The Mitochondrial Protease, Neurolysin (NLN), Regulates Respiratory Chain Supercomplex Formation and Represents a New Therapeutic Target for AML
Bibliographic record
Abstract
Abstract Our group and others have shown that acute myeloid leukemia (AML) cells have unique mitochondrial characteristics with an increased reliance on oxidative phosphorylation. Through an shRNA screen for new biological vulnerabilities in the mitochondria of AML cells, we identified the mitochondrial protease, neurolysin (NLN). NLN is a zinc metalloprotease whose mitochondrial function is not well understood and whose role in AML growth and viability has not been previously reported. We analyzed the expression of NLN in AML cells and normal hematopoietic cells. By immunoblotting, NLN was overexpressed in 80% of primary AML patient samples compared to normal hematopoietic cells. Likewise, in an analysis of gene expression databases, NLN mRNA was increased in a subset of AML patient samples, compared to normal hematopoietic cells. Next, we assessed the effects of knocking down NLN in AML cell lines (OCI-AML2, NB4, and MV4-11) using three independent shRNAs in lentiviral vectors. Target knockdown was confirmed by immunoblotting. NLN knockdown reduced growth in all three tested cell lines by 50-70%. NLN knockdown also targeted the leukemia initiating cells in vitro and in vivo as NLN knockdown reduced the clonogenic growth of AML cells (40-75%) and the engraftment of TEX cells into immune deficient mice by 85%. Taken together, these data suggest that NLN is necessary for the growth of AML cells. The role of NLN in the mitochondria is not well understood. To gain insight into NLN's mitochondrial function, we investigated NLN's protein interactors using proximity-dependent biotin labeling (BioID). The top hits in the protein-protein interaction screen were mitochondrial matrix proteins and respiratory chain subunits were particularly enriched. Therefore, we measured the effects of NLN knockdown on mitochondrial structure and function. Knockdown of NLN in AML cells reduced basal oxygen consumption without altering reactive oxygen species generation, mitochondrial membrane potential, or mitochondrial mass. No changes were seen in the total levels of respiratory chain complex subunits as measured by immunoblotting on denaturing gels. Respiratory chain complexes assemble into higher order supercomplex structures that maintain the integrity of the mitochondria and promote efficient oxidative metabolism. Therefore, we tested whether NLN is required for the formation of respiratory chain supercomplexes. As measured by blue native polyacrylamide gel electrophoresis, knockdown of NLN impaired the formation of respiratory chain supercomplexes. Through our BioID analysis, we also identified the mitochondrial Ca2+/H+ antiporter, LETM1 (leucine zipper-EF-hand containing transmembrane protein 1) as a top interactor with NLN. LETM1 is a known regulator of respiratory chain supercomplex formation. We showed that knockdown of NLN impaired LETM1 assembly, potentially explaining how NLN regulates supercomplex formation. Finally, we tested if hypoxia influences respiratory chain supercomplex formation and sensitivity to NLN inhibition. OCI-AML2 cells cultured for 72 hours under hypoxic conditions (0.2% O2) showed impaired assembly of respiratory chain supercomplexes, decreased levels of LETM1 protein, and resistance to NLN knockdown. Thus, we discovered that the mitochondrial protease NLN regulates oxidative metabolism by controlling the assembly of respiratory chain supercomplexes. Moreover, we highlight NLN as a potential new therapeutic target for AML. Disclosures Schimmer: Otsuka Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medivir AB: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.
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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".