Background: Endocrine therapy with a CDK4/6 inhibitor is standard of care for patients (pts) with estrogen-receptor-positive (ER+), HER2-negative MBC, yet resistance ultimately develops. We have shown that low doses of palbociclib activates autophagy, which reverses initial G1 cell cycle arrest. High concentrations of palbociclib induce senescence, but these are off target effects of the drug. The autophagy inhibitor hydroxychloroquine (HCQ) induces senescence at a lower (i.e. on-target) continuous dosing of palbociclib, in in vitro and in vivo models. This strategy is being tested in a phase I/II trial (NCT03774472). Results from the phase I portion are reported here. Methods: The phase I part of this study uses a dose escalation 3+3 design testing HCQ, 400, 600 and 800 mg daily (6 pts at 800 mg) with continuously dosed palbociclib at 75 mg and letrozole 2.5 mg daily. Dose limiting toxicity (DLT) includes any study drug-related grade ≥ 3 nonhematological (lab) toxicity. Responding pts may continue on therapy beyond 8 weeks for up to 52 weeks. Primary objective is to determine safety, tolerability and the recommended phase 2 dose (RP2D) of HCQ. Secondary objectives are overall tumor response and time to progression. Eligible pts are ≥18 years of age, postmenopausal (ovarian suppression allowed) with ER+/HER2-negative MBC, ECOG performance status score of ≤1 and with adequate renal, hepatic, and hematologic function. Response is assessed per RECIST v1.1. Results: Between 9/24/18 and 12/15/20, 14 pts were evaluable for safety. Median age was 41 with Asian (1, 7.1%), Black (2, 14.3%) White (11, 78.6%) patients enrolled. No DLTs were observed. One pt progressed during the DLT period and 2 withdrew consent (one during the DLT period); two pts were replaced for DLT assessment. Reasons for coming off study were grade 3 skin toxicity (1), per protocol at 8 weeks (non-measurable or pt/physician preference, 9), and (2) full duration treatment at 50 and 52 weeks. Adverse events (AEs) of grade ≥3 were hematologic (29), metabolism/nutrition (2), musculoskeletal/ connective tissue (1), and skin/subcutaneous tissue (3), with no serious AEs reported. The percent of palbociclib doses held per pt due to neutrophil level ranged from 0-37.5% with no apparent relation to HCQ dose. Best response was partial (2) stable (11); and progression (1). For measurable disease, tumor decreases of 11%, 12%, 21%, 26%, 30%, 55% and increase in 1 pt by 55% were seen. Conclusions: This phase I study showed acceptable safety and no HCQ dose-toxicity relationship. The RP2D of HCQ is 800 mg/day with continuous dosing palbociclib at 75 mg/day and letrozole at 2.5 mg/day. The phase 2 trial will proceed in the neoadjuvant setting, with Ki67 proliferative index response as the primary endpoint. Clinical trial information: NCT03774472.