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Methylation-associated partial down-regulation of mesothelin causes resistance to anti-mesothelin immunotoxins in a pancreatic cancer cell line.

Hollevoet K, Mason-Osann E, Müller F, Pastan I - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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.

No MeSH data available.


Related in: MedlinePlus

Protein synthesis inhibition and EF-2 ADP-ribosylation in KLM-1-R.A: Protein synthesis inhibition by RG7787 is limited in resistant KLM-1 (KLM-1-R). KLM-1 was incubated for 16 hrs with RG7787, and KLM-1-R for 16 and 48 hrs with RG7787 and anti-CD25 LMB-2. RG7787 induces a dose-dependent protein synthesis inhibition in KLM-1-R, which is absent in KLM-1-R. After 48 hrs, RGG778 induces some decrease in protein synthesis in KLM-1-R, which is also the case with LMB-2. Protein synthesis inhibition was evaluated by measuring [3H]leucine incorporation. B: Diphthamide Biosynthesis Protein (DPH) genes expression is not down-regulated in KLM-1-R, compared to KLM-1. Expression levels were evaluated with real time RT-PCR, standardized for ß-actin and presented relative to KLM-1 C: EF-2 ADP-ribosylation is functional in KLM-1-R. RIT-induced EF-2 ADP-ribosylation was evaluated by incubating cell lysate with ADP-ribosylation buffer, 6-Biotin-17-NAD and 10 ng of RG7787 for 0, 15, 30 and 60 min at 25°C. Samples were subjected to SDS/PAGE followed by Western blotting with streptavidin HRP conjugate to detect biotin ADP-ribosylated EF-2. The 0 min time point and the sample without RG7787 are negative controls. D: EF-2 protein levels are on average 2-fold higher in KLM-1-R compared to KLM-1. Western blot was done on cell lysate of KLM-1 and KLM-1-R. β-actin acts as loading control. Protein levels were quantified and adjusted for β-actin levels with Image J. K: KLM-1, R: KLM-1-R,—no RG7787, + with RG7787.
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pone.0122462.g004: Protein synthesis inhibition and EF-2 ADP-ribosylation in KLM-1-R.A: Protein synthesis inhibition by RG7787 is limited in resistant KLM-1 (KLM-1-R). KLM-1 was incubated for 16 hrs with RG7787, and KLM-1-R for 16 and 48 hrs with RG7787 and anti-CD25 LMB-2. RG7787 induces a dose-dependent protein synthesis inhibition in KLM-1-R, which is absent in KLM-1-R. After 48 hrs, RGG778 induces some decrease in protein synthesis in KLM-1-R, which is also the case with LMB-2. Protein synthesis inhibition was evaluated by measuring [3H]leucine incorporation. B: Diphthamide Biosynthesis Protein (DPH) genes expression is not down-regulated in KLM-1-R, compared to KLM-1. Expression levels were evaluated with real time RT-PCR, standardized for ß-actin and presented relative to KLM-1 C: EF-2 ADP-ribosylation is functional in KLM-1-R. RIT-induced EF-2 ADP-ribosylation was evaluated by incubating cell lysate with ADP-ribosylation buffer, 6-Biotin-17-NAD and 10 ng of RG7787 for 0, 15, 30 and 60 min at 25°C. Samples were subjected to SDS/PAGE followed by Western blotting with streptavidin HRP conjugate to detect biotin ADP-ribosylated EF-2. The 0 min time point and the sample without RG7787 are negative controls. D: EF-2 protein levels are on average 2-fold higher in KLM-1-R compared to KLM-1. Western blot was done on cell lysate of KLM-1 and KLM-1-R. β-actin acts as loading control. Protein levels were quantified and adjusted for β-actin levels with Image J. K: KLM-1, R: KLM-1-R,—no RG7787, + with RG7787.

Mentions: Protein synthesis inhibition is initiated after the toxin traffics from the cell surface to the cytosol and inactivates EF-2 by ADP-ribosylation. KLM-1 cells were incubated with RG7787 for 16 hrs, and KLM-1-R cells for 16 and 48 hrs, after which protein synthesis was examined by measuring [3H]leucine incorporation (Fig. 4A). After 16 hrs, RG7787 induced a dose-dependent decrease in protein synthesis in KLM-1, but not in KLM-1-R. After 48 hrs, KLM-1-R showed a small decrease in protein synthesis at the higher concentrations of RG7787, reminiscent of the growth inhibition observed with the ATP assay (Fig. 1A). LMB-2 caused protein synthesis inhibition above 100 ng/ml after 48 hrs, confirming that the protein synthesis inhibition in KLM-1-R at higher RG7787 concentrations is in part attributable to non-specific uptake. Next, we evaluated whether the dismal protein synthesis inhibition by RG7787 in KLM-1-R was due to a problem with EF-2 ADP ribosylation. The toxin inactivates EF-2 by ADP-ribosylation of the diphthamide residue on EF-2, which requires the activity of enzymes DPH1–5 and 7 [3]. We measured expression of these six diphthamide genes by RT-qPCR and found no meaningful decrease in KLM-1-R, compared to KLM-1 cells (Fig. 4B). To investigate the status of EF-2 in KLM-1-R, we examined EF-2 protein levels and the ability of RG7787 to ADP-ribosylate EF-2 in cell-free extracts at different times of RG7787 incubation. On average, EF-2 levels were 2-fold higher in KLM-1-R cells compared to KLM-1 cells (Fig. 4D). At each time point, the amount of EF-2 that was ADP-ribosylated by RG7787 was similar in KLM-1 and KLM-1-R (Fig. 4C). These data demonstrate that the dismal protein synthesis inhibition in KLM-1-R is not linked to downregulation of DPH enzymes or failure of the toxin to ADP-ribosylate EF-2. These data show that the anti-mesothelin RIT resistance is linked to events occurring upstream of protein synthesis inhibition, which is in agreement with the earlier findings that the resistant and KLM-1 cells were equally sensitive to the anti-CD71 HB21(Fv)-PE40.


Methylation-associated partial down-regulation of mesothelin causes resistance to anti-mesothelin immunotoxins in a pancreatic cancer cell line.

Hollevoet K, Mason-Osann E, Müller F, Pastan I - PLoS ONE (2015)

Protein synthesis inhibition and EF-2 ADP-ribosylation in KLM-1-R.A: Protein synthesis inhibition by RG7787 is limited in resistant KLM-1 (KLM-1-R). KLM-1 was incubated for 16 hrs with RG7787, and KLM-1-R for 16 and 48 hrs with RG7787 and anti-CD25 LMB-2. RG7787 induces a dose-dependent protein synthesis inhibition in KLM-1-R, which is absent in KLM-1-R. After 48 hrs, RGG778 induces some decrease in protein synthesis in KLM-1-R, which is also the case with LMB-2. Protein synthesis inhibition was evaluated by measuring [3H]leucine incorporation. B: Diphthamide Biosynthesis Protein (DPH) genes expression is not down-regulated in KLM-1-R, compared to KLM-1. Expression levels were evaluated with real time RT-PCR, standardized for ß-actin and presented relative to KLM-1 C: EF-2 ADP-ribosylation is functional in KLM-1-R. RIT-induced EF-2 ADP-ribosylation was evaluated by incubating cell lysate with ADP-ribosylation buffer, 6-Biotin-17-NAD and 10 ng of RG7787 for 0, 15, 30 and 60 min at 25°C. Samples were subjected to SDS/PAGE followed by Western blotting with streptavidin HRP conjugate to detect biotin ADP-ribosylated EF-2. The 0 min time point and the sample without RG7787 are negative controls. D: EF-2 protein levels are on average 2-fold higher in KLM-1-R compared to KLM-1. Western blot was done on cell lysate of KLM-1 and KLM-1-R. β-actin acts as loading control. Protein levels were quantified and adjusted for β-actin levels with Image J. K: KLM-1, R: KLM-1-R,—no RG7787, + with RG7787.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4372481&req=5

pone.0122462.g004: Protein synthesis inhibition and EF-2 ADP-ribosylation in KLM-1-R.A: Protein synthesis inhibition by RG7787 is limited in resistant KLM-1 (KLM-1-R). KLM-1 was incubated for 16 hrs with RG7787, and KLM-1-R for 16 and 48 hrs with RG7787 and anti-CD25 LMB-2. RG7787 induces a dose-dependent protein synthesis inhibition in KLM-1-R, which is absent in KLM-1-R. After 48 hrs, RGG778 induces some decrease in protein synthesis in KLM-1-R, which is also the case with LMB-2. Protein synthesis inhibition was evaluated by measuring [3H]leucine incorporation. B: Diphthamide Biosynthesis Protein (DPH) genes expression is not down-regulated in KLM-1-R, compared to KLM-1. Expression levels were evaluated with real time RT-PCR, standardized for ß-actin and presented relative to KLM-1 C: EF-2 ADP-ribosylation is functional in KLM-1-R. RIT-induced EF-2 ADP-ribosylation was evaluated by incubating cell lysate with ADP-ribosylation buffer, 6-Biotin-17-NAD and 10 ng of RG7787 for 0, 15, 30 and 60 min at 25°C. Samples were subjected to SDS/PAGE followed by Western blotting with streptavidin HRP conjugate to detect biotin ADP-ribosylated EF-2. The 0 min time point and the sample without RG7787 are negative controls. D: EF-2 protein levels are on average 2-fold higher in KLM-1-R compared to KLM-1. Western blot was done on cell lysate of KLM-1 and KLM-1-R. β-actin acts as loading control. Protein levels were quantified and adjusted for β-actin levels with Image J. K: KLM-1, R: KLM-1-R,—no RG7787, + with RG7787.
Mentions: Protein synthesis inhibition is initiated after the toxin traffics from the cell surface to the cytosol and inactivates EF-2 by ADP-ribosylation. KLM-1 cells were incubated with RG7787 for 16 hrs, and KLM-1-R cells for 16 and 48 hrs, after which protein synthesis was examined by measuring [3H]leucine incorporation (Fig. 4A). After 16 hrs, RG7787 induced a dose-dependent decrease in protein synthesis in KLM-1, but not in KLM-1-R. After 48 hrs, KLM-1-R showed a small decrease in protein synthesis at the higher concentrations of RG7787, reminiscent of the growth inhibition observed with the ATP assay (Fig. 1A). LMB-2 caused protein synthesis inhibition above 100 ng/ml after 48 hrs, confirming that the protein synthesis inhibition in KLM-1-R at higher RG7787 concentrations is in part attributable to non-specific uptake. Next, we evaluated whether the dismal protein synthesis inhibition by RG7787 in KLM-1-R was due to a problem with EF-2 ADP ribosylation. The toxin inactivates EF-2 by ADP-ribosylation of the diphthamide residue on EF-2, which requires the activity of enzymes DPH1–5 and 7 [3]. We measured expression of these six diphthamide genes by RT-qPCR and found no meaningful decrease in KLM-1-R, compared to KLM-1 cells (Fig. 4B). To investigate the status of EF-2 in KLM-1-R, we examined EF-2 protein levels and the ability of RG7787 to ADP-ribosylate EF-2 in cell-free extracts at different times of RG7787 incubation. On average, EF-2 levels were 2-fold higher in KLM-1-R cells compared to KLM-1 cells (Fig. 4D). At each time point, the amount of EF-2 that was ADP-ribosylated by RG7787 was similar in KLM-1 and KLM-1-R (Fig. 4C). These data demonstrate that the dismal protein synthesis inhibition in KLM-1-R is not linked to downregulation of DPH enzymes or failure of the toxin to ADP-ribosylate EF-2. These data show that the anti-mesothelin RIT resistance is linked to events occurring upstream of protein synthesis inhibition, which is in agreement with the earlier findings that the resistant and KLM-1 cells were equally sensitive to the anti-CD71 HB21(Fv)-PE40.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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.

No MeSH data available.


Related in: MedlinePlus