Background: Cell cycle deregulation is ubiquitous in HNSCC. In HPV-negative disease, the most common genomic alteration of cell-cycling included CDKN2A deletions (57%), mutations (27%) or hypermethylation (12%). Selective CDK4/6 inhibition arrested cell cycling and inhibited tumor growth in cell-line and xenograft models of HPV-negative HNSCC, and CDKN2A alterations were predictive of response. An exploratory analysis of a double-blind, randomized, phase 2 trial of patients with HPV-negative recurrent/metastatic HNSCC showed that CDKN2A alterations were associated with better overall survival (OS) with palbociclib and cetuximab vs placebo and cetuximab (median 9.7 vs 4.6 months, HR 0.38). OS was similar between the two arms in patients without CDKN2A alterations. A phase 2 basket trial of patients with CDKN2A-altered head and neck cancers observed that palbociclib resulted in a target lesion decrease in 25% of patients. Collectively, these data warrant further studies to delineate predictive biomarkers of response to selective CDK4/6 inhibitors in HPV-negative HNSCC. Methods: The primary aims of this single-arm, phase 2 trial were to determine the objective response rate (ORR) of HPV-negative, LA-HNSCC to palbociclib, and to correlate responses to somatic CDKN2A alterations. HPV-negative disease was defined as SCC of the larynx, hypopharynx or oral cavity, or SCC of the oropharynx if negative for p16 by IHC and/or high-risk HPV-RNA by ISH. Genome sequencing (FoundationOne CDx/Tempus xT) was performed on tumor tissue obtained before treatment. Patients received palbociclib 125 mg/d orally on days 1-21 of each 28-day cycle. Tumor response was assessed using RECIST 1.1 with CT scans performed pre and post two cycles of palbociclib. Patients then received CRT. A sample size of 24 patients yielded an 80% power if the ORR was ≥38%, using an exact binomial test of one sample proportion comparison with an upper one-sided nominal significance level of 0.05 and null ORR of ≤17%. Results: 24 patients enrolled and completed two cycles of palbociclib: primary site (larynx-15; hypopharynx-4; oropharynx-4, oral cavity-1), clinical stage (III-7; IV-17), and smoking history (yes-23; no-1). The ORR with palbociclib was 41.7%. Best tumor response included: CR (1), PR (9), SD (13), and PD (1). CDKN2A altered disease was identified in 15 patients (62.5%) [mutation: 8, deletion: 7]. Tumor response to palbociclib occurred in 10 of 15 patients (66.7%) with CDKN2A altered disease versus 0 of 9 patients (0%) without CDKN2A altered disease (p=0.002, Fisher’s Exact Test). Conclusions: The primary hypothesis was met: the ORR with palbociclib in HPV-negative, LA-HNSCC was 41.7%. The ORR with palbociclib was significantly higher in CDKN2A altered disease. Clinical trial information: NCT03389477.