Targeting H3K27-Altered Diffuse Midline Glioma with Combined MEK and HDAC Inhibition: Clinical Response and in Vitro Validation

1. Introduction

Histone H3 lysine 27 (H3K27)-altered diffuse midline gliomas (DMG) represent one of the most lethal central nervous system (CNS) malignancies, with a median overall survival rate of 14- 20 months [1,2,3,4]. The pathognomonic histone H3.3 (H3-3A) mutation at lysine 27 (K27) results in a pathologic epigenome characterized by a global reduction of H3 K27 trimethylation (H3K27me3) [5]. While DMGs primarily affect children, they also occur in adolescent and young adult (AYA) populations [6]. Standard of care remains maximally safe surgical resection followed by radiotherapy. Although temozolomide is frequently administered concurrently and adjuvantly for radiosensitization, its efficacy remains unproven [7,8]. Radiotherapy is palliative, and disease progression typically occurs within 6 to 12 months. Emerging treatment modalities, including oncolytic virotherapy, dopamine receptor antagonists, and chimeric antigen receptor T-cell therapies have shown efficacy signals in early clinical experiences but long-term disease control remains elusive [6,9,10,11]. In this report, we describe an adolescent with Neurofibromatosis Type 1 (NF1) and a spinal cord H3K27-altered diffuse midline glioma who achieved 22 months of radiotherapy-free, progression-free survival (PFS) using a novel combination of the histone deacetylase (HDAC) inhibitor vorinostat and the MEK inhibitor selumetinib.

Neurofibromatosis Type 1 (NF1), a multisystem disorder phenotypically characterized by café-au-lait macules, is the most common tumor predisposition syndrome [12], though the development of high-grade gliomas in the adolescent and young adult NF1 population is rare [13,14]. More common NF1-associated neoplasms in the AYA age group include low-grade gliomas, plexiform neurofibromas, and malignant peripheral nerve sheath tumors (MPNST) [15]. NF1-related tumors are characterized by hyperactivation of the RAS-mitogen-activated protein kinase (MAPK) [16–21pathway due to loss of neurofibromin-mediated negative regulation of Ras signaling. MAPK pathway inhibitors, including MEK inhibitors, are now standard in managing various NF1-related and sporadic pediatric neoplastic pathologies [16,17,18,19,20,21]. High-grade MPNSTs evolve from plexiform neurofibromas via additional somatic alterations, including loss-of-function mutations in the Polycomb Repressive Complex 2 (PRC2) [22,23]. This loss results in a global depletion of (H3K27me3) creating an epigenetic landscape analogous to H3K27-altered DMGs, where the K27M substitution itself antagonizes PRC2 function [3,24,25]. Preclinical models of H3K27me3-deficient MPNST demonstrate profound synergy between HDAC and MAPK inhibition [26], a finding currently being translated in a phase 0 clinical trial (NCT06693284). Guided by this biological rationale, and published safety of HDAC inhibition in the DMG population [27], combination therapy with selumetinib and vorinostat was initiated as a personalized strategy for an adolescent with NF1 and a spinal DMG who sought to avoid radiotherapy due to the risk of exacerbated toxicity in the setting of baseline paraplegia.

2. Materials and Methods

2.1. Clinical Assessment and Treatment

The clinical course was reviewed retrospectively, including diagnostic pathology, germline testing, serial MRI, treatment exposure, and reported adverse events.

2.2. Cell Models and Culture

To investigate whether the notable clinical activity of dual HDAC and MEK inhibition was unique to the patient’s underlying germline NF1 genetics or represented a broader therapeutic vulnerability driven by the H3K27me3-deficient epigenetic phenotype, we conducted an in vitro validation study using two independently derived H3K27M-mutant glioma cell lines, DIPG17 and SF8628, together with the isogenic K27-rescued DIPGWT derivative of DIPG17 and two H3-wildtype pediatric-type high-grade glioma lines, SF9427 and SJ-HGGx2.

Glioma cell lines were maintained as serum-free adherent cultures using glioma tumor stem cell medium based on established protocols.28,29 The basal medium consisted of a 1:1 mixture of KnockOut DMEM/F-12 (Thermo Fisher Scientific, Cat# 12660012) and phenol red-free Neurobasal Medium (Thermo Fisher Scientific, Cat# 12348017). This base was supplemented with 1× MEM Non-Essential Amino Acids (Thermo Fisher Scientific, Cat# 11140050), GlutaMAX (Thermo Fisher Scientific, Cat# 35050061), sodium pyruvate (Thermo Fisher Scientific, Cat# 11360070), and 1M HEPES (Thermo Fisher Scientific, Cat# 15630080). The medium was enriched with B-27 Supplement (vitamin A-free) (Thermo Fisher Scientific, Cat# 12587010), N-2 Supplement (Thermo Fisher Scientific, Cat# 17502048), StemPro Neural Supplement (Thermo Fisher Scientific, Cat# A1050801), 0.2% heparin sodium salt (StemCell Technologies, Cat# 07980), and Primocin (InvivoGen, Cat# ant-pm-1). Recombinant human growth factors were added at final concentrations of 20 ng/mL epidermal growth factor (PeproTech, Cat# AF-100-15), 20 ng/mL basic fibroblast growth factor (PeproTech, Cat# 100-18B), 10 ng/mL platelet-derived growth factor-AA (Cell Guidance Systems, Cat# GFH16AF-100), and 10 ng/mL platelet-derived growth factor-BB (PDGF-BB; Cell Guidance Systems, Cat# GFH18AF-100). For adherent monolayer growth, culture vessels were pre-coated with Corning hESC-Qualified Matrigel matrix (Corning, Cat# 354277) diluted in chilled, HEPES-buffered DMEM/F-12 (Thermo Fisher Scientific, Cat# 11330032) and allowed to polymerize at 37 °C for 2 hours prior to cell seeding.

2.3. Drug-Response Assay

Glioma cells were seeded onto matrix-coated 96-well plates at 10,000 cells per well. Following overnight attachment, culture medium was replaced with treatment medium containing three-fold serial dilutions of mirdametinib (Selleckchem, Cat# S1036), vorinostat (Selleckchem, Cat# S1047), or their fixed-ratio combination. Maximum concentrations were 1,000 µM mirdametinib and 800 µM vorinostat, with the combination maintaining a fixed 1.25:1 mirdametinib:vorinostat ratio. This protocol mirrored the optimized experimental conditions for our MPNST experience [26]. Vehicle control wells were treated with a DMSO concentration equivalent to the highest concentration present in the drug dilutions.

Following 48 hours of continuous drug exposure, alamarBlue HS cell viability reagent (Thermo Fisher Scientific, Cat# DAL1025) was added to each well according to the manufacturer’s protocol. After a 4-hour incubation at 37 °C, absorbance at 570 and 600 nm was measured using a Cytation 5 cell imaging multi-mode reader (BioTek). Percent reduction of alamarBlue was calculated using the manufacturer’s dual-wavelength extinction coefficient method. Media-only background was subtracted from each measurement, and relative viability was normalized to the mean blank-corrected signal of vehicle-treated wells, defined as 100% viability. Six technical replicate wells at each concentration were averaged within each independent biological experiment.

2.4. Statistical Analysis

Combination benefit across the complete dose-response range was quantified for each independent biological experiment using the difference in normalized area under the viability curve (ΔAUC), defined as AUCvorinostat − AUCcombination. Larger positive ΔAUC values therefore indicate greater reduction in viability following addition of mirdametinib to vorinostat. First, ΔAUC was compared between DIPG17 and its isogenic K27-rescued DIPGWT derivative using a two-sided unpaired Welch t-test. Second, cross-background activity was evaluated using an equal-weight Welch-Satterthwaite fixed contrast comparing the mean of the two independently derived H3K27M lines, DIPG17 and SF8628, with the mean of the two independently derived H3-wildtype lines, SF9427 and SJ-HGGx2. Dose-response relationships were fit separately for each biological experiment using a four-parameter log-logistic model, with lower and upper asymptotes fixed at 0% and 100% viability, respectively. Statistical analyses and graphics were performed in R using the drc, ggplot2, and ggprism packages.

3. Results

3.1. Patient Presentation, Treatment, and Outcome

An 11-year-old with Neurofibromatosis Type 1 (germline pathogenic NF1 c.1586T>G (p.Leu529Arg) mutation) presented with scoliosis, neck pain, urinary hesitancy, and gait instability. MRI revealed a thoracic spinal cord mass (T2-T3 to T9) with a syrinx extending to C2 and surrounding edema (Figure 1A). In April 2021, the patient underwent a gross-total surgical resection. Post-operative baseline neurologic deficits included lower extremity paresis and neurogenic bladder. Histopathology confirmed a diffuse midline glioma, H3K27-altered (WHO grade 4). Immunohistochemistry was positive for H3K27M, SOX10, GFAP, S100, and Olig2, and negative for both H3K27me3 and MGMT promoter methylation. A neurofilament stain confirmed an infiltrative pattern of growth. Germline testing for EPCAM, MLH1, MSH2, MSH6, and PMS2 returned with no alterations, ruling out constitutional mismatch repair deficiency disorders [14].

Post-operative imaging showed trace intramedullary contrast enhancement, consistent with post-operatives changes or residual disease (Figure 1B). To avoid the toxicities of spinal radiotherapy in the context of NF1 and existing neurological compromise, the patient began selumetinib (25 mg/m2 BID) and vorinostat (230 mg/m2, days 1-7 and 15-21) in May 2021. The regimen was well-tolerated, with side effects limited to grade 1 diarrhea and grade 1 dermatitis. The patient maintained typical development, including normal linear growth and initiation of menses. Follow-up MRI at three and five months showed a contrast enhancing focus on the superior margin of the previously resected lesion, concerning for residual disease (Figure 1C, D). After eight months of therapy, this focus of enhancement resolved and there was no radiographic evidence of disease from January 2022 through February 2023 (Figure 1E,F).

In April 2023, 23 months after initiating therapy, the patient presented with back pain and worsening of her baseline neurologic deficits. MRI confirmed tumor recurrence, and she was initiated on dexamethasone for management of spinal cord compression. The family subsequently reported decreased medication adherence in the preceding months, involving pill-hiding behavior in the context of an underlying anxiety disorder. Despite a second resection and salvage radiotherapy, the disease proved refractory, and the patient passed away in September 2023.

3.2. In Vitro Combination Activity Across H3K27-Altered and Comparator Models

Biallelic NF1 tumor status was unable to be determined in this case owing to tissue availability limitations. To assess whether the combination effect extended beyond the NF1-/- genotype, we evaluated H3K27M-mutant, isogenic K27-rescued, and H3-wildtype glioma models (Figure 2) using the MEK inhibitor mirdametinib and HDAC inhibitor vorinostat.

In two independently derived H3K27M-mutant lines, DIPG17 and SF8628, addition of mirdametinib enhanced vorinostat activity across the dose-response range, despite relatively limited single-agent activity of mirdametinib. Mean ΔAUC was 25.5 in DIPG17 and 3.1 in its isogenic K27-rescued DIPGWT derivative (mean difference, 22.4; 95% CI, 10.1–34.7; Welch p = 0.015). Across independent cell-line backgrounds, mean ΔAUC was 22.9 for the two H3K27M lines and 9.7 for the two H3-wildtype lines (difference, 13.2; 95% CI, 8.1–18.3; Welch–Satterthwaite fixed contrast p < 0.001). Both comparisons remained significant after Holm correction.

4. Discussion

The clinical course of this patient is noteworthy, as a progression-free survival (PFS) exceeding 20 months without upfront radiotherapy is exceptional in the management of DMG. This prolonged disease control must be interpreted in the context of the surgical accessibility of the spinal lesion, whereas most DMGs involve more eloquent structures and are ineligible for near-total resection. However, maximal safe resection alone is insufficient for durable control of DMG, particularly in the presence of residual contrast-enhancing disease. It remains unknown whether the patient stopped adhering to therapy, permitting disease regrowth, or if she stopped therapy without seeking medical advice owing to the return of neurologic symptoms; thus, whether further PFS would have been observed remains conjecture. Another important caveat is that the disease response observed here was achieved prior to the institution of radiotherapy, which could in theory modulate cellular responses to targeted therapies in unpredictable ways. Our institutional experience evaluating the synergy of histone deacetylase and MEK inhibition for H3K27me3-deficient MPNST (NCT06693284), combined with this signal of efficacy in a patient with DMG, carries significant implications not only for DMGs but also for Posterior Fossa Group A (PFA) ependymomas, another lethal childhood malignancy characterized by a global loss of H3K27me3.

While global loss of H3K27me3 in MPNSTs typically arise from inactivating mutations in the PRC2 subunits SUZ12 or EED, PFA ependymomas and DMGs achieve a similar H3K27me3-deficient phenotype through EZHIP overexpression or H3K27 alterations, respectively [2,30,31,32]. This depletion of repressive trimethylation facilitates a reciprocal gain in acetylation (H3K27ac) at the same residue, which promotes an active, enhancer-driven chromatin state [32]. HDAC inhibitors have emerged as a potent therapeutic strategy to exploit this vulnerability. By blocking the removal of acetyl groups, HDAC inhibitors induce hyperacetylation, leading to transcriptional dysregulation [33,34].

Importantly, H3K27me3 loss converges with heightened MAPK signaling across these malignancies, linking the altered epigenome to a therapeutically targetable kinase pathway. In MPNSTs, the loss of PRC2 acts as a potent amplifier of Ras-driven transcription, synergizing with NF1 loss to drive oncogenesis [35,36]. A similar phenomenon is observed in DMGs and PFA ependymomas, with observed MAPK pathway activation. H3K27-altered DMGs frequently exhibit functional hyperactivation of the RAS/MAPK signaling cascade as a direct consequence of their defining epigenetic state [37]. This epigenetically driven MAPK activation is further augmented by the activity of commonly co-occurring activating alterations of receptor tyrosine kinases such as PDGFRA [38,39,40]. Intriguingly, PFA ependymoma exhibits some of the highest functional phosphorylated ERK scores across pediatric central nervous system tumors despite absence of genomic mutations in the MAPK pathway [38,41,42]. Together, these data identify recurrent MAPK pathway activation across H3K27me3-deficient nervous system tumors and provide a rationale for extending dual HDAC/MEK inhibition beyond MPNST.

The cell-line results support testing H3K27me3 deficiency as a candidate marker of combination benefit. The well-tolerated nature of these agents, coupled with proven safety profiles and blood-brain-barrier penetrance [18,43,44], warrants independent in vivo and prospective clinical validation. The co-occurrence of MAPK activation and H3K27me3 loss suggest that dual inhibition of HDACs and the MAPK pathway may provide a synergistic therapeutic approach for these aggressive malignancies.

5. Conclusions

An adolescent with H3K27-altered spinal diffuse midline glioma experienced prolonged radiotherapy-free disease control while receiving selumetinib and vorinostat after gross-total resection. In vitro, adding mirdametinib to vorinostat produced greater viability reduction in H3K27M-mutant models than in the K27-rescued and H3-wildtype comparators. Together, these observations justify prospective testing of H3K27me3 loss as a candidate biomarker for combined MEK and HDAC inhibition.