O2144: PIM Kinase Inhibition Sensitizes Neuroblastoma to Doxorubicin

Background: Children with high-risk neuroblastoma continue to have significant rates of relapse despite the use of complex multi-modal therapies. Disease relapse in these patients may be attributed, in part, to the development of drug resistance. Cancer cells employ multiple mechanisms to acquire chemotherapeutic resistance, such as drug efflux pumps. In neuroblastoma, expression of the multidrug resistance-associated protein-1 (MRP1/ABCC1) drug efflux pump correlates with worse outcomes. Drug efflux pumps are regulated by PIM kinases, a family of serine-threonine kinases, overexpressed in some cancers including neuroblastoma. We hypothesized that PIM kinase inhibition would sensitize neuroblastoma cells to chemotherapeutic drugs through inhibition of the MRP1 drug efflux pump.

Methods: Kocak database query evaluated neuroblastoma patient data for ABCC1 expression, the gene regulating MRP1 protein expression, and the gene expression of PIM1, PIM2, and PIM3. SK-N-AS and SK-N-BE(2) human neuroblastoma cells were treated for 1-72 hours with doxorubicin, a drug commonly used to treat neuroblastoma, or AZD1208, a pan-PIM kinase inhibitor. Intracellular doxorubicin fluorescence was assessed using flow cytometry. Viability was measured with alamarBlue assay, the lethal dose 50% (LD50) of each drug and combination indices (CI) were calculated, and isobolograms constructed. Combination indices < 1 indicated synergy.

Results: Kocak database query demonstrated significant correlation between high ABCC1 expression and decreased overall neuroblastoma survival probability (p ≤ 4.9 x 10-21, Fig 1A). There was positive correlation between each of the individual PIM genes (PIM1, PIM2, PIM3) and ABCC1 (p ≤ 0.001, Fig 1B-D), suggesting PIM kinase regulation of MRP1. PIM kinase inhibition with AZD1208 led to accumulation of intracellular doxorubicin over time (1-72 hours) in both SK-N-AS (5.04 ± 1.76 vs. 1.94 ± 0.28, AZD1208 treatment vs. doxorubicin alone, 72 h, p ≤ 0.05, Fig 2A) and SK-N-BE(2) cell lines (26.2 ± 3.79 vs. 5.06 ± 0.56, AZD1208 treatment vs. doxorubicin alone, 72 h, p ≤ 0.01, Fig 2B). AZD1208 (10 μM and 20 μM) combined with doxorubicin, resulted in CIs of 0.64 and 0.82 in SK-N-AS cells (Fig 2C) and 0.30 and 0.65 in SK-N-BE(2) cells, respectively (Fig 2D), indicating synergy between the two compounds.

Conclusion: In the current study, we demonstrate that ABCC1 gene expression is associated with lower overall neuroblastoma survival and that PIM gene expression correlates with drug efflux pump gene expression, indicating PIM kinases may contribute to neuroblastoma chemotherapeutic resistance. PIM kinase inhibition increased intracellular doxorubicin accumulation and demonstrated synergy when combined with doxorubicin. These findings suggest combination therapy with PIM kinase inhibition and doxorubicin could enhance the efficacy of doxorubicin, and support the need for further investigations to explore PIM kinase inhibition as a therapeutic strategy in neuroblastoma.