BRAF/MEK inhibition induces cell state transitions boosting immune checkpoint sensitivity in BRAFV600E-mutant glioma

Abstract

Resistance to v-raf murine sarcoma viral oncogene homolog B1 (BRAF) plus mitogen-activated protein kinase kinase (MEK) inhibition (BRAFi+MEKi) in BRAF^V600E-mutant gliomas drives rebound, progression, and high mortality, yet it remains poorly understood. This study addresses the urgent need to develop treatments for BRAFi+MEKi-resistant glioma using preclinical mouse models and patient-derived materials. BRAFi+MEKi reveals glioma plasticity by heightening cell state transitions along glial differentiation trajectories, giving rise to astrocyte- and immunomodulatory oligodendrocyte (OL)-like states. PD-L1 upregulation in OL-like cells links cell state transitions to immune evasion, possibly orchestrated by Galectin-3. BRAFi+MEKi induces interferon response signatures, tumor infiltration, and suppression of T cells. Combining BRAFi+MEKi with immune checkpoint inhibition enhances survival in a T cell-dependent manner, reinvigorates T cells, and outperforms individual or sequential therapies in mice. Elevated PD-L1 expression in BRAF-mutant versus BRAF-wild-type glioblastoma supports the rationale for PD-1 inhibition in patients. These findings underscore the potential of targeting glioma plasticity and highlight combination strategies to overcome therapy resistance in BRAF^V600E-mutant high-grade glioma.

Keywords: BRAF V600E; BRAF and MEK inhibitor adaptation; Galectin-3; T cell modulation; cell state transitions; high-grade glioma; immune checkpoint inhibition; programmed death-ligand 1.

Graphical abstract

Figure 1

Patient BRAF^V600E-mutant HGG undergoes glial differentiation after treatment (A) Schematic of longitudinal analyses in two patients. (B) MRI scans of patient 49 showing tumor (left), post-surgical cavity (middle), and recurrence (right) after BRAFi+MEKi. (C) GO analysis showing gliogenesis pathway upregulation at second recurrence after BRAFi+MEKi in patient 12. (D) Scatterplot of glial and progenitor expression changes before and after BRAFi+MEKi in patient 12. (E) Representative IF images for GFAP, MBP, and OLIG2 before and after BRAFi+MEKi and chemoradiation in patient 49. High-magnification images of boxed areas are shown on the right. Scale bars: 180 μm (left) and 140 μm (right). Quantification of GFAP and MBP intensities and OLIG2+ cells in patient 49 (n = 5 tumor-containing images). ∗p < 0.05 and ∗∗p < 0.01 (Student’s t test). Mean ± SEM. (F) Representative H&E- and NESTIN-stained tumor sections before and after BRAFi+MEKi in patient 49. High-magnification images of boxed areas are shown on the right. Scale bars: 150 μm. See also Figure S1 and Table S1.

Figure 2

Orthotopic BRAF^V600E-mutant mouse models show tumor heterogeneity (A) Schematic of BRAF-2341 and BRAF-M34 model generation. (B) MRI scans showing tumor mass in BRAF-2341 and BRAF-M34 HGG mice (yellow dashed outlines). (C) Representative low- and high-magnification images of H&E-stained coronal sections of BRAF-2341 and BRAF-M34 HGGs. Scale bars: 80 μm (top) and 60 μm (bottom). (D) Representative IF images of BRAF-2341. GFP and/or CRE expression mark tumor cells. DAPI (cell nuclei). Left: GFP and PROM1/CD133 double-positive tumor cells (white arrowheads). Middle: OLIG2 and PROM1/CD133 double-positive/GFP-negative glioma-associated OLs (white arrowheads). Right: GFP and CRE double-positive tumor cells (white arrowheads); tumor cells co-express GFAP and OLIG2 (yellow arrowheads) or OLIG2 alone (blue arrowhead). Single-channel images of the white-boxed area are shown next to the merged images. Scale bars: 60 μm (left), 45 μm (middle), and 75 μm (right). (E) Representative IF images of BRAF-M34 HGG. Left: CRE, NESTIN, and PROM1/CD133 triple-positive NSPC/CSC-like tumor cell (white arrowheads). CRE and PROM1/CD133 double-positive (NESTIN negative) OL-like tumor cells (yellow arrowheads). Right: NeuN, OLIG2, and GFAP single-positive cells (white arrowheads) in the tumor core featuring hypercellularity. Single-channel images of the white-boxed area are shown next to the merged images. Scale bars: 110 μm (left) and 50 μm (right). (F) Schematic of RCAS (replication-competent avian retrovirus)-induced BRAF^V600E TP53-deleted (RCAS-BRAF) HGG and orthotopic, immunocompetent model development. (G) MRI scans showing the tumor infiltrating into the contralateral hemisphere (yellow dashed outlines). (H) Representative image of an H&E-stained coronal section of orthotopic RCAS-BRAF HGG showing hypercellular proliferation of malignant ovoid to spindled cells arranged in fascicles and sheets. Scale bar: 140 μm. (I) Representative IF images of RCAS-BRAF HGG. GFAP-positive astrocyte-like cells, infrequently double-positive for OLIG2 (green arrowhead). An abundance of NESTIN+ cells, frequently double-positive for OLIG2 (white arrowheads), and PDGFRα+ cells (white arrowheads). Single-channel images of the white-boxed area are shown next to the merged images. Scale bars: 90 μm (left) and 115 μm (right). See also Figure S2 and Table S2.

Figure 3

BRAFi+MEKi treatment promotes glial differentiation in BRAF^V600E-mutant HGG (A) Schematic of BRAFi+MEKi treatment timeline and analyses (RNA-seq, flow cytometry, [FC[, and immunofluorescence [IF]) of BRAF-2341 HGG. (B) Heatmap from bulk RNA-seq data depicting average expression levels of stem/progenitor cell and glial differentiation-associated transcripts between two replicates in BRAF-2341 HGG after 3-day BRAFi+MEKi treatment. (C) Schematic of 14-day BRAFi+MEKi treatment timeline and analyses (scRNA-seq and IF) of RCAS-BRAF HGG. (D) Heatmap from scRNA-seq data showing transcripts in the RCAS-BRAF tumor cluster. (E) Cnetplot showing enrichment of glial-related transcripts from GO analysis of the scRNA-seq dataset from RCAS-BRAF HGG. (F) Representative IF images of control or BRAFi+MEKi-treated RCAS-BRAF HGGs stained for GFAP, NESTIN, and DAPI. Scale bar: 120 μm. Quantification of marker-positive cells within the tumor field (right) ∗∗p < 0.01 (unpaired t test). n = 4 mice/group, mean ± SEM. (G) Representative IF images of control/BRAFi+MEKi-treated RCAS-BRAF HGGs stained for MBP, PDGFRα, and DAPI. Scale bar: 170 μm. High-magnification images of the white-boxed areas are shown on the right. Quantification of marker-positive cells within the tumor field (right). ∗p < 0.05 (unpaired t test). n = 4 mice/group, mean ± SEM. (H) Schematic of BRAFi+MEKi treatment timeline and analysis of BRAF^V600E HGG cell lines in vitro. (I and J) Representative ICC images of murine BRAF-M34 (I) and RCAS-BRAF (J) cell lines treated with control/BRAFi+MEKi and stained for MBP, OLIG2, NESTIN, and DAPI. Scale bars: 40 μm. ∗p < 0.05 (unpaired multiple t tests and Holm-Šídák method). n = 2 independent experiments, mean ± SEM. See also Figure S3.

Figure 4

BRAFi+MEKi treatment induces interferon response pathway signature and glial differentiation, with a PD-L1-expressing immunomodulatory subpopulation (A) Reactome pathway analysis revealed upregulated genes associated with interferon receptor signaling after 48-h BRAFi+MEKi treatment in the STN-49 cells. Numbers on the bar graph indicate the gene count. (B) Heatmap demonstrating expression changes of IFN-γ receptor-related transcripts after 24- and 48-h BRAFi+MEKi treatment to the STN-49 cell line. (C) Representative ICC images of murine and patient-derived cell lines treated with control/BRAFi+MEKi for 13 days. Scale bars: 40 μm. Quantification of (C). ∗p < 0.05 (multiple paired t tests). n = 2 independent experiments, mean ± SEM. (D) Representative IF images of BRAF-M34 HGGs stained for PD-L1, BRAF V600E, PLP1, and DAPI. Control panel: BRAF^V600E-mutated tumor cell negative for PD-L1 (arrow). PD-L1-positive non-tumor cell (arrowhead). BRAFi+MEKi panel: differentiated BRAF^V600E-mutant glioma cells with PLP1 and PD-L1 co-expression (white arrows). PLP1-negative, PD-L1-positive glioma cells (yellow arrows). PD-L1 only positive cells (arrowheads). Scale bars: 70 μm (left) and 80 μm (right). (E) Representative IF image of BRAFi+MEKi-treated BRAF-M34 HGG stained for PD-L1, Cre, PLP1, and DAPI. Differentiated Cre-positive glioma cell co-expressing PLP1 and PD-L1. Scale bar: 20 μm. (F) Quantification of (D). Proportions of BRAF^V600E and PLP1 double-positive differentiated glioma cells with OL-like state positive or negative for PD-L1 (left) and proportions of PLP1-positive cells co-expressing BRAF^V600E and/or PD-L1 (right) were measured and compared between groups. ∗p < 0.05 for BRAF^V600E+PLP1⁺PD-L1⁺ triple-positive cells between treatment groups (two-way ANOVA with Sidak’s multiple comparisons test). n = 3 mice/group, mean ± SEM. (G) Representative IF images of human BRAF^V600E-mutant HGG (patient 49) tissue before and after BRAFi+MEKi treatment, stained for PD-L1, Olig2, and DAPI. Scale bars, 180 μm. (H) Quantification of (G). n = 4 images from 2 consecutive sections of patient 49 tissue, ∗p<0.05 (unpaired t test), mean ± SEM. (I) The scatterplot of upregulated CD274 transcript (PD-L1) after BRAFi+MEKi treatment in patient 12. See also Figure S4.

Figure 5

PD-L1 expression is elevated in BRAF-mutant gliomas (A) Bar graph depicting percentage of patients with PD-L1+ tumors in the BRAF mutation/fusion versus BRAF-wild-type (no alterations) groups. n = 59 patients with BRAF mutation/fusion and 3,126 total patients (chi-squared tests). (B) Boxplot representing CD274 (PD-L1) transcripts per million in the BRAF mutation/fusion (n = 17) versus BRAF-wild-type (no alterations; n = 1,022) groups, from whole-transcriptome sequencing (Wilcoxon rank-sum test). (C) Representative IHC images for BRAF V600E and PD-L1 in consecutive sections of BRAF^V600E-mutant HGG. Scale bars: 250 μm. See also Table S4.

Figure 6

BRAFi+MEKi enhances T cell infiltration and synergizes with ICI to improve T cell-dependent survival (A) Experimental timeline (green: 12–14 days treatment for CyTOF, IF, and scRNA-seq analyses; pink: continuous treatment for survival analysis) in all three murine models. (B) Frequency of populations of tumor-infiltrating lymphocytes in BRAF-2341 tumors by CyTOF analysis. 3 mice/group and two independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001 (two-way ANOVA with Sidak’s multiple comparisons test), mean ± SEM. (C) Graphs depicting the frequency of PD-1+CD8⁺ and PD-1+CD4⁺ (left) or CTLA-4+CD8⁺ and CTLA-4+CD4⁺ (right) T cells normalized to control by CyTOF analysis. 3 mice/group and two independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (two-way ANOVA with Tukey’s multiple comparisons test), mean ± SEM. (D) Kaplan-Meier survival curve of orthotopic BRAF-2341 (left) or BRAF-M34 (right) HGG-bearing mice of all four treatment groups. Treatment initiation at day 30 (BRAF-2341) or day 14 (BRAF-M34) post-injection (black arrow). Data representative of two independent experiments and 5–6 mice/group (BRAF-2341) and 4–5 mice/group (BRAF-M34). p values are indicated in the graph and Tables S5 and S6 when compared to the control group. (E) Kaplan-Meier survival curve in immunocompetent C57Bl/6 mice with orthotopic BRAF-M34 HGG treated with BRAFi+MEKi and ICI, either concurrently (BRAFi+MEKi+ICI) or sequentially. Treatment initiation at day 14 post-injection (black arrow). 4–5 mice/group. p values are indicated in the graph and Table S7 when compared to the concurrent treatment group. (F) Kaplan-Meier survival curve in immunocompetent C57Bl/6 mice with orthotopic RCAS-BRAF HGG treated with BRAFi+MEKi and/or ICI for 14 days. Treatment period from day 22 to day 36 post-injection (shaded area). Data representative of two independent experiments and 7–10 mice/group. p values are indicated in the graph and Table S5 when compared to the control group. (G) Heatmaps of T cell inhibition- and apoptosis-associated transcripts in CD4⁺ and CD8⁺ T cells in the RCAS-BRAF model. (H) Densities of CD4+PD-L1+ and CD8+PD-L1+ T cells in BRAF-M34 and RCAS-BRAF (IF analysis). ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (two-way ANOVA with uncorrected Fisher’s LSD). n = 4 mice/group, mean ± SEM. (I and J) Representative IF images of RCAS-BRAF tissue stained for PD-L1, CD4 (I) and CD8 (J). CD4⁺ and CD8⁺ cells positive for PD-L1 (arrows); CD4⁺ and CD8⁺ cells negative for PD-L1 (arrowheads). Scale bars: (I) 20 μm (top), 15 μm (bottom), (J) 10 μm (top), and 20 μm (bottom). Quantifications of cell populations before versus after treatment. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (two-way ANOVA with Sidak’s multiple comparisons test). n = 4 mice/group, mean ± SEM. (K) Heatmaps of T cell activation-associated transcripts in CD4⁺ and CD8⁺ T cells in the RCAS-BRAF model. (L) Heatmaps of MHC class I (green), II (red), and IFN-γ (black)-related transcripts associated with antigen presentation in CD4⁺ and CD8⁺ T cells in the RCAS-BRAF model. (M) Densities of CD4⁺CD69⁺ and CD8⁺CD69⁺ T cells from BRAF-M34 and RCAS-BRAF (IF analysis). ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (two-way ANOVA with uncorrected Fisher’s LSD). n = 3–4 mice/group, mean ± SEM. See also Figures S5 and S6 and Tables S5–S7.

Figure 7

BRAFi+MEKi-induced Galectin-3 secretion links glial differentiation to PD-L1 upregulation (A) Timelines of in vitro experiments using STN-49 and aGBM5 cell lines. (B) Relative Galectin-3 concentrations measured over time with nELISA in supernatants of patient-derived cell lines treated with control/BRAFi+MEKi (two-way ANOVA with multiple comparisons). Simple linear regression ± SEM. (C and D) Scatterplots representing Galectin-3 expression levels in STN-49 (C) and aGBM5 (D) cell lines treated with DMSO or BRAFi+MEKi at 24 and 48 h (E and F) Representative ICC images of STN-49 (E) and aGBM5 (F) cell lines treated with BSA or recombinant Galectin-3 protein for 72 h and subsequently stained for PD-L1, MBP, or OPALIN, and DAPI. Scale bars: 40 μm. Bar graphs showing cellular quantification (% of total DAPI+ cells). ∗p < 0.05 (two-way ANOVA with Sidak’s multiple comparisons test). ns, not significant. n = 2–3 replicates from one independent experiment, mean ± SEM. See also Figure S7.