Many emerging and established anticancer therapies accumulate in lysosomes due to their physicochemical properties, independent of their intended targets. However, the relevance to anticancer activity remains understudied. Here, we use UNC0642, a G9a/GLP inhibitor, as a tool compound to investigate the impact of chronic lysosomal accumulation of a therapeutic molecule in pancreatic ductal adenocarcinoma (PDAC) cells.

We generated a transient G9a/GLP double knockout (DKO) in a murine PDAC cell line using CRISPR-Cas9 and validated the loss of protein expression and enzymatic activity via Western blot. This DKO cell line was used to differentiate the on-target versus off-target effects of UNC0642. Immunofluorescence was performed to assess the expression of lysosome-associated proteins. Additionally, RNA-Seq followed by quantitative real-time PCR was used to examine transcriptional changes induced by lysosomal-damaging agents.

UNC0642 was originally designed as a catalytic inhibitor of the methyltransferase G9a and its paralog GLP. However, using a G9a/GLP DKO PDAC cell line, we demonstrate that its primary anti-proliferative effect is mediated predominantly by an off-target mechanism. Lysotracker co-staining confirmed UNC0642 accumulation in lysosomes, leading to lysosomal swelling, as evidenced by increased LAMP-1- puncta and altered lysosomal morphology. Furthermore, UNC0642 induced chronic lysosomal damage, as demonstrated by increased galectin-3 puncta, a well-established marker of lysosomal membrane damage. Notably, UNC0642 triggered lysosomal damage to a similar extent as L-Leucyl-L-Leucine methyl ester (LLOMe), a known lysosomal detergent. Chronic lysosomal damage induced by UNC0642 and LLOMe correlated with reduced PDAC cell viability. Additionally, RNA-Seq revealed a novel transcriptional signature indicative of lysosomal damage, including upregulation of Serpinb9 family members, Car6, and Rgs1.

Our findings suggest that chronic lysosomal damage is a significant off-target effect that can create vulnerabilities in PDAC cells for targeted therapeutic treatment. These effects should be considered when evaluating the mechanisms of action of current and emerging anticancer therapies.