Functional characterization of NPM1-TYK2 fusion oncogene

Abstract

Gene fusions are known to drive many human cancers. Therefore, the functional characterization of newly discovered fusions is critical to understanding the oncobiology of these tumors and to enable therapeutic development. NPM1-TYK2 is a novel fusion identified in CD30 + lymphoproliferative disorders, and here we present the functional evaluation of this fusion gene as an oncogene. The chimeric protein consists of the amino-terminus of nucleophosmin 1 (NPM1) and the carboxyl-terminus of tyrosine kinase 2 (TYK2), including the kinase domain. Using in vitro lymphoid cell transformation assays and in vivo tumorigenic xenograft models we present direct evidence that the fusion gene is an oncogene. NPM1 fusion partner provides the critical homodimerization needed for the fusion kinase constitutive activation and downstream signaling that are responsible for cell transformation. As a result, our studies identify NPM1-TYK2 as a novel fusion oncogene and suggest that inhibition of fusion homodimerization could be a precision therapeutic approach in cutaneous T-cell lymphoma patients expressing this chimera.

Fig. 1. In vitro transformation potential of the NPM1–TYK2 fusion gene.

A Detection of overexpressed NPM1–TYK2 mRNA transcript levels in transformed Ba/F3 cells by qRT-PCR. Myla and SU-DHL-1 cell lines were used as positive and negative controls, respectively. Columns represent the mean of three independent experiments; bars represent the SEM. B Subcellular localization of NPM1–TYK2. Ba/F3-Vector and transformed Ba/F3 (NPM1–TYK2) cells were cytospun onto glass slides, fixed, permeabilized, and stained for FLAG antibody and DAPI. Images were acquired with a fluorescent microscope using a 60× oil immersion lens. C Transformation of Ba/F3 cells to IL-3 independent growth. Ba/F3-Vector and transformed Ba/F3 (NPM1–TYK2) cells were grown in RPMI medium in the absence of IL-3. Cell viability from each condition was determined daily by trypan blue exclusion assay. Points represent the mean of three independent experiments; bars represent SEM. ^∗∗∗P < 0.0005 was considered as statistically extremely significant. D The clonogenic potential of transformed Ba/F3 (NPM1–TYK2) cells. Ba/F3-Vector and transformed Ba/F3 NPM1–TYK2 cells were mixed with MethoCult medium and plated. After 7 days of incubation, the number of colonies was counted. Columns represent the mean of three independent experiments; bars represent the SEM.

Fig. 2. Fusion kinase NPM1–TYK2 oncogenic signaling drives the lymphoma cell transformation.

Ba/F3-Vector and transformed Ba/F3–NPM1–TYK2 cell lysates were made, subjected to immunoblot analysis for NPM1–TYK2 (FLAG), phospho-NPM1–TYK2, phospho-STAT1/3/5, and respective total proteins. β-Actin served as an internal control.

Fig. 3. NPM1–TYK2 fusion kinase is an oncogenic driver in lymphoid cell transformation.

A Fusion kinase-dead mutant blocks kinase activation and downstream signaling. Cell lysates were made from Ba/F3-vector, Ba/F3–NPM1–TYK2, and Ba/F3–NPM1–TYK2–K462R cell lines, and immunoblot analysis was performed for FLAG (NPM1–TYK2), phospho-NPM1–TYK2, phospho-STAT1/3/5, and respective total proteins. β-Actin served as an internal control. B Fusion kinase signaling drives cell transformation. Ba/F3-Vector, Ba/F3–NPM1–TYK2, and Ba/F3–NPM1–TYK2–K462R cells were grown in RPMI medium in the absence of IL-3. Cell viability from each condition was determined daily by trypan blue exclusion assay. Points represent the mean of three independent experiments; bars represent SEM. ^∗∗∗P < 0.0005 was considered as statistically extremely significant.

Fig. 4. In vivo tumorigenic potential of the NPM1–TYK2 fusion gene.

A Representative image of mice (n = 6) subcutaneously injected with Ba/F3-Vector showed no tumors. B Representative images of mice (n = 6) subcutaneously injected with transformed Ba/F3–NPM1–TYK2 show tumorigenesis. C Representative images of resected tumors with their respective tumor weight (grams). D Tumor volume was increased significantly in transformed Ba/F3–NPM1–TYK2 injected mice but not in Ba/F3-Vector mice. Bars represent the SEM. E, F In transformed Ba/F3 (NPM1–TYK2) mice, tumor growth was associated with hepatosplenomegaly.

Fig. 5. Detection of NPM1–TYK2 in tumors and lymphoma cell infiltration induced hepatosplenomegaly.

A, B Detection of NPM1–TYK2 (FLAG) in tumors by immunostaining and RT-qPCR. Columns represent the mean of three independent experiments; bars represent the SEM. C Hematoxylin and eosin (H&E) staining of sections from tumor tissue demonstrate lymphoma cell infiltration into tumor, spleen, and liver.

Fig. 6. Fusion kinase NPM1–TYK2 constitutive activation drives tumorigenicity in in vivo xenograft models.

Representative lysates were made from tissue collected from Ba/F3-Vector and transformed Ba/F3–NPM1–TYK2 injected mice. Western blot analysis was performed for NPM1–TYK2 (FLAG), phospho-NPM1–TYK2, phospho-STAT1/3/5, and respective total proteins. β-Actin served as an internal control.

Fig. 7. Fusion partner NPM1 is essential for NPM1–TYK2 mediated oncogenic signaling.

A, B Deletion of NPM1 partner in NPM1–TYK2 fusion gene inhibits activation of fusion kinase NPM1–TYK2 mediated signaling in HEK293T and Ba/F3 cells. Western blot analysis of NPM1–TYK2 (FLAG), phospho-NPM1–TYK2, phospho-STAT1/3/5, and respective total proteins. β-Actin served as an internal control.

Fig. 8. Loss of NPM1 partner abrogates NPM1–TYK2 transformation potential.

A Deletion of NPM1 portion in NPM1–TYK2 fusion gene abrogates the transformation potential of the NPM1–TYK2 fusion gene in Ba/F3 cells. Ba/F3 cells were transduced with lentiviral particles expressing vector, NPM1–TYK2, and ΔN-1–257-NPM1–TYK2. Cells were grown in RPMI medium without IL-3 growth factor for 10 days. Cell viability from each condition was determined daily by trypan blue exclusion assay. Points represent the mean of three independent experiments; bars represent SEM. ^∗∗∗P < 0.0005 was considered as statistically extremely significant. B The deletion of the NPM1 portion in the NPM1–TYK2 fusion gene inhibits the clonogenic potential. Columns represent the mean of three independent experiments; bars represent the SEM.

Fig. 9. Visual overview.

NPM1-TYK2 fusion mediated oncogenic signaling.