GRK2 suppresses lymphomagenesis by inhibiting the MALT1 proto-oncoprotein
Jing Cheng, … , Peter C. Lucas, Linda M. McAllister-Lucas
Jing Cheng, … , Peter C. Lucas, Linda M. McAllister-Lucas
Published January 21, 2020
Citation Information: J Clin Invest. 2020;130(2):1036-1051. https://doi.org/10.1172/JCI97040.
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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 8

shRNA knockdown or CRISPR/Cas9 knockout of GRK2 in ABC-DLBCL (OCI-Ly3) cells leads to enhanced MALT1-mediated IκB phosphorylation, RELB and CYLD cleavage, cytokine secretion, and cell proliferation, and GRK2 rescue reverses these effects.

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shRNA knockdown or CRISPR/Cas9 knockout of GRK2 in ABC-DLBCL (OCI-Ly3) c...
(A) GRK2 knockdown (KD) (left) or knockout (KO) (middle) leads to increased basal IκB phosphorylation in ABC-DLBCL line OCI-Ly3. Stable OCI-Ly3 cells with GRK2 KD were made using specific shRNA lentivirus. GRK2 KOs were made using Cas9/gRNA. GRK2 KD or KO was confirmed by Western blot. Rescue of GRK2 in GRK2-KO cells reverses this phenotype (right). Blots are representative of at least 3 experiments. (B) GRK2 KD (left) or KO (middle) in OCI-Ly3 cells leads to increased cleavage of RELB and CYLD. Rescue of GRK2 in GRK2-KO cells reverses this phenotype (right). Blots shown are representative of 3 experiments. Clv, cleaved; FL, full-length. (C and D) GRK2 KD (left) leads to increased IL-6 and IL-10 production in OCI-Ly3 cells. Control clones (1, 2, and 3) and GRK2-KD clones (1E, 2C, and 2D) were analyzed separately (total of n = 6 for both control and GRK2 KD). GRK2 KO (middle) leads to increased IL-6 and IL-10 production in OCI-Ly3 cells (n = 3). Rescue of GRK2 in GRK2-KO cells (right) reverses this phenotype (n = 3). IL-6 and IL-10 secretion was determined by ELISA. All values are represented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001, by unpaired, 2-tailed Student’s t test. (E) GRK2 KD (left) or KO (middle) leads to increased proliferation of OCI-Ly3 cells. Rescue of GRK2 in GRK2-KO cells reverses this phenotype (right) (n = 3). Two-way ANOVA and Šidák’s multiple-comparisons test were performed to show proliferation differences between the GRK2-KD or -KO and control groups (only significance for the end time points is shown). ***P < 0.001.