Chemotherapy is a harsh therapeutic option, which remains the gold standard in most cancer entities. Chemotherapeutic agents like platinum derivatives, anthracyclines or taxanes act unspecific on mitotic cells and affect somatic cells as well as cancer cells (Agarwal, 2016).
Common side effects of chemotherapy such as nausea, fatigue and impairments of the nervous system can be addressed by supportive therapy but remain a challenge. Targeted agents could help to reduce side effects of the treatment and increase selectivity towards cancer cells. In this study, temozolomide and doxorubicin from the RIST and NB2004 study protocols for treatment of neuroblastoma patients were used for the analyses. In addition, oxaliplatin as first-line chemotherapeutic and the preclinical compound I-BET762 were investigated as representatives for chemotherapeutic or targeted compounds used in first-line therapy or in preclinical models of high-risk neuroblastoma. Treatment of TERT-rearranged neuroblastoma cells with these compounds did not impair TERT expression or telomerase activity at clinically relevant concentrations, except for doxorubicin reducing TERT expression in GI-ME-N cells. Although reducing the cell viability, these agents display their cytotoxic potential independent of TERT.
The TERT-rearranged GI-ME-N cells are classified as mesenchymal-like neuroblastoma cells, whereas CLB-GA cells reflect an adrenergic-like cell profile (Boeva, 2017). Mesenchymal cells were demonstrated to be more resistant to chemotherapy and to probably account for minimal
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residue disease, probably giving to relapse after therapeutic intervention (van Groningen, 2017).
The antitumoral effect induced by treatment with the selected chemotherapeutic or targeted compounds showed to be more effective towards CLB-GA cells than GI-ME-N cells, and lower concentrations were used for treatment of CLB-GA cells. This could correlate with the observation that mesenchymal-like cells show to be more resistant to drugs applied in neuroblastoma therapy such as cisplatin, doxorubicin or etoposide, than an adrenergic counterpart (van Groningen, 2017). The importance of TERT in regard to therapy resistance was shown in osteosarcoma cells, where TERT was reported to inhibit cisplatin-induced apoptosis (Zhang, 2017b). Targeting TERT could increase vulnerability of cancer cells towards chemotherapeutic or target therapy. In pancreatic cancer, regulators of epithelial-mesenchymal transition (EMT) maintain drug resistance against chemotherapeutic agents and could serve as an interesting target for therapeutic intervention (Arumugam, 2009). It would be interesting to investigate the role of TERT repression after HDAC inhibition on regulators of EMT in preclinical models of high-risk neuroblastoma, and if resistant clones potentially giving rise to MRD can be diminished by targeting TERT and telomerase with HDACi treatment. Further, differentiation of neuroblastoma cells into benign forms is highly desirable and can be mediated by treatment with 13-cis-retinoic acid (Haas, 1988; Reynolds, 2003; Sidell, 1982; Thiele, 1985).
TERT is expressed in human stem cells, while repression of TERT during cell differentiation is observed (Liu, 2004). In neuroblastoma, inhibition of HDAC1/2 was shown to induce cell differentiation (Frumm, 2013), and in high-risk neuroblastoma, TERT expression was demonstrated to be reduced after induced cell differentiation, potentially contributing to reduce malignancy (Bui, 2019; Das, 2009). In this study, it was observed that the mesenchymal-like GI-ME-N cells changed their morphology towards a more differentiated phenotype after HDACi treatment (data not shown). It would be interesting to investigate whether inhibition of HDAC1/2 induces differentiation in mesenchymal-like TERT-rearranged neuroblastoma cells towards an adrenergic profile. This would open up new perspectives for subsequent combination therapy, as adrenergic cell lines are more vulnerable towards treatment with chemotherapeutic agents. Together, this study revealed that the analyzed standard antineoplastic and targeted agents applied in clinical or preclinical neuroblastoma treatment showed no reduction of TERT levels and telomerase activity in TERT-rearranged cell lines.
5.5 Panobinostat treatment represses TERT and telomerase in TERT-driven neuroblastoma xenograft mouse models
The assessment of novel drugs as stand-alone or combination therapy requires preclinical models that closely resemble the primary tumors. The antitumoral efficacy of panobinostat and other HDACi has been demonstrated in various cancer models including MYCN-amplified neuroblastoma (He, 2001; Helland, 2016; Lodrini, 2013). Xenograft mouse models of TERT-rearranged GI-ME-N and CLB-GA cells are underrepresented in neuroblastoma xenograft studies (Mandriota, 2015). This study demonstrates that panobinostat treatment reduces xenograft tumor growth, TERT expression and telomerase activity in TERT-rearranged xenograft models. This sustains data from the in vitro experiments of this study. The results of the GI-ME-N and CLB-GA xenograft mouse studies were confirmed in a second independent cohort of each model, highlighting the efficacy of panobinostat treatment on TERT and telomerase repression in TERT-rearranged high-risk neuroblastoma models. Reduced expression of TERT was also observed in MYCN-amplified xenograft tumors treated with panobinostat (Lodrini, 2013), highlighting the TERT-repressive efficacy of panobinostat in other telomerase-positive neuroblastoma high-risk groups (data not shown).
Mesenchymal-like neuroblastoma cells were demonstrated to be more resistant to chemotherapy and to probably account for minimal residue disease (van Groningen, 2017).
Regarding the different transcription profiles of the cell models, the mesenchymal-like GI-ME-N xenograft model showed good response to panobinostat therapy, offering a prospect to diminish the risk of relapse. Whether panobinostat treatment reduces the risk of relapse is to be investigated in future long-term experiments.
Panobinostat is one of the most potent pan-HDACi in clinical application, presented here to reduce TERT expression and telomerase activity in vitro and in vivo models of TERT-rearranged neuroblastoma. It has a long elimination time and causes prolonged hyperacetylation of histone proteins (Singh, 2016; Tate, 2012). Long-term treatment of panobinostat has been investigated in models of MYCN-amplified high-risk neuroblastoma. In the TH-MYCN mouse model, terminal-differentiation of tumors to benign ganglioneuromas after nine weeks of treatment and prolonged survival were demonstrated (Waldeck, 2016). The continuous treatment with panobinostat questions current therapy schedules including only short-period treatment with HDACi. Further, hydroxamate-based HDACi like panobinostat have been described to induce DNA mutations (Al-Hamamah, 2019; Munakata, 1980; Prince, 2009; Wang, 1977). Up to now, hydroxamates are a major class of HDACi because of their high zinc-chelating capability,
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isoform selectivity, and in vivo efficacy (Shen, 2016). Applying mutagen therapeutics to patients needs a careful risk-benefit assessment and their medical use should be weighted against the hazards of carcinogenesis. Panobinostat has been applied in preclinical models of neuroblastoma, but its application in models of the TERT-rearranged high-risk subgroup had not been evaluated before. Currently, there are several pediatric applications of panobinostat under investigation in clinical phase I-II trials (NIH, 2020), but its mutagenic potential still needs to be carefully evaluated. Addressing that issue, whole exome sequencing (WES) analyses of xenograft tumors of the preventive treatment schedules are currently under investigation in our laboratory. Alternatively, structurally different HDACi like the cyclic peptide romidepsin could serve as non-mutagenic therapeutic option. Specific HDACi targeting HDAC1/2 like romidepsin, mocetinostat and Santacruzamate A were demonstrated here to reduce TERT expression in vitro. Applying selective HDACi for the treatment of TERT-rearranged neuroblastoma provides an interesting approach to target TERT and telomerase on the one hand and reduce the toxic side effects of the treatment on the other hand.
Potential therapeutics often fail when translated from in vitro to in vivo models. A changed microenvironment, reduced uptake and metabolization of drugs diminish plasma levels and therapeutic success. Here, it was demonstrated that a reduction of the initial 15 mg/kg/d panobinostat to half of the dose resulted in comparable outcome, reducing toxic side effects of the treatment. The chosen concentration of 15 mg/kg/d panobinostat is in the spectrum of therapeutically effective doses of 5-25 mg/kg/d (Helland, 2016; Hennika, 2017; Shahbazi, 2016; Waldeck, 2016) and is achievable in patient plasma levels (Rathkopf, 2010b; Van Veggel, 2018). Upon panobinostat treatment, the observed side effects in xenografted mice here were weight loss of a maximal of about 5% and mild diarrhea, as described in previous studies (Floris, 2009; Hennika, 2017). In contrast, no significant bodyweight loss was described in mouse models receiving 7.5-15 mg/kg/d panobinostat (Helland, 2016). Reduction of bodyweight upon treatment may be strain-specific and might additionally depend on the administration route. Compared to other drugs used in cancer therapy, the side effects of panobinostat treatment are manageable and are usually reversible (Greig, 2016).
GI-ME-N xenograft tumor growth was slow and tumor engraftment was successful three month after transplantation, impeding the experimental schedule. This maybe explains that GI-ME-N xenograft models are underrepresented in the literature and showed slow growth in control-transfected GI-ME-N xenograft tumors (Mandriota, 2015). CLB-GA xenograft models have been investigated in few studies (Huang, 2020; Provost, 2016; Regairaz, 2016). Many studies
use xenograft tumor volumes of 50 mm³ at onset of treatment, but this may overestimate a potential effect of the treatment (Wong, 2019). On the one hand, smaller tumors might not have reached their replicative potential and show slow progression in cell cycle, being more vulnerable to external stressors. On the other hand, in large and highly proliferating tumors a potential effect of the drug might be extinguished. In the preventive treatment schedule of this study, a tumor volume of about 150 mm³ was chosen for induction of treatment, assuring successful tumor engraftment. In the therapeutic treatment schedule, tumor volumes of about 300 mm³ were used to investigate highly proliferating tumors. Each condition represented a balanced mean reflecting the desired clinical outset upon therapeutic intervention. Although tumors were larger and highly proliferative at onset of the therapeutic treatment schedule, panobinostat treatment repressed TERT expression and telomerase activity and arrested xenograft tumor growth.
Patient derived xenograft (PDX) models reflect the heterogeneous biology of patient tumors much closer than established cell culture models (Jung, 2018). In this study, a TERT-rearranged neuroblastoma PDX could not be investigated due to insufficient tumor growth. Instead, subcutaneous xenograft mouse models of TERT-rearranged neuroblastoma cells were evaluated. Further experiments in PDX models generated from patient tumor material could help to assess the TERT repressive phenotype and antitumoral efficacy of panobinostat in these heterogeneous models of patient tumors. In summary, this study demonstrates that panobinostat treatment represses TERT expression, telomerase activity and harbors antitumoral efficacy in TERT-rearranged high-risk neuroblastoma models following preventive and therapeutic treatment schedules. These effects of panobinostat treatment have not been previously described in models of TERT-rearranged high-risk neuroblastoma and might be translated to patients with TERT-rearranged neuroblastoma still facing poor survival rates today.