Raptor mediates the selective inhibitory effect of cardamonin on RRAGC-mutant B cell lymphoma

Abstract

Background: mTORC1 (mechanistic target of rapamycin complex 1) is associated with lymphoma progression. Oncogenic RRAGC (Rag guanosine triphosphatase C) mutations identified in patients with follicular lymphoma facilitate the interaction between Raptor (regulatory protein associated with mTOR) and Rag GTPase. It promotes the activation of mTORC1 and accelerates lymphomagenesis. Cardamonin inhibits mTORC1 by decreasing the protein level of Raptor. In the present study, we investigated the inhibitory effect and possible mechanism of action of cardamonin in RRAGC-mutant lymphoma. This could provide a precise targeted therapy for lymphoma with RRAGC mutations.

Methods: Cell viability was measured using a cell counting kit-8 (CCK-8) assay. Protein expression and phosphorylation levels were determined using western blotting. The interactions of mTOR and Raptor with RagC were determined by co-immunoprecipitation. Cells overexpressing RagC wild-type (RagC^WT) and RagC Thr90Asn (RagC^T90N) were generated by lentiviral infection. Raptor knockdown was performed by lentivirus-mediated shRNA transduction. The in vivo anti-tumour effect of cardamonin was assessed in a xenograft model.

Results: Cardamonin disrupted mTOR complex interactions by decreasing Raptor protein levels. RagC^T90N overexpression via lentiviral infection increased cell proliferation and mTORC1 activation. The viability and tumour growth rate of RagC^T90N-mutant cells were more sensitive to cardamonin treatment than those of normal and RagC^WT cells. Cardamonin also exhibited a stronger inhibitory effect on the phosphorylation of mTOR and p70 S6 kinase 1 in RagC^T90N-mutant cells. Raptor knockdown abolishes the inhibitory effects of cardamonin on mTOR. An in vivo xenograft model demonstrated that the RagC^T90N-mutant showed significantly higher sensitivity to cardamonin treatment.

Conclusions: Cardamonin exerts selective therapeutic effects on RagC^T90N-mutant cells. Cardamonin can serve as a drug for individualised therapy for follicular lymphoma with RRAGC mutations.

Keywords: Cardamonin; Follicular lymphoma; RagC; Raptor; mTORC1.

Fig. 1

Viability of SUDHL-4 and OCI-Ly7 cells treated with inhibitory cardamonin. SUDHL-4 (A) and OCI-Ly7 (B) cells were treated with the indicated concentrations of cardamonin for 24 and 48 h. Cell viability was determined by CCK-8 assay. Data are presented as mean ± SD (n = 3). ^*p < 0.05, ^**p < 0.01 compared with the control; ^##p < 0.01 compared at 24 h

Fig. 2

Cardamonin inhibits mTORC1 signalling and reduces the Raptor level. SUDHL-4 (A) and OCI-Ly7 (B) cells were treated with cardamonin at the indicated concentrations for 24 h. Phosphorylation rates of mTOR, mTOR signalling activity, and protein expression of mTORC1 components were analysed using immunoblotting (n = 3)

Fig. 3

Cardamonin disrupts the interaction between RagC and mTOR. SUDHL-4 (A) and OCI-Ly7 cells (B) were treated with cardamonin (12 µM) or everolimus (0.5 µM) for 24 h. Immunoprecipitates pulled down by an anti-RagC antibody were collected. Cell lysates and immunoprecipitates were analysed by western blotting (n = 3)

Fig. 4

Effects of the RagC^T90N mutant on mTORC1 signalling. HEK-293T or SUDHL-4 cells were transfected with lentiviruses expressing FLAG-GFP, FLAG-RagC^WT and FLAG-RagC^T90N. (A) Immunoprecipitates pulled down by an anti-FLAG antibody from cells expressing the indicated cDNAs were collected. Cell lysates and immunoprecipitates were analysed by western blotting (n = 3). (B, C) Cell viability of normal, FLAG-RagC^WT- and FLAG-RagC^T90N-expressing HEK-293T and SUDHL-4 cells was determined by CCK-8 assay. Data are presented as mean ± SD (n = 3). ^**p < 0.01 compared with the normal group; ^##p < 0.01 compared with the RagC^WT group

Fig. 5

RRAGC mutation renders SUDHL-4 sensitive to cardamonin. Normal, RagC^WT and RagC^T90N-mutant HEK-293T or SUDHL-4 cells were treated with cardamonin (12 µM). (A) Immunoprecipitates pulled down by an anti-FLAG antibody were collected from cells expressing the indicated cDNAs. Cell lysates and immunoprecipitates were analysed by western blotting (n = 3). (B) Cell viability of normal, RagC^WT and RagC^T90N-mutant SUDHL-4 cells was determined by CCK-8 assay. Data are presented as mean ± SD (n = 3). ^**p < 0.01 compared with the control group; ^##p < 0.01 compared with the cardamonin-treated RagC^WT group. (C) RagC^WT and RagC^T90N-mutant SUDHL-4 cells were treated with cardamonin (12 µM) or everolimus (0.5 µM). Cell lysates were analysed by western blotting to measure the level of the indicated proteins (n = 3)

Fig. 6

Raptor shRNA abolishes the inhibitory effect of cardamonin on mTOR signalling in RRAGC-mutant cells. Protein expression of Raptor was knocked down by shRNA in RagC^T90N-mutant SUDHL-4 cells. Then, cells were treated with cardamonin (12 µM). Cell lysates were analysed by immunoblotting to measure the levels of the indicated proteins (n = 3)

Fig. 7

Cardamonin inhibits the growth of the RagC^T90N-mutant SUDHL-4 in vivo. Mice bearing normal or RagC^T90N mutant xenograft tumours (n = 5 for each group) were randomly assigned to two different groups: (1) control and (2) cardamonin (15 mg/kg) group. Tumour size was measured with a calliper, and the tumour volume was calculated. Results are presented as the tumour volume (mm³) ± SD. ^**p < 0.01 compared with the control or RagC^T90N-mutant-bearing mice

Fig. 8

Inhibition mechanism of cardamonin on the RagC^T90N-mutant cell. (Left) In normal cells, the “active” RagA·GTP-RagC·GDP heterodimer binds with Raptor, following the activation of mTOR in the presence of amino acids. (Right) RagC mutation increases its binding capacity with Raptor, which ultimately elicits hyperactivated mTORC1 signalling and enhancing the cell proliferation of lymphoma cells. Cardamonin decreases the protein level of Raptor and further disrupts the formation of mTOR activating complex. Pharmacological inhibition of mTORC1 by cardamonin exerts selective therapeutic effects on the RagC^T90N-mutant lymphoma cells