Background: We demonstrated in mouse xenografts that CDK4/6 inhibition with palbociclib (P) is synergistic with 5FU in suppressing tumor growth. Based on these preclinical data, we initiated a Phase I trial to evaluate the safety and activity of P plus 5FU. Methods: Eligible patients (pts) had advanced, retinoblastoma (Rb)-positive (as demonstrated by IHC) solid tumors with an adequate performance status and intact organ function. The first stage of the study was a dose escalation to identify the recommended phase II dose (RP2D) of P in this combination (n = 21). The second stage is an ongoing schedule optimization, in which the timing of the P and 5FU are varied to identify the most effective sequence, as demonstrated by Ki67 suppression (n = 8 to date). All pts underwent tumor biopsies prior to treatment (tx), after 7 days of P, and again after the 5FU infusion. Results: Six pts were enrolled at a P dose of 50mg orally daily, Days 1-7, and IV 5FU as a 400mg/m² bolus Day 8, and 2400mg/m² continuous infusion Days 8-10, on an every 2 week cycle. However, 5/6 pts experienced significant delays in tx due to persistent myelosuppression, though only 1 met the definition of dose-limiting toxicity (DLT). The protocol was thus amended to remove the 5FU bolus. An additional 23 pts were enrolled. Most pts had colorectal (19) or breast (6) cancer; and pts had received a median 3 prior lines of Tx. 17 pts were DLT evaluable. The most common adverse events (AEs) were neutropenia (10), thrombocytopenia (4), nausea (N) (4), vomiting (V) (3), and mucositis (3). There were 4 DLTs (treatment delay (1), N/V (3)). The RP2D of P in this combination was 100mg orally daily. Efficacy was evaluable in 26/29 pts. One pt experienced a confirmed PR (Breast), and 6 pts had SD for a DCR of 27%. The median PFS in these 7 pts was 10 months, with 3 pts still on study. Tumor assessment of changes in Ki67 and Rb status are ongoing. Conclusions: The combination of P and 5FU is safe at a RP2D of P of 100mg. Accrual to schedule optimization is nearly complete, and the optimal schedule will be presented. This combination demonstrated promising disease control in this highly refractory population and should be tested in disease-specific Phase II trials. Clinical trial information: NCT01522989