Challenges in the discovery of tumor-specific alternative splicing-derived cell-surface antigens in glioma.

Despite advancements in cancer immunotherapy, solid tumors remain formidable challenges. In glioma, profound inter- and intra-tumoral heterogeneity of antigen landscape hampers therapeutic development. Therefore, it is critical to consider alternative sources to expand the repertoire of targetable (neo-)antigens and improve therapeutic outcomes. Accumulating evidence suggests that tumor-specific alternative splicing (AS) could be an untapped reservoir of antigens. In this study, we investigated tumor-specific AS events in glioma, focusing on those predicted to generate major histocompatibility complex (MHC)-presentation-independent, cell-surface antigens that could be targeted by antibodies and chimeric antigen receptor-T cells. We systematically analyzed bulk RNA-sequencing datasets comparing 429 tumor samples (from The Cancer Genome Atlas) and 9166 normal tissue samples (from the Genotype-Tissue Expression project), and identified 13 AS events in 7 genes predicted to be expressed in more than 10% of the patients, including PTPRZ1 and BCAN, which were corroborated by an external RNA-sequencing dataset. Subsequently, we validated our predictions and elucidated the complexity of the isoforms using full-length transcript amplicon sequencing on patient-derived glioblastoma cells. However, analyses of the RNA-sequencing datasets of spatially mapped and longitudinally collected clinical tumor samples unveiled remarkable spatiotemporal heterogeneity of the candidate AS events. Furthermore, proteomics analysis did not reveal any peptide spectra matching the putative antigens. Our investigation illustrated the diverse characteristics of the tumor-specific AS events and the challenges of antigen exploration due to their notable spatiotemporal heterogeneity and elusive nature at the protein levels. Redirecting future efforts toward intracellular, MHC-presented antigens could offer a more viable avenue.

Immunotherapy Approaches in Isocitrate-Dehydrogenase-Mutant Low-Grade Glioma.

Low-grade gliomas (LGGs) are slow-growing tumors in the central nervous system (CNS). Patients characteristically show the onset of seizures or neurological deficits due to the predominant LGG location in high-functional brain areas. As a molecular hallmark, LGGs display mutations in the isocitrate dehydrogenase (IDH) enzymes, resulting in an altered cellular energy metabolism and the production of the oncometabolite D-2-hydroxyglutarate. Despite the remarkable progress in improving the extent of resection and adjuvant radiotherapy and chemotherapy, LGG remains incurable, and secondary malignant transformation is often observed. Therefore, novel therapeutic approaches are urgently needed. In recent years, immunotherapeutic strategies have led to tremendous success in various cancer types, but the effect of immunotherapy against glioma has been limited due to several challenges, such as tumor heterogeneity and the immunologically "cold" tumor microenvironment. Nevertheless, recent preclinical and clinical findings from immunotherapy trials are encouraging and offer a glimmer of hope for treating IDH-mutant LGG patients. Here, we aim to review the lessons learned from trials involving vaccines, T-cell therapies, and IDH-mutant inhibitors and discuss future approaches to enhance the efficacy of immunotherapies in IDH-mutant LGG.

Correction: Gallus et al. Immunotherapy Approaches in Isocitrate-Dehydrogenase-Mutant Low-Grade Glioma. Cancers 2023, 15, 3726.

It has come to our attention that the previously published manuscript contained an outdated iteration of Table 1 [...].

Glioma-neuronal circuit remodeling induces regional immunosuppression.

Neuronal activity-driven mechanisms impact glioblastoma cell proliferation and invasion 1-7 , and glioblastoma remodels neuronal circuits 8,9 . Distinct intratumoral regions maintain functional connectivity via a subpopulation of malignant cells that mediate tumor-intrinsic neuronal connectivity and synaptogenesis through their transcriptional programs 8 . However, the effects of tumor-intrinsic neuronal activity on other cells, such as immune cells, remain unknown. Here we show that regions within glioblastomas with elevated connectivity are characterized by regional immunosuppression. This was accompanied by different cell compositions and inflammatory status of tumor-associated macrophages (TAMs) in the tumor microenvironment. In preclinical intracerebral syngeneic glioblastoma models, CRISPR/Cas9 gene knockout of Thrombospondin-1 (TSP-1/ Thbs1 ), a synaptogenic factor critical for glioma-induced neuronal circuit remodeling, in glioblastoma cells suppressed synaptogenesis and glutamatergic neuronal hyperexcitability, while simultaneously restoring antigen-presentation and pro-inflammatory responses. Moreover, TSP-1 knockout prolonged survival of immunocompetent mice harboring intracerebral syngeneic glioblastoma, but not of immunocompromised mice, and promoted infiltrations of pro-inflammatory TAMs and CD8+ T-cells in the tumor microenvironment. Notably, pharmacological inhibition of glutamatergic excitatory signals redirected tumor-associated macrophages toward a less immunosuppressive phenotype, resulting in prolonged survival. Altogether, our results demonstrate previously unrecognized immunosuppression mechanisms resulting from glioma-neuronal circuit remodeling and suggest future strategies targeting glioma-neuron-immune crosstalk may open up new avenues for immunotherapy.

Challenges in the discovery of tumor-specific alternative splicing-derived cell-surface antigens in glioma.

Background: Despite advancements in cancer immunotherapy, solid tumors remain formidable challenges. In glioma, profound inter-and intra-tumoral heterogeneity of antigen landscape hampers therapeutic development. Therefore, it is critical to consider alternative sources to expand the repertoire of targetable (neo-)antigens and improve therapeutic outcomes. Accumulating evidence suggests that tumor-specific alternative splicing (AS) could be an untapped reservoir of neoantigens. Results: In this study, we investigated tumor-specific AS events in glioma, focusing on those predicted to generate major histocompatibility complex (MHC)-presentation-independent, cell-surface neoantigens that could be targeted by antibodies and chimeric antigen receptor (CAR)-T cells. We systematically analyzed bulk RNA-sequencing datasets comparing 429 tumor samples (from The Cancer Genome Atlas [TCGA]) and 9,166 normal tissue samples (from the Genotype-Tissue Expression project [GTEx]), and identified 13 AS events in 7 genes predicted to be expressed in more than 10% of the patients, including PTPRZ1 and BCAN , which were corroborated by an external RNA-sequencing dataset. Subsequently, we validated our predictions and elucidated the complexity of the isoforms using full-length transcript amplicon sequencing on patient-derived glioblastoma cells. However, analyses of the RNA-sequencing datasets of spatially mapped and longitudinally collected clinical tumor samples unveiled remarkable spatiotemporal heterogeneity of the candidate AS events. Furthermore, proteomics analysis did not reveal any peptide spectra matching the putative neoantigens. Conclusions: Our investigation illustrated the diverse characteristics of the tumor-specific AS events and the challenges of antigen exploration due to their notable spatiotemporal heterogeneity and elusive nature at the protein levels. Redirecting future efforts toward intracellular, MHC-presented antigens could offer a more viable avenue.

Tumor-wide RNA splicing aberrations generate immunogenic public neoantigens.

T-cell-mediated immunotherapies are limited by the extent to which cancer-specific antigens are homogenously expressed throughout a tumor. We reasoned that recurrent splicing aberrations in cancer represent a potential source of tumor-wide and public neoantigens, and to test this possibility, we developed a novel pipeline for identifying neojunctions expressed uniformly within a tumor across diverse cancer types. Our analyses revealed multiple neojunctions that recur across patients and either exhibited intratumor heterogeneity or, in some cases, were tumor-wide. We identified CD8+ T-cell clones specific for neoantigens derived from tumor-wide and conserved neojunctions in GNAS and RPL22 , respectively. TCR-engineered CD8 + T-cells targeting these mutations conferred neoantigen-specific tumor cell eradication. Furthermore, we revealed that cancer-specific dysregulation in splicing factor expression leads to recurrent neojunction expression. Together, these data reveal that a subset of neojunctions are both intratumorally conserved and public, providing the molecular basis for novel T-cell-based immunotherapies that address intratumoral heterogeneity.

Targeting the TP53/MDM2 axis enhances radiation sensitivity in atypical teratoid rhabdoid tumors.

Atypical teratoid rhabdoid tumor (ATRT) is a highly aggressive pediatric brain tumor. Despite radiation, aggressive chemotherapy and autologous stem cell rescue, children usually have a poor survival time. In the present study, the role of TP53/MDM2 interaction in ATRT was investigated. A functional genomic screen identified the TP53/MDM2 axis as a therapeutic target in the central nervous system (CNS) ATRT. Gene expression analysis revealed that all ATRT sub­groups expressed high levels of MDM2, which is a negative regulator of TP53. Using cell viability, colony formation and methylcellulose assays it was found that genetic MDM2 inhibition with short hairpin RNA or chemical MDM2 inhibition with small molecule inhibitors, Nutlin3 and idasanutlin (RG7388) decreased the growth of ATRT cell lines. Furthermore, idasanutlin significantly decreased the growth of intracranial orthotopic ATRT brain tumors, as evaluated using T2 MRI, and prolonged survival time relative to control animals. MRI of intracranial tumors showed that diffusion coefficient, an effective marker for successful treatment, significantly increased with idasanutlin treatment showing tumor necrosis/apoptosis. Immunohistochemistry revealed an increased number of caspase­3­positive cells in the idasanutlin treatment group, confirming the induction of apoptosis in vivo. Using flow cytometry and western blot analysis we show that inhibition of MDM2 enhanced radiation sensitivity in vitro by potentiating DNA damage via the induction of the TP53/Bax/Puma proapoptotic axis. Furthermore, DNA damage was associated with increased mitochondrial reactive oxygen species accumulation. The present study demonstrated that MDM2 expression level was increased in ATRT patient samples and MDM2 inhibition suppressed ATRT cell growth in vitro, and leads to apoptosis in vivo. MDM2 inhibition potentiates DNA damage and sensitizes ATRT cells to radiation. These findings highlight the TP53/MDM2 axis as a rational therapeutic target in CNS ATRT.

Venetoclax Cooperates with Ionizing Radiation to Attenuate Diffuse Midline Glioma Tumor Growth.

PURPOSE: Tumor relapse after radiotherapy is a major hurdle in treating pediatric H3K27M-mutant diffuse midline gliomas (DMG). Radiotherapy-induced stress increases association of BCL2 family of proteins with BH3 pro-apoptotic activators preventing apoptosis. We hypothesized that inhibition of radiotherapy-induced BCL2 with a clinically relevant inhibitor, venetoclax, will block BCL2 activity leading to increased apoptosis. BCL2 has never been implicated in DMG as a radiotherapy-induced resistant mechanism. EXPERIMENTAL DESIGN: We performed an integrated genomic analysis to determine genes responsible for radioresistance and a targeted drug screen to identify drugs that synergize with radiation in DMG. Effect of venetoclax on radiation-naïve and 6 Gy radiation on cells was evaluated by studying cell death, changes in BCL2 phosphorylation, reactive oxygen species (ROS), and apoptosis, as well as BCL2 association with BH3 apoptosis initiators. The efficacy of combining venetoclax with radiation was evaluated in vivo using orthotopic xenograft models. RESULTS: BCL2 was identified as a key regulator of tumor growth after radiation in DMGs. Radiation sensitizes DMGs to venetoclax treatment independent of p53 status. Venetoclax as a monotherapy was not cytotoxic to DMG cells. Postradiation venetoclax treatment significantly increased cell death, reduced BCL2-BIM association, and augmented mitochondrial ROS leading to increased apoptosis. Combining venetoclax with radiotherapy significantly enhanced the survival of mice with DMG tumors. CONCLUSIONS: This study shows that venetoclax impedes the antiapoptotic function of radiation-induced BCL2 in DMG, leading to increased apoptosis. Results from these preclinical studies demonstrate the potential use of the BCL2 inhibitor venetoclax combined with radiotherapy for pediatric DMG.

New Insights into the Multifaceted Role of Myeloid-Derived Suppressor Cells (MDSCs) in High-Grade Gliomas: From Metabolic Reprograming, Immunosuppression, and Therapeutic Resistance to Current Strategies for Targeting MDSCs.

Cancer cells "hijack" host immune cells to promote growth, survival, and metastasis. The immune microenvironment of high-grade gliomas (HGG) is a complex and heterogeneous system, consisting of diverse cell types such as microglia, bone marrow-derived macrophages (BMDMs), myeloid-derived suppressor cells (MDSCs), dendritic cells, natural killer (NK) cells, and T-cells. Of these, MDSCs are one of the major tumor-infiltrating immune cells and are correlated not only with overall worse prognosis but also poor clinical outcomes. Upon entry from the bone marrow into the peripheral blood, spleen, as well as in tumor microenvironment (TME) in HGG patients, MDSCs deploy an array of mechanisms to perform their immune and non-immune suppressive functions. Here, we highlight the origin, function, and characterization of MDSCs and how they are recruited and metabolically reprogrammed in HGG. Furthermore, we discuss the mechanisms by which MDSCs contribute to immunosuppression and resistance to current therapies. Finally, we conclude by summarizing the emerging approaches for targeting MDSCs alone as a monotherapy or in combination with other standard-of-care therapies to improve the current treatment of high-grade glioma patients.

Loss of TP63 Promotes the Metastasis of Head and Neck Squamous Cell Carcinoma by Activating MAPK and STAT3 Signaling.

TP63 is frequently amplified or overexpressed in primary head and neck squamous cell carcinomas (HNSCC). Nevertheless, the role of TP63 in the initiation and progression of HNSCCs is not known. Using archival HNSCC tissue sections, we found that TP63 expression is often downregulated in late-stage human HNSCCs. To establish a causal link between TP63 loss and HNSCC tumorigenesis, we developed a genetically engineered mouse model in which Trp63 (the mouse homolog of human TP63) was ablated from head and neck epithelia. Upon exposure of the mice to a chemical carcinogen, we found that Trp63 ablation accelerated HNSCC initiation and progression. To determine whether these findings are relevant for human HNSCCs, we generated TP63 knockdown HNSCC cell lines. These cells were implanted into the tongue of athymic nude mice to generate orthotopic xenografts. We found that loss of TP63 promoted HNSCC progression and metastasis. Furthermore, we determined that tumor metastasis is dependent on MAPK activation in TP63 knockdown HNSCCs. The significance of these findings is underscored by our finding that pharmacologic inhibition of MAPK activity by trametinib drastically impaired HNSCC metastasis mediated by TP63 loss. In conclusion, our data provide novel mechanistic insights into the role of TP63 loss in HNSCC initiation and progression, and provide a rationale for the development of new therapeutic approaches specifically targeting TP63-dependent tumor pathways. IMPLICATIONS: Our findings uncover a novel functional role for TP63 loss in HNSCC metastasis and identify MAPK signaling as a potential therapeutic target for treating HNSCCs with low TP63 expression.