St. Jude Children's Research Hospital

Objective.The dose-weighted linear energy transfer (LETd) increases significantly at the distal edge of the proton Bragg peak, leading to distinct biological responses compared to proximal regions of the depth-dose profile. Normal tissue complications have been linked to composite LETd distributions from clinical treatment plans, but the associations between LETd and complications remain inconclusive. This study introduces a field-by-field analysis method to provide more detailed insights into these relationships.Approach. LETd in composite plans was reformulated to account for weighted contributions from individual fields. Dose and LETd distributions for each field were calculated using the MCsquare Monte Carlo code. Field-specific dose volume histograms and LETd volume histograms (LETdVHs) were generated for regions with elevated LETd in 101 pediatric craniopharyngioma cases treated with two-field plans (n= 98) or three-field plans (n= 3).Main results.Individual fields contribute high LETd at the distal edge of the spread-out Bragg peak (SOBP) and lateral field edges. High LET particles delivered physical doses of several Gy exceeding the threshold for non-stochastic acute and late effects. Composite LETd distributions, however, show lower LET at the SOBP distal edge due to contributions from low LET particles in opposing fields. Information about high LET dose deposited by individual beams is diluted in composite plan LETd distributions.Significance.Evaluating LETd distributions based solely on composite plans can obscure the contribution of high-LET regions from individual fields to the biological response of tissues. A field-by-field assessment may provide a more accurate understanding of LETd's role in normal tissue effects and may improve the prediction of complications in proton therapy.

Pineoblastoma is a clinically aggressive childhood brain tumor composed of distinct molecular subgroups with divergent driver genes, demographics, and clinical outcomes. To identify developmental origins and mechanisms governing disease pathogenesis, we derive single-cell transcriptomes from pineal parenchymal tumors, aligning malignant cells with developmental counterparts to retrace cellular origins. Integrative computational analyses map pineoblastoma origins to transient, cycling pinealocyte progenitors during development. Lineage-specific perturbation of suspected drivers in the early pineal gland yields preclinical models representative of consensus molecular subgroups. Multi-omic characterization of patient tumors and these models uncover a tumor-associated photoreceptor signature (TAPS) common to pineoblastoma, retinoblastoma, and Group 3 medulloblastoma. Transcriptional activity of this signature within respective cellular origins establishes a developmental basis for molecular similarities between entities. Photoreceptor signature constituents are selective dependencies across these anatomically distinct central nervous system malignancies, motivating future studies evaluating developmentally encoded programs of malignancy as potential therapeutic liabilities.

Structure and dynamics of proteins are key to understanding their roles in biological systems and provide a framework for rational development of novel therapeutics. Here, we combine NMR chemical shifts (CSs), X-ray crystal structures, and molecular dynamics (MD) simulations to characterize the extracellular domain of human tissue factor, i.e., soluble tissue factor (sTF), a protein that is involved in the initiation of the blood clotting process by forming a complex with the coagulation factor VIIa (fVIIa). Starting with the X-ray structures, solution NMR CSs were incorporated as restraints in CS-guided MD simulations to obtain structures in agreement with the NMR solution data of the protein. Our results reveal a dynamic ensemble of configurations in a loop that is key to sTF interaction with fVIIa. Key residues have been identified in the fVIIa-binding loop with divergent backbone and/or side-chain configurations to account for the loop dynamics. We demonstrate that the resulting structural ensemble from the incorporation of solution NMR CSs provides a better description of sTF dynamics in solution. The integrated approach used in this study can be applied to provide a better molecular guide for therapeutics that specifically target sTF.

ZFTA-RELA is the most recurrent genetic alteration seen in paediatric supratentorial ependymoma (EPN) and is sufficient to initiate tumours in mice1. Despite its oncogenic potential, ZFTA-RELA (ZR) is observed nearly exclusively in childhood EPN, with tumours located distinctly in the supratentorial brain of the central nervous system1. We proposed that specific chromatin modules accessible during brain development would render distinct cell lineage programs at direct risk of transformation by ZR. To test this hypothesis, we performed combined single-nucleus assay for transposase-accessible chromatin and RNA (snMultiome) sequencing of the developing mouse forebrain compared with ZR-driven mouse and human EPN. We demonstrated that specific developmental lineage programs present in transient progenitor cells and regulated by PLAG/L family transcription factors were at risk of neoplastic transformation. Binding of this chromatin network by ZR or other PLAG/L family motifs targeting fusion oncoproteins led to persistent chromatin accessibility at oncogenic loci and oncogene expression. Cross-species analysis of mouse and human ZR EPN revealed significant cell type heterogeneity indicating incomplete neurogenic and gliogenic differentiation, with a small percentage of cycling progenitor-like or radial glial-like cells that established a putative tumour cell hierarchy. In vivo lineage tracing studies identified neoplastic clones that aggressively dominated tumour growth and established the entire EPN cellular hierarchy. These findings identify developmental epigenomic states that are critical for fusion-oncoprotein-driven transformation and show how these states continue to shape tumour progression.

Cannabinoid receptors are therapeutically promising GPCRs that are also interesting test systems for structure-based methods, which have targeted them previously. Here we used the CB2 receptor as a template to explore several topical questions in library docking. Whereas an earlier campaign against the CB1 receptor led to potent but relatively non-selective ligands, here we found that targeting interactions with polar, orthosteric site residues led to subtype-selective ligands. Docking hit rate and especially hit affinity improved in moving from a 7 million to a 2.6 billion molecule library. Similar to earlier studies, docking against active and inactive states of the receptor did not reliably bias toward the discovery of agonists or inverse agonists. Cryo-EM structures of two of the new agonists, each in a different chemotype, superposed well on the docking predictions. Correspondingly, structure-based optimization led to 10- to 140-fold improvements within three different series, also consistent with well-behaved ligand families. Hit rates with a fully enumerated 2.6 billion molecule library resembled those of an implied 11 billion molecule library from a building-block method, consistent with the latter's ability to explore this space, though higher affinities were discovered from the fully enumerated set. Overall, eight diverse families of ligands, with potencies <100 nM and mostly unrelated to previously known ligands were found. Implications for future studies are considered.

Despite the well-known benefits of hospice care for seriously ill children and their families, pediatric hospice services remain difficult to access across community settings. The vast majority of hospice organizations utilize an adult-focused paradigm, in which adult-trained providers often lack the requisite pediatric-specific education to provide high-quality hospice care for children and families. Hospice nurses self-report significant discomfort with pediatric care provision, resulting in hesitation by hospice agencies to accept pediatric patients, further exacerbating deficits in pediatric hospice access across the nation. Given the growing numbers of children with serious illness eligible for hospice in the community, innovative strategies are urgently needed. To address this gap, a multidisciplinary team comprising physicians, nurses, psychosocial clinicians, community members, and bereaved parents was convened to develop the Regional Pediatric Education and Assistance Collaborative for Hospice Nurses (REACH) initiative. Using a community-based participatory research approach, a stakeholder-driven tele-educational intervention was designed, refined, and implemented as a pilot for hospice nurses across Tennessee. Pilot data showed this hub-and-spoke model to be feasible, acceptable, and impactful, increasing hospice nurses' knowledge and comfort with provision of pediatric care in the community. Future work will focus on the expansion of REACH across Louisiana, followed by other states.