The Lysosomal Sequestration of Tyrosine Kinase Inhibitors and Drug Resistance

Abstract

The Lysosomal sequestration of weak-base anticancer drugs is one putative mechanism for resistance to chemotherapy but it has never been directly proven. We addressed the question of whether the lysosomal sequestration of tyrosine kinase inhibitors (TKIs) itself contributes to the drug resistance in vitro. Our analysis indicates that lysosomal sequestration of an anticancer drug can significantly reduce the concentration at target sites, only when it simultaneously decreases its extracellular concentration due to equilibrium, since uncharged forms of weak-base drugs freely diffuse across cellular membranes. Even though the studied TKIs, including imatinib, nilotinib, and dasatinib, were extensively accumulated in the lysosomes of cancer cells, their sequestration was insufficient to substantially reduce the extracellular drug concentration. Lysosomal accumulation of TKIs also failed to affect the Bcr-Abl signaling. Cell pre-treatment with sunitinib significantly enhanced the lysosomal accumulation of the TKIs used; however, without apparent lysosomal biogenesis. Importantly, even increased lysosomal sequestration of TKIs neither decreased their extracellular concentrations nor affected the sensitivity of Bcr-Abl to TKIs. In conclusion, our results clearly show that the lysosomal sequestration of TKIs failed to change their concentrations at target sites, and thus, can hardly contribute to drug resistance in vitro.

Lysosomal Biogenesis and Implications for Hydroxychloroquine Disposition

“…Lysosomes have been observed as a drug sink for many lysosomotropic agents, including many prescribed drugs like doxorubicin, vinblastine, imipramine, and a variety of TKIs, like gefitinib (Skoupa et al, 2020), imatinib (Burger et al, 2015), and sunitinib (Ruzickova et al, 2019). In a recent study by Ruzickova et al the lysosomotropic agent sunitinib was shown to be capable of increasing overall lysosomal sequestration of the TKIs tested, although this effect was insufficient in reducing sensitivity to the TKIs by the hypothesized mechanism of sequestering them away from their mechanistic target sites as the concentrations tested in culture maintained a static extracellular concentration.…”

“…2C). Recent reports suggest that many weakly basic lipophilic compounds, including CQ, activate lysosomal biogenesis by activating the transcription factor EB (TFEB) (Lu et al, 2017;Zhitomirsky et al, 2018;Zhao et al, 2020), which ultimately increases the lysosomal volume fraction of treated cells and should allow them to sequester even more drug than their original baseline (Ruzickova et al, 2019). CQ has been observed to activate TFEB and increase lysosomal content of the cell, so we investigated the capability of HCQ to do the same.…”

Lysosomal Fusion: An Efficient Mechanism Increasing Their Sequestration Capacity for Weak Base Drugs without Apparent Lysosomal Biogenesis

“…However, it is probably not a universal mechanism that occurs in all types of cancer cells. In our laboratory, we have recently shown that A549, MCF7 and K562 tumor lines exposed to non-toxic sunitinib concentrations for three days, increased significantly their ability to sequester various TKIs by lysosomes without observable lysosomal biogenesis [31].…”

“…Absolute accumulation of TKIs in lysosomes was calculated as described previously [30,31]. Briefly, the value of intracellular accumulation of particular TKI in the presence of BafA1 was subtracted from the value of intracellular accumulation of particular TKI in the absence of BafA1.…”

“…Expression of acidic phosphatase (ACP) and β-d-galactosidase (GLP) was determined by measurement of their enzymatic activity in cell lysates [24,31]. Cells were washed in ice cold PBS and lysed in buffer (25 mM HEPES (pH 7.0), 0.5% CHAPS, 0,5% Triton X-100, 0.5 mM dithiothreitol, 2 mM EGTA, and protease cocktail inhibitors) on ice for 30min.…”