Hematopoietic stem cells (HSC) maintain blood homeostasis during steady state and stress through a balanced regulation of self-renewal and differentiation. While transient activation of interferon stimulated gene (ISG) is critical for HSC function during development and stress, chronic inflammation impairs HSC integrity. However, the intrinsic mechanisms that suppress aberrant inflammatory signaling in HSCs remain elusive and further studies are necessary for restoring HSC function in settings such as aging and cancer. We have identified a m6A-SON axis that governs HSC fate and controls inflammation (Cheng*, Luo* and Izzo* et al., 2019, Cell reports; Luo* and Lopez* et al., 2023, Cell Stem Cell). m6A is the most abundant RNA modification, and its loss leads to increased double-stranded RNA (dsRNA), ISG activation and HSC dysfunction. SON is a key m6A target, whose loss accounts for the observed defects. SON is a core component of the nuclear speckles that controls gene regulation through RNA processing. Importantly, individuals with heterozygous SON loss of function mutations (ZTTK syndrome), exhibit developmental delay and hematological symptoms including bone marrow failure. However, the molecular function of SON in HSCs and how it controls inflammation remains unclear. To investigate SON function in vivo, we generated the Mx1-cre Son conditional knockout mice. SON loss resulted in a severe reduction in bone marrow cellularity (WT 168.2 million vs Son cKO 73.71 million, p<0.0001) and reduced platelet count (WT 408 k/ul vs Son cKO 74.5 k/ul, p<0.0001). SON loss also resulted in depletion of myeloid progenitors (WT 2.51million vs Son cKO 1.01 million, p=0.0405) and an expansion of phenotypic HSCs (WT 11.21k vs Son cKO 38.4k, p=0.0312), indicating an early differentiation block. Functionally, SON cKO HSCs failed to engraft recipient mice (WT 26.4% vs Son cKO 3.8%, p<0.000001), suggesting that SON is essential for HSC maintenance. SON loss led to a 2-fold increase in dsRNA accumulation (p=0.0094), suggesting innate immune activation in SON cKO HSCs. Bulk RNA sequencing revealed 94 upregulated genes and 68 downregulated genes between SON cKO and WT HSCs (padj<0.05). Top enriched pathway among the upregulated genes upon SON loss is the interferon signaling (padj=8.46E-06) and RIG-I ligand pathways (padj=1.19E-11), indicating robust ISG induction. Major contributors to dsRNA accumulation include the derepression of endogenous retroelements (EREs) and aberrant RNA splicing (i.e retained introns). SON cKO hematopoietic stem and progenitor cells (HSPCs) exhibited increased ERE expression, including endogenous retroviruses (ERVs) and LINE elements (upregulated vs downregulated: 6 vs 0 in HSC; 72 vs 18 in MPP1; 108 vs 45 in MPP2; 70 vs 50 in MPP4, padj<0.05). We also observed widespread splicing abnormalities. In total, 6,330 splicing alterations were identified in SON-deficient HSCs: 50% intron retention, 25% exon skipping, and 25% alternative splice site usage (padj<0.05). Nearly 30% of upregulated genes, including numerous ISGs, exhibited differential splicing. To assess SON direct RNA binding targets, we performed SON CLIP-seq and identified 4337 genes as SON direct binding transcripts (padj<0.05). CLIP peak analysis showed that SON binds to preferentially to proximal introns and 3′ splice sites, with ~10% of ISG upregulation attributable to direct SON binding. These data suggest that ISG upregulation following SON loss may result both indirectly from dsRNA-mediated innate immune activation and directly from aberrant splicing of ISG transcripts. Together, our finding highlights SON controls innate immune response in HSCs through splicing control of ISGs and repression of EREs. Notably, SON loss resulted in elevated levels of serum inflammatory cytokines, implicating HSC-intrinsic inflammation as a contributor to systemic inflammation.

PAS domains are ubiquitous sensory modules that transduce environmental signals into cellular responses through tandem PAS folds and PAS-associated C-terminal (PAC) motifs. While this conserved architecture underpins their regulatory roles, here we uncover a structural divergence in the metazoan PAS domain-regulated kinase (PASK). By integrating evolutionary-scale domain mapping with deep learning-based structural models, we identified two PAS domains in PASK, namely PAS-B and PAS-C, in addition to the previously known PAS-A domain. Unlike canonical PAS domains, the PAS fold and PAC motif in the PAS-C domain are spatially segregated by an unstructured linker, yet a functional PAS module is assembled through intramolecular interactions. We demonstrate that this assembly is nutrient responsive and serves to remodel the quaternary structure of PASK that positions the PAS-A domain near the kinase activation loop. This nutrient-sensitive spatial arrangement stabilizes the activation loop, enabling catalytic activation of PASK. These findings revealed an alternative mode of regulatory control in PAS sensory proteins, where the structural assembly of PAS domains links environmental sensing to enzymatic activity. By demonstrating that PAS domains integrate signals through dynamic structural rearrangements, this study broadens the understanding of their functional and regulatory roles and highlights potential opportunities for targeting PAS domain-mediated pathways in therapeutic applications.

The ligand-regulated PAS domains are one of the most diverse signal-integrating domains found in proteins from prokaryotes to humans. By biochemically connecting cellular processes with their environment, PAS domains facilitate an appropriate cellular response. PAS domain-containing Kinase (PASK) is an evolutionarily conserved protein kinase that plays important signaling roles in mammalian stem cells to establish stem cell fate. We have shown that the nuclear translocation of PASK is stimulated by differentiation signaling cues in muscle stem cells. However, the mechanistic basis of the regulation of PASK nucleo-cytoplasmic translocation remains unknown. Here, we show that the PAS-A domain of PASK contains a putative monopartite nuclear localization sequence (NLS) motif. This NLS is inhibited in cells via intramolecular association with a short linear motif, termed the PAS Interacting Motif (PIM), found upstream of the kinase domain. The interaction between the PAS-A domain and PIM is evolutionarily conserved and serves to retain PASK in the cytosol in the absence of signaling cues. Consistent with that, we show that metabolic inputs induce PASK nuclear import, likely by disrupting the PAS-A: PIM association. We suggest that a route for such linkage may occur through the PAS-A ligand binding cavity. We show that PIM recruitment and artificial ligand binding to the PAS-A domain occur at neighboring locations that could facilitate metabolic control of the PAS-PIM interaction. Thus, the PAS-A domain of PASK integrates metabolic signaling cues for nuclear translocation and could be targeted to control the balance between self-renewal and differentiation in stem cells.

The Per-Arnt-Sim (PAS) domains are characterized by diverse sequences and feature tandemly arranged PAS and PAS-associated C-terminal (PAC) motifs that fold seamlessly to generate the metabolite-sensing PAS domain. Here, using evolutionary scale sequence, domain mapping, and deep learning-based protein structure analysis, we deconstructed the sequence-structure relationship to unearth a novel example of signal-regulated assembly of PAS and PAC subdomains in metazoan PAS domain-regulated kinase (PASK). By comparing protein sequence, domain architecture, and computational protein models between fish, bird, and mammalian PASK orthologs, we propose the existence of previously unrecognized third PAS domain of PASK (PAS-C) formed through long-range intramolecular interactions between the N-terminal PAS fold and the C-terminal PAC fold. We experimentally validated this novel structural design using residue-level cross-linking assays and showed that the PAS-C domain assembly is nutrient-responsive. Furthermore, by combining structural phylogeny approaches with residue-level cross-linking, we revealed that the PAS-C domain assembly links nutrient sensing with quaternary structure reorganization in PASK, stabilizing the kinase catalytic core of PASK. Thus, PAS-C domain assembly likely integrates environmental signals, thereby relaying sensory information for catalytic control of the PASK kinase domain. In conclusion, we theorize that during their horizontal transfer from bacteria to multicellular organisms, PAS domains gained the capacity to integrate environmental signals through dynamic modulation of PAS and PAC motif interaction, adding a new regulatory layer suited for multicellular systems. We propose that metazoan PAS domains are likely to be more dynamic in integrating sensory information than previously considered, and their structural assembly could be targeted by regulatory signals and exploited to develop therapeutic strategies.

Quiescent stem cells are activated in response to a mechanical or chemical injury to their tissue niche. Activated cells rapidly generate a heterogeneous progenitor population that regenerates the damaged tissues. While the transcriptional cadence that generates heterogeneity is known, the metabolic pathways influencing the transcriptional machinery to establish a heterogeneous progenitor population remains unclear. Here, we describe a novel pathway downstream of mitochondrial glutamine metabolism that confers stem cell heterogeneity and establishes differentiation competence by countering post-mitotic self-renewal machinery. We discovered that mitochondrial glutamine metabolism induces CBP/EP300-dependent acetylation of stem cell-specific kinase, PAS domain-containing kinase (PASK), resulting in its release from cytoplasmic granules and subsequent nuclear migration. In the nucleus, PASK catalytically outcompetes mitotic WDR5-anaphase-promoting complex/cyclosome (APC/C) interaction resulting in the loss of post-mitotic Pax7 expression and exit from self-renewal. In concordance with these findings, genetic or pharmacological inhibition of PASK or glutamine metabolism upregulated Pax7 expression, reduced stem cell heterogeneity, and blocked myogenesis in vitro and muscle regeneration in mice. These results explain a mechanism whereby stem cells co-opt the proliferative functions of glutamine metabolism to generate transcriptional heterogeneity and establish differentiation competence by countering the mitotic self-renewal network via nuclear PASK.

The 1,287 genes targeted by the shRNA of the leukemia library.

Next generation sequencing for 52 myeloid malignancies-related genes.

Patient sample information.

Blood stem cells intricately regulate their fate, balancing self-renewal and differentiation through symmetric and asymmetric divisions. When exposed to various stresses they can rapidly undergo symmetric commitment divisions to generate differentiated progenitors for immune regeneration or driving inflammation. In previous studies, we and others found that m 6A RNA methylation controls symmetric commitment and inflammation in hematopoietic stem cells (HSCs) (Cheng and Luo et al., Cell Reports 2019). However, the precise molecular mechanisms underlying the role of m 6A and its influence on the inflammatory program in stem cells remain elusive. To uncover the dynamic RNA methylation during HSC commitment, we employed a recently developed tool called DART-seq, that fuses the YTH domain of the m 6A reader protein YTHDF2 with the RNA editing protein APOBEC1. We identified 300 m 6A sites and approximately 250-300 gene targets within HSCs and MPPs (padj beta binomial < 0.05). We found m 6A sites in HSCs were enriched for: innate immune response pathways including NFκB and Toll-like receptor cascades, cellular signaling processes encompassing RhoGTPase and ERK/MAPK signaling targets, and metabolic pathways associated with amino acid transport and fatty acid oxidation. In contrast, the m 6A targets enriched in MPPs were associated with lineage differentiation and myeloid programs such as MLL, GATA1, PU.1, and CEBPD. By integrating our findings with two additional m 6A mapping datasets, Son was a shared m 6A target with increased m 6A modification during the transition from HSCs to MPPs. SON, an RNA binding protein residing in nuclear speckles, has been implicated in a myriad of cellular processes including splicing control and transcriptional repression. Notably, de novo heterozygous loss-of-function variants in SON (ZTTK Syndrome) results in hematological symptoms, neurological impairments, and developmental delays. The genetic deletion of METTL3 ( Mettl3 conditional knockout mice; Mettl3 cKO) or METTL3 inhibitor STM2457 treatment, resulted in a twofold increase in Son transcripts in both HSCs and MPPs, but reduced SON protein abundance by 50%. Notably, SON exhibited asymmetric segregation during HSC division, with its abundance displaying a strong correlation with the commitment marker NUMB. Collectively, these findings suggest that m 6A modification regulates the abundance of SON protein and implicates SON in HSC commitment fate. Reintroduction of SON in m 6A-deficient HSCs effectively rescued commitment defects (cKO EV 9.74% vs. cKO+SON 21.43%). Furthermore, SON overexpression partially restored in vivo engraftment defects in Mettl3 cKO LSKs (cKO EV 1.5% vs. cKO+SON 4.5%). Intriguingly, the functional rescue was accomplished using a fragment containing the RNA-binding domain of SON (cKO EV 1.2% vs. cKO+SON-RB 12.3%), highlighting the significance of SON's RNA-binding ability. Additionally, deletion of SON in RosaCas9 WT LSKs using sgRNAs resulted in a substantial reduction in engraftment (WT EV 35.4% vs. WT+sgSON 11%), whereas SON overexpression in WT LSKs enhanced engraftment (WT EV 6% vs. WT+SON 39%). These results demonstrate that SON is a positive regulator of stem cell engraftment and function. Moreover, SON overexpression effectively rescued specific clusters identified through scRNA-seq analysis in Mettl3 KO (KOsp1-1 and CCL5+) by 50%. In bulk RNA-seq analysis, we identified 257 upregulated genes and 227 downregulated genes between the Mettl3 cKO EV and Mettl3 cKO SON groups (padj<0.05). Remarkably, SON overexpression significantly downregulated inflammatory pathways that were aberrantly upregulated upon m 6A loss. Furthermore, SON overexpression indirectly rescued dsRNA formation in Mettl3 cKO LSKs, aligning with the role of m 6A in suppressing dsRNA formation in HSPCs. SON overexpression reduced Ccl5 through binding to its transcript and decreasing nascent transcription. Strikingly, depletion of CCL5 significantly rescued Mettl3 cKO LSK engraftment (cKO 1.6% vs. cKO+shCCL5 5.3%), indicating that CCL5 is sufficient to mediate the effects downstream of SON. Moreover, co-culturing WT HSCs with CCL5 resulted in a symmetric commitment defect similar to that observed with m 6A loss. In summary, our study delineates the critical m 6A-SON-CCL5 axis governing HSC symmetric commitment fate and inflammation control.

Ranked list of 1,287 genes from shRNA library screen in primary AML cells. The 1,287 genes assessed with an shRNA library screen were sorted by the second highest percentile fold change present in 2 shRNA and across 2 samples, with the 34 genes showing a fold change in the top 2 percent in more than 2 samples listed first.

Standard hematologic parameters in SIRT5-/- mice and SIRT5+/+ littermates.

All supplemental figures and legends.

Tissue homeostasis is maintained after stress by engaging and activating the hematopoietic stem and progenitor compartments in the blood. Hematopoietic stem cells (HSCs) are essential for long-term repopulation after secondary transplantation. Here, using a conditional knockout mouse model, we revealed that the RNA-binding protein SYNCRIP is required for maintenance of blood homeostasis especially after regenerative stress due to defects in HSCs and progenitors. Mechanistically, we find that SYNCRIP loss results in a failure to maintain proteome homeostasis that is essential for HSC maintenance. SYNCRIP depletion results in increased protein synthesis, a dysregulated epichaperome, an accumulation of misfolded proteins and induces endoplasmic reticulum stress. Additionally, we find that SYNCRIP is required for translation of CDC42 RHO-GTPase, and loss of SYNCRIP results in defects in polarity, asymmetric segregation, and dilution of unfolded proteins. Forced expression of CDC42 recovers polarity and in vitro replating activities of HSCs. Taken together, we uncovered a post-transcriptional regulatory program that safeguards HSC self-renewal capacity and blood homeostasis.

Stem cells regulate their self-renewal and differentiation fate outcomes through both symmetric and asymmetric divisions. m6A RNA methylation controls symmetric commitment and inflammation of hematopoietic stem cells (HSCs) through unknown mechanisms. Here, we demonstrate that the nuclear speckle protein SON is an essential m6A target required for murine HSC self-renewal, symmetric commitment, and inflammation control. Global profiling of m6A identified that m6A mRNA methylation of Son increases during HSC commitment. Upon m6A depletion, Son mRNA increases, but its protein is depleted. Reintroduction of SON rescues defects in HSC symmetric commitment divisions and engraftment. Conversely, Son deletion results in a loss of HSC fitness, while overexpression of SON improves mouse and human HSC engraftment potential by increasing quiescence. Mechanistically, we found that SON rescues MYC and suppresses the METTL3-HSC inflammatory gene expression program, including CCL5, through transcriptional regulation. Thus, our findings define a m6A-SON-CCL5 axis that controls inflammation and HSC fate.

The cell cycle offers a unique opportunity for stem cells to sample metabolic and signaling cues to establish cell fate. Molecular pathways that integrate and convey these signals to cell cycle machinery to license cell fate transitions and drive terminal differentiation remain unknown. Here, we describe a signaling role of mitochondrial glutamine metabolism in driving exit from cell cycle-linked self-renewal to generate differentiation competent progenitors. In proliferating stem cells, mitochondrial glutamine metabolism opposes the WDR5-linked self-renewal network via acetylation and nuclear translocation of its upstream regulator, PASK. Nuclear PASK disrupts the mitotic WDR5-anaphase-promoting complex (APC/C) interaction to drive exit from self-renewal. Consistent with these roles, loss of PASK or inhibition of glutamine metabolism preserves stemness in vitro and in vivo during muscle regeneration. Our results suggest a mechanism whereby the proliferative functions of glutamine metabolism are co- opted by stem cells to establish cell fate.

Activation of stem cell proliferation is a critical event in tissue regeneration. The metabolic switch in adult stem cells from the oxidative to the glycolytic mode of carbon utilization is essential for rapid proliferative bursts, but its impact on self-renewal is unclear. During the glycolytic mode of glucose utilization, glutamine-derived carbons drive the mitochondrial TCA cycle. While glutamine is required for stem cell proliferation burst, its role in maintaining stem cell self-renewal property remains unclear. Here, we show that withdrawal or chemical inhibition of mitochondrial glutamine metabolism blunted adult muscle stem cell proliferation, but also reactivated the transcription of self-renewal-associated transcripts, such as Pax7, to reduce stem cell heterogeneity and build the self-renewing stem cell population. Thus, surprisingly, glutamine withdrawal preserved and accentuated the self-renewing stem cell population. This effect of glutamine is mediated via reductive carboxylation of alpha-ketoglutarate. Mechanistically, we extensively show that glutamine inhibited cell-cycle linked self-renewing network during the G2-M phase of cell-cycle to drive the exit from self-renewal during the terminal mitosis phase before differentiation. Thus, we propose that glutamine metabolism plays an unexpected role in building the progenitor population that is uniquely primed for differentiation during tissue regeneration.

Abstract We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML. Significance: Reducing SIRT5 activity is detrimental to the survival of AML cells regardless of genotype, yet well tolerated by healthy hematopoietic cells. In mouse models, disrupting SIRT5 inhibits AML progression. SIRT5 controls several metabolic pathways that are required for leukemia cell survival. These results identify SIRT5 as a therapeutic target in AML. See related commentary by Li and Melnick, p. 198.

Abstract Standard of care for AML includes chemotherapy and stem cell transplant, with 5-year survival rates <30%. We sought to identify genes critical to AML cells, irrespective of mutational status, and performed an shRNA screen targeting 1,287 genes on 12 AML patient samples. This screen identified Sirtuin 5 (SIRT5) as a top candidate. SIRT5 is the only known enzyme with desuccinylase, demalonylase, and/or deglutarylase activity and we are the first to report the dependence of AML cells on SIRT5. Next, we stably transduced a panel of AML cell lines with doxycycline (dox)-inducible shSIRT5 (dox-shSIRT5). SIRT5 knockdown (KD) strongly inhibited cell growth, colony formation and increased apoptosis in 15/22 lines (SIRT5-dependent), while 7/22 lines were SIRT5-independent. SIRT5 dependence did not correlate with AML-related mutations nor basal SIRT5 expression. SIRT5 KD in primary AML samples (N=25) revealed a therapeutic window (~50% reduction), with no effect in CB samples (N=5). We examined the requirement of SIRT5 in vivo using three mouse models of leukemia. In a xenograft model with AML cell lines, SIRT5 KD indefinitely prolonged survival of mice injected with SIRT5-dependent cells with no sign of leukemia. Bone marrow transplant with transduced (MLL-AF9 or BCR-ABL1) SIRT5 null cells showed reduced leukemia cell burden and splenomegaly, and significantly prolonged survival. FLT3-ITD-driven disease was also blunted by the absence of SIRT5 in a genetic knockout mouse model. Mechanically, SIRT5 KD profoundly reduced oxidative phosphorylation (OXPHOS) and glycolysis. Additionally, SIRT5 KD increased mitochondrial superoxide selectively in annexin V-negative, SIRT5-dependent cells. Concomitant, ectopic expression of SOD2 abrogated the increase in superoxide, rescued cells from apoptosis, and rescued the colony formation deficit. Untargeted metabolomics revealed RNA charging and alanine and serine metabolism as top metabolic pathways regulated by SIRT5, with glutaminase (GLS) and α-ketoglutarate identified as potential upstream regulators. Metabolic tracing experiments with [13C5,15N2]-glutamine confirmed disrupted glutamine metabolism in SIRT5-dependent cells. Together, these results indicate that SIRT5 is required to regulate glutamine flux to sustain redox homeostasis and/or anabolism. NRD167, a novel SIRT5 inhibitor, was used to target SIRT5 in AML. NRD167 reduced cell proliferation, induced apoptosis, and reduced OXPHOS in SIRT5-dependent but not SIRT5-independent cells. NRD167 inhibited colony formation from AML patient samples, but not in CB samples. An AML patient-derived xenograft model trended toward prolonged survival following ex vivo treatment with NRD167. Our data suggest that the majority of AML samples are dependent on SIRT5 and that inhibition preferentially targets AML cells, implicating SIRT5 as a therapy target in AML. Citation Format: Dongqing Yan, Anca Franzini, Anthony D. Pomicter, Brayden J. Halverson, Orlando Antelope, Clinton C. Mason, Jonathan M. Ahmann, Anna V. Senina, Courtney L. L. Jones, Matthew S. Zabriskie, Hein Than, Michael J. Xiao, Alexandria van Scoyk, Ami B. Patel, William L. L. Heaton, Shawn C. Owen, Joshua L. Andersen, Christina M. Egbert, Julie A. Reisz, Angelo D'Alessandro, James E. Cox, Kevin C. Gantz, Hannah M. Redwine, Siddharth M. Iyer, Jamshid S. Khorashad, Nima Rajabi, Christian A. Olsen, Thomas O'Hare, Michael W. Deininger. A critical role for SIRT5 in acute myeloid leukemia metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB109.

Stem cell fate in the tissue niche is intimately connected with intracellular metabolic state and the extra‐cellular hormonal stimulations. We have shown that a sensory kinase, PAS domain Kinase (PASK) phosphorylates Wdr5, a member of COMPASS family of histone methyltransferases, to activate the stem cell differentiation program in multiple differentiation paradigms, in vivo and in vitro (eLife, 2016). PASK is expressed highly and exclusively in stem cells, yet the differentiation signaling cues are required to activate PASK and its downstream functions, suggesting PASK remains inaccessible to bind Wdr5 in proliferating stem cells. Here, we show that the mechanistic Target of Rapamycin (mTOR) phosphorylates PASK to promote Wdr5 recruitment and myogenesis in response to nutrient and hormonal signaling. How mTOR phosphorylation stimulates PASK‐Wdr5 interactions remains unknown but is a key mechanistic question that could help understand how nutrient and metabolic signaling can acutely control stem cell differentiation on demand. By using multi‐disciplinary approaches, we show that the PAS domain of PASK inhibits catalytic activity of the kinase domain. Interestingly, mTOR stimulated phosphorylation induces a conformational change resulting in the increased Wdr5 binding, and catalytic activity of PASK. Thus, our data show how information pertaining to nutrient availability is communicated to epigenetic complexes via sequential activation of the mTORC1‐PASK‐Wdr5 pathway.Support or Funding InformationNIH R01: Epigenetic Control of muscle stem cell function by PASK‐Wd5 signaling pathway.1R01AR073906‐01A1