Restoring the Lost B-Cell Phenotype Renders Classical Hodgkin's Lymphoma Susceptible to B-Cell-Targeting Therapies
Authors: Neuendorff Nina Rosa , Du Jing , Yu Yong , von Kries Jens Peter , Mathas Stephan , Dörken Bernd , Lee Soyoung , Schmitt Clemens A. 
Published: November 2019

Abstract
Introduction: Although originating from B-cells, classical Hodgkin's lymphoma (cHL) is characterized by the virtual lack of gene products whose expression constitutes the B-cell phenotype. Epigenetic repression of B-cell-specific genes via promoter hypermethylation and histone deacetylation as well as compromised expression of B-cell-committed transcription factors were previously reported to contribute to the lost B-cell phenotype in cHL. Restoration of the lost B cell phenotype may not only change one of the functional hallmarks of cHL but provide a new Achilles heel by sensitizing cHL to clinically established antibody therapies targeting B-cell surface receptors as well as small compounds interfering with B-cell receptor (BCR) signaling.
Methods: A high-throughput pharmacological screening based on more than 28,000 compounds was conducted in cHL cell lines carrying a CD19 reporter for identification of drugs promoting re-expression of the B-cell phenotype. Furthermore, the co-therapeutic potential of B-cell-targeting antibodies and small compounds was exploited. Pharmacological and genetic inhibition of EHMT2 was performed by exposure to BIX-01294 or shRNA-mediated knockdown.
Results: Three chemicals that robustly enhanced CD19 transcription were retrieved by our high-throughput pharmacological screening. Since two of these compounds reportedly interfere with epigenetic regulators, we subsequently performed chromatin immunoprecipitation (ChIP)-based analyses showing that these compounds lowered transcriptionally repressive lysine 9-trimethylated histone H3 (H3K9me3) levels at the CD19 promoter. Inhibition of the H3K9-methyltransferase EHMT2, a possible target structure of these two compounds, by BIX-01294 or shRNA-mediated knockdown resulted in increased CD19 transcript levels, suggesting that this enzyme might be involved in repression of the B-cell phenotype in cHL. Furthermore, the anti-leukemic and differentiation-promoting agents arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) were found to reconstitute the silenced B-cell transcriptional program and impair viability of cHL cell lines. In combination with a screening-identified chemical, ATO evoked re-expression of the CD20 surface receptor, which could be further therapeutically exploited by enabling CD20 antibody-mediated direct apoptosis and antibody-dependent cellular cytotoxicity of Hodgkin cells. Furthermore, restoration of the B-cell phenotype profoundly sensitized cHL cells towards the B-cell Non-Hodgkin's lymphoma (B-NHL)-tailored small compound inhibitors of BCR signaling, Ibrutinib and Idelalisib.
Conclusions: We identified several compounds inducing re-expression of B-cell-specific genes based on a high-throughput pharmacological screening followed by genetic and pharmacological validation. Restoration of components of the B-cell phenotype sensitized cHL cells to B-cell targeting therapeutics as a conceptually novel and promising non-chemotherapeutic treatment strategy for this lymphoma entity.

Disclosures
Schmitt: Roche: Consultancy, Honoraria.