Biology subjects

Williams, N. T.

Publications and source records attributed to Williams, N. T..

10 recordsLinked to original sources

bioRxiv2025

Oncogenic Ppm1d mutations deregulate the p53 pathway in primary mouse gliomas

Importance of StudyProtein phosphatase magnesium-dependent 1D (PPM1D) is frequently mutated in diffuse midline gliomas (DMGs). DMGs are rare pediatric brain tumors with limited treatment options. Due to the cancers rapid progression, patients usually survive 12-24 months after diagnosis. This underscores the critical need to better understand the molecular mechanisms driving DMGs. This study describes a novel mouse model that provides a powerful platform to investigate PPM1D-driven tumor biology and offers mechanistic insights into disease development and progression. Furthermore, it serves as a valuable preclinical system for evaluating therapeutic strategies and identifying translational opportunities to target Ppm1d-mutant tumors. BackgroundDiffuse midline gliomas (DMGs) are incurable brain tumors with limited treatment options. Approximately 20% of DMGs harbor truncating mutations in exon 6 of phosphatase PPM1D, which stabilize the protein and deregulate p53 signaling. However, the consequences of these mutations for tumor initiation, progression, and therapy remain unclear. MethodsWe developed a conditional Ppm1d-loxP-exon6-loxP-exon6-E518X-tag mouse allele (Ppm1d-flex-6) that enables lineage-, spatial-, and temporal-specific expression of a DMG-derived truncated Ppm1d protein from its endogenous locus in the presence of Cre-recombinase. Ubiquitous activation of mutant Ppm1d was modeled using the Meox2-Cre driver, and primary gliomas were modeled using the RCAS/tv-a system to introduce Cre and PDGFB co-drivers into Nestin-positive neural stem cells. Complementary studies were performed in mouse embryonic fibroblasts (MEFs) expressing truncated Ppm1d following Cre recombination. ResultsWhile Meox2-Cre-driven ubiquitous recombination of Ppm1d-flex-6 produced muted phenotypes, Ppm1d-flex-6 recombination in Nestin+ neural stem cells accelerated gliomagenesis. Its oncogenic effect was weaker than complete p53 loss, and it did not accelerate tumorigenesis further in p53-null tumors. Single-cell RNA-sequencing revealed that Ppm1d-flex-6 gliomas adopt more progenitor-like transcriptional states and upregulate p53- and cell cycle associated pathways. In MEFs, Ppm1d-flex-6 enhanced proliferation and shifted transcriptomic programs toward MAPK and PI3K-Akt signaling, while impairing DNA damage responses, including reduced {gamma}-H2AX induction after irradiation. These defects sensitized cells to radiation and decreased clonogenic survival after ionizing radiation and PARP inhibition. ConclusionsPpm1d mutations confer intermediate suppression of the p53 pathway, consistent with the clinical features of PPM1D-mutant DMGs and are associated with radiosensitivity and PARP inhibitor vulnerability.

genetics↗

bioRxiv2025

Differential Impact of Lymphocytes on Radiation Response in Autochthonous versus Transplant Sarcomas in Syngeneic Mice

Preclinical studies in transplant tumor models showing high cure rates with combined radiotherapy (RT) and immunotherapy have rarely translated to clinical success, and studies in autochthonous tumor models are limited. We hypothesized that lymphocytes differentially affect RT response in transplant versus autochthonous tumor models. Here, we compared tumor onset and growth delay after 0 or 20 Gy for autochthonous versus transplant soft tissue sarcomas in Rag2-/- mice lacking an adaptive immune system versus immune-intact Rag2+/- mice. While time to tumor onset did not differ between Rag2-/- and Rag2+/- mice in the autochthonous model, transplant tumor onset was significantly slower in immunocompetent mice. No transplant tumors in Rag2-/- mice or autochthonous tumors were cured by RT, whereas 30.4% of Rag2+/- mice with transplant tumors were cured. These results highlight the importance of including autochthonous tumors as complementary model systems to study the interplay between the immune system and RT response.

cancer biology↗

bioRxiv2025

An Investigation of mini-GRID Radiation therapy with Immune Checkpoint Blockade in a Murine Tumor Model

Spatially fractionated radiotherapy has shown potential to improve therapeutic outcomes possibly with an immunogenic mechanistic component. Here we report on in vivo mouse studies investigating mini-GRID pencil-beam radiotherapy combined with anti-PD-1 immune checkpoint blockade. Methods: GRID therapy was delivered at 225kV using the XStrahl Small Animal Radiation Research Platform with two custom lead mini-GRIDs, each consisting of an array of equally spaced holes: 1 mm diameter with 1mm spacing and 254 {micro}m diameter with 508 {micro}m spacing. GRID dosimetry was characterized using EBT3 film to determine peak-to-valley dose ratios and output. Two studies were performed with C57BL/6J mice bearing subcutaneous LLC1 flank tumors. In the first, mice (n=5/group) were treated in 3 groups with a single fraction: 15 Gy open field, 15 Gy 1 mm GRID, or 24 Gy 1 mm GRID. In the second, mice (n=6-7/group) were treated with fractionated GRID radiotherapy in 5 groups: 15 Gy open field x 3 fractions, 15 Gy hemi-irradiation x 3 fractions, (15 Gy 1 mm GRID x 3 fractions, or 15 Gy 254 {micro}m GRID x 3 fractions. All mice were treated with 200 g anti-PD-1 antibody on days 0, 3, and 6, then weekly until humane endpoint (tumor >15 mm in any dimension or ulceration). Results: Peak to valley ratios were 24.5 {+/-} 0.6 and 19.8 {+/-} 0.7 for the 1 mm and 254 {micro}m GRIDs, respectively. Tumor growth and mean survival times in both studies were significantly shorter for all non-open field arms (p < 0.05; Log Rank for survival; 2-way ANOVA for tumor growth). Conclusions: Two novel mini-GRIDs were characterized and tested in combination with anti-PD-1 therapy. In this study, neither single dose nor fractionated GRID therapy with anti-PD-1 improved tumor growth delay or survival. Similarly, hemi-irradiation resulted in worse tumor control compared to conventional open field radiotherapy.

cancer biology↗

bioRxiv2024

Enhancing radiotherapy response via intratumoral injection of the TLR9 agonist CpG to stimulate CD8 T cells in an autochthonous mouse model of sarcoma

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-{gamma}. 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.

cancer biology↗

bioRxiv2023

Ataxia-telangiectasia mutated (Atm) disruption sensitizes spatially-directed H3.3K27M/TP53 diffuse midline gliomas to radiation therapy

Diffuse midline gliomas (DMGs) are lethal brain tumors characterized by p53-inactivating mutations and oncohistone H3.3K27M mutations that rewire the cellular response to genotoxic stress, which presents therapeutic opportunities. We used RCAS/tv-a retroviruses and Cre recombinase to inactivate p53 and induce K27M in the native H3f3a allele in a lineage- and spatially-directed manner, yielding primary mouse DMGs. Genetic or pharmacologic disruption of the DNA damage response kinase Ataxia-telangiectasia mutated (ATM) enhanced the efficacy of focal brain irradiation, extending mouse survival. This finding suggests that targeting ATM will enhance the efficacy of radiation therapy for p53-mutant DMG but not p53-wildtype DMG. We used spatial in situ transcriptomics and an allelic series of primary murine DMG models with different p53 mutations to identify transactivation-independent p53 activity as a key mediator of such radiosensitivity. These studies deeply profile a genetically faithful and versatile model of a lethal brain tumor to identify resistance mechanisms for a therapeutic strategy currently in clinical trials.

cancer biology↗

bioRxiv2022

Temporary knockdown of p53 during focal limb irradiation increases the development of sarcomas

BackgroundApproximately half of all cancer patients receive radiotherapy and, as cancer survivorship rates increase with more effective therapies, the very low rate of radiation-associated sarcomas is rising. Radiation-associated sarcomas are life-threatening cancers, and radiation exposure is a primary risk factor for sarcoma development. During radiotherapy or other genotoxic cancer therapy for p53 mutant cancers, pharmacological inhibition of p53 has been proposed to ameliorate acute injury of normal tissues. However, enhancing the survival of normal cells that sustain DNA damage by temporarily inhibiting p53 has the potential to increase the risk of cancer development. Here, we use in vivo shRNA technology to examine the consequences of temporarily reducing p53 expression on radiation-induced sarcoma development. MethodsWe utilized a mouse model of radiation-induced sarcoma where mice express a doxycycline (dox)-inducible p53 shRNA to temporarily and reversibly reduce p53 expression. Mice were placed on a dox diet 10 days prior to receiving 30 or 40 Gy hind limb irradiation in a single fraction and then returned to normal chow. Mice were examined weekly for sarcoma development and scored for radiation-induced normal tissue injuries. Radiation-induced sarcomas were harvested and subjected to RNA sequencing. ResultsFollowing single high-dose irradiation, 21% of temporary p53 knockdown animals developed a sarcoma in the radiation field compared to 2% of control animals. Mice with more severe acute injuries in the first 3 months after irradiation had a significantly increased risk of developing late persistent wounds in the soft tissue and bone. Chronic radiation-induced wounds were associated with sarcomagenesis. Examination of muscle stem cells by flow cytometry following hind limb irradiation indicated p53 knockdown preserves muscle stem cells in the irradiated limb, supporting the notion that temporary p53 knockdown at the time of irradiation reduces death of cells with DNA damage which may then persist to develop into a sarcoma. We performed RNA sequencing on 16 radiation-induced sarcomas compared to normal muscle controls. Gene set enrichment analysis revealed upregulation in the sarcomas of genes related to translation, epithelial mesenchymal transition (EMT), inflammation, and the cell cycle versus downregulation of genes related to myogenesis and tumor metabolism. Furthermore, genes with increased copy number such as Met and Cdk4 were overexpressed in tumors. ConclusionsTemporary reduction of p53 during high-dose irradiation increases late effects including tissue injuries and sarcoma development.

cancer biology↗

bioRxiv2022

Atm loss does not radiosensitize a primary mouse model of Pten-deleted brainstem glioma

Diffuse midline gliomas arise in the brainstem and other midline brain structures and cause a large proportion of childhood brain tumor deaths. Radiation therapy is the most effective treatment option, but these tumors ultimately progress. Inhibition of the phosphoinositide-3-kinase (PI3K)-like kinase ataxia telangiectasia mutated (ATM), which orchestrates the cellular response to radiation-induced DNA damage, may enhance the efficacy of radiation therapy. Diffuse midline gliomas in the brainstem contain loss-of-function mutations in the tumor suppressor PTEN, or functionally similar alterations in the phosphoinositide-3-kinase (PI3K) pathway, at moderate frequency. Here, we sought to determine if Atm inactivation could radiosensitize a primary mouse model of brainstem glioma driven by Pten loss. Using Cre/loxP recombinase technology and the RCAS/TVA retroviral gene delivery system, we established a mouse model of brainstem glioma driven by Pten deletion. We find that Pten-null brainstem gliomas are relatively radiosensitive at baseline. In addition, we show that deletion of Atm in the tumor cells does not extend survival of mice bearing Pten-null brainstem gliomas after focal brain irradiation. These results characterize a novel primary mouse model of PTEN-mutated brainstem glioma and provide insights into the mechanism of radiosensitization by Atm deletion, which may guide the design of future clinical trials. Brief SummaryWe develop a mouse model of PTEN-mutated brainstem glioma and find that perturbation of the ATM does not enhance radiation efficacy in this model.

cancer biology↗

bioRxiv2021

The p53 transactivation domain 1-dependent response to acute DNA damage in endothelial cells protects against radiation-induced cardiac injury

Thoracic radiation therapy can cause endothelial injury in the heart, leading to cardiac dysfunction and heart failure. Although it has been demonstrated that the tumor suppressor p53 functions in endothelial cells to prevent the development of radiation-induced myocardial injury, the key mechanism(s) by which p53 regulates the radiosensitivity of cardiac endothelial cells is not completely understood. Here, we utilized genetically engineered mice that express mutations in p53 transactivation domain 1 (TAD1) (p5325,26) or mutations in p53 TAD1 and TAD2 (p5325,26,53,54) specifically in endothelial cells to study the p53 transcriptional program that protects cardiac endothelial cells from ionizing radiation in vivo. p5325,26,53,54 loses the ability to drive transactivation of p53 target genes after irradiation while p5325,26 can induce transcription of a group of non-canonical p53 target genes, but not the majority of classic radiation-induced p53 targets critical for p53-mediated cell cycle arrest and apoptosis. After 12 Gy whole-heart irradiation, we found that both p5325,26 and p5325,26,53,54 sensitized mice to radiation-induced cardiac injury, in contrast to wild-type p53. Histopathological examination suggested that mutation of TAD1 contributes to myocardial necrosis after whole-heart irradiation, while mutation of both TAD1 and TAD2 abolishes the ability of p53 to prevent radiation-induced heart disease. Taken together, our results show that the transcriptional program downstream of p53 TAD1, which activates the acute DNA damage response after irradiation, is necessary to protect cardiac endothelial cells from radiation injury in vivo.

genetics↗

bioRxiv2021

Atrx deletion impairs cGAS-STING signaling and increases response to radiation and oncolytic herpesvirus in sarcoma

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 are more sensitive to radiation therapy. In the absence of Atrx, irradiated sarcomas have increased persistent DNA damage, telomere dysfunction, and mitotic catastrophe. We find that Atrx deleted sarcomas have a reduced adaptive immune response, impaired cGAS-STING signaling, and increased sensitivity to an oncolytic herpesvirus therapy. Translation of these results to patients with ATRX mutant cancers could enable genomically-guided cancer therapeutic approaches that improve patient outcomes.

cancer biology↗

bioRxiv2020

Tumor genotype dictates radiosensitization after Atm deletion in brainstem gliomas

Diffuse intrinsic pontine glioma (DIPG) kills more children than any other type of brain tumor. Despite clinical trials testing many chemotherapeutic agents, 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 following radiotherapy. Similar to patients with DIPG, mice with p53 wild-type tumors had improved survival after radiotherapy independent of Atm deletion. p53 wild-type tumor cell lines induced proapoptotic genes after radiation and repressed the NRF2 target, 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.

cancer biology↗