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. 2025 Mar 14;27(7):1787–1800. doi: 10.1093/neuonc/noaf068

Dual inhibition of MAPK and TORC1 signaling retards development of radiation resistance in pediatric BRAFV600E glioma models

Fuyang Li 1,2,✉, Kathryn M Bondra 3, Hanzhou Wang 4, Dias Kurmashev 5, Bipasha Mukherjee 6, Suman Kanji 7, Amyn A Habib 8, Yidong Chen 9,10, Siyuan Zheng 11,12, Sandeep Burma 13,✉, Peter J Houghton 14,15,✉
PMCID: PMC12417835  PMID: 40083135

Abstract

Background

MAPK pathway inhibitors (MAPKi) have shown significant efficacy in treating childhood BRAF-activated brain tumors. For tumors harboring BRAFV600E mutations, the drugs are rarely curative, and patients can become refractory to treatment. MAPKi combining X-radiation therapy (XRT) may improve cure rate, but the development of XRT resistance is a challenge.

Methods

XRT resistance was induced by multiple XRT cycles in pediatric BRAFV600E glioma patient-derived xenograft (PDX) models. RNA sequencing was performed to identify differentially expressed genes and pathways potentially contributing to XRT resistance. Cells isolated from PDXs were used to test the contribution of specific genes and pathways to XRT resistance. PDX models were used to evaluate the efficacy of targeted treatments combined with XRT.

Results

Tumors developed resistance after multiple cycles of XRT. MEK inhibition combining XRT significantly improved tumor control compared to XRT alone, but resistance to combined therapy developed rapidly. RNA sequencing analysis revealed up-regulation of MAPK and PI3K-mTOR signaling in the XRT-resistant tumors. Isolated cells showed in vitro resistance to XRT, which was partially reversed by inhibiting PI3K-mTOR. Up-regulation of TORC1 signaling in XRT naïve tumor cells, via constitutively active Akt or TSC2 deletion, conferred in vitro XRT resistance. The pro-survival gene BIRC5 (Survivin), a target of TORC1 signaling, contributed to XRT resistance. Combining trametinib-rapamycin with XRT significantly enhanced therapeutic efficacy in PDX models and prevented or delayed resistance development.

Conclusion

PI3K-mTOR activation promotes the development of XRT resistance in pediatric BRAFV600E glioma. Dual targeting of MAPK and TORC1 signaling significantly enhances the therapeutic efficacy of XRT and can potentially prevent the development of XRT resistance.

Keywords: BRAF V600E mutation, MAPK inhibitor, pediatric low-grade glioma, therapeutic resistance, X-radiation therapy (XRT)

Key Points.

  1. Combined MAPKi/XRT significantly improves tumor control in pediatric BRAFV600E glioma PDX models, but tumors eventually develop resistance to treatment.

  2. PI3K/mTOR activation underlies the development of radiation resistance in pediatric BRAFV600E gliomas.

  3. Dual targeting of MAPK and TORC1 signaling significantly enhances the therapeutic efficacy of XRT, and can potentially prevent the development of radiation resistance.

Importance of the Study.

Inhibitors of the MAPK pathway (MAPKi) have shown significant activity in the treatment of childhood BRAF-activated brain tumors. Recently, dabrafenib and trametinib combination has been approved by the FDA for treating pediatric patients with low-grade glioma driven by BRAFV600E mutation. However, the drugs are rarely curative, and patients can become refractory to treatment. Our results propose a promising solution to enhance the current MAPK-targeted therapy and prevent the development of therapeutic resistance.

Brain tumors are the most common childhood solid tumors, and the leading cause of childhood cancer-related deaths, with low-grade gliomas constituting 30% to 40% of all childhood central nervous system tumors.1,2 For low-grade tumors, the overall survival for patients is good when complete resection is possible. Deep-seated or infiltrative tumors that are not amenable to complete resection have a worse prognosis than superficial lesions.3 Unresectable tumors or higher grade tumors require intensive chemoradiation therapy that leads to cognitive decline, secondary malignancies, and other life-debilitating or life-threatening sequelae.4 Long-term adverse outcomes, such as hearing loss, obesity, and hormonal imbalance, affect 18% to 53% of patients, with ~34% experiencing cognitive decline. The most common cause of death is progression to therapy-resistant disease.5

Genomic studies reveal that in pediatric low-grade glioma, the predominant genomic alteration is a tandem duplication involving the KIAA1549 and BRAF genes that generates constitutively active BRAF::KIAA1549 fusions.6,7 Activating point mutations of BRAF, most frequently the V600E variant, are common in higher grade gliomas, such as diffuse astrocytoma (23%),8 glioblastomas (10%-15%), pleomorphic xanthoastrocytoma (PXA) (70%), and gangliogliomas (33%).9

BRAF activation drives the MAPK pathway in these tumors.8 Our initial data showed the sensitivity of a BRAFV600E astrocytoma patient-derived xenograft (PDX) model to the MEK inhibitor selumetinib, while another astrocytoma with wild-type BRAF was intrinsically resistant to the same treatment.10 Subsequent clinical trials showed selumetinib’s effectiveness in both fusion-positive and BRAF-mutant disease, though BRAF-mutant tumors had a higher progression rate.11,12 Two more recent trials confirmed the efficacy of another MEK inhibitor, trametinib, alone or in combination with the BRAF inhibitor, dabrafenib.13,14 In the latter trial in newly diagnosed low-grade BRAFV600E-driven gliomas, the clinical benefit rate and time to progression with MEK/BRAF-targeted therapy were significantly greater than with conventional chemotherapy. Recently, the FDA has approved vertical targeting of the MAPK pathway with dabrafenib (BRAFi) and trametinib (MEKi) for pediatric patients with low-grade glioma harboring a BRAFV600E mutation. However, as denoted by the time to progression, the failure rate for this therapy is still quite significant.

X-radiation therapy (XRT) is an integral part of therapeutic modalities for higher grade pediatric glioma.15 For unresectable low-grade gliomas, XRT has proven to be effective for long-term disease control but poses risks of late side effects which are directly related to irradiated tissue volume and dose.16 Combining MAPK-targeted therapies with XRT might enhance cure rates by sensitizing tumors to XRT or preventing XRT resistance. In this study, using BRAFV600E-driven pediatric gliomas implanted in mice, we have examined the rate at which XRT resistance emerges without or with concomitant targeting of the MAPK pathway or combined targeting of MAPK and TORC1 signaling. Here we report the mechanism of XRT resistance and the identification of an effective combinatorial treatment that improves disease control significantly, and more importantly, delays/prevents the development of XRT resistance in these pediatric BRAFV600E glioma models.

Materials and Methods

Reagents

RPMI-1640 medium, fetal bovine serum, Crystal Violet, and Alamar Blue (AB) were purchased from Invitrogen. Primary antibodies, if not indicated, were all purchased from Cell Signaling Technology. Trametinib was purchased from MedChem Express and dissolved in dimethyl sulfoxide (DMSO) at 10 mmol/L. Rapamycin and dabrafenib were purchased from LC Laboratories and dissolved in DMSO at a concentration of 10 mmol/L. Details of antibodies and their source used are given in Supplementary Table S1. Details of plasmids and other constructs used are also given in Supplementary Methods.

Childhood Brain Tumor PDX Models

BT-40, established at diagnosis from a leptomeningeal mass in a 14-year-old male, is an anaplastic astrocytoma grade II/III.10 NCH-MN-1 was established in mice from an occipital mass in a 16-year-old female at diagnosis.17 IC-3635 was established from a cerebral mass diagnosed as a PXA from a 10-year-old female.18 All models were directly implanted into mice without culture on plastic. Each PDX model was confirmed as having BRAFV600E mutation by sequencing.19,20

Western Blotting

Protein extraction from xenograft tissue and in vitro cultured cells, cell lysis, and western blotting were performed as described.21 Immunoreactive bands were visualized by using Super Signal Chemiluminescence substrate (Pierce) and ChemiDoc MP Imaging System (Bio-Rad). Images were processed by ImageJ.

Tumor Cell Isolation and Culture from PDX

Primary BT-40 and XRT-resistant (BT-40X) cells were developed as described below. Tumor cells were isolated and grown in neurostem cell culture medium as described.22 After ~30 days of culture, cells were plated on Matrigel-coated plates supplemented with full culture media (RPMI-1640 with 10% FBS). LDH assay was applied to check the human/mouse origin of the cells. Immunostaining with antibodies specific for murine vimentin and human EpCAM antibody was performed to verify the human origin. The cells’ origin was ultimately confirmed by short tandem repeat (STR) analysis.

In Vivo Tumor Growth Inhibition/Irradiation Studies

C.B.17SC scid−/− female mice (C.B-17-/IcrHsd-Prkdcscid, Envigo, Indianapolis, IN) were used to propagate subcutaneously implanted tumors as previously described.10 All mice were maintained under barrier conditions, and experiments were carried out using protocols and conditions approved by the institutional animal care and use committee of UT Health San Antonio. Mice were randomized into groups of 5 or 10 when tumors were 200-400 mm3. Drugs (trametinib and rapamycin) were formulated as described previously and administered P.O. daily for 42 days.21 Dabrafenib was formulated in 20% Tween 80 in sterile water and administered at 30 mg/kg daily for 42 days P.O. XRT was started when the tumors implanted on the hind limbs reached 200-400 mm3 (XRAD320).22,23 Subcutaneous tumor volumes were determined weekly using digital calipers to measure perpendicular diameters, as previously described.10 Irradiation was performed as described.21,24 XRT treatment was started when the tumors implanted on the hind limbs reached 200-400 mm3. Briefly, 5~10 mice from the group were placed in the lead shielding chamber and maintained there for an additional ~30-60 s to assure that they were sufficiently anesthetized before the chamber was moved to the XRAD320. The table in the irradiator was adjusted to 43 cm SSD/offset 0; 320 kV and 12.5 mA; Filter 1 was set in place, and mice are irradiated for 40 s to receive a dose of 2 Gy at a 3 Gy/min dose rate. After irradiation, anesthesia was turned off, and mice were returned to the biosafety cabinet and immediately removed from the shielding chamber and placed back in a cage to recover from anesthesia.

Orthotopic Xenograft Studies

Female athymic nude mice (FOXN1nu Envigo, IN) were implanted with primary tumor cells isolated from mice xenograft (BT-40 or BT-40X or BT-40TRX [trametinib/rapamycin/XRT]) as described.21 Three weeks after injection, mice were subjected to 10 Gy XRT (2 Gy fraction × 5 days). Mice were euthanized as an endpoint when neurological symptoms, visible tumor growth, or less than 10% body weight decrease were observed.

RNA Sequencing and Analysis

Primary tumor or XRT-resistant tumor subjected to 3 cycles of XRT were transplanted subcutaneously to naïve 6- to 8-week-old female scid mice. Tumors were collected when tumor volume reached 200~400 mm3. Total RNA was isolated and applied to subsequent 100 bp paired read sequencing (Illumina NovaSeq 6000) run with the 100 bp paired-end module. Details for RNA sequencing analysis are given in Supplementary Methods.

Statistical Analysis

Group difference was analyzed by student unpaired t-test using Prism 9.

Results

Development of XRT Resistance in Pediatric BRAFV600E Glioma Models

To develop XRT resistance models, scid mice were implanted subcutaneously with BT-40 tumor21 that harbors BRAFV600E and CDKN2A homozygous deletions.19 Mice were treated with 2 Gy daily fractions for 5 days; the first tumor to reach 400% of the day 1 treatment volume was transplanted into 5 recipient mice, and the process repeated until the tumor line was significantly resistant to XRT (Figure 1A). 10 Gy XRT resulted in partial regression of the BT-40 tumors (Figure 1B). In the second-round treatment, the tumors became less responsive to 10 Gy XRT, with slow progression but no regression (Figure 1B, center lower panel). During the third cycle of XRT, BT-40 tumors became completely resistant, showing progressive disease. The least responsive tumor (designated BT-40X) was transplanted into scid mice, and the responses of parental BT-40 (XRT naïve) and BT-40X tumors were evaluated at higher doses of XRT. BT-40X tumors were unresponsive at doses up to 30 Gy, whereas the responses of parental BT-40 xenografts ranged from partial to complete regression (Figure 1C). Event-free survival (EFS) for BT-40 (cycle 1, 20 Gy) was 93.2 ± 4 days, whereas for BT-40X (20 Gy), EFS was 56.0 ± 4.4 days (P = .0001).

Figure 1.

Figures showing development of radiation resistance in a pediatric glioma model. A schematic illustrates a stepwise experimental approach in which tumors from BT-40 xenografts are irradiated and re-transplanted across multiple cycles to generate radiation-resistant tumors. A line graph shows decreasing tumor response to 10 Gy radiation across three treatment cycles. Another graph shows parental tumors respond to 20 or 24 Gy radiation with regression, while resistant tumors do not regress after 20 or 30 Gy radiation.

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Pediatric BRAFV600E glioma PDX model rapidly develops resistance to XRT. (A) Schematic illustration of the experimental design to observe the response of tumors to XRT and development of resistance. (B) Response of BT-40 PDX to XRT (10 Gy) on cycles 1-3. (C) Response of BT-40 PDXs to 20 and 24 Gy on cycle 1, and for XRT-resistant xenograft (BT-40X) to 20 and 30 Gy. The number of mice (n = 10) is indicated in the graph. The number of mice (n = 10) is indicated where applicable, with a default of n = 5 for unspecified graphs.

To determine whether rapid development of XRT resistance occurred in other BRAFV600E-driven glioma models, we examined sensitivity to XRT in IC-3635 PXA18 and NCH-MN-1 meningioma.17 The meningioma line was intrinsically resistant to XRT (Supplementary Figure S1). In contrast, the IC-3635 line showed sensitivity to XRT (10 Gy), with partial or complete regression (Supplementary Figure S2). During XRT cycles 2-6, tumors showed less volume regression and reduced EFS compared to parental XRT naïve IC-3635 tumors (EFScycle 1 = 115.6 ± 7.7 days vs EFScycle 2 = 72.6 ± 8.2 days; P = .0001). Thus, in 2 BRAFV600E-driven PDX models, the emergence of resistance to XRT was relatively rapid.

Combined MAPKi/XRT Improves Therapeutic Efficacy, But Does Not Delay Development of Resistance

The above results suggested that glioma cells rapidly become resistant to XRT and that this leads to tumor progression. Therefore, we wanted to determine whether MAPKi and XRT combination could retard the development of resistance. As before, subcutaneous tumors were subjected to localized 10 Gy XRT and trametinib was administered orally at 1 mg/kg for 42 consecutive days starting on day 1 of XRT. The combination treatment led to complete regression of BT-40 tumors (Figure 2A), significantly extended EFS (EFS Tram + XRTcycle 1 = 129.1 ± 34.3 days, vs EFS XRTcycle 1 = 56 ± 11 days, P = .0001; vs EFS Tram cycle 1 = 86 ± 5.4 days,21  P = .003). Notably, 4 out of 10 mice did not have detectable relapsed tumors by week 24, when the experiments ended. However, the relapsed tumors progressed through treatment during cycle 4 (Figure 2A). Direct targeting of BRAF (dabrafenib) combined with XRT was less efficacious in the BT-40 model (Figure 2B). The response of BT-40 to the XRT/dabrafenib/trametinib triple combination was similar to that of the XRT/trametinib combination (Figure 2C). While the EFS of the triple combination was significantly greater than XRT/trametinib (157 ± 8.6 days vs 129.1 ± 34.3 days; P = .04), resistance developed at a similar rate for both treatments. This suggests that vertical targeting of MAPK signaling may not be able to prevent the development of XRT resistance in this tumor model.

Figure 2.

Figures showing how targeting the MAPK pathway affects tumor growth in a pediatric glioma model. Line graphs track tumor responses across treatment cycles. Trametinib plus radiation causes tumor regression initially but resistance develops by cycle four. Dabrafenib with radiation does not delay resistance. A combination of trametinib, dabrafenib, and radiation improves response, but resistance still emerges.

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The effects of single-agent targeting or vertical targeting of MAPK signaling on therapeutic efficacy of XRT. (A) Response of BT-40 xenografts to trametinib + XRT. Data show responses of individual BT-40 xenografts on cycles 1 through 4. (B) The same study design was used for dabrafenib (30 mg/kg daily × 42 days P.O.). Results show the responses to dabrafenib + 10 Gy XRT on cycles 1 and 2. (C) A similar study was conducted using trametinib (1 mg/kg daily × 6 weeks) + dabrafenib (30 mg/kg daily × 42 days P.O.) + 10 Gy. Results show responses of individual tumors on cycles 1 through 4.

In the IC-3635 PXA model, combining trametinib with XRT led to rapid and complete tumor regression within 2-3 weeks. Tumors regrew 12 weeks posttreatment cessation. At the end of the 24-week experiment, tumor in only 1/5 mice reached 400% of the day 1 treatment volume (Supplementary Figure S3). In the cycle 2 treatment, tumors continued to exhibit a rapid response, but with incomplete regression (Supplementary Figure S3). Compared to first cycle treatment, the EFS was significantly decreased (EFS Tram + XRTcycle 1 = 163 ± 10.7 days vs EFScycle 2 = 118.5 ± 10.7 days; P = .0002).

Results from both PDX models showed that combining MAPK inhibition with XRT significantly enhanced therapeutic efficacy but did not prevent resistance development.

Alterations in DNA Damage Response (DDR) Do Not Contribute to XRT Resistance

The DDR and repair capacity of tumor cells are critical determinants of their response to XRT.24 To investigate if DDR contributes to radiation resistance in these tumor models, we compared DNA damage checkpoint signaling between parental and XRT-resistant tumors after XRT exposure. Both BT-40 and BT-40X tumor xenografts showed robust activation of the G1 checkpoint via Ataxia-Telangiectasia mutated (ATM), evidenced by phosphorylation of ATM and Chk2, leading to p53 activation and subsequent upregulation of p21, a CDK inhibitor that arrests cell cycle progression (Supplementary Figure S4A). BT-40X tumors exhibited stronger ATM phosphorylation and higher p53/p21 induction compared to BT-40, although Chk2 phosphorylation was similar. Visualizing 53BP1 foci in irradiated tumors revealed near-complete repair of DNA double-strand breaks within 24 h for both BT-40 and BT-40X (Supplementary Figure S4B). Similarly, analysis of IC-3635 and IC-3635X tumors exposed to 6 Gy XRT showed no significant differences in ATM/Chk2/p53/p21 checkpoint signaling (Supplementary Figure S5A) or in 53BP1 foci formation and resolution (Supplementary Figure S5B), indicating comparable efficiency of DNA repair in primary and radiation-resistant tumors.

Upregulation of MAPK and PI3K signaling pathways in XRT-resistant tumors.

To investigate the mechanisms contributing to XRT resistance, we analyzed the expression profiles of parental (XRT naïve) BT-40 and BT-40X PDX xenografts by RNA-seq. 847 differentially expressed genes (log2FC > 2, Padj < .01) were identified between BT-40 parental and its XRT-resistant derivative, with 394 genes upregulated, and 455 genes downregulated in XRT-resistant tumors, respectively (Supplementary Figure S6A, B). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed enrichment of MAPK and PI3K signaling pathways among differentially expressed genes (Figure 3A). Significant upregulation of MET, FGFR1, TGFBR2, TEK, and corresponding ligands in XRT-resistant tumor tissues (Supplementary Figure S6C). Higher levels of Met, TrkB, EGFR, and TGFBR2 proteins were detected in BT-40X tumor tissues, supporting the results from RNA sequencing. In alignment with the upregulated expression of receptor tyrosine kinases (RTKs), higher phosphorylation of RTK, such as Met, and downstream Akt phosphorylation signal was detected in the radiation-resistant tumor tissue (Figure 3B). FGFR1 protein abundance was relatively low in BT-40 and BT-40X tumor tissues, and its phosphorylation was not detectable.

Figure 3.

Image showing activation of MAPK and PI3K pathways in radiation-resistant glioma tumors. A volcano plot highlights increased signaling in resistant BT-40X tumors compared to parental BT-40 tumors. Western blot images show elevated receptor tyrosine kinases, ERK1/2, and AKT-TORC1 signaling in BT-40X and IC-3635X tumors. Bar graphs quantify increased phospho-AKT and RTK expression levels, with significant differences between parental and resistant tumors.

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Upregulation of MAPK and PI3K signaling in XRT-resistant tumors. RNA-seq was performed on parental BT-40 xenografts and XRT-resistant (BT-40X) tumors. (A) Volcano Plot of the enriched pathways in BT-40X tumors. (B) Upper panel: protein levels of RTKs and ERK1/2/Akt-TORC1 signaling detected by Western blotting in BT-40/BT-40X tumors. Lower panel: quantitation of phospho-Akt. (C) Left panel: protein levels of RTKs and ERK1/2/Akt-TORC1 signaling detected by Western blotting in parental IC-3635 tumors and IC-3635X; right panel: quantitation of RTKs and phospho-Akt. Each lane in panels B and C represents the results from an individual tumor. The numbers above each lane correspond to the serial number of each individual mouse xenograft.

To further investigate the involvement of RTKs in radiation resistance, IC-3635 and XRT-resistant tumors (IC-3635X) were examined by RNA sequencing. KEGG pathway enrichment analysis showed multiple pathways enriched (Supplementary Figure S6F), including PI3K/ATK/mTOR. RTK expression and downstream signaling were also detected by western blotting. Interestingly, FGFR1 and PDGFRA were highly expressed in IC-3635X tumor tissue along with higher level of phosphorylated Akt, supporting the upregulation of PI3K/Akt signaling in IC-3635 XR-resistant xenografts (Figure 3C).

PI3K/Akt/TORC1 signaling drives XRT resistance in BT-40X tumor cells.

RTKs, including Met, EGFR, AXL, FGFR1, and TrkB, have been reported to play critical roles in XRT resistance in different cancers.24–31 To determine whether these upregulated RTKs contribute to XRT resistance in pediatric BRAFV600E glioma, tumor cells were first isolated from BT-40 primary and XRT-resistant xenografts. Compared to the parental tumor cells (BT-40), the XRT-resistant cells (BT-40X) showed increased growth rates, with cell doubling times for BT-40 and BT-40X cells being approximately 53 h versus 33 h, respectively (Figure 4A). While BT-40X cells could form colonies when plated as single cells, primary BT-40 cells isolated from the parental tumor lacked colony-forming capacity. Therefore, cell proliferation monitored by Incucyte was utilized to quantify XRT resistance. BT-40X cells were resistant to XRT (5 Gy) in vitro relative to cells derived from parental BT-40 xenografts (Figure 4B), although both lines remained sensitive to trametinib (Figure 4C).

Figure 4.

Image showing in vitro analysis of cell proliferation in BT-40 and BT-40X glioma cells. Line graphs demonstrate that BT-40X cells grow faster and are resistant to radiation compared to parental BT-40 cells. Both cell types respond similarly to trametinib, indicating drug sensitivity is retained in radiation-resistant cells.

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Primary cells from BT-40X xenografts are resistant to XRT but remain sensitive to trametinib in vitro. (A) Proliferation of BT-40 (blue) and BT-40X (orange) cells in vitro (Incucyte zoom); (B) Proliferation of BT-40 and BT-40X exposed to 5 Gy XRT (Incucyte zoom); (C) Responses of BT-40 cells (left) and BT-40X cells (right) to trametinib (1.25 nM).

Specific RTK inhibitors were applied to test if blocking the RTK activity would sensitize BT-40X cells to XRT. XRT (5 Gy) reduced the overall growth/proliferation of BT-40X cells. Inhibition of FGFR1, EGFR, or TrkB with the corresponding inhibitors alone at a concentration of 0.25 µM did not affect cell proliferation. However, these inhibitors enhanced the effect of radiation on cell proliferation when combined with 5 Gy XRT. Crizotinib, a Met inhibitor, required a higher concentration (0.5 µM) to significantly enhance the effects of 5 Gy XRT (Supplementary Figure S7A). These results suggested that multiple RTKs function redundantly in promoting XRT resistance, and, therefore, targeting the common downstream PI3K/TORC1 signaling could be more effective. To test this, we evaluated BEZ235, a dual PI3K/mTOR kinase inhibitor, on BT-40X cells. At a low concentration (12 nM), BEZ235 alone did not significantly affect BT-40X cell proliferation but markedly enhanced the radiation effect when combined with 5 Gy XRT. Targeting TORC1/2 activity with AZD8055 achieved a similar effect to BEZ235 (Supplementary Figure S7B), supporting the hypothesis that multiple RTK signaling merges into PI3K/TOR signaling to promote XRT resistance. Inhibition of TORC1 activity alone with a low concentration of rapamycin or the rapamycin derivative everolimus also effectively sensitized BT-40X cells to XRT (Figure 5A; Supplementary Figure S7C), indicating that RTK signaling promotes XRT resistance through TORC1 complex activity.

Figure 5.

Image illustrating the role of TORC1 signaling in radiation resistance. Graphs show rapamycin sensitizes BT-40X cells to radiation and inhibits tumor growth in vivo. Genetic activation of the TORC1 pathway confers resistance in naïve cells. Tumors treated with trametinib, rapamycin, and radiation do not develop resistance after multiple cycles. Survival curves compare intracranial tumor models, showing that cycle-5 treated tumors remain responsive to radiation, with statistical results from log-rank tests included.

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TORC1 signaling is required for radiation resistance in BT-40 tumor cells. (A) Cell proliferation was monitored with Incucyte. Suppressing TORC1 with rapamycin (Rapa, 25 nM) sensitized XRT-resistant tumor cells (BT-40X) to 5 Gy XRT in vitro. (B) Expression of constitutively active Myr-Akt3, knockdown of TSC2, or expression of TSC2GAP in XRT naïve cell BT-40 confers resistance to XRT in vitro. (C) The response of BT-40 astrocytoma xenografts to rapamycin + XRT in treatment cycles 1-3 and 5. (D) Combined trametinib (0.5 mg/kg daily × 6 weeks P.O.) and rapamycin (5 mg/kg daily × 42 days P.O.) blocks development of XRT resistance. Results show responses of individual BT-40 tumors to trametinib/rapamycin/10 Gy XRT on treatment cycles 1, 2, 3, and 6 (cycles 4 and 5 are not shown). Growth of tumors in untreated mice was shown in Supplementary Figure S8B). (E) BT-40 xenografts retain XRT sensitivity after 5 cycles of trametinib + rapamycin + 10 Gy therapy. Tumor from cycle 5 of treatment (trametinib + rapamycin + XRT) was transplanted into a new cohort of mice and randomized to receive no treatment (control) or 10 Gy XRT. (F) Kaplan-Meier curve of parental BT-40 tumor, XRT-resistant tumor (BT-40X), and BT-40TRX (cycle 5)-bearing athymic nude mice subjected to different treatments. 3 × 105 tumor cells isolated from fresh resected PDX tumor tissue were tagged with luciferase and inoculated by intracranial injection into athymic mice. At 2~3 weeks postinjection, mice were randomly assigned to control (no treatment) or 10 Gy treatment. Mice were euthanized as an endpoint when neurological symptoms, visible tumor growth, or less than 10% body weight decrease were observed. Log-rank test values are shown for the comparisons between BT-40X 10 Gy vs BT-40 10 Gy and BT-40TRX 10 Gy vs BT-40 10 Gy. HR: hazard ratio.

Upregulation of TORC1 Signaling Confers Resistance to XRT

We examined whether upregulating TORC1 signaling in parental BT-40 cells by expressing constitutively active Myr-Akt3, knockout of TSC2, or expressing TSC2 dominant-negative mutant32 affected radiation resistance. We found that upregulation of TORC1 signaling induced resistance to 3 Gy of XRT (Figure 5B).

To further understand the contribution of TORC1 signaling to XRT resistance in vivo, we first tested rapamycin in the BT-40 PDX model. Daily rapamycin (5 mg/kg daily for 42 days) did not significantly enhance the therapeutic effects of XRT but delayed the emergence of XRT resistance. By cycle 3 of XRT + rapamycin, tumors remained responsive to treatment with reduced regression. By cycle 5, tumors progressed through combination treatment compared to cycle 1, but with no significant decrease in EFS (EFS Rapa + XRTcycle 1 = 73.2 ± 20.4 days vs EFS Rapa + XRTcycle 5 = 66.5 ± 6.0 days, P = .5, Figure 5C). These results indicated that TORC1 inhibition by rapamycin can delay the development of XRT resistance in the BT-40 tumor model.

Dual Inhibition of MAPK and TORC1 Signaling Enhances the Therapeutic Effect of XRT, Prevents or Retards the Development of XRT Resistance

In pediatric BRAFV600E brain tumors, TORC1 signaling is also controlled by MAPK signaling through phosphorylation of TSC2-Serine 644.21 In our prior study, trametinib combined with intermittent low-dose rapamycin led to more efficient blockade of TORC1 signaling and prevented emergence of trametinib resistance.22 We investigated whether this combination could also inhibit the development of resistance to XRT. As shown in Figure 5D, BT-40 xenografts responded consistently to trametinib + rapamycin + XRT from cycle 1 to 6, achieving complete regression. The least responsive tumor from cycle 5 was transplanted to mice and irradiated (10 Gy) to compare with naïve BT-40 tumors in cycle 1. After 5 cycles of trametinib + rapamycin + 10 Gy XRT, BT-40 tumors remained as sensitive to XRT as naïve BT-40 xenografts to 10 Gy alone (Figure 5E). The results from the BT-40 PDX model indicated that dual targeting of MAPK and TORC1 signaling not only improved the therapeutic efficacy of XRT but also prevented the development of radiation resistance.

To further validate the in vivo observations from the BT-40 model, we also applied the trametinib + rapamycin + 10 Gy treatment to the IC-3635 PDX model. Tumors responded with rapid and complete regression 2-3 weeks after the start of treatment, with detectable regrowth after 20-23 weeks (Supplementary Figure S9). At the end of the experiment (24 weeks), no tumors reached 400% of the day 1 treatment volume. On cycle 2 treatment, tumors were responsive with complete regression, and a slight but significant decrease in EFS (EFS Tram + Rapa + XRT cycle 1 >168 days vs EFScycle 2 146.2 ± 14.1 days, P < .026). Compared to Tram + XRT treatment, adding rapamycin to the treatment significantly extended the EFS (EFS Tram + XRT cycle 2 = 118.5 ± 10.7 vs EFS Tram + Rapa + XRT cycle 2 = 146.2 ± 14.1, P = .009). On cycles 3 and 4 of treatment, tumors remained responsive to treatment with rapid and nearly complete regression during treatment (Supplementary Figure S9) but with gradual decrease of EFS (EFS Tram + Rapa + XRT cycles 3, 4 = 119 ± 7.4 and 121.5 ± 7.8, respectively), indicating delayed development of resistance.

To evaluate whether the results acquired from subcutaneous PDX model predict the tumor response to treatment in the brain, intracranial experiments were performed. Tumor cells were isolated from parental tumor BT-40, XRT-resistant tumor BT-40X, and tumor that had undergone 5 cycles of trametinib/rapamycin/XRT (TRX) treatment, respectively. Cells from each BT-40 derivative were implanted intracranially into athymic nude mice, and mice received 10 Gy XRT over 5 days. Event-free survival was determined and plotted as Kaplan-Meier curve (Figure 5F). Log-rank test was performed between each group (Supplementary Table S4). XRT alone extended the event-free survival time of BT-40-bearing mice to 35 days, compared to 14 days without treatment (log-rank test P = .0001). BT-40X tumors exhibited reduced sensitivity to XRT, with an average EFS of 21 days compared to 35 days for BT-40 tumors following 10 Gy XRT (log-rank test, P < .0001), indicating resistance. Notably, tumors that had undergone multiple cycles of TRX treatment remained sensitive or even showed increased sensitivity to XRT compared to parental tumor, with EFS significantly extended (log-rank test, P = .0002). Remarkably, 8 out of 9 mice showed no tumor-related events within the 60-day observation period (Figure 5F).

Dual inhibition of MAPK and TORC1 signaling selectively enhanced the therapeutic efficacy of XRT in BRAF-mutant gliomas, with no effect on BT-35, BRAF wild-type gliomas (Supplementary Figure S10A, B). Similarly, rapamycin, trametinib, or the combination did not enhance XRT activity in 2 additional lines with wild-type BRAF (SJ-GBM2 human glioblastoma and 3T3 mouse fibroblasts in vitro; Supplementary Figure S10C). Additionally, trametinib/rapamycin did not cause greater damage to normal brain tissue compared to XRT alone (Supplementary Figure S11). In vitro data further confirmed that dual inhibition of MAPK and TORC1 selectively enhances tumor cell killing (BT-40X) while sparing normal astrocytes (Supplementary Figure S12).

Survivin, Downstream of TORC1 Signaling, Contributes to XRT Resistance

TORC1 signaling is a central regulator of cell growth, proliferation, and survival.33,34 Some pro-survival factors, such as Mcl-1 and Survivin (BIRC5), have been identified as downstream targets.34,35 In BT-40 cells, upregulating PI3K-TORC1 signaling resulted in an increase of Mcl-1 and Survivin levels (Figure 6A). On the other hand, TORC1 inhibition with rapamycin led to a decrease of Mcl-1 and Survivin (Figure 6B). BT-40X cells exhibited higher levels of Survivin and Mcl-1 compared to BT-40 cells, and rapamycin treatment decreased both proteins in a concentration-dependent manner in both cell lines (Figure 6C). S6K1 knockdown by siRNA in BT-40X cells abrogated Survivin and Mcl1 expression (Supplementary Figure S13A) and sensitized cells to XRT (Supplementary Figure S13B, C). These results suggest that TORC1 signaling regulates Survivin expression via TORC1/S6K1 sub-pathway, which may contribute to the mechanism of radiation resistance mediated by TORC1 signaling.

Figure 6.

Image showing that TORC1 signaling regulates survival proteins in glioma cells. Western blot data show that activating TORC1 increases Survivin and Mcl-1 expression. Rapamycin suppresses these proteins over time and in a dose-dependent manner. Knockdown of Survivin reduces proliferation and increases radiation sensitivity. Inducing Survivin expression causes resistance to radiation. Dual inhibition of MEK and TORC1 leads to more effective suppression of pro-survival proteins and enhanced radiation response.

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TORC1 signaling regulates pro-survival proteins and response to XRT. (A) Activation of TORC1 signaling by expressing Myr-Akt3, TSC2 knockdown, or expressing TSC2GAP increases Mcl-1 and Survivin in BT-40 cells in vitro. (B) Response to 5 Gy XRT in control or rapamycin-treated BT-40X cells. Cell pellets were derived from 0.5 to 72 h postradiation and processed as described in Materials and Methods. Rapamycin notably suppressed Survivin and Mcl-1. (C) Rapamycin suppresses Survivin and Mcl-1 in both BT-40 and BT-40X cells. BT-40 and BT-40X cells were exposed to increasing concentration of rapamycin (0-20 nM) for 24 h. (D) Knockdown of Survivin in BT-40X cells reduced cell proliferation and increased sensitivity to XRT. Left panel: siRNA-mediated knockdown of Survivn in BT-40X cells; Right panel: proliferation of BT-40X cells with downregulated Survivin (BIRC5) without XRT treatment (control) or after exposure to 5 Gy XRT. (E) Induction of Survivin confers resistance to XRT. Upper panel: survivin level over 48 h in BT-40 cells engineered to express Survivin under control of a doxycycline-inducible TETON promoter; lower panel: response to XRT is dependent on Survivin expression. Cells were grown for 48 h in the absence of doxycycline (DOX-) or presence of doxycycline (DOX+) and then left unirradiated or radiated with 5 Gy of XRT and plated at equal cell density. Proliferation was determined by cell confluency over 300 h (Incucyte zoom). (F) Combined treatment of BT-40X cells with trametinib (10 nM, 24 h) and rapamycin (10 nM, 24 h) was more effective than individual drugs in suppressing Survivin and Mcl-1 in BT-40X cells. (G) Dual inhibition of MEK and TORC1 enhanced the cell killing of radiation on BT-40X cells in vitro. BT-40X cells were seeded at 5 × 105 cells/6 cm plate and exposed to trametinib (10 nM) and rapamycin (10 nM)

The above results suggested the potential contribution of one or both pro-survival factors to XRT resistance. However, treating BT-40X cells with Mcl-1 inhibitor S63845 had a mild effect on cell survival after exposure to XRT (Supplementary Figure S14), suggesting a minor role of Mcl-1 or functional redundancy with other factors in XRT resistance. The role of Survivin in XRT resistance has been well documented in adult cancers.36–41 In BT-40X cells, siRNA-mediated knockdown of Survivin resulted in decreased proliferation and increased sensitivity to XRT (Figure 6D), while doxycycline-induced overexpression of Survivin conferred resistance to XRT in BT-40 cells (Figure 6E).

In vitro, inhibition of MAPK signaling with trametinib reduced TORC1 signaling and partially decreased Survivin levels, whereas combined treatment with trametinib + rapamycin completely inhibited TORC1 and depleted Survivin (Figure 6F). Next, we examined the combination of trametinib + rapamycin with 10 Gy XRT treatment of BT-40X cells in vitro. This treatment resulted in complete loss of Survivin, increased phosphorylated H2AX levels, elevated Bim expression, and PARP1 cleavage, indicating enhanced apoptosis (Figure 6G). These results suggest that dual targeting of MAPK and TORC1 signaling enhances the therapeutic effect of XRT and can prevent the development of resistance by promoting apoptosis of irradiated tumor cells via downregulation of pro-survival protein Survivn and upregulation of apoptotic activator Bim.

Discussion

MEK inhibitors, alone or with BRAF inhibitors, have transformed childhood BRAF-driven brain tumor treatment, but those drugs are not curative in BRAFV600E glioma. Further studies with cytotoxic treatments are urgently needed to develop therapeutic strategies that could lead to cures.42 XRT is an integral part of treatment for pediatric higher grade gliomas, offering good long-term survival but with risk of serious long-term side effects.16 Even with advances in conformal radiation, the risk of side effects is directly correlated with the tissue volume irradiated and dose delivered.43 Minimizing XRT dose without losing therapeutic efficacy remains a priority. Moreover, cancers developing resistance to XRT remain a challenge.

Using 2 pediatric BRAFV600E glioma PDX models that are sensitive to XRT, we first examined the tumor response to low-dose XRT (10-30 Gy in 2 Gy fractions). Tumors initially responded to XRT, with significant regression and delayed regrowth. To study emergence of resistance, tumors were exposed to 10 Gy, and the least responsive tumor was transplanted, and the process was repeated until tumors demonstrated significant XRT resistance. Both models developed resistance after 2 cycles of 10 Gy XRT. BT-40X tumors were completely resistant, with tumor progression during treatment, while IC-3635 remained partially responsive to XRT after 6 cycles of XRT. Of note, when BT-40 tumors became resistant to lower-dose XRT (10 Gy), they were also resistant to higher-dose XRT (20-30 Gy). These results exemplify the challenge of XRT resistance in pediatric glioma and underscore the critical need to identify agents that prevent the development of resistance. To explore if MAPKi + XRT can improve the therapeutic efficacy of trametinib, we evaluated the effect of trametinib + 10 Gy XRT on the BT-40 PDX model. After the first cycle of treatment, tumors completely regressed to undetectable levels. Tumor regrowth was detected around 11 weeks after the end of treatment. The EFS was significantly extended compared to XRT (P = .0001) alone or trametinib alone (P = .003). Notably, 4 of 10 mice had no tumor relapse after treatment. However, when the relapsed tumors were transplanted and subjected to subsequent cycles of combination treatment, tumors rapidly developed resistance to the treatment, suggesting that tumors developed resistance to both MEK inhibitors and XRT. We next examined vertical inhibition of MAPK signaling by combining dabrafenib (BRAF inhibitor) with trametinib, a combination shown to improve outcome in pediatric patients with low-grade glioma harboring BRAFV600E mutation.13,14 The addition of dabrafenib to trametinib increased tumor response significantly (P = .0008), however, resistance to XRT emerged as rapidly as with trametinib alone. These results suggest vertical targeting of MAPK signaling cannot prevent the development of XRT resistance. We previously reported that low-dose intermittent administration of rapamycin prevented or retarded emergence of trametinib resistance in BRAFV600E PDX models.21 Rapamycin as monotherapy delayed emergence of XRT resistance and, in combination with trametinib, prevented XRT resistance altogether. BT-40 tumors treated with 5 cycles of trametinib-rapamycin + XRT (10 Gy) remained as sensitive to XRT alone as XRT-naïve BT-40 xenografts.

To decipher the mechanism(s) of radiation resistance, we performed RNA sequencing to compare the global gene expression alterations between primary tumors and XRT-resistant tumors. Pathway enrichment analysis showed that MAPK, PI3K, and Ras signaling pathways were among the top enriched pathways. Interestingly, upregulated genes were shared among these 3 pathways, including MET, FGFR1, NTRK2 (TrkB), TEK (TIE2), and their corresponding ligands. Altered expression of several RTKs was confirmed by western blotting. Upregulation of RTK expression was associated with increased Akt phosphorylation in XRT-resistant tumor samples. RTKs, such as Met, FGFR1, and Axl, have been reported to be associated with XRT resistance in adult cancers, and inhibition of the implicated RTKs re-sensitized tumors to radiation.24,27–29 Inhibition with chemical inhibitors mildly but significantly re-sensitized the XRT-resistant BT-40X tumor cells to XRT in vitro. In these pediatric glioma PDX models driven by oncogenic BRAFV600E, multiple RTKs might work redundantly to contribute to radiation resistance. Nonetheless, the extracellular signals from different RTKs ultimately merge into MAPK and PI3K/Akt/mTOR signaling, which is targetable. As predicted, inhibition of either PI3K/TORC1/2 signaling enhanced XRT-induced cell killing, although these inhibitors had modest effects on tumor cell proliferation or survival. On the other hand, upregulating TORC1 signaling in BT-40 cells (from primary tumor) by expressing constitutively active Akt, or knockout/inactivation of TSC2, a negative regulator of TORC1, all conferred resistance to XRT. Therefore, we propose that upregulation of multiple RTKs merge into PI3K/Akt/mTOR signaling to contribute to the development of XRT resistance in the brain tumor models, possibly by promoting pro-survival signaling.

mTOR signaling has been implicated in the DDR44 and promoting cell survival by regulating apoptotic protein function or protein levels.33–35,45 Our results from in vitro examination focused on the pro-survival function of mTOR signaling by controlling the expression of Survivin and Mcl-1. Survivin appeared to play a major role over Mcl-1, as knockdown of Survivin alone in radiation-resistant tumor cells resulted in decreased cell proliferation and increased sensitivity to XRT, while inhibition of Mcl-1 function with a specific inhibitor had marginal effects on XRT-induced cell killing. Overexpression of Survivin in parental BT-40 cells conferred resistance to XRT. In BRAFV600E-driven pediatric glioma, MAPK signaling positively controls TORC1 signaling by phosphorylating TSC2-S664 and inactivating the TSC complex.21 As expected, MEK inhibition with trametinib repressed TORC1 signaling and Survivin expression, while combining trametinib and rapamycin blocked TORC1 signaling more completely, with Survivin protein being further depleted. Our study thus identified Survivin as one of the important pro-survival factors regulated by TORC1 signaling that contributes to XRT resistance.

ERK1/2 promotes cell survival by a dual mechanism including posttranslational modification and inactivation of apoptotic proteins like Bim and Bad and transcriptional regulation of pro-survival genes.46 Dual inhibition of ERK1/2 and TORC1 was expected to enhance the XRT-induced cell killing and overcome XRT resistance. This prediction was affirmed by in vitro experiments, and subsequently, xenograft studies. Importantly, the triple combination treatment in BT-40 tumor-bearing mice led to complete regression, significantly longer EFS, and fewer tumors demonstrating regrowth during the period of observation than other treatments in this study. More importantly, after multiple cycles of treatment (up to 6), the tumors remained completely responsive to the combination treatment and remained responsive to XRT alone, indicating no development of resistance to either trametinib or XRT per se.

In summary, our data suggest that TORC1 signaling from upregulated RTKs contributed to development of XRT resistance in 2 pediatric glioma PDX models driven by BRAFV600E by promoting pro-survival signaling. Survivin was identified as an important pro-survival factor controlled by TORC1 signaling to promote XRT resistance. The PDX results suggest that dual inhibition of MAPK and TORC1 signaling could enhance the therapeutic efficacy of XRT and potentially prevent or delay the development of resistance to the combination treatment. These results may also be applicable to pediatric low-grade brain tumors with BRAF::KIAA1549. While combined trametinib-rapamycin plus XRT was well tolerated in mice, the utility and safety of this combination in human glioma patients should be carefully evaluated in clinical trials in the future.

Supplementary material

Supplementary material is available online at Neuro-Oncology (https://academic.oup.com/neuro-oncology).

noaf068_suppl_Supplementary_Tables_S1-S4_Figures_S1-S14
noaf068_suppl_supplementary_tables_s1-s4_figures_s1-s14.zip (44.8MB, zip)

Acknowledgments

RNA sequencing data were generated in the Genome Sequencing Facility, which is supported by UT Health San Antonio, NIH-NCI P30 CA054174 (Cancer Center at UT Health San Antonio), and NIH Shared Instrument grant S10OD030311 (S10 grant to NovaSeq 6000 System), and CPRIT Core Facility Award (RP220662).

Contributor Information

Fuyang Li, Department of Molecular Medicine, UT Health San Antonio, San Antonio, Texas, USA; Greehey Children’s Cancer Research Institute, UT Health San Antonio, San Antonio, Texas, USA.

Kathryn M Bondra, Greehey Children’s Cancer Research Institute, UT Health San Antonio, San Antonio, Texas, USA.

Hanzhou Wang, Greehey Children’s Cancer Research Institute, UT Health San Antonio, San Antonio, Texas, USA.

Dias Kurmashev, Greehey Children’s Cancer Research Institute, UT Health San Antonio, San Antonio, Texas, USA.

Bipasha Mukherjee, Department of Neurosurgery, UT Health San Antonio, San Antonio, Texas, USA.

Suman Kanji, Department of Neurosurgery, UT Health San Antonio, San Antonio, Texas, USA.

Amyn A Habib, Department of Neurology, UT Southwestern Medical Center, Dallas, Texas, USA.

Yidong Chen, Department of Population Health Sciences, UT Health San Antonio, San Antonio, Texas, USA; Greehey Children’s Cancer Research Institute, UT Health San Antonio, San Antonio, Texas, USA.

Siyuan Zheng, Department of Population Health Sciences, UT Health San Antonio, San Antonio, Texas, USA; Greehey Children’s Cancer Research Institute, UT Health San Antonio, San Antonio, Texas, USA.

Sandeep Burma, Department of Neurosurgery, UT Health San Antonio, San Antonio, Texas, USA.

Peter J Houghton, Department of Molecular Medicine, UT Health San Antonio, San Antonio, Texas, USA; Greehey Children’s Cancer Research Institute, UT Health San Antonio, San Antonio, Texas, USA.

Funding

This study was supported by the National Cancer Institute (NCI), National Institutes of Health (NIH) under grant numbers CA258381, CA169368, CA199297, CA246807, and CA244212, as well as NS119225 from the National Institute of Neurological Disorders and Stroke (NINDS), NIH. Additional support was provided by the Cancer Prevention and Research Institute of Texas (CPRIT) under grant RP160732.

Conflict of interest statement

The authors declare no potential conflicts of interest.

Authorship statement

Conceptualization: P.J.H., F.L., and S.B. Experiment design: P.J.H. and F.L. Experiment conduction: F.L., K.M.B., and S.K. Data analysis: F.L., K.M.B., H.W., D.K., Y.C., S.Z., A.A.H., B.M., and P.J.H. The study was supervised by S.B. and P.J.H. The manuscript and figures were prepared by F.L., S.B., and P.J.H., with input from all authors.

Data availability

The generated RNA sequencing data are available in GEO profile (GSE271064 and GSE284577 with token for reviewer access: kxmnauccdtwfhmp and czihwqauxdczviz, respectively).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

noaf068_suppl_Supplementary_Tables_S1-S4_Figures_S1-S14
noaf068_suppl_supplementary_tables_s1-s4_figures_s1-s14.zip (44.8MB, zip)

Data Availability Statement

The generated RNA sequencing data are available in GEO profile (GSE271064 and GSE284577 with token for reviewer access: kxmnauccdtwfhmp and czihwqauxdczviz, respectively).

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