A Germline Mutation in Cathepsin B in a Child with ALL Points towards a Key Role for This Enzyme in L-Asparaginase Pharmacokinetics.
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. 
Published: October 2019

Abstract 2458

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 actively secreted or released into the serum as a result of cell lysis. Although we find a variable low but detectable activity of Cathepin B in serum samples, all tested preparations of ASNase were stable upon prolonged incubation in serum, suggesting that serum components are not contributing to ASNase clearance in vivo. Hence, we propose that cellular uptake and subsequent proteolytic degradation of ASNase is the primary mechanism of clearance.

In conclusion, we have identified a mutation in protease Cathepsin B and provide evidence that this mutation results in a loss of protease function towards ASNase, which can explain the strongly delayed clearance of ASNase in the patient. Our data suggest that differences in Cathepsin B activity may contribute to the large inter-patient variability in ASNase pharmacokinetics. Furthermore, given the role of Cathepsin proteases in antigen presentation, Cathepsin B may not only provide a target for predicting or controlling ASNase clearance kinetics but inhibition of Cathepsin may also prevent or delay the formation of inhibitory antibodies.

Disclosures:

Boos: European Erwinase Providers (EUSAPharm): Speakers Bureau; Medac: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lanvers-Kaminsky:Medac: Speakers Bureau.