A Perspective on the CD47-SIRPA Axis in High-Risk Neuroblastoma.

Neuroblastoma is a pediatric cancer with significant clinical heterogeneity. Despite extensive efforts, it is still difficult to cure children with high-risk neuroblastoma. Immunotherapy is a promising approach to treat children with this devastating disease. We have previously reported that macrophages are important effector cells in high-risk neuroblastoma. In this perspective article, we discuss the potential function of the macrophage inhibitory receptor SIRPA in the homeostasis of tumor-associated macrophages in high-risk neuroblastoma. The ligand of SIRPA is CD47, known as a "don't eat me" signal, which is highly expressed on cancer cells compared to normal cells. CD47 is expressed on both tumor and stroma cells, whereas SIRPA expression is restricted to macrophages in high-risk neuroblastoma tissues. Notably, high SIRPA expression is associated with better disease outcome. According to the current paradigm, the interaction between CD47 on tumor cells and SIRPA on macrophages leads to the inhibition of tumor phagocytosis. However, data from recent clinical trials have called into question the use of anti-CD47 antibodies for the treatment of adult and pediatric cancers. The restricted expression of SIRPA on macrophages in many tissues argues for targeting SIRPA on macrophages rather than CD47 in CD47/SIRPA blockade therapy. Based on the data available to date, we propose that disruption of the CD47-SIRPA interaction by anti-CD47 antibody would shift the macrophage polarization status from M1 to M2, which is inferred from the 1998 study by Timms et al. In contrast, the anti-SIRPA F(ab')2 lacking Fc binds to SIRPA on the macrophage, mimics the CD47-SIRPA interaction, and thus maintains M1 polarization. Anti-SIRPA F(ab')2 also prevents the binding of CD47 to SIRPA, thereby blocking the "don't eat me" signal. The addition of tumor-opsonizing and macrophage-activating antibodies is expected to enhance active tumor phagocytosis.

Macrophage-mediated anti-tumor immunity against high-risk neuroblastoma.

Neuroblastoma is the most common extracranial childhood solid tumor. The majority of high-risk neuroblastoma is resistant/refractory to the current high intensity therapy. Neuroblastoma lacks classical HLA Class I expression and exhibits low mutation burden, allowing neuroblastoma cells to evade CD8+ T cell-mediated immunity. Neuroblastoma cells do not express PD-L1, and tumor-associated macrophages are the predominant PD-L1+ cells in the tumor. In this study, we performed gene expression profiling and survival analyses on large neuroblastoma datasets to address the prognostic effect of PD-L1 gene expression and the possible involvement of the SLAMF7 pathway in the anti-neuroblastoma immunity. High-level expression of PD-L1 was found significantly associated with better outcome of high-risk neuroblastoma patients; two populations of PD-1+ PD-L1+ macrophages could be present in high-risk tumors with PD-1/PD-L1 ratios, ≈1 and >1. Patients with the PD-1/PD-L1 ratio >1 tumor showed inferior survival. High-level co-expression of SLAMF7 and SH2D1B was significantly associated with better survival of the high-risk neuroblastoma patients. Together, this study supports the hypothesis that macrophages are important effector cells in the anti-high-risk neuroblastoma immunity, that PD-1 blockade therapy can be beneficial to the high-risk neuroblastoma subset with the PD-1/PD-L1 expression ratio >1, and that SLAMF7 is a new therapeutic target of high-risk neuroblastoma.

Clinical Relevance of CD4 Cytotoxic T Cells in High-Risk Neuroblastoma.

Neuroblastoma is the most common extracranial childhood solid tumor. The majority of high-risk neuroblastoma is resistant/refractory to the current high intensity therapy, and the survival of these patients remains poor for the last three decades. To effectively treat these extremely unfavorable neuroblastomas, innovative immunotherapy approaches would be the most promising. In this article, we discuss the identity of tumor-infiltrating effector cells and immunosuppressive cells in high-risk neuroblastoma. Neuroblastoma is unique in that it expresses little or no classical HLA Class I and II. In contrast, high-risk neuroblastomas express the stress-responsive non-classical Class I, HLA-E molecule. HLA-E is the ligand of activating receptors NKG2C/E that are expressed on memory NK cells, CD8+T cells and CD4 CTLs. By examining a comprehensive RNA-seq gene expression dataset, we detected relatively high levels of CD4 expression in high-risk neuroblastoma tissues. The majority of CD4+ cells were CD3+, and thus they were likely tumor-associated CD4+T cells. In addition, high-level of both CD4 and NKG2C/E expression was associated with prolonged survival of the high-risk neuroblastoma patients, but CD8 levels were not, further suggesting that the CD4+ NKG2C/E+ T cells or CD4 CTL conferred cytotoxicity against the neuroblastoma cells. However, this T cell mediated- "protective effect" declined over time, in part due to the progressive formation of immunosuppressive tumor microenvironment. These observations suggest that to improve survival of high-risk neuroblastoma patients, it is essential to gain insights into how to enhance CD4 CTL cytotoxicity and control the immunosuppressive tumor microenvironment during the course of the disease.

MYC-family protein overexpression and prominent nucleolar formation represent prognostic indicators and potential therapeutic targets for aggressive high-MKI neuroblastomas: a report from the children's oncology group.

Neuroblastomas with a high mitosis-karyorrhexis index (High-MKI) are often associated with MYCN amplification, MYCN protein overexpression and adverse clinical outcome. However, the prognostic effect of MYC-family protein expression on these neuroblastomas is less understood, especially when MYCN is not amplified. To address this, MYCN and MYC protein expression in High-MKI cases (120 MYCN amplified and 121 non-MYCN amplified) was examined by immunohistochemistry. The majority (101) of MYCN-amplified High-MKI tumors were MYCN(+), leaving one MYC(+), 2 both(+), and 16 both(-)/(+/-), whereas non-MYCN-amplified cases appeared heterogeneous, including 7 MYCN(+), 36 MYC(+), 3 both(+), and 75 both(-)/(+/-) tumors. These MYC-family proteins(+), or MYC-family driven tumors, were most likely to have prominent nucleolar (PN) formation (indicative of augmented rRNA synthesis). High-MKI neuroblastoma patients showed a poor survival irrespective of MYCN amplification. However, patients with MYC-family driven High-MKI neuroblastomas had significantly lower survival than those with non-MYC-family driven tumors. MYCN(+), MYC-family protein(+), PN(+), and clinical stage independently predicted poor survival. Specific inhibition of hyperactive rRNA synthesis and protein translation was shown to be an effective way to suppress MYC/MYCN protein expression and neuroblastoma growth. Together, MYC-family protein overexpression and PN formation should be included in new neuroblastoma risk stratification and considered for potential therapeutic targets.

Destabilization of MYC/MYCN by the mitochondrial inhibitors, metaiodobenzylguanidine, metformin and phenformin.

In the present study, we investigated the anticancer effects of the mitochondrial inhibitors, metaiodobenzylguanidine (MIBG), metformin and phenformin. 131I-MIBG has been used for scintigraphic detection and the targeted radiotherapy of neuroblastoma (NB), a pediatric malignancy. Non-radiolabeled MIBG has been reported to be cytotoxic to NB cells in vitro and in vivo. However, the mechanisms behind its growth suppressive effects have not yet been fully elucidated. Metformin and phenformin are diabetes medications that are being considered in anticancer therapeutics. We investigated the anticancer mechanisms of action of MIBG and metformin in NB. Our data revealed that both drugs suppressed NB cell growth and that the combination drug treatment was more potent. MIBG reduced MYCN and MYC expression in MYCN-amplified and non-MYCN-amplified NB cells in a dose- and time-dependent manner. Metformin was less effective than MIBG in destabilizing MYC/MYCN. The treatment of NB cells with metformin or MIBG resulted in an increased expression of genes encoding biomarkers for favorable outcome in NB [(ephrin (EFN)B2, EFNB3, EPH receptor B6 (EPHB6), neurotrophic tyrosine kinase, receptor, type 1 (NTRK1), CD44 and Myc-interacting zinc finger protein (MIZ-1)] and tumor suppressor genes [(early growth response 1 (EGR1), EPH receptor A2 (EPHA2), growth arrest and DNA-damage-inducible, beta (GADD45B), neuregulin 1 (NRG1), TP53 apoptosis effector (PERP) and sel-1 suppressor of lin-12-like (C. elegans) (SEL1L)]. Accordingly, metformin and MIBG augmented histone H3 acetylation in these cells. Phenformin also exhibited histone modification and was more effective than metformin in destabilizing MYC/MYCN in NB cells. Our data suggest that the destabilization of MYC/MYCN by MIBG, metformin and phenformin and their effects on histone modification are important mechanisms underlying their anticancer effects.

S(+)-ibuprofen destabilizes MYC/MYCN and AKT, increases p53 expression, and induces unfolded protein response and favorable phenotype in neuroblastoma cell lines.

Neuroblastoma is a common pediatric solid tumor that exhibits a striking clinical bipolarity: favorable and unfavorable. The survival rate of children with unfavorable neuroblastoma remains low among all childhood cancers. MYCN and MYC play a crucial role in determining the malignancy of unfavorable neuroblastomas, whereas high-level expression of the favorable neuroblastoma genes is associated with a good disease outcome and confers growth suppression of neuroblastoma cells. A small fraction of neuroblastomas harbors TP53 mutations at diagnosis, but a higher proportion of the relapse cases acquire TP53 mutations. In this study, we investigated the effect of S(+)-ibuprofen on neuroblastoma cell lines, focusing on the expression of the MYCN, MYC, AKT, p53 proteins and the favorable neuroblastoma genes in vitro as biomarkers of malignancy. Treatment of neuroblastoma cell lines with S(+)-ibuprofen resulted in a significant growth suppression. This growth effect was accompanied by a marked decrease in the expression of MYC, MYCN, AKT and an increase in p53 expression in neuroblastoma cell lines without TP53 mutation. In addition, S(+)-ibuprofen enhanced the expression of some favorable neuroblastoma genes (EPHB6, CD44) and genes involved in growth suppression and differentiation (EGR1, EPHA2, NRG1 and SEL1L). Gene expression profile and Ingenuity pathway analyses using TP53-mutated SKNAS cells further revealed that S(+)-ibuprofen suppressed molecular pathways associated with cell growth and conversely enhanced those of cell cycle arrest and the unfolded protein response. Collectively, these results suggest that S(+)-ibuprofen or its related compounds may have the potential for therapeutic and/or palliative use for unfavorable neuroblastoma.

Transient treatment with epigenetic modifiers yields stable neuroblastoma stem cells resembling aggressive large-cell neuroblastomas.

Cancer stem cells (CSCs) are plastic in nature, a characteristic that hampers cancer therapeutics. Neuroblastoma (NB) is a pediatric tumor of neural crest origin, and half of the cases are highly aggressive. By treating NB cell lines [SKNAS, SKNBE(2)C, CHP134, and SY5Y] with epigenetic modifiers for a short time, followed by sphere-forming culture conditions, we have established stem cell-like NB cells that are phenotypically stable for more than a year. These cells are characterized by their high expression of stemness factors, stem cell markers, and open chromatin structure. We referred to these cells as induced CSCs (iCSCs). SKNAS iCSC and SKNBE(2)C iCSC clones (as few as 100 cells) injected s.c. into SCID/Beige mice formed tumors, and in one case, SKNBE(2)C iCSCs metastasized to the adrenal gland, suggesting their increased metastatic potential. SKNAS iCSC xenografts showed the histologic appearance of totally undifferentiated large-cell NBs (LCNs), the most aggressive and deadly form of NB in humans. Immunohistochemical analyses showed that SKNAS iCSC xenografts expressed high levels of the stem cell marker CXCR4, whereas the SKNAS monolayer cell xenografts did not. The patterns of CXCR4 and MYC expression in SKNAS iCSC xenografts resembled those in the LCNs. The xenografts established from the NB iCSCs shared two common features: the LCN phenotype and high-level MYC/MYCN expression. These observations suggest both that NB cells with large and vesicular nuclei, representing their open chromatin structure, are indicative of stem cell-like tumor cells and that epigenetic changes may have contributed to the development of these most malignant NB cells.

Hsp90 inhibition increases p53 expression and destabilizes MYCN and MYC in neuroblastoma.

Neuroblastoma is a childhood cancer that exhibits either a favorable or an unfavorable phenotype. MYCN and MYC are oncoproteins that play crucial roles in determining the malignancy of unfavorable neuroblastoma. The Hsp90 superchaperone complex assists in the folding and function of a variety of oncogenic client proteins. Inhibition of Hsp90 by small molecule inhibitors leads to the destabilization of these oncogenic proteins and consequently suppresses tumor malignancy. Nonetheless, little is known about the effect of Hsp90 inhibition on the stability of MYCN and MYC proteins. In this study, we investigated the effect of Hsp90 inhibition on the phenotype of unfavorable neuroblastoma cells including its effect on MYCN and MYC expression. Two MYCN-amplified neuroblastoma cell lines (IMR5 and CHP134) and two non-MYCN-amplified cell lines (SY5Y and SKNAS) were used to address the effect of Hsp90 inhibition on the malignant phenotype of neuroblastoma. It was found that Hsp90 inhibition in neuroblastoma cell lines resulted in significant growth suppression, a decrease in MYCN and MYC expression, and an increase in the expression of p53. In the TP53-mutated SKNAS cell line, Hsp90 inhibition enhanced the expression of the favorable neuroblastoma genes EFNB2, MIZ-1 and NTRK1 (TrkA). In addition, Hsp90 inhibition reduced HDAC6 expression and enhanced tubulin acetylation. Together our data suggest that Hsp90 inhibition suppresses the growth of neuroblastoma through multiple cellular pathways and that MYC/MYCN destabilization is among the important consequences of Hsp90 inhibition.

Biological effects of induced MYCN hyper-expression in MYCN-amplified neuroblastomas.

Neuroblastoma is a childhood malignancy of the sympathetic nervous system. The tumor exhibits two different phenotypes: favorable and unfavorable. MYCN amplification is associated with rapid tumor progression and the worst neuroblastoma disease outcome. We have previously reported that inhibitors of histone deacetylase (HDAC) and proteasome enhance favorable neuroblastoma gene expression in neuroblastoma cell lines and inhibit growth of these cells. In this study, we investigated the effect of trichostatin A or TSA (an HDAC inhibitor), and epoxomycin (a proteasome inhibitor) on MYCN and p53 expression in MYCN-amplified neuroblastoma cells. It was found that TSA down-regulated MYCN expression, but Epoxomycin and the TSA/Epoxomycin combination led to MYCN hyper-expression in MYCN-amplified neuroblastoma cell lines. Despite their contrasting effects on MYCN expression, TSA and Epoxomycin caused growth suppression and cell death of the MYCN-amplified cell lines examined. Consistent with these data, forced hyper-expression of MYCN in MYCN-amplified IMR5 cells via transfection resulted in growth suppression and the increased expression of several genes known to suppress growth or induce cell death. Furthermore, Epoxomycin as a single agent and its combination with TSA enhance p53 expression in the MYCN-amplified neuroblastoma cell lines. Unexpectedly, co-transfection of TP53 and MYCN in IMR5 cells resulted in high p53 expression but a reduction of MYCN expression. Together our data suggest that either down regulation or hyper-expression of MYCN results in growth inhibition and/or apoptosis of MYCN-amplified neuroblastoma cells. In addition, elevated p53 expression has a suppressive effect on MYCN expression in these cells.

Biological significance of EPHA2 expression in neuroblastoma.

Neuroblastoma is a pediatric solid tumor that exhibits striking clinical bipolarity. Despite extensive efforts to treat unfavorable neuroblastoma, survival rate of children with the disease is among the lowest. Previous studies suggest that EPHA2, a member of the EPH family receptor kinases, can either promote or suppress cancer cell growth depending on cellular contexts. In this study, we investigated the biological significance of EPHA2 in neuroblastoma. It was found that tumorigenic N-type neuroblastoma cell lines expressed low levels of EPHA2, whereas hypo-tumorigenic S-type neuroblastoma cell lines expressed high levels of EPHA2 (p<0.005). Notably, inhibitors of DNA methylation and histone deacetylase enhanced EPHA2 expression in N-type cells, suggesting that EPHA2 is epigenetically silenced in unfavorable neuroblastoma cells. Furthermore, ectopic high-level expression of EPHA2 in N-type neuroblastoma cell lines resulted in significant growth suppression. However, Kaplan-Meier survival analysis showed that high EPHA2 expression was not associated with a good disease outcome of neuroblastoma, indicating that EPHA2 is not a favorable neuroblastoma gene, but a growth suppressive gene for neuroblastoma. Accordingly, EPHA2 expression was markedly augmented in vitro in neuroblastoma cells treated with doxorubicin, which is commonly used for treating unfavorable neuroblastoma. Taken together, EPHA2 is one of the effectors of chemotherapeutic agents (e.g., gene silencing inhibitors and DNA damaging agents). EPHA2 expression may thus serve as a biomarker of drug responsiveness for neuroblastoma during the course of chemotherapy. In addition, pharmaceutical enhancement of EPHA2 by non-cytotoxic agents may offer an effective therapeutic approach in the treatment of children with unfavorable neuroblastoma.

De novo identification of MIZ-1 (ZBTB17) encoding a MYC-interacting zinc-finger protein as a new favorable neuroblastoma gene.

PURPOSE: Neuroblastoma is a childhood cancer that exhibits either a favorable or an unfavorable phenotype. Favorable neuroblastoma genes (EPHB6, EFNB2, EFNB3, NTRK1, and CD44) are genes whose high-level expression predicts favorable neuroblastoma disease outcome. Accordingly, the forced expression of these genes or their reactivation by gene silencing inhibitors in unfavorable neuroblastoma cells results in suppression of tumor growth and metastases. This study was undertaken to design an experimental strategy to identify additional favorable neuroblastoma genes. EXPERIMENTAL DESIGN: Favorable neuroblastoma gene candidates were first identified by gene expression profiling analysis on IMR5 neuroblastoma cells treated with inhibitors of DNA methylation and histone deacetylase against the untreated control cells. Among the candidates, we focused on MIZ-1, which encodes a MYC-interacting zinc-finger protein, because it is known to enhance the expression of growth suppressive genes, such as CDKN1A. RESULTS: High-level MIZ-1 expression was associated with favorable disease outcome of neuroblastoma (P = 0.0048). Forced MIZ-1 expression suppressed in vitro growth of neuroblastoma cell lines. High MIZ-1 expression was correlated with the small-size neuroblastoma xenografts treated with gene silencing inhibitors or a glucocorticoid. In addition, forced MIZ-1 expression enhanced the expression of CD44 and EFNB2 in neuroblastoma cell lines in vitro. Furthermore, MIZ-1 expression was positively correlated with the expression of favorable neuroblastoma genes (EFNB2, EFNB3, EPHB6, and NTRK1) in the human neuroblastoma xenograft therapeutic models. CONCLUSION: MIZ-1 is a new favorable neuroblastoma gene, which may directly or indirectly regulate the expression of other favorable neuroblastoma genes.

The MYCN enigma: significance of MYCN expression in neuroblastoma.

MYCN amplification strongly predicts adverse outcome of neuroblastoma. However, the significance of MYCN expression in the clinical and biological behavior of neuroblastoma has been unclear. To address this question, we first examined the expression of MYCN in combination with TrkA (a favorable prognostic indicator of neuroblastoma) in 91 primary neuroblastoma by quantitative reverse transcription-PCR and investigated the relationship among patient survival, MYCN, and TrkA expressions. Three subsets of neuroblastoma were defined based on MYCN and TrkA expression. Neuroblastoma expressing the highest level of MYCN but little TrkA were MYCN-amplified cases, which had a 5-year survival of 9.3%. Interestingly, MYCN and TrkA expression showed a linear correlation (r = 0.5664, P < 0.00005) in neuroblastoma lacking MYCN amplification, and the 5-year survival of neuroblastoma patients with low MYCN and low TrkA expressions was 63.7%, whereas those with high expression of both had a 5-year survival of 88.1% (P < 0.00005). This nonlinear distribution of disease outcome relative to MYCN expression in neuroblastoma explains why MYCN expression is not predictive of neuroblastoma disease outcome by dichotomous division of the neuroblastoma cohort. However, high-level MYCN expression is associated with favorable outcome in neuroblastoma lacking MYCN amplification. Furthermore, forced expression of MYCN significantly suppresses growth of neuroblastoma cells lacking MYCN amplification by inducing apoptosis and enhancing favorable neuroblastoma gene expression. Collectively, these data suggest that high-level MYCN expression in neuroblastoma lacking MYCN amplification results in a benign phenotype. Thus, the high MYCN expression confers the opposite biological consequence in neuroblastoma, depending on whether or not MYCN is amplified.

Favorable neuroblastoma genes and molecular therapeutics of neuroblastoma.

PURPOSE AND EXPERIMENTAL DESIGN: Neuroblastoma (NB) is a common pediatric solid tumor that exhibits a striking clinical bipolarity: favorable and unfavorable. Favorable NB genes (EPHB6, EFNB2, EFNB3, NTRK1, and CD44) are genes whose high-level expression predicts favorable NB outcome, and forced expression of these genes inhibits growth of unfavorable NB cells. In this study, we investigated whether favorable NB gene expression could be augmented in unfavorable NB cells by chemical compounds and whether an increased expression of these genes was associated with suppression of NB growth and metastasis. RESULTS: We found that inhibitors of DNA methylation [5-aza-2'-deoxycytidine (5AdC)], histone deacetylase (HDAC) [4-phenylbutyrate (4PB)], and proteasome (MG262) enhanced the expression of favorable NB genes in NB cell lines and inhibited the growth of these cells in vitro (P < 0.0005). The growth-inhibitory effects of 5AdC and 4PB in vitro were in part due to caspase-dependent cell death and inhibition of DNA synthesis. Administration of 5AdC and/or 4PB also suppressed growth of subcutaneous NB xenografts in nude mice (P < 0.001), which was accompanied by enhanced favorable NB gene expression and an increase in apoptosis. Moreover, 4PB suppressed bone marrow and liver metastases of NB cells in severe combined immunodeficient/Beige mice (P = 0.007 and P = 0.008, respectively). The growth-suppressive activity of HDAC inhibitors on NB was further confirmed by the efficacy of trichostatin A, a potent and specific HDAC inhibitor. CONCLUSIONS: Collectively, these observations further emphasize the link between the elevated favorable NB gene expression and a benign phenotype of NB.