Robert T. Galvin

Background/Objectives: Histone H3 K27-altered diffuse midline gliomas (DMGs) have poor outcomes and are characterized by global loss of H3K27 trimethylation (H3K27me3). Evidence from other H3K27me3-deficient tumors suggests a potential vulnerability to combined histone deacetylase (HDAC) and MEK inhibition. We examined a clinical experience with this combination and tested its activity in glioma models. Methods: We reviewed the course of an 11-year-old with neurofibromatosis type 1 and a thoracic spinal DMG treated with selumetinib and vorinostat after gross-total resection, without upfront radiotherapy. In post hoc viability assays, we tested HDAC and MEK inhibition in two H3K27M-mutant glioma lines, an isogenic K27-rescued derivative, and two H3-wildtype lines. Combination benefit was measured as the reduction in area under the viability curve relative to vorinostat alone. Results: The patient experienced 22 months of radiotherapy-free, progression-free survival before recurrence, with a postoperative enhancing focus resolving during treatment. Treatment was associated with grade 1 diarrhea and dermatitis. In vitro, combination benefit was greater in the H3K27M-mutant lines than in the H3-wildtype lines (mean difference, 13.2; 95% confidence interval, 8.1–18.3; p < 0.001). Benefit was also greater in DIPG17 than in its K27-rescued derivative (mean difference, 22.4; 95% confidence interval, 10.1–34.7; p = 0.015). Conclusions: This clinical observation and the post hoc validation experiments support prospective study of H3K27me3 loss as a candidate biomarker for combined HDAC and MEK inhibition.

Background: High-grade malignant peripheral nerve sheath tumors (MPNST) frequently lose Polycomb Repressive Complex 2 (PRC2), reducing the repressive histone mark, H3K27me3. We hypothesized that H3K27me3 loss sensitizes MPNST to oncolytic HSV due to impaired transcriptional resistance. HSV replication is also restricted by the integrated stress response, normally countered by HSV ICP34.5, representing an additional resistance pathway to ICP34.5 -attenuated HSV. Methods: PRC2 loss-of-function was evaluated as a biomarker of HSV G47Δ ( ΔICP34.5 ) susceptibility in MPNST using isogenic, pharmacologic inhibition, and genetic rescue models. CUT&RUN and RNA-sequencing defined PRC2-dependent viral and host responses. We engineered a G47Δ-derived vector (G47Δ ₂ ) with tumor-regulated ICP34.5 expression [G47Δ ₂ ,ins( BIRC5>ICP34.5 )] and evaluated efficacy in MPNST xenografts. Results: H3K27me3 deficiency increased G47Δ sensitivity and viral spread in vitro . CUT&RUN demonstrated PRC2-dependent H3K27me3 accumulation on the viral genome, explaining G47Δ activity in H3K27me3-deficient MPNST. RNA sequencing of G47Δ infected MPNST cells revealed heightened integrated stress response activity and blunted induction of translation-associated programs in the PRC2-deficient state (both p 2 (8.3%; 4.5–12.2%) and controls (22.5%; 17.4–27.8%). A murine gastrointestinal toxicity phenotype reduced all-cause event-free survival, indicating vector and dosing optimizations are needed to widen the therapeutic window. Conclusions: H3K27me3 loss creates a paradoxical antiviral state that tumor-selective ICP34.5 overcomes to rescue HSV anti-tumor activity, supporting biomarker-driven viral engineering to exploit the permissive transcriptional state of H3K27me3-deficient MPNSTs. Key Points: Loss of a key epigenetic mark (H3K27me3) MPNST is a biomarker of susceptibility to oncolytic HSV H3K27me3-deficient MPNST mounts a compensatory stress response that limits HSV propagation. Engineered viral gene therapy to overcome the stress response rescues antitumor activity. Importance of the Study: High-grade MPNST lacks effective systemic therapies, and biomarkers that can guide oncolytic virotherapy are poorly defined. This study identifies loss of PRC2-dependent H3K27me3 as a marker of susceptibility to oncolytic HSV but shows that this same epigenetic state also primes a compensatory integrated stress response that limits productive viral propagation at the level of protein translation. These findings connect a tumor-specific chromatin defect to both enhanced viral permissiveness and a compensatory resistance mechanism. By restoring tumor-selective expression of ICP34.5 , an HSV-encoded antagonist of the integrated stress response frequently removed to promote oncolytic vector attenuation, we demonstrate that this second barrier can be therapeutically targeted to improve antitumor activity. The work therefore provides a mechanistic framework for matching viral engineering to tumor epigenetic state. Robust anti-tumor activity must be balanced with therapeutic safety, providing avenues for further vector optimization. This biomarker-driven strategy may be relevant to other H3K27me3-deficient nervous system tumors.

Background/Objectives: Histone H3 K27-altered diffuse midline gliomas (DMGs) have poor outcomes and are characterized by global loss of H3K27 trimethylation (H3K27me3). Evidence from other H3K27me3-deficient tumors suggests a potential vulnerability to combined histone deacetylase (HDAC) and MEK inhibition. We examined a clinical experience with this combination and tested its activity in glioma models. Methods: We reviewed the course of an 11-year-old with neurofibromatosis type 1 and a thoracic spinal DMG treated with selumetinib and vorinostat after gross-total resection, without upfront radiotherapy. In post hoc viability assays, we tested HDAC and MEK inhibition in two H3K27M-mutant glioma lines, an isogenic K27-rescued derivative, and two H3-wildtype lines. Combination benefit was measured as the reduction in area under the viability curve relative to vorinostat alone. Results: The patient experienced 22 months of radiotherapy-free, progression-free survival before recurrence, with a postoperative enhancing focus resolving during treatment. Treatment was associated with grade 1 diarrhea and dermatitis. In vitro, combination benefit was greater in the H3K27M-mutant lines than in the H3-wildtype lines (mean difference, 13.2; 95% confidence interval, 8.1–18.3; p < 0.001). Benefit was also greater in DIPG17 than in its K27-rescued derivative (mean difference, 22.4; 95% confidence interval, 10.1–34.7; p = 0.015). Conclusions: This clinical observation and the post hoc validation experiments support prospective study of H3K27me3 loss as a candidate biomarker for combined HDAC and MEK inhibition.

Map demonstrating the countries represented by contributing institutions.

Long-term survivors ≥ 5 years (n = 12)

Patients with G34V mutation subtype (n = 9)

Initial chemotherapy combinations

Kaplan-Meier survival plots demonstrating overall survival. (A). Initial chemotherapy (all ages): TMZ-based vs TMZ/CCNU. (B). Initial chemotherapy (under 18 years old): TMZ-based vs TMZ/CCNU. (C). Re-irradiation: yes vs no. (D). ATRX mutation: yes vs no. (E). PDGFRA amplification: yes vs no. (F) TP53 mutation: yes vs no. (G). PDGFRA mutation: yes vs no.

Purpose: Knowledge of prognostic factors and long-term survival in patients with diffuse hemispheric glioma, H3 G34–mutant (DHG, H3 G34), remains limited in this tumor with a poor prognosis.Experimental Design:This retrospective, multi-institutional study investigated prognostic variables for patients with DHG, H3 G34, and their association with progression-free survival (PFS) and overall survival (OS). Uni- and multivariable Cox proportional hazard models were applied with multiple imputed datasets.Results:A total of 153 patients (142 G34R, 9 G34V, 2 via DNA methylation) were included. The median age at diagnosis was 17 years (range, 2–45). Initial gross/near total resection (GTR/NTR) was achieved in 43% of patients. Radiation was given in 91% (85% focal irradiation), and initial chemotherapy was given in 87% [70% temozolomide-based (TMZ), 25% TMZ/lomustine, 5% non-TMZ]. Median OS was 24 months [interquartile range (IQR), 22–28] with a median PFS of 14 months (IQR, 12–19). Twelve patients (8%) were found to be long-term survivors (≥5 years). Exploratory multivariable analysis showed that adjuvant radiotherapy [HR, 0.076; 95% confidence interval (CI), 0.033–0.17] and achieving GTR/NTR compared with < NTR (HR, 0.51; 95% CI, 0.33–0.78) were associated with improved PFS. Multivariable analysis showed improved OS with increasing age at diagnosis (HR, 0.70; 95% CI, 0.57–0.87), initial radiotherapy (HR, 0.38; 95% CI, 0.15–0.96), and initial GTR/NTR compared with < NTR (HR, 0.60; 95% CI, 0.37–0.97).Conclusions:This cohort highlights prognostic factors for patients with DHG, H3 G34, and describes relapse patterns and therapy approaches. Clinical trials and prospective registries are needed to improve outcomes.