Resistance to the Antibody–Drug Conjugate T-DM1 Is Based in a Reduction in Lysosomal Proteolytic Activity

Abstract

Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) that was approved recently to treat HER2 breast cancers. Despite its impressive clinical efficacy in many patients, intrinsic and acquired resistance to T-DM1 has emerged as a challenge. To identify mechanisms of T-DM1 resistance, we isolated several resistant HER2 clones exhibiting stable drug refractoriness and Genomic comparisons showed substantial differences among three of the isolated clones, indicating several potential mechanisms of resistance to T-DM1. However, we observed no differences in HER2 levels and signaling among the resistant models and parental HER2 cells. Bioinformatics studies suggested that intracellular trafficking of T-DM1 could underlie resistance to T-DM1, and systematic analysis of the path followed by T-DM1 showed that the early steps in the internalization of the drug were unaltered. However, in some of the resistant clones, T-DM1 accumulated in lysosomes. In these clones, lysosomal pH was increased and the proteolytic activity of these organelles was deranged. These results were confirmed in T-DM1-resistant cells from patient-derived HER2 samples. We postulate that resistance to T-DM1 occurs through multiple mechanisms, one of which is impaired lysosomal proteolytic activity. Because other ADC may use the same internalization-degradation pathway to deliver active payloads, strategies aimed at restoring lysosomal functionality might overcome resistance to ADC-based therapies and improve their effectiveness. .

The Second Generation Antibody-Drug Conjugate SYD985 Overcomes Resistances to T-DM1

“…Both lysosomal inhibitors reduced the efficacy of T-DM1 ( Figure 3B). Thus, an increase in lysosomal pH, similar to that previously shown in R44 and R55 cells [11], partially explains the resistance of these cells. Quantification of the levels of HER2 (mRNA and protein) showed a~50% downmodulation in R44 and R55 cells ( Figure 3C).…”

“…These experiments show that downmodulation of HER2 contributes to the resistance of R44 and R55 cells. Thus, both impairment of lysosomal function [11] and downmodulation of HER2 ( Figure 3C) account for the resistance to T-DM1 of these cells.…”

“…We have previously partially characterized two of the resistant models (R44 and R55) and found a defect in lysosomal function that results in a higher lysosomal pH that may partially explain their resistance to T-DM1 [11]. To further support that compromising the lysosomal function affects the efficacy of T-DM1, we treated parental cells with the lysosomal inhibitors chloroquine and bafilomycin [24,25].…”

“…Using established breast cancer cell lines, several groups have identified mechanisms of resistance to T-DM1. These include downregulation of HER2 [7][8][9], activation of alternative RTKs or intracellular signaling pathways downstream of HER2 [7], upregulation of Bcl-2/X L [8], defective intracellular routing [9] defective lysosomal function [10][11][12] and upregulation of multidrug resistance transporters [13][14][15].…”

Systematic identification of regulators of antibody-drug conjugate toxicity using CRISPR-Cas9 screens

“…Moreover, a major mechanism of acquired resistance to ADCs shown in in vitro models is altered ADC trafficking and lysosomal function 47,53,54 , a signature that is also identified in our screens.…”