Inhibition of D-2HG leads to upregulation of a proinflammatory gene signature in a novel HLA-A2/HLA-DR1 transgenic mouse model of IDH1R132H-expressing glioma

Abstract

Background: Long-term prognosis of WHO grade II, isocitrate dehydrogenase (IDH)-mutated low-grade glioma (LGG) is poor due to high risks of recurrence and malignant transformation into high-grade glioma. Immunotherapy strategies are attractive given the relatively intact immune system of patients with LGG and the slow tumor growth rate. However, accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) in IDH-mutated gliomas leads to suppression of inflammatory pathways in the tumor microenvironment, thereby contributing to the 'cold' tumor phenotype. Inhibiting D-2HG production presents an opportunity to generate a robust antitumor response following tumor antigen vaccination and immune checkpoint blockade.

Methods: An IDH1^R132H glioma model was created in syngeneic HLA-A2/HLA-DR1-transgenic mice, allowing us to evaluate the vaccination with the human leukocyte antigens (HLA)-DR1-restricted, IDH1^R132H mutation-derived neoepitope. The effects of an orally available inhibitor of mutant IDH1 and IDH2, AG-881, were evaluated as monotherapy and in combination with the IDH1^R132H peptide vaccination or anti-PD-1 immune checkpoint blockade.

Results: The HLA-A2/HLA-DR1-syngeneic IDH1^R132H cell line expressed the IDH1 mutant protein and formed D-2HG producing orthotopic gliomas in vivo. Treatment of tumor-bearing mice with AG-881 resulted in a reduction of D-2HG levels in IDH1^R132H glioma cells (10 fold) and tumor-associated myeloid cells, which demonstrated high levels of intracellular D-2HG in the IDH1^R132H gliomas. AG-881 monotherapy suppressed the progression of IDH1^R132H gliomas in a CD4⁺ and CD8⁺ cell-dependent manner, enhanced proinflammatory IFNγ-related gene expression, and increased the number of CD4⁺ tumor-infiltrating T-cells. Prophylactic vaccination with the HLA-DR1-restricted IDH1^R132H peptide or tumor-associated HLA-A2-restricted peptides did not enhance survival of tumor-bearing animals; however, vaccination with both HLA-A2-IDH1^R132H and DR1-IDH1^R132H peptides in combination with the IDH inhibitor significantly prolonged survival. Finally, tumor-bearing mice treated with both AG-881 and a PD-1 blocking antibody demonstrated improved survival when compared with either treatment alone.

Conclusion: The development of effective IDH1^R132H-targeting vaccine may be enhanced by integration with HLA class I-restricted cytotoxic T cell epitopes and AG-881. Our HLA-A2/HLA-DR1-syngeneic IDH1^R132H glioma model should allow us to evaluate key translational questions related to the development of novel strategies for patients with IDH-mutant glioma.

Keywords: Brain Neoplasms; Central Nervous System Neoplasms; Drug Evaluation, Preclinical; Programmed Cell Death 1 Receptor; Vaccination.

Figure 1

Generation and validation of the new dTG glioma model. (A) Schematic description of the genetic background of the dTG mouse strain and the workflow to generate the dTG glioma model. The transgenic mouse strain used in the current study lacks expression of mouse MHC class I (${\displaystyle \beta }$2 m) and class II (H-2 IA${\displaystyle \beta }$^b) molecules. However, all nucleated cells (both immune cells and tumor cells) can express the human HLA-A*0201 and the human HLA-DR1*0101. (B) Immunofluorescent staining of DTG- IDH1^R132H cells. Nuclei were visualized with DAPI (blue); intracellular IDH1^R132H protein in yellow (bottom image); isotype control for primary antibody (top image; scale bar, 50 µm). (C) Cell surface expression of HLA-A2 (left panel) and HLA-DR1 (right) on IFNɣ treatment. DTG-R132H cells were cultured in the presence of 50 ng/mL IFNɣ (or solvent only media) for 72 hours and analyzed by FC. (D) Extracellular 2-HG produced by dTG-IDH1^R132H cells in vitro. Within a 6-well plate, 1.5×10⁵ normal human astrocytes (WT astrocytes) and 1.5×10⁵ dTG-IDH1^R132H cells were plated in 2 mL of media and cultured for 72 hours. Levels of 2-HG in the supernatant of each cell line were measured by LC-MS/MS. Data represent technical triplicates of each condition. (E) High levels of 2-HG produced by orthotopically inoculated dTG-IDH1^R132H cells. Tumors were collected from mice injected with dTG-wt (n=5) or dTG-IDH1^R132H (n=4) when tumor burden reached protocol endpoint. Single-cell suspensions were analyzed for the presence of 2-HG. dTG, double transgenic; FC, flow cytometry; HG, hydroxyglutarate; HLA, human leukocyte antigens; MHC, major histocompatibility complex.

Figure 2

Inhibition of IDH1^R132H suppresses 2-HG production and tumor growth in vivo. (A) Inhibition of IDH1^R132H activity by AG-120 and AG-881 in vitro. Levels of 2-HG in the supernatant of dTG-IDH1^R132H cells cultured in different concentrations of AG-881 (green) or AG-120 (purple) were examined by LC-MS/MS to determine the IC50 values for each inhibitor’s activity. (B) Levels of AG-881 (black) and AG-120 (pink) detected in the brain of dTG mice bearing dTG-IDH1^R132H tumors. Beginning 7 days after the tumor inoculation, animals received oral administrations of vehicle or the corresponding inhibitor for 4 days. Samples were collected 1, 4, 12, and 24 hours after final drug administration and analyzed for the concentration of AG-881 and AG-120. (C) Both AG-881 and AG-120 suppressed intratumoral levels of 2-HG. Pharmacodynamics of AG-881 (black) and AG-120 (pink) in vivo were determined by analyzing the levels of 2-HG in samples from (B). (D, E) Tumor growth was significantly delayed by AG-881 treatment. Tumor-bearing mice were randomized to receive daily p.o. administration of AG-881 (n=5) or vehicle (n=4) beginning on day 7 post-tumor inoculation. (D) The tumor size, determined by BLI, and (E) weight of each mouse were measured every 3–4 days after treatment initiation and ended when control mice reached the protocol-specified endpoint due to tumor burden. BLI, bioluminescent imaging; dTG, double transgenic; HG, hydroxyglutarate.

Figure 3

Inhibition of 2-HG production by AG-881 does not alter the tumor-infiltrating myeloid cell population but results in increased recruitment of CD4⁺ T-cells. (A) Mice bearing dTG-IDH1^R132H tumors were treated daily with 10 mg/kg AG-881 or vehicle. Levels of 2-HG in the tumor-bearing hemisphere after 14 days of treatment were measured by LC-MS/MS. (B–D) A separate cohort of mice were treated as described in (A), and single-cell suspensions of the tumors were either analyzed by FC to determine the frequency of CD11b⁺ (B) cells among tumor-infiltrating leukocytes or used to isolate CD11b⁺ cells using a column separation protocol (C, D). 2-HG levels were significantly reduced in CD11b⁺ cells from AG-811-treated mice (C); however, their ability to suppress CD8⁺ ((D), left panel) and CD4⁺ ((D), right panel) T-cell proliferation ex vivo was unchanged. T-cell proliferation as a result of polyclonal CD3/CD28 activation was measured by dilution of CFSE 72 hours post-coculture initiation. (E) Single-cell suspensions generated in (B) were used to examine the frequency for CD8⁺ and CD4⁺ T-cells by FC. In 2-HG quantification and T-cell suppression studies n=3 mice per group. For FC analyses, n=4 in control group and n=5 in AG-881 groups. dTG, double transgenic; FC, flow cytometry; HG, hydroxyglutarate.

Figure 4

Enhanced survival outcomes, resulting from IDH1^R132H inhibition, depend on activity of both CD4⁺ and CD8⁺ T-cells. (A) Schematic description of the T-cell depletion treatment regimen. (B) Kaplan-Meier survival curve. *P<0.05, **p<0.002; log-rank test.

Figure 5

Prophylactic IDH1^R132H peptide vaccine extends survival only when combined with class I peptide vaccine and IDH1^R132H inhibition. (A) 9-week-old dTG mice received s.c. vaccinations with 100 µg of R132Hp emulsified in IFA and 20 µg intramuscular (i.m.) poly-ICLC on days 0 and 11. On day 21, mice were euthanized, and their spleens were harvested. IFNγ secretion by splenocytes stimulated ex vivo for 72 hours with peptide-pulsed syngeneic CD11b⁺ cells as indicated. Cells from three individual mice were plated in technical triplicates. Media only, no CD11b⁺ cells negative control. (B) Schematic representation of the treatment protocol for prophylactic vaccinations. 9–10 week-old dTG mice received s.c. vaccinations as described in (A) followed by an intracerebral challenge with dTG-IDH1^R132H cells. After 7 days, tumor-bearing mice were randomized to receive daily oral administrations of 10 mg/kg AG-881 or vehicle control for up to 35 consecutive days. Kaplan-Meier curves demonstrate survival outcomes: Sham, Vehicle (n=4); Sham, AG-881 (n=6); R132Hp, Vehicle (n=6), R132Hp, AG-881 (n=6). (C) Diagram representing the retroviral vector used to transduce the dTG-IDH1^R132H cells with HLA class I TAA epitopes derived from gp100, tyrosinase-related protein 2 (Trp2), and tyrosinase (Tyr). (D) Schematic representation of the treatment protocol. 7–10-week-old dTG mice received s.c. vaccinations with the class II R132Hp, peptides gp100_209-217, Trp2_180-188, Tyr_368-376 (class I vacc), or PBS (Sham Vacc) emulsified in IFA and 20 µg i.m. poly-ICLC. Vaccines were boosted 10 and 15 days later. And 21 days after initial vaccine administration, all mice received an intracerebral challenge with the dTG-IDH1^R132H; Class I TAA⁺ cells tumor cells (described in (C)). Seven days later, tumor-bearing mice started receiving daily oral administration of 10 mg/kg AG-881 or vehicle control for up to 35 days. Kaplan-Meier curves demonstrate survival outcomes: Sham, Vehicle (n=4); Class I Vacc, Vehicle (n=4); R132Hp, AG-−881 (n=5), Class I Vacc, AG881 (n=5), and Class I+R132 Hp, AG-881 (n=5). *P<0.05; long-rank test. dTG, double transgenic; HLA, human leukocyte antigens; IFA, incomplete Freund adjuvant; TAA, tumor-associated antigen.

Figure 6

Inhibition of IDH1^R132H function in vivo results in an enhanced IFNɣ signature response and therapeutic efficacy of PD-1 blockade. (A) Mice bearing dTG-IDH1^R132H tumors were treated with vehicle (n=3) or 10 mg/kg AG-881 (n=3) for 14 days. mRNA from tumor samples was collected and analyzed using the Mouse Immunology v2 panel (Nanostring) for significant changes in gene expression resulting from IDH1^R132H inhibition. (B) The top 5 (out of 136 gene sets) significantly upregulated pathways following AG-881 treatment. (C) CD45⁺ single-cell suspensions derived from the dTG-IDH1^R132H tumors of mice treated as in (A) were analyzed by FC for the expression of HLA-DR and PD-L1. *P<0.05, **p<0.002; Student t-test. (D) Schematic representation of the combined AG-881 and PD-1 blockade treatment protocol and Kaplan-Meier survival curves. Vehicle control and isotype antibody (Veh, IgG; n=8), vehicle and PD-1 blocking antibody (Veh, α-PD-1, n=9), AG-881 and isotype antibody (AG-881, IgG; n=9), AG-881 and PD1 blocking antibody (AG-881, α-PD-1, n=8). *p<0.05; long-rank test. dTG, double transgenic; HLA, human leukocyte antigens; NES, normalized enrichment score; padj, adjusted p values.