Sumoylation Sustains the Stability of NPM-ALK Oncogenic Protein and Facilitates Its Nuclear Accumulation in T-Cell Anaplastic Large-Cell Lymphoma

Abstract

Anaplastic lymphoma kinase-expressing anaplastic large-cell lymphoma (ALK+ ALCL) is an aggressive type of T-cell non-Hodgkin lymphoma. The expression of ALK in T-cell lymphoma is an aberrant event that results from one of several chromosomal abnormalities. The t(2;5)(p23;q35) chromosomal translocation constitutes 85% of these abnormalities, and leads to the fusion of the nucleophosmin (NPM) gene on 2p23 and the ALK gene on 5q35. This translocation induces the generation of NPM-ALKoncogene, which encodes the expression of NPM-ALK chimeric tyrosine kinase. NPM-ALK induces lymphomagenic effects through the formation of the constitutively activated NPM-ALK/NPM-ALK homodimers, which phosphorylate downstream survival-promoting proteins including JAK/STAT, PI3K/AKT, and MAP kinase. Although it resides in the cytoplasm, NPM-ALK is also capable of forming the wild-type ALK/NPM-ALK heterodimers that translocate to the nucleus. Notably, the mechanisms that promote the stability and mediate the translocation of NPM-ALK from the cytoplasm to the nucleus are not completely understood.SUMOylation is a post-translational modification that is characterized by the covalent and reversible binding of small ubiquitin-like modifiers (SUMO), including SUMO-1, SUMO-2/3, and SUMO-4, with their target proteins. Although SUMOylation has some similarities with ubiquitination, it has been shown that SUMO proteins compete with ubiquitin for substrate binding and thus SUMOylation protects target proteins from proteasomal degradation. In addition to enhancing protein stability, SUMO proteins are involved in nuclear translocation of target proteins, which affects processes essential for cellular homeostasis. SUMO family of proteins also includes the sentrin-specific proteases (SENPs): SENP1-3 and SENP5-7. The roles of SENPs encompass removal of SUMO from target proteins; thus suppressing protein stabilization induced by SUMOylation. It has been recently demonstrated that SUMOylation plays a key role in the progression of cancer. For instance, SUMOylation inhibits cancer development through stabilization of tumor suppressor genes or promote cancer development through stabilization of oncogenes. Because it is also involved in DNA damage repair mechanisms that ensure maintenance and stabilization of genomic integrity, it is thought that SUMOylation has the ability to promote tumor development and to initiate chemo-resistance in cancer cells.In this study, we hypothesized that defects in SUMOylation system exist in NPM-ALK+ T-cell lymphoma and contribute to the stabilization of NPM-ALK protein and its accumulation in the nucleus. Using the online software SUMOplot (http://www.abgent.com/sumoplot), we identified potential SUMO consensus binding sites within NPM-ALK amino acid sequence. Analyzing the expression levels of SUMO proteins by Western blotting demonstrated universal upregulation of the SUMO proteins and substantial decrease in the deSUMOylating protein SENP1 in 5 NPM-ALK+ T-cell lymphoma cell lines compared with normal human T lymphocytes. Furthermore, similar patterns of SUMO and SENP1 expressions were detected in 15 primary ALK+ T-cell lymphoma tumors from patients. Immunoprecipitation showed binding between the SUMO and NPM-ALK proteins. In vitro SUMOylation assays provided novel evidence that SUMO-1 and SUMO-3 induce SUMOylation of NPM-ALK at Lys24 and Lys32, and these findings were further confirmed using a protein degradation assay. In addition, transfection of the NPM-ALK+ T-cell lymphoma cells with a SENP1 expression plasmid decreased the expression levels of the SUMO and NPM-ALK proteins, and abrogated the accumulation of NPM-ALK in the nucleus. SENP1-induced effects were associated with decreased cell viability, proliferation, migration, and anchorage-independent colony formation of NPM-ALK+T-cell lymphoma cells.In summary, we identify for the first time defects in the SUMOylation system as novel mechanisms contributing to the pathogenesis of NPM-ALK+ T-cell lymphoma. Unraveling such mechanisms is expected to advance current understanding of the pathobiology of this lymphoma and may lead to devising novel strategies to eradicate this aggressive form of cancer. DisclosuresNo relevant conflicts of interest to declare.