Abstract Triple-negative breast cancer (TNBC) is an aggressively metastatic subtype that can only be treated by chemotherapy. Nearly 50% of patients have residual disease after neoadjuvant chemotherapy (NACT) and have extremely poor prognoses. Recent genome sequencing studies have revealed extensive intratumoral heterogeneity (ITH) in treatment-naïve TNBC. However, the functional contribution of ITH to chemoresistance in TNBC is unknown. To understand how this occurs, we are generating patient derived xenograft (PDX) models from treatment-naïve TNBC in order to identify and characterize tumor cell populations that are responsible for chemoresistance with the ultimate goal of selectively targeting resistant tumor clones. Through an IRB-approved clinical trial using standard techniques, samples (primary tumor, skin metastasis, and blood for germline reference) were obtained from a patient with newly diagnosed, untreated metastatic TNBC. Subsequently, this patient was found to have disease resistant to chemotherapy. Tumor cells were implanted into the humanized mammary fat pad of NOD/SCID mice to establish PDX models of the primary (PIM1-P) and metastatic (PIM1-M) tumors. RNA sequencing and whole-exome sequencing (∼300X) were performed on the patient's primary and metastatic tumors and the first and third passage PDX tumors. Mouse sequences were computationally subtracted from the PDX data and which was then processed according to Genome Analysis Toolkit best practices workflow. Variants were called using MuTect and copy number alterations were estimated by ExomeCN. This revealed high concordance between the genomic profiles of the patient and PDX models. While there were 81 somatic non-silent mutations shared in the patient and PDX, only a few fell in known cancer genes, including TP53 (V143fs), ELF4 (L593H), and ARID3A (R351P). Modeling of clonal clusters with ABSOLUTE revealed ITH in the patient tumor that is preserved in the PDX. Both the patient and PDX model (PIM1-P) exhibited progressive disease when treated with paclitaxel. Only partial responses were observed in mice treated with doxorubicin plus cyclophosphamide (AC), consistent with residual disease after standard NACT for patients with TNBC. AC treatment resulted in an ∼60% reduction in tumor volume which was not enhanced by repeated cycles. Tumors rapidly re-grew when treatment was halted or if treated tumor cells were engrafted into the mammary fat pads of new recipient mice. This suggests that a subpopulation of AC-resistant tumor cells is present in PIM1-P tumors. To dissect this subpopulation, we established conditions for simultaneous tracking of thousands of PIM1-P tumor clones in vivo using a high-complexity library of up to 30 million unique DNA barcodes. Mice engrafted with barcoded PIM-1P tumors are being treated with AC to identify and characterize AC-resistant tumor cells present in PIM1. Citation Format: Gloria V. Echeverria, Sahil Seth, Shirong Cai, Stacy Moulder, William F. Symmans, Timothy P. Heffernan, Jeffrey Chang, Helen Piwnica-Worms. Identifying and targeting chemoresistant subclones in triple negative breast cancer. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2406.