Simple SummaryBlocking telomerase to drive telomere erosion-dependent antiproliferative effects in cancer cells appears impractical. However, the evidence of extra-telomeric functions of TERT, the catalytic component of telomerase, in promoting tumour growth/progression strongly supports the potential telomere length-independent therapeutic effects of TERT inhibition. The mechanism(s) underlying these effects need to be explored to identify cellular pathways being (de)regulated by telomerase during the oncogenic process to establish how the selective targeting of TERT can rapidly interrupt the expansion of tumour cells, regardless of telomere length and erosion. Using in vitro models of B-cell lymphoproliferative disorders and B-cell malignancies, we found that TERT inhibition impairs the NF-κB p65 pathway, resulting in decreased MYC expression and a consequent P21-mediated cell cycle arrest. The in vivo results in the zebrafish model confirm the in vitro data and prompt an evaluation of strategies combining TERT inhibition with chemotherapeutic agents to enhance the therapeutic benefits of current treatment modalities.AbstractTelomerase reverse transcriptase (TERT), the catalytic component of telomerase, may also contribute to carcinogenesis via telomere-length independent mechanisms. Our previous in vitro and in vivo studies demonstrated that short-term telomerase inhibition by BIBR1532 impairs cell proliferation without affecting telomere length. Here, we show that the impaired cell cycle progression following short-term TERT inhibition by BIBR1532 in in vitro models of B-cell lymphoproliferative disorders, i.e., Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines (LCLs), and B-cell malignancies, i.e., Burkitt’s lymphoma (BL) cell lines, is characterized by a significant reduction in NF-κB p65 nuclear levels leading to the downregulation of its target gene MYC. MYC downregulation was associated with increased expression and nuclear localization of P21, thus promoting its cell cycle inhibitory function. Consistently, treatment with BIBR1532 in wild-type zebrafish embryos significantly decreased Myc and increasedp21expression. The combination of BIBR1532 with antineoplastic drugs (cyclophosphamide or fludarabine) significantly reduced xenografted cells’ proliferation rate compared to monotherapy in the zebrafish xenograft model. Overall, these findings indicate that short-term inhibition of TERT impairs cell growth through the downregulation of MYC via NF-κB signalling and supports the use of TERT inhibitors in combination with antineoplastic drugs as an efficient anticancer strategy.