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Multiple Sclerosis (MS) is an autoimmune disease characterized by inflammation and neurodegeneration. Current MS treatments were designed to reduce inflammation in MS, rather than to directly prevent neurodegeneration. Estrogen has well-documented neuroprotective effects in a variety of disorders of the CNS, including experimental autoimmune encephalomyelitis (EAE), the most widely used mouse model of MS. Treatment with an estrogen receptor-beta (ERβ) ligand is known to effectively ameliorate clinical disease and provide neuroprotection in EAE. However, the protective effects of this ERβ ligand have only been demonstrated when administered prior to disease (prophylactically). Here, we tested whether ERβ ligand treatment could provide clinical protection when treatment was initiated after onset of disease (therapeutically). We found that therapeutic treatment effectively ameliorated clinical disease in EAE. Specifically, ERβ ligand-treated animals exhibited preserved axons and myelin as compared to vehicle treated animals. We observed no difference in the number of T lymphocytes, macrophages, or microglia in the CNS of vehicle versus ERβ ligand-treated animals. Our findings show that therapeutically administered ERβ ligand successfully treats clinical EAE, bearing translational relevance to MS as a candidate neuroprotective agent.

Radiation therapy 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 radiation therapy (RT) in transplanted tumors, but the mechanism(s) 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 two doses of CpG significantly enhanced tumor response, which was abrogated by genetic or immunodepletion of CD8+ T cells. To characterize the immune response to RT + CpG, 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 interferon-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 RT + CpG 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., Competing Interests: DGK is a co-founder of Xrad Therapeutics, which is developing radiosensitizers, and serves on the Scientific Advisory Board of Lumicell, which is commercializing intraoperative imaging technology. DGK is a co-inventor on patents for radiosensitizers and an intraoperative imaging device. DGK also receives funding for a clinical trial from a Stand Up To Cancer (SU2C) Catalyst Research Grant with support from Merck. The laboratory of DGK received funding from Xrad Therapeutics, Merck, Bristol-Myers Squibb, but this did not support the research described in this manuscript. DGK received funding from Varian Medical Systems and an antibody to OX-40 from Bristol-Myers Squibb, which were used to support experiments in this manuscript. The other authors have declared that no conflict of interest exists.

The development of a telomere maintenance mechanism is essential for immortalization in human cancer. While most cancers elongate their telomeres by expression of telomerase, 10-15% of human cancers use a pathway known as alternative lengthening of telomeres (ALT). In this work, we developed a genetically engineered primary mouse model of sarcoma in CAST/EiJ mice which displays multiple molecular features of ALT activation after CRISPR/Cas9 introduction of oncogenic Kras G12D and loss of function mutations of Trp53 and Atrx . In this model, we demonstrate that the loss of Atrx contributes to the development of ALT in an autochthonous tumor, and this process occurs independently of telomerase function by variation of mTR alleles. Furthermore, we find that telomere shortening from the loss of telomerase leads to higher chromosomal instability while loss of Atrx and activation of ALT lead to an increase in telomeric instability, telomere sister chromatid exchange, c-circle production, and formation of ALT-associated promyelocytic leukemia bodies (APBs). The development of this primary mouse model of ALT could enable future investigations into therapeutic vulnerabilities of ALT activation and its mechanism of action., Competing Interests: DGK is a cofounder of Xrad Therapeutics, which is developing radiosensitizers, and serves on the Scientific Advisory Board of Lumicell, which is commercializing intraoperative imaging technology. DGK is a coinventor on patents for radiosensitizers and an intraoperative imaging device. DGK also receives funding for a clinical trial from a Stand Up To Cancer (SU2C) Catalyst Research Grant with support from Merck. Amgen provided the mouse variant TVEC used in this study. The laboratory of DGK currently receives funding or reagents from Xrad Therapeutics, Merck, Bristol-Myers Squibb, Varian Medical Systems, and Calithera, but these did not support the research described in this manuscript.

The adaptive immune system plays an essential anti-tumor role through immunosurveillance and response to immunotherapies. Characterizing phenotypic features and mechanisms of dysfunction of tumor-specific T cell populations may uncover novel immunotherapeutic targets and biomarkers of response. To study tumor-specific T cell responses in vivo , a tumor model must express a known neoantigen. While transplant models with known neoantigen expression are widely available, autochthonous tumor models in which the tumor coevolves with the immune system are limited. In this study, we combined CRISPR/Cas9 and sleeping beauty transposase technology to develop an autochthonous orthotopic murine sarcoma model with oncogenic Kras G12D , functionally impaired p53, and expression of known MHCI and MHCII sarcoma neoantigens. Using MHC tetramer flow cytometry, we identified a tumor-specific immune response in the peripheral blood as early as 10 days after tumor induction leading to tumor clearance. Tumors developed at high penetrance after co-depletion of CD8 and CD4 T cells, but depletion of either CD8 or CD4 T cells alone was insufficient to permit tumor growth. These results suggest that CD8 and CD4 T cells can independently contribute to immunosurveillance leading to clearance of sarcomas expressing MHCI and MHCII neoantigens., Competing Interests: Competing Interests: DGK is a cofounder of and stockholder in XRAD Therapeutics, which is developing radiosensitizers. DGK is a member of the scientific advisory board and owns stock in Lumicell Inc, a company commercializing intraoperative imaging technology. None of these affiliations represents a conflict of interest with respect to the work described in this manuscript. DGK is a coinventor on a patent for a handheld imaging device and is a coinventor on a patent for radiosensitizers. XRAD Therapeutics, Merck, Bristol Myers Squibb, and Varian Medical Systems provide research support to DGK, but only funds from Merck supported the research described in this manuscript. The other authors have no conflicting financial interests.