Interfering with resistance to smoothened antagonists by inhibition of the PI3K pathway in medulloblastoma

Abstract

The malignant brain cancer medulloblastoma is characterized by mutations in Hedgehog (Hh) signaling pathway genes, which lead to constitutive activation of the G protein (heterotrimeric guanosine triphosphate-binding protein)-coupled receptor Smoothened (Smo). The Smo antagonist NVP-LDE225 inhibits Hh signaling and induces tumor regression in animal models of medulloblastoma. However, evidence of resistance was observed during the course of treatment. Molecular analysis of resistant tumors revealed several resistance mechanisms. We noted chromosomal amplification of Gli2, a downstream effector of Hh signaling, and, more rarely, point mutations in Smo that led to reactivated Hh signaling and restored tumor growth. Analysis of pathway gene expression signatures also, unexpectedly, identified up-regulation of phosphatidylinositol 3-kinase (PI3K) signaling in resistant tumors as another potential mechanism of resistance. Probing the relevance of increased PI3K signaling, we demonstrated that addition of the PI3K inhibitor NVP-BKM120 or the dual PI3K-mTOR (mammalian target of rapamycin) inhibitor NVP-BEZ235 to the initial treatment with the Smo antagonist markedly delayed the development of resistance. Our findings may be useful in informing treatment strategies for medulloblastoma.

Figure 1. Antagonism of Smo inhibits Hh signaling and growth of Ptch^+/−p53^−/− tumors, but induces resistance

Nude mice subcutaneously implanted with medulloblastoma tumors were treated with vehicle (control) or NVP-LDE225 at different doses (indicated in mg/kg/day once a day (qd) or split in two doses (bid)). (A) Gli1 mRNA levels in tumors quantified at day 3 (black) or day 26 (gray, vehicle control taken on day 11 when tumor size limit was reached) post treatment, normalized to β-actin and blotted as % of matching vehicle for the respective study, and tumor volume over time (B) are shown. Data are expressed as mean ± s.e.m, (n=8). C, NVP-LDE225 inhibits proliferation of sensitive tumors (filled circles) but not of resistant tumors cells ex-vivo (filled triangles) at low nM concentration, as measured in vitro by ³H-thymidine uptake. Data expressed as mean ± s.d, (n=3). Results are shown for one sensitive and one resistant tumors but similar results were obtained for multiple different tumors.

Figure 2. Smo resistant Ptch^+/−p53^−/− tumors acquire Gli2 amplifications and are sensitive to Gli2 inhibition

RNA from sensitive tumors treated with either vehicle or NVP-LDE225 (20mg/kg/day) for 4, 16 and 48 hours, and resistant tumors treated with 20 or 160 mg/kg/day (mpk) for 26 days (d) was profiled on Affymetrix mouse gene expression arrays. A, Pattern of genes initially inhibited by NVP-LDE225 but reemerging in resistant tumors. Numbers express relative rank in expression. B, Top ranking genes by Spearman correlation of expression matching with the pattern in 2A. C, Heat map of the top ranked genes associated with the pattern in 2A. Each gene’s expression values are z-transformed for comparability, with red indicating relatively high expression and green indicating relatively low expression. D, CGH analysis using the Agilent Mouse Genome CGH Microarray Kit 244A of three resistant (LDE 1,2,3) and three sensitive (Veh 1,2,3) tumors identified a focal amplification in the region containing Gli2 on Chromosome 1 in 2 out of 3 resistant tumors. Copy number changes are expressed as log2. E, Additional DNA from normal liver (NL), vehicle (controls) and resistant tumors was analyzed by quantitative PCR for the Gli2 locus. F, Correlation between levels of Gli2 mRNA expression and Gli2 copy number is shown for vehicle-treated control tumors (open square) and resistant tumors that emerged after treatment with NVP-LDE225 at 10 bid (green square), 20 qd (red diamond), 40 bid (brown triangle), 80 bid (blue circle) and 160 bid (black inverse triangle) mg/kg/day. Axis in log10 scale. G, Inhibition of Gli2 mRNA levels by siRNA knock-down of Gli2 was associated with decreased proliferation and Gli1 mRNA expression in resistant Gli2 amplified (copy number:20) and Gli2-overexpressing (17-fold) medulloblastoma tumors. Two independent Gli2 siRNAs (Gli2-1 and Gli2-1) were used.

Figure 3. The PI3K/mTor pathway is upregulated in NVP-LDE225 resistant Ptch^+/−p53^−/− tumors, and emergence of resistance is delayed by combination treatment with the Smo and PI3K inhibitors, NVP-LDE225 and NVP-BKM120

A, Affymetrix gene expression data were mined for genes not affected by short-term NVP-LDE225 treatment, but upregulated in resistant tumors. B, Pathway categories upregulated in resistant tumors ranked by FDR scores C, Heat map of expression values of upregulated genes inAkt, PIP3 and Igf-1R pathway category.. Data normalized as in Fig. 2C. D,E Nude mice subcutaneously implanted with Ptch^+/−p53^−/− tumors were treated with vehicle (control), NVP-LDE225 (80mg/kg/day qd), NVP-BKM120 (30mg/kg/day qd) or NVP-LDE225 in combination with NVP-BKM120 at the same doses and schedules starting on day 9 post-implant. Tumor volume (mean ± s.e.m. (n=8)) (D) and time to end point (tumor volume reaching 700 mm³) (E) are shown.. F, Total protein isolated from tumors at the end of the study was evaluated for phospho-S6 (S235/236), phospho-4EBP1 (T37/36) and total S6 and 4EPB1.

Figure 4. The emergence of resistance in Ptch^+/−Hic^+/− tumors is suppressed by combination treatment with the Smo and PI3K/mTor inhibitors, NVP-LDE225 and NVP-BEZ235

A, Nude mice subcutaneously implanted with Ptch^+/−Hic^+/− tumors were treated with vehicle (control), NVP-LDE225 (80mg/kg/day qd), NVP-BEZ235 (40mg/kg/day qd) or NVP-LDE225 in combination with NVP-BEZ235 at the same doses and schedules starting on day 8 post-implant. Tumor volume (mean ± s.e.m. (n=8)) (A) and time to end point (tumor volume reaching 700 mm³) (B) are shown. C), Total protein isolated from tumors at the end of the study was evaluated for phospho-S6 (S235/236), phospho-4EBP1 (T37/36) and total S6 and 4EPB1.