Search

ATRX is one of the most frequently altered genes in solid tumors, and mutation is especially frequent in soft tissue sarcomas. However, the role of ATRX in tumor development and response to cancer therapies remains poorly understood. Here, we developed a primary mouse model of soft tissue sarcoma and showed that Atrx-deleted tumors were more sensitive to radiation therapy and to oncolytic herpesvirus. In the absence of Atrx, irradiated sarcomas had increased persistent DNA damage, telomere dysfunction, and mitotic catastrophe. Our work also showed that Atrx deletion resulted in downregulation of the CGAS/STING signaling pathway at multiple points in the pathway and was not driven by mutations or transcriptional downregulation of the CGAS/STING pathway components. We found that both human and mouse models of Atrx-deleted sarcoma had a reduced adaptive immune response, markedly impaired CGAS/STING signaling, and increased sensitivity to TVEC, an oncolytic herpesvirus that is currently FDA approved for the treatment of aggressive melanomas. Translation of these results to patients with ATRX-mutant cancers could enable genomically guided cancer therapy approaches to improve patient outcomes., Introduction ATRX, or [alpha]-thalassemia/mental retardation, X-linked, is a chromatin remodeling protein and tumor suppressor, with mutations or copy number alterations occurring in approximately 6% of the more than 65,163 tumors [...]

Diffuse intrinsic pontine glioma (DIPG) kills more children than any other type of brain tumor. Despite clinical trials testing many chemotherapeutic agents, palliative radiotherapy remains the standard treatment. Here, we utilized Cre/loxP technology to show that deleting Ataxia telangiectasia mutated (Atm) in primary mouse models of DIPG can enhance tumor radiosensitivity. Genetic deletion of Atm improved survival of mice with p53-deficient but not p53 wild-type gliomas after radiotherapy. Similar to patients with DIPG, mice with p53 wild-type tumors had improved survival after radiotherapy independent of Atm deletion. Primary p53 wild-type tumor cell lines induced proapoptotic genes after radiation and repressed the NRF2 target, NAD(P)H quinone dehydrogenase 1 (Nqo1). Tumors lacking p53 and Ink4a/Arf expressed the highest level of Nqo1 and were most resistant to radiation, but deletion of Atm enhanced the radiation response. These results suggest that tumor genotype may determine whether inhibition of ATM during radiotherapy will be an effective clinical approach to treat DIPGs., Introduction Diffuse intrinsic pontine glioma (DIPG), also referred to as highgrade brainstem glioma, is an incurable cancer that originates in the pons and occurs primarily in children. Focal radiation therapy [...]

Radiation therapy (RT) is frequently used to treat cancers, including soft-tissue sarcomas. Prior studies established that the toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine oligodeoxynucleotide (CpG) enhances the response to RT in transplanted tumors, but the mechanisms of this enhancement remain unclear. Here, we used CRISPR/Cas9 and the chemical carcinogen 3-methylcholanthrene (MCA) to generate autochthonous soft-tissue sarcomas with high tumor mutation burden. Treatment with a single fraction of 20 Gy RT and 2 doses of CpG significantly enhanced tumor response, which was abrogated by genetic or immunodepletion of CD8+ T cells. To characterize the immune response to CpG+RT, we performed bulk RNA-Seq, single-cell RNA-Seq, and mass cytometry. Sarcomas treated with 20 Gy and CpG demonstrated increased CD8 T cells expressing markers associated with activation and proliferation, such as Granzyme B, Ki-67, and IFN-γ. CpG+RT also upregulated antigen presentation pathways on myeloid cells. Furthermore, in sarcomas treated with CpG+RT, TCR clonality analysis suggests an increase in clonal T cell dominance. Collectively, these findings demonstrate that CpG+RT significantly delays tumor growth in a CD8 T cell-dependent manner. These results provide a strong rationale for clinical trials evaluating CpG or other TLR9 agonists with RT in patients with soft-tissue sarcoma.

This study aims to investigate whether adding neoadjuvant radiotherapy (RT), anti-programmed cell death protein-1 (PD-1) antibody (anti-PD-1), or RT + anti-PD-1 to surgical resection improves disease-free survival for mice with soft tissue sarcomas (STS). We generated a high mutational load primary mouse model of STS by intramuscular injection of adenovirus expressing Cas9 and guide RNA targeting Trp53 and intramuscular injection of 3-methylcholanthrene (MCA) into the gastrocnemius muscle of wild-type mice (p53/MCA model). We randomized tumor-bearing mice to receive isotype control or anti-PD-1 antibody with or without radiotherapy (20 Gy), followed by hind limb amputation. We used micro-CT to detect lung metastases with high spatial resolution, which was confirmed by histology. We investigated whether sarcoma metastasis was regulated by immunosurveillance by lymphocytes or tumor cell-intrinsic mechanisms. Compared with surgery with isotype control antibody, the combination of anti-PD-1, radiotherapy, and surgery improved local recurrence-free survival (P = 0.035) and disease-free survival (P = 0.005), but not metastasis-free survival. Mice treated with radiotherapy, but not anti-PD-1, showed significantly improved local recurrence-free survival and metastasis-free survival over surgery alone (P = 0.043 and P = 0.007, respectively). The overall metastasis rate was low (∼12%) in the p53/MCA sarcoma model, which limited the power to detect further improvement in metastasis-free survival with addition of anti-PD-1 therapy. Tail vein injections of sarcoma cells into immunocompetent mice suggested that impaired metastasis was due to inability of sarcoma cells to grow in the lungs rather than a consequence of immunosurveillance. In conclusion, neoadjuvant radiotherapy improves metastasis-free survival after surgery in a primary model of STS., (©2022 American Association for Cancer Research.)

Soft-tissue sarcomas (STS) are rare malignancies showing lineage differentiation toward diverse mesenchymal tissues. Half of all high-grade STSs develop lung metastasis with a median survival of 15 months. Here, we used a genetically engineered mouse model that mimics undifferentiated pleomorphic sarcoma (UPS) to study the molecular mechanisms driving metastasis. High-grade sarcomas were generated with Cre recombinase technology using mice with conditional mutations in Kras and Trp53 (KP) genes. After amputation of the limb bearing the primary tumor, mice were followed for the development of lung metastasis. Using RNA-sequencing of matched primary KP tumors and lung metastases, we found that the long noncoding RNA (lncRNA) Nuclear Enriched Abundant Transcript 1 ( Neat1 ) is significantly upregulated in lung metastases. Furthermore, NEAT1 RNA ISH of human UPS showed that NEAT1 is upregulated within a subset of lung metastases compared with paired primary UPS. Remarkably, CRISPR/Cas9-mediated knockout of Neat1 suppressed the ability of KP tumor cells to colonize the lungs. To gain insight into the underlying mechanisms by which the lncRNA Neat1 promotes sarcoma metastasis, we pulled down Neat1 RNA and used mass spectrometry to identify interacting proteins. Interestingly, most Neat1 interacting proteins are involved in RNA splicing regulation. In particular, KH-Type Splicing Regulatory Protein (KHSRP) interacts with Neat1 and is associated with poor prognosis of human STS. Moreover, depletion of KHSRP suppressed the ability of KP tumor cells to colonize the lungs. Collectively, these results suggest that Neat1 and its interacting proteins, which regulate RNA splicing, are involved in mediating sarcoma metastasis. IMPLICATIONS: Understanding that lncRNA NEAT1 promotes sarcoma metastasis, at least in part, through interacting with the RNA splicing regulator KHSRP may translate into new therapeutic approaches for sarcoma., (©2020 American Association for Cancer Research.)