Supplementary Figure S5. Intracellular localization of Frag J and Frag J-Δ.

Supplementary Figure S1. Purification of GRAIL1 C-terminal fragment.

Supplementary Figure S4. Frag J/Jm mediated loss of misfolded Fluc-DM is independent of mutant p53 status.

Supplementary Figure S7. Design and testing of scrambled peptides as negative controls.

Supplementary Figure S3. GRAIL1 is a glycosylated protein that undergoes processing.

Supplementary Figure S2. Full-length GRAIL1 does not interact with DNAJA1-GFP.

Supplementary Table S1. Frag J-Jm Interactors

Supplementary Figure S6. Metabolic stability of Pep J.

Background Radiotherapy (RT) is the primary treatment for diffuse midline glioma (DMG), a lethal pediatric malignancy defined by histone H3 lysine 27-to-methionine (H3K27M) mutation. Based on the loss of H3K27 trimethylation producing broad epigenomic alterations, we hypothesized that H3K27M causes a functional double-strand break (DSB) repair defect that could be leveraged therapeutically with PARP inhibitor and RT for selective radiosensitization and antitumor immune response. Methods H3K27M isogenic DMG cells and orthotopic brainstem DMG tumors in immune deficient and syngeneic, immune competent mice were used to evaluate the efficacy and mechanisms of PARP1/2 inhibition by olaparib or PARP1-selective inhibition by AZD9574 with concurrent RT. Results H3K27M mutation caused a homologous recombination repair (HRR) defect characterized by impaired RT-induced K63-linked polyubiquitination of histone H1 and inhibition of HRR protein recruitment. H3K27M DMG cells were selectively radiosensitized by olaparib in comparison to isogenic controls, and this effect translated to efficacy in H3K27M orthotopic brainstem tumors. Olaparib and RT induced an innate immune response and induction of NK cell (NKG2D) activating ligands leading to increased NK cell-mediated lysis of DMG cells. In immunocompetent syngeneic orthotopic DMG tumors, either olaparib or AZD9574 in combination with RT enhanced intratumoral NK cell infiltration and activity in association with NK cell-mediated therapeutic responses and favorable activity of AZD9574. Conclusions The HRR deficiency in H3K27M DMG can be therapeutically leveraged with PARP inhibitors to radiosensitize and induce an NK cell-mediated antitumor immune response selectively in H3K27M DMG, supporting the clinical investigation of PARP1 inhibitors with RT in DMG patients.

H3K27 mutant(H3K27M) tumors are the most common type of diffuse midline gliomas and are lethal childhood tumors with a median survival of less than 12 months. Radiation is a critical treatment, yet these tumors recur rapidly within the radiation field due to resistance to radiation-induced DNA damage, allowing the tumor cells to survive. However, the interactions mediating efficient DNA repair, radiation resistance, and recurrence are poorly understood. Our goal was to identify mediators of treatment resistance in H3K27M tumors for therapeutic targeting to improve survival. We developed an in vivo recurrence model to assess mediators of treatment resistance. Bulk RNA, single cell RNA, and spatial transcriptomics analyses were used to determine drivers of resistance, and genes of interest were mechanistically assessed for their role in resistance. We determined that H3K27M epigenetic reprogramming results in activation of cell-surface protein THY1 which is highly upregulated in all H3K27M tumors. We then confirmed in numerous in vitro and in vivo models that THY1 mediates significant treatment resistance. We determined that THY1 mediates resistance through rapid and efficient DNA repair. We then performed extensive mechanistic analyses which revealed that THY1 signaling activates Fyn, a Src family kinase, resulting in downstream DNA repair. Remarkably both biological knockdown of Fyn and pharmacological Fyn inhibition, with brain-penetrating saracatinib, result in delayed DNA repair and tumor cell death. We have uncovered that the Thy1-Fyn signaling cascade mediates rapid DNA repair and treatment resistance. Pharmacological Fyn inhibition successfully delays DNA repair which could improve patient survival. As saracatinib is highly brain penetrant(0.5:1 Brain: Plasma ratio) and has demonstrated safety in Phase I/II Alzheimer’s clinical trials, it is an ideal drug for clinical translation. We are currently designing a Phase 0/1 study to assess drug penetration, efficacy, and safety with saracatinib and radiation.

Purpose Biologically-informed radiation therapy (RT) targeting an adversely prognostic hypercellular/hyperperfused imaging phenotype in patients with newly diagnosed glioblastoma (GBM) may improve outcomes by identifying emerging regions of treatment resistance associated with overall survival, and is under investigation in an ongoing phase 2 trial (NCT04574856) of individualized, response-adaptive RT. Methods and Materials In this single-arm phase 2 study, patients with newly diagnosed GBM after resection undergo dose-intensified chemoradiation targeting the residual hypercellular (TVHCV, 2 SD above mean intensity contralateral normal brain) and hyperperfused tumor volume (TVCBV, 1 SD above contralateral normal frontal lobe gray matter) identified using high b-value diffusion-weighted and dynamic contrast-enhanced perfusion magnetic resonance imaging. The combination of TVHCV and TVCBV (TVHCV/TVCBV) is treated to 50 Gy in 20 fractions (2.5 Gy/fraction), and after mid-RT reassessment, the persistent and developing TVHCV/TVCBV is treated to 30 Gy in 10 fractions (3 Gy/fraction). The primary endpoint is improvement in overall survival, with planned interim safety analysis. Results At interim analysis, 16 of 30 patients were enrolled. Median age was 58 years (range, 29-75) and 69% were male. No patient underwent biopsy only, and 50% had gross total resection; 19% had O6-methylguanine-DNA methyltransferase methylated tumors. Median TVHCV/TVCBV was 6.9 cc (range, 1.9-42.8) pre-RT and 30% (range, 1%-72%) was nonenhancing. By mid-RT, TVHCV/TVCBV was reduced to 4.2 cc (range, 0.8-34.3) and 47% (range, 3%-74%) was nonenhancing. The TVHCV/TVCBV persisting from pre-RT to mid-RT was 2.3 cc (range, 0-24.2), with an additional 1.8 cc (range, 0.3-20.6) newly developing outside of the initial region. All patients underwent adaptive replanning for boost without interruption. Planned interim analysis determined an acceptable rate of neurologic toxicity and safety to continue enrollment. Conclusions Individualized, response-adaptive chemoradiation using an advanced imaging biomarker to assess emerging and especially nonenhancing regions of treatment resistance in patients with GBM is feasible, with short-term safety and longer-term efficacy outcomes anticipated with completion of accrual.

Odds ratios (OR) and p values for multi-variable models for acute and late toxicity outcomes including covariates of p16+ OPSCC vs LAHNSCC, primary tumor volume (GTVp) and RT boost volume treated.

Linear mixed model results for arm*time interaction EORTC QLQ30/HN35 PRO subscales.

The brain avidly consumes glucose to fuel neurophysiology1. Cancers of the brain, such as glioblastoma, relinquish physiological integrity and gain the ability to proliferate and invade healthy tissue2. How brain cancers rewire glucose use to drive aggressive growth remains unclear. Here we infused 13C-labelled glucose into patients and mice with brain cancer, coupled with quantitative metabolic flux analysis, to map the fates of glucose-derived carbon in tumour versus cortex. Through direct and comprehensive measurements of carbon and nitrogen labelling in both cortex and glioma tissues, we identify profound metabolic transformations. In the human cortex, glucose carbons fuel essential physiological processes, including tricarboxylic acid cycle oxidation and neurotransmitter synthesis. Conversely, gliomas downregulate these processes and scavenge alternative carbon sources such as amino acids from the environment, repurposing glucose-derived carbons to generate molecules needed for proliferation and invasion. Targeting this metabolic rewiring in mice through dietary amino acid modulation selectively alters glioblastoma metabolism, slows tumour growth and augments the efficacy of standard-of-care treatments. These findings illuminate how aggressive brain tumours exploit glucose to suppress normal physiological activity in favour of malignant expansion and offer potential therapeutic strategies to enhance treatment outcomes.

Purpose: Contouring targets for stereotactic body radiation therapy (SBRT) requires expertise for each body site. Likewise, peer reviewers require sufficient expertise to provide an adequate review. In this work, we investigate physician self-reported expertise for performing peer review by body site and how the quality of SBRT peer review is impacted by the expertise of the reviewer. Methods and Materials: The results of 7 years of SBRT rounds, which included information on body site, attending and reviewing physicians, changes to targets, prescriptions, and planning target volume, were analyzed. We surveyed physicians on their expertise for reviewing each body site and defined them as being an expert by body site if they indicated a moderate or high level of competence. Multivariable logistic regression models were used to assess the association between reviewing physician expertise and planning data changes, and whether this varied by body site or by presenting physician expertise. Models were adjusted for physician and case characteristics, and generalized estimating equations were used to account for the correlation of cases reviewed by the same physician. Results: The survey response rate was 95% (20/21) with 4103 cases for analysis. Reviewing physician experts were more likely to make any change, gross target volume, and prescription compared with reviewing physicians who were nonexperts. Controlling for physician expertise and case characteristics, brain, liver, spine, and stereotactic radiosurgery cases have an increased odds of any change being made when compared to lung cases, with odds ratios of 2.42 (95% CI, 1.78-3.30), 1.55 (95% CI, 1.19-2.01), 1.7 (95% CI, 1.31-2.20), and 2.18 (95% CI, 1.73-2.77), respectively. Conclusions: The extent to which changes are made during contour review is associated with both peer reviewer disease-site expertise and disease site. In larger radiation oncology departments relying on a general coverage model, rather than review by disease-site experts, peer review results in variations in the outcome of the preplanning review.