The Metabolic Enzyme Hexokinase 2 Localizes to the Nucleus in AML and Normal Hematopoietic Stem/Progenitor Cells to Maintain Stemness
Bibliographic record
Abstract
Hematopoietic cells are arranged in a hierarchy where mature blood cells arise from stem and progenitor precursors. AML is also hierarchical with differentiated blasts arising from leukemic stem/progenitor cells. Recent studies show that metabolites can affect epigenetic marks; however, it is unknown whether metabolic enzymes can directly localize to the nucleus to regulate stemness in AML and normal hematopoietic cells. Here, we discovered that the mitochondrial enzyme, Hexokinase 2, localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stemness. Metabolic enzymes that localize to nucleus of stem cells were identified by evaluating stem and bulk fractions of OCI-AML-8227 leukemia cells, which are arranged in a hierarchy with functionally defined stem cells. We separated OCI-AML-8227 cells into CD34+38- and CD34-38+ populations by FACS and prepared nuclear and cytoplasmic lysates. Immunoblotting of the lysates revealed that the metabolic enzyme Hexokinase 2 (HK2) was increased in the nuclear fraction of 8227 stem cells compared to bulk cells. In contrast, other mitochondrial enzymes such as Enolase1, Aconitase2, and Succinate Dehydrogenase A & B, were not detected in the nuclear lysates. HK2 is an outer mitochondrial membrane protein that phosphorylates glucose to glucose-6-phosphate, initiating glycolysis. We confirmed nuclear HK2 in OCI-AML-8227 stem cells by confocal microscopy and also demonstrated nuclear HK2 in AML cell lines (OCI-AML2, NB4, K563, and MV411) and in 7 of 9 primary AML samples. We FACS sorted normal cord blood into populations of stem/progenitor (HSC, MPP, MLP, CMP, GMP and MEP) and differentiated (Monocytes, Granulocytes, B, T, and NK) cells. The localization of HK2 in these cells was analysed and quantified by immunofluorescence. Nuclear HK2 was detected in the stem/progenitor cells and progressively declined to minimal levels as cells matured. Next, we explored mechanisms that regulate nuclear localization of HK2. AKT-mediated phosphorylation of HK2 promoted localization to mitochondria while inhibition of phosphorylation increased its nuclear levels. Moreover, the nuclear import of HK2 was dependent on IPO5, a member of b-importin family that imports protein to the nucleus; CRM1 was responsible for HK2 nuclear export. We tested whether the nuclear localization of HK2 was functionally important to maintain stemness. We overexpressed HK2 tagged with nuclear localizing signals (PKKKRKV or PAAKRVKLD) in 8227 and NB4 leukemia cells. Selective overexpression of HK2 in the nucleus did not alter the rate of proliferation of the cells, however there was enhanced clonogenic growth and inhibition of retinoic acid-mediated cell differentiation. Conversely, we selectively reduced nuclear HK2 by expressing HK2 with an outer mitochondrial localization signal while knocking down endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 in AML cells did not alter growth rate, but did reduce clonogenic growth and increased differentiation after treatment with retinoic acid. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2(D209A D657A). Nuclear kinase dead HK2 increased clonogenic growth and inhibited differentiation after retinoic acid treatment, demonstrating that HK2 maintains stemness independent of kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry to identify proteins that interact with nuclear HK2. A top hit in our screen was Exonuclease 3'-5' domain containing 2 (EXD2), involved in DNA repair. Of note, DNA damage induces differentiation of AML cells. In 8227 cells, nuclear EXD2 was higher in the stem cell fraction compared to the bulk fraction. Moreover, knockdown of EXD2 reduced AML growth, clonogenic growth and decreased nuclear HK2 levels. Finally, nuclear HK2 overexpression conferred resistance to the PARP inhibitor, olaparib. In summary, we discovered that unphosphorylated HK2 localizes to the nucleus in malignant and normal hematopoietic stem cells. Through mechanisms independent of its kinase function, nuclear HK2 maintains AML cells in their stem/progenitor state potentially by regulating DNA damage and repair. Thus, we define a new role for a mitochondrial enzyme in the regulation of stemness and differentiation. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Schimmer:Medivir Pharmaceuticals: Research Funding; Otsuka Pharmaceuticals: Consultancy; Novartis Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".