The protein arginine methyltransferase PRMT5 confers therapeutic resistance to mTOR inhibition in glioblastoma

Abstract

Introduction: Clinical trials directed at mechanistic target of rapamycin (mTOR) inhibition have yielded disappointing results in glioblastoma (GBM). A major mechanism of resistance involves the activation of a salvage pathway stimulating internal ribosome entry site (IRES)-mediated protein synthesis. PRMT5 activity has been implicated in the enhancement of IRES activity.

Methods: We analyzed the expression and activity of PRMT5 in response to mTOR inhibition in GBM cell lines and short-term patient cultures. To determine whether PRMT5 conferred resistance we used genetic and pharmacological approaches to ablate PRMT5 activity and assessed the effects on in vitro and in vivo sensitivity. Mutational analyses of the requisite IRES-trans-acting factor (ITAF), hnRNP A1 determined whether PRMT5-mediated methylation was necessary for ITAF RNA binding and IRES activity.

Results: PRMT5 activity is stimulated in response to mTOR inhibitors. Knockdown or treatment with a PRMT5 inhibitor blocked IRES activity and sensitizes GBM cells. Ectopic expression of non-methylatable hnRNP A1 mutants demonstrated that methylation of either arginine residues 218 or 225 was sufficient to maintain IRES binding and hnRNP A1-dependent cyclin D1 or c-MYC IRES activity, however a double R218K/R225K mutant was unable to do so. The PRMT5 inhibitor EPZ015666 displayed synergistic anti-GBM effects in vitro and in a xenograft mouse model in combination with PP242.

Conclusions: These results demonstrate that PRMT5 activity is stimulated upon mTOR inhibition in GBM. Our data further support a signaling cascade in which PRMT5-mediated methylation of hnRNP A1 promotes IRES RNA binding and activation of IRES-mediated protein synthesis and resultant mTOR inhibitor resistance.

Keywords: Drug resistance; EPZ015666; Glioblastoma; PP242; PRMT5; Rapamycin; mTOR.

Fig. 1

PRMT5 expression and activity in response to mTOR inhibition. a Expression of PRMT5 mRNA (left panel) and protein (right panel) in LN229 cells following treatment with rapamycin (rapa) or PP242 (50 nM) for the indicated time points. b Effects of mTOR inhibitors on PRMT5 mRNA (left panel) and protein (right panel) in short-term patient derived GBM39 cells. Cells were treated for 24 h at 50 nM with each inhibitor. c LN229 cells expressing Flag-PRMT5 were treated with the indicated concentrations of either rapa or PP242 (0–100 nM) for 4 h and subsequently PRMT5/MEP50 complexes were immunoprecipitated using Flag antibodies. In vitro methylation activity of the complexes was then determined utilizing H4 as a substrate and activity detected by immunoblotting reactions with symmetric dimethylarginine (R3me2s)-specific antibodies. Reactions were also probed for H4 and PRMT5. NE, no extract used in reactions (negative control); recombinant PRMT5/MEP50 was added to positive control reactions (left panel). In vitro PRMT5 activity of LN229 cells treated with mTOR inhibitors displayed graphically (right panel). d LN229 or GBM39 cells were treated with mTOR inhibitors (50 nM, 24 h) and extracts immunoblotted with pan-SDMA motif and actin antibodies.

Fig. 2

PRMT5 knockdown sensitizes GBM and patient-derived lines to mTOR-targeted therapies. a Cell proliferation was determined following treatment of lines with rapa or PP242 (100 nM, 48 h) as indicated in the context of PRMT5 knockdown via siRNAs. non-targeting siRNAs (siRNA scr) were utilized as a control. Data are means +S.D., n=3. * P < 0.05. b Effects of PRMT5 knockdown and mTOR inhibitors on LN229, LN18, GBM6 and GBM39 cell line death as determined by trypan blue exclusion assays. Data are means +S.D., n=3. * P < 0.05.

Fig. 3

Methylation of hnRNP A1 is required for mTOR-inhibitor induced IRES-binding and activity. a RNA-pull down assays utilizing biotinylated cyclin D1 and c-MYC IRES RNAs. Cytoplasmic extracts of LN229 or GBM39 cells in which hnRNP A1 has been knocked down via siRNA and expressing the indicated wt hnRNP A1 or non-methylatable hnRNP A1 mutant (A1-R218K, A1-R225K or A1-R218K/R225K) constructs in the absence or presence of PP242 (50 nM, 24 h) were incubated with biotinylated cyclin D1 (top panels) or c-MYC (bottom panels) IRES RNAs and precipitated with streptavidin-Sepharose beads. AS, antisense RNA sequence. Input and bound fractions were analyzed by immunoblotting using hnRNP A1 antibodies. b LN229 or GBM39 cells were transfected with control siRNA or siRNA targeting hnRNP A1 and 24 h later either co-transfected with the control pRF, cyclin D1 or c-MYC IRES reporters and the indicated non-methylatable hnRNP A1 mutant (A1-R218K, A1-R225K or A1-R218K/R225K) in the absence or presence of PP242 (50 nM, 24 h). Relative Renilla and firefly luciferase activities were subsequently determined. Data are means + S.D., n = 3.

Fig. 4

Synergistic anti-GBM effects of mTORC1/2 and PRMT5 inhibitors in vitro. a Viability of LN229, LN18 and GBM39 cells following 48 h in culture in EPZ015666 at the indicated concentrations. Data represent ±S.D. of 3 independent experiments. b Combination analysis of PP242 and EPZ015666 in LN229, LN18 and GBM39 cells. Cells were treated with the indicated doses PP242 alone or in combination with EPZ015666 for 48 h and percent cell viability was determined relative to control cultures. Control cells were treated with DMSO vehicle. Data are means ±S.D., n = 3. c Percent apoptotic cells as determined via annexin V-FITC staining in LN229, LN18 and GBM39 cells treated with the indicated inhibitors for 48 h (PP242, 50 nM; EPZ015666, 5 µM; PP242, 50 nM + EPZ015666, 5 µM). Data are means +S.D., n = 3. d Protein levels of cyclin D1, c-MYC and actin in LN229, LN18 and GBM39 cells following the indicated treatments with PP242, EPZ015666 or both compounds at 24 h.

Fig. 5

Co-therapy of GBM xenografts with mTOR and PRMT5 inhibitors. a Xenograft tumor volume in SCID mice implanted with LN229 cells and treated with the indicated schedules for 10 consecutive days and tumor growth assessed every two days following the initiation of treatment (start, day 0). *, P < 0.05 (significantly different then double vehicle) n=6 mice per treatment group. b Overall survival of mice with subcutaneous implanted LN229 tumors receiving the indicated treatment schedules. Treatments were initiated upon tumors reaching ~ 100 mm³ in size. *, P < 0.05, n=6 mice per treatment group. c Apoptotic cells were identified by TUNEL assays of sections prepared from harvested tumors at day 12 following initiation of treatment regimens. Data are expressed as the number of positive apoptotic bodies divided by high power field (hpf; 10–12 hpf/tumor). Values are means +S.D., * P < 0.05. d Cyclin D1(left panel) and c-MYC (right panel) protein levels in tumors. Values are means ±S.D. *, P < 0.05 (significantly different from vehicle). Protein levels were quantified by Western analyses of harvested tumors from mice with the corresponding treatments as indicated. Band intensities were quantified by densitometry analyses via ImageJ software. e Polysome distributions of cyclin D1, c-MYC and actin transcripts from tumors harvested from mice receiving the indicated treatments. Tumor cell extracts were separated via sucrose density gradient centrifugation, fractionated and pooled into nonribosomal, monosomal fraction (N, white bars) and polysomal fraction (P, black bars). Purified mRNAs were subsequently used in qrt-PCR analyses to determine distributions of the indicated mRNAs across the gradients. Means and +S.D. values are shown for quadruplicate measurements. *, P < 0.05. f mTOR inhibition leads to activation of PRMT5 and symmetric dimethylation of hnRNP A1 at arginine residues 218 and 225. Either of these methylation events is sufficient to promote hnRNP A1 binding to cyclin D1 and c-MYC IRESs resulting in enhanced protein synthesis of these determinants and mTOR inhibitor resistance in GBM.