The Achilles’ heel of cancer

Human cells

Genoscience Pharma’s Philippe Halfon and Eric Raymond share their expertise on targeting the recycling of unsustainable production of palmitoylated cancer-associated proteins.

Cancer relies on palmitoylation for progression

Palmitoylation is a global post-translational lipid modification, that controls the localisation, stability, and function of several proteins. For instance, protein palmitoylation involves the covalent attachment of fatty acyl chains, typically a palmitate to internal cysteine residues of a protein via labile thioester linkages. Palmitoyl transferases are a zinc finger Asp-His-His-Cystype (ZDHHC) family containing 23 distinct mammalian genes (excluding ZDHHC10) that catalyse this reaction1. In cancer cells, protein localisation in membrane rafts play a central role in the capacity of activating cell signal transduction for several transmembrane protein G-protein coupled receptors and for oncogenic kinases that require attachment to the inner leaflet membrane such as oncogenic kinases-like Ras. Although all Ras proteins are prenylated, their association with the plasma membrane also requires more fine-tuned palmytoylation for membrane partitioning either to lipid raft (doubly palmitoylated H-Ras) or domain boundary (singly palmitoylated N-Ras). Conformational changes in the Ras membrane proximal domain facilitate the phosphorylation of GDP to GTP and the kinase domain activation. Interestingly, the recycling of H- and N-Ras proteins need to be de-palmitoylated by thioestherases (PPT) to cycle back to Golgi membranes, where they are re-palmitoylated (i.e., recycle) by Golgi resident enzymes to translocate back to the plasma membrane. Involvement of palmitoylation/depalmitoylation in cancer cell progression is reflected through the aberrant expression patterns of ZDHHCs/PPTs and through the changes in palmitoylation levels of cancer-associated proteins, which in turn affect their functions in tumour cell proliferation, adhesion, migration and metastasis, but also in autophagy and apoptosis2.

PPT-1 as a primary target to normalise palmitoyl-related functions

PPT1 (palmitoyl-protein thioesterase 1) is a lysosomal enzyme that plays a critical role in the hydrolysis of palmitoylated proteins. The complete PPT1 deficiency is induced by knocking out gene function in cellular and animal models or related to human mutation in neuronal ceroid lipofuscinosis-1, lead to neurodegeneration by the abnormal accumulation of unfolded palmitoylated proteins. This role in protein recycling is also observed in cancer cells, where the concomitant high expression levels of both palmitoyl-protein transferases and thioestherases, such as PPT1, emphasise the importance of palmitoylation for the membrane trafficking of major oncogeneic proteins (about a quarter of oncogenes) and the role associated with the recycling of proteins through the Golgi and the lysosomes to sustain signalling without inducing an ‘overflow’ of unusable proteins that may trigger autophagy and apoptosis. While a complete knockdown of PPT1 in human cells may be impossible and likely undesirable, normalising palmitoyl functions using enzymatic inhibitors that balance the high level of palmitoylation may be desirable to impair the enhanced oncogenic cell signalling.

The improved knowledge of PPT1 three-dimensional protein conformation and enzymatic substrates led to the development of allosteric and substrate competitive inhibitors. As a result, seminal works performed using GNS561 and didemnin B have shown that PPT1 inhibition may cause the disruption of lysosome function, which could occur through the inhibition of PPT1 and/or the accumulation of excess palmitoylated proteins, to make the unsustainable choking palmitoyl runaway in cancer cell.

Figure 1: Molecular model of GNS561 bound to site 5 (Met112) of PPT1. (a) A CPK model of GNS561 inserted above the palmitate site. (b) A detailed view of the drug binding site with the hydrophobicity area (colour code indicated).

PPT1 inhibitors for the treatment of patients with cancer

Contemplating the potential of PPT1 inhibition in cancer led to the development of small molecules with high PPT1 affinity and limited toxicity for clinical drug development. Ezurpimtrostat (GNS561) was identified from a large library of small molecules as a lead compound for drug development. Inhibiting PPT1 using ezurpimtrostat led to growth suppression in a large variety of tumours3 with no significant toxicity in animal models. The palmitate binding site was early defined as a putative binding site for various PPT1 inhibitors. Therein is an accessible molecular cavity, sufficiently large to accommodate many small molecules4. There is also a solvent-exposed lipid-binding groove in PPT13, corresponding to a hydrophobic channel, relatively narrow but sufficiently long to incorporate extended molecules. Ezurpimtrostat was found to be well-suited to binding to the Met112 site. The final representation in Figure 1 illustrates the co-binding of palmitate and GNS561 to this site in an ideal position to interfere with the enzymatic activity. Binding of the drug to this site would likely inhibit the enzyme activity, restraining access to adjacent catalytic triad of Ser115, His289, and Asp233. Upon binding to Met112, the drug establishes contacts with neighbour residues (such as Leu290, Leu292), potentially hindering the access to and correct functioning of the enzyme catalytic site.

Based on this data, ezurpimtrostat completed a first-in-human clinical trial for patients with advanced cancer5 showing sustained stabilisation in heavily pre- treated patient with mild toxicity. Those results led to further clinical development in ongoing larger studies in patients with advanced hepatocellular carcinoma and cholangiocarcinoma.

Figure 2: GNS561 bound to palmitoylated PPT1 at site 5 (Met 112).

Overcoming PD-L1- mediated immune evasion in cancer by inhibiting PPT1

Immune checkpoint inhibitors targeting the membrane programmed death-1 (PD-1) receptor and death-ligand 1 (PD-L1) have been successfully used in the clinic to restore the tumour-specific cytotoxic T cell functions. The lack of response to checkpoint inhibitors remains largely unknown, requiring further investigation into a novel area where palmitoylation may play an important role. The intracellular storage of PD-L1 and its active redistribution to the cell membrane was shown to play an important role in response to checkpoint inhibitors. Recent studies have suggested that palmitoylation can regulate the expression and activity of PD-L16. For example, palmitoylation of PD-L1 on cysteine residues near its transmembrane domain was shown to promote its stability and cell surface expression in cancer cells. PD-L1 is palmitoylated in its cytoplasmic domain, and this lipid modification stabilises PD- L1 by blocking its ubiquitination, consequently suppressing PD-L1 degradation by lysosomes. Palmitoyltransferase ZDHHC3 (DHHC3) is the main acetyltransferase required for the palmitoylation of PD-L1, which shows that the inhibition of PD-L1 palmitoylation via 2-bromopalmitate, or the silencing of DHHC3, activates antitumour immunity in vitro and in mice bearing MC38 tumour cells. Exposure to small molecules inhibiting PD-L1 palmitoylation also decreases PD-L1 expression in tumour cells and were shown to enhance T-cell activation in tumours7.

These findings suggest that palmitoylation does not only affect cancer cells, but also plays a critical role in regulating the expression and activity of PD-L1 in the tumour microenvironment.

This led us to investigate if targeting palmitoylation could be a potential strategy for modulating PD-L1 expression and enhancing anti-tumour immune responses. We recently showed that ezurpimtrostat can potentiate the anti-tumour effects of immune checkpoint inhibitors through increasing expression of major histocompatibility complex (MHC)-I proteins at the surface of cancer cells, further modulating immunity by the recolonisation and the activation of cytotoxic CD8+ lymphocytes. Our results highlight that the inhibition of PPT1 sensitive tumours in immunotherapy by switching cold tumours to hot tumours can provide a clinical rationale for combining PPT1 inhibitors and checkpoint inhibitors in clinical trials for patients with cancer.

DDW Volume 24 – Issue 2, Spring 2023 – Global Cancer Research Guide


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  2. Zhuang Liu, Mingming Xiao, Yaqi M, et al. Emerging roles of protein palmitoylation and its modifying enzymes in cancer cell signal transduction and cancer therapy. Molecular Diagnostics and Therapeutics 2021;8, Int J Biol Sci 2022;18.
  3. Brun S, Bestion E, Raymond E, et al. GNS561, a clinical-stage PPT1 inhibitor, is efficient against hepatocellular carcinoma via modulation of lysosomal functions. Autophagy 2022;18:678-94.
  4. Biehl E, Clardy J, Hofmann SL. Structural basis for the insensitivity of a serine enzyme (palmitoyl-protein thioesterase) to phenylmethylsulfonyl fluoride. J Biol Chem 2000;275:23847-23851.
  5. Harding JJ, Awada A, Roth G, et al. First-In-Human Effects of PPT1 Inhibition Using the Oral Treatment with GNS561/Ezurpimtrostat in Patients with Primary and Secondary Liver Cancers. Liver Cancer 2022;11(3):268-277.
  6. Lan J, Li C, et al. Inhibiting PD-L1 palmitoylation enhances T-cell immune responses against tumours. Nat Biomed Eng 2019;3:306-317.
  7. Yang Y, Hsu JM, Sun L, et al. Palmitoylation stabilizes PD-L1 to promote breast tumor growth. Cell Res 2019;29:83-86.

About the authors:

Eric RaymondEric Raymond, Pr, MD, PhD is the Chairman of Medical Oncology at Saint-Joseph Hospital in Paris, France. He previously developed a Phase I and translational unit at Gustave Roussy. His research focuses on anticancer drug development, including experimental pharmacology, translational research, molecular selection and monitoring.


Philippe HalfonPhilippe Halfon, Pr, MD, PharmD, PhD, founded Genoscience Pharma in 2001, a clinical stage biotech initially oriented in hepatic diseases due to Halfon’s international expertise in this matter. He is Head of Internal Medicine at the European Hospital in Marseille, France, and a Professor at the Medical College, Paris, France.


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