Cell lines and primary cultures

Tumor-derived fresh tissues utilized for meningioma primary cultures are described in Supplementary Table 1. The cultures were established as previously reported [10]. Tumor tissue was enzymatically dissociated and unsorted cells were cultured in neurobasal medium with 4% FCS. Subconfluent cultures were expanded by trypsinization and replating. After attachment, the media was changed to Neurobasal Medium with N2 and B27 for subsequent assays. The meningioma-derived C157-MN and F5 cell lines were generated and shared by Robert Martuza (Massachusetts General Hospital, Boston, MA, USA). Tumor-derived fresh-frozen tissues used for RNA sequencing and previously analyzed by genome sequencing [6] are described in Supplementary Table 2. HepG2 (RRID:CVCL 0027) was purchased from ATCC (HB-8065, ATCC, Manassas, VA). The AC007T immortalized human arachnoid cell line was generated and shared by Vijaya Ramesh (Massachusetts General Hospital, Boston, MA, USA). AC007T, C157-MN and F5 were authenticated commercially by short tandem repeat DNA profiling (Labcorp, Burlington, NC). All cell cultures were tested [19] and were mycoplasma-free.

Meningioma small RNA sequencing

RNA was isolated from tissue sections obtained from fresh-frozen blocks, using column purification (PureLink RNA Mini, ThermoFisher Scientific, Waltham, MA, USA). Small RNA sequencing was performed at the Center for Cancer Computational Biology, Dana Farber Cancer Institute. Libraries were prepared using a multiplex small RNA kit (NEBNext, E7300, New England Biolabs, Ipswich, MA, USA) followed by column purification and size selection (AMPure XP, Beckman Coulter, Indianapolis, IN, USA). Sequencing was performed using a flow cell with 11 samples in one lane and 10 samples in the second lane (SR50, HiSeq, Illumina, San Diego, CA, USA).

Transfection and metabolic assay

Meningioma tumor-derived cultures were transfected as previously described [10], using 0.22 nmol of oligonucleotides and 1.25 µl transfection reagent per well (Lipofectamine 2000, 11,668,027, ThermoFisher Scientific, Waltham, MA, USA). The medium was changed after six hours to Neurobasal Medium with N2 and B27 (ThermoFisher Scientific, Waltham, MA, USA). Forty-eight hours after transfection the medium was replaced with DMEM containing 10% WST-1 (11,644,807,001, Roche, Mannheim, Germany). The plates were assayed after 20 min at 450 nm using an optical reader (GloMax Multi+, Promega, Madison, WI, USA).

Oligonucleotides and small molecule inhibitors

Hsa-miR-483-5p mimic, YM00473215-ADA, mimic control, YM00479902-ADA, inhibitors hsa-miR-675-5p, 427419-00, hsa-miR-675-3p, 427420-00, hsa-miR-483-5p, 427155-00, hsa-miR-483-3p, 427154-00, hsa-miR-204-5p, 426934-00, hsa-miR-135-5p, 426790-00, hsa-miR-10a-5p, 426651-00, hsa-miR-9-5p, 427460-00, hsa-miR-9-3p, 427461-00, Control B, 199021-00 are from Qiagen, Germantown, MD, USA. Ceritinib, S7083, linsitinib, S1091, entrectinib, S7998, GSK1904529A, S1093, picropodophyllin, S7668 were obtained from Selleck Chemicals, Houston, TX, USA; GSK1838705A, SML0995, was purchased from Millipore-Sigma, Burlington, MA, USA. The primers used for miRNA and mRNA quantification were miR-99b-5p, 4,427,975, miR-483-5p, 4,427,975, miR-675-5p, 4,427,975, IGF-2, 4,331,182, GAPDH, 4,331,182, ThermoFisher Scientific, Waltham, MA, USA.

Analysis of IGF-2 expression

IGF-2 expression microarray data from 109 samples including 96 tumors representing the major meningioma mutation groups (NF2/chr22 loss, POLR2A, KLF4/TRAF7, PI3K mutant, and Sonic Hedgehog mutant) and 13 adult meninx tissue controls are displayed. This data is publicly available [20]. The center line of the box shows the median expression, box limits indicate the 25th and 75th percentiles, and whiskers extend to the minimum and maximum values.

Inhibition of Akt phosphorylation

HepG2 cells were plated in 100 mm dishes and grown in DMEM with 10% FCS. Subconfluent cultures were washed three times with PBS and serum-starved for one hour in DMEM. Small molecule inhibitors were added at 100 times the IC50 concentration for 30 min. The cell-free assay IC50 values for each inhibitor are summarized in Table 1, along with the observed cell-based assay IC50 values. Human recombinant IGF-2 (292-G2, R&D Systems, Minneapolis, MN, USA) was added at a concentration of 200 ng/ml for 15 min.

Table 1 Summary of the half-inhibitory concentrations of the IGF1R inhibitors used. The cell-free IC50 values were provided by the drug supplier. The cell-based IC50 values were determined experimentally. The average and the range are listed from three cultures tested (MEN049, MEN051, MEN052). The peak plasma concentrations were published elsewhere [28,29,30] Western blot

Cultured cells and tumor samples were processed for western blot as previously described [10], except the membrane was incubated in 1 M DTT solution at room temperature for 5 min then washed with TBS-T three times before adding IGF-2 antibody. Phosphatase inhibitors (P2850, P5726, Millipore-Sigma, Burlington, MA, USA) were added to the lysis buffer for P-Akt. The antibodies used were IGF-2, 8H1, MA5-17096, 1:1000, ThermoFisher Scientific, Waltham, MA, USA, α-tubulin, Ab7291, 1:10,000, Abcam, Cambridge, UK, GAPDH, sc-47,724, 1:1000, Santa Cruz Biotechnology, Dallas, Texas, USA, Akt, 2938, P-Akt, 4058, 1:1000, secondary antibodies 7074 and 7076 S, 1:10,000, Cell Signaling Technology, Danvers, MA.

Inhibition of meningioma cell growth

Primary cells were seeded in 96-well plates in Neurobasal Media with N2, B27 and 10% FCS at 10–50% confluence. For neutralizing antibody treatment, the cells were washed with DMEM twice then with Neurobasal Media once, then 100 µl of Neurobasal Media was added to each well. The antibodies were washed to remove sodium azide by adding 0.5 ml protein G magnetic particles (Dynabeads, 10003D, ThermoFisher Scientific, Waltham, MA, USA) in PBS, washed once with PBS and eluted with 90 µl 0.1 M citric acid pH 2.0, neutralized with 10 µl 1 M TRIS base, and the concentration was adjusted to 400 ng per µl. Antibody (400 ng) was added to each well. The cells were washed twice with PBS, then assayed 24 h later. For small molecule inhibitor assays, the compounds were added directly to the media 24 h after plating, with serial dilution starting at 500 times the IC50 concentration. After 72 h, cells were washed with PBS twice, the media was replaced with DMEM plus 10% WST-1 and assayed after 20 min at 450 nm.

Chromatin mark analysis

ChIP-Seq data for H3K27Ac, H3K4Me3, H3K9Ac, H3K27Me3 and H3K9Me3 chromatin marks covering the H19-IGF2 locus in meningioma were retrieved from a public repository (https://dataverse.tdl.org) and signal tracks were assembled using an open source application [21].

Real-time PCR

Total RNA (3 ng) was used for reverse transcription and real-time PCR assays (Superscript III First-Strand Synthesis, 11,752,250, Taqman Universal Master Mix II, 4,440,040, Quantstudio 7 Flex, ThermoFisher Scientific, Waltham, MA, USA). Cycle threshold values for miR-483-5p and miR-675-5p were normalized to those of miR-99b-5p which was uniformly and highly expressed in all meningioma tumor samples. The IGF-2 mRNA expression was normalized to that of GAPDH.

Data analysis

Small RNA quantification was performed using an open-source algorithm [22]. Unpaired two-tailed t-tests and one-way analysis of variance tests were performed using commercial software (GraphPad Prism 8.3.0). Linear regression was used for the expression analysis. The standard error of the mean is shown for each category In the bar graphs. A heatmap with hierarchical clustering was generated using open-source software (heatmap.2, gplots, R 3.1.3).