Effect of sulfasalazine on human neuroblastoma: analysis of sepiapterin reductase (SPR) as a new therapeutic target

SPR mRNA expression in NB

We have previously reported on the role of SPR in NB proliferation 32], where we demonstrated a deleterious effect of RNAi-mediated SPR expression knockdown
in the MYCN2 NB cell line. We also showed that high SPR mRNA expression was correlated
to poor patient prognosis in Kaplan-Meier analysis in the Versteeg-88 NB dataset in
the public domain. We now present SPR mRNA expression analysis on all 12 NB cohorts
in the public domain (Table 1). We find that high SPR expression is significantly correlated in all four NB cohorts
annotated for patient survival and/or prognosis. While in our previous study 32] we could only show a trend for a correlation between SPR expression and tumor MYCN
gene amplification in the Versteeg-88 set (P?=?0.06), we can now state that SPR expression is significantly higher in patients
with tumor MYCN gene amplification in 6 of 8 datasets with MYCN amplification annotation.
Considering the different compositions of these datasets with respect to patient age,
MYCN amplification, and INSS stage, together with the different array platforms used
for the generation of these data, this is a very robust finding. In Fig. 2, we show the results for the largest NB cohort in the public domain, the Kocak-649
dataset. Although this dataset does not contain survival data, the correlations between
SPR expression and three important clinical NB parameters are highly significant (Fig. 2, a-c): age at diagnosis (P?=?1.9?·?10
^?23
, MYCN tumor amplification (P?=?7.9?·?10
^?15
, and INSS stage (various P values??0.05). In addition, the Kocak-649 dataset shows a significant correlation
between SPR and ODC mRNA expression (Fig. 3, R?=?0.225, P?=?6.5?·?10
^?9
). This association, although highly significant, has a relatively low R value. However,
since we previously found a similar association (R?=?0.289, P?=?6.2?·?10
^?3
) in the Versteeg-88 cohort 32], we felt strengthened in our argument that this correlation is meaningful.

Table 1. SPR mRNA correlations in public NB mRNA expression datasets

Fig. 2. SPR mRNA expression correlation with NB clinical parameters. Differential expression
of SPR mRNA expression in the Kocak-649 cohort upon separation of patient samples
into clinically important groups. (a) SPR expression is significantly higher in older than in younger patients (age at
diagnosis ?18 months versus 18 months; P?=?1.9?·?10
^?23
), (b) SPR expression is significantly higher in patients with than in patients without
tumor MYCN gene amplification (P?=?7.9?·?10
^?15
), and (c) SPR expression is significantly higher in high than in low stage tumors (INSS stage
3 and 4 versus stage 1, 2, and 4S; various P??0.05). For all three parameters, SPR expression is highest in the poor outcome
group. Statistical analysis was performed using the non-parametric Kruskal-Wallis
tests

Fig. 3. SPR expression correlation with ODC expression in NB. SPR and ODC mRNA expression
correlation in the Kocak-649 NB cohort: visual representation of SPR and ODC expression
in all 649 NB tumor samples, ranked horizontally from left to right according to their
SPR expression. SPR and ODC (2log) expression values for each sample are visualized
with red circles and black rectangles, respectively. The correlation between SPR and
ODC expression is r?=?0.225, with a P value of 6.5?·?10
^?9
(2log Pearson). Symbols representing the clinical values of the tumor samples: age
at diagnosis, MYCN amplification, and INSS stage, are listed below the graph, together
with their legend

These results show that SPR mRNA expression is highest in all NB clinical groups with
poor outcome: high age at diagnosis, tumors with MYCN oncogene amplification, and
patients with high INSS tumor stage. Its expression pattern therefore resembles that
of ODC, and indeed we found a tentative correlation between SPR and ODC expression.
Together, these results prompted us to investigate the specific targeting of SPR alone
or together with targeting of ODC as novel NB therapy.

The effect of Sulfasalazine (SSZ) treatment on NB cell proliferation and survival

A recent study by Chidley et al. revealed that SSZ blocks BH4 biosynthesis through inhibition of SPR 30]. To examine the inhibitory effects of SSZ in NB cells, we treated SK-N-Be(2)c, SK-N-SH,
and LAN-5 cells with increasing concentrations of SSZ (0–400 ?M) and measured cell
viability 48 h after treatment. As shown in Fig. 4, SSZ decreased the cell viability of all three NB cell lines in a dose-dependent
manner. We did not observe overt apoptosis (data not shown), suggesting that SSZ inhibits
cell proliferation of NB cells without cytotoxic effects.

Fig. 4. Effect of Sulfasalazine (SSZ) on the viability of NB cells using the MTS cell viability
assay. NB cell lines SK-N-Be(2)c, SK-N-SH, and LAN-5 were treated with increasing
concentrations of SSZ for 48 hours. Dose-dependent inhibition of cell viability was
observed. Statistically significant differences between values obtained from DMSO-treated
control cells and SSZ-treated cells are indicated with an asterisk (*P??0.05) or solid triangle (?P??0.005). Data represent the average of three independent experiments (n?=?3); bars, mean?±?SEM

To investigate potential signaling molecules and pathways involved in SSZ-mediated
cell death, we tested the expression levels of several proteins that regulate cell
proliferation, including p27
^Kip1
, retinoblastoma tumor suppressor protein Rb, Akt/PKB, and p44/42 MAPK (Erk1/2). Western
blot analysis did not reveal any significant protein expression differences between
SSZ-treated and untreated NB cells (data not shown), suggesting that additional, alternative
signaling pathways are activated by SSZ.

Computational modeling and docking of SSZ into SPR

To examine if SPR binds SSZ, we performed computational docking simulations. SSZ is
an amino-salicylate, specifically 5-((4- (2- Pyridylsulfamoyl) phenyl)azo) salicylic
acid (Fig. 1). SSZ has one canonical conformer with an MMFF94-minimized (Merck Molecular Force
Field) energy of 83.9 kcal/mol, which was used in the docking simulations 33]. Under physiological conditions the molecule carries a negative charge which may
have a role in the interaction with the receptor.

The human SPR crystal structure is available in complex with NADP+ in a hexameric
assembly (unpublished data, PDB: 1Z6Z). This biologically active, functional form
of SPR exists as a dimer and has 2-fold (180°) rotational symmetry. The SPR monomer
is an alpha and beta (a/b) class protein with a 3-layer (aba) sandwich architecture
and Rossmann fold topology, and it contains an NADP- binding Rossmann-like domain
34].

We explored feasible binding modes both for the SPR monomer and the dimer. The docking
computations were carried out on each binding mode by geometric complementarity and
semi-flexible docking to allow for inherent receptor flexibility. From each computation,
the 50 lowest energy-docking positions were saved for further analysis. The presumed
SSZ-binding sites were ranked by conservation score, specifically by the frequency
of occurrence of a residue in a contact surface. The contact surface was delimited
as an area consisting of the residues inside a 3.6 Å radius of the ligand.

Based on the conservation scores of all the residues, we identified the main binding
location within the NADP-binding Rossmann-like domain. A consensus of five binding
regions constituted the receptor pocket comprising residues Gly11, Ser13, Arg14, Phe16
(Region 1), Ala38, Arg39 (Region 2), Asn97, Ala98, Gly99, Ser100 (Region 3), Tyr167
(Region 4), and Leu198, Thr200, Met202 (Region 5). Thus, the binding pocket appeared
to contain 2 basic polar residues, 5 neutral polar residues, and 7 neutral non-polar
residues. Due to the presence of 2 arginine residues, the site has a basic, positively
charged character which may be essential for SSZ binding. Most or all of SSZ exists
in a non-protonated, negatively charged state at neutral pH, as the acidic pK
_a
of carboxylic acid is 2.3 and the pK
_a
of the sulfonamide nitrogen is 6.5, i.e. less than half-protonated at pH 7.0 35].

The same residues listed above are involved in NADP+ binding, but the complete NADP+
binding site extends beyond these residues (Table 2). The monomeric or dimeric state of SPR did not affect the location of the SSZ binding
site in the simulations, indicating that dimerization does not directly block the
access of ligand to the receptor. Table 2 also lists the dimer interface residues. Indeed, the interface residues do not share
common elements with the SSZ/NADPH+ binding pocket. Only Tyr167, which is part of
both ligand sites, is found in the vicinity of an interface residue, i.e. Cys168.

Table 2. Amino acid residues at the binding sites of SPR-SSZ, SPR-NADP+, and SPR-SPR complexes

Figure 5 shows the binding of SSZ to SPR monomer and dimer, respectively. Both chains were
found to simultaneously bind ligands in the dimer. While the SSZ site is close to
the N-terminus in the primary structure, it appears near the middle of the protein
in the 3D fold. The binding pocket is not in very close contact with the dimerization
interface and only a few side chains project into the joint neighborhood. The figure
also shows the NADP+ binding site of SPR in side-by-side comparison and overlay mode
with SSZ. The superimposition of the ligands clearly illustrates that the two binding
sites are essentially the same. The geometric center of SSZ and NADP+ is separated
only by about 0.5 Å from each other in the superimposed binding pockets. Thus, from
Fig. 5 and Table 2 it appears that the binding site for SSZ coincides with the region previously identified
in NADP+ binding in the X-ray structure. As a consequence, this could help elucidate
the interaction between SSZ and SPR in in vitro and in vivo studies.

Fig. 5. Binding of SSZ to SPR. (a) SPR dimer front view (C2 axis). Both chains bind SSZ independently. (b) SPR dimer in complex with NADP+. (c) SPR monomer close-up front view of the SSZ binding pocket: (d) SPR monomer close-up front view of the NADP+ binding pocket. (e) Overlay view of SSZ and NADP+ binding sites. The two binding sites overlap upon
3D alignment of the SPR protein chains. The amino acid residues involved in SSZ and
NADP binding are listed in Table 2. Color scheme for the molecular constituents: Protein chain ribbon – rainbow spectrum
from N-terminus (blue) to C-terminus (red); SSZ space fill – amber; NADP+ spacefill
– cyan

Synergism of SSZ and DFMO combination treatment in NB cells

To test whether the combined treatment with SSZ and DFMO induces synergistic cell
death in NB, we treated SK-N-Be(2)c and LAN-5 cells with different concentrations
of SSZ and DFMO. We used two common methods to analyze drug-drug interactions, the
isobologram and the combination index (CI) analysis. For both combination analyses,
we measured the SSZ and DFMO interaction at 50 % effect level. We first determined
the single-agent IC
₅₀
concentration for SSZ and DFMO in NB cell lines SK-N-Be(2)c and LAN-5 (Fig. 6, a and b) using an MTS cell viability assay after 48 h of treatment. SSZ exhibited an IC
₅₀
value of 133.1 ?M for SK-N-Be(2)c and 337.2 ?M for LAN-5 cells. DFMO showed an IC
₅₀
value of 4.07 mM for SK-N-Be(2)c and 5.79 mM for LAN-5 cells. Subsequently, we combined
SSZ and DFMO at different concentrations based on each IC
₅₀
value to treat the two NB cell lines, generated isobolograms, and calculated the CI
values illustrating the observed synergy. As shown in Fig. 6c and Table 3, SSZ and DFMO combinations revealed slight synergism in SK-N-Be(2)c cells when drug
concentrations were below 29.64 ?M and 1.80 mM, respectively. Strikingly, SSZ and
DFMO showed strong synergism in LAN-5 cells when drug concentrations were below 1.20 ?M
and 1.21 mM, respectively.

Fig. 6. Isobologram analysis for SSZ and DFMO in NB. Isobolograms were prepared to determine
synergisms between SSZ and DFMO. NB cell lines SK-N-Be(2)c and LAN-5 were used to
determine the inhibitory concentration at which 50 % of cells are dead (IC
₅₀
) after 48 h of treatment with (a) SSZ and (b) DFMO. (c) Isobologram analysis to determine the combined cytotoxicity of SSZ and DFMO using
the IC
₅₀
values from (a and b). The IC
₅₀
value of SSZ and DFMO used in combination provides the connective points for the line
of additive. Synergy, additivity, or antagonism is indicated below, on, or above the
line, respectively. The data present the average of three independent experiments
in duplicate (n?=?6); points, mean?±?SEM

Table 3. Combination treatment of SSZ and DFMO in SK-N-Be(2)c and LAN-5 cells for 48 h