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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 [...]

Purpose. Recently, the association between ATRX and a more aggressive sarcoma phenotype has been shown. We performed a retrospective study of sarcomas from an individual institution to evaluate ATRX as a prognosticator in soft tissue sarcoma. Experimental Design. 128 sarcomas were collected from a single institution and stained for ATRX. The prognostic significance of these markers was evaluated in a smaller cohort of primary soft tissue sarcomas (n = 68). Kaplan–Meier curves were created for univariate analysis, and Cox regression was utilized for multivariate analysis. Results. High expression of ATRX was found to be a positive prognostic indicator for overall survival and metastasis-free survival in our group of soft tissue sarcomas both in univariate analysis and multivariate analysis (HR: 0.38 (0.17–0.85), P = 0.02 and HR: 0.49 (0.24–0.99), P = 0.05 , respectively). Conclusions. High expression of ATRX is a positive prognostic indicator of overall survival and metastasis-free survival in patients with STS. This is consistent with studies in osteosarcoma, which indicate possible mechanisms through which loss of ATRX leads to more aggressive phenotypes. Future prospective clinical studies are required to validate the prognostic significance of these findings. [ABSTRACT FROM AUTHOR]

Cooperating gene mutations are typically required to transform normal cells enabling growth in soft agar or in immunodeficient mice. For example, mutations in Kras and transformation-related protein 53 (Trp53) are known to transform a variety of mesenchymal and epithelial cells in vitro and in vivo. Identifying other genes that can cooperate with oncogenic Kras and substitute for Trp53 mutation has the potential to lead to new insights into mechanisms of carcinogenesis. Here, we applied a genome-wide CRISPR/Cas9 knockout screen in KrasG12D immortalized mouse embryonic fibroblasts (MEFs) to search for genes that when mutated cooperate with oncogenic Kras to induce transformation. We also tested if mutation of the identified candidate genes could cooperate with KrasG12D to generate primary sarcomas in mice. In addition to identifying the well-known tumor suppressor cyclin dependent kinase inhibitor 2A (Cdkn2a), whose alternative reading frame product p19 activates Trp53, we also identified other putative tumor suppressors, such as F-box/WD repeat-containing protein 7 (Fbxw7) and solute carrier family 9 member 3 (Slc9a3). Remarkably, the TCGA database indicates that both FBXW7 and SLC9A3 are commonly co-mutated with KRAS in human cancers. However, we found that only mutation of Trp53 or Cdkn2a, but not Fbxw7 or Slc9a3 can cooperate with KrasG12D to generate primary sarcomas in mice. These results show that mutations in oncogenic Kras and either Fbxw7 or Slc9a3 are sufficient for transformation in vitro, but not for in vivo sarcomagenesis. [ABSTRACT FROM AUTHOR]

Cooperating gene mutations are typically required to transform normal cells enabling growth in soft agar or in immunodeficient mice. For example, mutations in Kras and transformation-related protein 53 (Trp53) are known to transform a variety of mesenchymal and epithelial cells in vitro and in vivo. Identifying other genes that can cooperate with oncogenic Kras and substitute for Trp53 mutation has the potential to lead to new insights into mechanisms of carcinogenesis. Here, we applied a genome-wide CRISPR/Cas9 knockout screen in KrasG12D immortalized mouse embryonic fibroblasts (MEFs) to search for genes that when mutated cooperate with oncogenic Kras to induce transformation. We also tested if mutation of the identified candidate genes could cooperate with KrasG12D to generate primary sarcomas in mice. In addition to identifying the well-known tumor suppressor cyclin dependent kinase inhibitor 2A (Cdkn2a), whose alternative reading frame product p19 activates Trp53, we also identified other putative tumor suppressors, such as F-box/WD repeat-containing protein 7 (Fbxw7) and solute carrier family 9 member 3 (Slc9a3). Remarkably, the TCGA database indicates that both FBXW7 and SLC9A3 are commonly co-mutated with KRAS in human cancers. However, we found that only mutation of Trp53 or Cdkn2a, but not Fbxw7 or Slc9a3 can cooperate with KrasG12D to generate primary sarcomas in mice. These results show that mutations in oncogenic Kras and either Fbxw7 or Slc9a3 are sufficient for transformation in vitro, but not for in vivo sarcomagenesis. [ABSTRACT FROM AUTHOR]

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.

Purpose: To investigate whether TP53 variants may be correlated with overall survival and local control following stereotactic radiosurgery (SRS) for brain metastases (BMs) from non-small cell lung cancer (NSCLC)., Methods: Patients undergoing an initial course of SRS for NSCLC brain metastases between 1/2015 and 12/2020 were retrospectively identified. Overall survival and freedom from local intracranial progression (FFLIP) were estimated via Kaplan-Meier method. Cox models assessed TP53 variant status (pathogenic variant, PV; variant not detected, ND)., Results: 255 patients underwent molecular profiling for TP53, among whom 144 (56%) had a TP53 PV. Median follow-up was 11.6 months. OS was not significantly different across TP53 status. A trend toward superior FFLIP was observed for PV (95% CI 62.9 months-NR) versus ND patients (95% CI 29.4 months-NR; p=0.06). Superior FFLIP was observed for patients with one TP53 variant versus those with TP53 ND., Conclusion: Among NSCLC patients with BMs, the potential association between TP53 status and post-SRS FFLIP warrants further investigation in a larger prospective cohort., Competing Interests: We thank Karen Allen for her assistance maintaining the prospective QA database. Authors’ disclosure of potential conflicts of interest The funding sources for this study (NIH grant R38CA24520401 to DJC, K08CA256045 to ZJR and NIH 5R38CA245204 to EJV and an Office of Physician Scientist Development Technician Award to EJV) had no role in the collection, analysis, or interpretation of the data, the writing of the manuscript, or the decision to submit it for publication. ZJR has intellectual property managed by Duke University that has been licensed to GeneTron Health related to brain tumor genomic profiling tests, and has received honoraria from Eisai Pharmaceuticals and Oakstone Publishing. JK reported receiving grants from Varian Medical Systems and BioMimetix SBR, receiving personal fees from Monteris Medical, and owning ClearSight LLC outside the submitted work. None of the above entities played a role in the collection, analysis, or interpretation of the data, the writing of the manuscript, or the decision to submit it for publication., (© 2024 Old City Publishing, Inc.)

Purpose: Genetic variants affecting the radiation response protein ataxia-telangiectasia mutated (ATM) have been associated with increased adverse effects of radiation but also with improved local control after conventional radiation therapy. However, it is unknown whether ATM variants affect rates of radionecrosis (RN) and local intracranial progression (LIP) after stereotactic radiosurgery (SRS) for brain metastases., Methods and Materials: Patients undergoing an initial course of SRS for non-small cell lung cancer (NSCLC) brain metastases at a single institution were retrospectively identified. Kaplan-Meier estimates were calculated and Cox proportional hazards testing was performed based on ATM variant status., Results: A total of 541 patients completed SRS for brain metastasis secondary to NSCLC, of whom 260 completed molecular profiling. Variants of ATM were identified in 36 cases (13.8%). Among patients who completed molecular profiling, RN incidence was 4.9% (95% CI, 1.6%-8.2%) at 6 months and 9.9% (95% CI, 4.8%-15.0%) at 12 months. Incidence of RN was not significantly increased among patients with ATM variants, with an RN incidence of 5.3% (95% CI, 0.0%-15.3%) at both 6 and 12 months ( P  = .46). For all patients who completed genomic profiling, LIP was 5.4% (95% CI, 2.4%-8.4%) at 6 months and 9.8% (5.5%-14.1%) at 12 months. A significant improvement in LIP was not detected among patients with ATM variants, with an LIP incidence of 3.1% (0.0%-9.1%) at both 6 and 12 months ( P  = .26). Although differences according to ATM variant type (pathologic variant or variant of unknown significance) did not reach significance, no patients with ATM pathologic variants experienced LIP., Conclusions: We did not detect significant associations between ATM variant status and RN or LIP after SRS for NSCLC brain metastases. The current data set allows estimation of patient cohort sizes needed to power future investigations to identify genetic variants that associate with significant differences in outcomes after SRS., Competing Interests: Eugene Vaios reported receiving research grants from the National Institutes of Health (NIH) and consulting fees from Glasshouse Health, United Kingdom, and having leadership roles in the American Society for Radiation Oncology Finance/Audit Committee. Scott R. Floyd reported receiving research grants from and/or having contracts with the NIH and the American Cancer Society and having a US patent (9,610,332). John P. Kirkpatrick reported having research grants and/or contracts with SBIR Project Funding, Varian Medical Systems, and BioMimetix JV LLC; receiving consulting fees and speaking honoraria from Monteris Medical; and being the owner and president of Clearsight RT LLC. Michelle Green reported receiving a research grant from Bayer Medical Education and receiving royalties from SciMed Solutions. Zachary J. Reitman reported receiving research grants from and/or having contracts with St. Baldrick's Foundation, the Pediatric Brain Tumor Foundation, the Defeat DIPG Foundation, the SoSo Strong Foundation, the ChadTough Foundation, and the National Cancer Institute; receiving royalties from Genetron Health; receiving payment or honoraria for presentations from Oakstone Publishing and Eisai Pharmaceuticals; and having patents (10711310, 11306364, and 8691960) for brain tumor diagnostic technology and biocatalyst technology, managed by the Duke Office of Licensing and Ventures. All other authors declare no conflicts of interest. None of the organizations or institutions had any role in the collection, analysis, or interpretation of the data, the writing of the manuscript, or the decision to submit it for publication., (© 2023 The Authors.)

Osteosarcoma (OS) is a lethal disease with few known targeted therapies. Here, we show that decreased ATRX expression is associated with more aggressive tumor cell phenotypes, including increased growth, migration, invasion, and metastasis. These phenotypic changes correspond with activation of NF-κB signaling, extracellular matrix remodeling, increased integrin αvβ3 expression, and ETS family transcription factor binding. Here, we characterize these changes in vitro, in vivo, and in a data set of human OS patients. This increased aggression substantially sensitizes ATRX-deficient OS cells to integrin signaling inhibition. Thus, ATRX plays an important tumor-suppression role in OS, and loss of function of this gene may underlie new therapeutic vulnerabilities. The relationship between ATRX expression and integrin binding, NF-κB activation, and ETS family transcription factor binding has not been described in previous studies and may impact the pathophysiology of other diseases with ATRX loss, including other cancers and the ATR-X α thalassemia intellectual disability syndrome.

Purpose: To evaluate the effect of prostate volume on outcomes after moderately hypofractionated radiation therapy (mHFRT) for prostate cancer., Methods and Materials: Prostate cancer patients treated with mHFRT at a Veteran's Affairs Medical Center from August 20, 2008, to January 31, 2018, were identified. Patients were placed into a large prostate planning target volume (LPTV) cohort if their prostate PTV was in the highest quartile. Acute/late genitourinary (GU) and gastrointestinal toxicity events among patients with and without LPTV were compared. Multivariable analyses estimated the effect of factors on toxicity. Overall survival, biochemical recurrence-free survival, and freedom from late GU/gastrointestinal toxicity of patients with and without LPTV were estimated via Kaplan-Meier., Results: Four hundred and seventy-two patients were included. Ninety-three percent received 70 Gy in 2.5 Gy fractions; 75% received androgen deprivation therapy. Median follow-up was 69 months. Patients with LPTV (PTV >138.4 cm 3 ) had a higher late 2 + GU toxicity compared with those without (59% vs 48%, P  = .03). Earlier time to late 2 + GU toxicity was associated with LPTV (hazard ratio 1.36; 95% confidence interval [CI], 1.00-1.86; P  = .047), androgen deprivation therapy use (hazard ratio 1.60; 95% CI, 1.13-2.27; P  = .01), and higher baseline American Urologic Association symptom score (odds ratio 1.03; 95% CI, 1.02-1.05; P < .001). At 2 years, freedom from late 2 + GU toxicity was 46% (95% CI, 47%-54%) for those with LPTV versus 61% (95% CI, 55%-65%) for those without ( P  = .04). Late grade 3 GU toxicity was 7% for those with LPTV and 4% for those without. No differences in overall survival or biochemical recurrence-free survival were observed between patients with or without LPTV., Conclusions: LPTV did not affect efficacy of mHFRT for prostate cancer; however, it was associated with increased risk and earlier onset of late grade 2 + GU toxicity., (© 2021 The Authors.)