Cleavage of NIK by the API2-MALT1 fusion oncoprotein leads to noncanonical NF-kappaB activation

Abstract

Proper regulation of nuclear factor κB (NF-κB) transcriptional activity is required for normal lymphocyte function, and deregulated NF-κB signaling can facilitate lymphomagenesis. We demonstrate that the API2-MALT1 fusion oncoprotein created by the recurrent t(11;18)(q21;q21) in mucosa-associated lymphoid tissue (MALT) lymphoma induces proteolytic cleavage of NF-κB-inducing kinase (NIK) at arginine 325. NIK cleavage requires the concerted actions of both fusion partners and generates a C-terminal NIK fragment that retains kinase activity and is resistant to proteasomal degradation. The resulting deregulated NIK activity is associated with constitutive noncanonical NF-κB signaling, enhanced B cell adhesion, and apoptosis resistance. Our study reveals the gain-of-function proteolytic activity of a fusion oncoprotein and highlights the importance of the noncanonical NF-κB pathway in B lymphoproliferative disease.

Fig. 1

API2-MALT1 induces noncanonical NF-κB signaling through NIK. (A and B) HEK293T cells were transfected as indicated, and p100 processing to p52 was assessed by Western blot (WB) with α-Flag (A) or α-p100/52 (to detect endogenous p100/52) (B). Where indicated, cells were treated with proteasome inhibitor, MG132. (C) After transfection of HEK293T cells, nuclear extracts were prepared and analyzed for p52 and RelB by WB. (D) p100 processing in lysates from control SSK41 cells or SSK41 cells stably expressing API2-MALT1 was analyzed by WB. (E and F) HEK293T cells were transfected with API2-MALT1 in the absence or presence of NIK mutants. Endogenous p100 processing was analyzed by WB (E), or nuclear extracts were analyzed for the presence of p52 (F). * = nonspecific band, HDAC1 = histone deacetylase 1 (loading control for nuclear extract). Data are representative of at least three separate experiments.

Fig. 2

API2-MALT1 induces NIK cleavage, a phenomenon requiring both the API2 moiety and MALT1 protease activity. (A) HEK293T cells were transfected as indicated and NIK cleavage fragments were detected by WB. (B) BJAB cells expressing API2-MALT1 from a tetracycline inducible promoter were treated with doxycycline. WB with an antibody raised against a C-terminal NIK sequence revealed time-dependent generation of an endogenous 70 kD NIK cleavage fragment. To enhance detection of full-length (FL) NIK, cells were incubated with 25 µM MG132. (C) HEK293T cells were transfected as indicated and the presence of the N-terminal 37 kD and the C-terminal 70 kD NIK cleavage fragments was analyzed by WB. (D) Recombinant purified NIK-V5-bioC and StrepII-flag-tagged MALT1 were incubated in kosmotropic salt buffer for 6 h at 37°C, with or without 100µM MALT1 protease inhibitor, Ac-LSSR-CHO, and analyzed by WB. (E) HEK293Tcells were transfected as indicated and the 37 kD NIK cleavage fragment was detected by WB. (F) HEK293T cells were transfected, and immunoprecipitations were carried out using α-Flag-agarose. For a detailed description of API2-MALT1 mutants, see the legend to fig. S5. * = nonspecific band. Data are representative of at least three separate experiments.

Fig. 3

API2-MALT1-dependent cleavage of NIK at R325 generates an active C-terminal fragment. (A) HEK293T cells were transfected as indicated, and the 37 kD N-terminal NIK cleavage fragment was detected by WB. (B) HEK293T cells were transfected as indicated and nuclear translocation of p52 and p65 NF-κB subunits was assessed. (C and D) HEK293T cells were transfected as indicated, and endogenous p100 processing (C) and nuclear translocation of NF-κB subunits (D) were assessed. (E) HEK293T cells were transfected as indicated, and the ability of endogenous TRAF3 or IKKα to co-immunoprecitate with each NIK protein was assessed. (F) HEK293T cells were transfected as indicated and then incubated in the absence or presence of 25 µM MG132. The presence of NIK was detected by WB. (G) Cell lysates were prepared in the absence of MG132 and analyzed by WB to detect full length NIK and the 70 kD C-terminal NIK cleavage fragment. Data are representative of at least three separate experiments.

Fig. 4

API2-MALT1-dependent NIK cleavage is associated with upregulation of noncanonical NF-κB target genes, results in an altered B-cell phenotype, and occurs in t(11;18)-positive MALT lymphoma. (A) API2-MALT1 expressing SSK41 cells were transiently transfected with control or NIK siRNA, and p100 processing was assessed by WB. (B and C) API2-MALT1 expressing SSK41 cells were stably infected with control or NIK shRNA lentiviral particles. The 70 kD NIK cleavage fragment, Pim-2 and phospho-Ser¹¹²-BAD levels were compared by WB (B). Cells were treated for 48 hours with dexamethasone and percent viability was compared (C). Data are expressed as average +/− SEM for four separate experiments. *p<0.005, **p<0.0001. (D) API2-MALT1 expressing BJAB cells were transfected with control or NIK siRNA, and adhesion to VCAM-1-coated plates was assessed. Data are representative of three separate experiments. (E) Expression of API2-MALT1 in two t(11;18)-positive MALT lymphoma samples was confirmed by WB. p52/p100 ratios were quantified using densitometry. Both t(11;18)-positive cases had the same breakpoint in the API2 gene (between exons 7 and 8), but different breakpoints in the MALT1 gene [between exons 4 and 5 (case 1), and exons 6 and 7 (case 2)]. A Follicular lymphoma and two t(11;18)-negative MALT lymphoma tumor specimens were used as controls. (F) GSEA of NF-κB target genes was performed in a set of MALT lymphomas with t(11;18) vs. without translocation from Collection #1 (34). The distribution of the genes are listed according to rank position, and known noncanonical NF-κB gene targets are highlighted in yellow. Absolute enrichment gave p=0.0021 and FDR=0.0021. (G) Comparison of noncanonical target gene expression in t(11;18)-positive vs. negative cases from Collection #2 (34). Statistical testing for genes differentially expressed between the two types of MALT lymphomas was done by t-test. #p<0.05, ##p<0.01, *p < 0.005, **p < 0.001.