| Yan Xiao
Acute Myeloid Leukemia (Acute Myeloid Leukemia, AML) is characterized by clonal proliferation of immature myeloid precursors accompanied by blocked differentiation. Similar to the role of normal hematopoietic , AML is organized in a hierarchical structure. leukemic stem cells (leukemic stem cells, LSCs) are responsible for replenishing large populations of AML cells and driving long-term clonal proliferation [1-2] . stem cells play a very important role in both normal and malignant hematopoiesis, but the factors regulating the growth and differentiation of LSCs and normal stem cells have not been fully elucidated.
The metabolic intermediate produced in mitochondria (including acetyl-CoA, α-ketoglutarate , S-adenosylmethionine , and nicotinamide adenine dinucleotide) is expressed by acting as the nuclear gene Genetically modified cofactors to regulate stem cell function and differentiation [3-4] . Mutations in the metabolic enzyme (such as IDH1 and IDH2) will produce tumor metabolites, leading to an increase in histone and DNA methylation , promoting the occurrence of leukemia and inhibiting differentiation [5-6] . Although studies have confirmed that metabolites regulate stem cell function, little is known about the mitochondrial metabolic enzymes located in the nucleus that directly affect gene expression, cell differentiation , and stem cell function.
Recently, Aaron D. Schimmer team from the Princess Margaret Cancer Center in Canada published online in the journal Nature Cell BiologyThe metabolic enzyme hexokinase 2 localizes to the nucleus in AML and normal haematopoietic and stem stem cells. genitor cells to maintain stemness article. The researchers found that hexokinase 2 (hexokinase 2, HK2), the initiating enzyme and rate-limiting enzyme in the glycolysis pathway, can be localized in the nuclei of AML and normal hematopoietic stem and progenitor cells. Nuclear HK2 regulates stem/progenitor cell function and differentiation in a manner independent of its kinase and metabolic functions. This study describes a non-classical mechanism by which a mitochondrial enzyme regulates gene expression and stem cell function.
In order to explore whether those metabolic enzymes that are traditionally located in mitochondria can moonlight in the nucleus and directly affect stem cell function and differentiation, the authors conducted experiments on 8227 cells (which is a low-passage primary AML model 【7】) Mitochondrial glycolytic enzymes and tricarboxylic acid cycle enzymes in were analyzed, and HK2 (the first enzyme in the glycolytic pathway, which converts glucose into ) was detected in the nucleus Glucose-6-phosphate ) . In contrast, other metabolic enzymes (including phosphofructokinase , fumarase, pyruvate kinase 2, etc.) were not detected in nuclear lysate. Subsequently, the authors also detected nuclear HK2 in seven primary AML samples. Furthermore, based on the level of reactive oxygen species , the authors subdivided primary AML samples into leukemia stem cells (stem population) and bulk population (bulk population) , and detected nuclei in these two groups of samples. HK2 level. Nuclear HK2 was significantly enhanced in stem cells compared with bulk cells.
So is nuclear HK2 required for AML stem and progenitor cell function? The authors constructed c-Myc (NLS1, PAAKRVKLD) or SV40 (NLS2, PKKKRKV) nuclear localization signal-tagged HK2 to selectively increase HK2 in the nucleus and found Nuclear HK2 overexpression can promote the implantation of 8227 cells into mouse bone marrow.8227 cells were collected from the bone marrow of the first recipient and transplanted to the second recipient. Overexpression of nuclear HK2 could still improve the implantation efficiency. OMMLS-HK2 (outer mitochondrial membrane-localizing signal, OMMLS) can selectively localize to mitochondria. Cells expressing OMMLS-HK2 were more resistant to 2-deoxy-D-glucose (2-DG) than control cells (Fig. 1a and b), indicating that the mitochondrial-tagged protein is metabolically active . Subsequently, the authors transduced cells overexpressing OMMLS-HK2 with shRNA targeting endogenous HK2 to eliminate nuclear HK2 while retaining HK2 in mitochondria (Fig. 1c). The results showed that after nuclear HK2 knockdown, (1) reduced the clonal growth of NB4 cells (Figure 1d) and 8227 LSCs (CD34+CD38-) number (Figure 1f) ; (2) reduced the number of AML cells Growth and transplantation in vivo; (3) Reduced expression of genes related to primitive/stem cell-like AML components and increased cell sensitivity to ATRA (all-trans-retinoic acid, all-trans- retinoic acid ) (Fig. 1d and e) ; (4) reduced the percentage of CD34+ CD38− stem cells after ATRA treatment (Fig. 1f) . These data demonstrate that nuclear HK2 is critical for stem cell function and differentiation in AML.
Figure 1. Knockdown of nuclear HK2 reduces stem and progenitor cell function
The authors also explored the nuclear expression and function of HK2 in normal hematopoietic stem and progenitor cells. The nuclear expression level and total level of HK2 in hematopoietic stem cells (hematopoietic stem cells, HSCs) and multipotent progenitor cells are higher, and decrease as the cells mature, and the expression of nuclear HK2 in differentiated cells is the smallest. Overexpression of nuclear HK2 in normal cord blood increases the rate of primary and secondary engraftment of these cells into mice. Furthermore, the authors constructed transgenic mice (Vav-NLS-HK2) , which overexpressed nuclear HK2, to selectively increase HK2 in of the hematopoietic system. The abundance and proliferation capacity of HSCs in the bone marrow of Vav-NLS-HK2 mice were significantly increased, indicating that nuclear HK2 is also important for the function of normal HSCs.
HK2 is a transferase kinase that phosphorylates glucose at the outer mitochondrial membrane to glucose-6-phosphate, initiating glycolysis . So is HK2 kinase activity required to maintain stem cell properties? Double mutations of Asp209 and Asp657 (D209A/D657A) will completely lose the kinase activity of HK2. The authors constructed a c-Myc NLS-tagged HK2 mutant (NLS1-HK2 D209A/D567A) . After overexpressing NLS1-HK2 D209A/D567A in cells, treatment with ATRA can still enhance clonal growth and block cell differentiation, which is similar to the phenotype of overexpressing wild-type nuclear HK2. Thus, nuclear HK2 maintains stem and progenitor cell function in a manner that is independent of its kinase and metabolic activities.
Finally, the authors analyzed the mechanism by which HK2 maintains cell stemness. Through BioID (proximity-dependent biotin labelling) combined with protein profiling, 12 proteins that interact with nuclear HK2 were initially screened, which are related to chromatin organization and regulation (CTR9, MAX, PHF8, PHF10 and SPIN1) , and transcriptional regulation (AASDH, CCNL2, IWS1 and ZNF136) and DNA damage response (SIRT1, TDP2 and UBR5) are related. Further analysis of (Proximity ligation assay, PLA) in situ confirmed the interaction between HK2 and MAX, SIRT1, IWS1, CTR9 and SPIN1. Subsequently, the authors found through ATAC-seq that overexpression of nuclear HK2 increased chromatin accessibility. HK2 interacts with MAX, which is related to cell proliferation, stem cell maintenance and differentiation, and DNA damage response. The authors therefore examined the DNA damage response in stem and bulk AML populations and explored whether nuclear HK2 affects DNA damage repair.After overexpressing nuclear HK2 in cells, cells were treated with daunorubicin (an intercalative chemotherapeutic agent that causes double-stranded DNA breaks); then, γH2AX (a surrogate marker for double-strand breaks) (a surrogate marker for double-stranded breaks), 53BP1 (the mediator of Guided non-homologous end joining ( repair) and RAD51HT3 ( homologous recombination to ) levels, double-stranded DNA breaks and DNA repair protein expression were detected at multiple time points after daunorubicin treatment. Results showed that nuclear HK2 overexpression increased 53BP1 and RAD51 levels and reduced the number of double-strand breaks. The authors also compared DNA damage responses in AML stem and bulk cells. Leukemic stem cells respond to DNA damage with increased expression of genes associated with homologous recombination and nonhomologous end joining.
In summary, This study describes the non-classical functions (part-time "moonlighting" function) of the mitochondrial metabolic enzyme HK2: (1) HK2 localizes to the nucleus of leukemia and normal hematopoietic stem cells; (2) nuclear HK2 overexpression can increase Leukemia stem cell properties and reduced differentiation; knockdown of nuclear HK2 promotes differentiation and reduces stem cell function; (3) Nuclear HK2 regulates stem cell function and differentiation independent of its kinase activity and metabolic function; (4) HK2 interacts with , which regulates chromatin openness The nuclear protein interacts to increase chromatin accessibility at DNA repair sites; (5) Nuclear HK2 overexpression reduces DNA double-strand breaks and confers chemical resistance, which helps to understand the mechanism by which leukemia stem cells resist DNA damaging agents. .
Original link:
https://doi.org/10.1038/s41556-022-00925-9
Plate maker: Eleven
References
1. Kreso, A. & Dick, J. E. Evolution of the cancer stem cell model. Cell Stem Cell 14, 275-291 (2014).
2. Gut, P. & Verdin, E Nature 502, 489-498 (2013).
4. Moussaieff, A. et al. Glycolysis-mediated changes in acetyl-CoA and histone acetylation control the early differentiation of embryonic stem cells. Cell Ward, P. S. et al. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting α-ketoglutarate to 2-hydroxyglutarate. Cancer Cell 17, 225 Lechman, E. Lechman, E. R. et al. miR-126 regulates distinct self-renewal outcomes in normal and malignant hematopoietic stem cells. Cancer Cell 29, 214-228 (2016).
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