Background: The RB-CDK4/6 and MAPK signaling pathways are dysregulated in glioblastoma (GBM). Our recent phase 0 study of ribociclib in recurrent GBM patients suggested that CDK4/6 inhibitor monotherapy is not durable. In this ongoing, dual-drug phase 0 study (NCT04391595), we evaluate the tumor pharmacokinetics (PK) and tumor pharmacodynamics (PD) of abemaciclib, a selective CDK4/6-inhibitor, plus LY3214996, a selective ERK1/2 inhibitor, in recurrent GBM patients. Methods: Adult patients eligible for this open-label, multi-center phase 0 protocol had recurrent GBM with (1) intact RB expression, (2) >30% pERK expression, and (3) CDKN2A/B deletion or CDK4/6 amplification. Prior to a planned resection, patients received six days of abemaciclib (150mg BID) plus LY3214996 (200mg QD). In a Time-Escalation Arm, ten patients were assigned to 3-5 hour or 7-9 hour intervals from final drug dose to tumor removal. Tumor tissue (gadolinium [Gd]-enhancing and nonenhancing regions), cerebrospinal fluid (CSF), and plasma were collected. Total and unbound drug concentrations were measured using validated LC-MS/MS methods. Tumor PD effects, including RB and RSK phosphorylation, were compared to matched archival or pre-treatment biopsied tissue. A PK ‘trigger’ (i.e., unbound concentration > 5x biochemical IC₅₀) was set for each drug. Gd-nonenhancing tumor tissue exhibiting abemaciclib and LY3214996 concentrations in excess of their trigger threshold qualified patients for postoperative dual-drug therapy. Results: In this interim analysis, no dose-limiting toxicities were observed. In Gd-nonenhancing tumor regions, median unbound concentrations of abemaciclib (including its equipotent M2 and M20 metabolites) were 31.2 nM (3-5 hour cohort) and 25.1 nM (7-9 hour cohort). In the same tissue, median unbound concentrations of LY3214996 were 52.0 nM (3-5 hour cohort) and 10.2 nM (7-9 hour cohort). Tumor RB and RSK phosphorylation decreased in 6/10 and 2/10 patients, respectively. Gd-enhancing tumor proliferation (MIB-1) was decreased in 8/10 patients. 5/10 patients exceeded PK thresholds for both abemaciclib (12 nM) and LY3214996 (25 nM), thereby entering the study’s therapeutic expansion phase. Conclusion: Abemaciclib and LY3214996 achieve pharmacologically-relevant concentrations in Gd-non-enhancing GBM tissue and are associated with suppression of the RB pathway and tumor proliferation. Following 6 days of presurgical drug exposure, the Optimal Time Interval (OTI) for tissue sampling was 3-5 hours after the final drug dose. Based on this interim analysis, the trial will accrue an additional 25 patients at this OTI. Clinical trial information: NCT04391595