ABSTRACT Hematopoietic stem cells (HSCs) are maintained in a hypoxic niche to limit oxidative stress. Although iron elicits oxidative stress, the importance of iron homeostasis in HSCs has

been unknown. Here we show that iron regulation by the F-box protein FBXL5 is required for HSC self-renewal. Conditional deletion of _Fbxl5_ in mouse HSCs results in cellular iron overload

and a reduced cell number. Bone marrow transplantation reveals that FBXL5-deficient HSCs are unable to reconstitute the hematopoietic system of irradiated recipients as a result of stem cell

exhaustion. Transcriptomic analysis shows abnormal activation of oxidative stress responses and the cell cycle in FBXL5-deficient mouse HSCs as well as downregulation of _FBXL5_ expression

STEM CELLS PROMOTES TET2-DEPENDENT ERYTHROID REGENERATION Article Open access 15 January 2024 RNA BINDING PROTEIN SYNCRIP MAINTAINS PROTEOSTASIS AND SELF-RENEWAL OF HEMATOPOIETIC STEM AND

PROGENITOR CELLS Article Open access 21 April 2023 FERRITIN-MEDIATED MITOCHONDRIAL IRON HOMEOSTASIS IS ESSENTIAL FOR THE SURVIVAL OF HEMATOPOIETIC STEM CELLS AND LEUKEMIC STEM CELLS Article

24 February 2024 INTRODUCTION Hematopoietic stem cells (HSCs) are the most undifferentiated cells in the mammalian hematopoietic system, which they maintain throughout life. At steady state,

HSCs are quiescent and reside in their hypoxic niche. They expend energy mostly via anaerobic metabolism by maintaining a high rate of glycolysis. These characteristics promote HSC

maintenance by limiting the production of reactive oxygen species (ROS)1, to which HSCs are highly vulnerable compared with other hematopoietic cells2. Homeostasis of cellular iron, which is

a major elicitor of ROS production, is thus likely to be strictly regulated in HSCs in order for them to maintain their stemness. Iron is essential for fundamental metabolic processes in

cells and organisms, and it is incorporated into many proteins in the form of cofactors such as heme and iron–sulfur clusters. Iron also readily participates in the Fenton reaction, however,

resulting in uncontrolled production of the hydroxyl radical, which is the most harmful of ROS _in vivo_ and damages lipid membranes, proteins and DNA. It is therefore important that

cellular iron levels are subject to regulation3. We previously showed that iron homeostasis _in vivo_ is regulated predominantly by F-box and leucine-rich repeat protein 5 (FBXL5) and iron

regulatory protein 2 (IRP2)4. IRP2 functions as an RNA binding protein to regulate the translation and stability of mRNAs that encode proteins required for cellular iron homeostasis. IRP2

thereby increases the size of the available iron pool under iron-limiting conditions. In contrast, under iron-replete conditions, FBXL5, which is the substrate recognition component of the

SCFFBXL5 E3 ubiquitin ligase, mediates ubiquitylation and degradation of IRP2. Whereas FBXL5 is unstable under iron-deficient conditions, direct binding of iron to its hemerythrin domain

stabilizes the protein, with this iron-sensing ability allowing FBXL5 to control the abundance of IRP2 in an iron-dependent manner5,6. Disruption of the _Fbxl5_ gene in mice results in the

failure of cells to sense increased cellular iron availability, which leads to constitutive accumulation of IRP2 and misexpression of its target genes. FBXL5-null mice die during

embryogenesis as a result of overwhelming oxidative stress, indicating the vital role of FBXL5 in cellular iron homeostasis during embryogenesis4. A substantial proportion of iron in the

adult body is present in the liver and hematopoietic system. Excess iron in the liver is clinically important given that cirrhosis and hepatocellular carcinoma often develop in individuals

with systemic iron-overload disorders7. Conditional FBXL5 deficiency in mouse liver was found to result in iron accumulation and mitochondrial dysfunction in hepatocytes, leading to the

development of steatohepatitis4. In contrast, hematopoiesis is sensitive to iron deficiency, with an insufficiency of available iron in the body being readily reflected as iron-deficiency

anaemia8. Iron overload in the haematopoietic system is also clinically important, however. Systemic iron overload is thus frequently associated with hematologic diseases such as

myelodysplastic syndrome (MDS), a clonal HSC disorder characterized by hematopoietic failure as a result of ineffective hematopoiesis9,10,11. Such iron overload is a consequence of the

inevitability of frequent blood transfusions and suppression of hepcidin production as a result of ineffective erythropoiesis12. Clinical evidence suggests that systemic iron overload has a

suppressive effect on hematopoiesis in individuals with MDS or aplastic anaemia, and that iron-chelation therapy often improves this situation13,14,15. These observations thus imply that

hematopoietic failure promotes systemic iron overload, which in turn exacerbates hematopoietic failure, with the two conditions forming a vicious cycle. Oxidative stress was found to be

increased in bone marrow (BM) cells of patients with iron overload, and the impaired hematopoietic function of these individuals was partially rescued by treatment with an antioxidant or

iron chelator, suggestive of the initial presence of ROS-induced cellular injury16. However, the molecular mechanisms underlying hematopoietic suppression by systemic iron overload in

patients as well as the cell-autonomous effect of cellular iron overload on HSC stemness have remained largely unknown. Here, we show that cellular iron homeostasis governed by the

FBXL5–IRP2 axis is integral to the maintenance of HSCs. Ablation of FBXL5 specifically in the hematopoietic system of mice resulted in cellular iron overload in HSCs and impaired their

ability to repopulate BM. FBXL5 was also found to be indispensable for the resistance of HSCs to stress induced by myelotoxic agents. FBXL5-deficient HSCs manifested oxidative stress,

increased exit from quiescence and eventual exhaustion. Of note, _FBXL5_ expression was shown to be downregulated in HSCs of some MDS patients, suggesting that disruption of cellular iron

homeostasis contributes to hematopoietic failure in such individuals by compromising HSC function. Suppression of IRP2 activity in FBXL5-deficient HSCs restored stem cell function,

implicating IRP2 as a potential novel therapeutic target in stem cell diseases such as MDS that are associated with cellular iron overload. RESULTS CELLULAR IRON HOMEOSTASIS IS ESSENTIAL FOR

HSC MAINTENANCE We first examined the expression of _Fbxl5_ in various hematopoietic cell lineages of wild-type mice by reverse transcription (RT) and real-time polymerase chain reaction

(rtPCR) analysis. FBXL5 mRNA was detected in many cell lineages including HSCs (Supplementary Fig. 1a). Among differentiated cells, FBXL5 mRNA was most abundant in myeloid (Gr1+Mac1+) cells

and least abundant in the erythroid (Ter119+) lineage. These findings are largely consistent with the results of previous microarray17 (Supplementary Fig. 1b) and RNA-sequencing18,19

(Supplementary Fig. 1c) analyses. To explore the role of cellular iron homeostasis in the maintenance and function of HSCs, we generated mice in which deletion of _Fbxl5_ is inducible in the

hematopoietic system. Crossing of _Fbxl5_F/F mice (which harbour floxed alleles of _Fbxl5_) with _Mx1-Cre_ transgenic mice (which express Cre recombinase under the control of the _Mx1_ gene

promoter) followed by intraperitoneal injection of the resulting offspring (_Mx1-Cre/Fbxl5_F/F mice) with poly(I)–poly(C) [poly(I:C)] gives rise to the _Fbxl5_Δ/Δ genotype in HSCs.

_Mx1-Cre/Fbxl5_+/+ mice injected with poly(I:C) were examined as controls. Deletion of _Fbxl5_ alleles resulted in increased expression of transferrin receptor 1 (TfR1, also known as CD71)

at the cell surface for both hematopoietic progenitors (c-Kit+Sca-1+Lin–, or KSL, cells) and HSCs (CD150+CD48–KSL cells) (Fig. 1a). To examine the intracellular abundance of iron in

hematopoietic progenitors, we loaded hematopoietic cells with the iron-sensitive fluorophore calcein-AM, the fluorescence of which is quenched on binding to ferrous iron (Fe2+)20. The

intensity of calcein fluorescence was significantly lower in _Fbxl5_Δ/Δ KSL cells than in control KSL cells (Fig. 1b). Exposure of the cells to the cell-permeable Fe2+ chelator

2,2′-bipyridyl abolished the difference in calcein fluorescence intensity between the two genotypes, confirming that the abundance of Fe2+ was increased in the _Fbxl5_Δ/Δ KSL cells. These

data collectively suggested that FBXL5 governs cellular iron homeostasis in HSCs. We next examined the role of FBXL5 in the maintenance of HSCs. Flow cytometric analysis revealed a slight

but significant reduction in the frequency of KSL cells or HSCs in BM of _Fbxl5_Δ/Δ mice 4 weeks after the last poly(I:C) injection, whereas this reduction was more prominent at 20 weeks

(Fig. 1c–e). The frequency of annexin V+ apoptotic cells did not differ between control and _Fbxl5_Δ/Δ HSCs at 4 weeks (Fig. 1f), indicating that FBXL5 loss impaired HSC maintenance without

a significant effect on their survival. These results thus suggested that FBXL5 is required for the maintenance of HSCs. We further evaluated whether the decrease in the number of _Fbxl5_Δ/Δ

hematopoietic progenitors was exacerbated under iron-overload conditions. To avoid a deleterious effect of iron overload on the liver4, we transplanted BM cells from either

_Mx1-Cre/Fbxl5_F/F or _Mx1-Cre/Fbxl5_+/+ mice not treated with poly(I:C) into lethally irradiated recipients and then injected these animals with poly(I:C). The recipients were fed a

high-iron diet for 4 weeks after the last poly(I:C) injection. The frequency of _Fbxl5_Δ/Δ KSL cells or HSCs in BM was found to be markedly reduced compared with that of the corresponding

control cells after this 4-week period (Fig. 1g), suggesting that FBXL5 deficiency results in exhaustion of HSCs in an iron-dependent manner. To examine whether the reduced frequency of

_Fbxl5_Δ/Δ HSCs in poly(I:C)-treated _Mx1-Cre/Fbxl5_F/F mice compromised hematopoietic capacity _in vivo,_ we evaluated the survival rate of the mice after repeated injection with

5-fluorouracil (5-FU) at 10-day intervals to eliminate differentiated hematopoietic cells and induce activation and proliferation of HSCs. The survival rate of the _Fbxl5_Δ/Δ mice began to

decline rapidly at ∼15 days after the first 5-FU injection, whereas no control mice died for at least 30 days after the first treatment (Fig. 2), suggesting that stress-induced hematopoiesis

is defective in the FBXL5-deficient animals. We also tested the _in vitro_ colony formation capacity of KSL cells in a serial replating assay. Whereas FBXL5 ablation did not affect such

capacity at the first plating, the number of clonogenic progenitor cells was markedly reduced for _Fbxl5_Δ/Δ KSL cells at the second and subsequent platings (Fig. 3a), suggesting that this

impairment is induced in a cell-autonomous manner. Even at the first plating, iron overload induced by the presence of ferric ammonium citrate (FAC) reduced the colony formation capacity of

_Fbxl5_Δ/Δ KSL cells compared with control cells (Fig. 3b). In contrast, _Fbxl5_Δ/Δ and control KSL cells showed a similar colony formation capacity at the second plating in the presence of

the ferric iron (Fe3+) chelator deferoxamine (DFO). These observations suggested that FBXL5 deficiency results in a deterioration in HSC function due to cellular iron overload. EFFECT OF

CELLULAR IRON OVERLOAD ON DIFFERENTIATED CELLS We next checked whether ablation of FBXL5 in the hematopoietic system affects systemic iron homeostasis and the maintenance of differentiated

hematopoietic cells. Measurement of serum iron parameters revealed that the serum iron concentration and transferrin saturation were not altered in _Fbxl5_Δ/Δ mice (Fig. 4a), suggesting that

the loss of FBXL5 in the hematopoietic system had no substantial effect on systemic iron homeostasis. Haematologic parameters of _Fbxl5_Δ/Δ mice at 4 weeks after poly(I:C) injection were

also similar to those of control mice, with the exception of a slight decrease in the number of platelets (Fig. 4b). We further examined the effect of FBXL5 ablation on the differentiation

of hematopoietic cells. The frequency of differentiated cells (Gr1+Mac1+ myeloid cells, B220+ B cells or CD3+ T cells) in peripheral blood (PB), BM or the spleen of _Fbxl5_Δ/Δ mice was

similar to that in control animals, with the exception of a small increase in the frequency of myeloid cells and decrease in the frequency of B cells in BM (Fig. 4c–e). In addition,

erythropoietic parameters of _Fbxl5_Δ/Δ mice did not differ from those of control mice at 20 weeks after poly(I:C) injection (Fig. 4f). The frequency of Ter119+ (erythroid) cells in BM was

also not affected by FBXL5 ablation (Fig. 4g). Together, these observations suggested that cellular iron overload induced by FBXL5 ablation had only a small effect on the maintenance of

differentiated cells. CELLULAR IRON OVERLOAD IMPAIRS HSC SELF-RENEWAL CAPACITY Given that FBXL5 deficiency results in a decline in the number of HSCs, we postulated that FBXL5 is essential

for the self-renewal capacity of these cells. To examine this possibility, we first assessed the repopulation capacity of _Fbxl5_Δ/Δ BM cells in a noncompetitive setting. Most lethally

irradiated mice transplanted with BM cells (1 × 106) from _Fbxl5_Δ/Δ mice died by ∼20 days after BM transfer, whereas those transplanted with control BM cells survived (Fig. 5a), suggesting

that the repopulation capacity of _Fbxl5_Δ/Δ BM cells was significantly impaired. To examine the long-term repopulation capacity of _Fbxl5_Δ/Δ HSCs, we performed a competitive reconstitution

assay in which _Fbxl5_Δ/Δ or control BM cells were transplanted into lethally irradiated C57BL/6 congenic recipient mice together with competitor cells. Flow cytometric analysis of the

resulting chimerism in PB of the recipients until 16 weeks after the BM transfer revealed that the long-term repopulation capacity of _Fbxl5_Δ/Δ HSCs was indeed markedly compromised (Fig.

5b). We further confirmed that _Fbxl5_Δ/Δ hematopoietic cells manifested almost no reconstitution capacity after a second BM transfer (Fig. 5c). The number of KSL cells derived from

_Fbxl5_Δ/Δ donor cells was greatly reduced in BM of the initial recipient mice at 16 weeks after BM transfer (Fig. 5d). To characterize the repopulation defect in _Fbxl5_Δ/Δ HSCs, we

examined the homing capacity of _Fbxl5_Δ/Δ hematopoietic progenitor cells after transplantation. _Fbxl5_Δ/Δ or control KSL cells were sorted, labelled with carboxyfluorescein succinimidyl

ester (CFSE) and transplanted into lethally irradiated recipients, and the recipient BM was analysed 16 h after transplantation. The homing capacity of the transplanted CFSE+ cells for BM

was similar for the two genotypes (Fig. 5e), excluding the possibility that a homing defect is responsible for the repopulation defect of _Fbxl5_Δ/Δ HSCs. To evaluate the stem cell capacity

of _Fbxl5_Δ/Δ HSCs excluding homing and engraftment, we transplanted BM cells from either _Mx1-Cre/Fbxl5_F/F or _Mx1-Cre/Fbxl5_+/+ mice not treated with poly(I:C) into lethally irradiated

recipients together with competitor cells. Four weeks after BM transfer, we confirmed that donor cells were reconstituted in the recipient BM and then injected the recipient mice with

poly(I:C). _Fbxl5_Δ/Δ HSCs gradually lost long-term repopulation capacity (Fig. 5f), showing that such capacity after homing and engraftment was impaired in _Fbxl5_Δ/Δ HSCs. The

differentiation of _Fbxl5_Δ/Δ HSCs appeared normal in this setting of competitive repopulation (Fig. 5g). These results collectively indicated that cellular iron homeostasis is essential for

the self-renewal capacity of HSCs. ROLE OF IRP2 IN THE EFFECT OF FBXL5 ON HSC STEMNESS Given that impaired degradation of IRP2 is primarily responsible for the embryonic mortality of

_Fbxl5_–/– mice4, we hypothesized that the defective stem cell capacity of _Fbxl5_Δ/Δ HSCs might also be due to IRP2 accumulation. We therefore evaluated whether suppression of IRP2 is

required for the repopulation capacity of HSCs with the use of a competitive reconstitution assay. Both the repopulation and differentiation capacities of _Irp2_–/– HSCs were similar to

those of control (_Irp2_+/+) HSCs (Fig. 6a–c). We next prepared _Mx1-Cre/Fbxl5_F/F/_Irp2_–/– and _Mx1-Cre/Fbxl5_+/+/_Irp2_+/+ mice to be able to analyse _Fbxl5_Δ/Δ_/Irp2_–/– HSCs after

poly(I:C) injection. The competitive reconstitution assay revealed that the long-term repopulation capacity of _Fbxl5_Δ/Δ_/Irp2_–/– HSCs did not differ significantly from that of control

HSCs after the first or second BM transfer (Fig. 6d–f), suggesting that aberrant IRP2 activity is responsible for the deleterious effect of FBXL5 ablation on the repopulation capacity of

HSCs. FBXL5 thus protects HSC stemness through suppression of IRP2. CELLULAR IRON OVERLOAD DISRUPTS REDOX REGULATION IN HSCS To investigate further the mechanism by which cellular iron

overload impairs HSC function, we profiled gene expression in _Fbxl5_Δ/Δ HSCs. Microarray analysis identified 1,128 differentially expressed genes (686 downregulated and 442 upregulated;

fold change of >1.5 or <−1.5 and _P_ value of <0.05) in _Fbxl5_Δ/Δ HSCs compared with control cells (Supplementary Data 1 and 2). A complete list of these genes has been deposited

in the Gene Expression Omnibus (GEO) database under the accession number GSE93649. As expected, expression of genes related to cellular iron metabolism such as _Hbb_, _Slc48a1_, _Ftl1_,

_Lcn2_ and _Abcb6_ was upregulated. The upregulated genes also included many genes important for redox regulation, such as _Mt1_, _Mt2_, _Hmox1_, _Gstm2_, _Slc7a11_, _Gclm_, _Gsta4_, _Cat_,

_Txn1_, _Nqo1_ and _Sod1_. Ingenuity pathway analysis (IPA) revealed that the differentially expressed genes were most highly associated with the NRF2-mediated oxidative stress response

(Fig. 7a). Gene set enrichment analysis (GSEA) also confirmed that the antioxidant defense system is activated in _Fbxl5_Δ/Δ HSCs (Fig. 7b,c). Changes in the expression of several genes

related to oxidative stress responses were validated by RT and rtPCR analysis (Fig. 7d). On the basis of these results, we concluded that FBXL5 ablation evokes oxidative stress in HSCs.

Oxidative stress in HSCs is also known to give rise to phosphorylation (activation) of p38 mitogen-activated protein kinase (MAPK), which results in aberrant cell proliferation and

exhaustion1,2. Intracellular flow cytometric analysis revealed that the frequency of cells positive for phosphorylated p38 MAPK was greater among _Fbxl5_Δ/Δ HSCs than among control HSCs

(Fig. 7e). The mean fluorescence intensity (MFI) of phospho-p38 for _Fbxl5_Δ/Δ HSCs also tended to be greater than that for control HSCs, although this difference did not achieve statistical

significance (_P_=0.062). Given that both extrinsic factors including various cytokines as well as intrinsic factors such as oxidative stress influence p38 MAPK phosphorylation status21, we

sought to examine the influence of only intrinsic factors on p38 phosphorylation in HSCs. To this end, we transplanted BM cells from _Mx1-Cre/Fbxl5_F/F or _Mx1-Cre/Fbxl5_+/+ mice into

lethally irradiated recipients and then injected these animals with poly(I:C). The frequency of _Fbxl5_Δ/Δ HSCs in BM of the recipients was significantly reduced compared with that of

control HSCs (Fig. 7f), as was the case for _Mx1-Cre/Fbxl5_F/F mice treated with poly(I:C) (Fig. 1e). The frequency of cells positive for phosphorylated p38 MAPK as well as the MFI for

phospho-p38 were also significantly increased in _Fbxl5_Δ/Δ HSCs compared with control HSCs in the recipient mice (Fig. 7g). These results thus also confirmed that _Fbxl5_Δ/Δ HSCs are

exposed to intense oxidative stress. CELLULAR IRON HOMEOSTASIS IS ESSENTIAL FOR HSC DORMANCY GSEA also showed that the gene expression profile of _Fbxl5_Δ/Δ HSCs was shifted toward

proliferation compared with that of control HSCs (Fig. 8a). Given that the loss of dormancy in HSCs leads to their exhaustion, we hypothesized that FBXL5 ablation might promote exit from the

dormant state in HSCs. We therefore evaluated the cell cycle kinetics of _Fbxl5_Δ/Δ HSCs by intracellular staining of the proliferation marker Ki-67 and analysis of DNA ploidy by staining

with Hoechst 33342. The frequency of cells in the dormant state (Ki-67– fraction) was reduced for _Fbxl5_Δ/Δ HSCs compared with control cells (Fig. 8b). A similar difference in the frequency

of dormant HSCs was also observed in poly(I:C)-treated recipients of transplanted BM cells from _Mx1-Cre/Fbxl5_F/F or _Mx1-Cre/Fbxl5_+/+ mice (Fig. 8c). GSEA also revealed loss of an

HSC-specific gene signature in _Fbxl5_Δ/Δ HSCs (Fig. 8d). The top 30 downregulated genes in our microarray analysis (Supplementary Data 2) include the HSC-specific gene _Necdin_22, whose

downregulation in _Fbxl5_Δ/Δ HSCs was confirmed by RT and rtPCR analysis (Fig. 8e). We also confirmed the downregulation of the HSC-specific gene _p57_ (ref. 23), whereas the expression of

other such genes including _Hoxb5_ (ref. 24) and _Hif1a_ (ref. 25) was not affected (Fig. 8e). These results suggested that cellular iron overload induces exit of HSCs from the dormant state

and loss of an HSC-specific gene expression signature. DISRUPTED IRON HOMEOSTASIS IN HSCS IS ASSOCIATED WITH MDS The results of our mouse experiments together indicated that cellular iron

homeostasis governed by FBXL5 plays an essential role in the maintenance and function of HSCs. We finally examined whether FBXL5 deficiency might be associated with human hematopoietic

diseases. Impaired HSC function can result in the development of MDS, a clonal HSC disorder characterized by ineffective hematopoiesis9,10,11. Expression of _FBXL5_ was shown to be

differentially downregulated in CD34+CD38–CD90+ HSCs from MDS patients with deletion of chromosome 5q relative to those from healthy control subjects26. Analysis of a published set of

microarray data revealed that _FBXL5_ expression was also significantly downregulated in Lin–CD34+CD38–CD90+CD45RA– HSCs from eight MDS patients without deletion of chromosome 5q compared

with 11 age-matched healthy control samples27 (Fig. 9a). It is of note that the expression of both _TFR1_ and _DMT1_, which is upregulated by IRP2 and therefore represents an index of IRP2

activity, was also increased in the MDS patients (Fig. 9b,c). To determine whether _FBXL5_ expression is also downregulated in more differentiated hematopoietic progenitor cells in MDS

patients, we evaluated a published set of microarray data for CD34+ hematopoietic progenitor cells from 183 MDS patients with various cytogenetic abnormalities and 17 healthy control

subjects28. Expression of _FBXL5_ was significantly downregulated in the CD34+ cells of patients with refractory anaemia with ringed sideroblasts (RARS), a subgroup of MDS characterized by

iron deposition and apoptosis in hematopoietic progenitor cells (Fig. 9d). Consistent with this finding, _TFR1_ expression was also significantly upregulated in the CD34+ cells from the RARS

patients (Fig. 9e). These findings implicate cellular iron overload due to FBXL5 downregulation in the pathogenesis of human hematopoietic failure. Given that suppression of aberrant IRP2

activity cancelled the deleterious effect of FBXL5 ablation on the repopulation capacity of HSCs (Fig. 6), IRP2 is a potential therapeutic target for cellular iron overload in HSCs due to

FBXL5 downregulation, including that in patients with MDS. Consistent with this notion, a published microarray data set revealed that an increased IRP2 mRNA abundance in CD34+ hematopoietic

progenitor cells was related to reduced survival in MDS patients without deletion of chromosome 5q (ref. 29) (Fig. 9f). DISCUSSION We have here discovered a previously unrecognized role for

cellular iron homeostasis in the maintenance of HSCs with the use of mouse models of conditional _Fbxl5_ deletion. Mechanistically, cellular iron homeostasis in HSCs regulates oxidative

stress, quiescence and self-renewal capacity. Analysis of public data sets revealed that downregulation of _FBXL5_ expression was associated with MDS, a disease characterized by BM failure.

Suppression of IRP2 activity in FBXL5-deficient HSCs restored stem cell function, implicating IRP2 as a potential therapeutic target for cellular iron overload in HSCs with FBXL5 deficiency

(Fig. 10). FBXL5 is a master regulator of cellular iron metabolism by virtue of its role as the substrate recognition component of the SCFFBXL5 E3 ubiquitin ligase for IRP2 degradation5,6.

Other proteins targeted by FBXL5 for proteasomal degradation include p150Glued, cortactin and single-stranded DNA binding protein 1 (SSB1)30,31,32. FBXL5 has also been shown to interact with

Snail1 (refs 33, 34) and CBP/p300-interacting transactivator 2 (CITED2)35, leading to their degradation. CITED2 was shown to control the proliferation of mouse embryonic fibroblasts by

promoting expression of the Polycomb group genes _Bmi1_ and _Mel18_ (ref. 36) as well as to selectively maintain adult HSC function at least in part through regulation of p16 and p53 (ref.

37). If CITED2 accumulates in FBXL5-deficient HSCs, it might also promote their proliferation and exhaustion. However, FBXL5-deficient mice die during embryogenesis and their mortality is

prevented by additional ablation of IRP2, suggesting that impaired IRP2 degradation is primarily responsible for the embryonic death4. Our data now provide evidence that IRP2 is also the

major target of SCFFBXL5 in HSCs, given that the defect in repopulation capacity of FBXL5-deficient HSCs was rescued by additional ablation of IRP2. These lines of evidence also suggest that

the contribution of CITED2 to the phenotype of FBXL5-deficient HSCs is limited. We therefore conclude that FBXL5 plays an essential role in the maintenance of HSCs through suppression of

IRP2 activity. Our present study shows that disruption of cellular iron homeostasis by FBXL5 ablation in HSCs resulted in cellular iron overload, oxidative stress responses, exit from

dormancy and eventual exhaustion in these cells. Increased ROS levels promote the proliferation and differentiation of HSCs, primarily via modulation of p38 MAPK and the forkhead box O

(FOXO) family of transcription factors1. Many mutations that result in aberrantly high ROS levels in HSCs also lead to impairment of quiescence and self-renewal potential as a result of

enhanced differentiation38. We now show that FBXL5 is also an essential ROS regulator in HSCs and plays a key role in the maintenance of stemness. The expression of many genes that

contribute to oxidative stress responses was found to be upregulated in FBXL5-deficient HSCs: The most upregulated ROS-related genes included those for metallothionein (MT) 1 and MT2, which

are small, cysteine-rich, and heavy metal-binding proteins that participate in an array of protective stress responses39. These proteins thus protect cells from exposure to oxidants and

electrophiles, which react readily with sulfhydryl groups. Moreover, they play a key role in regulation of cellular zinc levels by binding and releasing zinc. The marked upregulation of MT

gene expression in FBXL5-deficient HSCs might thus also modify cellular zinc metabolism. Another such upregulated gene was _Slc7a11_, which encodes a component of system Xc–. System Xc–

contributes to the maintenance of redox homeostasis by importing cystine for synthesis of the major cellular antioxidant glutathione. Inhibition of system Xc– by erastin in cancer cells

triggers ferroptosis, a recently recognized form of iron-dependent cell death40. Upregulation of _Slc7a11_ expression might therefore represent a mechanism to protect HSCs with cellular iron

overload from ferroptosis. Indeed, FBXL5 ablation was shown to promote erastin-induced ferroptosis in cultured cells40. These changes in gene expression in FBXL5-deficient HSCs are thus

indicative of iron-mediated cellular damage and disruption of redox homeostasis. In contrast to the deleterious effect of iron overload evoked by FBXL5 loss on HSC function, FBXL5 deficiency

did not substantially affect differentiated hematopoietic cells. Although cross talk between systemic iron homeostasis and erythropoiesis is well established41, detailed analysis of

erythropoiesis in FBXL5-deficient mice indicated that FBXL5 has a limited role in erythropoiesis, consistent with the finding that the amount of FBXL5 mRNA is smallest in the erythroid

lineage among differentiated haematopoietic cells. In general, erythroid cells require large amounts of iron to sustain haemoglobin synthesis41, suggesting that the importance of FBXL5 as a

brake on iron uptake might be rather limited in erythropoiesis. Systemic iron overload is sometimes a complication of hematopoietic failure such as that associated with MDS as a result of

the required frequent blood transfusions and the suppression of hepcidin production due to ineffective erythropoiesis12. Systemic iron overload in turn has a suppressive effect on

hematopoiesis in patients with hematopoietic failure, with iron-chelation therapy having been found to be beneficial in these patients13,14,15. However, iron overload has little effect on

hematopoiesis in patients with hereditary hemochromatosis, which is also a major cause of systemic iron overload due to hepcidin deficiency. These various observations suggest that the

hematopoietic system in some patients with BM failure is intrinsically vulnerable to iron. By analysing public data sets, we found that _FBXL5_ expression was significantly downregulated in

HSCs in a subset of patients with MDS. Reduced expression of _FBXL5_ might promote loss of quiescence and exhaustion in HSCs of such patients. Similar to the _Fbxl5_Δ/Δ HSCs examined in the

present study, HSCs of MDS patients with low _FBXL5_ expression might also be vulnerable to systemic iron overload. The expression of _FBXL5_ was also found to be significantly downregulated

in CD34+ progenitor cells of patients with RARS, a subtype of MDS characterized by iron deposition in hematopoietic progenitor cells. Downregulation of _FBXL5_ expression in RARS is of

interest given that RARS progenitor cells are loaded with excess iron in mitochondria and are vulnerable to ROS-induced apoptosis9. FBXL5 deficiency might thus exacerbate the cellular and

mitochondrial iron overload in RARS progenitor cells, contributing to disease pathogenesis. Our findings raise the possibility that FBXL5 plays a key role in the pathogenesis of BM failure

syndromes including MDS, a possibility that warrants further investigation. Our finding that suppression of aberrant IRP2 activity rescued the defect in repopulation capacity of HSCs induced

by FBXL5 ablation suggests that targeting of IRP2 is effective for mitigation of cellular iron overload and is therefore a candidate for therapeutic application. This notion is further

supported by the finding that an increased abundance of IRP2 mRNA in CD34+ hematopoietic progenitor cells was related to reduced clinical survival in MDS patients without deletion of

chromosome 5q. Given that complete loss of IRP2 gives rise to microcytic anaemia42,43, however, an appropriate level of IRP2 suppression would be needed. Another limitation of such a

therapeutic strategy is that an IRP2 inhibitor has not yet been developed. Given that IRP2 is an mRNA binding protein, inhibition of such binding with antisense oligonucleotides is a

possible approach. Despite these limitations, inhibition of IRP2 is a potentially novel approach to the treatment of hematopoietic failure associated with FBXL5 downregulation and cellular

iron overload. METHODS MICE Generation of _Fbxl5_F/F mice was described previously4. These mice were crossed with _Mx1-Cre_ transgenic mice44 or _Irp2_−/− mice45 to generate

_Mx1-Cre/Fbxl5_F/F and _Mx1-Cre/Fbxl5_F/F/_Irp2_−/− mice. All of these mice were backcrossed with C57BL/6 mice for more than six generations. Expression of Cre recombinase in mice harbouring

the _Mx1-Cre_ transgene was induced by intraperitoneal injection of poly(I:C) (R&D Systems, Minneapolis, MN) at a dose of 20 mg kg−1 on seven alternate days beginning at 8 weeks of age.

_Mx1-Cre/Fbxl5_F/F mice and _Mx1-Cre/Fbxl5_F/F/_Irp2_−/− mice were analysed 4 weeks after the last poly(I:C) injection unless indicated otherwise. _Irp2_−/− mice were analysed at 14 weeks

of age. C57BL/6-Ly5.1 congenic mice were obtained from The Jackson Laboratory (Bar Harbor, ME). For some experiments, mice were injected intraperitoneally with 5-FU (Sigma, St Louis, MO) at

a dose of 150 mg kg−1 or fed a high-iron diet formulated by supplementation of CA-1 (containing 0.03% ferric citrate; CLEA, Tokyo, Japan) with 2% ferric citrate. All mouse experiments were

approved by the Animal Ethics Committee of Kyushu University. FLOW CYTOMETRIC ANALYSIS AND CELL SORTING Flow cytometric analysis and cell sorting were performed with the use of FACSVerse or

FACSAria instruments (BD Biosciences, San Jose, CA). Mouse antibodies to CD45.1 (A20), CD45.2 (104), Sca-1 (E13-161.7), c-Kit (2B8) or CD34 (RAM34) were obtained from BD Biosciences; those

to CD3ɛ (145-2C11), CD4 (L3T4), CD8 (53-6.7), B220 (RA3-6B2), CD16/32 (93) or Mac1 (M1/70) were from eBioscience (San Diego, CA); and those to CD48 (HM48-1), Ter119 (TER119), Gr1 (RB6-8C5),

CD71 (RI7217), CD150 (TC15-12F12.2) or Ki-67 (16A8) were from BioLegend (San Diego, CA). Antibodies to phosphorylated (Thr180/Tyr182) p38 MAPK (12F8) were from Cell Signaling Technology

(Danvers, MA). CD4, CD8, B220, Ter119, Gr1 and Mac1 were used as lineage markers. For analysis of HSCs, antibodies except those to lineage markers were used at a 1:50 dilution and those to

lineage markers were used at a 1:60 dilution. For analysis of differentiated cells, all antibodies were used at a 1:100 dilution. BM mononuclear cells flushed from the tibia and femur,

thymocytes, or splenocytes of mice were suspended in phosphate-buffered saline (PBS) supplemented with 2% heat-inactivated fetal bovine serum, incubated with the indicated antibodies for 30 

min on ice, washed and then analysed. For intracellular staining with antibodies to Ki-67 or to phosphorylated p38 MAPK, cells were stained for surface markers, fixed in PBS containing 2%

paraformaldehyde for 20 min, permeabilized with PBS containing 0.5% saponin and 0.5% bovine serum albumin for 10 min at room temperature and then incubated with these antibodies. For

detection of phosphorylated p38 MAPK, cells were further stained with Alexa Fluor 488-conjugated goat antibodies to rabbit immunoglobulin G (A11034, Molecular Probes, Eugene, OR) at a 1:100

dilution for 30 min at room temperature. For analysis of the cell cycle, cells stained with antibodies to Ki-67 were briefly exposed to Hoechst 33342 (Sigma) before analysis. EVALUATION OF

INTRACELLULAR IRON STATUS BM mononuclear cells were stained with 0.25 μM calcein-AM (Molecular Probes) in PBS for 5 min at 37 °C and then washed with PBS. The calcein-loaded cells were

further stained with antibodies to surface markers for 30 min on ice, washed, incubated for 20 min on ice in PBS with or without 100 μM 2,2′-bipyridyl (Sigma), and analysed with the FACSAria

instrument. DETECTION OF APOPTOSIS For assay of apoptosis, BM cells stained with antibodies to cell surface markers were further stained for 15 min at room temperature with annexin V and

propidium iodide with the use of an Annexin V-FITC Apoptosis Detection Kit (BD Biosciences). COMPETITIVE RECONSTITUTION ASSAY Unfractionated BM cells (4 × 105) isolated from

_Mx1-Cre/Fbxl5_F/F, _Mx1-Cre/Fbxl5_F/F/_Irp2_−/− or _Mx1-Cre/Fbxl5_+/+ mice (CD45.2) were transplanted into lethally irradiated C57BL/6 congenic (CD45.1) recipients together with competitor

BM cells (4 × 105) from C57BL/6 congenic (CD45.1) mice. BM cells (1 × 106) isolated from the recipient mice at 16 weeks after the first BM transfer were transplanted into a second set of

lethally irradiated mice (second BM transfer). HOMING ASSAY Sorted KSL cells were incubated with 2 μM CFSE (Molecular Probes) in PBS for 12 min at 37 °C and then washed. The cells (2 × 104)

were then transplanted into lethally irradiated mice. After 16 h, BM cells were isolated from the recipient mice and analysed with the use of the FACSVerse instrument. COLONY FORMATION ASSAY

Colony formation capacity was examined for 500 KSL cells per dish with Methocult medium (MethoCult GF M3434; Stem Cell Technologies, Vancouver, BC, Canada). For a serial replating assay,

cells from the first plating were collected and counted, and 1 × 104 of the cells were replated. In some experiments, FAC (100 μg ml−1) or 10 μM DFO was added to the medium. RT AND RTPCR

ANALYSIS Total RNA isolated from sorted cells with the use of Isogen and Ethachinmate (Nippon Gene, Tokyo, Japan) was subjected to RT with ReverTra Ace (Toyobo, Tokyo, Japan), and the

resulting cDNA was subjected to rtPCR analysis with SYBR Green PCR Master Mix and specific primers in a Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA). Data were

normalized by the abundance of β-actin mRNA (Figs 7d and 8e) or attachment region-binding protein (ARBP) mRNA (Supplementary Fig. 1a). The sequences of the various primers (sense and

antisense, respectively) were as follows: 5′-AGGTGACAGCATTGCTTCTG-3′ and 5′-GGGAGACCAAAGCCTTCATA-3′ for β-actin, 5′-GGACCCGAGAAGACCTCCTT-3′ and 5′-GCACATCACTCAGAATTTCAATGG-3′ for ARBP,

5′-TCTTCCTCCTGAGGTAATGCTGTCC-3′ and 5′-CACAAAGATCCTGTTTTTGCCAGC-3′ for FBXL5, 5′-GCACCTGAGGCTGACCAATC-3′ and 5′-CATGGGCATACGGTTGTTGAG-3′ for necdin, 5′-GCGCAAACGTCTGAGATGAGT-3′ and

5′-AGAGTTCTTCCATCGTCCGCT-3′ for p57, 5′-CCGGACTATCAGTTGCTAA-3′ and 5′-GGACGTCGCCTGCCTGAA-3′ for HoxB5, 5′-GCTGTCCTCTAAGCGTCACC-3′ and 5′-AGGAGCAGCAGCTCTTCTTG-3′ for MT1,

5′-CAAACCGATCTCTCGTCGAT-3′ and 5′-AGGAGCAGCAGCTTTTCTTG-3′ for MT2, 5′-TGGGTGGAACTGCTCGTAAT-3′ and 5′-AGGATGTAGCGTCCAAATGC-3′ for Slc7a11, 5′-AAGCCGAGAATGCTGAGTTCA-3′ and

5′-GCCGTGTAGATATGGTACAAGGA-3′ for Hmox1 and 5′-CGGCGAGAACGAGAAGAA-3′ and 5′-AAACTTCAGACTCTTTGCTTCG-3′ for Hif1α. MICROARRAY ANALYSIS HSCs (3,000 cells) were sorted directly into Trizol (Life

Technologies, Carlsbad, CA), and total RNA was subjected to mRNA amplification, RT, fragmentation and labelling with the use of a Genechip WT Pico Kit (Affymetrics, Santa Clara, CA).

Labelled single-stranded cDNA from each sample was subjected to hybridization with a GeneChip Mouse Transcriptome Array 1.0 (Affymetrics). Gene expression data were imported and analysed

with the use of Transcriptome Analysis Console (TAC) Software (Affymetrics). Normalized expression data were analysed with the use of GSEA v2.0.13 software (Broad Institute, Cambridge, MA).

All gene sets were obtained from the Molecular Signatures Database v4.0 distributed at the GSEA Web site (http://www.broadinstitute.org/gsea/index.jsp). Data were also analysed with IPA

Software (Ingenuity Systems, Redwood City, CA). HAEMATOLOGIC AND BIOCHEMICAL ANALYSES Haematologic parameters were determined with the use of a Sysmex K-4500 automatic analyser. Serum iron

concentration and total iron binding capacity were measured with a standard clinical autoanalyser. Transferrin saturation was calculated from serum iron concentration and total iron binding

capacity. HUMAN DATA ANALYSIS Microarray data for Lin–CD34+CD38–CD90+CD45RA– HSCs from eight MDS patients without deletion of chromosome 5q and 11 age-matched healthy control subjects were

accessed at GEO with the reference series tag GSE30201 (ref. 27); those for CD34+ hematopoietic progenitor cells from 183 MDS patients with various cytogenetic abnormalities and 17 healthy

control subjects were accessed at GEO with the tag GSE19429 (ref. 28); and those for CD34+ hematopoietic progenitor cells from MDS patients with survival data were accessed at GEO with the

tag GSE58831 (ref. 29). In the latter instance, the data for 108 MDS patients without deletion of chromosome 5q and without the WHO category ‘AML-MDS’ who survived for >1 week were

analysed. All data were downloaded for analysis of _FBXL5_ (209004_s_at), _TFR1_ (237214 _at), _DMT1_ (1555116_s_at) or _IRP2_ (214666_x_at) signal intensity. ANALYSIS OF PUBLISHED MOUSE

DATA Microarray or RNA-sequencing data (GSE60101)19 for FBXL5 mRNA abundance in various hematopoietic cell types are available online at Gene Expression Commons (https://gexc.riken.jp)17 or

BloodSpot (http://servers.binf.ku.dk/bloodspot)18. STATISTICAL ANALYSIS No statistical methods were used to predetermine sample size. Experiments were not randomized, and investigators were

not blinded to allocation during experiments and outcome assessment. Quantitative data are presented as means±s.d. as indicated and were compared between groups with the two-tailed Student’s

_t_-test as performed with Microsoft Excel software. Survival curves were analysed with the log-rank nonparametric test. The cutoff value to determine whether the level of _IRP2_ expression

was high or low in a sample (Fig. 9f) was designed by the minimal _P_ value approach46. A _P_ value of <0.05 was considered statistically significant. DATA AVAILABILITY The microarray

data were deposited in GEO under the accession number GSE93649. All other relevant data are available from the corresponding authors on reasonable request. ADDITIONAL INFORMATION HOW TO CITE

THIS ARTICLE: Muto, Y. _et al_. Essential role of FBXL5-mediated cellular iron homeostasis in maintenance of hematopoietic stem cells. _Nat. Commun._ 8, 16114 doi: 10.1038/ncomms16114

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application enabling rapid biomarker cutoff optimization. _PLoS ONE_ 7, e51862 (2012). ADS  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We thank K.

Miyawaki for a technical suggestion regarding microarray analysis; A. Matsumoto for a technical suggestion regarding bone marrow transplantation experiments; E. Koba, K. Tsunematsu and other

laboratory members for technical assistance; and A. Ohta for help with preparation of the manuscript. This study was funded in part by KAKENHI grants (25221303 and 26640080) from the

Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan. AUTHOR INFORMATION Author notes * Masaaki Nishiyama and Akihiro Nita: These authors contributed equally to

this work. AUTHORS AND AFFILIATIONS * Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582,

Fukuoka, Japan Yoshiharu Muto, Masaaki Nishiyama, Akihiro Nita, Toshiro Moroishi & Keiichi I. Nakayama Authors * Yoshiharu Muto View author publications You can also search for this

author inPubMed Google Scholar * Masaaki Nishiyama View author publications You can also search for this author inPubMed Google Scholar * Akihiro Nita View author publications You can also

search for this author inPubMed Google Scholar * Toshiro Moroishi View author publications You can also search for this author inPubMed Google Scholar * Keiichi I. Nakayama View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Y.M. planned and performed all experiments. M.N. provided materials and intellectual support. A.N.

assisted with BM transplantation experiments. T.M. generated _Fbxl5_F/F mice. K.I.N. coordinated the study, oversaw collection and analysis of the results, and wrote the manuscript. All

authors discussed the data and commented on the manuscript. CORRESPONDING AUTHORS Correspondence to Masaaki Nishiyama or Keiichi I. Nakayama. ETHICS DECLARATIONS COMPETING INTERESTS The

authors declare no competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION (PDF 375 KB) SUPPLEMENTARY DATA 1 (XLSX 89 KB) SUPPLEMENTARY DATA 2 (XLSX 127 KB) PEER

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CITE THIS ARTICLE Muto, Y., Nishiyama, M., Nita, A. _et al._ Essential role of FBXL5-mediated cellular iron homeostasis in maintenance of hematopoietic stem cells. _Nat Commun_ 8, 16114

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