A Germline Mutation in Cathepsin B in a Child with ALL Points towards a Key Role for This Enzyme in L-Asparaginase Pharmacokinetics

• Published in: Blood 2012-11, Vol.120 (21), p.2458-2458
• Main Authors: van der Meer, Laurens T., Waanders, Esmé, Levers, Marloes, Venselaar, Hanka, Roeleveld, Debbie, Boos, Joachim, Lanvers-Kaminsky, Claudia, Kuiper, Roland P., Hoogerbrugge, Peter M., van Leeuwen, Frank N., te Loo, D. Maroeska W.M.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V120.21.2458.2458

Abstract 2458▪▪This icon denotes a clinically relevant abstract The bacterially derived enzyme L-Asparaginase (ASNase) is a key component in the multidrug therapy regimens used worldwide to treat pediatric and adult patients with acute lymphoblastic leukemia (ALL), however little is known about the molecular mechanisms that control the pharmacokinetics of this therapeutic protein. As a result, many patients who receive a standardized dose either exceed or do not reach the desired serum concentration. While elevated serum levels are associated with an increase in treatment related morbidity, underexposure seriously compromises therapeutic benefits. In search of molecular factors that determine ASNase turnover in vivo, we investigated a patient with strongly aberrant clearance kinetics. This 3-year old female diagnosed with common ALL suffered from severe ASNase-induced adverse events upon treatment with ErwiniaSNase as a result of strongly elevated serum ASNase levels. Pharmacokinetics data showed a severely delayed ASNase clearance. As a result, serum ASNase levels accumulated to intolerable levels upon repeated administration of the drug. We isolated DNA from peripheral blood mononuclear cells and buccal cells of this patient and performed targeted sequencing on genes suggested to be involved in ASNase clearance. We identified a novel heterozygous mutation in the gene encoding Cathepsin B in the germline of this patient. The mutant allele shows a deletion of a single codon, leading to a deletion of a lysine residue in the C terminus of the protein. We generated an EBV LCL cell line from this patients which showed a 75% reduction in Cathepsin B activity, relative to controls, indicating that this heterozygous mutation has a profound effect on the total Cathepsin B activity. Cathepsin B is normally synthesized as a 37 kD pre-pro enzyme and is processed in a two step process into a mature 2-chain active form. During this process, the protein is transported to the lysosome where it exerts its primary function. Using a combination of biochemical and imaging experiments we show that the mutant Cathepsin B cannot be processed into the mature form and is retained in the endoplasmatic reticulum. ASNase degradation assays demonstrate that this mutant form of Cathepsin B shows a diminished protease activity towards both E.coli and Erwinia ASNase, consistent with the reduced clearance observed in our patient. Cathepsin B and other cellular proteases are either active