O1089: PIM Kinase Inhibition Results in Neuroblastoma Differentiation

Background: Neuroblastoma is the most common extracranial solid tumor in children. Prognosis for children with high-risk disease remains poor. An important component of high risk therapy is promoting cellular differentiation with the use of retinoic acid (RA) in the maintenance phase. Unfortunately, up to 50% of children suffer disease relapse despite the use of RA. It has been demonstrated that neuroblastoma cells treated with RA have increased HOXC9 expression and HOXC9 is associated with improved neuroblastoma survival. We have previously shown that PIM kinase inhibition promotes cellular differentiation and increased mRNA and protein levels of HOXC9. We aimed to determine whether combining PIM kinase inhibition with a novel rexinoid agonist, UAB30, could further promote differentiation in neuroblastoma.

Methods: We queried the Versteeg (R2) gene database to compare PIM1 and the neural differentiation marker, HOXC9, in neuroblastoma. We used SK-N-AS human neuroblastoma cells and human neuroblastoma patient derived xenograft (PDX129). Cells were treated with the pan-PIM inhibitor, AZD1208, a novel rexinoid agonist, UAB30, or a combination of pre-treatment with AZD1208 for 72 hours followed by 72 hours of UAB30 treatment. Real time PCR identified changes in HOXC9 mRNA. Differentiation was measured by neurite outgrowth and quantified by comparing cell body length to neurite length. Data were reported as mean ± SEM, with two-tailed t-test, p≤0.05 considered significant.

Results: R2 database revealed a negative correlation between PIM1 and HOXC9 (Figure 1A), indicating PIM1 was associated with less differentiation. Abundance of HOXC9 mRNA was increased with PIM inhibition in PDX129 cells (Figure 1B). Treatment with the combination of AZD1208 and UAB30 significantly increased neurite outgrowth. Representative photomicrographs are presented in Figure 1C (left and middle panels) with black arrows demonstrating neurites. Quantification of neurite outgrowth confirmed that PIM inhibition combined with UAB30 significantly increased differentiation.

Conclusion: Neuroblastoma cells treated with the combination of a PIM inhibitor and a rexinoid agonist demonstrated a more differentiated phenotype. These findings are potentially mediated through increased HOXC9. These data support the need for further studies of the role of PIM kinase inhibition as a therapeutic strategy to improve RA-induced differentiation, potentially mitigating disease relapse in high risk neuroblastoma.