Biology subjects

Reitman, Z. J.

Publications and source records attributed to Reitman, Z. J..

5 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

Investigating Rat-Brain Normal Tissue and Tumor FLASH Effects with a Novel Very High Energy Electron Beam

PurposeUltra-high dose rate (FLASH) irradiation is reported to reduce normal tissue toxicity while maintaining tumor control, however mechanism(s) remain obscure. To study FLASH mechanisms in brain tissue, we developed a novel experimental platform featuring a unique high-energy electron linear accelerator (High Intensity Gamma Source, HIGS) paired with an organotypic ex vivo brain metastasis model. MethodsWe varied inter-pulse spacing to modulate the mean dose rate (MDR) of our unique 35 MeV electron beam, while maintaining extremely high instantaneous dose rate (IDR), and used film dosimetry to characterize dosimetry and targeting accuracy. We combined this HIGS-FLASH beam with an organotypic rat brain slice/breast carcinoma co-culture model of brain metastasis to assess effects on normal and neoplastic tissues. Live cell and bioluminescence imaging demonstrated cancer cell growth effects, while normal tissue responses and immune activation were assessed with live cell imaging, cytokine profiles, and confocal microscopy. We performed comparison experiments with 20 MeV electrons from a Varian clinical linear accelerator (VCLA) operating at conventional dose rate. ResultsThe highest IDR of the HIGS-FLASH beam to-date was 20.7 {+/-} 0.6 MGy/s, with maximum MDR of 20.7 MGy/s (1 s pulse of 20.7 Gy). Beam targeting was accurate to < 1 mm and reproducible. HIGS-FLASH and VCLA dose rates equivalently decreased cancer cell growth. HIGS-FLASH irradiation significantly increased TNF and fractalkine levels and confocal microscopy revealed distinct changes in microglial morphology in normal brain slices, suggesting microglia activation following HIGS-FLASH irradiation. ConclusionsOur novel experimental platform produces extremely high dose rates and rapid normal/neoplastic tissue readouts for mechanistic research into the effects of FLASH radiation on the brain. HIGS-FLASH irradiation induces comparable cancer cell growth inhibition but differential effects on cytokines and microglial morphology, suggesting that acute innate immune responses may be involved in FLASH normal tissue effects in the brain.

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

Ganglioglioma deep transcriptomics reveals primitive neuroectoderm neural precursor-like population

Gangliogliomas are brain tumors composed of neuron-like and macroglia-like components that occur in children and young adults. Gangliogliomas are often characterized by a rare population of immature astrocyte-appearing cells expressing CD34, a marker expressed in the neuroectoderm (neural precursor cells) during embryogenesis. New insights are needed to refine tumor classification and to identify therapeutic approaches. We evaluated five gangliogliomas with single nucleus RNA-seq, cellular indexing of transcriptomes and epitopes by sequencing, and/or spatially-resolved RNA-seq. We uncovered a population of CD34+ neoplastic cells with mixed neuroectodermal, immature astrocyte, and neuronal markers. Gene regulatory network interrogation in these neuroectoderm-like cells revealed control of transcriptional programming by TCF7L2/MEIS1-PAX6 and SOX2, similar to that found during neuroectodermal/neural development. Developmental trajectory analyses place neuroectoderm-like tumor cells as precursor cells that give rise to neuron-like and macroglia-like neoplastic cells. Spatially-resolved transcriptomics revealed a neuroectoderm-like tumor cell niche with relative lack of vascular and immune cells. We used these high resolution results to deconvolute clinically-annotated transcriptomic data, confirming that CD34+ cell-associated gene programs associate with gangliogliomas compared to other glial brain tumors. Together, these deep transcriptomic approaches characterized a ganglioglioma cellular hierarchy - confirming CD34+ neuroectoderm-like tumor precursor cells, controlling transcription programs, cell signaling, and associated immune cell states. These findings may guide tumor classification, diagnosis, prognostication, and therapeutic investigations.

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↗