Rachael Vaubel’s research while affiliated with Mayo Clinic - Rochester and other places

ATM inhibitors are being developed as radiosensitizers to improve the antitumor effects of radiotherapy, but ATM inhibition can also radiosensitize normal tissues. Therefore, understanding the elevated risk for normal tissue toxicities is critical for radiosensitizer development. This study focused on modeling the relationship between acute mucosal toxicity, radiation dose, fractionation schedule, and radiosensitizer exposure. The ATM inhibitor WSD0628 was combined with single or fractionated doses of radiation delivered to the oral cavity or esophagus of FVB mice. The potentiation by WSD0628 was quantified by a sensitizer enhancement ratio (SER), which describes the changes in radiation tolerance for radiation combined with WSD0628 relative to radiation-only regimens. WSD0628 profoundly enhanced radiation-induced acute oral and esophageal toxicities. For oral mucosal toxicity, the enhancement by WSD0628 with 3 fractions of radiation resulted in an SER ranging from 1.3 (0.25 mg/kg) to 3.1 (7.5 mg/kg). For the 7.5 mg/kg combination, the SER increased with increasing number of fractions from 2.2 (1 fraction) to 4.3 (7 fractions) for oral toxicity and from 2.2 (1 fraction) to 3.6 (3 fractions) for esophageal toxicity, which reflects a loss of the normal tissue sparing benefit of fractionated radiation. These findings were used to develop a modified biologically effective dose model to determine alternative radiation schedules with or without WSD0628 that result in similar levels of toxicity. Successful radiosensitizer dose-escalation to maximally effective therapeutic dose will require careful deliberation of tumor site and reduction of radiation dose-volume limits for organs at risk.

BACKGROUND Patients with meningiomas who have progressed after multimodal treatments face a significant shortage of viable therapeutic options. Recent advancements in the molecular understanding of meningiomas have identified the FAK and RAF/MEK signaling pathways as potential promising targets for progressive meningiomas. METHODS In vitro, we tested two human meningioma cell lines, IOMM-Lee (NF2 wild-type and CDKN2A loss) and CH157-MN (NF2 mutant). The cells were plated in Matrigel-coated wells, treated with defactinib (FAK inhibitor), VS-6766 (RAF/MEK inhibitor, avutometinib) and the combination over a 7-day period and assessed for cell viability using the CellTiter-Glo 3D assay. In vivo, we tested the efficacy of the FAK inhibitor VS-4718 (possessing favorable pharmacokinetic properties in mice) and RAF/MEK inhibitor VS-6766, both given daily via oral gavage, in athymic nude mice bearing IOMM-Lee or CH157-MN tumors. RESULTS In the matrigel assay, VS-6766 suppressed IOMM-Lee and CH157-MN cells in a dose dependent manner. The drug combination was mostly additive in IOMM-Lee cells. However, CH157-MN cells showed a strong synergistic response across all drug concentrations tested. In the IOMM-Lee flank tumor model, VS-4718 alone had no effect, while VS-6766 alone or in combination significantly reduced tumor growth and extended median survival from 25 days to 47 days (p < 0.05). In the CH157 flank tumor model, VS-4718 monotherapy did not affect tumor growth or survival, but VS-6766 monotherapy slowed tumor growth and increased median survival from 10 days to 31 days (p < 0.001). Combining both drugs further reduced tumor growth and extended median survival to 42 days (p < 0.001). CONCLUSION Treatment with FAK and RAF/MEK inhibitors showed synergistic or combination effects in the NF2 mutant CH157-MN model in vitro and in vivo. RAF/MEK inhibitor monotherapy was effective for the IOMM-Lee model (NF2 wild-type), without clear benefit of combining with the FAK inhibitor. Investigations in intracranial tumor models are underway.

Most TERT promoter (TERTp) clinically relevant sequence variants in CNS tumors are the canonical C228T and C250T mutations. Non-canonical TERTp mutations have been reported mainly in non-CNS tumors. We describe 12 CNS tumors with non-canonical TERTp variants interpreted as clinically relevant. Using clinical targeted NGS panels (2020-2024), we detected c.-100_-79dup (n=2), c.-92_-91insGGCGGCCCCGCCCCTTCCTTTC (n=1), c.-118_-117insTCCCCGGCCCAGCCCCTTCCGGG (n=1), c.-123_-76dup (n=1), c.-110_-89dup (n=2), and c.-57A>C (n=5). Most cases had chromosomal microarray (n=11) and/or methylation array data analyzed using the NCI/Bethesda v.2 classifier (n=9). All non-canonical TERTp variants occurred at a variant allele frequency suggestive of somatic clonal origin (mean, 34%; range, 18-46), and in adults (mean age, 60 years; range, 36-88). Except for the c.-57A>C variant, all non-canonical TERTp variants occurred in tumors with histological and/or molecular diagnostic features of glioblastoma, IDH-wildtype (GBM), often epigenetically aligning to this tumor type. All five tested cases matched to superfamily glioblastoma (score ≥ 0.9), with three to class GBM_RTK_I (>0.9 in 2, 0.86 in 1) and two to GBM_RTK_II (>0.9). The size and sequence of such variants suggest that these may be functionally equivalent to canonical mutations or generate favorable TERTp activation motif conformations like previously reported activating duplications. The c.-57A>C variant is a well-established oncogenic mutation observed in other cancer types but has not been reported in CNS tumors. We describe five cases with the c.-57A>C variant: one GBM matching to methylation class GBM_MES_TYP (0.99); two oligodendroglioma, IDH-mutant and 1p/19q-codeleted, one of which had methylation data matching to class O_IDH (0.99); one diffuse glioma, IDH-mutant, which was partially 1p/19q-codeleted and matched to class O_IDH (0.99); and one anaplastic meningioma, aligning to superfamily meningioma (0.99)/MNG_MAL (0.79). Our findings show that non-canonical TERTp variants typically occur in tumor types that often harbor TERTp canonical mutations, expanding the spectrum of TERTp clinically relevant variants that are molecular biomarkers in CNS tumors.

BACKGROUND Mouse double minute 2 (MDM2) inhibits the tumor suppressor protein 53 (p53). Brigimadlin, a potent oral MDM2-p53 antagonist, restores wild-type (wt) p53 function and has shown early efficacy in patients with solid tumors. Glioblastoma (GBM) is an area of unmet need with 5-year survival at <10%. In p53 wt GBM patient-derived xenograft models, brigimadlin promotes tumor cell apoptosis and extends survival in combination with radiotherapy (RT). METHODS NCT05376800 is a Phase 0/Ia non-randomized, open-label, single-arm trial that aims to measure brigimadlin concentration in brain tumor tissue in patients with histologically or radiologically newly diagnosed GBM eligible for resection (Phase 0), and determine the maximum tolerated dose of brigimadlin plus RT in patients with TP53 wt, IDH wt, MGMT promoter unmethylated GBM (Phase Ia). In Phase 0, patients received one brigimadlin dose (30 mg or 45 mg) ~12-24 hours before resection. Phase 0 primary endpoints are the measured total concentration and the calculated unbound concentration of brigimadlin in brain tissue homogenate from non-contrast enhancing (NCE) and contrast enhancing (CE) regions. The predefined threshold for trial continuation is 0.5 nmol/L (corresponding to IC50 in GBM cell lines) unbound brigimadlin in CE samples in ≥50% of patients. Brigimadlin concentration was measured using liquid chromatography-mass spectrometry and corrected for amount of brigimadlin in residual blood. Unbound concentration was calculated using an in vitro estimate of unbound fraction (fu): 0.654% (rat brain slice). fu in human plasma was 0.22%. Ratio of unbound concentration in brain versus plasma (Kp,uu) was calculated. Biomarker testing was performed. RESULTS Data are available for 11 patients (brigimadlin 30 mg: n=6; 45 mg: n=5). In the 30 mg group, median total brigimadlin concentration in NCE samples was 267 nmol/L (4 samples, range 86-316 nmol/L) and 332 nmol/L (6 samples, range 272-952 nmol/L) in CE samples. In the 45 mg group, median total brigimadlin concentration in NCE samples was 197 nmol/L (5 samples, range 140-347 nmol/L) and 603 nmol/L (5 samples, range 441-905 nmol/L) in CE samples. Unbound concentration exceeded the 0.5 nmol/L threshold in most cases. Post-brigimadlin, an increase in selected p53 target gene expression was observed in CE versus NCE tissue. CONCLUSION Unbound brigimadlin concentrations in CE regions in all patients receiving the low dose of 30 mg brigimadlin exceeded the 0.5 nmol/L threshold. Kp,uu in most patients was close to 1 in CE regions. Biomarker data support target engagement in brain tissue. Our findings support continued investigation of brigimadlin in GBM. Recruitment is ongoing. Updated data will be presented.The trial is funded by Boehringer Ingelheim.

Pleomorphic xanthoastrocytomas (PXAs) harbor CDKN2A homozygous deletion in >90% of cases, resulting in loss of p16 expression by immunohistochemistry. Considering the proximity of MTAP to CDKN2A and their frequent concurrent deletions, loss of MTAP expression may be a surrogate for CDKN2A homozygous deletion. We evaluated p16 and MTAP expression in 38 patient PXAs (CNS WHO grade 2: n = 23, 60.5%; grade 3: n = 15, 39.5%) with available chromosomal microarray data to determine whether MTAP can be utilized independently or in combination with p16 to predict CDKN2A status. CDKN2A, CDKN2B, and MTAP homozygous deletion were present in 37 (97.4%), 36 (94.7%), and 25 (65.8%) cases, respectively. Expression of p16 was lost in 35 (92.1%) cases, equivocal in one (2.6%), and failed in 2 (5.3%), while MTAP expression was lost in 27 (71.1%) cases, retained in 10 (26.3%), and equivocal in one (2.6%). This yielded a sensitivity of 94.6% for p16 and 73.0% for MTAP in detecting CDKN2A homozygous deletion through immunohistochemistry. MTAP expression was lost in the 2 cases with failed p16 staining (combined sensitivity of 100%). Our findings demonstrate that combined p16 and MTAP immunostains correctly detect CDKN2A homozygous deletion in PXA, while MTAP expression alone shows reduced sensitivity.

Background The optimal diagnosis and management of patients with brain tumors currently uses the 2021 WHO integrated diagnosis of histomorphologic and molecular features. However, neuro-oncology practice in resource-limited settings usually relies solely on histomorphology. This study aimed to classify glioma cases diagnosed in the Department of Anatomic and Molecular Pathology, Lagos University Teaching Hospital, using the 2021 WHO CNS tumor classification. Methods Fifty-six brain tumors from 55 patients diagnosed with glioma between 2013 and 2021 were reevaluated for morphologic diagnosis. Molecular features were determined from formalin-fixed paraffin-embedded (FFPE) tissue using immunohistochemistry (IHC) for IDH1-R132H, ATRX, BRAF-V600E, p53, Ki67, and H3-K27M, OncoScan chromosomal microarray for copy number, targeted next generation sequencing for mutation and fusion and methylation array profiling. Results Of 55 central nervous system tumors, 3 were excluded from histomorphologic reevaluation for not being of glial or neuroepithelial origin. Of the remaining 52 patients, the median age was 20.5 years (range: 1 to 60 years), 38(73%) were males and 14(27%) were females. Seventy-one percent of the gliomas evaluated provided adequate DNA from archival FFPE tissue blocks. After applying the 2021 WHO diagnostic criteria the initial morphologic diagnosis changed for 35% (18/52) of cases. Diagnoses of 5 (9.6%) gliomas were upgraded, and 7 (14%) were downgraded. Conclusions This study shows that the incorporation of molecular testing can considerably improve brain tumor diagnoses in Nigeria. Furthermore, this study highlights the diagnostic challenges in resource-limited settings and what is at stake in the global disparities of brain tumor diagnosis.

2071 Background: Brigimadlin (BI 907828) is a potent small molecule inhibitor of the p53-MDM2 pathway currently in a phase 0/1 clinical trial in combination with radiotherapy in newly diagnosed MGMTunmethylated glioblastoma (GBM). To inform development of brigimadlin for GBM, we developed a pharmacokinetic (PK)/ pharmacodynamic (PD)/ efficacy model using GBM patient derived xenografts (PDXs). Methods: Brigimadlin PK parameters were evaluated in FVB wild-type and knockout (Mdr1a/b -/- Bcrp1 -/- ) mice and in athymic nude mice. Drug binding was measured by rapid equilibrium dialysis, and drug levels quantified by LC/MS-MS. Dose-response relationships were evaluated in subcutaneous and orthotopic PDXs across a range of doses. Response to brigimadlin (2 mg/kg weekly) +/- RT (2 Gy x 10 fractions) was evaluated in orthotopic PDXs. Results: Brigimadlin significantly delayed median time to regrowth of GBM108 ( MDM2amplified) subcutaneous tumors (vs. placebo; p<0.05) at dose levels of 0.25 mg/kg (1.3-fold), 0.5 mg/kg (1.5-fold), 1 mg/kg (2.6-fold), and 2 mg/kg (5.3-fold). A single dose of brigimadlin resulted in corresponding intra-tumoral drug levels 24 hours post-dose: 84 nM (0.25 mg/kg), 175 nM (0.5 mg/kg), 398 nM (1 mg/kg) and 924 nM (2 mg/kg) and dose-dependent upregulation of p53 target genes (p21 and PUMA). In contrast, GBM14 ( MDM2non-amplified) showed regrowth delay (1.6-fold) only at 2 mg/kg (p=0.008). Brigimadlin was highly bound (fraction unbound = 0.0006 in brain; 0.0017 in GBM tumor tissue), and CNS distribution was limited by efflux; in wild-type mice Kp,uu_brain = 0.002 ± 0.003; in Mdr1a/b -/- Bcrp1 -/- mice Kp,uu = 0.043 ± 0.025. Efficacy of brigimadlin was compared in orthotopic GBM108 PDXs grown in athymic nude mice vs. immuno/efflux-deficient (Rag2 -/- Mdr1a -/- Bcrp1 -/- ) mice. In nude mice, median survival was extended at 2 mg/kg brigimadlin (1.4-fold, p=0.009), with corresponding intra-tumoral drug levels of 29 nM. In efflux deficient mice, median survival was extended (p<0.05) at 0.25 mg/kg (1.5-fold), 0.5 mg/kg (1.5-fold), 1 mg/kg (1.8-fold), and 2 mg/kg (>4-fold). Measurement of intra-tumoral drug levels is in progress. In combination with RT in GBM108, brigimadlin (2 mg/kg x 2 weeks) extended median survival vs. vehicle (66 vs. 50 days; p=0.012) and enhanced median survival following RT (128.5 vs. 82 days; p=0.009). In GBM14, 2 mg/kg brigimadlin (2 mg/kg x 12 weeks) extended median survival (39.5 vs. 32.5 days; p=0.0004) and enhanced survival following RT (97 vs. 81 days; p<0.0001). Conclusions: Efficacy of brigimadlin is dependent on adequate CNS delivery. Studies in subcutaneous PDX demonstrated intra-tumoral total drug levels in the mid-nanomolar range provide meaningful tumor effects in a highly sensitive, MDM2 amplified PDX. In orthotopic PDXs with and without MDM2 amplification, brigimadlin provided further benefit in combination with RT. These data support ongoing clinical evaluation of brigimadlin in GBM.