Abstract Background: Targeted therapies directed against oncogenic signaling addictions, including one resulting from EML4-ALK fusions, can induce strong and durable clinical responses. However, some tumors do not respond (intrinsic resistance), while the initially responsive advanced cancers eventually relapse (acquired resistance). Our earlier studies with experimental models of ALK+ lung cancer revealed that acquired resistance to ALK inhibitors represents a gradual, multifactorial adaptation that progresses through multiple intermediate stages. These evolutionary intermediates can display unique collateral sensitivities to pharmacological inhibitors, including high sensitivity to a KDM histone demethylase inhibitor JIB-04. Results: To explore the utility of using JIB04 to suppress the evolution of resistance in vivo, we examined its ability to enhance responses to clinical ALKi alectinib and lorlatinib in mouse xenograft models. Surprisingly, JIB04 blunted the in vivo tumor responses and accelerated their relapse. The discrepancy between the in vitro and in vivo effects of JIB04 implied environmental mediation. Therefore, we examined the potential involvement of several mechanisms of environment-mediated resistance. We found that rather than directly decreasing cell-intrinsic therapy sensitivity, JIB04 altered ALKi pharmacokinetics. Using mass spectrometry measurements, we found a strong reduction in systemic and intratumor levels of the ALKi. This reduction is attributable to an accelerated metabolic conversion and excretion of metabolites. Our ongoing and planned efforts focus on the elucidation of mechanistic details and the exploration of clinical correlation. Significance: Both intrinsic and acquired resistance are assumed to reflect reduced sensitivities of tumor cells due to mutational, plasticity-related, and microenvironmental mechanisms. Pharmacokinetics studies are the essential component of drug development and clinical trial pipelines. However, after clinical approval, drug concentrations are not routinely examined in clinics. Whereas JIB04 is not used in clinics, its effects are mediated by mechanisms that might be triggered by more relevant dietary or pharmacological factors. Our results highlight the potential danger of blind spots in consideration of causes of therapy resistance and warrant the inclusion of pharmacokinetics analyses in diagnosing/treating therapy resistance. Citation Format: Pragya Kumar, Robert Vander Velde, Virginia Turati, Andriy Marusyk. Cell-extrinsic mechanisms of accelerated therapy resistance mediated by altered pharmacokinetics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A013.