Contrasting molecular and pharmacodynamic signatures of response and resistance to sequential bortezomib and temozolomide in recurrent IDH-wildtype glioblastoma and grade 4 IDH-mutant astrocytoma: an exploratory interim analysis of NCT03643549 phase IB/II trial

Background The revision of the WHO classification of CNS tumours now distinguishes grade 4 diffuse gliomas by their molecular features, but tailored treatment is yet to be standardised. Glioblastoma (isocitrate dehydrogenase ( IDH ))-wildtype tumours harbouring an unmethylated O 6 -methylguanine-DNA methyltransferase “(u MGMT )” promoter are inherently resistant to temozolomide (TMZ). However, no standardised treatment recommendations are available for grade 4 astrocytoma IDH -mutant. We previously demonstrated that administration of bortezomib (BTZ) 48 h prior to TMZ depletes MGMT protein and sensitizes u MGMT glioma cells to TMZ. Based on these findings, a phase IB/II trial was initiated to evaluate the safety and clinical activity of sequential BTZ and TMZ. This interim analysis aimed to determine a futility safeguard, identify molecular and pharmacodynamic determinants of treatment response and failure in the first 15 patients consecutively treated. Methods Adult patients with recurrent grade 4 IDH- mutant astrocytoma or IDH- wildtype glioblastoma, u MGMT promoter, measurable disease, Karnofsky Performance Status ≥ 70, and disease progression ≥ 12 weeks after radiotherapy were enrolled. Patients received intravenous BTZ 1.3 mg/m² on days 1, 4, and 7 of each 28-day cycle, followed by oral TMZ 200 mg/m²/day for 5 consecutive days beginning on day 3. Targeted sequencing of glioma-associated genes and longitudinal plasma proteomic profiling using LC-MS/MS were performed before, during, and after treatment. Results Fifteen patients (median age 52 years) comprised 10 patients with IDH - wildtype glioblastoma and 5 patients with grade 4 IDH -mutant astrocytoma. Disease control (partial response and stable disease) was observed in 4/10 (40%) IDH - wildtype glioblastoma patients, comprising one PR and three cases of SD. In contrast, all five patients with grade 4 IDH -mutant astrocytoma experienced rapid progressive disease. Notably, non-responding IDH -wildtype glioblastomas and all IDH -mutant tumours harboured alterations affecting the CDKN2A/RB1 -cell-cycle regulatory axis, including CDKN2A and/or RB1 loss, with one IDH -mutant patient additionally harbouring PIK3CA gain. By comparison, responding IDH- wildtype glioblastomas were characterized by amplified EGFR , nuclear NF-κB (p65/RelA) activation, and absence of RB- pathway alterations. Longitudinal proteomic analyses demonstrated comparatively attenuated treatment-induced changes in proteins associated with invasion and extracellular matrix remodelling in responders, whereas proteins involved in damage clearance immune phenotype, platelet activation, aggregation, and degranulation were significantly altered during disease control. Conclusions Sequential BTZ and TMZ therapy was feasible with manageable hematologic and non-hematologic toxicities and met prespecified interim futility criteria, supporting continued recruitment to the phase II study. Disease control was restricted to a molecularly defined subset of IDH - wildtype glioblastoma characterized by EGFR amplification and NF-κB activation. In contrast, universal treatment failure among grade 4 IDH -mutant astrocytomas was associated with alterations of the CDKN2A/RB1 cell-cycle checkpoint pathway, suggesting that advanced molecular evolution and cell-cycle deregulation may override the survival advantage typically associated with IDH -mutant gliomas and limit the effectiveness of MGMT-targeted sensitization strategies. Registry ClinicalTrials.gov trial registration number: NCT03643549, date of registration: 2018-08-21.