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Dissection of CD20 regulation in lymphoma using RNAi

Leukemia volume 30, pages 2409–2412 (2016)Cite this article

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CD20, encoded by MS4A1, is a cell surface glycoprotein involved in calcium signaling,1 expressed on normal and malignant B cells. Antibodies targeting CD20 (for example, rituximab) are used for the treatment of B-cell lymphoma, and mediate antitumor response by complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity and direct induction of apoptosis.2 Resistance to anti-CD20 antibodies remains a clinical challenge. Transcription factors like SPI1/PU.1,3 Oct2(ref. 4) and Pax5(ref. 5) have been described in MS4A1 regulation, however, other factors (e.g., epigenetic changes, somatic mutations, noncoding RNA) could also affect the CD20 expression levels.

CREM (cAMP-responsive element modulator) was identified as a top candidate for CD20 suppression (Figures 1b and c). Three non-overlapping shRNAs targeting CREM efficiently decreased CREM mRNA and protein levels, and resulted in upregulation of surface CD20 levels (Supplementary Figures S6 and S7) across 6 out of 7 lymphoma cell lines (Supplementary Figure S8). To assess the specificity of CREM knockdown, we examined additional B-cell surface receptors (CD81, CD79b, CD19, CD22, CD38 and CD72). Only the level of CD72 expression also increased after CREM knockdown, albeit to a lower extent (Supplementary Figure S7c) suggesting a specific role of CREM for CD20 regulation. As the promoter region of CD20 contains three half-cAMP response elements sites, which were previously reported as binding motives for CREM in primary T cells6 (Supplementary Figure S9a), we investigated CREM-binding capacity to the CD20 promoter. We used two different CREM antibodies and SPI1 antibodies as a positive control.3 Our ChIP-PCR showed that CREM binds the MS4A1 promoter region (Supplementary Figure S9b); however, we did not observe an increase in promoter activity upon CREM knockdown (Supplementary Figure S9c). Taken together, our work associates the transcriptional repressor CREM with the regulation of MS4A1.

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Acknowledgements

We thank the FACS Core Facility, and DKFZ Genomics and Proteomics Core Facility, especially Steffen Schmitt and Sabine Schmidt for their technical support and expertise; Marc S Raab for the multiple myeloma cell lines; Christof von Kalle, Claudia Scholl, Hanno Glimm, Holger Bierhoff, Ali Nowrouzi, Taronish Dubash, Klara Gießler and Katarzyna Mleczko-Sanecka for the discussion. Lentiviral shRNA libraries were kindly provided and developed by Cellecta Inc. based on NIH-funded research grant support 44RR024095, 44HG003355. This study makes use of data generated by the Blueprint Consortium. A full list of the investigators who contributed to the generation of the data is available from www.blueprint-epigenome.eu. Funding for the project was provided by the ‘Deutsche Krebshilfe’ (Mildred Scheel Professur), the Helmholtz association (Helmholtz Virtual Institute) and the European Union’s Seventh Framework Programme (FP7/2007-2013) under the grant agreement no 282510 BLUEPRINT.

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Authors and Affiliations

  1. Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany

    M Słabicki, K S Lee, A Jethwa, L Sellner, T Walther, J Hüllein, S Dietrich, B Wu, S Fröhling & T Zenz

  2. Department of Molecular Therapy in Haematology and Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany

    M Słabicki, K S Lee, A Jethwa, L Sellner, T Walther, J Hüllein, S Dietrich, B Wu & T Zenz

  3. Department of Medicine V, University Hospital Heidelberg, Heidelberg, Germany

    L Sellner, S Dietrich & T Zenz

  4. Department of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Martinsried, Germany

    F Sacco

  5. Department of Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

    S Dietrich, M Oleś, W Huber & T Zenz

  6. Department of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany

    D B Lipka, C C Oakes & C Plass

  7. Department of Internal Medicine, Division of Hematology, The Ohio State University, Columbus, OH, USA

    C C Oakes

  8. Department of Immunology, University of Heidelberg, Heidelberg, Germany

    S Mamidi & M Kirschfink

  9. Department of Immunology, Center for Biostructure Research, Medical University of Warsaw, Warsaw, Poland

    B Pyrzyńska, M Winiarska & J Gołąb

  10. Department of Cell Biology (Cancer Research), Medical School, University of Duisburg-Essen, Essen, Germany

    M Seifert

  11. Department of Functional Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany

    M Boettcher

  12. Department of Microbiology and Immunology, UCSF Diabetes Center, University of California, San Francisco, CA, USA

    M Boettcher

  13. Section for Personalized Oncology, Heidelberg University Hospital, Heidelberg, Germany

    S Fröhling

  14. DKTK, Heidelberg, Germany

    S Fröhling

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  1. M Słabicki
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  20. J Gołąb
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  21. W Huber
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Corresponding author

Correspondence to T Zenz.

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Additional information

Supplementary Information accompanies this paper on the Leukemia website

Supplementary information

Supplementary Information (DOC 51 kb)

Supplementary Materials and Methods (DOC 163 kb)

Supplementary Figure S1 (PDF 171 kb)

Supplementary Figure S2 (PDF 66 kb)

Supplementary Figure S3 (PDF 792 kb)

Supplementary Figure S4 (PDF 9 kb)

Supplementary Figure S5 (PDF 74 kb)

Supplementary Figure S6 (PDF 253 kb)

Supplementary Figure S7 (PDF 41 kb)

Supplementary Figure S8 (PDF 582 kb)

Supplementary Figure S9 (PDF 47 kb)

Supplementary Figure S10 (PDF 83 kb)

Supplementary Figure S11 (PDF 253 kb)

Supplementary Figure S12 (PDF 61 kb)

Supplementary Figure S13 (PDF 216 kb)

Supplementary Figure S14 (PDF 41 kb)

Supplementary Figure S15 (PDF 57 kb)

Supplementary Figure S16 (PDF 77 kb)

Supplementary Figure S17 (PDF 93 kb)

Supplementary Figure S18 (PDF 35 kb)

Supplementary Table S1 (XLS 38205 kb)

Supplementary Table S2 (XLS 318 kb)

Supplementary Table S3 (XLS 3441 kb)

Supplementary Table S4 (XLS 5859 kb)

Supplementary Table S5 (XLS 87 kb)

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Słabicki, M., Lee, K., Jethwa, A. et al. Dissection of CD20 regulation in lymphoma using RNAi. Leukemia 30, 2409–2412 (2016). https://doi.org/10.1038/leu.2016.230

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