Neurolysin (NLN) is a zinc metallopeptidase whose mitochondrial function is unclear. We found that NLN was overexpressed in almost half of patients with acute myeloid leukemia (AML), and inhibition of NLN was selectively cytotoxic to AML cells and stem cells while sparing normal hematopoietic cells. Mechanistically, NLN interacted with the mitochondrial respiratory chain. Genetic and chemical inhibition of NLN impaired oxidative metabolism and disrupted the formation of respiratory chain supercomplexes (RCS). Furthermore, NLN interacted with the known RCS regulator, LETM1, and inhibition of NLN disrupted LETM1 complex formation. RCS were increased in patients with AML and positively correlated with NLN expression. These findings demonstrate that inhibiting RCS formation selectively targets AML cells and stem cells and highlights the therapeutic potential of pharmacologically targeting NLN in AML.

Mitochondrial metabolites regulate leukaemic and normal stem cells by affecting epigenetic marks. How mitochondrial enzymes localize to the nucleus to control stem cell function is less understood. We discovered that the mitochondrial metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus in leukaemic and normal haematopoietic stem cells. Overexpression of nuclear HK2 increases leukaemic stem cell properties and decreases differentiation, whereas selective nuclear HK2 knockdown promotes differentiation and decreases stem cell function. Nuclear HK2 localization is phosphorylation-dependent, requires active import and export, and regulates differentiation independently of its enzymatic activity. HK2 interacts with nuclear proteins regulating chromatin openness, increasing chromatin accessibilities at leukaemic stem cell-positive signature and DNA-repair sites. Nuclear HK2 overexpression decreases double-strand breaks and confers chemoresistance, which may contribute to the mechanism by which leukaemic stem cells resist DNA-damaging agents. Thus, we describe a non-canonical mechanism by which mitochondrial enzymes influence stem cell function independently of their metabolic function.

A number of unique proteases localize to specific sub‐compartments of the mitochondria, but the functions of these enzymes are poorly defined. Here, in vivo proximity‐dependent biotinylation (BioID) is used to map the interactomes of seven proteases localized to the mitochondrial intermembrane space (IMS). In total, 802 high confidence proximity interactions with 342 unique proteins are identified. While all seven proteases co‐localized with the IMS markers OPA1 and CLPB, 230 of the interacting partners are unique to just one or two protease bait proteins, highlighting the ability of BioID to differentiate unique interactomes within the confined space of the IMS. Notably, high‐temperature requirement peptidase 2 (HTRA2) interacts with eight of 13 components of the mitochondrial intermembrane space bridging (MIB) complex, a multiprotein assembly essential for the maintenance of mitochondrial cristae structure. Knockdown of HTRA2 disrupts cristae in HEK 293 and OCI‐AML2 cells, and leads to increased intracellular levels of the MIB subunit IMMT. Using a cell‐free assay it is demonstrated that HTRA2 can degrade recombinant IMMT but not two other core MIB complex subunits, SAMM50 and CHCHD3. The IMS protease interactome thus represents a rich dataset that can be mined to uncover novel IMS protease biology.