Zebrafish polg2 knock-out recapitulates human POLG-disorders; implications for drug treatment.

The human mitochondrial DNA polymerase gamma is a holoenzyme, involved in mitochondrial DNA (mtDNA) replication and maintenance, composed of a catalytic subunit (POLG) and a dimeric accessory subunit (POLG2) conferring processivity. Mutations in POLG or POLG2 cause POLG-related diseases in humans, leading to a subset of Mendelian-inherited mitochondrial disorders characterized by mtDNA depletion (MDD) or accumulation of multiple deletions, presenting multi-organ defects and often leading to premature death at a young age. Considering the paucity of POLG2 models, we have generated a stable zebrafish polg2 mutant line (polg2ia304) by CRISPR/Cas9 technology, carrying a 10-nucleotide deletion with frameshift mutation and premature stop codon. Zebrafish polg2 homozygous mutants present slower development and decreased viability compared to wild type siblings, dying before the juvenile stage. Mutants display a set of POLG-related phenotypes comparable to the symptoms of human patients affected by POLG-related diseases, including remarkable MDD, altered mitochondrial network and dynamics, and reduced mitochondrial respiration. Histological analyses detected morphological alterations in high-energy demanding tissues, along with a significant disorganization of skeletal muscle fibres. Consistent with the last finding, locomotor assays highlighted a decreased larval motility. Of note, treatment with the Clofilium tosylate drug, previously shown to be effective in POLG models, could partially rescue MDD in Polg2 mutant animals. Altogether, our results point at zebrafish as an effective model to study the etiopathology of human POLG-related disorders linked to POLG2, and a suitable platform to screen the efficacy of POLG-directed drugs in POLG2-associated forms.

The Use of Hexokinase 2-Displacing Peptides as an Anti-Neoplastic Approach for Malignant Peripheral Nerve Sheath Tumors.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are aggressive sarcomas that can arise both sporadically and in patients with the genetic syndrome Neurofibromatosis type 1 (NF1). Prognosis is dismal, as large dimensions, risk of relapse, and anatomical localization make surgery poorly effective, and no therapy is known. Hence, the identification of MPNST molecular features that could be hit in an efficient and selective way is mandatory to envision treatment options. Here, we find that MPNSTs express high levels of the glycolytic enzyme Hexokinase 2 (HK2), which is known to shield cancer cells from noxious stimuli when it localizes at MAMs (mitochondria-associated membranes), contact sites between mitochondria and endoplasmic reticulum. A HK2-targeting peptide that dislodges HK2 from MAMs rapidly induces a massive death of MPNST cells. After identifying different matrix metalloproteases (MMPs) expressed in the MPNST microenvironment, we have designed HK2-targeting peptide variants that harbor cleavage sites for these MMPs, making such peptides activatable in the proximity of cancer cells. We find that the peptide carrying the MMP2/9 cleavage site is the most effective, both in inhibiting the in vitro tumorigenicity of MPNST cells and in hampering their growth in mice. Our data indicate that detaching HK2 from MAMs could pave the way for a novel anti-MPNST therapeutic strategy, which could be flexibly adapted to the protease expression features of the tumor microenvironment.