NPM1-TYK2 Fusion Is an Oncogene and a Novel HSP90-Client Protein

Cutaneous CD30-positive lymphoproliferative disorders (LPDs), the second most common type of cutaneous T-cell lymphoma include a clinicopathologic spectrum of benign lymphomatoid papulosis (LYP) and primary cutaneous anaplastic large-cell lymphoma (ALCL). NPM1-TYK2 is the first identified tyrosine kinase chromosomal translocation in CD30-positive LPDs. Understanding the deregulated functions resulting from new chromosomal translocations in these neoplasms is essential to discover novel therapeutic approaches. Nucleophosmin 1 (NPM1) is a nucleolar phosphoprotein, which functions as a molecular chaperone for proteins and nucleic acids. Tyrosine kinase 2 (TYK2) is a non-receptor tyrosine kinase that belongs to the family of Janus Kinases (JAKs). NPM1-TYK2 is an 81 kDa fusion protein comprising of NPM1 (1-257 amino acids) and the kinase domain of TYK2 (726-1187 amino acids). Since NPM1-TYK2 is a recently discovered fusion gene, we conducted experiments to determine its oncogenic potential. Transduction of lentiviral particles bearing NPM1-TYK2 gene transformed the interleukin-3 (IL-3) dependent Ba/F3 cell line to IL-3 independent growth through constitutive activation of TYK2 kinase and downstream STAT signaling. We further evaluated the role of NPM1 as part of fusion protein in NPM1-TYK2 mediated oncogenicity. Transfection of NPM1-TYK2 deletion construct lacking NPM1 in HEK293 cells resulted in significant decrease in TYK2 kinase activity in the cells compared to full-length NPM1-TYK2 transfected cells. Currently, there are no effective small molecule inhibitors for NPM1/ TYK2 in the clinical trials. Hence, there is a clinical unmet need to develop novel, targeted therapies for NPM1-TYK2 driven lymphomas. As TYK2 is an established HSP90 client protein, the rationale for this study is to investigate the ability of HSP90 to regulate the stability and kinase activity of NPM1-TYK2 fusion protein. HSP90 is a master chaperone involved in the proper folding and maturation of a variety of oncogenic kinases and perturbing HSP90 function is emerging as a promising approach for cancer therapy. We used Myla cells (expressing endogenous NPM1-TYK2 fusion gene) to investigate the status of NPM1-TYK2 as a HSP90 client protein. Here we report that the treatment of cells with small molecule HSP90 inhibitor, 17-AAG promotes degradation of NPM1-TYK2 fusion protein resulting in downregulation of its oncogenically induced downstream STAT signaling pathways. Mechanistically, dephosphorylation of STAT1, 3, and 5 following 17-AAG treatment resulted in apoptotic cell death of the lymphoma cells. The results obtained using 17-AAG, were further corroborated using other HSP90 small molecule inhibitors AUY922 and PU-H71. Immunoprecipitation studies clearly demonstrate that 17-AAG treatment disrupts interaction between HSP90 and NPM1-TYK2 chimeric protein. Collectively, these findings provide an evidence for NPM1-TYK2 oncogenicity and therapeutic potential of HSP90 inhibitors for the treatment of a subset of cutaneous CD30-positive lymphoproliferative disorder patients expressing NPM1-TYK2 chimeric gene.