Epigenetic upregulation of Schlafen11 renders WNT- and SHH-activated medulloblastomas sensitive to cisplatin

Abstract

Background: Intensive chemotherapeutic regimens with craniospinal irradiation have greatly improved survival in medulloblastoma patients. However, survival markedly differs among molecular subgroups and their biomarkers are unknown. Through unbiased screening, we found Schlafen family member 11 (SLFN11), which is known to improve response to DNA damaging agents in various cancers, to be one of the top prognostic markers in medulloblastomas. Hence, we explored the expression and functions of SLFN11 in medulloblastoma.

Methods: SLFN11 expression for each subgroup was assessed by immunohistochemistry in 98 medulloblastoma patient samples and by analyzing transcriptomic databases. We genetically or epigenetically modulated SLFN11 expression in medulloblastoma cell lines and determined cytotoxic response to the DNA damaging agents cisplatin and topoisomerase I inhibitor SN-38 in vitro and in vivo.

Results: High SLFN11 expressing cases exhibited significantly longer survival than low expressing cases. SLFN11 was highly expressed in the WNT-activated subgroup and in a proportion of the SHH-activated subgroup. While WNT activation was not a direct cause of the high expression of SLFN11, a specific hypomethylation locus on the SLFN11 promoter was significantly correlated with high SLFN11 expression. Overexpression or deletion of SLFN11 made medulloblastoma cells sensitive and resistant to cisplatin and SN-38, respectively. Pharmacological upregulation of SLFN11 by the brain-penetrant histone deacetylase-inhibitor RG2833 markedly increased sensitivity to cisplatin and SN-38 in SLFN11-negative medulloblastoma cells. Intracranial xenograft studies also showed marked sensitivity to cisplatin by SLFN11-overexpression in medulloblastoma cells.

Conclusions: High SLFN11 expression is one factor which renders favorable outcomes in WNT-activated and a subset of SHH-activated medulloblastoma possibly through enhancing response to cisplatin.

Keywords: DNA damaging agent; SLFN11; medulloblastoma.

Figure 1.

SLFN11 mRNA is highly expressed in WNT-activated and subset of SHH-activated medulloblastoma and confers a favorable prognosis. Kaplan-Meier analysis comparing the top 15%, intermediate, and bottom 15% SLFN11 (A), EYS (EGFL11) (B) and GABRA5 (C). (D) Kaplan–Meier analysis of 612 cases with available survival data. (E) SLFN11 mRNA expression was almost universally high in the WNT-activated subgroup and a proportion of SHH-activated subgroup. (F) Subtype analysis showed that SLFN11 high cases were distributed between SHH-α, -β, -γ subtypes but not -δ subtype. Analysis of Kool (G) and Fattet (H) datasets in Oncomine.

Figure 2.

SLFN11 is highly expressed in WNT-activated medulloblastoma samples. (A–C) WNT-activated medulloblastoma with high expression of SLFN11. (D–F) SHH-activated medulloblastoma with moderate expression of SLFN11. (G–I) Non-WNT/non-SHH cases with negative or low expression of SLFN11. (J) SLFN11 H-scores in WNT-activated, SHH-activated and Group 3/4. (K) SLFN11 is highly expressed in the nuclei of tumor cells in the Ymed4sc xenograft established from a WNT-activated medulloblastoma. (L) Intranuclear β-catenin staining found in the same xenograft. (M) Weak SLFN11 expression in D425 intracranial xenograft. Scale bars: (A, B, D, E, H, I, M) = 100 μm; (C, G, K) = 200 μm; (F, L) = 50 μm.

Figure 3.

SLFN11 sensitizes medulloblastoma cells to DNA damaging agents. (A) Western blot analysis of SLFN11 in medulloblastoma cell lines (DAOY, UW228, ONS-76, and D425). (B) Sensitivity curves of SLFN11 expressing (red) and non-SLFN11 expressing (blue) medulloblastoma cell lines to cisplatin. (C, D) SLFN11-KO cells displayed markedly reduced sensitivity to DNA damaging agents CPT or cisplatin (D). (E) Western blot analysis of the indicated proteins in cells treated with vehicle or 5 μM cisplatin for 48 h. Cleaved-PARP is a marker of apoptosis. (F) Western blot analysis of the indicated proteins after treatment with 5 μM cisplatin or 10 nM SN-38 for 48 h. (G–J) Viability curves were of the indicated cells after treatment with SN-38 or cisplatin for 72 h.

Figure 4.

SLFN11 overexpression sensitizes D425 cells to cisplatin treatment in intracranial xenografts. Kaplan–Meier survival curves are plotted for D425 intracranial xenografts and show prolonged survival in DOX + cisplatin group (P < .0001, Mantel-Cox test) (A). Bioluminescence studies show increased sensitivity to cisplatin treatment by induction of SLFN11 (P = .0005, t-test, cisplatin vs DOX + cisplatin) after 3 weeks of treatment (B). (C) Representative IVIS images of D425 xenografts. (D) Kaplan–Meier analysis of ONS-76 cisplatin ± DOX treatment, in which treatment was discontinued after 10 weeks because of toxicity (P = .0006). (E) SLFN11 immunohistochemistry showing increased SLFN11 expression in DOX-induced D425 xenografts.

Figure 5.

Expression of SLFN11 correlates with SLFN11 promoter hypomethylation and is activated by Class I HDAC inhibitor RG2833. Methylation (β-value) of 18 probes on chromosome 17, encompassing the SLFN11 gene, was assessed in normal cerebella and medulloblastomas samples of the four molecular subgroups. These showed similar methylation patterns (A), except for the area 33701776–33704725, located at the SLFN11 promoter and H3K4me3 mark in the SHH-activated medulloblastoma cell line D341-Med and 7 other cancer cell lines (B), which was hypomethylated in most WNT-activated cases and some SHH-activated cases (A, C). (D) Correlation (r = −0.376) between DNA methylation (β-value) and SLFN11 mRNA expression. (E) Western blots show a dose-dependent increase in SLFN11 expression after treatment with RG2833, but not 5-AZA EPZ6438 or GSK343 in low SLFN11 expressing cells. (F) Real-time qPCR analysis of SLFN11 mRNA expression after 24- and 48-h treatment with RG2833.

Figure 6.

Brain-penetrant Class I HDAC inhibitor RG2833 synergizes with cisplatin. Synergistic effects are observed with combined cisplatin and RG2833 treatment in D425 (HSA synergy score 11.713) (A) and ONS-76 (20.949) (B). (C) A summary of the HSA synergy scores.