Active TERT expression is detected in about 80-90% of tumors across cancer entities and is commonly associated with poor prognosis (Kim, 1994a; Shay, 1997). Telomere maintenance is
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mandatory for cancer cell’s survival, and activation or reactivation of TERT enables tumor cells to overcome the Hayflick limit to achieve replicative immortality, which is considered as a central hallmark of cancer (Hayflick, 1965). In earlier studies of neuroblastoma, telomerase activity has been correlated with unfavorable outcome (Hiyama, 1995). More recently, in about 30% of high-risk neuroblastoma, genomic TERT rearrangements have been described as a major driver of malignancy by demonstrating a strong correlation between high TERT-expression and telomerase activity and poor survival rates (Ackermann, 2018; Peifer, 2015;
Valentijn, 2015). As demonstrated in colorectal carcinomas and neuroblastoma, there was no correlation between TERT copy number and its expression levels or telomerase activity, indicating that the high TERT expression is caused by the genomic repositioning and subsequent transcriptional upregulation of TERT, rather than by gain of TERT copy numbers (Palmqvist, 2005; Peifer, 2015; Valentijn, 2015). The lack of telomere maintenance mechanisms in neuroblastoma tumors correlates with a more benign prognosis (Ackermann, 2018). The study presented here reveals that treatment of TERT-rearranged neuroblastoma models highly expressing TERT with the pan-HDACi panobinostat reduced TERT expression by 2-4.5-fold.
The on-target effect of HDACi treatment on TERT expression was demonstrated by treatment with structurally different pan-HDACi and HDAC1/2 inhibitors, showing that TERT repression is a common event upon HDAC inhibition in TERT-rearranged neuroblastoma cell lines. In normal human renal cortical epithelial cells and foreskin fibroblasts, trichostatin A treatment transcriptionally activated TERT expression and induced telomerase activity (Takakura, 2001).
TERT downregulation after treatment with HDACi trichostatin A was shown in prostate cancer (Suenaga, 2002), liver cancer (Nakamura, 2001) and brain tumor cell lines (Rahman, 2010).
Treatment with HDACi B1 repressed telomerase in lung adenocarcinoma cells via downregulation of STAT3, a transcription factor known to regulate TERT expression (Cheng, 2017b). Together, this points to a major role of HDACs in the regulation of TERT in healthy and malignant tissue.
In this study, TERT protein levels were demonstrated to decrease after panobinostat treatment.
Although several publications present TERT immunoblotting (Bui, 2019; Iannilli, 2013), western blot analysis of the full length protein of about 127 kDa is technically challenging. The protein seems to degrade easily in absence of its native template TERC and was shown to form functional dimers, impeding analysis of the TERT monomer (Sauerwald, 2013). By using a TERT overexpressing plasmid, antibody specificity was validated. Decreasing TERT levels were demonstrated after pan-HDACi treatment in rat aortic vascular smooth muscle cells (Qing, 2016). More importantly, the functional consequence of decreasing TERT levels after HDACi
treatment was demonstrated here by detection of reduced activity of the telomerase holoenzymatic complex.
Telomere maintenance is a major driver of malignancy in neuroblastoma (Ackermann, 2018) and the pharmacologic targeting of TERT and telomerase with small-molecules is currently under clinical investigation (Relitti, 2020; Salloum, 2016; Thompson, 2013). Most of these inhibitors directly bind to the catalytic center or the periphery of TERT or the telomerase holoenzymatic complex (Lavanya, 2018; Yan, 2019). Imetelstat (GRN163L) is a specific and one of the most developed telomerase inhibitors in phase I and II clinical trials targeting the TERC RNA. In a clinical phase II study in children with recurrent CNS malignancies, treatment with imetelstat before surgical resection of the tumors resulted in grade 3/4 toxicities including thrombocytopenia, lymphopenia and neutropenia, and overall proved too toxic (Salloum, 2016). During embryogenesis and development, TERT and telomerase are important for normal development, but in most adult somatic tissue, TERT expression and telomerase activity are repressed and are only detectable in stem and germline cells (Collins, 2002). There are additional cell survival and stress resistance beneficial roles of telomerase in the cell, which are abrogated after its repression, probably explaining the observed toxicities upon pharmacological inhibition of telomerase (Ahmed, 2008). For neuroblastoma patients harboring TERT-rearranged tumors with high TERT levels and telomerase activity, HDACi treatment presents a promising therapeutic option to reduce TERT expression and telomerase activity. In this study, telomerase repression was similar in the TERT-rearranged and MYCN-amplified neuroblastoma cell lines treated with panobinostat, suggesting that panobinostat treatment could also be beneficial for patients with tumors harboring MYCN amplification.
TERT is the major component of the telomerase holoenzymatic complex and contains the catalytic subunit. In breast cancer cells, transient knockdown of TERT expression resulted in reduced telomerase activity, while knockdown of other telomerase subunits as the RNA template TERC or TP1 was not decreasing activity of the complex (Rubis, 2013). Telomerase repression was demonstrated in liver cancer cells treated with HDACi (Nakamura, 2001) and in brain tumor cells treated with HDACi trichostatin A, with no toxicity to normal brain tissue function (Rahman, 2010). This highlights the potential utility of panobinostat treatment in patients presenting with telomerase-positive tumors. Reduced activity of the telomerase complex might result in limited ability of cancer cells to undergo cell division and replicative immortality may be lost, reducing tumorigenicity. In this study, telomerase activity was stronger repressed after panobinostat treatment in TERT-rearranged xenograft tumors than in
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the cell culture models, although reduction of TERT mRNA expression was comparably reduced in the TERT-rearranged in vitro and in vivo neuroblastoma models. There might be additional factors like posttranscriptional modifications of the TERT protein or of other subunits of the telomerase complex (Kharbanda, 2000; Kim, 2005a), probably explaining the strong reduction of telomerase activity after several weeks of panobinostat treatment. The xenograft tumors were treated for about three to four weeks with panobinostat, whereas this long treatment was not applicable in cell culture at comparable concentrations. Post-transcriptional and -translational modifications, targeted decay of the TERT protein, repression of further subunits of the telomerase complex or other factors might contribute to reduced telomerase activity after long-term treatment with panobinostat. As demonstrated in breast cancer cells, alternative splicing events result in a product termed β-deletion of the TERT transcript, coding for a truncated TERT protein that lacks most of the reverse transcriptase activity (Listerman, 2013; Wong, 2013). Phosphorylation (Kharbanda, 2000) or ubiquitination (Kim, 2005a) of TERT were found to be associated with decreased telomerase activity.
Alternative splicing or post-translational modifications of TERT might contribute to decreasing TERT and telomerase levels after HDACi treatment. Further studies investigating the fate of the TERT mRNA transcript are necessary to address these possible regulatory pathways induced by panobinostat treatment. Taken together, the evidence obtained here demonstrate the reduction of TERT levels and telomerase activity by panobinostat treatment in vitro and in vivo models of TERT-rearranged neuroblastoma. Panobinostat treatment could present a potential way to target telomerase in neuroblastoma patients presenting with telomerase-positive tumors.
As telomere maintenance is given in most cancers and generally considered as one of the hallmarks of cancer, it would be worthwhile to investigate if panobinostat treatment reduces TERT levels and telomerase activity in other telomerase-positive cancer entities as well.