PMON148 ACQUIRED TEMOZOLOMIDE RESISTANCE IN CORTICOTROPE PITUITARY TUMOR CELLS
Authors: Silva Ticiana , Dogan Fadime , van Koetsveld Peter M , Feelders Richard A , Hofland Leo J 
Published: November 2022

Abstract

Introduction
Temozolomide (TMZ) treatment is an effective treatment for aggressive pituitary tumors, leading to tumor regression or stabilization of tumor progression in the majority of cases. In cases of disease recurrence, however, TMZ treatment does not control tumour regrowth. The expression of O6-methyl-guanine-DNA-methyltransferase (MGMT) protein and mismatch repair components (MMR) are implicated in the mechanism of TMZ resistance. In the present study, we aimed to develop an in vitro model of acquired TMZ resistance and to describe its mechanism.

Methods
The mouse AtT20 corticotrope pituitary tumor cell line was used. Two protocols were developed, one based on high dose of TMZ (high TMZ-challenge) and another one using lower TMZ concentrations (low TMZ-challenge) that are clinically achievable after a standard TMZ schedule. Both protocols consisted in pulse exposures to increasing concentrations of TMZ for 24rhs. TMZ concentrations in the high TMZ-challenge were 100, 200 and 300 µM while in the low TMZ-challenge were 20, 40, 60 and 80 µM. AtT20 cells treated with challenges of the vehicle dimethyl sulfoxide (vehicle-challenged cells) were used as control.

Results
The TMZ challenges induced a significant increase in EC50 (decreased sensitivity) of the growth inhibitory effect of TMZ in TMZ-challenged cells compared to vehicle-challenged cells in both protocols (high TMZ-challenge, EC50 = 123 µM vs 19 µM, p&lt;0.001; low TMZ-challenge, EC50 = 69 µM vs 21 µM, p&lt;0.001). The TMZ-challenged cells remained resistant compared to vehicle-challenged cells even after 9 weeks following the last TMZ challenge. The apoptotic cell death was higher in vehicle-challenged cells compared to TMZ-challenged cells in both protocols, albeit not statistically significant in the low TMZ-challenge protocol (high TMZ-challenge, p=0.003; low TMZ-challenge, p=0.169). TMZ-induced changes in cell cycle phases were more significant in vehicle-challenged cells compared to TMZ-challenged cells. Differences in cell morphology were observed between vehicle- and TMZ-challenged cells in the high dose protocol. In vehicle-challenged cells, small rounded and fusiform-shaped cells were observed, whereas in the TMZ-challenged cells only rounded cells were detected in culture. mRNA expression of MGMT was significantly higher in TMZ-challenged cells compared to vehicle-challenged cells. MMR components expression was not affected by TMZ challenges.

Conclusion
The present study describes an acquired TMZ-resistant corticotrope pituitary tumor cell model. TMZ resistance was demonstrated in both protocols by a sustained increase in TMZ EC50, small TMZ-induced cell cycle changes, and lower increase in TMZ-induced apoptosis. Additionally, TMZ challenges affected AtT20 cell morphology. This was evident only in the high TMZ-challenge protocol. Acquired TMZ resistance was associated with a strong increase in MGMT expression.
Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.