Palmitoylation of GNA13 Is Essential for Its Tumor Suppressor Function and Its Inhibition Confers Hypersensitivity of B-Cell Lymphoma to BCL2 Inhibitors

GNA13, encoding G alpha subunit 13 protein, is a major tumor suppressor gene frequently mutated in germinal center B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) and Burkitt's lymphoma (BL). GNA13 is one of the key mediators of sphingosine-1-phosphate (S1P) signaling to confine B cells in germinal center. Inactivation of GNA13 coupled signaling could release tumor B cells from germinal center of lymphoid organs to peripheral blood and promote lymphomagenesis. Due to the "undruggable" nature of GNA13 as a tumor suppressor, targeted therapy remains an unsolved clinical issue.

Protein palmitoylation is a unique post-translational lipid modification in regulating protein trafficking, stability and functionality. Emerging evidence reveals that palmitoylation plays vital roles in control of G-protein-coupled receptor (GPCR) signaling transduction. In this study, we discovered that GNA13 protein relies on amino-terminal palmitoylation for its membrane association and signal transduction, which is essential for its tumor suppressor activities. By using a Isobaric iodoTMT switch labeling based mass spectrometry method, we firstly identified that palmitoylation mainly occurs on two cysteine residues, Cys14 and Cys18, one of which site was also found mutated in DLBCL patients. To validate this result, we genetically mutated the cysteine to serine residues on these two potential palmitoylation sites, respectively. We found that both single and double mutants could largely reduce the palmitoylation level of GNA13. We also found that the loss of palmitoylation dissociates GNA13 protein from cell plasma membrane. Similar results were obtained using a pan-palmitoylation inhibitor, 2-bromopalmitate (2-BP).

In order to examine the role of GNA13 palmitoylation in human DLBCL cells, we reintroduced either wildtype (wt) GNA13 or palmitoylation mutant GNA13 into Su-DHL-4 lymphoma cells after knocking down the endogenous wt GNA13 expression with short hairpin RNA interference. Our data show that the loss of palmitoylation promotes GCB-DLBCL cell proliferation and tumor growth both in vitro and in vivo, indicating that palmitoylation of GNA13 is essential to its tumor suppressor function. Mechanically, inactivation of GNA13, either by knocking down GNA13 expression or mutating the palmitoylation sites of GNA13, leads to phosphoinositide 3-kinase (PI3K)/AKT activation and BCL2 overexpression. Consistent to the increased BCL2 expression, we found that BCL2 inhibitors are among the most effective drugs to kill GNA13-deficient cells in a high-throughput chemical screen. Furthermore, we show that inhibition of palmitoylation by 2-BP enhances the drug sensitivity of GNA13 wildtype GCB-DLBCL cells to BCL2 inhibitors. These results indicate that the palmitoylation of GNA13 could serve as a target for treating B-cell lymphoma in combination with a BCL2 inhibitor.