Local Targeting of NAD+ Salvage Pathway Alters the Immune Tumor Microenvironment and Enhances Checkpoint Immunotherapy in Glioblastoma

Abstract

The aggressive primary brain tumor glioblastoma (GBM) is characterized by aberrant metabolism that fuels its malignant phenotype. Diverse genetic subtypes of malignant glioma are sensitive to selective inhibition of the NAD⁺ salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT). However, the potential impact of NAD⁺ depletion on the brain tumor microenvironment has not been elaborated. In addition, systemic toxicity of NAMPT inhibition remains a significant concern. Here we show that microparticle-mediated intratumoral delivery of NAMPT inhibitor GMX1778 induces specific immunologic changes in the tumor microenvironment of murine GBM, characterized by upregulation of immune checkpoint PD-L1, recruitment of CD3⁺, CD4⁺, and CD8⁺ T cells, and reduction of M2-polarized immunosuppressive macrophages. NAD⁺ depletion and autophagy induced by NAMPT inhibitors mediated the upregulation of PD-L1 transcripts and cell surface protein levels in GBM cells. NAMPT inhibitor modulation of the tumor immune microenvironment was therefore combined with PD-1 checkpoint blockade in vivo, significantly increasing the survival of GBM-bearing animals. Thus, the therapeutic impacts of NAMPT inhibition extended beyond neoplastic cells, shaping surrounding immune effectors. Microparticle delivery and release of NAMPT inhibitor at the tumor site offers a safe and robust means to alter an immune tumor microenvironment that could potentiate checkpoint immunotherapy for glioblastoma. SIGNIFICANCE: Microparticle-mediated local inhibition of NAMPT modulates the tumor immune microenvironment and acts cooperatively with anti-PD-1 checkpoint blockade, offering a combination immunotherapy strategy for the treatment of GBM.

Figure 1.. NAMPT inhibitor-induced NAD+ depletion upregulates PD-L1 on mouse and human glioblastoma cells.

A, Left, NAD+ assay showing treatment with FK866 and GMX1778 for 72 h induced a decrease in NAD+ levels in GL261 cells. Middle and right, Flow cytometry analysis showing dose dependent upregulation of PD-L1 levels in NAMPT inhibitor-treated GL261 cells after 72 h. *p＜0.05, ***p＜0.01 for the difference between drug-treated and untreated cells. B, Nicotinamide mononucleotide (NMN, 1 mM) reverses NAD+ levels (Left) and PD-L1 levels (Middle and right) in GL261 cells treated with NAMPT inhibitors for 72 h. FK-866, 100 nM; GMX1778, 1000 nM. ***p＜0.01 for the difference between indicated two groups. C, Left, quantitative RT-PCR showing upregulation of PD-L1 (Cd274) mRNA by FK866 (upper) and GMX1778 (lower) in a time dependent manner. Middle and right, Flow cytometry confirms corresponding PD-L1 upregulation. D and E, Dose-dependent upregulation of cell surface PD-L1 after 72-h exposure to NAMPT inhibitors FK866 (upper panels) and GMX1778 (lower panels) in MGG23 GBM (IDH wild-type) cells (D) and MGG119 GBM (IDH1-mutant) cells (E). *p＜0.05, ***p＜0.01 for the difference between drug-treated and untreated cells, and between two groups indicated. Error bars, SEM. Drug doses include IC₁₀ (10% inhibitory concentration), IC₅₀ and IC₈₀ or IC₉₀.

Figure 2.. Local treatment with GMX1778 microparticles upregulates PD-L1 in glioblastoma in vivo.

A, Three ul microliter of coumarin-6/GMX1778 loaded microparticles (MP) (Upper left, In vitro) were injected into intracranial GL261 tumors, and distribution of MP was observed under fluorescence microscopy (Upper right, In vivo). Lower panels, immunofluorescence for glioblastoma (GBM) marker nestin (red) revealed MP spread within the GL261 brain tumor. B, Mice bearing GL261 GBM were treated with intratumoral injections of PBS, blank MP or GMX1778 MP on day 16, and the brains were collected on day 20 (N=3/group). C, Representative microscopic pictures of immunohistochemistry for PD-L1, and quantification on the right. D and E, double immunofluorescence for PD-L1 (green) and Nestin (red) (D) and PD-L1 (green) and Arg1 (red) (E). Representative picture from each treatment group is presented. Number of double positive cells for each treatment group is plotted on the right (D, E). *, P<0.05.

Figure 3.. Local treatment with GMX1778 microparticles activates immune microenvironment in glioblastoma.

Immunofluorescence for CD3 (A), CD4 (B), Foxp3 (C), CD8 (D), CD68 (E), iNOS (F), and Arg1 (G) in orthotopic GL261 glioblastoma treated with PBS, blank microparticles (MP) and GMX1778 MP. Brains were removed 4 days after treatment of 16-day-old tumors. *, P<0.05; ***, P<0.01.

Figure 4.. NAMPT inhibitor is cytotoxic to glioblastoma-associated macrophages.

Freshly isolated cells from GL261 glioblastoma tissue were subjected to culture and untreated or treated with NAMPT inhibitors in vitro (FK866, 100 nM; GMX1778, 1000 nM). Cells were stained for nestin (A), Arg1 (B) and CD11b (C) at 24, 48 and 72 hours after treatment. Positive cells were quantified. Error bars, SEM. *, P<0.05. Here representative microscopic pictures on Day 3 (72 h) are shown. See Supplementary Fig. S7 for staining on Day 0, 1 and 2. *, P<0.005 (Control versus FK866 and Control versus GMX1778).

Figure 5.. Combination therapy of GMX1778 microparticles and systemic anti-PD-1 enhances efficacy.

A, Experimental protocol for survival analysis. Microparticles, MP; anti-PD-1, aPD1. B, Kaplan-Meier survival plots of mice bearing orthotopic GL261 tumors treated with or without GMX1778 microparticles (intratumoral) and anti-PD-1 antibody (systemic). C, Changes in body weight of animals in the survival study. D, Experimental protocol for immune profiling analysis. E-K, Immunohistochemical (for PD-L1) or immunofluorescence (all others) analysis of a panel of immune markers in GL261 glioblastoma treated with or without GMX1778 microparticles (intratumoral) and anti-PD-1 antibody (systemic). E, PD-L1. F, CD3. G, CD4. H, CD8. I, CD68. J, iNOS. K, Arg1. Blank, Blank microparticles; GMX1778, GMX1778 microparticles; aPD-1, anti-PD-1. *, P<0.05. See data of PD-L1 immunohistochemistry in Supplemental Fig. S8 and immunofluorescence in Supplemental Fig. S9.