Expression-based screening identifies the combination of histone deacetylase inhibitors and retinoids for neuroblastoma differentiation

Abstract

The discovery of new small molecules and their testing in rational combination poses an ongoing problem for rare diseases, in particular, for pediatric cancers such as neuroblastoma. Despite maximal cytotoxic therapy with double autologous stem cell transplantation, outcome remains poor for children with high-stage disease. Because differentiation is aberrant in this malignancy, compounds that modulate transcription, such as histone deacetylase (HDAC) inhibitors, are of particular interest. However, as single agents, HDAC inhibitors have had limited efficacy. In the present study, we use an HDAC inhibitor as an enhancer to screen a small-molecule library for compounds inducing neuroblastoma maturation. To quantify differentiation, we use an enabling gene expression-based screening strategy. The top hit identified in the screen was all-trans-retinoic acid. Secondary assays confirmed greater neuroblastoma differentiation with the combination of an HDAC inhibitor and a retinoid versus either alone. Furthermore, effects of combination therapy were synergistic with respect to inhibition of cellular viability and induction of apoptosis. In a xenograft model of neuroblastoma, animals treated with combination therapy had the longest survival. This work suggests that testing of an HDAC inhibitor and retinoid in combination is warranted for children with neuroblastoma and demonstrates the success of a signature-based screening approach to prioritize compound combinations for testing in rare diseases.

Fig. 1.

GE-HTS identifies the combination of VPA and ATRA for the induction of neuroblastoma differentiation. (A) Ten neuroblastoma differentiation marker genes were chosen to distinguish undifferentiated neuroblastoma from differentiated cells. Blue represents poorly expressed genes and red depicts highly expressed genes. (B) Distribution of the screening data is shown for the summed-score and weighted summed-score metrics. Undifferentiated BE (2)-C controls (DMSO treated) included in the screen are shown in yellow and differentiated controls (5 μM ATRA treated) in gray. The black dots represent the compounds that did not score as hits with either of these two measurements. Hits scoring as differentiated based on the overall scores of all replicates are ATRA (red), cytarabine (blue), flumequine (green), and penicillamine (purple). Each unfiltered replicate is depicted in the figure.

Fig. 2.

The combination of VPA and ATRA induces increased differentiation. (A) BE (2)-C cells were treated with combinations of 1 mM VPA, 10 nM LD-ATRA, and 5 μM HD-ATRA and the neuroblastoma differentiation signature evaluated. A box-and-whisker plot demonstrates the distribution of weighted summed scores for each sample type where the heavy lines inside the box show the median, the boxes show the quartiles, and the whiskers show the extremes of the observed distribution of scores. (B) May Grunwald Giemsa staining of BE (2)-C cells treated for 5 days reveals maximal differentiation with both agents in combination. Images were acquired with an Olympus CK40 microscope, 400× magnification, and Qcapture software. (C) Western immunoblot of BE (2)-C cells treated with either vehicle, ATRA (10 nM or 5 μM), VPA (1 mM), or both agents in combination for 5 days and analyzed with antibody to neurofilament medium (NF-M) and GAPD as a control.

Fig. 3.

VPA and ATRA show synergistic effects on cell viability and cell death. (A) The combined effect of VPA and ATRA on BE (2)-C (Left) and IMR-32 (Right) cell viability at 5 days, as determined by ATP level, is shown by isobologram. Synergy appears as points below the line of additivity. (B) BE (2)-C cells were treated with compounds for 3 days. Combination treatment induced increased annexin V positive cells consistent with apoptosis with an additive interaction at the low-dose ATRA and synergistic interaction with high-dose ATRA as evaluated by excess over Bliss independence. (C) Western blot analysis of histone acetylation at 6 h in BE (2)-C cells treated with VPA 1 mm (V), ATRA 10 nM (A), and both agents in combination. HeLa cell controls ± butyrate are included.

Fig. 4.

Differentiation precedes apoptosis in combination-treated neuroblastoma cells. (A) BE (2)-C cells were treated with either vehicle (veh) or the combination of 5 μM ATRA (A) and 1 mM VPA (V) for 6, 24, or 72 h and the effects on the 14-gene differentiation signature were evaluated. *, statistical significance in a pairwise t test comparing vehicle with drug treatment at each time point. Duplicate biological replicates and 16 technical replicates for each time point and condition were evaluated. BE (2)-C cells were treated in triplicate as above and the effects on early apoptosis (annexin V-FITC positive and PI negative) (B) and late apoptosis (annexin V-FITC positive and PI positive) (C) were evaluated. *, statistical significance in a pairwise t test comparing vehicle with drug treatment at each time point.

Fig. 5.

ATRA and SAHA have enhanced activity in an in vivo model of neuroblastoma. (A) BE (2)-C xenografts were established for 10 days in NCr nude mice. Animals received vehicle, ATRA 2.5 mg/kg IP daily, SAHA 25 mg/kg IP daily, or a combination of ATRA and SAHA for up to 21 days. Error bars show standard error of the mean across six or seven replicates. The x axis represents the days since the initiation of treatment. (B) Percentage of surviving animals is shown. The x axis represents the days since the initiation of treatment. The two-tailed P values of the survival curves were determined by logrank test for pairwise comparisons: vehicle vs. ATRA = NS, vehicle vs. SAHA = 0.05, vehicle vs. combo = 0.003, ATRA vs. SAHA = NS, ATRA vs. combination = 0.001 and SAHA vs. combination = 0.009. (C) BE (2)-C xenografts in NCr nude mice were treated with either vehicle, ATRA 2.5 mg/kg IP daily, SAHA 25 mg/kg IP daily, or a combination of ATRA and SAHA for 4 days. Tumors treated with the combination of drugs demonstrated marked increased areas of cell death by hematoxylin and eosin staining. Images were acquired with an Olympus BX41 microscope, 40× magnification, and Qcapture software. (D) As in C, BE (2)-C xenografts were established and treated. Three to five tumors from each class were harvested, and the neuroblastoma differentiation signature measured for each sample with 16 technical replicates. All drug treatments were statistically elevated compared with vehicle, and combination treatment was statistically elevated compared to single agent treatment (P < 0.001 by t test).