Activated c-Jun N-Terminal Kinase Is a Therapeutic Target in Advanced Stage ALK-Negative Anaplastic Large Cell Lymphoma.
Authors: Nakamaki Tsuyoshi , Fukuchi Kunihiko , Yamochi-Onizuka Toshiko , Ota Hidekazu , Tomoyasu Shigeru 
Published: October 2019

Abstract
Anaplastic large cell lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) is an aggressive T cell lymphoma. To find a novel therapeutic target for aggressive ALK-negative ALCL, we studied two ALK-negative CD30-positive ALCL cell lines, N1 and N2, which were previously reported. In vitro culture, N1 and N2 represent early and advanced stage lymphoma phenotype, respectively. Increased tumorigenicity of N2 was confirmed by its xenotransplantability in SCID mice, whereas N1 was not transplantable either in SCID or in NOD/SCID mice. Subcutaneous tumors formed in the flank of SCID mice inoculated with N2 consisted of large basophilic lymphoma cells with prominent nucleoli, strongly positive for CD2 and CD30. Subcutaneous tumor showed TCR γ rearrangement identical with those in N2, and expressed aberrant CD30 protein with molecular weight 100 Kd, which was expressed only in N2, but not in N1. Those suggested subcutaneous tumors had characteristics of advanced stage ALCL. We hypothesized that constitutively activated c-Jun N-terminal kinase (JNK) is a therapeutic target in advanced stage ALK-negative ALCL, because, (1) JNKs were highly phosphorylated in N2 compared to in N1 studied by immunoblotting using phospho-specific antibodies against JNKs (N1=1.2±0.1 and N2=2.9±0.3, p=0.01, signal intensity); (2)specific JNK inhibitor, SP600125(SP) significantly inhibited cell growth of N2 compared to those of N1in culture for three days(N1=95±1.8 and N2=58±1.6 at 5μM SP, p<0.05; N1=89±0.1 and N2=29±2.4 at 50μM SP, p<0.01, % of viable cell No. of control). Incubation with SP restored the expression of co-stimulatory molecule, CD86 protein, and putative tumor suppressor gene, JunB protein, both of which were strongly downregulated in N2 compared with in N1. Despite of significant growth inhibition, constitutive activation of AP-1 was still demonstrated in N2 incubated with 50μM SP, whereas it was completely inhibited in N1 in electro mobility shift assay using AP-1 DNA consensus sequence(N1=0.2±0.1 and N2=8.3±0.2, p<0.01, arbitrary density units). It suggested that SP exhibited its effect in pathway(s) other than transcriptional regulation through AP-1 complex in ALCL cell lines, N2. In vivo model, intraperitoneal injection of SP (30mg/weight) alternate days for 22days significantly suppressed the growth of subcutaneous tumors (DMSO=1.4±0.5 and SP=0.2±0.1, p<0.05, tumor weight[g] at day26). Tumors in SP-treated mice were exclusively localized in subcutaneous region, in contrast, tumors infiltrated and produced signs of hindlimb paralysis in two of three DMSO-treated mice. Histopathologically tumors in SP-treated showed increase of cells with nuclear condensation, suggesting apoptotic cell death. Furthermore, in addition to partial inhibition of phosphorylation of JNK, tumors in SP-treated mice showed significant decrease of levels of both mRNA (64±13%, p<0.05, real-time PCR) and protein (62±2%, p=0.02, signal intensity by immunoblotting) of BCL2, compared with those in DMSO-treated mice. Whereas no change was found in Bcl-XL expression. No change was found in CD30 protein expression. Taken together, the present study showed that aberrantly activated JNK(s) is a therapeutic target in some types of advanced stage ALK-negative ALCL, although details of signaling pathways triggered by JNK(s) in ALCL cells remained to be elucidated.