Mechanism of resistance to lysosomotropic treatments: inhibition of autophagy and metabolic adaptations – LYSORES

The project combined complementary experimental approaches to investigate the biological consequences of lysosomal drug sequestration.

A first work package focused on the design and evaluation of fluorescent probes aimed at detecting lysosomotropic compounds and monitoring lysosomal alterations. These probes were developed in collaboration with medicinal chemists (ICN) and tested in cancer cell models.

The second work package investigated the adaptive mechanisms induced by lysosomal dysfunction. We combined cell biology, molecular biology, transcriptomic and metabolomic analyses to characterize the metabolic rewiring associated with lysosomotropic treatments.

The project initially focused on renal cell carcinoma models exposed to sunitinib, a tyrosine kinase inhibitor displaying lysosomotropic properties. Subsequently, the analyses were extended to additional lysosomotropic compounds and to breast and lung cancer models in order to assess the broader relevance of the identified mechanisms.

The project also enabled the implementation of zebrafish tumor xenograft technology at IRCAN. This model provides a rapid in vivo platform to evaluate tumor growth, dissemination and therapeutic responses.

The project generated important insights into the mechanisms by which cancer cells adapt to lysosomotropic therapies.

Although the development of fluorescent probes did not result in a sufficiently robust tool for routine applications, this work provided valuable information regarding the technical challenges associated with monitoring lysosomal alterations.

The major scientific achievement of the project was the identification of de novo serine biosynthesis as a key metabolic adaptation to lysosomal dysfunction. We demonstrated that cancer cells rely on this pathway to maintain their growth and survival following lysosomotropic treatment and that targeting this metabolic vulnerability restores sensitivity to therapy. These findings led to a publication in Cancer Research.

The project also enabled the establishment of zebrafish xenograft expertise at IRCAN, providing a new experimental platform for in vivo studies.

As the project progressed, we observed that several lysosomotropic drugs induced similar adaptive responses across different tumor models. These observations suggested that lysosomal sequestration may promote common resistance mechanisms across cancer types.

We identified profound alterations in lipid metabolism, including cholesterol accumulation, following exposure to lysosomotropic therapies. This unexpected finding opened a new research direction that is now being actively pursued.

One of the most significant outcomes of the project was the discovery that lysosomotropic therapies profoundly affect lipid metabolism. In particular, we observed substantial cholesterol accumulation following treatment with multiple lysosomotropic compounds.

Current research efforts aim to understand how these alterations arise and how they influence tumor cell behavior, therapeutic response and disease progression. This research program has now expanded to several cancer models, including renal, breast and lung cancers.

By uncovering the links between lysosomal dysfunction and metabolic rewiring, these studies may reveal novel therapeutic opportunities and improve our understanding of resistance mechanisms shared across different anticancer treatments.

State of art
The physicochemical properties of drugs can lead to a therapeutic failure. Physicochemical properties can predictably influence intracellular distribution of a drug, an important consideration for its efficacy. In particular, drugs with LogP>2 and pKa>6 can be trapped in acidic intracellular compartments such as lysosomes away from their targets. Their protonation at acidic pH causes their sequestration and subsequent limitations of drug efficacy. These drugs are qualified as lysosomotropic.

Preliminary results
We demonstrated that lysosomal sequestration results in an increase of the lysosome pH, which inhibits lysosomal proteases and leads to an incomplete autophagic process [1]. The physio-pathological consequences of sequestration in lysosomes and the adaptation to autophagic defects need better understanding. Our recent results reveal that lysosomotropic-dependent inhibition of autophagy results in the activation of the serine biosynthetic pathway leading to a metabolic adaptation and sustained proliferation. The comprehensive investigations of metabolic alterations in response to lysosomotropic drugs resulting in acquired resistance have never been conducted.

Objectives
Mainly dedicated to sunitinib, the standard care of clear cell Renal Cell Carcinoma (ccRCC), our work will be extended to lysosomotropic drugs such as chloroquine (anti-malaria) or azithromycin (antibiotic, asthma).
The objectives of our project are:

(1) To detect the lysosomotropic potential of any drugs
To develop a "biotest" probe for the detection of lysosomotropic properties. In collaboration with the Institute of Chemistry of Nice, we will produce fluorescent probes for the accurate detection of lysosome pH variations. This biotest, which can be used routinely, will detect the lysosomotropic potential of any drugs and will therefore assess the efficacy of the drug before any drug development. This test will be patented and proposed to pharmaceutical companies.

(2) To understand the consequences of lysosome sequestration of drugs on metabolomic adaptations
We have identified the serine biosynthetic pathway as a potential mechanism of resistance (transcriptomic and metabolomic). We will characterize and validate the importance of this pathway in drug resistance (cell lines, patient primary cells and organoids). Also, we will test the therapeutic interest of targeting the actors of this metabolic pathway, an alternative strategy that we can propose to patients in therapeutic impasses.

Expected results
This project will evidence mechanistic and clinical concepts counteracting resistance to lysosomotropic drugs. The applications of this project are for diagnostic, prognostic and therapeutic purposes. A biotest, used to evaluate the lysosomotropic potential of drugs currently on the market or under development in preclinical studies, could anticipate failures of phase I/II clinical trials. A better knowledge of the mechanisms of resistance will promote; a) therapeutic strategies combining lysosomotropic drugs with inhibitors of metabolic enzymes; b) the identification of predictive markers of resistance.