Context-Dependent Modification of PFKFB3 in Hematopoietic 1 Stem Cells Promotes Anaerobic Glycolysis and Ensures Stress 2 Hematopoiesis

67 Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current 68 metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic 69 changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed 70 to identify the key metabolic enzymes that define metabolic differences between steady-state and 71 stress conditions in HSCs and elucidate their regulatory mechanisms. Through quantitative 13 C 72 metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and 73 mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme 74 phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) 75 inhibition. Real-time measurement of adenosine triphosphate (ATP) levels in single HSCs 76 demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via 77 increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to 78 meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1- 79 dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during 80 OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted 81 differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study 82 reveals the flexible and multilayered regulation of HSC metabolism to sustain hematopoiesis under 83 stress and provides techniques to better understand the physiological metabolism of rare 84 hematopoietic cells.

techniques. In this study, we aimed to identify the key metabolic enzymes that define metabolic 132 differences between steady-state and stress conditions in HSCs and elucidate their regulatory 133 mechanisms using a quantitative and mathematical approach. Our findings provide a platform for 134 quantitative metabolic analysis of rare cells such as HSCs, characterize the overall metabolic 135 reprogramming of HSCs during stress loading, and highlight the key enzyme involved in this process.

138
Mice 139 C57BL/6 mice (7-16 weeks old, Ly5.2 + ) were purchased from Japan SLC (Shizuoka, Japan). 140 C57BL/6 mice (Ly5.1 + ) were purchased from CLEA Japan (Shizuoka, Japan). Knock-in mice 141 harboring GO-ATeam2 51-53 in the ROSA26 locus were generated in the Yamamoto laboratory. The 153 For metabolome analysis focused on glycolytic metabolites and nucleotides, anionic metabolites 154 were measured using an orbitrap-type MS (Q-Exactive Focus; Thermo Fisher Scientific, Waltham, 155 MA, USA) connected to a high-performance IC system (ICS-5000+, Thermo Fisher Scientific), 156 enabling highly selective and sensitive metabolite quantification owing to the IC-separation and 157 Fourier Transfer MS principle 56 . The IC instrument was equipped with an anion electrolytic 158 suppressor (Dionex AERS 500; Thermo Fisher Scientific) to convert the potassium hydroxide 159 gradient into pure water before the sample entered the mass spectrometer. Separation was performed  All relevant data are available from the corresponding author upon reasonable request. 1F-G). Second, we used a G 0 marker mouse line 55 . These mice expressed a fusion protein of the p27 216 inactivation mutant p27Kand the fluorescent protein mVenus (G 0 marker), allowing prospective 217 identification of G 0 cells. We tested whether the expression of G 0 marker in HSCs was altered after 218 5-FU administration to the G 0 marker mice (Supplemental Figure 1H) and found that 5-FU treatment 219 reduced the frequency of G 0 marker-positive HSCs, regardless of the EPCR expression 220 (Supplemental Figure 1I-J). This was not observed in the PBS group. These results indicated that 5-

221
FU administration induced cell cycle progression of entire HSCs in mice.

222
HSC cell cycling is preceded by the activation of intracellular ATP-related pathways that 223 metabolize extracellular nutrients, including glucose 39,40 , which are utilized in both ATP-producing 224 and -consuming pathways, determining cellular ATP levels. Therefore, we examined the metabolic 225 flux of glucose by performing in vitro IC-MS tracer analysis with uniformly carbon-labeled (U-13 C 6 ) 226 glucose to determine the pathways driving changes in ATP in 5-FU-treated HSCs ( Figure 1A; Table   227 S2). To avoid metabolite changes, samples were continuously chilled on ice during cell preparation, 228 and the process from euthanasia to cell preparation was performed in the shortest possible time (see 229 "Preparation and storage of in vitro U-13 C 6 -glucose tracer samples" section under 230 "Supplementary Methods" for more information). We found that changes in metabolite levels 231 before and after sorting were present but limited (Supplemental Figure 2A). This result is consistent 232 with the finding that the cell purification process does not significantly affect metabolite levels when 233 sufficient care is taken in cell preparation 50 . In 5-FU-treated HSCs, the levels of glycolytic 234 metabolites derived from U-13 C 6 -glucose were double those observed in PBS-treated HSCs ( Figure   235 1B-C; Supplemental Figure 2B). The total levels of TCA cycle intermediates derived from U-13 C 6 -236 glucose were similar between PBS-and 5-FU-treated cells ( Figure 1D; Supplemental Figure 2B).

237
Levels of U-13 C 6 -glucose-derived intermediates involved in the pentose phosphate pathway (PPP) 238 and nucleic acid synthesis (NAS) were two-fold higher in 5-FU-treated than in PBS-treated HSCs, 239 whereas no significant differences in the levels of metabolites were observed between both groups 240 ( Figure 1E-F; Supplemental Figure 2B). Notably, the labeling rate of metabolites during the first half 241 of glycolysis was almost 100% in both groups, allowing us to easily track the labeled metabolites 242 (Supplemental Figure 2C-E). This was thought to be due to the rapid replacement of unlabeled 243 metabolites with labeled metabolites during exposure to U-13 C 6 -glucose because of the generally 244 rapid glycolytic reaction. Next, we evaluated whether glucose uptake in HSCs after 5-FU 245 administration was differentially affected by the expression of EPCR. The fluorescent analog of 246 glucose, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), was 247 administered intravenously to mice 50 and its uptake in EPCR + and EPCR -HSCs was assayed ( Figure   248 1G). Regardless of the EPCR expression, the 2-NBDG uptake was greater in HSCs treated with 5-FU 249 than in those treated with PBS ( Figure 1H-J). Furthermore, compared with HSCs cultured under 250 quiescence-maintaining conditions, those cultured under proliferative conditions were more resistant 251 to OXPHOS inhibition by oligomycin (Supplemental Figure 1H; Table S1). Overall, the results

252
showed that 5-FU-treated HSCs exhibited activated glycolytic flux, increasing the turnover of ATP.

253
Moreover, glycolytic flux into mitochondria was equally unchanged in PBS-and 5-FU-treated-HSCs, 254 supporting that 5-FU activated anaerobic glycolysis in HSCs. oligomycin-treated HSCs ( Figure 2A; Table S3). Similar to 5-FU-treated HSCs (Figure 1 Table S4). This increase in metabolic flux upstream of the 295 glycolytic pathway was also supported by our in vitro tracer analysis ( Figure 1B and Figure 2B), 296 suggesting that 13 C-MFA was a valid metabolic simulation. Among the reactions in the first half of   Figure 4B-I, Table S5). When the amount of U-13 C 6 -glucose-derived labeled metabolites in each 313 pathway was calculated, more glucose-derived metabolites entered TCA cycle in the 5-FU-treated 314 group than PBS-treated group (Supplemental Figure 4J). Thus, although short-term (10-30 min) in 315 vitro tracer analysis showed that HSCs exhibited more potent activation of anaerobic glycolysis than 316 of other pathways in response to 5-FU administration, long-term (approx.   Figure 6G). These analyses suggest that ATP was produced by mitochondrial 384 OXPHOS in steady-state HSCs, and that only HSCs, but not HPCs, maintained ATP production by 385 glycolysis when OXPHOS was compromised.  Figure 6L). This may explain differences in AMPK-dependent ATP production between proliferative 399 HSCs and HSCs under OXPHOS inhibition.

473
Pfkfb3-or Rosa-KO HSPC-derived blood cells were almost equally present, suggesting that the loss 474 of PFKFB3 did not affect steady-state blood cell production ( Figure 7E). However, after 5-FU 475 administration, Pfkfb3-KO HSPC-derived blood cell abundance was reduced compared to that in the 476 Rosa group ( Figure 7E). This change occurred on day 6 after 5-FU administration (day 1), when the   probably because they regain a quiescent state. This is consistent with the fact that inhibition of 559 PFKFB3 in quiescent HSCs does not reduce the ATP concentration ( Figure 5F, H) suggesting that even if PFKFB3 is activated by one stress (in this case, proliferative), it has the 571 activation capacity to respond to a different stress (i.e., mitochondrial). Therefore, the functions of 572 phosphorylated and methylated forms of PFKFB3 are to some extent interchangeable, and either 573 modification can be used to handle diverse stresses.

574
In summary, we found that HSCs exhibit a highly dynamic range of glycolytic flux. Our study 575 highlights glycolysis as a pivotal source of energy production in stressed HSCs, and indicates that