSC. Thus, both intrinsic and extrinsic factors support the great heterogeneity of the HSC compartment in terms of transcriptional and functional lineage priming, as demonstrated by the varied outcomes of transplanted individual HSC1. The HSC niche refers to cell-to-cell interactions, secreted factors, inflammation, and metabolic signals such as hypoxia, among others2. The hypoxic niche allows the fine-tuned metabolic control required to maintain HSC long-term quiescence and self-renewal, with participation of hypoxia-inducible factor-1 (HIF-1)2,3. HSC generate energy mainly via glycolysis3, while leukemia stem cells (LSC) in AML contain greater mitochondrial mass, have higher oxygen consumption but display a low reactive oxygen species (ROS) content, and are sensitive to inhibition of mitochondrial translation4, indicating that they are dependent on mitochondrial metabolism including the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OxPHOS). Reduced OxPHOS through BCL-2 inhibition selectively eradicated human quiescent LSC in xenografts5.

Under physiological hypoxia, low oxygen causes the intermediate metabolite of the TCA cycle succinate to accumulate. Succinate enables ATP generation by OxPHOS6 and it can be released from mitochondria into cytoplasm, nucleus and extracellular space, where it exerts pleiotropic roles7–9. In myeloid cells, cytoplasmic succinate results in stabilization of HIF-1α subunit10,11, which then induces interleukin-1β (IL-1β) expression11. One of the first evidences for a role of succinate in cancer development was the discovery of pseudohypoxia; activation of hypoxia signaling under normal oxygen levels12. This can happen for example as a result of succinate dehydrogenase (Sdh) inhibiting mutations and lead to increased expression of genes that facilitate angiogenesis, metastasis, and glycolysis, ultimately leading to tumor progression mediated through HIF-1α10. Global inducible deletion of the mitochondrial Sdhd led to hematopoietic defects, including depletion of bone marrow (BM) progenitors and differentiated cells13, using a mouse model that did not allow distinction of potential hematopoietic and stromal effects8. Succinate is a metabolic signal that links hypoxia to IL-1β and cell responses in myeloid cells through intracellular mechanisms, but also autocrine/paracrine processes through Sucnr18,11,14–16. Sucnr1 seems to have different functions depending on the context, and has been proposed as inflammatory in dendritic cells but anti-inflammatory in neural stem cells and both inflammatory or anti-inflammatory in macrophages14–17. Cancer cells release succinate and locally polarize macrophages into tumor-associated macrophages through Sucnr1 activation18. Despite these intriguing precedents, succinate has been scarcely explored in hematopoiesis. Human CD34+ progenitors express SUCNR1 and its activation has been suggested to induce proliferation in vitro19.

Here, we investigate the in vivo role and the cellular and molecular mechanisms of succinate and Sucnr1 signaling in normal and malignant hematopoiesis. Low SUCNR1 represents a marker for poor prognosis in AML patients. In mouse models of pre-leukemic myelopoiesis, AML and AML xenografts that express low SUCNR1, treatment with succinic acid results in disease progression. To study the effect of low Sucnr1 in the hematopoietic system, we used global or hematopoietic deletion of Sucnr1, which induce expansion of HSPC and hematopoiesis. Conversely, we generated a Sucnr1-tdTomato mouse line which informed that Sucnr1-tomato+ HSPC form a subset of HSPC with restricted engraftment potential. Importantly, activation of Sucnr1 counterbalances the stimulatory effect of succinate in HSPC and preserves transcriptional programs characteristic of HSPC via control of S100a8/S100a9 under steady-state. We further show therapeutic value for a drug combination blocking S100a9 and activating Sucnr1 in AML mice, and confirm SUCNR1 as a potential therapeutic vulnerability in human AML. Thus, our data support dysregulation of Sucnr1 as a contributor to AML pathogenesis that show promise for future therapeutic exploitation.

Results
Low SUCNR1 predicts acute myeloid leukemia progression
We first studied the prognostic value of SUCNR1 in publicly available arrays from purified AML blasts in a published cohort of AML patients (GSE1446820–22). Low levels of expression of SUCNR1 were associated with reduced overall survival (Fig. 1A) and progression-free survival (Fig. 1B). We performed subsequent Cox regression analysis, which uncovered SUCNR1 expression as an independent predictor of overall survival and progression-free survival after adjusting for age, gender and French-American-Bristish (FAB) classification (Supplementary Table S1–S2). We separately analyzed less differentiated AML profiles according to FAB and found that low SUCNR1 is a marker of poor prognosis in M0–M3 AML patients (n = 224) (Supplementary Fig. 1A). In parallel, we identified M4–M5 patients according to FAB as those patients with lowest SUCNR1 expression, versus M0–M3 (Fig. 1C). In the same cohort, the expression of SUCNR1 was heterogeneous among AML patients with various molecular signatures. CEBPA, FLT3-ITD and NPM1 mutations were associated with higher SUCNR1 expression in AML blasts, IDH1, IDH2 and FLT3-TKD had no impact on SUCNR1 expression, and NRAS mutations were linked to a trend towards reduced levels of SUCNR1 expression (Supplementary Fig. 1B). We then used the dataset TARGET AML at BloodSpot (GSE4251923 and GSE1315924,25) that confirmed great heterogeneity in SUCNR1 expression in primary AML and uncovered overall lower expression values compared to controls (Fig. 1D). To understand how low expression of SUCNR1 is functionally linked to poor survival in AML patients in vivo, we used succinic acid injections in AML patient xenografts, as succinic acid takes the form of the endogenous anion succinate in living organisms. Using CD34+ progenitors obtained from one M1 AML patient with adverse risk and no detectable expression of SUCNR1 by digital droplet (dd) PCR (Supplementary Fig. 1C), we found that in vivo treatment with succinic acid increased the leukemic output, as measured by numbers of human CD33+ myeloid progenitors, in the BM of xenograft recipients versus vehicle-treated mouse recipients (Fig. 1E and Supplementary 1D).

We then used the Mx1-Cre NRASG12D mouse model of aberrant pre-leukemic myelopoiesis26,27, from now on referred to as NRAS-G12D+. Primary NRAS-G12D+ mice exhibit a mild hematopoietic phenotype, characterized as chronic myelomonocytic leukemia or myeloproliferative neoplasm (MPN)28,29. BM nucleated cells from NRAS-G12D+ expressed less Sucnr1 than their control counterparts (Fig. 2A). Early stages of NRAS-G12D+-driven disease are characterized by a selective expansion of BM HSPC in the absence of other remarkable hematopoietic events, including unchanged monocytes and BM cellularity (Supplementary Fig. 2A). Intracellular succinate was similar in NRAS-G12D+ Lin−Sca-1+c-Kit+ (LSK) and monocytes versus control cells, but LSK cells showed 3-fold higher succinate content than monocytes (Fig. 2B). At early stages of disease, we found higher content of succinate in the BM extracellular fluid (BMEF) of pre-leukemic NRAS-G12D+ mice compared to control mice (Fig. 2C). In turn, succinic acid injection in vivo increased succinate content in BMEF (Supplementary Fig. 2B) and aggravated disease in NRAS-G12D+ mice, promoting myeloid bias and B cell development deficiency (Fig. 2D and Supplementary Fig. 2C) together with expansion of the LSK cell compartment in the BM analyzed by fluorescence-activated cell sorting (FACS) (Fig. 2E and Supplementary Fig. 2D). Analysis of the stem and progenitor cell subsets corresponding to HSC and MPP1-MPP630–32 revealed a selective expansion of MPP3 and MPP4 HSPC (Fig. 2F and Supplementary Fig. 2D). Within lineage-negative progenitors, we found altered fractions of Lin−c-Kit+Sca-1− (LK) cells which were increased and common lymphoid progenitors (CLP) that were reduced after succinic acid treatment (Supplementary Fig. 2E and F). LK subsets were slightly biased towards common myeloid progenitors (CMP) whereas megakaryocyte erythroid progenitors (MEP) and granulocyte monocyte progenitors (GMP) were unchanged after succinic acid treatment (Supplementary Fig. 2E and G).

We then used an aggressive leukemic mouse model heterozygous for Mll-AF9 knock-in (KI) fusion transgene or wild-type (WT) control male littermates33. Expression of the MLL-AF9 fusion protein results in development of leukemia beginning around six months of age, mainly AML. BM nucleated cells from mice transplanted with BM of MLL-AF9+ leukemic donors expressed less Sucnr1 than their healthy counterparts (Fig. 2G). Succinic acid administration reduced survival in mice transplanted with BM of MLL-AF9+ leukemic donors versus vehicle-treated mice (Fig. 2H). MLL-AF9+ mice had overt AML and the lineage-negative compartment was expanded in the BM of succinic acid- versus vehicle-treated mice (Fig. 2I), with an observed trend toward increased LK fraction and LSK cells that did not reach statistical significance (Supplementary Fig. 2H). The leukemic burden by means of increased counts of abnormal monocytes in extramedullary sites was higher in leukemic mice treated with succinic acid (Fig. 2J).

To sum up, AML patients show heterogeneous expression of SUCNR1 and low SUCNR1 represents a prognostic marker for reduced overall and progression-free survival. Conversely, in AML mouse models and patient xenografts with low SUCNR1 expression, succinate accumulates in BM and contributes to leukemia progression.

Sucnr1 restricts HSPC and hematopoiesis under steady-state
To understand the uncovered protective effect of succinate signaling via Sucnr1 on hematopoiesis, we next characterized the immunophenotype of Sucnr1 knock-out (KO) mice, which have not been used before in studies of hematopoiesis. Young adult Sucnr1-KO mice had increased circulating red blood cells, hemoglobin and hematocrit, but no other remarkable events were observed compared to WT mice in peripheral blood (PB), BM or spleen (SP) (Supplementary Fig. 3A–D). Sucnr1-KO mice were analyzed at later middle-aged stages and then showed an increase in circulating leukocytes, particularly monocytes and B cells (Fig. 3A), in addition to expanded reticulocytes (Supplementary Fig. 3E). In the BM, Sucnr1 deletion induced hypercellularity with expansion of myeloid cells, B cells and HSPC identified as LSK cells (Fig. 3B–D). The fractions of LSK cells were altered in Sucnr1-KO versus WT mice with expansion of long-term (LT)-HSC, short-term (ST)-HSC and multipotent progenitors (MPP), analyzed by FACS (Supplementary Fig. 4A). Further analysis of the stem and progenitor cell subsets corresponding to HSC and MPP1-MPP630–32 revealed absolute expansion of HSC, MPP1, MPP4, MPP5 and MPP6, and a trend toward expansion of MPP3 (Fig. 3E). We also analyzed the population percentages of LSK cells, which was increased in the BM of Sucnr1-KO versus WT mice (Supplementary Fig. 4B). Analysis of HSPC subsets corresponding to HSC and MPP1-MPP6 uncovered increased relative frequencies of HSC, MPP1, MPP4, MPP5 and MPP6 (Supplementary Fig. 4B), supporting for an HSPC origin of the stimulated hematopoiesis in the BM of Sucnr1-KO mice. Numbers of colonies formed ex vivo by HSPC were higher in Sucnr1-KO mice (Fig. 3F). We performed serial replatings and found that HSPC from Sucnr1-KO mice generated higher numbers of secondary colonies in a non-significant trend, and similar numbers of tertiary colonies compared to WT (Fig. 3F). Numbers of LK progenitors, particularly MEP and GMP, as well as CLP were increased in the BM of Sucnr1-KO versus WT mice (Fig. 3G and H).

Consistent with the increased numbers of HSPC, analysis of the LSK cell compartment in the BM of Sucnr1-KO mice showed reduced numbers of early apoptotic BM LSK and a trend towards increased numbers of live cells (Supplementary Fig. 4C). Conversely, we analyzed LSK cell cycle and the proliferative fraction in the G1 phase was slightly reduced (Supplementary Fig. 4D).

Succinate levels were reduced in the BMEF of Sucnr1-KO versus WT mice (Supplementary Fig. 4E), whilst intracellular succinate levels in HSC were increased (Supplementary Fig. 4F). Given that succinate is an intermediate metabolite of the TCA cycle, we studied its potentially increased cycling which would result in metabolic changes. Sucnr1 deletion resulted in increased baseline oxygen consumption rate in lineage-negative progenitors (Supplementary Fig. 4G). This effect was specific and reversible because the maximal oxygen consumption rate and the glycolytic flux remained unchanged (Supplementary Fig. 4G). Consistent with these data, both total and mitochondrial ROS levels were higher in LSK cells from Sucnr1-KO mice (Supplementary Fig. 4H).

In view of the stimulated hematopoiesis in the BM of Sucnr1-KO mice, we also analyzed the SP as an extramedullary site of hematopoiesis. The total numbers of nucleated cells in SP were increased in Sucnr1-KO mice, due to higher numbers of myeloid cells, including both granulocytes and monocytes, and B cells (Supplementary Fig. 4I). Numbers of secondary colonies generated ex vivo by HSPC were higher in Sucnr1-KO mice (Supplementary Fig. 4J). Conversely, in vivo treatment of mice with one single injection of succinic acid did not affect mobilization of progenitors from BM (Supplementary Fig. 5A).

To dissect the role of Sucnr1 on healthy hematopoiesis via the hematopoietic compartment, we transplanted Sucnr1fl/fl control or Mx1-Cre Sucnr1fl/fl experimental BM cells into C57BL/6J WT mice, which were later injected with poly-inosine:poly-cytosine (pIpC; Fig. 4A) to induce Sucnr1 deletion from hematopoietic cells (Supplementary Fig. 6A). The end-point analysis was performed 47 weeks (about 11 months) after pIpC induction, to make it a time-point comparable to the global deletion of Sucnr1. In vivo deletion of Sucnr1 from hematopoietic cells resulted in early expansion of the numbers of circulating total leukocytes, i.e., myeloid cells, monocytes and B lymphocytes (Fig. 4B), which was maintained over the course of the experiment (Fig. 4C). In BM, in vivo deletion of Sucnr1 from hematopoietic cells led to an expansion of MPP versus intact recipients, particularly MPP4 (Fig. 4D). Thus, Sucnr1 effects on hematopoiesis can be explained greatly by intrinsic effects to hematopoietic cells.

Together, these results indicate that Sucnr1 restricts HSPC and hematopoiesis in vivo, mainly cell-autonomously.

Sucnr1 is expressed in HSPC and its activation restricts their functional response
In view of the lack of commercially available mouse Sucnr1-specific antibody, we generated a Sucnr1-tdTomato reporter mouse line (Supplementary Fig. 7A–D) and used it to study the expression of Sucnr1 protein by BM HSPC subsets. FACS-sorted LSK cells from Sucnr1-tdTomato and WT mice were imaged by imaging flow cytometry, which revealed presence of SUCNR1 in a subset of LSK cells (Fig. 5A). HSPC subsets, committed hematopoietic progenitors and differentiated cells obtained from the mouse BM were further analyzed by spectral flow cytometry (Supplementary Fig. 8A). After spectral unmixing, we quantified the Sucnr1-tdTomato+ cell fractions (Fig. 5B and Supplementary Fig. 9A–E). Our analysis revealed that all HSPC subsets show a varying fraction of Sucnr1+ cells and the majority of Sucnr1+ HSPC were identified as MPP3, with an average of 0.191% of Sucnr1+ cells, followed by MPP4 (Fig. 5B and Supplementary Fig. 9C). Importantly, although Sucnr1+ cells were found across all hematopoietic cell subsets analyzed, including all lineage-positive cell types (Supplementary Fig. 9C and D), the majority of Sucnr1+ cells in the hematopoietic system were identified as lineage-negative progenitors, including all LK progenitors particularly GMP, LSK cells and CLP (Supplementary Fig. 9E). In parallel, we confirmed SUCNR1 mRNA expression across all hematopoietic cell subsets analyzed in normal human hematopoiesis, using expression data from the HemaExplorer collection at Blood Spot (GSE1705434, GSE1959935, GSE1186436 and E-MEXP-124237), including HSC and HSPC (Supplementary Fig. 9F). In humans, CMP, GMP and differentiating promyelocytes and myelocytes display remarkably high levels of SUCNR1 mRNA expression (Supplementary Fig. 9F).

We then tested mouse HSPC functional response to cell-permeable succinate ex vivo by adding monomethyl succinate, inactive on Sucnr138, which enhanced the colony-forming potential in total BM (TBM) in a dose-response manner (Fig. 5C). Conversely, succinic acid, which displays both Sucnr1-dependent and independent effects38,39, resulted in unchanged colony-forming potential (Fig. 5C). Taken together, these results suggest that Sucnr1 activation represses the intracellular effect of succinate. We further explored the effect of succinic acid and monomethyl succinate combination, which resulted in a partial increase in colony-forming potential in TBM versus succinic acid alone and a partial reduction versus monomethyl succinate alone, confirming that Sucnr1 activation represses the intracellular effect of succinate (Fig. 5D).

To dissect a potentially direct effect of Sucnr1 activation on HSC function, FACS-sorted HSC immunophenotypically defined as Lin−c-Kit+Sca-1+CD34−Flt3−CD150+CD48− were tested for their colony-forming potential in serial replatings in the presence of monomethyl succinate or cis-epoxy succinate, 10- to 20-fold more potent than succinic acid on Sucnr139. In the second replating, monomethyl succinate showed a trend towards stimulation but cis-epoxy succinate reduced the colony-forming potential of HSC, with no effect on long-term colony forming potential measured after the third plating of the cells (Fig. 5E).

To confirm a direct role of Sucnr1 on HSPC function, FACS-sorted HSC from Sucnr1-KO mice were tested for their colony-forming potential in serial replatings in the presence of monomethyl succinate or cis-epoxy succinate. In the first and second replating, both monomethyl succinate and cis-epoxy succinate stimulated or showed a trend towards stimulation of the colony-forming potential of Sucnr1-KO HSC, suggesting loss of colony-forming potential repression by Sucnr1 activation in the absence of Sucnr1 (Fig. 5F). No changes were observed at third plating, indicative of no impact on long-term colony forming potential.

We then performed competitive repopulation assays of BM nucleated cells isolated from WT or Sucnr1-KO mice (Ly5.2) into lethally irradiated Ly5.1 congenic recipients. Surprisingly, peripheral blood analysis from recipient mice 16 weeks after transplantation revealed a mild reduction in HSPC engraftment in competition, in recipients transplanted with Sucnr1-KO cells compared to recipients transplanted with WT control cells (Fig. 5G and Supplementary Fig. 9G). To substantiate the role of Sucnr1 in HSPC function in vivo, we performed competitive repopulation assays using 200 Sucnr1-tdTomato+ or Sucnr1-tdTomato− FACS-sorted HSPC (containing a mix of 40 HSC, 80 MPP3 and 80 MPP4) isolated from Ly5.2 Sucnr1-tdTomato mice into lethally irradiated Ly5.1 congenic recipients. Peripheral blood analysis from recipient mice 10 weeks after the transplant showed a reduction of circulating leukocytes and donor-derived monocytes in recipients of Sucnr1+ cells compared to recipients of Sucnr1− cells (Fig. 5H and Supplementary Fig. 9H).

Thus, Sucnr1 is expressed in HSPC mainly in MPP3 and its activation restricts their functional response to succinate ex vivo. Notably, although BM cells from Sucnr1-KO mice are less fit in competition with WT cells, HSPC transplantation experiments uncovered that Sucnr1 expression traces a subset of HSPC with restricted engraftment potential in vivo versus Sucnr1− HSPC.

Sucnr1 preserves transcriptional programs characteristic of HSPC
To identify specific changes in HSPC driven by Sucnr1 deletion, we next performed gene expression profiling by bulk RNA sequencing (RNA-Seq) of WT and Sucnr1-KO LSK cells. We identified a significant impact of Sucnr1 genetic abrogation on the transcriptional program of the LSK cell compartment, with 4585 differentially expressed genes (adjusted p<0.05) (Fig. 6A). Gene Set Enrichment Analysis (GSEA) according to two different databases, Chemical and Genetic Perturbations (CGP) and Gene Ontology Biological Processes (GOBP) revealed coordinated changes in a variety of genes associated with reduced stemness and primitive HSC, and instead enrichment in functional categories related to myeloid activation, inflammatory response and ROS as well as signatures characteristic of late progenitors and mature hematopoietic cells, in Sucnr1-KO HSPC compared to WT HSPC (Fig. 6B and Supplementary Fig. 10A and B). Various RNA-Seq hits involving some of the most differentially expressed genes in LSK cells from Sucnr1-KO versus WT mice and related to enhanced inflammation (Il1b, S100a8, S100a9)40 and reduced self-renewal (Procr, Hoxa9) were confirmed by qRT-PCR (Fig. 6C). We then analyzed the levels of expression of Procr and Hoxa9 in different HSPC subsets by ddPCR, and found that they were significantly reduced only in the MPP subset, but not in the LT-HSC and ST-HSC compartments (Fig. 6D).

Given the increased mRNA expression of Il1b in Sucnr1-KO versus WT mice, we analyzed the intracellular levels of pro-IL-1β protein and found them slightly but significantly increased in Sucnr1-KO compared to WT mice (Fig. 6E). This effect seemed to be specific of the HSPC compartment, as we observed no differences in the intracellular levels of pro-IL-1β in total live cells, lineage-positive cells, lineage-negative progenitors or LK progenitors (Supplementary Fig. 11A).

In fact, consistent with the reduced levels of succinate, we found that the protein levels of IL-1β were lower in the BM extracellular fluid of Sucnr1-KO versus WT mice, with no changes in IL-1α (Fig. 6F). To capture the global inflammatory status in the BM of Sucnr1-KO mice, we then studied additional pro-inflammatory cytokines, including IL-6 which showed no changes and chemokines like MIP-1β, MCP-1 and RANTES, and growth factors like GM-CSF, which were all reduced compared to WT (Fig. 6F). In turn, anti-inflammatory cytokines such as IL-9 and IL-13 were increased in the BM of Sucnr1-KO versus WT mice (Fig. 6G).

Conversely, consistent with the increased mRNA expression of S100a8, S100a9 in HSPC from Sucnr1-KO versus WT mice, the protein levels of the alarmin complex S100A8/S100A9 were increased in the BMEF (Fig. 6H).

Taken together, in vivo deletion of Sucnr1 impacts gene expression programs related to impaired function and promotion of inflammation in HSPC, and particularly in the MPP compartment. Despite complex changes taking place in the global cytokine status of the BM in Sucnr1-KO mice, S100a8 and S100a9 are consistently increased in expression in HSPC and in levels in BMEF.

Sucnr1 restricts HSPC and hematopoiesis via control of S100a8/S100a9
We next asked the potential functional link between Sucnr1 and S100a8/S100a9 by in vivo treatment of Sucnr1-KO mice with tasquinimod (Fig. 7A), a small-molecule that inhibits S100a9 signaling41. Tasquinimod treatment in Sucnr1-KO mice reduced the expanded numbers of circulating monocytes as early as 3 weeks after the start of the treatment. Further reductions in circulating leukocytes, including both overall myeloid cells as well as B cells, followed at 6 weeks after the start of tasquinimod administration (Fig. 7B). Consistent with these observations, tasquinimod administration reduced the numbers of monocytes and B cells in BM and SP of Sucnr1-KO mice, as well as overall myeloid cells in SP (Fig. 7C and D). The effect on both the myeloid and lymphoid lineages seemed to originate from the reduction of the expanded HSC, MPP3 and MPP6 subsets in Sucnr1-KO mice after treatment with tasquinimod as compared to vehicle-treated mice (Fig. 7E). HSPC from Sucnr1-KO mice treated with tasquinimod generated less primary and secondary colonies, but similar numbers of tertiary colonies, compared to vehicle-treated mice (Fig. 7F and G). These data suggest that Sucnr1 restricts HSPC function and myeloid and lymphoid development through inhibition of S100a8/S100a9 signaling.

To substantiate a selective role of S100a8/S100a9 in the hematopoietic system via Sucnr1 deletion, we performed an independent experiment treating both Sucnr1-KO and WT mice with tasquinimod or vehicle in parallel (Supplementary Fig. 12A). Confirming our previous results, tasquinimod treatment in Sucnr1-KO mice reduced the expanded numbers of circulating leukocytes to the numbers observed in WT mice, including both overall myeloid cells as well as B cells and with no significant effect observed in WT mice (Supplementary Fig. 12B). Tasquinimod administration reduced the expanded numbers of monocytes in BM of Sucnr1-KO mice to the numbers found in WT mice, whilst no effect was observed in WT mice (Supplementary Fig. 12C and D). Conversely, treatment with tasquinimod reduced the numbers of BM B cells to similar levels in both Sucnr1-KO and WT mice (Supplementary Fig. 12C and D). The expansion of BM HSC in Sucnr1-KO mice was reduced by treatment with tasquinimod, but a minor reduction in HSC numbers was also observed in WT mice, bringing HSC values in both tasquinimod-treated groups to similar level (Supplementary Fig. 12C and E). In turn, the increased colony-forming potential in Sucnr1-KO mice was reduced to WT values, with no effect seen in samples obtained from WT mice (Supplementary Fig. 12F). Of note, this assay is optimal for the growth of primitive erythroid progenitor cells, granulocyte-macrophage progenitor cells, and multi-potential granulocyte, erythroid, macrophage, megakaryocyte progenitor cells, but not for the growth of lymphoid progenitor cells. Thus, these data suggest that tasquinimod effect originates in the HSC compartment for both lineages, and whereas the effect on the myeloid lineage is fully dependent on Sucnr1 deletion, the effect on the B cell lineage is only partially dependent on the loss of Sucnr1 signaling.

To confirm a direct role of Sucnr1 and S100a8/S100a9 on HSPC function, FACS-sorted HSC from Sucnr1-KO mice were tested for their colony-forming potential in serial replatings in the presence of tasquinimod. Tasquinimod inhibited the colony-forming potential of Sucnr1-KO HSC at first plating compared to vehicle, confirming the functional role of elevated S100a8/S100a9 expression in Sucnr1-KO HSPC (Fig. 7H). No changes were observed at second and third plating, suggesting no impact on long-term colony forming potential.

Together, Sucnr1 restricts haematopoiesis through inhibition of S100a8/S100a9 signaling in HSPC.

Therapeutic potential of Sucnr1 and S100a8/S100a9 targeting in acute myeloid leukemia
To understand the effect of Sucnr1 via control of S100/a8 and S100/a9 in malignant hematopoiesis, we first analyzed the expression level of S100a8 and S100a9 mRNA in mouse NRAS-G12D+ HSPC. In pre-leukemic NRAS-G12D+ HSC, MPP1 and MPP2-3 versus control cells, low Sucnr1 expression correlated with S100a8 and S100a9 upregulation (Supplementary Fig. 13A). To study the therapeutic potential of targeting Sucnr1 signaling pathway in AML, we turned to the more aggressive Mll-AF9 mouse model, which expressed very low levels of Sucnr1 (Fig. 2G). We transplanted MLL-AF9+ BM into lethally irradiated recipients and treated them with tasquinimod, tasquinimod in combination with cis-epoxy succinate, or the corresponding vehicles, for a maximum of 24 weeks (Fig. 8A). The combination of tasquinimod and cis-epoxy succinate reduced the numbers of circulating leukocytes (Fig. 8B) and Gr-1hi blasts in spleen (Fig. 8C), compared to mice receiving the vehicle for both drugs. In the BM, we found that tasquinimod alone decreased the numbers of c-Kit+FcRγ+ GMP-like leukemic blasts42 versus mice treated with vehicle (Fig. 8D and E). Further, tasquinimod reduced the number of BM c-Kit+FcRγ−CD34+CD48+ lymphoid-primed multipotent progenitor (LMPP) LSC43,44 when administered alone or in combination with cis-epoxy succinate, versus the corresponding vehicle-treated mice (Fig. 8D and E). These data indicate that activating Sucnr1 signaling with a potent Sucnr1 agonist and its downstream pathway through S100A9 targeting shows therapeutic value in MLL-AF9+ mice, reducing the numbers of immunophenotypically-defined LSC in the BM and leukemic burden in periphery.

In humans, low SUCNR1 expression associated with high S100A8 and S100A9 expression in M4–M5 patients versus M0–M3, in publicly available arrays from purified AML blasts in a published cohort of AML patients (GSE14468; n=543) (Fig. 9A). We then analyzed single-cell RNA-sequencing (scRNA-seq) data from 2 AML patient-derived xenografts (PDX) before and 8 days after the start of cytarabine (AraC), following a 5-day treatment starting at diagnosis (GSE17891245; Fig. 9B and Supplementary Fig. 14A). After integration of the data with the Harmony algorithm, we performed unbiased clustering using K-nearest neighbor analysis (Fig. 9B), which identified 7 clusters (0–6). Following AraC treatment, clusters 0 and 1 expanded markedly (Fig. 9B). Together with cluster 5, these clusters displayed a shared transcriptional program characterized both by the residual disease signature post-AraC (AraC_UP; Farge et al.)46 and by an inflammatory/senescence–stress signature (CISG_UP; Duy et al.)47 (Fig. 9C). Importantly, we found S100A8 and S100A9 greatly expressed in these clusters of persistent cells enriched post-AraC (clusters 0, 1, 5) (Fig. 9D). Their expression was significantly increased post-AraC compared to diagnosis (Fig. 9E and F) and correlated with the residual disease signature46 (Fig. 9G). In contrast, SUCNR1 was expressed at low levels in cluster 3, which decreased in numbers after AraC treatment (Fig. 9B) and showed low expression of S100A8 and S100A9 (Fig. 9D and E).

Functional enrichment analysis revealed that cluster 3 was enriched for mitochondrial and metabolic processes (Supplementary Fig. 14B). This finding is consistent with previous studies showing that AraC treatment induces a metabolic rewiring characterized by enhanced mitochondrial activity, a key driver of chemoresistance48. Thus, the reduction of cluster 3 post-AraC suggests that SUCNR1-expressing cells represent a metabolically active population with a distinct role in shaping treatment response. In this context, SUCNR1 expression could represent both an important determinant of chemotherapy response and a potential therapeutic vulnerability.

To test the role of SUCNR1 in chemotherapy response, we treated NOMO-1 or Kasumi-1 AML cell lines with AraC alone or in combination with cis-epoxy succinate. Cis-epoxy succinate increased AraC-induced apoptosis rate in both cell lines (Supplementary Fig. 15A and B). The combination of cis-epoxy succinate but not monomethyl succinate and AraC reduced the expression of S100A9, versus AraC alone (Supplementary Fig. 15C). In view of these data, we then asked the potential of SUCNR1 activation to induce apoptosis in human AML cell lines. To answer this question, we used a lentivector to overexpress SUCNR1 in Kasumi-1 cells and treated the cells with cis-epoxy succinate. Notably, cis-epoxy succinate treatment alone was able to induce apoptosis, and the rate of apoptosis was significantly stronger in cells overexpressing SUCNR1 versus cells transfected with an empty vector (Supplementary Fig. 15D and E).

Collectively, these data indicate that SUCNR1 and S100A8/S100A9 are linked and functionally relevant in human AML. Whereas S100A8 and S100A9 are markers of residual disease after chemotherapy in AML PDX models, SUCNR1 selectively marks a metabolically active cell subset, and its activation induces apoptosis and potentiates chemotherapy response in vitro.

Methods
Ethic statement
Our research complies with all relevant ethical regulations. This human study was approved by the Regional Committee for Medical Research Ethics North Norway (REC North 2015/1082). Written informed consent was obtained in accordance with the Norwegian legislation and the Declaration of Helsinki. Participants received no compensation. Mouse experiments were conducted with the ethical approval of the Norwegian Food and Safety Authority (8660, 13407, 24739, 30992), the local Animal Care and Ethics Committees at UiT–The Arctic University of Norway (09/16 and 09/21) and University of Oslo (Norway; A022). The maximal leukemic burden permitted was leukocytosis 5-fold over normal healthy values and it was not exceeded. Criteria applied to define humane endpoints for mouse termination before the established endpoint were in accordance with the Norwegian Food and Safety Authority. Briefly, we used a cumulative score sheet to evaluate welfare that included parameters related to appearance, discomfort symptoms, eyes appearance, body weight, and injection site reaction when applicable. Human endpoints were applied under a total score > 9, and euthanasia was considered under total scores of 6–9.

Humans
Two females, one healthy donor and one AML patient, aged 48–62 years old, were included in the study. The healthy donor donated peripheral blood and the AML patient donated peripheral blood and bone marrow. Sex was not considered in the study design. The AML patient was classified as M1 with adverse risk and was selected based on the low expression of SUCNR1. The healthy individual was selected according to similarity to the AML patient regarding age and sex. The diagnosis of AML was established according to the revised criteria of the World Health Organization. Cytogenetic risk group was established according to Dohner and colleagues. Myeloblasts were calculated by morphological evaluation of BM smears, complemented by regular flow cytometry.

Mice
Age and sex matched C57BL/6J WT (The Jackson Laboratory, strain 000664), immunodeficient NOD Scid Gamma (NSG)60 (The Jackson Laboratory, strain 005557), Mll-AF960 (The Jackson Laboratory, strain 009079), Mx1-Cre NRASG12D26,61 (gift from P. García), Sucnr1-tdTomato (generated in this paper), Sucnr1−/−52 and Sucnr1fl/fl (gift from A. Cervera), Mx1-Cre62 (The Jackson Laboratory, strain 003556) and C57BL/6J Ly5.1 (Charles River Laboratories, strain 708) were used in experiments. NSG mice are on a NOD/ShiLtJ background, Mll-AF9 are on a mixed genetic background (C57BL/6, 129P and possibly other contributions) and all other mice are on a C57BL6/J background. The sex of the mice used in each experiment is indicated. Briefly, either female or male mice were used in most experiments, and in experiments where both females and males were used, no sex-based analysis was conducted to maintain statistically relevant experimental group sizes. Phenotyping of Sucnr1 mice versus C57BL/6J WT mice was performed in males of different age groups: 10-17 weeks and 38–54 weeks, unless otherwise indicated. Cre expression in Mx1-Cre NRASG12D was induced by intraperitoneal (i.p.) injection of two doses (in two consecutive days) of 300μg of poly-inosine:poly-cytosine (pIpC; Sigma-Aldrich Merck, P9582). Experimental animals were housed with food and water ad libitum, with a 12h day/night cycle, under specific and opportunistic pathogen free (SOPF) environment at 21.5±1.5°C and 55±5% humidity at the Section of Comparative Medicine at University of Oslo or at 21±1°C and 55±10% humidity at the Unit of Comparative Medicine at UiT–The Arctic University of Norway, where experiments were conducted. Ages and sex of animals in each experiment are provided in supplementary Data 2.

Generation of Sucnr1-tdTomato mice
Sucnr1-tdTomato reporter mice were generated by gene targeting at the Pluripotent Cell Technology and Transgenic Units of CNIC. Targeting vector was assembled by Gene Bridges GmbH (The recombineering company, Germany). Briefly, a 5’-T2A-tdTomato-3’ followed by bGH polyadenylation signal was placed in front of the stop codon of Sucnr1, and an FRT-Neo-FRT cassette was inserted downstream. In the construct, right and left homologous arms were 7.0 Kb in length. The backbone of Sucnr1-tdTomato-FRT-Neo-FRT targeting vector was eliminated by AscI-MluI digestion and gene targeting performed as previously described63, using hybrid mouse embryonic stem cells (mESC) 129×C57BL/6. Neomycin-resistant ES cell clones were screened for homologous recombination (HR) by Southern blot of BamHI digested DNA using an external probe 5’ to the left arm. Of 288 clones, 10 were confirmed as positive also for HR at 3’ side, using an external probe 3’ to the right arm and PstI digested DNA. Both probes were amplified by PCR using the following primer sequences: 5’-CAAACC-CACAAAAGACACAGTAAG-3’ and 5’-TTCCTTTTTCAGCCCATCTATATC-3’ (5’ probe-BamHI); 5’-TCAAAAACTACCTCATTTCCAAGA-3’ and 5’-TAATCCTGCTATGTTTTCCTCTCC-3’ (3’ probe-PstI).
Targeted clones were expanded and injected into C57BL/6 blastocysts, and microinjected embryos were transferred to pseudopregnant females using standard procedures. Germline transmission of Sucnr1-tdTomato-FRT-Neo-FRT allele was confirmed with 2 of the targeted clones by PCR using the following primer sequences: 5’-TAGTGAGACGTGCTACTTCCATTT-3’, and 5’-TTTCAGATCCTAAGTGTCCAAACA-3’.
Then, the Neo resistance cassette was eliminated by breeding with a Flp deleter line. The mice were backcrossed to C57BL/6J for more than 10 generations, until a pure genetic background was achieved in the colony.
For colony genotyping we used the following primer sequences: 5’-TATATACACACTGACACGGCCTCT-3’ (P1) and 5’-ATGAACCCATCTTCAGCATATTTT-3’ (P2), amplifying a region of size 900 bp for the WT allele; 5’-TAGTGAGACGTGCTACTTCCATTT-3’ (P3) and 5’-TTTCAGATCCTAAGTGTCCAAACA-3’ (P4), amplifying a region of size 600 bp for the targeted allele; 5’-GGGGAGGATTGGGAAGACAATA-3’ (P5) and 5’-GGTGGAAAGCTAAAGCCTAGAAATG-3’ (P6), amplifying a region of 250 bp for Sucnr1-tdTomato allele after deletion of the Neo resistance cassette.

Mouse hematopoietic cell fraction extraction
Blood samples, bones and spleens were processed as previously described64. Briefly, peripheral blood was obtained from the saphenous vein and analyzed with either ABX Pentra XL80, HORIBA (software v2.2.1), ProCyte Dx Hematology Analyzer, IDEXX (software v:00-34 Build 57) or HT5 element, Antech (software v01.16.00.5281), red blood cells were afterwards lysed for flow cytometry analysis. Bone marrow hematopoietic cells fraction was obtained from hips, femurs, tibias, humerus, sternum and spine, pooled together from each mouse. Bone marrow nucleated cells (BMNC) were obtained by crushing bones in PBS and filtered on a 40µm mesh. Spleens were weighed, crushed and filtered on a 40µm mesh. Red blood cells from blood, BM and spleens were lysed with 0.15M NH4Cl (Sigma-Aldrich Merck, A9434) for 10 min at 4°C. Cells were counted on a CASY cell counter (OMNI Life Science) for subsequent methods.

Fluorescence-activated cell sorting (FACS)
Immunophenotype of HSPC was defined as LSK. LSK were further defined as LT-HSC (LSK CD34−Flt3−), ST-HSC (LSK CD34+Flt3−), and MPP (LSK CD34+Flt3+). Detailed FACS analysis of the stem and progenitor cell subsets corresponding to HSC and MPP1-MPP6 was performed as previously described30–32. Briefly, LT-HSC were further defined as HSC (LT-HSC CD150+CD48−) and MPP6 (LT-HSC CD150−CD48−). ST-HSC were further defined as MPP1 (ST-HSC CD150+CD48−), MPP2 (ST-HSC CD150+CD48+), MPP3 (ST-HSC CD150−CD48+) and MPP5 (ST-HSC CD150−CD48−). MPP were further defined as MPP4 (MPP CD150−CD48+). Lin− hematopoietic progenitor subsets were defined as committed CLP; (c-KitlowSca-1lowCD127+), CMP (Lin−c-Kit+Sca-1− (LK) CD34+FcRγ−), MEP (LK CD34−FcRγ−), and GMP (LK CD34+FcRγ+). Differentiated hematopoietic cells of CD11b+ myeloid lineage were defined as CD11b+Gr1hiF4/80− granulocytes and CD11b+Gr1hiF4/80+ monocytes, cells of lymphoid lineage were defined as CD3+ T cells and B220+ B cells. In Mll-AF9 leukemic mice, GMP-like leukemic blasts were defined as c-Kit+FcRγ+ and LMPP LSC were identified as c-Kit+FcRγ−CD34+CD48+. For pro-IL-1β intracellular staining, mouse TBM was stained for LSK, then fixed and permeabilized with eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (ThermoFischer, 88-8824-00). Briefly, cells were fixed in 100µL of intracellular fixation buffer for 40 min at room temperature (RT), then washed and incubated in 100µL of permeabilization buffer with 4% of anti-IL1β (pro-form) monoclonal antibody (ThermoFischer, 12-7114-82) or corresponding isotype for 40 min at RT protected from light, then washed. Samples were analyzed on a Beckton Dickinson (BD) Canto or LSR Fortessa analyzer with DIVA software v6.1.3, v8.0.1 and v9.0, or a SONY ID7000 spectral flow cytometer with ID7000 software v2.02.17121. For transplant of HSPC from Sucnr1-tdTomato donors, cells were sorted on an Aria II in PBS supplemented with 2% heat-inactivated FBS. For colony-forming unit cell (CFU-C) assays, cells were sorted on a FACSMelody (BD) with FACSChorus software v1.1.2 in PBS supplemented with 2% heat-inactivated fetal bovine serum (FBS; ThermoFisher, 10082147). For molecular biology analyses, cells were FACS-sorted on an Aria II and Aria III cell sorter (BD). For qRT-PCR and ddPCR, cells were sorted in Lysis/Binding Buffer from Dynabeads™ mRNA DIRECT™ Purification Kit (ThermoFischer, 61012) or PicoPure™ RNA Isolation Kit (ThermoFischer, KIT0204). For RNA-sequencing, cells were sorted in extraction buffer from PicoPure™ RNA Isolation Kit. Antibodies used are listed in Supplementary Data 3.

Imaging flow cytometry
Bone marrow cells from Sucnr1-tdTomato mice or C57BL6/J WT littermates were magnetically enriched for Lin− progenitors using Mouse Hematopoietic Progenitor (Stem) Cell Enrichment Set–DM (Beckton Dickinson, 558451). LSK cells were FACS-sorted on an Aria III (BD) in PBS from Lin− enriched cells and concentrated in 20µl of PBS. Samples were visualized and raw data recorded on an Amnis® ImageStream® X MkII imaging cytometer using INSPIRE® software v200.1.620.0. Single-stained BMNC from C57BL6/J WT mice and non-stained BMNC from Sucnr1-tdTomato mice were used for compensation controls. Raw data were processed and analyzed with IDEAS® software version 6.2, single cells were gated using Area and Aspect ratio features followed by gating of Sucnr1-tdTomato+ LSK cells.

Cell culture
Colony-forming unit cell (CFU-C) assay was performed as previously described65. Briefly, cultures were performed at 37°C, 20% O2 and 5% CO2 water-jacketed incubator (Thermo Scientific). 20 x 103 and 200×103 cells were plated in duplicate in MethoCult™ GF M3434 (StemCell Technologies, 03434) for BM and spleen, respectively. CFU-C were counted once a week for 3 weeks, replating the first 2 weeks. For replating, CFU-C were resuspended in PBS at RT, methylcellulose was washed twice with PBS, and the cells were then seeded in fresh Methocult™ GF M3434. For in vitro treatment, succinic acid (Sigma-Aldrich Merck, S9512) or monomethyl succinate (Sigma-Aldrich Merck, M81101) were added to the methylcellulose at 0.05, 2, or 10 mM at each plating. Serial CFU-C assays from 300 FACS-sorted HSC from magnetically enriched bone marrow Lin− progenitors were performed as described previously66, colonies were scored and 10,000 cells replated after 7, 12 and 17 days, 10 mM of monomethyl succinate or cis-epoxy succinate (Glixx Laboratories, GLXC-22816) or 25µM tasquinimod (MedChemExpress, HY-10528) were added at each plating. For cell lines, Kasumi-1 (provided by Dr Paloma Garcia, DSMZ, ACC 220, authentication from supplier) and NOMO-1 (DSMZ, ACC 542, authentication from supplier) were seeded at 0.5×106 cells per ml in RPMI medium without phenol red (ThermoFisher, 11835030) supplemented with 10% heat-inactivated FBS and 1% Penicillin-Streptomycin-Glutamine (ThermoFischer, 10378016). Cells were passed every 2–3 days. 0.5×106 Kasumi-1 and NOMO-1 cells were treated for 48h with 1.77µM of cytarabine (AraC, MedChemExpress, HY-13605) or vehicle together with 10 mM of cis-epoxy succinate or vehicle. Kasumi-1 cells transduced with a lentivector overexpressing SUCNR1 or by an empty vector were treated with 10 mM of cis-epoxy succinate or vehicle without AraC. After 48h, cells were washed and pelleted for apoptosis assay.

Lentivirus production and transduction
Lentiviruses encoding SUCNR1-GFP or empty vector (EV-GFP) were produced by transfecting HEK293FT (ThermoFischer, R70007, authentication from supplier) cells cultured in DMEM (ThermoFischer, 41966029) containing 1% Penicillin/Streptomycin (ThermoFischer, 15140122) and 10% FBS with VSV-G, psPAX2, and expression constructs designed and produced using VectorBuilder at a 1:2:2 ratio, using Lipofectamin 2000 (ThermoFischer, 11668019). SUCNR1-GFP construct was pLV[Exp]-Hygro-EF1A>hSUCNR1[NM_033050.6] (ns):P2A:EGFP and EV-GFP construct was pLV[Exp]-Hygro-EF1A>EGFP. After 8h, the cells were washed and cultured in Kasumi-1 medium. Virus-containing supernatant was harvested at 48h, filtered through a 0.45µm membrane, and used for transduction. Kasumi-1 cells were transduced by spinoculation; 1×10⁶ cells in 2ml were mixed with 2ml of virus with polybrene (Sigma-Aldrich, H9268) at 8µg per ml and centrifuged at 1000g for 90 min at RT, then incubated overnight. Medium was refreshed the next day. Transduced cells were expanded and sorted with a BD FACS Melody for GFP expression after 2–3 passages. EV-GFP and SUCNR1-GFP vector summaries are provided in Supplementary Data 4.

Cell cycle and apoptosis assays
For cell cycle analysis, freshly isolated total bone marrow cells were magnetically enriched for Lin− population. Cells were resuspended at 2×106 cells per ml of Hank’s Balanced Salt Solution (HBSS; ThermoFisher, 14175095) supplemented with 10 % heat-inactivated FBS, 20mM HEPES (ThermoFisher, 15630056), 50µM Verapamil (Sigma-Aldrich, V4629) and 5µg of Hoescht 33342 (Sigma-Aldrich Merck, 14533) per ml. After 45 min of incubation at 37°C under orbital agitation, Pyronin Y (Sigma-Aldrich Merck, 83200) was added at 1µg per ml and cells were incubated for 45 additional min at 37°C under agitation. Analysis of apoptotic cells was performed as previously described64 through Pacific Blue™ Annexin V/SYTOX™ AADvanced™ Apoptosis Kit for flow cytometry (ThermoFischer, A35136), using BMNC to study apoptosis in LSK cells, or in AML cell lines after in vitro treatment with SUCNR1 agonist and/or cytarabine. Briefly, BMNC were stained for LSK, washed and incubated in 1X Annexin binding buffer (ThermoFisher, V13246) with Pacific Blue-conjugated anti-Annexin V antibody (ThermoFisher, A35122) and SYTOX™ AADvanced™ Dead Cell Stain Kit (ThermoFisher, S10274) for 15 min at RT, before flow cytometry analysis, according to the manufacturer instructions.

Succinate measurements
FACS-sorted LSK and CD11b+Gr-1+F4/80+ monocytes were analyzed using Succinate colorimetric assay kit (Sigma-Aldrich Merck, MAK 184-1KT) according to manufacturer instructions. Intracellular succinate measurement in HSC was done by liquid chromatography-coupled mass spectrometry (LC-MS) after FACS-sorting of 5000–10000 Lin−Sca-1+c-Kit+CD150+CD48− or 5000 Lin−Sca-1+c-Kit+CD34−Flt3−CD150+CD48− HSC after c-Kit+ magnetic enrichment using CD117 microbeads (Miltenyi biotec, 130-091-224), directly into acetonitrile (Sigma-Aldrich, 1000291000) as previously described67. Briefly, for LC-MS cell lysates were centrifuged at 17,000g for 30 min at −9°C, followed by transfer of supernatants to auto-sampler vials (SureSTART, ThermoFisher Scientific). The supernatants were analyzed on a Vanquish™ Neo UHPLC (ThermoFisher Scientific) with SeQuant ZIC-HILIC™ trap column (0.3×5mm, 5μm particle size; Merck, 1.50492) and SeQuant ZIC-HILIC™ capillary separation column (0.3×150mm, 3.5μm particle size; Merck, 1.50479). We injected 5μl of sample, blank or succinic acid standard (Sigma-Aldrich, S9512) onto the trap column in 30s, followed by gradient elution. HILIC gradient mobile phase A was 5% acetonitrile and 95% water containing 5mM ammonium acetate; mobile phase B was 90% acetonitrile and 10% water. To elute nonpolar compounds, mobile phase B was kept at 100% from 0 to 6.5 min, then it was ramped linearly from 100 to 42.5% from 6.5 to 26.5 min, and decreased to 10% at 27.5 min. To remove salt from the column, mobile phase B was held at 10% from 27.5 to 37.5 min B and increased back to 100% at 37.5 min. From 37.5 to 41.5 min, mobile phase B was held at 100% to regenerate the water layer of the column. Solvent flow rate was set at 5μl per min for the gradient and at 4μl per min for the salt elution phase (waste). The temperature of the column was maintained at 45°C. MS data were acquired on an Orbitrap Exploris™ 240 mass spectrometer (ThermoFisher Scientific) equipped with Easy-Spray™ source (ThermoFisher Scientific, ES081) with capillary emitter (ThermoFisher Scientific, ES994) in full scan MS mode with control software version 4.4. Negative polarity was acquired at a resolution of 60,000 (at m/z=200); automatic gain control target and maximum injection time were set by default; microscans and scan range were set to 2 and 80–800 Daltons, respectively. MS source parameters were 2300V (negative ion mode) for capillary voltage and 320°C for the temperature of the ion transfer tube. Raw data were loaded into Thermo XCalibur Qual Browser and the total ion chromatogram (TIC) and succinic acid peak (m/z 117.0194 with 5ppm window, retention time 2.7–3.3 min; identity confirmed by standard) were integrated. Succinic acid peak areas were normalized to the TIC area and batch corrected.
Succinate was measured in BMEF by colorimetric assay with normalization to the amount of protein measured by Pierce™ BCA Protein Assay Kit (ThermoFisher, 23225); and by nuclear magnetic resonance (NMR) as previously described68. For the latter, BMEF samples were prepared by mixing 1:1 with phosphate buffer prepared in D2O, containing the internal reference TMSP ((3-trimethylsilyl)propionic-(2,2,3,3-d4)-acid sodium salt) and NaN3 to yield final concentrations of 0.1M phosphate, 0.5mM TMSP and 1.5mM NaN3 (all from Sigma-Aldrich Merck). Samples were then transferred into 3mm NMR tubes and measured on a 600 MHz Bruker spectrometer equipped with an Avance-III console and a 5mm triple resonance cryoprobe (TCI) z-axis pulsed field gradient (PFG) cryogenic probe, a cooled SampleJet autosampler (Bruker), and automated tuning and matching. Spectra were acquired at 300K with a 1D 1H-NOESY (Nuclear Overhauser effect spectroscopy) pulse sequence with pre-saturation water suppression (noesygppr1d, Bruker standard pulse program), with a spectral width of 12ppm, 32768 data points, and an interscan delay of 4s. The 1H carrier frequency was set on water resonance, and the 1H 90° pulse was calibrated at a power of 0.256W with a typical length of about 7–8 μs. A total of 64 transients and 8 steady state scans were acquired, resulting in a total experimental time of 7.5 min. Spectral processing was performed using MetaboLab69 software v2023.0529. Free induction decays were apodized with a 0.3Hz exponential line-broadening function and zero-filled to 131072 points prior to Fourier transformation. Chemical shifts were referenced to the TMSP signal at 0 ppm, and spectra were manually phase corrected. A spline baseline correction was applied, and the water and spectral edge regions were excluded prior to probabilistic quotient normalization (PQN). Succinate was assigned using Chenomx NMR Suite (v7.0, Chenomx Inc.) in conjunction with the Human Metabolome Database (HMDB). Succinate intensities were obtained directly from the spectra. Succinate intensities were normalized to the reference (TMSP) intensity, total spectral area of the sample and BMEF volume obtained.

Cell energy phenotype
Freshly isolated total bone marrow cells from Sucnr1-KO and C57BL/6J WT males aged 26–52 weeks were magnetically enriched for Lin− progenitors and 0.5×106 Lin− cells were immobilized in triplicates to XF96 microplates (Agilent, 101085-004) pretreated with Cell-Tak (Corning; 354240). Measurement of intact cellular respiration was performed using the Seahorse XFe 96 analyzer (Agilent) with Wave controller software controller 2.4.2 and the XF Cell Energy Phenotype Test Kit (Agilent, Cat. 103325-100) according to manufacturer instructions. Medium assay consisted of XF Base Medium (Agilent, 102353-100) supplemented with 10mM glucose, 1mM pyruvate and 2mM glutamine. Respiration was measured under basal conditions, and in response to a stressor mix of Oligomycin (ATP synthase inhibitor; 0.3μM) and the electron transport chain accelerator ionophore Trifluorocarbonylcyanide Phenylhydrazone (FCCP; 0.15μM).

Reactive oxygen species
Quantification of reactive oxygen species (ROS) was performed as previously described70. Briefly, 10x106 BMNC were stained for ROS levels (superoxide anion) with 5μM hydroethidine (DHE; ThermoFisher D23107) for cellular ROS or 10μM MitoSOX (ThermoFisher, M36008) for mitochondrial ROS, by 20 min incubation at 37°C. Cells were then washed once with PBS and stained for LSK. Fluorescence of oxidized DHE or MitoSOX was determined by flow cytometry. Antibodies used are listed in Supplementary Data 3.

RNA isolation, reverse transcription and droplet digital PCR
RNA was isolated using TriReagent (Sigma-Aldrich Merck, T9424) or RNeasy Plus Mini Kit (QIAGEN, 74134) according to manufacturer protocol for mouse BMNC, with Dynabeads™ mRNA DIRECT™ Purification Kit (ThermoFischer, 61012) for ddPCR, or using PicoPure™ RNA Isolation Kit (ThermoFischer, KIT0204) for RNA-seq on FACS-sorted mouse LSK cells. Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (ThermoFischer, 4368814), following the manufacturer recommendations. ddPCR was performed with ddPCR™ Supermix for Probes (No dUTP) (Bio-Rad 186-3023) according to the manufacturer instruction, using custom-designed probes. Droplets were generated and read on a QX200 Droplet Digital™ PCR Systems with QuantaSoft software v1.7.4. The expression level of each gene was calculated by absolute quantification of copies. All values in mice were normalized with mouse Gapdh as endogenous housekeeping gene. Probes used are listed in Supplementary Data 3.

Cytokine analyses
BMEF was obtained as previously described and used for cytokine analyses64. Briefly, BMEF was isolated from one femur and one tibia of each mouse by flushing both bones together with 150µL of PBS. The cell suspension was then centrifuged 10min at 15,000g and the supernatant collected. Multiplex immunoassays were performed using Bio-Plex Pro Mouse Cytokine 23-plex Assay kit (Bio-Rad, M60009RDPD) and Bio-Plex Mouse Cytokine IL-6 set (Bio-Rad, 171G5007M) on a Bio-Plex 200 System (Bio-Rad) with Bio-Plex Manager v6.2 software. Proteins were quantified with Pierce™ BCA Protein Assay Kit (ThermoFisher, 23225) using a CLARIOStar plate reader with CLARIOStar plus v6.20 software. Results were expressed as pg per mg of protein. S100A8/S100A9 were quantified by sandwich ELISA as previously described71,72. The ELISA system detects both active heterodimers and inactive tetramers formed by S100A8 and S100A9. Increased levels of S100A8/A9 complexes represent biological active S100A8/A9 heterodimers73. Briefly, 20μl of BMEF were diluted 1/100, 1/200, and 1/400 and measured in duplicate. If all values coincided after recalculation, the value was accepted. If needed, another dilution range was run in the linear 1–30 ng per ml range to allow calculation of exact S100A8/A9 levels. Antibodies used for ELISA were prepared as previously described74. Each plate included a reference sample as positive control. Total protein amount was measured with Pierce™ Coomassie (Bradford) Protein Assay kit (ThermoFisher, 23200). Results were expressed as ng per μg of protein.

In vivo pharmacological treatments
For the mobilization study, C57BL6/J mice aged 51 weeks were injected with 2mg per kg of succinic acid and killed 1h or 6h after to study CFU-C proportion in BM, SP and PB. For S100A9 inhibitor tasquinimod, Sucnr1-KO male mice aged 28–41 weeks or C57BL6/J WT mice aged 26-39 weeks were administered with vehicle or tasquinimod at 30mg per kg per day in drinking water for 9–16 weeks as previously described41. Briefly, tasquinimod was dissolved in DMSO, the required volume of tasquinimod solution was added to the bottle, then top up with DMSO to reach a 2% DMSO concentration in the final volume of drinking solution. The drinking solution was completed by adding polyethylene glycol 300 (PEG300, Sigma-Aldrich Merck, 8.07484) for a final concentration of 2%, then drinking water containing 3% sucrose. The vehicle for tasquinimod was 2% DMSO, 2% PEG300 and water containing 3% sucrose. Water bottles were changed twice per week. Effect of acute succinic acid administration in human AML xenografts was studied through short-term daily i.p. injections of vehicle (saline) or succinic acid at 2mg per kg neutralized with NaOH to pH 7.3, for 10 days. Effect of chronic succinic acid administration in leukemia models was evaluated by long-term i.p. injections of succinic acid. C57BL6/J WT female mice transplanted with BMNC from leukemic Mll-AF9 mice, were treated with vehicle or incrementing doses of succinic acid at 2, 20, 100, 200 and 400mg per kg daily over intervals of 2–4 weeks, then at 400mg per kg 3 times a week on alternated days for 8 additional weeks. The treatment started 4 weeks after the transplant. The total treatment duration was 42 weeks. For tasquinimod and cis-epoxy succinate in recipients of MLL-AF9+ bone marrow, treatment started 5 weeks after transplantation and mice were euthanized and used for endpoint analyses at the maximum period of 24 weeks of treatment or before, when they exhibited signs of frailty according to humane endpoints or reached a threshold of 100×106 WBC per ml in peripheral blood. Mice were treated with vehicle or tasquinimod at 30mg per kg per day in drinking water for 17 weeks, then 20mg per kg per day in drinking water for 7 weeks, as previously described41. Mice received daily i.p. injection of vehicle or 2mg per kg of cis-epoxy succinate for a maximum of 24 weeks. 22–37 weeks old Mx1-Cre NRASG12D male mice were treated daily with vehicle or incrementing doses of succinic acid at 100, 200, 400, 800 and 1200mg per kg over intervals of 2–4 weeks, starting 7 weeks after pIpC induction. The total treatment duration was 27 weeks.

Transplantation assays
For human xenografts, myeloablation of 8–12-week-old female NSG mice was performed through i.p. injection of Busulfan (Busilvex, Pierre Fabre Pharmaceuticals, 130309) at 25mg per kg. Mice were transplanted 24h later with 2×106 CD34+ cells isolated from the BM of one AML patient. Animals showing human CD33 engraftment <0.1% in BM were considered negative and excluded from the study. Mll-AF9 knock-in mice BM transplantation was performed through the tail vein after myeloablation of 12-week-old female mice for succinic acid treatment or 10-week-old male mice for tasquinimod and cis-epoxy succinate treatment, whole body irradiated with 9Gy (in 2 doses separated by 3h) using an X-RAY source (RadSource’s RS2000 or Xstrahl RS320, respectively). 4h after, mice were transplanted intravenously (i.v.) with 2×106 BMNC. To study the function of Sucnr1 through the hematopoietic compartment, we transplanted 2×106 BMNC from Mx1-Cre Sucnr1fl/fl or Sucnr1fl/fl mice into 15-week-old male C57BL/6J recipients, previously irradiated as described (RadSource’s RS2000). Expression of Cre recombinase was induced by i.p. injection of two doses (in two consecutive days) of 300μg of pIpC 4 weeks after the transplant. For competitive transplants, 16-week-old Ly5.1 C57BL6/J mice were whole body irradiated (Xstrahl RS320) and injected i.v. in the tail vein with 1×106 BMNC from either Sucnr1-KO or C57BL6/J Ly5.2 mice aged 42–43 weeks, together with 1×106 BMNC from 16-week-old Ly5.1 C57BL6/J mice. For transplants of HSPC from Sucnr1-tdTomato donors, 8-week-old Ly5.1 C57BL6/J were whole body irradiated (Xstrahl RS320) and injected in the tail vein with 200 Ly5.2 Sucnr1-tdTomato+ or Sucnr1-tdTomato− FACS-sorted mix of HSC, MPP3 and MPP4 (40 HSC, 86 MPP3 and 74 MPP4), together with 2.5×105 BMNC from Ly5.1 C57BL6/J mice.

Human CD34+ cell enrichment
Peripheral blood mononuclear cells (PBMC) and BM nucleated cells from one AML patient and PBMC from one healthy donor, were obtained by density gradient centrifugation (Lympholyte, Cedarlane, CL5015) and used for CD34+ cell enrichment using immune magnetic technology (StemCell Technologies, 15086).

RNA sequencing and bioinformatic data analysis
For calculation of survival probability, normalized gene expression was retrieved from GEO GSE14468 as previously described75. Briefly, probe “223939_at” specific to the SUCNR1 transcript, NM_033050, was used in the analysis of expression for survival analysis of 426 AML patients, containing 224 M0-M3 and 171 M4-M5 AML patients, 216 females (50.7%) and 210 males (49.3%), aged 19–61 years old. Survival data were gently provided by contributor author Dr. P.J.M. Valk (Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands). The median expression of SUCNR1 for the whole cohort (n=426) was used to dichotomize its expression into High and Low levels. Then, Kaplan–Meier curves were produced for the overall and progression-free survival of the individuals. Log-rank p-value is reported. Two-tailed Wald test without Benjamini-Hochberg correction for multiple comparisons was used for Cox regression of human survival. SUCNR1 expression in healthy individuals and AML patients were obtained in BloodSpot and retrieved from GSE42519 and GSE13159, respectively.
RNA-Seq data from FACS-sorted LSK cells obtained from the BM of Sucnr1-KO and C57BL/6J WT male mice aged 27–30 weeks have accession number GSE215255. RNA amplification and RNA-Seq library production were performed at the Genomics Support Centre Tromsø (GSCT, UiT–The Arctic University of Norway). Total RNA was isolated using the Arcturus PicoPure™ RNA Isolation Kit (ThermoFischer, KIT0204) from small numbers of FACS-sorted cells (27000-75000). RNA was amplified with the NEBNext Single cell/Low input RNAlibray kit v. 3.0 (NEB#E6420) following the protocol to the fragmentation step. cDNA was fragmented to target size of 150bp with Covaris M220. Libraries were prepared with TruSeq RNA library preparation kit v. 2.0 (RS#122-2001, Illumina) starting the protocol from “perform end repair” to final library. The quantity of the final library was determined by dsDNA-Qubit High Sensitivity and fragment size was determined by Agilent Technologies 2100 Bioanalyzer using the Agilent DNA 1000 chip. Libraries were sequenced with the NextSeq 550 instrument (Illumina) with System Suite software v4.0 at the GSCT (UiT–The Arctic University of Norway). At least 3 biological replicates were used per experimental group (4 WT, 3 Sucnr1-KO).
Sequencing reads were pre-processed by means of a pipeline that used FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) with software v0.11.9, to assess read quality, and Cutadapt76 v5.2 to eliminate Illumina adaptor remains, to trim the first 6 nucleotides, and to discard reads that were shorter than 30bp after trimming. Resulting reads were mapped against reference transcriptome GRCm38.99 and quantified using RSEM77. Expected expression counts calculated with RSEM were then processed with an analysis pipeline that used the Bioconductor package Limma78 v3.17 for normalization (using TMM method) and differential expression testing, for contrast Sucnr1-KO versus C57BL/6J WT mice. The test focused on 15224 genes expressed with at least 1 count per million (CPM) in at least three samples, and used a random variable to define groups of samples that belonged to the same batch (date of FACS-sorting). Changes in gene expression were considered significant if associated to Benjamini and Hochberg adjusted p<0.05. 4585 differentially expressed genes were detected. Functional enrichment analyses were performed with Ingenuity Pathway Analysis (IPA) from QIAGEN Inc. (https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) and GSEA79. IPA was performed on 3841 differentially expressed genes with Benjamini-Hochberg adjusted p<0.05 and absolute log2 FC>1. GSEA was performed against two different gene set collections from the Molecular Signatures Database (MSigDB)80: Chemical and Genetic Perturbations (CGP) and Gene Ontology Biological Processes (GOBP). FDR q values <0.25 were used to filter enrichment results. Additional graphical representations (bar plots, volcano plots) were generated with R.
The scRNA-seq data from GSE178912 were aligned and quantified using the Cell Ranger Single-Cell toolkit (v. 3.0.2) against the GRCh38 human reference genome. The pre-filtered output generated by Cell Ranger was used for all downstream analyses. Count matrices were imported into the R environment (v4.4.2) using the Seurat package (v5.3.0), and all visualizations were generated with ggplot2 (v4.0.0). A Seurat object was created using the CreateSeuratObject function with the min.cells parameter. Low-quality cells were removed based on quality control thresholds: fewer than 200 or more than 6000 detected genes, and a mitochondrial RNA content exceeding 15%. On average, 21650 cells were retained, with a median of 19065 detected genes per cell across all conditions (CTL and ARAC D8) from TUH07 and TUH69 PDX samples (GSE178912).
Data normalization was performed using the NormalizeData function with a scaling factor of 10,000 and logarithmic transformation. The 2000 most highly variable genes (HVGs) were identified using the FindVariableFeatures function with the selection method set to Variance Stabilizing Transformation (vst). The merged dataset was then scaled with ScaleData. Principal component analysis (PCA) was performed using the RunPCA function (Seurat) with 30 principal components (npcs=30), followed by batch correction and integration using the Harmony algorithm.
Dimensionality reduction was conducted with Uniform Manifold Approximation and Projection (UMAP) using RunUMAP with dims=1:30. A K-nearest neighbor (KNN) graph was constructed using FindNeighbors (dims=1:20), and clustering was performed using the Louvain algorithm implemented in Seurat via FindClusters, with a resolution parameter of 0.1.
Gene signature scores were computed using the AddModuleScore function from Seurat and visualized through UMAP and violin plots. Functional enrichment analysis was performed with RunEnrichment function of the single-cell pipeline (scp) package v0.5.6. Sourced data from analysis of GSE178912 are provided as Supplementary Data 1.

Statistics and reproducibility
Statistical analyses were performed using Prism 9 software (GraphPad). Data are biologically independent humans samples or animals, and means±SEM for bar plots or medians for violin plots. Box plots show median as centerline; upper and lower quartiles as box limits; 1.5x interquartile range as whiskers. All biologically independent data points are shown unless otherwise indicated for better visualization. A p value of less than 0.05 was considered significant. Significant p values are reported in the figures and non-significant p values are reported in the Source Data file. Statistical significance was evaluated by unpaired two-tailed t-test or two-tailed Mann–Whitney U test where appropriate. Survival curve in mice was analyzed by log-rank (Mantel-Cox) test. Two-tailed Wald test with or without Benjamini-Hochberg correction for multiple comparisons was used for gene expression in bulk RNA-seq and Cox regression of human survival, respectively. For scRNA-seq data, the 2000 most HVGs were identified using the FindVariableFeatures function with the selection method set to vst. PCA was performed using the RunPCA function (Seurat) with 30 principal components (npcs=30), followed by batch correction and integration using the Harmony algorithm. Gene signature scores were computed using the AddModuleScore function from Seurat and visualized through UMAP and violin plots. Functional enrichment analysis was performed with RunEnrichment function of the scp package v0.5.6. The differential gene expression was analyzed using two-tailed t-test.
In most experiments and unless otherwise indicated, mice of the same sex and approximate similar age were used to control for covariates. Mice were randomized to treatment groups. The investigators were not blinded to allocation during experiments and outcome assessment. Animals that showed symptoms of disease or health issues unrelated to aberrant myelopoiesis were excluded from the study, i.e., obesity, tumor masses, etc. Criteria applied for mouse termination before the established endpoint were in accordance with the Norwegian Food and Safety Authority. Outliers in mouse studies were excluded using Grubbs test, and ROUT test was used when several outliers were suspected. For in vivo experiments, sample size was calculated based on estimation of the minimum number of animals required to obtain biologically meaningful results. Where appropriate, experimental group size was increased by adding results from different mice of similar characteristics analyzed on different days based on availability of new experimental and wild-type or control mice. Most in vivo experiments were performed over two or more days, to ensure replication of results. Experiments replicated independently confirmed previous results. Cohort size and numbers of cells transplanted in xenografts were informed by the total number of available cells. Animals showing human CD33 engraftment <0.1 % in BM were considered negative and excluded from the study. Data of ex vivo cultures, cytokine analyses, molecular biology assays, from each mouse are the mean result of at least two or three technical replicates, and most of these assays were performed over two or more days with samples from different mice, to ensure replication of results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The article presents a clear and well-structured exploration of Sucnr1’s role in hematopoiesis, offering valuable insights into a complex and underexplored area of stem cell biology. The use of comprehensive experimental approaches—ranging from genetic manipulations to transcriptomic analysis—enhances the clarity of the conclusions and strengthens the overall impact of the study. The figures are well-organized and effectively support the narrative, providing clear visual representations of the data. Overall, the manuscript is excellently presented, with a clear focus on the novelty of the findings and their potential implications in the context of hematopoiesis. However, the manuscript would benefit by addressing certain key issues.\n1) The global knockout model used may limit the interpretation of Sucnr1’s role in hematopoiesis, as indirect effects arising from systemic Sucnr1 deficiency could influence hematopoietic stem and progenitor cell (HSPC) responses. To clarify whether Sucnr1’s effects are intrinsic to hematopoietic cells or arise from changes in the microenvironment, a lineage-specific knockout approach would be insightful. This strategy could help differentiate between direct roles of Sucnr1 within hematopoietic cells and broader physiological or paracrine influences, thereby enhancing our understanding of its role in self-renewal and differentiation dynamics in hematopoiesis.\n2) The effects of Sucnr1 deficiency on hematopoiesis appear contradictory. Sucnr1 deletion results in both an expansion of HSPCs and increased hematopoietic activity in the bone marrow and spleen, yet, paradoxically, it also shows reduced transcriptomic markers of stemness and self-renewal. Could the authors provide a more in-depth discussion to explain this finding? Additionally, targeted experiments investigating differential effects across specific HSPC subpopulations (e.g., long-term vs. short-term HSCs, or lineage-specific progenitors) could help clarify whether Sucnr1’s impact varies across these subsets, potentially reconciling the observed expansion with the reduction in stemness markers.\n3) Section: Low SUCNR1 Predicts Acute Myeloid Leukemia Progression\nIn lines 141-143, it is mentioned that \"MLL-AF9+ mice had overt AML, and the lineage-negative compartment was expanded in the bone marrow of succinic acid-treated mice compared to vehicle-treated controls (Figure 1L), showing an increase in the LK fraction but no changes in LSK cells (Supplemental Figure 2E).\" While there was an observed trend toward an increase in both the LK fraction and LSK cells in succinic acid-treated mice, it is crucial to emphasize that none of these differences reached statistical significance. Adding this clarification in the text would provide a more accurate and nuanced interpretation of the data.\nIn line 143, the statement that 'low SUCNR1 represents a prognostic marker for reduced survival in AML patients' would benefit from further statistical validation to strengthen its impact. To rigorously establish SUCNR1 as an independent prognostic factor, a Cox regression analysis should be performed. This would allow the authors to assess the effect of SUCNR1 on survival while adjusting for other known prognostic variables in AML. This addition would significantly enhance the robustness of the study's conclusions regarding the prognostic value of SUCNR1 in AML.\n4) Section: Sucnr1 is expressed in HSPC, and its activation restricts their functional response to succinate ex vivo\nIn line 223, the authors suggest that Sucnr1 activation suppresses succinate's intracellular effects, supported by their observations with monomethyl succinate, which increased colony-forming potential in total bone marrow (TBM) in a dose-dependent manner. However, this conclusion is not entirely convincing. If Sucnr1 activation indeed dampens succinate’s effects, one would expect to see a reduction in CFU-C in TBM with succinate treatment (Figure 4A), but no such decrease is observed. This raises questions about the robustness of the authors’ claim. Additionally, to strengthen their argument, the authors could have tested the combination of monomethyl succinate with succinic acid to explore potential synergistic effects, which would provide more clarity on their hypothesis.\nAdditionally, clarification is needed on why monomethyl succinate increased CFU-C in FACS-sorted HSC from Sucnr1-KO mice in the first plating (Figure 4C), while this effect was not seen in cells from WT mice (Figure 4B). Could the difference be attributed to varying intracellular succinate levels between the two mouse strains? Has the possibility of altered succinate accumulation been considered? Without addressing this, the interpretation of the results remains incomplete and raises doubts about the underlying mechanisms.\n5) Section: Sucnr1 restricts HSPC and hematopoiesis via control of S100a8/S100a9\nTo better elucidate the role of Sucnr1 in hematopoiesis through S100a8/S100a9 regulation, it would be valuable to conduct a direct comparison between tasquinimod-treated WT mice and tasquinimod-treated Sucnr1-KO mice. This comparison would clarify whether the observed hematopoietic effects, including changes in HSPC populations and lineage distribution, are specifically mediated by Sucnr1.\nAdditionally, investigating the combined effects of succinic acid treatment and tasquinimod in mouse models of pre-leukemic myelopoiesis and AML could provide a more comprehensive understanding of S100a8/S100a9 signaling in leukemogenesis and its potential modulation by Sucnr1. This approach would help identify the therapeutic relevance of targeting Sucnr1 and its downstream pathways in both pre-leukemic and leukemic hematopoiesis.\n6) Minor point: please correct “succinate” in Supplementary Figure 2 to “succinic acid.”", "reviewer_2": "Starting from the observation that low succinate receptor 1 (SUCNR1) expression is associated with AML subtypes M0-M3 and correlates with poorer outcomes, Cuminetti et al. explore SUCNR1 role in hematopoiesis and its impact on AML progression. The authors utilize a Sucnr1-tdTomato reporter mouse to analyze its expression across hematopoietic compartments. This model reveals that SUCNR1 is predominantly expressed in LSK, HSC and MPP cells subsets, while other hematopoietic progenitors, suggesting a role for SUCNR1 in early hematopoietic differentiation.\nTo assess SUCNR1 function, the authors develop a KO model. In SUCNR1 KO mice, immature progenitor within LSK cells expand, but other populations increase in frequency, including mature B cells. This LSK expansion coincides with a gene expression shift toward an inflammatory profile and a reduced stem cell signature, including increase expression of S100A8/A9. Interestingly, progenitor-expanding phenotype is partly reversed by tasquinimod, an S100A8/A9 inhibitor, suggesting that S100A8/A9 signaling may be mediating these effects.\nThe study extends these findings into AML models (NRASG12D, MLL-AF9), where succinate treatment promotes myeloid output and accelerates disease progression. Pre-leukemic HSC/MPP subsets in Mx1-Cre NRASG12D show increased S100A8/A9 expression, mirroring the inflammatory state seen in SUCNR1 KO animals.\nIn human AML patient-derived xenografts (PDX) from two patients, chemoresistant cells exhibit increased inflammation and S100A8/A9 expression, while SUCNR1 expression is mainly observed in pre-treatment samples, suggesting possible downregulation in response to therapy.\nOverall Comments\nWhile the manuscript presents interesting data and some novel characterizations of SUCNR1 role in hematopoiesis, a lack of proper controls and incomplete analyses make data interpretation difficult.\n- Manuscript Structure: The narrative is challenging to follow and at times counterintuitive, frequently shifting between AML models, human AML, healthy hematopoiesis, and KO mouse models. To improve readability, the structure should follow a linear narrative. This could start with SUCNR1 expression profiling in healthy mouse and human hematopoiesis (using gene expression databases, data from mouse models, and the Sucnr1-tdTomato reporter model). Profiling should cover both immature and mature myeloid cells, as some mature cells are also affected in the KO. Additionally, data on SUCNR1 expression in non-hematopoietic tissues (e.g., stromal cells) would help interpret findings from the KO mouse model. AML datasets could be used to differentiate SUCNR1 expression between healthy and malignant tissues. Conditional Sucnr1 deletions in hematopoietic cells could clarify whether the observed hematopoietic phenotypes stem directly from HSPC-autonomous functions of Sucnr1 or indirect effects via niche cells.\n- Figure Titles: Figure titles often do not accurately reflect the content, making it hard for readers to follow the data being presented.\n- FAB Classification: The FAB classification is mentioned but not systematically incorporated. Patient and PDX sample classifications by FAB subtype should be included for clarity.\n- Inflammatory Cytokine Levels (Fig 3): The increase in mRNA for certain pro-inflammatory cytokines is not substantiated by corresponding protein levels in bone marrow fluid. Intracellular cytokine detection by flow cytometry would be more appropriate. Given the inflammatory phenotype, a panel to detect immune cell activation in the KO model would be important.\n- Focus on S100A8/A9: Among various inflammation-related factors, the rationale for focusing on S100A8/A9 as a primary mechanistic candidate is unclear.\n- Colony Assay (Fig 4): The CFU assay results suggest that succinic acid has no effect on colony output in healthy BM, while monomethyl succinate (which bypasses SUCNR1) increases CFU output. Conversely, cis-epoxy succinate reduces colony formation in healthy LSK cells, with no effect in SUCNR1 KO cells. This implies that SUCNR1 activation may inhibit LSK proliferation while alternative pathways enhance progenitor potential. However, these hypotheses completely lack mechanistic insights and are somewhat counterintuitive.\n- Controls in Fig 5: Healthy controls are missing in Fig 5. Without WT references, it is impossible to (1) measure the non-specific effects of tasquinimod on WT HSPC function and (2) evaluate to what extent the KO phenotype is reversed by tasquinimod.\n- Functional Assays for SUCNR1 Role in HSCs: To substantiate the role of SUCNR1 in restricting HSC numbers and function, serial replating is suboptimal in mouse models. A competitive bone marrow transplant assay would more releavant to test SUCNR1 effect on HSC engraftment potential, especially given the inflammatory gene expression signature observed, which typically impair HSC function in WT. The Sucnr1-tdTomato reporter mouse could also be employed to compare the differential repopulating potential of SUCNR1-positive versus SUCNR1-negative HSCs.\n- Gene Expression Levels in Fig 6A: Gene expression levels of S100A8/9 in Mx1-Cre NRASG12D/NRASG12D mice are less informative without corresponding SUCNR1 measurements in the same cell populations. Crossing this model with the reporter mouse or performing qPCR for SUCNR1 would add clarity.\n- Single-Cell Data Presentation (Fig 6): The single-cell data presentation lacks depth, with SUCNR1 and S100A8/9 correlations presented visually but without quantification. Cluster annotations by sample, timepoint, or cell type are absent, making interpretation difficult. While S100A8/A9 are identified as markers of residual disease post-cytarabine treatment, it remains unclear if they actively contribute to chemoresistance or are upregulated as a stress response.\n- Role in Metabolism: The manuscript suggests a role for SUCNR1 in hematopoietic metabolism, but this lacks direct experimental support.", "reviewer_3": "The manuscript presented by Cuminetti et al. offers a compelling study, demonstrating for the first time a direct influence of succinate and its receptor, SUCNR1, on cellular hematopoiesis, with implications for hematological malignancies. While prior research suggested that succinate/SUCNR1 might impact hematopoiesis indirectly, no studies to date have documented a direct effect, lending this study considerable novelty. Overall, the publication is well-structured and clearly written. However, for enhanced clarity to a broader audience, adding some contextual information in the results section could be beneficial. In certain areas, the reasoning behind specific experiments and the implications of the findings remain somewhat unclear (see comments below). Briefly outlining the observations could enrich the reader’s experience significantly.\nThis study is extensive and detailed, and its results, along with the newly developed mouse model, hold promise for future mechanistic studies aimed at identifying targetable factors for potential therapeutic applications.\nThat said, I have several specific comments and questions that may enhance the already high quality of the manuscript:\n•Figure 1A: Could you specify the type of survival displayed? It appears to be overall survival, but progression-free survival would also be informative.\n•Figure 1D:\no What was the rationale for treating mice with succinic acid? Thus far, the indication for succinic acid involvement derives only from SUCNR1 expression differences in the patient cohort.\no How was succinic acid administered to the animals? Does the chosen route allow succinic acid to reach the cells directly, or might this be an indirect effect? Drawing conclusions about cellular metabolism from systemic metabolic changes can be challenging. Are elevated succinic acid levels detectable in the serum or other compartments in the mouse model?\no Considering the heterogeneity of AML, it is unfortunate that only one patient sample was used. Could this patient fall within either the SUCNR1 high or low expression groups?\n•Figure 1G:\no The authors state that succinic acid injection worsens the disease. However, the administration route remains unclear (see above), and an explanation for this aggravation is lacking. Are elevated succinic acid levels observable in serum, other body fluids, organs, or cells?\no While significant, the increase in myeloid frequency is marginal. Could the authors comment on whether such a small difference holds biological significance? It appears the range in myeloid frequency in the vehicle group is wider than the mean difference between the vehicle and succinic acid groups.\n•Figure 1J: It would be insightful to see corresponding protein levels.\n•The concluding statement in the first Results section could benefit from added context. The link between low SUCNR1 expression and succinate as a contributor to leukemia progression is not fully clear.\n•Figure 2A: Could you specify the exact mouse ages corresponding to “young adult” and “later middle-aged stage”?\n•Supplemental Figure 4B: The claimed increase in live cell count is minimal and not statistically significant. Displaying actual frequencies instead of percentage changes from WT may be more illustrative. Also, the event count in the representative plots appears low (below 10,000 total events), which is below the standard for reliable flow cytometry.\n•Supplemental Figure 4D: How can the knockout of SUCNR1 impact succinate levels in BMEF? As I understand it, SUCNR1 is activated by succinate but does not regulate succinate availability or metabolism. I would anticipate succinate accumulation due to receptor absence.\n•Supplemental Figure 4E: Background controls for OCR and ECAR measurements, such as rotenone/antimycin A for OCR and 2-DG for ECAR, are absent. Without these, it is challenging to isolate mitochondrial respiration from other oxidative processes. Could such controls be included?\n•Figure 3E: How do you explain the reduced IL-1β protein levels in BMEF when IL-1β gene expression is significantly increased in Sucnr1-KO versus WT animals? Consequently, the concluding statement of this section may require modification or expansion.\n•Supplemental Figure 6E: The intent of this raw spectral ribbon plot is unclear. Without spectral unmixing, assigning signals to specific fluorochromes or fluorescent proteins is not possible.\n•In the discussion, the authors note “overexpression of S100a8 and S100a9 in HSPC subsets […].” How does this align with observations of increased S100A8/S100A9 in AML blasts in bone marrow compared to peripheral blood and the identified pro-inflammatory triggers reported in Blood Advances 2022 6(21)?", "reviewer_4": "The manuscript by Cuminetti et al. examines the role of Succinate receptor (Suncr1) in hematopoiesis. The authors show that low Suncr1 expression correlates with poor survival in AML. Further, treatment of human and leukemia cells in vivo with succinic acid promotes leukemia progression. In the context of normal hematopoiesis sucnr1 knockout resulted in the expansion of several stem and progenitor populations determined by cell number in aged mice. The increased in HSPC cell numbers correlated with an increase in gene expression in signatures associated with HSCs and a decreased gene expression in signatures associated with decreased differentiation and inflammation. Expression of s100a8 and s100a9 were increased in sucnr1 knockdown mice. Treatment with s100a8/9 inhibitor results in reduced hematopoietic cells in the sucnr1 knockdown mice. Finally, the authors show that sucnr1 low expressing cells have high S100a8/9 in single-cell RNA-sequencing post cytarabine treatment (derived from PDX models). Overall, of the manuscript highlights several intriguing findings. However, several additional experiments/analyses should be performed to address the concerns below.\nMajor Concerns:\n1. In Figure 2, the conclusions about changes in HSPC and mature cells are made based on cell numbers only. Most of the populations have increased numbers including total WBCs. Are there any changes in population percentages which may help to tease about if any one population is expanded upon sucnr1 knockout?\n2. The authors use the steady-state populations and gene expression analysis to conclude changes in HSPC function. These conclusions should be supported by performing competitive bone marrow transplants.\n3. Figure 5 would benefit from including wild-type mice. This would allow the assessment of tasquinimod on hematopoiesis in the normal setting and enable the researchers to determine if treatment restores cell numbers back to the wild type levels.\n4. Do monomethyl succinate and succinic acid have the same degree of effect on the intracellular biology of succinate? For example, to they increase oxidative phosphorylation to the same level. Differences between the compounds could be a confounding factor in the interpretation of results in figure 4.\n5. Using a genetic approach like overexpressing sucnr1 would help to solidify the conclusions from figure 4.\n6. Further, overexpressing sucnr1 and measuring the consequence on leukemia progression would enhance the study.\n7. Figure 6 suggests that expression of sucnr1 may influence the response to chemotherapy. It would strengthen the results by functionally testing this.\nMinor:\n1. In Figure 1D what are the patient characteristics? Specifically it would be helpful to include FAB and mutation status as the authors show these are related to sucnr1 expression.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We would like to thank all 5 Referees for the time you have spent with the revision of our work, your positive comments and constructive criticism. We believe our work is substantially improved and hope you will agree with us.\nReviewer #1 (Remarks to the Author): expert in Succinate and Sucnr1\nThe article presents a clear and well-structured exploration of Sucnr1’s role in hematopoiesis, offering valuable insights into a complex and underexplored area of stem cell biology. The use of comprehensive experimental approaches—ranging from genetic manipulations to transcriptomic analysis—enhances the clarity of the conclusions and strengthens the overall impact of the study. The figures are well-organized and effectively support the narrative, providing clear visual representations of the data. Overall, the manuscript is excellently presented, with a clear focus on the novelty of the findings and their potential implications in the context of hematopoiesis. However, the manuscript would benefit by addressing certain key issues.\n1) The global knockout model used may limit the interpretation of Sucnr1’s role in hematopoiesis, as indirect effects arising from systemic Sucnr1 deficiency could influence hematopoietic stem and progenitor cell (HSPC) responses. To clarify whether Sucnr1’s effects are intrinsic to hematopoietic cells or arise from changes in the microenvironment, a lineage-specific knockout approach would be insightful. This strategy could help differentiate between direct roles of Sucnr1 within hematopoietic cells and broader physiological or paracrine influences, thereby enhancing our understanding of its role in self-renewal and differentiation dynamics in hematopoiesis.\nWe thank the Reviewer for this comment. To dissect the role of Sucnr1 on healthy hematopoiesis via the hematopoietic compartment, we transplanted Sucnr1fl/fl control or Mx1-Cre Sucnr1fl/fl experimental BM cells into C57BL/6J WT mice, which were later injected with poly-inosine:poly-cytosine (pIpC; please, see schematics in Figure 2I). In vivo deletion of Sucnr1 from hematopoietic cells resulted in early expansion of the numbers of circulating total leukocytes, i.e. myeloid cells, monocytes and lymphocytes (Figure 2J), which was maintained over the course of the experiment (Figure 2K). In BM, in vivo deletion of Sucnr1 from hematopoietic cells led to an expansion of MPP versus intact recipients, particularly MPP4 (Figure 2L). This means that Sucnr1 effects can be explained greatly by intrinsic effects to hematopoietic cells. These ideas have been included in the manuscript.\nThe end-point analysis of this experiment was performed 47 weeks (about 11 months) after the transplant, to make it a time-point comparable to the global deletion of Sucnr1. The differences compared with the results obtained in Sucnr1-KO may arise from the experimental conditions (irradiation, pIpC-induced recombination, etc) and/or a potential contribution from the stem cell microenvironment and/or other systemic effects that should be subject of future work.\n2) The effects of Sucnr1 deficiency on hematopoiesis appear contradictory. Sucnr1 deletion results in both an expansion of HSPCs and increased hematopoietic activity in the bone marrow and spleen, yet, paradoxically, it also shows reduced transcriptomic markers of stemness and self-renewal. Could the authors provide a more in-depth discussion to explain this finding? Additionally, targeted experiments investigating differential effects across specific HSPC subpopulations (e.g., long-term vs. short-term HSCs, or lineage-specific progenitors) could help clarify whether Sucnr1’s impact varies across these subsets, potentially reconciling the observed expansion with the reduction in stemness markers.\nThank you for bringing up this interesting topic. We have performed new FACS-sorting of long-term HSC, short-term HSC and MPP obtained from the BM of WT and Sucnr1-KO mice, and performed ddPCR on two important genes related to stemness (Procr, Hoxa9).\nThe expression of both genes was reduced in the gene expression profiling by RNA-Seq of Sucnr1-KO LSK cells versus WT cells, and their reduced expression levels were also confirmed by ddPCR in independent samples. Importantly, when analyzing different HSPC subsets, we find that the levels of expression of Procr and Hoxa9 are significantly reduced only in the MPP subset, but not in the long-term and short-term HSC compartments (new Figure 4D).\nWe are also providing new data on the functionality of HSPC in vivo analyzed by competitive repopulation assays of BM nucleated cells isolated from WT or Sucnr1-KO mice (Ly5.2) into lethally irradiated Ly5.1 congenic recipients. Peripheral blood analysis from recipient mice 16 weeks after transplantation revealed a mild reduction in HSPC engraftment in recipients transplanted with Sucnr1-KO cells compared to recipients transplanted with WT control cells (please see new Figure 3G).\nAs discussed in the manuscript, these seemingly paradoxical effects of Sucnr1 deletion on HSPC with increased numbers/survival/colony-forming potential versus reduced engraftment in competition/proliferation/gene expression markers of stemness may be important factors underlying the chronic nature of the hematopoietic abnormalities displayed by Sucnr1-KO mice, which become symptomatic only at the middle-age.\nThis paradox between numbers and function is common to other chronic conditions, like ageing and related chronic inflammatory conditions, where the HSPC pool is increased in numbers but reduced in function, representing a shift towards a biased, less effective and abnormal hematopoietic system. The potential role of Sucnr1 in the ageing hematopoietic system should be subject of future work. We have included these ideas in the Discussion section.\nFurther, although Sucnr1-KO BM cells are less fit in competition with WT cells, as explained in the point above, primary transplants of Sucnr1fl/fl or Mx1-Cre Sucnr1fl/fl BM cells into C57BL/6J WT mice, induced after stable engraftment, result in expanded hematopoiesis and MPP compartment, greatly recapitulating the hematopoietic defects of Sucnr1-KO mice (Figure 2I-2L).\nTo further substantiate the role of Sucnr1 in HSPC function in vivo, we performed competitive repopulation assays using 200 Sucnr1-tdTomato+ or Sucnr1-tdTomato− FACS-sorted HSPC (containing a mix of 40 HSC, 86 MPP3 and 74 MPP4) isolated from Ly5.2 Sucnr1-tdTomato mice into lethally irradiated Ly5.1 congenic recipients. Peripheral blood analysis from recipient mice 10 weeks after the transplant showed a reduction of circulating leukocytes and donor-derived monocytes in recipients of Sucnr1+ cells compared to recipients of Sucnr1− cells (Figure 3H).\nThus, Sucnr1 expression marks a subset of HSPC with restricted engraftment potential in vivo.\n3) Section: Low SUCNR1 Predicts Acute Myeloid Leukemia Progression\nIn lines 141-143, it is mentioned that \"MLL-AF9+ mice had overt AML, and the lineage-negative compartment was expanded in the bone marrow of succinic acid-treated mice compared to vehicle-treated controls (Figure 1L), showing an increase in the LK fraction but no changes in LSK cells (supplemental Figure 2E).\" While there was an observed trend toward an increase in both the LK fraction and LSK cells in succinic acid-treated mice, it is crucial to emphasize that none of these differences reached statistical significance. Adding this clarification in the text would provide a more accurate and nuanced interpretation of the data.\nThanks for pointing out this error, which has now been corrected and reads “with an observed trend toward increased LK fraction and LSK cells that did not reach statistical significance”.\nIn line 143, the statement that 'low SUCNR1 represents a prognostic marker for reduced survival in AML patients' would benefit from further statistical validation to strengthen its impact. To rigorously establish SUCNR1 as an independent prognostic factor, a Cox regression analysis should be performed. This would allow the authors to assess the effect of SUCNR1 on survival while adjusting for other known prognostic variables in AML. This addition would significantly enhance the robustness of the study's conclusions regarding the prognostic value of SUCNR1 in AML.\nWe have now performed Cox regression analysis, which uncovered SUCNR1 expression as an independent predictor of both overall survival and progression-free survival after adjusting for age, gender and FAB classification. Please, see results in new Table 1.\n4) Section: Sucnr1 is expressed in HSPC, and its activation restricts their functional response to succinate ex vivo\nIn line 223, the authors suggest that Sucnr1 activation suppresses succinate's intracellular effects, supported by their observations with monomethyl succinate, which increased colony-forming potential in total bone marrow (TBM) in a dose-dependent manner. However, this conclusion is not entirely convincing. If Sucnr1 activation indeed dampens succinate’s effects, one would expect to see a reduction in CFU-C in TBM with succinate treatment (Figure 4A), but no such decrease is observed. This raises questions about the robustness of the authors’ claim. Additionally, to strengthen their argument, the authors could have tested the combination of monomethyl succinate with succinic acid to explore potential synergistic effects, which would provide more clarity on their hypothesis.\nWe appreciate this comment. Monomethyl succinate is inactive on Sucnr1 and enhances the colony-forming potential in total BM (TBM), in a dose-response manner. Conversely, succinic acid, which displays both Sucnr1-dependent and independent effects, results in unchanged colony-forming potential. Taken together, we reasoned that these results suggest that Sucnr1 activation represses the intracellular effect of succinate. We have reformulated these results for clarity. Further, we have followed the Referee suggestion and explored the effect of succinic acid and monomethyl succinate combo, which results in a partial increase in colony-forming potential in TBM versus succinic acid alone and a partial reduction versus monomethyl succinate alone, providing robust evidence that Sucnr1 activation represses the intracellular effect of succinate. Please, see the results in new Figure 3D.\nAdditionally, clarification is needed on why monomethyl succinate increased CFU-C in FACS-sorted HSC from Sucnr1-KO mice in the first plating (Figure 4C), while this effect was not seen in cells from WT mice (Figure 4B). Could the difference be attributed to varying intracellular succinate levels between the two mouse strains? Has the possibility of altered succinate accumulation been considered? Without addressing this, the interpretation of the results remains incomplete and raises doubts about the underlying mechanisms.\nThis is a legit consideration, and we had indeed taken into account the possibility of increased intracellular succinate levels in Sucnr1-KO mice versus WT mice. In fact, as stated in the Results section of the previous version of our manuscript, “Given that succinate is an intermediate metabolite of the TCA cycle, we studied its potentially increased cycling which would result in metabolic changes. Sucnr1 deletion resulted in increased baseline oxygen consumption rate in lineage-negative progenitors (supplemental Figure 4G). This effect was specific and reversible because the maximal oxygen consumption rate and the glycolytic flux remained unchanged (supplemental Figure 4G). Consistent with these data, both total and mitochondrial reactive oxygen species (ROS) levels were higher in LSK cells from Sucnr1-KO mice (supplemental Figure 4H).” We have now established a new collaboration that has enabled us to provide direct evidence using metabolomics in HSC, which confirms increased intracellular succinate levels in Sucnr1-KO versus WT mice (please see new supplemental Figure 4F). The regulation of succinate localization by Sucnr1 should be subject of future work. Whereas it could simply be a matter of increased succinate availability in the absence of Sucnr1 binding, active regulatory mechanisms could additionally take place. We have added these ideas to the Discussion.\nIn parallel, we provide compelling evidence showing that activation of Sucnr1 counterbalances the stimulatory effect of intracellular succinate in HSPC, via control of S100a8/S100a9. Consistent with the increased mRNA expression of S100a8, S100a9 in HSPC from Sucnr1-KO versus WT mice, the protein levels of the alarmin complex S100A8/S100A9 were increased in the BMEF. Notably, intracellular succinate activates metabolism and may have multiple additional effects, but treatment with tasquinimod, S100a9 blocker, rescued the hematopoietic defects of Sucnr1-KO HSPC both added ex vivo and in vivo.\nA remaining open question was whether S100a9 pathway could be both repressed by Sucnr1 signaling and activated by increased intracellular succinate. To answer this question, we have used available material from cell lines, where we show that the combination of cis-epoxy succinate but not monomethyl succinate and AraC reduces the expression of S100A9, versus AraC alone (new supplemental Figure 11C). Thus, S100a9 is an alternative pathway controlled by Sucnr1, independent of intracellular succinate, which counterbalances the stimulatory effect of intracellular succinate.\n5) Section: Sucnr1 restricts HSPC and hematopoiesis via control of S100a8/S100a9\nTo better elucidate the role of Sucnr1 in hematopoiesis through S100a8/S100a9 regulation, it would be valuable to conduct a direct comparison between tasquinimod-treated WT mice and tasquinimod-treated Sucnr1-KO mice. This comparison would clarify whether the observed hematopoietic effects, including changes in HSPC populations and lineage distribution, are specifically mediated by Sucnr1.\nAs suggested by Reviewers #1, #2 and #5, and #4, to substantiate a selective role of S100a8/S100a9 in the hematopoietic system via Sucnr1 deletion, we performed an independent experiment treating both Sucnr1-KO and WT mice with tasquinimod or vehicle in parallel (supplemental Figure 8A). Confirming our previous results, tasquinimod treatment in Sucnr1-KO mice reduced the expanded numbers of circulating leukocytes to the numbers observed in WT mice, including both overall myeloid cells as well as B cells and with no significant effect observed in WT mice (supplemental Figure 8B). Tasquinimod administration reduced the expanded numbers of monocytes in BM of Sucnr1-KO mice to the numbers found in WT mice, whilst no effect was observed in WT mice (supplemental Figure 8C). Conversely, treatment with tasquinimod reduced the numbers of BM B cells to similar levels in both Sucnr1-KO and WT mice (supplemental Figure 8C). The expansion of BM HSC in Sucnr1-KO mice was reduced by treatment with tasquinimod, but a minor reduction in HSC numbers was also observed in WT mice, bringing HSC values in both tasquinimod-treated groups to similar level (supplemental Figure 8D). In turn, the increased colony-forming potential in Sucnr1-KO mice was reduced to WT values, with no effect seen in samples obtained from WT mice (supplemental Figure 8E). Of note, this assay is optimal for the growth of primitive erythroid progenitor cells, granulocyte-macrophage progenitor cells, and multi-potential granulocyte, erythroid, macrophage, megakaryocyte progenitor cells, but not for the growth of lymphoid progenitor cells. Thus, these data suggest that tasquinimod effect originates in the HSC compartment for both lineages, and whereas the effect of tasquinimod on the myeloid lineage is fully dependent on Sucnr1 deletion, the effect on the B cell lineage is only partially dependent on the loss of Sucnr1 signaling.\nAdditionally, investigating the combined effects of succinic acid treatment and tasquinimod in mouse models of pre-leukemic myelopoiesis and AML could provide a more comprehensive understanding of S100a8/S100a9 signaling in leukemogenesis and its potential modulation by Sucnr1. This approach would help identify the therapeutic relevance of targeting Sucnr1 and its downstream pathways in both pre-leukemic and leukemic hematopoiesis.\nThank you for the opportunity to address this question. To study the therapeutic potential of targeting Sucnr1 signaling pathway in AML, we turned to the more aggressive Mll-AF9 mouse model, which express low levels of Sucnr1 (Figure 1L). We transplanted Mll-AF9+ BM into lethally irradiated recipients and treated them with tasquinimod, tasquinimod in combination with cis-epoxy succinate, or the corresponding vehicles, for a maximum of 24 weeks (Figure 6A). The combination of tasquinimod and cis-epoxy succinate reduced the numbers of circulating leukocytes (Figure 6B) and Gr-1hi blasts in spleen (Figure 6C), compared to mice receiving the vehicle for both drugs. In the BM, we found that tasquinimod alone decreased the numbers of c-Kit+FcRγ+ GMP-like leukemic blasts versus mice treated with vehicle (Figure 6D-6E). Further, tasquinimod reduced the number of BM c-Kit+FcRγ−CD48+CD34+ lymphoid-primed multipotent progenitor (LMPP) LSC when administered alone or in combination with cis-epoxy succinate, versus the corresponding vehicle-treated mice (Figure 6D-6E). These data indicate that activating Sucnr1 signaling with a potent Sucnr1 agonist and its downstream pathway through S100A9 blocking shows therapeutic value in Mll-AF9+ mice, reducing immunophenotypically-defined LSC in the BM and peripheral leukemic burden.\n6) Minor point: please correct “succinate” in Supplementary Figure 2 to “succinic acid.”\nThanks for noticing, we have corrected this mistake.\nReviewer #2 (Remarks to the Author): expert in RNA-seq and scRNA-seq analysis, AML and hematopoiesis\nStarting from the observation that low succinate receptor 1 (SUCNR1) expression is associated with AML subtypes M0-M3 and correlates with poorer outcomes, Cuminetti et al. explore SUCNR1 role in hematopoiesis and its impact on AML progression. The authors utilize a Sucnr1-tdTomato reporter mouse to analyze its expression across hematopoietic compartments. This model reveals that SUCNR1 is predominantly expressed in LSK, HSC and MPP cells subsets, while other hematopoietic progenitors, suggesting a role for SUCNR1 in early hematopoietic differentiation.\nTo assess SUCNR1 function, the authors develop a KO model. In SUCNR1 KO mice, immature progenitor within LSK cells expand, but other populations increase in frequency, including mature B cells. This LSK expansion coincides with a gene expression shift toward an inflammatory profile and a reduced stem cell signature, including increase expression of S100A8/A9. Interestingly, progenitor-expanding phenotype is partly reversed by tasquinimod, an S100A8/A9 inhibitor, suggesting that S100A8/A9 signaling may be mediating these effects.\nThe study extends these findings into AML models (NRASG12D, MLL-AF9), where succinate treatment promotes myeloid output and accelerates disease progression. Pre-leukemic HSC/MPP subsets in Mx1-Cre NRASG12D show increased S100A8/A9 expression, mirroring the inflammatory state seen in SUCNR1 KO animals.\nIn human AML patient-derived xenografts (PDX) from two patients, chemoresistant cells exhibit increased inflammation and S100A8/A9 expression, while SUCNR1 expression is mainly observed in pre-treatment samples, suggesting possible downregulation in response to therapy.\nOverall Comments\nWhile the manuscript presents interesting data and some novel characterizations of SUCNR1 role in hematopoiesis, a lack of proper controls and incomplete analyses make data interpretation difficult.\n- Manuscript Structure: The narrative is challenging to follow and at times counterintuitive, frequently shifting between AML models, human AML, healthy hematopoiesis, and KO mouse models. To improve readability, the structure should follow a linear narrative.\nWe appreciate there are various ways to effectively convey a scientific report. We prefer to start with the most important and clinically relevant information to catch the attention of the reader, then continue with the body of the work where the mechanisms (cellular and molecular) are uncovered, mainly by use of biologically relevant mouse models and paralleled validations in humans where possible. In this work, we end by using the mechanistic information back in AML mouse models, patients and human cell lines.\nThis is the only comment referring to “challenging” structure out of four Reviewer reports, and two other Reviewers (#1 and #3) in fact refer to the manuscript as “well-structured”, “clear” and “well-written”. However, given the extensive revisions, we have made a small reorganization of the narrative from “Sucnr1 restricts HSPC and hematopoiesis under steady-state” to “Sucnr1 is expressed in HSPC and its activation restricts their functional response”, followed by “Sucnr1 preserves transcriptional programs characteristic of HSPC” and “Sucnr1 restricts HSPC and hematopoiesis via control of S100a8/S100a9”.\nThis could start with SUCNR1 expression profiling in healthy mouse and human hematopoiesis (using gene expression databases, data from mouse models, and the Sucnr1-tdTomato reporter model). Profiling should cover both immature and mature myeloid cells, as some mature cells are also affected in the KO.\nTo answer these questions, HSPC subsets, committed hematopoietic progenitors and differentiated cells obtained from the mouse BM were analyzed by spectral flow cytometry. After spectral unmixing, we quantified the Sucnr1-tdTomato+ cell fractions (Figure 3A and supplemental Figure 6A-6D). Our analysis revealed that all HSPC subsets show a varying fraction of Sucnr1+ cells and the majority of Sucnr1+ HSPC were identified as MPP3, with an average of 0.191% of Sucnr1+ cells, followed by MPP4 (Figure 3B and supplemental Figure 6C). Importantly, although Sucnr1+ cells were found across all hematopoietic cell subsets analyzed, including all lineage-positive cell types (supplemental Figure 6C-6D), the majority of Sucnr1+ cells in the hematopoietic system were identified as lineage-negative progenitors, including all LK progenitors particularly GMP, LSK cells and CLP (supplemental Figure 6E).\nIn view of the expression of Sucnr1 across lineage-positive cell in the BM, future studies should explore its potential role regulating their primary and effector functions. This idea has been included in the Discussion.\nIn parallel, we confirmed SUCNR1 mRNA expression across all hematopoietic cell subsets analyzed in normal human hematopoiesis, using expression data from the HemaExplorer collection at BloodSpot (GSE17054, GSE19599, GSE11864 and E-MEXP-1242), including HSC and HSPC (supplemental Figure 6F). In humans, CMP, GMP and differentiating promyelocytes and myelocytes display remarkably high levels of SUCNR1 mRNA expression. The expression of SUCNR1 in CLP was not available (supplemental Figure 6F).\nAdditionally, data on SUCNR1 expression in non-hematopoietic tissues (e.g., stromal cells) would help interpret findings from the KO mouse model.\nPlease, see answer below referring to “conditional Sucnr1 deletions in hematopoietic cells could clarify whether the observed hematopoietic phenotypes stem directly from HSPC-autonomous functions of Sucnr1 or indirect effects via niche cells”. Sucnr1 effects on hematopoiesis can be explained greatly by intrinsic effects to hematopoietic cells.\nAs stated in the Discussion, although partial contribution of the BM stem cell microenvironment or metabolic systemic effects with potential to influence hematopoiesis will require future investigation, taken together our data provide compelling evidence that supports for a direct, selective role of Sucnr1 on HSPC.\nAML datasets could be used to differentiate SUCNR1 expression between healthy and malignant tissues.\nPlease, see new Figure 1D, where we are showing now data obtained from the dataset TARGET AML at BloodSpot (www.fobinf.com) confirming great heterogeneity in SUCNR1 expression in primary AML and overall lower expression values compared to controls (GSE42519 and GSE13159).\nConditional Sucnr1 deletions in hematopoietic cells could clarify whether the observed hematopoietic phenotypes stem directly from HSPC-autonomous functions of Sucnr1 or indirect effects via niche cells.\nWe thank the Reviewers for this comment. To dissect the role of Sucnr1 on healthy hematopoiesis via the hematopoietic compartment, we transplanted Sucnr1fl/fl control or Mx1-Cre Sucnr1fl/fl experimental BM cells into C57BL/6J WT mice, which were later injected with poly-inosine:poly-cytosine (pIpC; please, see schematics in Figure 2I). In vivo deletion of Sucnr1 from hematopoietic cells resulted in early expansion of the numbers of circulating total leukocytes, i.e. myeloid cells, monocytes, or lymphocytes (Figure 2J), which was maintained over the course of the experiment (Figure 2K). In BM, in vivo deletion of Sucnr1 from hematopoietic cells led to an expansion of MPP versus intact recipients, particularly MPP4 (Figure 2L). This means that Sucnr1 effects can be explained greatly by intrinsic effects to hematopoietic cells. The end-point analysis of this experiment was performed 47 weeks (about 11 months) after the transplant, to make it a time-point comparable to the global deletion of Sucnr1.\n- Figure Titles: Figure titles often do not accurately reflect the content, making it hard for readers to follow the data being presented.\nFor clarity, the main Figure titles now match the heading of each part of the Results section. The Supplemental Figure titles have been improved accordingly.\n- FAB Classification: The FAB classification is mentioned but not systematically incorporated. Patient and PDX sample classifications by FAB subtype should be included for clarity.\nThe patient and PDX sample were classified as M1. This information has now been included in the manuscript.\n- Inflammatory Cytokine Levels (Fig 3): The increase in mRNA for certain pro-inflammatory cytokines is not substantiated by corresponding protein levels in bone marrow fluid. Intracellular cytokine detection by flow cytometry would be more appropriate. Given the inflammatory phenotype, a panel to detect immune cell activation in the KO model would be important.\nThank you for this important question. Given the increased mRNA expression of Il1b in HSPC from Sucnr1-KO versus WT mice, we analyzed the intracellular levels of IL-1β protein and confirmed them slightly but significantly increased in Sucnr1-KO compared to WT mice (Figure 4E). This effect seemed to be specific of the HSPC compartment, as we observed no differences in the intracellular levels of IL-1β in total live cells, lineage-positive cells, lineage-negative progenitors or LK progenitors (supplemental Figure 7C). This is in contrast with the levels of IL-1β, which we found to be reduced in the BM of Sucnr1-KO versus WT mice. Thus, in vivo deletion of Sucnr1 promotes inflammation specifically in HSPC. We have included these ideas in the Results and Discussion sections.\n- Focus on S100A8/A9: Among various inflammation-related factors, the rationale for focusing on S100A8/A9 as a primary mechanistic candidate is unclear.\nDespite complex changes taking place in the global cytokine status of the BM in Sucnr1-KO mice, S100a8 and S100a9 were consistently increased in their mRNA expression in HSPC and in their protein levels in BMEF, making it a promising mechanistic candidate to follow up. We have included this idea in the corresponding Results section.\n- Colony Assay (Fig 4): The CFU assay results suggest that succinic acid has no effect on colony output in healthy BM, while monomethyl succinate (which bypasses SUCNR1) increases CFU output. Conversely, cis-epoxy succinate reduces colony formation in healthy LSK cells, with no effect in SUCNR1 KO cells. This implies that SUCNR1 activation may inhibit LSK proliferation while alternative pathways enhance progenitor potential. However, these hypotheses completely lack mechanistic insights and are somewhat counterintuitive.\nWe appreciate this comment, which is similar to comment 4) from Reviewer #1. Briefly, monomethyl succinate is inactive on Sucnr1 and enhances the colony-forming potential in total BM (TBM), in a dose-response manner. Conversely, succinic acid, which displays both Sucnr1-dependent and independent effects, results in unchanged colony-forming potential. Taken together, we reasoned that these results suggest that Sucnr1 activation represses the intracellular effect of succinate. We have reformulated these results for clarity. Further, we have followed Referee #1 suggestion and explored the effect of succinic acid and monomethyl succinate combination, which results in a partial increase in colony-forming potential in TBM versus succinic acid alone and a partial reduction versus monomethyl succinate alone, confirming that Sucnr1 activation represses the intracellular effect of succinate. Please, see the results in new Figure 3D.\nOf note, cis-epoxy succinate, 10‐ to 20‐fold more potent than succinic acid on Sucnr1, reduced the colony-forming potential of healthy HSC (Figure 3E) whereas it stimulated the colony-forming potential of Sucnr1-KO HSC (Figure 3F), suggesting loss of colony-forming potential repression by Sucnr1 activation in the absence of Sucnr1. These data were present in the previous version of the manuscript.\nIn parallel, we provide compelling evidence showing that activation of Sucnr1 counterbalances the stimulatory effect of intracellular succinate in HSPC, via control of S100a8/S100a9. Notably, treatment with tasquinimod, S100a9 blocker, rescued the hematopoietic defects of Sucnr1-KO HSPC both added ex vivo and in vivo.\nA remaining open question was whether S100a9 pathway could be both repressed by Sucnr1 signaling and activated by increased intracellular succinate. To answer this question, we have used available material from cell lines, and we show now that the combination of cis-epoxy succinate but not monomethyl succinate and AraC reduces the expression of S100A9, versus AraC alone (new supplemental Figure 11C). Thus, S100a9 seems to be an alternative pathway controlled by Sucnr1, independent of intracellular succinate, which counterbalances the stimulatory effect of intracellular succinate.\n- Controls in Fig 5: Healthy controls are missing in Fig 5. Without WT references, it is impossible to (1) measure the non-specific effects of tasquinimod on WT HSPC function and (2) evaluate to what extent the KO phenotype is reversed by tasquinimod.\nAs suggested by Reviewers #1, #2 and #5, and #4, to substantiate a selective role of S100a8/S100a9 in the hematopoietic system via Sucnr1 deletion, we performed an independent experiment treating both Sucnr1-KO and WT mice with tasquinimod or vehicle in parallel (supplemental Figure 8A). Confirming our previous results, tasquinimod treatment in Sucnr1-KO mice reduced the expanded numbers of circulating leukocytes to the numbers observed in WT mice, including both overall myeloid cells as well as B cells and with no significant effect observed in WT mice (supplemental Figure 8B). Tasquinimod administration reduced the expanded numbers of monocytes in BM of Sucnr1-KO mice to the numbers found in WT mice, whilst no effect was observed in WT mice (supplemental Figure 8C). Conversely, treatment with tasquinimod reduced the numbers of BM B cells to similar levels in both Sucnr1-KO and WT mice (supplemental Figure 8C). The expansion of BM HSC in Sucnr1-KO mice was reduced by treatment with tasquinimod, but a minor reduction in HSC numbers was also observed in WT mice, bringing HSC values in both tasquinimod-treated groups to similar level (supplemental Figure 8D). In turn, the increased colony-forming potential in Sucnr1-KO mice was reduced to WT values, with no effect seen in samples obtained from WT mice (supplemental Figure 8E). Of note, this assay is optimal for the growth of primitive erythroid progenitor cells, granulocyte-macrophage progenitor cells, and multi-potential granulocyte, erythroid, macrophage, megakaryocyte progenitor cells, but not for the growth of lymphoid progenitor cells. Thus, these data suggest that tasquinimod effect originates in the HSC compartment for both lineages, and whereas the effect of tasquinimod on the myeloid lineage is fully dependent on Sucnr1 deletion, the effect on the B cell lineage is only partially dependent on the loss of Sucnr1 signaling.\n- Functional Assays for SUCNR1 Role in HSCs: To substantiate the role of SUCNR1 in restricting HSC numbers and function, serial replating is suboptimal in mouse models. A competitive bone marrow transplant assay would more releavant to test SUCNR1 effect on HSC engraftment potential, especially given the inflammatory gene expression signature observed, which typically impair HSC function in WT. The Sucnr1-tdTomato reporter mouse could also be employed to compare the differential repopulating potential of SUCNR1-positive versus SUCNR1-negative HSCs.\nWe agree with the Reviewers. We are now providing new data on the functionality of HSPC in vivo analyzed by competitive repopulation assays of BM nucleated cells isolated from WT or Sucnr1-KO mice (Ly5.2) into lethally irradiated Ly5.1 congenic recipients. Peripheral blood analysis from recipient mice 16 weeks after transplantation revealed a mild reduction in HSPC engraftment in recipients transplanted with Sucnr1-KO BM cells compared to recipients transplanted with WT BM control cells (please see new Figure 3G).\nAlthough Sucnr1-KO BM cells seem to be less fit in competition with WT cells, as explained in the point above, primary transplants of Sucnr1fl/fl or Mx1-Cre Sucnr1fl/fl BM cells into C57BL/6J WT mice greatly recapitulate the hematopoietic expansion of Sucnr1-KO mice (Figure 2I-2L).\nTo further substantiate the role of Sucnr1 in HSPC function in vivo, we then performed competitive repopulation assays using 200 Sucnr1-tdTomato+ or Sucnr1-tdTomato− FACS-sorted HSPC (containing a mix of 40 HSC, 86 MPP3 and 74 MPP4) isolated from Ly5.2 Sucnr1-tdTomato mice into lethally irradiated Ly5.1 congenic recipients. Peripheral blood analysis from recipient mice 10 weeks after the transplant showed a reduction of circulating leukocytes and donor-derived monocytes in recipients of Sucnr1+ cells compared to recipients of Sucnr1− cells (Figure 3H). Thus, Sucnr1 expression marks a subset of HSPC with restricted engraftment potential in vivo.\nThe conclusions on functionality drawn from the experiments on serial replatings have also been toned down throughout the manuscript.\n- Gene Expression Levels in Fig 6A: Gene expression levels of S100A8/9 in Mx1-Cre NRASG12D/NRASG12D mice are less informative without corresponding SUCNR1 measurements in the same cell populations. Crossing this model with the reporter mouse or performing qPCR for SUCNR1 would add clarity.\nWe have recently moved our lab and activities from the University of Tromsø to the University of Oslo and unfortunately, we do not have available animals of these strains to perform cell sortings at this time. However, we have been able to quantify Sucnr1 expression in frozen total BM nucleated cell samples, and found it reduced in samples obtained from pI-pC-induced Mx1-Cre NRAS-G12D versus NRAS-G12D controls (see new Figure 1F).\n- Single-Cell Data Presentation (Fig 6): The single-cell data presentation lacks depth, with SUCNR1 and S100A8/9 correlations presented visually but without quantification. Cluster annotations by sample, timepoint, or cell type are absent, making interpretation difficult. While S100A8/A9 are identified as markers of residual disease post-cytarabine treatment, it remains unclear if they actively contribute to chemoresistance or are upregulated as a stress response.\nWe apologize for the lack of clarity regarding the correlation and annotations in our single-cell analysis, and we thank the reviewer for the opportunity to clarify our results. Annotation by sample and timepoint is now provided in supplemental Figure 10A. The single-cell RNA-sequencing (scRNA-seq) dataset corresponds to two AML patient-derived xenografts (PDX) analyzed before and after 8 days of a 5-day cytarabine (AraC) treatment (GSE178912). After integration with the Harmony algorithm, we performed unbiased clustering using K-nearest neighbor analysis (Figure 7B), which identified seven clusters (0–6).\nFollowing AraC treatment, clusters 0 and 1 expanded markedly. Together with cluster 5, these clusters displayed a shared transcriptional program characterized both by the residual disease signature post-AraC (AraC_UP)1 and by an inflammatory/senescence–stress signature (CISG_UP)2 (Figure 7C). Notably, S100A8 and S100A9 were highly expressed in these persisting clusters (0, 1, 5) (Figure 7D). Their expression was significantly increased post-AraC compared to diagnosis (Figure 7E–7F) and correlated with the residual disease signature (Figure 7G).\nIn contrast, SUCNR1 was expressed at low levels in cluster 3, which decreased in numbers after AraC treatment and showed low expression of S100A8 and S100A9 (Figure 7D–7E). Functional enrichment analysis (RunEnrichment, SCP package) revealed that cluster 3 was enriched for mitochondrial and metabolic processes (supplemental Figure 10B). This finding is consistent with previous studies showing that AraC treatment induces a metabolic rewiring characterized by enhanced mitochondrial activity, a key driver of chemoresistance3.\nThus, the reduction of cluster 3 post-AraC suggests that SUCNR1-expressing cells represent a metabolically active population with a distinct role in shaping treatment response. The metabolic adaptation triggered by increased mitochondrial activity may sustain chemopersistence in vivo. In this context, SUCNR1 expression, as a marker of this cell subset, could represent both an important determinant of chemotherapy response and a potential therapeutic vulnerability.\n- Role in Metabolism: The manuscript suggests a role for SUCNR1 in hematopoietic metabolism, but this lacks direct experimental support.\nWe agree with the Reviewers that our data provides evidence for a role of Sucnr1 in HSPC metabolism. As stated in the Results section of the previous version of our manuscript, “Given that succinate is an intermediate metabolite of the TCA cycle, we studied its potentially increased cycling which would result in metabolic changes. Sucnr1 deletion resulted in increased baseline oxygen consumption rate in lineage-negative progenitors (supplemental Figure 4G). This effect was specific and reversible because the maximal oxygen consumption rate and the glycolytic flux remained unchanged (supplemental Figure 4G). Consistent with these data, both total and mitochondrial reactive oxygen species (ROS) levels were higher in LSK cells from Sucnr1-KO mice (supplemental Figure 4H).” We have now established a new collaboration that has enabled us to provide direct evidence using metabolomics in HSC, which confirms increased intracellular succinate levels in Sucnr1-KO versus WT mice (please see new supplemental Figure 4F). The regulation of succinate localization by Sucnr1 should be subject of future work. Whereas it could simply be a matter of increased succinate availability in the absence of Sucnr1 binding, active regulatory mechanisms could additionally take place. We have added these ideas to the Discussion.\nHowever, in parallel, we provide compelling evidence showing that activation of Sucnr1 counterbalances the stimulatory effect of intracellular succinate in HSPC, via control of S100a8/S100a9. Consistent with the increased mRNA expression of S100a8, S100a9 in HSPC from Sucnr1-KO versus WT mice, the protein levels of the alarmin complex S100A8/S100A9 were increased in the BMEF. Notably, intracellular succinate activates metabolism and may have multiple additional effects, but treatment with tasquinimod, S100a9 blocker, rescued the hematopoietic expansions of Sucnr1-KO HSPC both added ex vivo and in vivo.\nA remaining open question was whether S100a9 pathway could be both repressed by Sucnr1 signaling and activated by increased intracellular succinate. Using available material from cell lines, we show that the combination of cis-epoxy succinate but not monomethyl succinate and AraC reduces the expression of S100A9, versus AraC alone (new supplemental Figure 11C). Thus, S100a9 seems to be an alternative pathway controlled by Sucnr1, independent of intracellular succinate, which counterbalances the stimulatory effect of intracellular succinate.\nThe single-cell RNA-sequencing (scRNA-seq) dataset from PDX analyzed before and after 8 days of a 5-day cytarabine (AraC) treatment (GSE178912) further supported for a role of SUCNR1 in metabolism of human AML blasts. As previously stated, SUCNR1 was expressed at low levels in cluster 3, which decreased in numbers after AraC treatment and showed low expression of S100A8 and S100A9 (Figure 7D–7E). Cluster 3 was enriched for mitochondrial and metabolic processes (supplemental Figure 10B), finding consistent with previous studies showing that AraC treatment induces a metabolic rewiring characterized by enhanced mitochondrial activity, a key driver of chemoresistance3. These findings will require future in-depth exploration, but this is out of the scope of our present manuscript.\nReviewer #3 (Remarks to the Author): expert in S100A8/S100A9, AML\nThe manuscript presented by Cuminetti et al. offers a compelling study, demonstrating for the first time a direct influence of succinate and its receptor, SUCNR1, on cellular hematopoiesis, with implications for hematological malignancies. While prior research suggested that succinate/SUCNR1 might impact hematopoiesis indirectly, no studies to date have documented a direct effect, lending this study considerable novelty. Overall, the publication is well-structured and clearly written. However, for enhanced clarity to a broader audience, adding some contextual information in the results section could be beneficial. In certain areas, the reasoning behind specific experiments and the implications of the findings remain somewhat unclear (see comments below). Briefly outlining the observations could enrich the reader’s experience significantly.\nThis study is extensive and detailed, and its results, along with the newly developed mouse model, hold promise for future mechanistic studies aimed at identifying targetable factors for potential therapeutic applications.\nThat said, I have several specific comments and questions that may enhance the already high quality of the manuscript:\n•Figure 1A: Could you specify the type of survival displayed? It appears to be overall survival, but progression-free survival would also be informative.\nAs stated in the Methods, we reported overall survival, but we have now additionally confirmed that low levels of expression of SUCNR1 are associated with reduced progression-free survival in the same dataset (new Figure 1B). Following the recommendation of Reviewer #1, we performed subsequent Cox regression analysis, which uncovered SUCNR1 expression as an independent predictor of overall survival and progression-free survival after adjusting for age, gender and FAB classification.\n•Figure 1D:\no What was the rationale for treating mice with succinic acid? Thus far, the indication for succinic acid involvement derives only from SUCNR1 expression differences in the patient cohort.\nAs stated in the Introduction, “Succinate is a metabolic signal that links hypoxia to IL-1β and cell responses in myeloid cells through intracellular mechanisms, but also autocrine/paracrine processes through Sucnr1. Sucnr1 seems to have different functions depending on the context, and has been proposed as inflammatory in dendritic cells but anti-inflammatory in neural stem cells and both inflammatory or anti-inflammatory in macrophages. Cancer cells release succinate and locally polarize macrophages into tumor-associated macrophages through Sucnr1 activation. Despite these intriguing precedents, succinate has been scarcely explored in hematopoiesis. Human CD34+ progenitors express SUCNR1 and its activation has been suggested to induce proliferation in vitro. Here we investigated the in vivo role and the cellular and molecular mechanisms of succinate and Sucnr1 signaling in normal and malignant hematopoiesis.”\nWe first uncovered that low levels of expression of SUCNR1 were associated with reduced survival in AML patients. To understand how low expression of SUCNR1 is functionally linked to poor survival in AML patients in vivo, we used succinic acid injections in AML patient xenografts, as succinic acid takes the form of the anion succinate in living organisms. This clarification has been included in the manuscript (Page 5).\no How was succinic acid administered to the animals? Does the chosen route allow succinic acid to reach the cells directly, or might this be an indirect effect? Drawing conclusions about cellular metabolism from systemic metabolic changes can be challenging. Are elevated succinic acid levels detectable in the serum or other compartments in the mouse model?\nAs stated in the Methods, “Effect of acute succinic acid administration in human AML xenografts was studied through short-term daily i.p. injections of vehicle (saline) or succinic acid at 2 mg per kg neutralized with NaOH to pH 7.3, for 10 days.” Unfortunately, we did not have biological material available to answer this question for this experiment. Please, see similar comment below referred to Figure 1G, where we have been able to answer this question experimentally.\no Considering the heterogeneity of AML, it is unfortunate that only one patient sample was used. Could this patient fall within either the SUCNR1 high or low expression groups?\nThe patient was classified as M1, with adverse risk, and we found no detectable expression of SUCNR1 by ddPCR. The data on SUCNR1 expression has been included in new supplemental Figure 1C.\n•Figure 1G:\no The authors state that succinic acid injection worsens the disease. However, the administration route remains unclear (see above), and an explanation for this aggravation is lacking. Are elevated succinic acid levels observable in serum, other body fluids, organs, or cells?\nAs stated in the Methods, “Effect of chronic succinic acid administration in leukemia models was evaluated by long-term i.p. injections of succinic acid.”\nSuccinic acid is cleared from plasma and distributed to tissues quickly after administration through various routes4, so we measured succinic acid levels in BM extracellular fluid and found that they were increased in succinic acid-treated versus vehicle-treated pre-leukemic NRAS-G12D+ mice. This result links succinate with the aggravation of disease in a direct way. The data have been included in new supplemental Figure 2B.\no While significant, the increase in myeloid frequency is marginal. Could the authors comment on whether such a small difference holds biological significance? It appears the range in myeloid frequency in the vehicle group is wider than the mean difference between the vehicle and succinic acid groups.\nWe agree that the increase in the already expanded myeloid cell frequency is marginal if considered as an isolated event. However, this happens coupled to further hampering of the B cell lineage and expansion of the HSPC compartment shown in the same figure. Together, it is possible to conclude aggravation of various hallmarks of NRAS-G12D-driven disease and hence biological significance. We have reformulated these Results so that they are considered together, removing any potential emphasis on the effect on the myeloid bias alone.\n•Figure 1J: It would be insightful to see corresponding protein levels.\nWe agree but this is challenging, given the lack of commercially available mouse Sucnr1-specific antibody stated in the manuscript and the limited availability of Sucnr1-tomato mice. However, our data in Figure 1 show that SUCNR1 mRNA expression is biologically relevant for disease progression in AML patients, as shown by the association of low SUCNR1 mRNA expression with reduced overall survival and progression-free survival (Figure 1A-1B).\n•The concluding statement in the first Results section could benefit from added context. The link between low SUCNR1 expression and succinate as a contributor to leukemia progression is not fully clear.\nWe have reformulated this statement as recommended, it reads as follows: “To sum up, AML patients show heterogeneous expression of SUCNR1 and low SUCNR1 represents a prognostic marker for reduced overall and progression-free survival. Conversely, in AML mouse models and patient xenografts with low SUCNR1 expression, succinate accumulates in BM and contributes to leukemia progression”.\n•Figure 2A: Could you specify the exact mouse ages corresponding to “young adult” and “later middle-aged stage”?\nThis information was included in the Legends of the corresponding Figures in the previous version of the manuscript. Briefly, supplemental Figure 3 “young adult” – (A-E) 10-17 weeks of age and figure 2 “middle-aged” – (A, peripheral blood) 38-41 weeks old, (B-H, bone marrow) 53-54 weeks old.\n•Supplemental Figure 4B: The claimed increase in live cell count is minimal and not statistically significant. Displaying actual frequencies instead of percentage changes from WT may be more illustrative. Also, the event count in the representative plots appears low (below 10,000 total events), which is below the standard for reliable flow cytometry.\nWe have reformulated the Results section, which now reads: “analysis of the LSK cell compartment in the BM of Sucnr1-KO mice showed reduced numbers of early apoptotic BM LSK and a trend towards increased numbers of live cells”.\nBoth frequency and percentage of change are illustrative and accepted as valid ways to display results. In this case, we chose to express the results as percentage of change because it reduces variability in the analysis and helps place the focus in the actual changes caused by Sucnr1 deletion, which we agree are mild as shown by the data.\nRegarding event count, in these experiments around 1.5x10e6 total events from total bone marrow per sample were recorded in the flow cytometer, and from those, 1000 to 2000 gated-LSK were used to determine the proportion of live and apoptotic LSK cells per sample. This experimental setup is standard in studies involving subsets of HSPC by flow cytometry. Further, although lineage depletion or LSK sorting would increase the numbers of LSK cells analyzed, those protocols are known to reduce cell viability which could result in misleading observations.\n•Supplemental Figure 4D: How can the knockout of SUCNR1 impact succinate levels in BMEF? As I understand it, SUCNR1 is activated by succinate but does not regulate succinate availability or metabolism. I would anticipate succinate accumulation due to receptor absence.\nAs stated in the Results section of the previous version of our manuscript, succinate levels were reduced in the BM extracellular fluid (BMEF) of Sucnr1-KO versus WT mice (supplemental Figure 4E). Given that succinate is an intermediate metabolite of the TCA cycle, we studied its potentially increased cycling which would result in metabolic changes. Sucnr1 deletion resulted in increased baseline oxygen consumption rate in lineage-negative progenitors (supplemental Figure 4G). This effect was specific and reversible because the maximal oxygen consumption rate and the glycolytic flux remained unchanged (supplemental Figure 4G). Consistent with these data, both total and mitochondrial reactive oxygen species (ROS) levels were higher in LSK cells from Sucnr1-KO mice (supplemental Figure 4H). We have now established a new collaboration that has enabled us to provide direct evidence using metabolomics in HSC, which confirms increased intracellular succinate levels in Sucnr1-KO versus WT mice (please see new supplemental Figure 4F). The regulation of succinate localization by Sucnr1 should be subject of future work. Whereas it could simply be a matter of increased succinate availability in the absence of Sucnr1 binding, in otherwise steady-state/healthy conditions, active regulatory mechanisms could additionally take place. We have added these ideas to the Discussion.\n•Supplemental Figure 4E: Background controls for OCR and ECAR measurements, such as rotenone/antimycin A for OCR and 2-DG for ECAR, are absent. Without these, it is challenging to isolate mitochondrial respiration from other oxidative processes. Could such controls be included?\nWe agree, but it is not possible to repeat these experiments to include these controls due to mice availability. The main result of these experiments is that lineage-negative progenitors from Sucnr1-KO mice have more oxygen consumption in basal conditions and this is concomitant with increased ROS levels in LSK cells. Hence, we have toned down the specific participation of mitochondrial respiration in these processes.\n•Figure 3E: How do you explain the reduced IL-1β protein levels in BMEF when IL-1β gene expression is significantly increased in Sucnr1-KO versus WT animals? Consequently, the concluding statement of this section may require modification or expansion.\nGiven the increased mRNA expression of Il1b in HSPC from Sucnr1-KO versus WT mice, we analyzed the intracellular levels of IL-1β protein and confirmed them slightly but significantly increased in Sucnr1-KO compared to WT mice (Figure 4E). This effect seemed to be specific of the HSPC compartment, as we observed no differences in the intracellular levels of IL-1β in total live cells, lineage-positive cells, lineage-negative progenitors or LK progenitors (supplemental Figure 7C). This is indeed in contrast with the levels of IL-1β protein in BMEF, which we found to be reduced in Sucnr1-KO versus WT mice. Thus, in vivo deletion of Sucnr1 promotes inflammation specifically in HSPC, as explained in the concluding statement of this section. We have also added that there are complex changes taking place in the global cytokine status of the BM in Sucnr1-KO mice. Discrepancies may be caused by multiple factors, and due to the complexity of the question, we believe that it falls outside the scope of this work and should be subject of future work. However, despite this complexity, it is important to note that we were able to uncover S100a8 and S100a9 as consistently increased in their mRNA expression in HSPC and in their protein levels in BMEF, and demonstrated their primary functional/mechanistic role in Sucnr1-KO mice. We have included these ideas in the Discussion section.\n•Supplemental Figure 6E: The intent of this raw spectral ribbon plot is unclear. Without spectral unmixing, assigning signals to specific fluorochromes or fluorescent proteins is not possible.\nWe agree and this panel has been removed.\n•In the discussion, the authors note “overexpression of S100a8 and S100a9 in HSPC subsets […].” How does this align with observations of increased S100A8/S100A9 in AML blasts in bone marrow compared to peripheral blood and the identified pro-inflammatory triggers reported in Blood Advances 2022 6(21)?\nThank you for this comment. Our work aligns but deepen the understanding provided by previous reports showing that S100A8/A9+ AML cells display a distinct metabolic, differentiation, and chemoresistance profile, compared to S100A8/A9− AML cells, which are therapy-sensitive and found mainly in periphery. Using cells lines and in vitro cultures, the authors of this work identified stromal cell–derived IL-6 induced expression of S100A8/A9 in AML cells in a JAK/STAT3-dependent manner. We are now providing new measurements showing similar levels of IL-6 in the BMEF of WT and Sucnr1-KO mice. Thus, our data in vivo supports Sucnr1 as an alternative pathway controlling S100A8/A9 independently of IL-6. Boosting Sucnr1 and blocking S100A9 thus may open new potential therapeutic avenues in the treatment of myeloid malignancies. We have included the new results in Figure 4F and these ideas in the Discussion.\nReviewer #4 (Remarks to the Author): expert in succinate AML, hematopoiesis, mouse models\nThe manuscript by Cuminetti et al. examines the role of Succinate receptor (Suncr1) in hematopoiesis. The authors show that low Suncr1 expression correlates with poor survival in AML. Further, treatment of human and leukemia cells in vivo with succinic acid promotes leukemia progression. In the context of normal hematopoiesis sucnr1 knockout resulted in the expansion of several stem and progenitor populations determined by cell number in aged mice. The increased in HSPC cell numbers correlated with an increase in gene expression in signatures associated with HSCs and a decreased gene expression in signatures associated with decreased differentiation and inflammation. Expression of s100a8 and s100a9 were increased in sucnr1 knockdown mice. Treatment with s100a8/9 inhibitor results in reduced hematopoietic cells in the sucnr1 knockdown mice. Finally, the authors show that sucnr1 low expressing cells have high S100a8/9 in single-cell RNA-sequencing post cytarabine treatment (derived from PDX models). Overall, of the manuscript highlights several intriguing findings. However, several additional experiments/analyses should be performed to address the concerns below.\nMajor Concerns:\n1. In Figure 2, the conclusions about changes in HSPC and mature cells are made based on cell numbers only. Most of the populations have increased numbers including total WBCs. Are there any changes in population percentages which may help to tease about if any one population is expanded upon sucnr1 knockout?\nWe focused this figure on absolute numbers as these are the accurate way to understand real expansions/reductions in cell subsets. Relative frequencies are not measurements of real cell numbers and in fact an increased proportion of a cell subset can be associated to reduced absolute numbers. However, we do agree with the Referee on the value of understanding the changes in cell population percentages in the global pathophysiological context, as they provide valuable information of which cell subset/s may be most affected by the intervention compared to others.\nAs requested by the Reviewer, we are now providing the population percentages of LSK cells, which was increased in the BM of Sucnr1-KO versus WT mice (new supplemental Figure 4B). Analysis of HSPC subsets corresponding to HSC and MPP1-MPP6 revealed increased relative frequencies of HSC, MPP1, MPP4, MPP5 and MPP6 (supplemental Figure 4B). These data further support that the stimulated hematopoiesis in the BM of Sucnr1-KO mice originates in the HSPC compartment. We have included these ideas in the Results and Discussion sections.\n2. The authors use the steady-state populations and gene expression analysis to conclude changes in HSPC function. These conclusions should be supported by performing competitive bone marrow transplants.\nWe agree with the Reviewers, and we are now providing new data on the functionality of HSC in vivo analyzed by competitive repopulation assays of BM nucleated cells isolated from WT or SUCNR1-KO mice (Ly5.2) into lethally irradiated Ly5.1 congenic recipients. Peripheral blood analysis from recipient mice 16 weeks after transplantation revealed a mild reduction in engraftment in recipients transplanted with SUCNR1-KO cells compared to recipients transplanted with WT control cells (please see new Figure 3G).\nAlthough Sucnr1-KO BM cells seem to be less fit in competition with WT cells, primary transplants of Sucnr1fl/fl or Mx1-Cre Sucnr1fl/fl BM cells into C57BL/6J WT mice, induced after stable engraftment, greatly recapitulate the hematopoietic expansions of Sucnr1-KO mice (Figure 2I-2L).\nThus, to further substantiate the role of Sucnr1 in HSPC function in vivo, we performed competitive repopulation assays using 200 Sucnr1-tdTomato+ or Sucnr1-tdTomato− FACS-sorted HSPC (containing a mix of 40 HSC, 86 MPP3 and 74 MPP4) isolated from Ly5.2 Sucnr1-tdTomato mice into lethally irradiated Ly5.1 congenic recipients. Peripheral blood analysis from recipient mice 10 weeks after the transplant showed a reduction of circulating leukocytes and donor-derived monocytes in recipients of Sucnr1+ cells compared to recipients of Sucnr1− cells (Figure 3H). This experiment allowed us to conclude that Sucnr1 expression marks a subset of HSPC with restricted engraftment potential in vivo.\n3. Figure 5 would benefit from including wild-type mice. This would allow the assessment of tasquinimod on hematopoiesis in the normal setting and enable the researchers to determine if treatment restores cell numbers back to the wild type levels.\nAs suggested by Reviewers #1, #2 and #5, and #4, to substantiate a selective role of S100a8/S100a9 in the hematopoietic system via Sucnr1 deletion, we performed an independent experiment treating both Sucnr1-KO and WT mice with tasquinimod or vehicle in parallel (supplemental Figure 8A). Confirming our previous results, tasquinimod treatment in Sucnr1-KO mice reduced the expanded numbers of circulating leukocytes to the numbers observed in WT mice, including both overall myeloid cells as well as B cells and with no significant effect observed in WT mice (supplemental Figure 8B). Tasquinimod administration reduced the expanded numbers of monocytes in BM of Sucnr1-KO mice to the numbers found in WT mice, whilst no effect was observed in WT mice (supplemental Figure 8C). Conversely, treatment with tasquinimod reduced the numbers of BM B cells to similar levels in both Sucnr1-KO and WT mice (supplemental Figure 8C). The expansion of BM HSC in Sucnr1-KO mice was reduced by treatment with tasquinimod, but a minor reduction in HSC numbers was also observed in WT mice, bringing HSC values in both tasquinimod-treated groups to similar level (supplemental Figure 8D). In turn, the increased colony-forming potential in Sucnr1-KO mice was reduced to WT values, with no effect seen in samples obtained from WT mice (supplemental Figure 8E). Of note, this assay is optimal for the growth of primitive erythroid progenitor cells, granulocyte-macrophage progenitor cells, and multi-potential granulocyte, erythroid, macrophage, megakaryocyte progenitor cells, but not for the growth of lymphoid progenitor cells. Thus, these data suggest that tasquinimod effect originates in the HSC compartment for both lineages, and whereas the effect of tasquinimod on the myeloid lineage is fully dependent on Sucnr1 deletion, the effect on the B cell lineage is only partially dependent on the loss of Sucnr1 signaling.\n4. Do monomethyl succinate and succinic acid have the same degree of effect on the intracellular biology of succinate? For example, to they increase oxidative phosphorylation to the same level. Differences between the compounds could be a confounding factor in the interpretation of results in figure 4.\nThanks for this very good question, challenging to answer in the light of the various variables to consider. Monomethyl succinate is a dicarboxylic acid monoester that is succinic acid in which one of the carboxy groups has been converted to its methyl ester (succinic acid monomethyl ester). Addition of ester moieties to molecules serves as an efficient means to help internalizing compounds across the plasma membrane. In living organisms, succinic acid takes the form of the anion succinate, which would be the naturally occurring endogenous form, and requires transporters for its internalization. Thus, monomethyl succinate is cell-permeable and it allows bypassing the permeability limitation of succinate. However, monomethyl succinate is considered a prodrug and it requires hydrolysis by esterases inside the cell that convert it to succinate, making it available for intracellular metabolic and signaling functions. In parallel, cis-epoxy succinate is 10-20x more potent than succinic acid on Sucnr1, and it did not change SDH activity in previous work at concentrations up to 500 μM5. Importantly, in our work, we are using concentrations up to 10mM, and uncover a similar CFU-C output for both monomethyl succinate and cis-epoxy succinate in Sucnr1-KO HSPC (whilst their effect is opposite in WT cells in presence of Sucnr1). Thus, we can conclude that the same concentration of monomethyl succinate and cis-epoxy succinate induce the same functional output in the absence of Sucnr1, but we cannot say that the underlying mechanisms are exactly the same. However, we think that these results add clarity and rule out potential differences between compounds as a confounding factor for interpretation of the results in figure 4. Given the multi-layer intracellular functions of succinate, the question raised by the Reviewer is out of the scope of the present manuscript, but we do agree that it should be subject of future work. We have added this idea in the Discussion section.\n5. Using a genetic approach like overexpressing sucnr1 would help to solidify the conclusions from figure 4.\nMonomethyl succinate is inactive on Sucnr1 and enhances the colony-forming potential in total BM (TBM), in a dose-response manner. Conversely, succinic acid, which displays both Sucnr1-dependent and independent effects, results in unchanged colony-forming potential. Taken together, we reasoned that these results suggest that Sucnr1 activation represses the intracellular effect of succinate. We have reformulated these results for clarity. Further, we have followed the suggestion of Referee #1 and explored the effect of succinic acid and monomethyl succinate combination, which results in a partial increase in colony-forming potential in TBM versus succinic acid alone and a partial reduction versus monomethyl succinate alone, providing robust evidence that Sucnr1 activation represses the intracellular effect of succinate. Please, see the results in new Figure 3D. In parallel, the previous version of our work already showed a similar CFU-C output for both monomethyl succinate and cis-epoxy succinate in Sucnr1-KO HSPC, whilst their effect is opposite in WT cells (in presence of Sucnr1). We agree with the Reviewer that using a complementary strategy to the in vivo deletion of Sucnr1 would add valuable information, but we think the conclusions from this figure are solid, particularly after the addition of the new experiments mentioned above. We hope the Referee will agree with us.\n6. Further, overexpressing sucnr1 and measuring the consequence on leukemia progression would enhance the study.\nTo answer this question, we have used the human AML cell line Kasumi. Overexpression of SUCNR1 with a lentivector and treatment with cis-epoxy succinate increased apoptosis in comparison with the apoptotic rate of Kasumi cells transduced with an empty vector and treated with cis-epoxy succinate. Please, see the results in new supplemental Figure 11C-11D.\n7. Figure 6 suggests that expression of sucnr1 may influence the response to chemotherapy. It would strengthen the results by functionally testing this.\nUsing NOMO-1 and Kasumi human AML cell lines, we observed that 10mM treatment with cis-epoxy succinate improved chemotherapy induced apoptosis versus chemotherapy alone (supplemental Figure 11A-11B). The results are particularly remarkable for Kasumi (M2) versus NOMO-1 (M5).\nMinor:\n1. In Figure 1D what are the patient characteristics? Specifically it would be helpful to include FAB and mutation status as the authors show these are related to sucnr1 expression.\nThe patient was classified as M1, with adverse risk, and we found no detectable expression of SUCNR1 by ddPCR. The data on SUCR1 expression has been included in supplemental Figure 1C.\nReviewer #5 (Remarks to the Author): ECR, co-reviewed with Rev#2\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReferences"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed the reviewer’s previous comments in a general and satisfactory manner, significantly improving the clarity and rigor of the manuscript. The experimental design, data presentation, and interpretation have been appropriately revised, and the manuscript is now substantially strengthened.\nHowever, a few minor points remain that should be clarified or corrected to avoid potential confusion:\n- It is not clear whether the efficiency of Sucnr1 deletion was verified in the Mx1-Cre model, both in the bone marrow of the primary mice and in the transplanted recipients reconstituted with these BM cells. It is recommended to confirm the deletion by assessing Sucnr1 expression levels (at the mRNA level) to ensure that the observed phenotypes are indeed due to effective loss of Sucnr1 in hematopoietic cells.\n- In the merged manuscript (lines 243–246), the authors state that the majority of Sucnr1⁺ HSPCs were identified as MPP3, followed by MPP4, with an average of 0.191% of Sucnr1⁺ cells (Figure 3B and Supplemental Figure 6C). Could the authors clarify that this order is correct, and not MPP2? In the figure, it appears that MPP2 might have a higher fraction than MPP4, so confirmation would help avoid confusion.\n- The authors mention that in the first and second replating, both monomethyl succinate and cis-epoxy succinate stimulated the colony-forming potential of Sucnr1-KO HSCs, suggesting a loss of SUCNR1-mediated repression of this potential (Figure 3F). The Discussion ends with the sentence “Given the multi-layer intracellular functions of succinate, the question should be subject of future work” (lines 533–534), which could be refined for greater clarity and precision.\nThe authors’ results suggest that, in the absence of SUCNR1, cis-epoxy succinate may enhance colony-forming potential through receptor-independent mechanisms, possibly related to intracellular metabolic modulation. Have the authors assessed intracellular succinate levels to support this interpretation? Such data would help to substantiate the proposed mechanism and clarify whether the effects of the two compounds truly involve similar intracellular pathways.\n- Please ensure that all figures include legends indicating the meaning of the colors used. In several cases (e.g., Figures 1F, 1K, 2I, and others), the color legends are missing. We recommend revising all figures to maintain consistency and clarity across the manuscript.", "reviewer_2": "Thanks for convincingly addressing my concerns", "reviewer_3": "The authors have thoroughly addressed my initial concerns, questions, and comments, which has further improved the overall already high quality of the manuscript. While most points were addressed experimentally, some were also resolved through well-reasoned justification and discussion. Well done, and thank you for this very nice piece of science.", "reviewer_4": "The authors have gone to great lengths to address concerns from five reviewers and significantly strengthened their manuscript. I have no additional concerns."}, "author_response": "REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author): expert in Succinate and Sucnr1\nThe authors have addressed the reviewer’s previous comments in a general and satisfactory manner, significantly improving the clarity and rigor of the manuscript. The experimental design, data presentation, and interpretation have been appropriately revised, and the manuscript is now substantially strengthened.\nHowever, a few minor points remain that should be clarified or corrected to avoid potential confusion:\n- It is not clear whether the efficiency of Sucnr1 deletion was verified in the Mx1-Cre model, both in the bone marrow of the primary mice and in the transplanted recipients reconstituted with these BM cells. It is recommended to confirm the deletion by assessing Sucnr1 expression levels (at the mRNA level) to ensure that the observed phenotypes are indeed due to effective loss of Sucnr1 in hematopoietic cells.\nThe Mx1-Cre mouse model is broadly used in the hematopoietic field to induce recombination in a variety of genes in the hematopoietic system during adulthood, allowing fine-tuned controlled levels of Cre selectively in blood cells when used under transplantation. One of the main drawbacks of Mx1-Cre is that the model is inducible with an inflammatory signal that may impact HSCs. However, bone marrow isolated from mice treated three times with similar doses of pIpC (followed by 10 days recovery) was serially transplanted into irradiated recipients, showing no differences in the repopulation activity (Essers et al, 2009). Thus, transient activation of IFNα signaling does not affect the number of functional HSC, as opposed to chronic activation. The mice in this experiment were monitored for a long period of time (47 weeks after pIpC induction) and the controls used were Sucnr1fl/fl with no Cre treated with pIpC, to reduce any potential confounding effect derived from pIpC-induced inflammation.\nIn addition, please note that all our mice are kept in SOPF status, to prevent spontaneous Cre activation before pIpC induction.\nTo answer to the Reviewer question, we have now assessed Sucnr1 expression in frozen total BM nucleated cell samples from primary mice and from the transplanted mouse recipients shown in Figure 2I. As expected, BM nucleated cells from Sucnr1fl/fl and Mx1-Cre Sucnr1fl/fl primary mutant mice displayed similar expression of Sucnr1 prior to pIpC induction. In turn, in vivo deletion of Sucnr1 from hematopoietic cells was confirmed by virtual absence of Sucnr1 expression in WT recipients of Mx1-Cre Sucnr1fl/fl experimental BM cells versus recipients of control Sucnr1fl/fl BM cells, injected with pIpC 4 weeks after the transplant and analyzed 47 weeks after pIpC induction. Of note, Sucnr1 expression in BM cells from WT recipients of control Sucnr1fl/fl BM cells injected with pIpC 4 weeks after the transplant and analyzed 47 weeks after pIpC induction showed no significant differences compared to Sucnr1fl/fl or Mx1-Cre Sucnr1fl/fl primary mutant mice. Please, see the results in new Supplementary Figure 4L.\n- In the merged manuscript (lines 243–246), the authors state that the majority of Sucnr1⁺ HSPCs were identified as MPP3, followed by MPP4, with an average of 0.191% of Sucnr1⁺ cells (Figure 3B and Supplemental Figure 6C). Could the authors clarify that this order is correct, and not MPP2? In the figure, it appears that MPP2 might have a higher fraction than MPP4, so confirmation would help avoid confusion.\nOur statement reads: “Our analysis revealed that all HSPC subsets show a varying fraction of Sucnr1+ cells and the majority of Sucnr1+ HSPC were identified as MPP3, with an average of 0.191% of Sucnr1+ cells, followed by MPP4 (Figure 3B and supplemental Figure 6C).” We confirm that this statement is correct.\nThe bar chart of Figure 3B (upper panel) shows the fraction of Sucnr1+ cells within each HSPC subset, as we thought it was important to provide these quantifications per cell subset. From the Source Data file:\n% of Sucnr1+ cells\nLSK HSC MPP1 MPP2 MPP3 MPP4 MPP5 MPP6\n0.120 0.000 0.100 0.090 0.200 0.060 0.090 0.070\n0.080 0.000 0.000 0.000 0.110 0.030 0.000 0.150\n0.060 0.000 0.000 0.000 0.130 0.000 0.000 0.000\n0.150 0.200 0.130 0.000 0.210 0.060 0.000 0.130\n0.120 0.000 0.000 0.080 0.170 0.000 0.000 0.000\n0.039 0.000 0.000 0.000 0.195 0.071 0.000 0.000\n0.190 0.000 0.000 0.000 0.429 0.118 0.000 0.000\n0.033 0.000 0.000 0.000 0.198 0.000 0.000 0.000\n0.059 0.000 0.000 0.000 0.272 0.069 0.000 0.000\n0.027 0.568 0.000 0.568 0.000 0.000 0.000 0.127\nAs you can see, all HSPC subsets showed a varying fraction of Sucnr1+ cells and the average fraction of Sucnr1+ cells in MPP3 was 0.191%. Sucnr1 was expressed consistently in the MPP3 subset in 9 out of 10 animals.\nFigure 3B (upper panel) shows that only 3 out 10 animals displayed detectable % of Sucnr1+ cells within MPP2, versus 6 out of 10 within MPP4. The fraction of Sucnr1+ cells within MPP4 showed also less standard deviation across animal samples in the group compared to MPP2, although it was high in both cases. Because of these reasons, we did not provide the average fraction in the text, but the raw values are shown in the figure and provided in the Source Data file.\nThe pie chart of Figure 3B (lower panel) represents the distribution of Sucnr1+ cells in the total HSPC compartment. Here, you can see how the majority of Sucnr1+ HSPC were identified as MPP3, followed by MPP4. From the Source Data file:\nPie chart % distribution of Sucnr1+ events\nHSC MPP1 MPP2 MPP3 MPP4 MPP5 MPP6\n6.333 1.051 5.214 71.516 10.783 0.577 4.526\nTo calculate the % shown above and represented in the pie chart, we considered each fraction of Sucnr1+ HSPC subset in absolute numbers, summed all of them, and calculated the distribution of each subset in the total Sucnr1+ HSPC population, in %. Thus, the numbers in the pie chart depend on the % of Sucnr1+ cells and the absolute numbers of each HSPC subset. MPP2 is a rare population in the bone marrow, found in lower numbers than MPP4 (0.00955% versus 0.0636% of total bone marrow cells, respectively; in this experiment, 6.7 times lower). Hence, when considering all HSPC subsets together to understand the distribution of Sucnr1+ cells, the majority of Sucnr1+ HSPC are identified as MPP3, followed by MPP4, HSC, MPP2 and MPP6, in that order.\n- The authors mention that in the first and second replating, both monomethyl succinate and cis-epoxy succinate stimulated the colony-forming potential of Sucnr1-KO HSCs, suggesting a loss of SUCNR1-mediated repression of this potential (Figure 3F). The Discussion ends with the sentence “Given the multi-layer intracellular functions of succinate, the question should be subject of future work” (lines 533–534), which could be refined for greater clarity and precision.\nThe authors’ results suggest that, in the absence of SUCNR1, cis-epoxy succinate may enhance colony-forming potential through receptor-independent mechanisms, possibly related to intracellular metabolic modulation. Have the authors assessed intracellular succinate levels to support this interpretation? Such data would help to substantiate the proposed mechanism and clarify whether the effects of the two compounds truly involve similar intracellular pathways.\nThese data were present in the previous version of the manuscript, and Reviewer 4 formulated a similar comment in the previous round of revision (point 4: “Do monomethyl succinate and succinic acid have the same degree of effect on the intracellular biology of succinate? For example, to they increase oxidative phosphorylation to the same level. Differences between the compounds could be a confounding factor in the interpretation of results in figure 4”).\nMonomethyl succinate is a dicarboxylic acid monoester that is succinic acid in which one of the carboxy groups has been converted to its methyl ester (succinic acid monomethyl ester). Addition of ester moieties to molecules serves as an efficient means to help internalizing compounds across the plasma membrane. In living organisms, succinic acid takes the form of the anion succinate, which would be the naturally occurring endogenous form, and requires transporters for its internalization. Thus, monomethyl succinate is cell-permeable and it allows bypassing the permeability limitation of succinate. However, monomethyl succinate is considered a prodrug and it requires hydrolysis by esterases inside the cell that convert it to succinate, making it available for intracellular metabolic and signaling functions. In parallel, cis-epoxy succinate is 10-20x more potent than succinic acid on Sucnr1, and it did not change SDH activity in previous work at concentrations up to 500 μM (Geubelle et al., 2017). Importantly, in our work, we are using concentrations up to 10mM, and uncover a similar CFU-C output for both monomethyl succinate and cis-epoxy succinate in Sucnr1-KO HSPC whilst their effect is opposite in WT cells in presence of Sucnr1. Thus, we can conclude that the same high concentration of monomethyl succinate and cis-epoxy succinate induce the same functional output in the absence of Sucnr1, but we cannot say that the underlying mechanisms are exactly the same. We think that these results rule out potential differences between compounds as a confounding factor for interpretation of the functional role of Sucnr1, and hope the Referee will agree with us.\nFurther, please note the experimental conditions of the technique used in these assays, where succinate analogues are added only at the time of plating. From the Methods: “Serial colony-forming-unit assays from 300 FACS-sorted HSC from magnetically enriched bone marrow Lin- progenitors were performed as described previously, colonies were scored and 10 000 cells replated after 7, 12 and 17 days, 10mM of monomethyl succinate or cis-epoxy succinate were added at each plating”. Measuring succinate in colonies may not be reflective of the initial effect of succinate analogues on HSC, but rather on the differential cell subsets present at the end of the culture procedure. The increase in intracellular succinate after a single addition of succinate analogues in vitro could also be transient and/or have differential dynamics for different analogues because of the structural differences mentioned above.\nTogether, we think that this would be a time-consuming experiment that would require a considerable use of animals and other resources, whilst producing an output that would have significant caveats and will not effectively convince whether the effects of the two compounds truly involve similar intracellular pathways.\nBesides, although certainly interesting, dissecting the potential intracellular differences between different succinate analogues is not the purpose of the present work and would not influence our main message, which robustly and convincingly shows that activation of Sucnr1 counterbalances the stimulatory effect of succinate in HSPC. We hope that the Referee will agree with us.\nHowever, as suggested by the Reviewer, we have reformulated the Discussion, which now reads: “In turn, similar concentrations of monomethyl succinate and cis-epoxy succinate induced the same increased functional output in HSC in the absence of Sucnr1, but it is unknown whether the underlying mechanisms are exactly the same for both compounds. Thus, in the absence of Sucnr1, cis-epoxy succinate enhances colony-forming potential through receptor-independent mechanisms, possibly with contribution to intracellular metabolic modulation. However, given the structural differences among different succinate analogues and the multi-layer intracellular functions of succinate, this question should be subject of future work.”\n- Please ensure that all figures include legends indicating the meaning of the colors used. In several cases (e.g., Figures 1F, 1K, 2I, and others), the color legends are missing. We recommend revising all figures to maintain consistency and clarity across the manuscript.\nWe have gone through all the figures, and all of them showed the colors used in all panels in the previous version of our manuscript, either in the corresponding panel or in its vicinity when the panel is part of a subgroup of panels that belong to a similar experimental design. This way, there is just one legend for Figure 1F, 1G and 1H (under 1G); Figure 1I, 1J and 1K (under 1I); 2A-2H (right of 1A); 2I-2L (colored mice in 2I), etc. This saves space in the figure while maintaining clarity.\nReviewer #2 (Remarks to the Author): expert in RNA-seq and scRNA-seq analysis, AML and hematopoiesis\nThanks for convincingly addressing my concerns.\nWe would like to thank the Reviewer again for the important points raised that have helped us strengthen our work.\nReviewer #3 (Remarks to the Author): expert in S100A8/S100A9, AML\nThe authors have thoroughly addressed my initial concerns, questions, and comments, which has further improved the overall already high quality of the manuscript. While most points were addressed experimentally, some were also resolved through well-reasoned justification and discussion. Well done, and thank you for this very nice piece of science.\nThank you for your kind feed-back. We would like to take the opportunity to thank you once more for the important input that we have received from you.\nReviewer #4 (Remarks to the Author): expert in succinate AML, hematopoiesis, mouse models\nThe authors have gone to great lengths to address concerns from five reviewers and significantly strengthened their manuscript. I have no additional concerns.\nWe would like to thank the Reviewer again for the constructive and important input that has helped us strengthen our manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_1": "Thank you for addressing the remaining minor points. I have no further comments."}, "author_response": "We would like to thank the Reviewer again for the points raised that helped us strengthen our work."}]

Decision letter