Evaluation of an EZH2 inhibitor in patient-derived orthotopic xenograft models of pediatric brain tumors alone and in combination with chemo- and radiation therapies

Abstract

Brain tumors are the leading cause of cancer-related death in children. Tazemetostat is an FDA-approved enhancer of zeste homolog (EZH2) inhibitor. To determine its role in difficult-to-treat pediatric brain tumors, we examined EZH2 levels in a panel of 22 PDOX models and confirmed EZH2 mRNA over-expression in 9 GBM (34.6 ± 12.7-fold) and 11 medulloblastoma models (6.2 ± 1.7 in group 3, 6.0 ± 2.4 in group 4) accompanied by elevated H3K27me3 expression. Therapeutic efficacy was evaluated in 4 models (1 GBM, 2 medulloblastomas and 1 ATRT) via systematically administered tazemetostat (250 and 400 mg/kg, gavaged, twice daily) alone and in combination with cisplatin (5 mg/kg, i.p., twice) and/or radiation (2 Gy/day × 5 days). Compared with the untreated controls, tazemetostat significantly (P_corrected < 0.05) prolonged survival times in IC-L1115ATRT (101% at 400 mg/kg) and IC-2305GBM (32% at 250 mg/kg, 45% at 400 mg/kg) in a dose-dependent manner. The addition of tazemetostat with radiation was evaluated in 3 models, with only one [IC-1078MB (group 4)] showing a substantial, though not statistically significant, prolongation in survival compared to radiation treatment alone. Combining tazemetostat (250 mg/kg) with cisplatin was not superior to cisplatin alone in any model. Analysis of in vivo drug resistance detected predominance of EZH2-negative cells in the remnant PDOX tumors accompanied by decreased H3K27me2 and H3K27me3 expressions. These data supported the use of tazemetostat in a subset of pediatric brain tumors and suggests that EZH2-negative tumor cells may have caused therapy resistance and should be prioritized for the search of new therapeutic targets.

Fig. 1. Expression of EZH2 mRNA in a panel of xenograft mouse models of pediatric cancers.

Data were extracted from RNAseq analysis of PPTC xenograft tumors (see ref. ) and presented as FPKM (Fragments Per Kilobase of transcript per Million mapped reads). Models with truncating (black filled circle) or missense (green filled circle) mutations were shown.

Fig. 2. EZH2 overexpression in pediatric brain tumors.

A, B. High levels of EZH2 mRNA expression in high-grade glioma (upper panel) and MB (lower panel) patient tumors (Pt tum) and PDOX models during serial in vivo subtransplantation from passage I (Xeno-I) up to passage VIII (Xeno-VIII). Normal brain tissues were included as references. The data from glioma were extracted from gene expression profiling using Affy 133 array, and MB from illumine array. C Representative immunohistochemical staining images showing increased expression of H3K27me3 in MB models. D Western hybridization showing elevated H3K27me3 expression in xenograft tumors of PDOX models. Normal human cerebellar tissues obtained from warm autopsy were included as references.

Fig. 3. In vivo therapeutic efficacy of tazemetostat in PDOX models of childhood brain tumors.

SCID mice were implanted with xenograft cells from the four models, IC-L1115ATRT (A), IC-2305GBM (B), group 3 (ICb-1572MB) (C) and group 4 (ICb-1078MB) (D) MBs, were allowed to grow for 14 days to form solid intra-cerebral (IC) or intra-cerebellar (ICb) xenograft tumors before being treated with tazemetostat alone and in combinations with chemo- and/or radiation as highlighted in the figures. Kaplan-Meier estimate of median time-to-event, ratio in median time to event between the treated and control groups (EFS T/C), and EFS p values were calculated and compared between the treatment groups.

Fig. 4. Histological changes induced by tazemetostat.

A Schematic illustration of the timing of mouse brain collection after in vivo treatment. B H&E staining showing the in vivo growth of PDOX tumors (*) from low (4×) to high (40×). Compared with the untreated control, the remnant tumor of IC-L1115ATRT remained highly invasive although the number of satellite foci (red arrowhead) was slightly reduced (upper panel). The local invasion of IC-1078MB was not significantly affected (lower panel). Cell proliferation (Ki-67) in the remnant tumor was similar to the untreated controls.

Fig. 5. Immunohistochemical staining of molecular targets of tazemetostat in vivo.

A In IC-L1117ATRT, strong positive staining (arrow) of EZH2, H3K27me2 and H3K27me3 were significantly reduced in the tumor core (upper panel) and in the remaining invasive foci (lower panel) accompanied by increased number of cells with low or no expression. Note that the same foci surround a micro-blood vessel (red circle) from the consecutive sections of the same tumor (arrow) were imaged and shown. B In IC-1078MB, the reduction of EZH2 strong positivity (arrow) was accompanied by the decrease of H3K27m3 expression in the remnant tumor.