EACR25-1416
High-risk neuroblastoma (NB) is an aggressive pediatric malignancy with poor prognosis, as the 5-year disease-free survival rate remains below 50% despite intensive therapy. Developing novel combination treatments is crucial to improving outcomes. Isotretinoin (13cRA), a key agent in post-consolidation NB therapy, exhibits anti-proliferative and pro-differentiative effects. To identify compounds that enhance its efficacy, we screened a library of 169 natural polyphenols and found that isorhamnetin (ISR) synergizes with 13cRA, reducing cell viability by 80%. Mechanistically, this effect correlates with upregulation of the adrenergic receptor α1B (ADRA1B), a member of the catecholamine receptor superfamily. This study investigates ADRA1B’s role in mediating the synergistic effects of 13cRA and ISR, as well as the broader impact of adrenergic receptor modulation on NB growth in combination with 13cRA.
We generated ADRA1B-knockout (KO) NB cell lines using CHP134 (13cRA-sensitive) and SK-N-AS (13cRA-resistant) cells. Cellular responses to 13cRA and isorhamnetin, alone or in combination, were assessed via metabolic assays, apoptosis assays, differentiation markers, and gene expression analysis. Additionally, a high-throughput screen using an adrenergic receptor ligand library evaluated cell viability upon co-administration with 13cRA. Finally, the combinatorial approach was tested in vivo using NB xenograft models.
CHP134 ADRA1B-KO cells treated with 13cRA showed reduced viability and increased Caspase-3/7 activation, comparable to wild-type cells treated with the combination of 13cRA and ISR. Furthermore, 13cRA exposure in CHP134 KO cells induced neurite outgrowth and upregulation of neural differentiation. In contrast, SK-N-AS cells (WT or ADRA1B-KO) showed no changes in viability or differentiation marker expression following 13cRA treatment. Pharmacological inhibition of α1B-AR and 13cRA administration to the cells, mirrored the genetic KO results. Treating a panel of 11 NB cell lines with α1/α1B-AR antagonists revealed that the presence of MYCN-amplification is essential to observe the sensitization over the cell viability reduction and neural differentiation induced by 13cRA, thus mimicking the ISR activity. As expected, the addition of α1/α1B-AR agonists led to opposite effects. In vivo, co-treatment with 13cRA and the FDA-approved α1 AR antagonist doxazosin significantly slowed tumor progression in NB xenograft models.
Our findings suggest that α1-AR antagonism sensitizes NB cells to 13cRA by facilitating the pro-differentiative and pro-apoptotic effects, potentially disrupting an autocrine catecholamine-driven pro-survival circuit. Given the druggable nature of ARs, α1-AR inhibition emerges as new a promising pharmacological therapeutic strategy for neuroblastoma treatment.