title: Methylation-associated partial down-regulation of mesothelin causes resistance to anti-mesothelin immunotoxins in a pancreatic cancer cell line.
abstract: Anti-mesothelin Pseudomonas exotoxin A-based recombinant immunotoxins (RITs) present a potential treatment modality for pancreatic ductal adenocarcinoma (PDAC). To study mechanisms of resistance, the sensitive PDAC cell line KLM-1 was intermittently exposed to the anti-mesothelin SS1-LR-GGS RIT. Surviving cells were resistant to various anti-mesothelin RITs (IC50s >1 μg/ml), including the novel de-immunized RG7787. These resistant KLM-1-R cells were equally sensitive to the anti-CD71 HB21(Fv)-PE40 RIT as KLM-1, indicating resistance was specific to anti-mesothelin RITs. Mesothelin gene expression was partially down-regulated in KLM-1-R, resulting in 5-fold lower surface protein levels and decreased cellular uptake of RG7787 compared to KLM-1. Bisulfite sequencing analysis found that the mesothelin promoter region was significantly more methylated in KLM-1-R (59 ± 3.6%) compared to KLM-1 (41 ± 4.8%), indicating hypermethylation as a mechanism of mesothelin downregulation. The DNA methyltransferase inhibitor 5-azacytidine restored original mesothelin surface expression to more than half in KLM-1-R and increased sensitivity to RG7787 (IC50 = 722.4 ± 232.6 ng/ml), although cells remained significantly less sensitive compared to parental KLM-1 cells (IC50 = 4.41 ± 0.38 ng/ml). Mesothelin cDNA introduction in KLM-1-R led to 5-fold higher surface protein levels and significantly higher RG7887 uptake compared to KLM-1. As a result, the original sensitivity to RG7787 was fully restored (IC50 = 4.49 ± 1.11 ng/ml). A significantly higher RG7787 uptake was thus required to reach the original cytotoxicity in resistant cells, hinting that intracellular RIT trafficking is also a limiting factor. RNA deep sequencing analysis of KLM-1 and KLM-1-R cells supported our experimental findings; compared to KLM-1, resistant cells displayed differential expression of genes linked to intracellular transport and an expression pattern that matched a more general hypermethylation status. In conclusion, resistance to anti-mesothelin RITs in KLM-1 is linked to a methylation-associated down-regulation of mesothelin, while aberrations in RIT trafficking could also play a role.
authors: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States of America; Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven University, Leuven, Belgium.
authors: Kevin
authors: K
authors: Hollevoet
authors: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States of America.
authors: Emily
authors: E
authors: Mason-Osann
authors: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States of America.
authors: Fabian
authors: F
authors: Müller
authors: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States of America.
authors: Ira
authors: I
authors: Pastan
mesh_terms: D036002:ADP Ribose Transferases / Q000276:immunology; D017209:Apoptosis / Q000187:drug effects; D001427:Bacterial Toxins / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D002452:Cell Count; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019175:DNA Methylation; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D005098:Exotoxins / Q000276:immunology; D005434:Flow Cytometry; D058851:GPI-Linked Proteins / Q000276:immunology / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology; D000922:Immunotoxins / Q000276:immunology / Q000494:pharmacology*; D000090204:Mesothelin; D008969:Molecular Sequence Data; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D037521:Virulence Factors / Q000276:immunology; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D058851:GPI-Linked Proteins; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D000090204:Mesothelin; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A