Protocol to assess the antitumor efficacy of an immunotherapeutic peptide in syngeneic orthotopic glioma mouse models.

Understanding the glioblastoma (GBM) immune microenvironment and development of clinical treatment drugs rely on suitable preclinical GBM models. Here, we present a protocol to establish syngeneic orthotopic glioma mouse models. We also describe the steps to intracranially deliver immunotherapeutic peptides and monitor the treatment response. Finally, we show how to assess the tumor immune microenvironment with treatment outcomes. For complete details on the use and execution of this protocol, please refer to Chen et al. (2021).1.

Loss of MAT2A compromises methionine metabolism and represents a vulnerability in H3K27M mutant glioma by modulating the epigenome.

Diffuse midline gliomas (DMGs) bearing driver mutations of histone 3 lysine 27 (H3K27M) are incurable brain tumors with unique epigenomes. Here, we generated a syngeneic H3K27M mouse model to study the amino acid metabolic dependencies of these tumors. H3K27M mutant cells were highly dependent on methionine. Interrogating the methionine cycle dependency through a short-interfering RNA screen identified the enzyme methionine adenosyltransferase 2A (MAT2A) as a critical vulnerability in these tumors. This vulnerability was not mediated through the canonical mechanism of MTAP deletion; instead, DMG cells have lower levels of MAT2A protein, which is mediated by negative feedback induced by the metabolite decarboxylated S-adenosyl methionine. Depletion of residual MAT2A induces global depletion of H3K36me3, a chromatin mark of transcriptional elongation perturbing oncogenic and developmental transcriptional programs. Moreover, methionine-restricted diets extended survival in multiple models of DMG in vivo. Collectively, our results suggest that MAT2A presents an exploitable therapeutic vulnerability in H3K27M gliomas.

Novel theranostic agent for PET imaging and targeted radiopharmaceutical therapy of tumour-infiltrating immune cells in glioma.

BACKGROUND: Malignant gliomas are deadly tumours with few therapeutic options. Although immunotherapy may be a promising therapeutic strategy for treating gliomas, a significant barrier is the CD11b+ tumour-associated myeloid cells (TAMCs), a heterogeneous glioma infiltrate comprising up to 40% of a glioma's cellular mass that inhibits anti-tumour T-cell function and promotes tumour progression. A theranostic approach uses a single molecule for targeted radiopharmaceutical therapy (TRT) and diagnostic imaging; however, there are few reports of theranostics targeting the tumour microenvironment. METHODS: Utilizing a newly developed bifunctional chelator, Lumi804, an anti-CD11b antibody (αCD11b) was readily labelled with either Zr-89 or Lu-177, yielding functional radiolabelled conjugates for PET, SPECT, and TRT. FINDINGS: 89Zr/177Lu-labeled Lumi804-αCD11b enabled non-invasive imaging of TAMCs in murine gliomas. Additionally, 177Lu-Lumi804-αCD11b treatment reduced TAMC populations in the spleen and tumour and improved the efficacy of checkpoint immunotherapy. INTERPRETATION: 89Zr- and 177Lu-labeled Lumi804-αCD11b may be a promising theranostic pair for monitoring and reducing TAMCs in gliomas to improve immunotherapy responses. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

TIGIT and PD-1 Immune Checkpoint Pathways Are Associated With Patient Outcome and Anti-Tumor Immunity in Glioblastoma.

Glioblastoma (GBM) remains an aggressive brain tumor with a high rate of mortality. Immune checkpoint (IC) molecules are expressed on tumor infiltrating lymphocytes (TILs) and promote T cell exhaustion upon binding to IC ligands expressed by the tumor cells. Interfering with IC pathways with immunotherapy has promoted reactivation of anti-tumor immunity and led to success in several malignancies. However, IC inhibitors have achieved limited success in GBM patients, suggesting that other checkpoint molecules may be involved with suppressing TIL responses. Numerous IC pathways have been described, with current testing of inhibitors underway in multiple clinical trials. Identification of the most promising checkpoint pathways may be useful to guide the future trials for GBM. Here, we analyzed the The Cancer Genome Atlas (TCGA) transcriptomic database and identified PD1 and TIGIT as top putative targets for GBM immunotherapy. Additionally, dual blockade of PD1 and TIGIT improved survival and augmented CD8+ TIL accumulation and functions in a murine GBM model compared with either single agent alone. Furthermore, we demonstrated that this combination immunotherapy affected granulocytic/polymorphonuclear (PMN) myeloid derived suppressor cells (MDSCs) but not monocytic (Mo) MDSCs in in our murine gliomas. Importantly, we showed that suppressive myeloid cells express PD1, PD-L1, and TIGIT-ligands in human GBM tissue, and demonstrated that antigen specific T cell proliferation that is inhibited by immunosuppressive myeloid cells can be restored by TIGIT/PD1 blockade. Our data provide new insights into mechanisms of GBM αPD1/αTIGIT immunotherapy.

A multi-herb-combined remedy to overcome hyper-inflammatory response by reprogramming transcription factor profile and shaping monocyte subsets.

Traditional Chinese multi-herb-combined prescriptions usually show better performance than a single agent since a group of effective compounds interfere multiple disease-relevant targets simultaneously. Huang-Lian-Jie-Du decoction is a remedy made of four herbs that are widely used to treat oral ulcers, gingivitis, and periodontitis. However, the active ingredients and underlying mechanisms are not clear. To address these questions, we prepared a water extract solution of Huang-Lian-Jie-Du decoction (HLJDD), called it as WEH (Water Extract Solution of HLJDD), and used it to treat LPS-induced systemic inflammation in mice. We observed that WEH attenuated inflammatory responses including reducing production of cytokines, chemokines and interferons (IFNs), further attenuating emergency myelopoiesis, and preventing mice septic lethality. Upon LPS stimulation, mice pretreated with WEH increased circulating Ly6C- patrolling and splenic Ly6C+ inflammatory monocytes. The acute myelopoiesis related transcriptional factor profile was rearranged by WEH. Mechanistically we confirmed that WEH interrupted LPS/TLR4/CD14 signaling-mediated downstream signaling pathways through its nine principal ingredients, which blocked LPS stimulated divergent signaling cascades, such as activation of NF-κB, p38 MAPK, and ERK1/2. We conclude that the old remedy blunts LPS-induced "danger" signal recognition and transduction process at multiple sites. To translate our findings into clinical applications, we refined the crude extract into a pure multicomponent drug by directly mixing these nine chemical entities, which completely reproduced the effect of protecting mice from lethal septic shock. Finally, we reduced a large number of compounds within a multi-herb water extract to seven-chemical combination that exhibited superior therapeutic efficacy compared with WEH.

Identification of Novel RAS Signaling Therapeutic Vulnerabilities in Diffuse Intrinsic Pontine Gliomas.

Diffuse intrinsic pontine gliomas (DIPG) are incurable brain tumors with an aggressive onset. Apart from irradiation, there are currently no effective therapies available for patients with DIPG, who have a median survival time of less than one year. Most DIPG cells harbor mutations in genes encoding histone H3 (H3K27M) proteins, resulting in a global reduction of H3K27 trimethylation and activation of oncogenic signaling pathways. Here we show that the H3K27M mutations contribute to RAS pathway signaling, which is augmented by additional RAS activators including PDGFRA. H3K27M mutation led to increased expression of receptor tyrosine kinases (RTK). A RAS pathway functional screen identified ERK5, but not ERK1/2, as a RAS pathway effector important for DIPG growth. Suppression of ERK5 decreased DIPG cell proliferation and induced apoptosis in vitro and in vivo. In addition, depletion or inhibition of ERK5 significantly increased survival of mice intracranially engrafted with DIPG cells. Mechanistically, ERK5 directly stabilized the proto-oncogene MYC at the protein level. Collectively, our data demonstrate an underappreciated role of H3K27M in RAS activation and reveal novel therapeutic targets for treating DIPG tumors. SIGNIFICANCE: These findings identify the H3K27M mutation as an enhancer of RAS activation in DIPG and ERK5 as a novel, immediately actionable molecular target. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/16/4026/F1.large.jpg.