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P019 (0158) FUNCTIONAL ANALYSIS OF CD30 IN HODGKIN AND ANAPLASTIC LARGE CELL LYMPHOMA CELL LINES BY CRISPR/CAS9-MEDIATED GENE KNOCKOUT

doi: 10.1097/01.HS9.0000547868.14041.f9
Biology and Microenvironment

Annika L. Weiß, Anna Lollies, Freya Kretzmer, Marc A. Weniger, Ralf Küppers

Institute for Cell Biology (Cancer Research), University of Duisburg-Essen, Essen, Germany

The tumor necrosis factor receptor family member CD30 is highly expressed on the malignant Hodgkin- and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL), but its role in the pathogenesis of this disease is controversially discussed. Based on the postulation of ligand-independent activity of CD30 signaling in HRS cells, high activity of the NF-κB and AP-1 signaling pathways has been linked to constitutive CD30 signaling. However, knockdown of CD30 in HRS cells lines led to contradicting results regarding its effect on cell viability, questioning the impact of CD30 signaling on this lymphoma entity.

To resolve this issue, we established and optimized the CRISPR/Cas9 system in cHL and anaplastic large cell lymphoma (ALCL) cell lines to achieve a complete knockout of CD30 and characterize the phenotype of CD30-depleted lymphoma cells. Flow cytometric analysis confirmed downregulation of CD30 surface expression. Genetic alterations were proven by T7 endonuclease assay and the type of mutation was analyzed on single alleles by cloning and sequencing, confirming homozygous knockout of CD30. An increased fraction of apoptotic cells was found in cHL and ALCL cell cultures after knockout of CD30 compared to CD30-positive HRS cells in the same culture as well as compared to cultures treated with a non-targeting sgRNA.

These results are evidence of the importance of CD30 for cHL and ALCL. Based on this we will examine the molecular and functional mechanisms of CD30 signaling and its interaction with the main pathways driving cHL lymphomagenesis.

Copyright © 2018 The Authors. Published by Wolters Kluwer Health Inc., on behalf of the European Hematology Association.