Adapting a Combination of Intrinsic and Extrinsic Cellular Factors to Generate an Improved Model of Tumor Exposure and Sensitivity to Chloroquine Derivatives

Abstract

In cancer clinical trials hydroxychloroquine (HCQ) is the only approved autophagy inhibitor for use in treatment. Pharmacokinetic data from clinical trials comparing tumor vs. blood concentrations of HCQ suggest that there is a significant disconnect between drug levels within these compartments [Barnard RA, Wittenburg LA, Amaravadi RK, Gustafson DL, Thorburn A, Thamm DH (2014) “Phase I Clinical Trial and Pharmacodynamic Evaluation of Combination Hydroxychloroquine and Doxorubicin Treatment in Pet Dogs Treated for Spontaneously Occurring Lymphoma Phase I Clinical Trial and Pharmacodynamic Evaluation of Combination Hydroxychloroquine.” Autophagy 10 (8): 1415–25]. Attempts to improve prediction of drug kinetics in these compartments have been made through development of individual patient‐focused physiologically‐based models, but these models fall short in predicting dynamic tumor cell‐specific intrinsic and extrinsic factors that dictate the amount of HCQ that sequesters within its ultimate pharmacodynamic target – the lysosome. Two factors intrinsic to tumor cells suggest that HCQ exposure should be predicted on a tumor‐by‐tumor basis. The first factor being basal levels of HCQ uptake drivers in the context of lysosomal burden, broken up into overall lysosomal volume and pH in a cell population. Different cell lines exhibit different pre‐exposure lysosomal burdens, modifying their overall initial exposure to HCQ both in vitro and in tumor‐bearing mice in vivo. Basal lysosomal burden is coupled to an adaptive response to HCQ exposure as well. This adaptive response is broken up into a short‐term component, driven primarily through lysosomal swelling, and a longer‐term component wherein TFEB‐activation induces biogenesis of functional lysosomes capable of sequestering HCQ. These intrinsic components result in a continual increase in cell uptake of HCQ over a 24‐hour period, split into rapid uptake within the short term (1st hour) and a slower, steadier uptake in the long term (1–24 hours). In addition, the main extrinsic factor that modulates HCQ exposure is extracellular pH. We observed a 5‐fold decrease in total cellular drug uptake and up to 10‐fold decrease in sensitivity to HCQ in media pH 6.8 vs. 7.4 across 4 cell lines. Combining these intrinsic and extrinsic factors we generate a cell‐based pharmacokinetic simulation model capable of predicting HCQ exposure to cell lines, and then translate it to next‐generation chloroquine derivatives through modification of compound‐specific physicochemical parameters. The power of this model is in its ability to account for dynamic systems within the tumor cell, giving a more accurate picture of how the tumor interacts with the drug, accounting for a major form of HCQ resistance through the non‐homogenous acidic tumor microenvironment in vivo, and pushing forward the development of next generation lysosomotropic autophagy inhibitors that may interact with the system similarly.Support or Funding InformationNational Cancer Institute [Grant R01CA190170 Therapeutic Targeting of Autophagy‐Dependent Cancer] and by Department of Defense CDMRP Breast Cancer Research Program [Grant BC130103P1 Identifying and Targeting Autophagy Dependence to Eliminate Metastatic Breast Cancer]