Distinct nuclear localization signals confer differential microtubule dependence among ZFTA fusion oncoproteins

Ependymomas are glial tumors that exhibit high resistance to chemotherapy. ZFTA-fusion-positive supratentorial ependymoma (ST-EPN-ZFTA) represents a subclass with a poor prognosis. ST-EPN-ZFTA expresses fusion proteins, which consist of zinc finger translocation-associated (ZFTA) combined with a transcriptional regulator, predominantly RELA. Unlike wild-type RELA, which translocates to the nucleus in a stimulus-dependent manner, ZFTA-RELA constitutively localizes to the nucleus and induces oncogenic gene expression. To identify inhibitors of this aberrant gene expression, 9600 structurally diverse compounds were screened using a ZFTA-RELA-responsive luciferase reporter system. The screen identified a colchicine derivative, and subsequent evaluation revealed colchicine, a microtubule polymerization inhibitor, as the most potent inhibitor (IC₅₀ of 90 nM). Colchicine attenuated ZFTA-RELA-upregulated endogenous genes, including L1CAM. Colchicine and another microtubule polymerization inhibitor, vinblastine, partially inhibited ZFTA-RELA nuclear localization, similar to the dynein motor protein inhibitors, ciliobrevin D and dynarrestin. This inhibition was mediated through the RELA region of ZFTA-RELA, indicating that RELA retains its microtubule-dependent nuclear import machinery, even in a fusion scenario. Among other ZFTA-fusion proteins, colchicine reduced nuclear localization of ZFTA-NCOA2 and ZFTA-MKL2, but not ZFTA-MAML2 and ZFTA-MAML3. In these fusion proteins, we identified three candidate nuclear localization signals (NLS) exhibiting colchicine-sensitive nuclear localization activity, two within the ZFTA regions absent in ZFTA-RELA and one within MKL2. The results indicate that the microtubule dependence of nuclear localization varies among different ZFTA-fusion partners, which may be attributable to unique NLS sequences in each fusion protein. Our results suggest that microtubule-dependent nuclear transport represents a therapeutic target in ZFTA-fusion-positive tumors.