GRK2 suppresses lymphomagenesis by inhibiting the MALT1 proto-oncoprotein
Jing Cheng, Linda R. Klei, Nathaniel E. Hubel, Ming Zhang, Rebekka Schairer, Lisa M. Maurer, Hanna B. Klei, Heejae Kang, Vincent J. Concel, Phillip C. Delekta, Eric V. Dang, Michelle A. Mintz, Mathijs Baens, Jason G. Cyster, Narayanan Parameswaran, Margot Thome, Peter C. Lucas, Linda M. McAllister-Lucas
Jing Cheng, Linda R. Klei, Nathaniel E. Hubel, Ming Zhang, Rebekka Schairer, Lisa M. Maurer, Hanna B. Klei, Heejae Kang, Vincent J. Concel, Phillip C. Delekta, Eric V. Dang, Michelle A. Mintz, Mathijs Baens, Jason G. Cyster, Narayanan Parameswaran, Margot Thome, Peter C. Lucas, Linda M. McAllister-Lucas
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Research Article Immunology Oncology

GRK2 suppresses lymphomagenesis by inhibiting the MALT1 proto-oncoprotein

Abstract

Antigen receptor–dependent (AgR-dependent) stimulation of the NF-κB transcription factor in lymphocytes is a required event during adaptive immune response, but dysregulated activation of this signaling pathway can lead to lymphoma. AgR stimulation promotes assembly of the CARMA1-BCL10-MALT1 complex, wherein MALT1 acts as (a) a scaffold to recruit components of the canonical NF-κB machinery and (b) a protease to cleave and inactivate specific substrates, including negative regulators of NF-κB. In multiple lymphoma subtypes, malignant B cells hijack AgR signaling pathways to promote their own growth and survival, and inhibiting MALT1 reduces the viability and growth of these tumors. As such, MALT1 has emerged as a potential pharmaceutical target. Here, we identified G protein–coupled receptor kinase 2 (GRK2) as a new MALT1-interacting protein. We demonstrated that GRK2 binds the death domain of MALT1 and inhibits MALT1 scaffolding and proteolytic activities. We found that lower GRK2 levels in activated B cell–type diffuse large B cell lymphoma (ABC-DLBCL) are associated with reduced survival, and that GRK2 knockdown enhances ABC-DLBCL tumor growth in vitro and in vivo. Together, our findings suggest that GRK2 can function as a tumor suppressor by inhibiting MALT1 and provide a roadmap for developing new strategies to inhibit MALT1-dependent lymphomagenesis.

Authors

Jing Cheng, Linda R. Klei, Nathaniel E. Hubel, Ming Zhang, Rebekka Schairer, Lisa M. Maurer, Hanna B. Klei, Heejae Kang, Vincent J. Concel, Phillip C. Delekta, Eric V. Dang, Michelle A. Mintz, Mathijs Baens, Jason G. Cyster, Narayanan Parameswaran, Margot Thome, Peter C. Lucas, Linda M. McAllister-Lucas

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Figure 5

GRK2 CRISPR/Cas9 knockout leads to enhanced MALT1-dependent activities in Jurkat T cells, and rescue of GRK2 reverses this phenotype.

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GRK2 CRISPR/Cas9 knockout leads to enhanced MALT1-dependent activities i...
(A) GRK2 knockout (KO) leads to increased IκB phosphorylation after P/I (left) or anti-CD3/CD28 (middle) but not after TNF (right) stimulation in Jurkat T cells. GRK2-KO Jurkat T cells were made using Cas9/gRNA. GRK2 knockout was confirmed by Western blot. Blots are representative of at least 3 experiments. (B) GRK2 KO in Jurkat T cells leads to increased cleavage of RELB after P/I (top) or anti-CD3/CD28 (bottom) stimulation. Blots shown are representative of n = 4. Clv, cleaved; FL, full-length. Quantification of cleavage is shown to the right of the blots. *P < 0.05, **P < 0.01, ****P < 0.0001. (C) GRK2 KO leads to increased IL-2 production in Jurkat T cells after P/I (left) or anti-CD3/CD28 (right) stimulation. IL-2 secretion was determined by ELISA (n = 3). ****P < 0.0001. (D) GRK2 rescue in GRK2-KO Jurkat T cells reverses the enhanced IκB phosphorylation after P/I (left) or anti-CD3/CD28 (middle) but has no effect on the TNF response (right). GRK2-KO Jurkat T cells were rescued using lentivirus expressing WT GRK2, and stable cell lines were made by selection using puromycin. GRK2 rescue was confirmed by Western blot. Blots are representative of at least 3 experiments. (E) Rescue of GRK2 in GRK2-KO Jurkat T cells reverses the enhanced cleavage of RELB caused by GRK2 KO. Blots shown are representative of n = 4. Quantification of cleavage is shown to the right of the blots. *P < 0.05, ***P < 0.001. (F) GRK2 rescue leads to reduced IL-2 production in GRK2-KO Jurkat T cells. IL-2 secretion was determined by ELISA (n = 3–4). All values are represented as mean ± SEM. ****P < 0.0001. Statistical significance for B, C, E, and F was evaluated by 2-way ANOVA, followed by Tukey’s multiple-comparisons test.