NPM-ALK and the JunB transcription factor regulate the expression of cytotoxic molecules in ALK-positive, anaplastic large cell lymphoma

Abstract

Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) is an aggressive non-Hodgkin lymphoma of T/null immunophenotype that is most prevalent in children and young adults. The normal cellular counterpart of this malignancy is presumed to be the cytotoxic T lymphocyte (CTL), and this presumption is partly based on the observation that these tumour cells often express cytotoxic granules containing Granzyme B (GzB) and Perforin. Chromosomal translocations involving the gene encoding for the ALK tyrosine kinase are also characteristic of ALK+ ALCL, and the resulting fusion proteins (e.g. NPM-ALK) initiate signalling events important in ALK+ ALCL pathogenesis. These events include the elevated expression of JunB; an AP-1 family transcription factor that promotes ALK+ ALCL proliferation. In this report we demonstrate that JunB is a direct transcriptional activator of GzB and that GzB transcription is also promoted by NPM-ALK. We found that Perforin expression was not regulated by JunB, but was promoted by NPM-ALK in some cell lines and inhibited by it in others. In conclusion, our study makes the novel observation that signalling through NPM-ALK and JunB affect the expression of cytotoxic molecules in ALK+ ALCL. Moreover, these findings demonstrate the expression of GzB and Perforin in this lymphoma is not solely due its presumed CTL origin, but that oncogenic signalling is actively influencing the expression of these proteins.

Keywords: ALK+ ALCL; JunB; NPM-ALK; granzyme B; perforin.

Figure 1

JunB promotes GzB protein expression in ALK+ ALCL. A. Western blot analysis of JunB (upper panel) and GzB (middle panel) expression in cell lysates of cells transfected with 100 nM of pooled non-targeting (control) or JunB siRNAs. The anti-actin western blot (lower panel) demonstrates equivalent protein loading. B. Quantification of western blots in A using the LI-COR Odyssey Infrared imager. The results shown are the mean and standard deviation of three independent experiments. p values were obtained using paired, one-tailed t-tests. C. Western blot analysis of JunB (upper panel) and GzB (middle panel) expression in lysates from Karpas 299 cells transfected with 100 nM of a non-targeting (control) or individual JunB siRNAs. The anti-actin western blot (lower panel) demonstrates equivalent protein loading. Molecular mass markers are indicated to the left of the blots.

Figure 2

JunB promotes GzB transcription in ALK+ ALCL. A. qRT-PCR analysis of GzB mRNA levels in Karpas 299 (left) and SUP-M2 (right) cells transfected with 100 nM of the indicated pooled siRNAs. Note: these experiments were performed on the mRNA extracted from cells transfected in Figure 1. B. Luciferase activity was measured in Karpas 299 cells transfected with a promoter-less luciferase construct (pGL2 basic) or a luciferase construct under control of the GzB proximal promoter (pGL2-GzB promoter) and 100 nM of the indicated pooled siRNAs. Luciferase activity is expressed relative to the activity present in cells co-transfected with the pGL2-GzB promoter and control siRNA (which was set at 100%). A western blot demonstrating the efficiency of JunB knock-down is shown to the right. C. Karpas 299 cells were transfected with the pGL2 basic, pGL2-GzB promoter, or AP-1 mutant pGL2-GzB promoter luciferase constructs and luciferase activity was measured 24 h post-transfection. Luciferase activity is expressed relative to the activity present in the pGL2-GzB promoter transfected cells (which was set at 100%). D. Luciferase activity was measured in Karpas 299 cells transfected with the pGL2-GzB promoter luciferase construct and Flag-tagged JunB (+) or empty vector (-) (top). Luciferase activity is expressed relative to the activity present in the pGL2-GzB promoter and empty vector–transfected cells (which was set at 100%). Anti-Flag and anti-JunB western blots demonstrate JunB over-expression (bottom). Molecular mass markers are indicated to the left of blots. E. EMSAs were performed with Karpas 299 nuclear extracts using a biotinylated probe based on the sequence of the AP-1 site in the GzB proximal promoter (GzB AP-1 probe). For competitor experiments, a 200-fold molar excess of unlabeled GzB AP-1 probe (wt competitor) or an unlabeled GzB AP-1 probe with a mutation in the AP-1 binding site (AP-1 mutant competitor) were included in the reaction. For super-shift experiments, 1 µg of the indicated antibody (Ab) was included in the reaction. In all experiments, the quantification represents the mean and standard deviation of three independent experiments. p values were determined using paired, one-tailed t-tests.

Figure 3

JunB does not regulate Perforin expression in ALK+ ALCL. Western blot (left) and quantification (right) of Perforin expression in cell lysates from Karpas 299 (A) and SUP-M2 (B) cells transfected with the 100 nM of the indicated pooled siRNAs. Results are the mean and standard deviation of three independent experiments. There was no statistical difference in Perforin levels between control and JunB siRNA-treated samples (paired, two-tailed t-tests; p > 0.15).

Figure 4

NPM-ALK promotes GzB expression in ALK+ ALCL. A. Western blots (left) and quantification (right) of GzB and JunB expression in lysates from Karpas 299 or SUP-M2 cells transfected with 100 nM of the indicated pooled siRNAs. Note: the anti-NPM blots demonstrate NPM-ALK silencing. Molecular mass markers are indicated to the left of the blots. B. qRT-PCR analysis of GzB mRNA expression in Karpas 299 and SUP-M2 cells transfected with the indicated siRNAs. Note: these experiments were performed on the mRNA extracted from cells transfected in A. C. GzB promoter luciferase activity was measured in Karpas 299 cells transfected with 100nM of non-targeting (control) or ALK siRNA. Luciferase activity is expressed relative to the activity present in cells transfected with the pGL2-GzB promoter and control siRNA (which was set at 100%). In all experiments, the quantification represents the mean and standard deviation of three independent experiments. p values were obtained using paired, one-tailed t-tests.

Figure 5

NPM-ALK regulates Perforin expression in ALK+ ALCL. A, Western blots (left) and quantification (right) of Perforin expression in lysates from Karpas 299 or SUP-M2 cells transfected with 100 nM of non-targeting (control) or ALK siRNA. B, qRT-PCR analysis of Perforin mRNA expression in Karpas 299 and SUP-M2 cells transfected with 100 nM of non-targeting (control) or ALK siRNA. Note: these experiments were performed on the mRNA extracted from cells transfected in A. C, Western blots (left) and quantification (right) of the indicated proteins in lysates from SU-DHL -1 cells transfected with 100 nM of non-targeting (control) or ALK siRNA. D, qRT-PCR analysis of Perforin and GzB mRNA expression in SU-DHL-1 cells transfected with 100 nM of non-targeting (control) or ALK siRNA. Note: these experiments were performed on the mRNA extracted from cells transfected in C. Molecular mass markers are indicated to the left of the western blots. In all experiments, the quantification represents the mean and standard deviation of three independent experiments. p values were obtained using paired, one-tailed t-tests. Note: the anti-NPM blots demonstrate NPM-ALK silencing.