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Amyotrophic lateral sclerosis (ALS) is a heterogenous neurodegenerative disorder that affects motor neurons and voluntary muscle control1. ALS heterogeneity includes the age of manifestation, the rate of progression and the anatomical sites of symptom onset. Disease-causing mutations in specific genes have been identified and define different subtypes of ALS1. Although several ALS-associated genes have been shown to affect immune functions2, whether specific immune features account for ALS heterogeneity is poorly understood. Amyotrophic lateral sclerosis-4 (ALS4) is characterized by juvenile onset and slow progression3. Patients with ALS4 show motor difficulties by the time that they are in their thirties, and most of them require devices to assist with walking by their fifties. ALS4 is caused by mutations in the senataxin gene (SETX). Here, using Setx knock-in mice that carry the ALS4-causative L389S mutation, we describe an immunological signature that consists of clonally expanded, terminally differentiated effector memory (TEMRA) CD8 T cells in the central nervous system and the blood of knock-in mice. Increased frequencies of antigen-specific CD8 T cells in knock-in mice mirror the progression of motor neuron disease and correlate with anti-glioma immunity. Furthermore, bone marrow transplantation experiments indicate that the immune system has a key role in ALS4 neurodegeneration. In patients with ALS4, clonally expanded TEMRA CD8 T cells circulate in the peripheral blood. Our results provide evidence of an antigen-specific CD8 T cell response in ALS4, which could be used to unravel disease mechanisms and as a potential biomarker of disease state.

Supplementary Figures, legends and References

Supplementary Table S1. GBM-sLCR features

Glioblastoma (GBM) is the most frequent and aggressive primary tumor type in the central nervous system in adults. Resistance to chemotherapy remains one of the major obstacles in GBM treatment. Identifying and overcoming the mechanisms of therapy resistance is instrumental to develop novel therapeutic approaches for patients with GBM. To determine the major drivers of temozolomide (TMZ) sensitivity, we performed shRNA screenings in GBM lines with different O6-methylguanine-DNA methyl-transferase (MGMT) status. We then evaluated dianhydrogalactitol (Val-083), a small alkylating molecule that induces interstrand DNA crosslinking, as a potential treatment to bypass TMZ-resistance mechanisms. We found that loss of mismatch repair (MMR) components and MGMT expression are mutually exclusive mechanisms driving TMZ resistance in vitro. Treatment of established GBM cells and tumorsphere lines with Val-083 induces DNA damage and cell-cycle arrest in G2–M phase, independently of MGMT or MMR status, thus circumventing conventional resistance mechanisms to TMZ. Combination of TMZ and Val-083 shows a synergic cytotoxic effect in tumor cells in vitro, ex vivo, and in vivo. We propose this combinatorial treatment as a potential approach for patients with GBM.

Screening results U251 TMZ

Combined supplementary Figures

Glioblastoma is a lethal brain tumor that exhibits heterogeneity and resistance to therapy. Our understanding of tumor homeostasis is limited by a lack of genetic tools to selectively identify tumor states and fate transitions. Here, we use glioblastoma subtype signatures to construct synthetic genetic tracing cassettes and investigate tumor heterogeneity at cellular and molecular levels, in vitro and in vivo. Through synthetic locus control regions, we demonstrate that proneural glioblastoma is a hardwired identity, whereas mesenchymal glioblastoma is an adaptive and metastable cell state driven by proinflammatory and differentiation cues and DNA damage, but not hypoxia. Importantly, we discovered that innate immune cells divert glioblastoma cells to a proneural-to-mesenchymal transition that confers therapeutic resistance. Our synthetic genetic tracing methodology is simple, scalable, and widely applicable to study homeostasis in development and diseases. In glioblastoma, the method causally links distinct (micro)environmental, genetic, and pharmacologic perturbations and mesenchymal commitment.Significance:Glioblastoma is heterogeneous and incurable. Here, we designed synthetic reporters to reflect the transcriptional output of tumor cell states and signaling pathways' activity. This method is generally applicable to study homeostasis in normal tissues and diseases. In glioblastoma, synthetic genetic tracing causally connects cellular and molecular heterogeneity to therapeutic responses.This article is highlighted in the In This Issue feature, p. 521

Supplementary Tables

Supplementary Figures 1-6

The tumor microenvironment plays an essential role in supporting glioma stemness and radioresistance. Following radiotherapy, recurrent gliomas form in an irradiated microenvironment. Here we report that astrocytes, when pre-irradiated, increase stemness and survival of cocultured glioma cells. Tumor-naïve brains increased reactive astrocytes in response to radiation, and mice subjected to radiation prior to implantation of glioma cells developed more aggressive tumors. Extracellular matrix derived from irradiated astrocytes were found to be a major driver of this phenotype and astrocyte-derived transglutaminase 2 (TGM2) was identified as a promoter of glioma stemness and radioresistance. TGM2 levels increased after radiation in vivo and in recurrent human glioma, and TGM2 inhibitors abrogated glioma stemness and survival. These data suggest that irradiation of the brain results in the formation of a tumor-supportive microenvironment. Therapeutic targeting of radiation-induced, astrocyte-derived extracellular matrix proteins may enhance the efficacy of standard-of-care radiotherapy by reducing stemness in glioma.Significance:These findings presented here indicate that radiotherapy can result in a tumor-supportive microenvironment, the targeting of which may be necessary to overcome tumor cell therapeutic resistance and recurrence.

Extended experimental details to supplement the main article file methods section

XLS file - 102K, TCGA data for TP53BP1 in the GBM dataset

PDF file - 132K

PDF file - 130K, Cell cycle alteration in 53BP1 silenced cells, upon exposure to IR

PDF file - 310K, 53BP1 null mouse cells, but not heterozygous, and robust silencing in GBM cells lead to high sensitivity to IR

XLS file - 66K, Co-occurrence of TP53BP1 and TP53 alterations in the TCGA dataset for GBM

PDF file - 205, 53BP1 modulates NHEJ in glioma cell lines

PDF file - 22K, 53BP1 silencing does not increase TMZ sensitivity in T98G cells

PDF file, 906K, Supplementary Materials and Methods Flow cytometry. Colony formation assay. Supplementary Tables Supplementary Table 1. siRNAs and shRNAs constructs used in this study. Supplementary Table 2. Expression primers and ChIP primers used in this study. Supplementary Table 3. Antibodies used in this study. Supplementary Figures Supplementary Figure S1. Analysis of siRNA screening for acetyltransferases. Supplementary Figure S2. Validation of candidate genes from the siRNA screening. Supplementary Figure S3. Stable depletion of Pcaf does not affect DNA synthesis in NIH3T3 cells. Supplementary Figure S4. Inhibition of PCAF expression affects neurosphere formation and proliferation of GBM543 cells. Supplementary Figure S5. Depletion of PCAF and inhibitors to PCAF induce apoptosis. Supplementary Figure S6. Inhibition of Pcaf expression affects proliferation of tNSCs.

BACKGROUND Currently approved Poly ADP-Ribose Polymerase (PARP) inhibitors (PARPi) selectively inhibit and trap both PARP1 and PARP2 (PARP1/2) at sites of single strand (ss) deoxyribonucleic acid (DNA) (ssDNA) damage, preventing ssDNA repair and leading to replication-dependent DNA double strand breaks. Recent data suggest that only inhibition of PARP1 is required for anti-proliferative effects, while PARP2 functions in the survival of haematopoietic stem and progenitor cells. These observations suggest that the inhibition and trapping of PARP 2 is not needed for anti-cancer effects, and may be a major driver of haematological toxicity observed. AZD9574 is a novel brain-penetrant PARPi that potently and selectively inhibits and traps PARP1, with the goal of delivering efficacious, less toxic, and more combinable PARPi. Furthermore, owing to its central nervous system penetration capability, AZD9574 may provide a new treatment option for patients with CNS malignancies or patients with brain metastases characterized by homologous recombination deficiency (HRD). METHODS This is a first-in-human modular study primarily designed to evaluate the safety and tolerability of AZD9574 as monotherapy and in combination with anti-cancer agents at increasing dose levels in patients with advanced solid malignancies, followed by expansion cohorts in specific indications. The study will also characterize the pharmacokinetics of AZD9574 and explore potential biological activity by assessing pharmacodynamic and exploratory biomarkers and anti-tumour activity. Module 1 will enrol patients with advanced breast, ovarian, pancreatic or prostate tumours harbouring homologous recombination deficiencies. Module 2 will enrol patients with isocitrate dehydrogenase (IDH)1/2 mutated glioma.

Immunotherapy is a promising treatment modality for highly invasive gliomas; however, clinical trials thus far have failed to provide significant clinical benefit to most GBM patients. GBM is one of the “immune cold” tumors characterized by poor infiltration of T cells even though greater than 40% of glioma cells are composed of immune cells in some patients. The majority of immune cells in GBM are immune-suppressive myeloid cells that block T cell infiltration and/or activation. To elucidate the mechanisms of immune evasion and to understand how the immune system interacts with glioma and stromal cells that shape the immune-suppressive landscape in GBM, there is an urgent need for immune-competent preclinical models that recapitulate the human disease. Human GBM is divided into three molecular subtypes (proneural:PR, classical:CL, and mesenchymal:MES) based on specific gene expression patterns and signature mutational profiles. Here, we report multi-dimensional analyses of six different transplantable mouse glioma models in the C57BL6/J background that represent all three human GBM molecular subtypes. We performed whole-exome sequencing as well as STR fingerprinting of each primary tumorsphere line and also performed immune phenotyping of each glioma model with flow cytometry. To gain molecular insights and determine cellular heterogeneity, we also performed single-cell RNA sequencing from the six models. Glioma cell analysis at the single-cell level revealed that cell-of-origin rather than the oncogenic driver (such as EGFRviii) plays a dominant role in determining the molecular phenotypes of glioma cells, driving their classification into a molecular subtype defined by human studies. In addition, we identified eight molecular subtypes of glioma-associated myeloid (GAMs), seven different subtypes of T cells, and provide molecular definitions of glioma-associated pericytes and endothelial cells in murine gliomas. In addition, we performed cross-species comparisons of glioma and immune cell subtypes between humans and mice at the single-cell level. Furthermore, we report qualitative and quantitative differences in the cell-to-cell communication among different stromal cells and glioma cells in each model, and propose that these interactions shape the local niche and functional neighborhoods. Finally, we leverage these preclinical models to elucidate underlying molecular mechanisms that drive differential sensitivities of each model to immunotherapies: anti-PD1, CTLA4, and 4-1BB in vivo. In summary, we report deeply characterized mouse models of human GBM subtypes and highlight their utility as preclinical models for immunotherapy evaluation and foundational tumor immunology studies. Citation Format: Caiyi Wang, Jose Maldonado, Thomas D. Gallup, Nourhan Abdelfattah, Jia-Shiun Leu, Nithin Joshy, joshy George, Jihye Paik, Massimo Squatrito, Kyuson Yun. Elucidating cell-to-cell communication and immunotherapy responses in deeply characterized mouse models of human glioma subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1331.