Dual IGF1R/IR inhibitors in combination with GD2-CAR T-cells display a potent anti-tumor activity in diffuse midline glioma H3K27M-mutant

Abstract

Background Diffuse midline gliomas (DMG) H3K27M-mutant, including diffuse intrinsic pontine glioma (DIPG), are pediatric brain tumors associated with grim prognosis. Although GD2-CAR T-cells demonstrated significant anti-tumor activity against DMG H3K27M-mutant in vivo, a multimodal approach may be needed to more effectively treat patients. We investigated GD2 expression in DMG/DIPG and other pediatric high-grade gliomas (pHGG) and sought to identify chemical compounds that would enhance GD2-CAR T-cell anti-tumor efficacy. Methods Immunohistochemistry in tumor tissue samples and immunofluorescence in primary patient-derived cell lines were performed to study GD2 expression. We developed a high-throughput cell-based assay to screen 42 kinase inhibitors in combination with GD2-CAR T-cells. Cell viability, western blots, flow-cytometry, real time PCR experiments, DIPG 3D culture models and orthotopic xenograft model were applied to investigate the effect of selected compounds on DIPG cell death and CAR T-cell function. Results GD2 was heterogeneously, but widely, expressed in the tissue tested, while its expression was homogeneous and restricted to DMG/DIPG H3K27M-mutant cell lines. We identified dual IGF1R/IR antagonists, BMS-754807 and linsitinib, able to inhibit tumor cell viability at concentrations that do not affect CAR T-cells. Linsitinib, but not BMS-754807, decreases activation/exhaustion of GD2-CAR T-cells and increases their central memory profile. The enhanced anti-tumor activity of linsitinib/GD2-CAR T-cell combination was confirmed in DIPG models in vitro, ex vivo and in vivo. Conclusion Our study supports the development of IGF1R/IR inhibitors to be used in combination with GD2-CAR T-cells for treating patients affected by DMG/DIPG and, potentially, by pHGG.

Generation of immunocompetent syngeneic allograft mouse models for pediatric diffuse midline glioma

“…To verify that these immunocompetent DMG mouse models can be employed for the development of immunotherapies, we analyzed the expression of the disialoganglioside GD2 and Interleukin-13 receptor subunit alpha-2 (IL13Rα2), two antigens highly expressed in DMG tissues and identified as promising targets for immunotherapy in DMG. 28 , 31–34 For Il13rα2, we could confirm expression in both histone 3 wildtype and K27M-mutant DMG tissues, although a slightly lower and more heterogenous expression was observed for histone 3 wildtype tumors ( Supplementary Figure 9A ). For GD2, flow cytometry analysis indicated that this antigen is also expressed in our murine DMG cells, irrespective of their histone mutational status ( Supplementary Figure 9B ).…”

“…GD2 immunophenotyping was performed by incubating cells for 30 min at 4°C with PE-conjugated anti-GD2 antibody (clone 14.G2a) (1:100; #562100, BD Biosciences) in staining buffer (0.2% BSA in PBS) in the dark, as described previously. 28 Flow cytometric analysis was performed on a CytoFLEX S Flow Cytometer (Beckman Coulter).…”

Receptor tyrosine kinase (RTK) targeting in pediatric high-grade glioma and diffuse midline glioma: Pre-clinical models and precision medicine

“…de Billy et al. combined IGF1R/IR inhibitors with GD2-CAR-T cells and saw increased antitumor activity from the combination in DMG cell lines ( 126 ). Exploring the combination of RTK therapy with MDSC inhibition, immune checkpoint inhibition, or CAR-T cell therapy could effectively increase the activation of CD8+ T-cells and change the TME to favor anti-tumor responses.…”

Ferroptosis-related gene signatures in neuroblastoma associated with prognosis

“…PROM2 is sensitive to insulin-like growth factor I receptor inhibitor linsitinib ( Fernando et al, 2021 ), insulin-like growth factor 1 inhibitor GSK1904529A ( Zeng et al, 2021 ), the estrogen receptors alpha antagonist AZD9496 ( Cani et al, 2021 ), and the dual-acting estrogen action inhibitor SR16157 ( Rausch et al, 2011 ). Among them, linsitinib has been reported to have potent antitumor activity in diffuse midline glioma when combined with modified chimeric antigen receptor T-cells ( de Billy et al, 2021 ); GSK1904529A has also been found to inhibit glioma tumor growth, induce apoptosis, and inhibit migration ( Zhou et al, 2015 ), and AZD9496; and SR16157 are potential drugs for treating breast cancer that are present in the early stages of clinical research ( Zhang et al, 2021 ) ( Rausch et al, 2011 ). GCH1 is sensitive to ribavirin, a synthetic nucleoside analogue with broad antiviral activity ( Jurković et al, 2021 ).…”