Abstract Studies of therapeutic resistance in cancer have conventionally focused on identification of acquired exome mutations in tissue or circulating DNA at progression. However, this strategy has generated limited insights into resistance to CDK4/6 inhibitor and endocrine therapy combinations, which are the key first-line treatment modality in ER+ breast cancer. We utilized an alternative approach of integrated proteogenomic analysis of 8 pairs of pre- and post-treatment biopsies, 67 rapid autopsy samples, and 17 plasma samples from 12 ER+ breast cancer patients treated with CDK4/6 inhibitor combinations at a large academic center. In addition to whole exome sequencing, RNA sequencing, and immunohistochemistry (IHC) profiling on all tissue samples, we performed deep-scale mass spectrometry-based proteomics and phospho-proteomics on 35 rapid autopsy samples from 5 patients that had sufficient protein yields for analysis. We describe a patient with acquired Rb IHC loss after treatment progression without a Rb1 genetic alteration detected in exome sequencing or Rb transcript loss at her post-progression biopsy. Integrated proteogenomic analysis of ten autopsy lesions from this patient revealed convergent Rb protein loss across all tumor lesions, including lesions with and without Rb1 exome alterations. ESR1 mutations were frequently acquired at post-treatment biopsies at high cancer cell fractions, but a pre-existing ESR1-mutant subclone was nevertheless lost in a patient who acquired a concurrent Rb1-mutant subclone in the same tumor lesion. This suggests ESR1 mutations confer a relative fitness advantage that can nevertheless be mitigated in the presence of more potent synchronous resistance mechanisms. To investigate whether circulating tumor DNA reflects DNA shed specifically by progressing tumor lesions, we modeled the DNA shedding of tumor lesions into the plasma for 3 patients with both rapid autopsy and serial plasma samples available. In some instances, new lesions began shedding significant amounts of DNA months prior to their initial radiographic appearance. Together, these results illustrate the value of integrated multi-omics interrogation of different sample types to investigate therapeutic resistance and advance precision oncology. Citation Format: Christopher T. Chen, Ignaty Leshchiner, Liz Martin, Harry Kane, Kahn Rhrissorrakrai, Filippo Utro, Chaya Levovitz, Michael Gillette, Shankha Satpathy, Christopher Pinto, Daniel McLoughlin, Read Allen, Brian P. Danysh, Kara Slowik, Raquel A. Jacobs, Steven Carr, Laxmi Parida, Gad Getz, Dejan Juric. Proteogenomic characterization of CDK4/6 inhibitor-resistant ER+ breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P065.