Abstract
Purpose :
The majority of uveal melanomas (UM) is managed using radiation based procedures. However, radiation retinopathy lead to irreversible vision loss within few years after treatment. BAP1 tumor suppressor gene is inactivated in up to 45% of UM patients. BAP1 is important in DNA repair and it could modify the tumor response to radiation. Here, we investigated the effect of BAP1 inactivation on response to radiation in a melanoma in-vitro model.
Methods :
To assess the BAP1 role in response to treatment of melanoma cells we used CRISPR-Cas9 to knock out (KO) BAP1 in mouse melanoma cell line B16F10 and two UM cell lines 92.1 and Mel202. B16F10 cell line was selected as it has low mutation burden similar to UM cells and it could be used in-vivo tumor models in syngeneic C57BL6 mice without immunosuppression. We used RS-2000 irradiator to deliver various doses of ionizing radiation (IR) ranging from 10-80 Gy. For PARP inhibitor we used rucaparib at the average IC50 dose within 1h of IR. After 5 days, surviving cells were washed with PBS and fixed with 3% PFA and stained with 0.2% crystal violet then stain was extracted with 10% acetic acid and the optical density (OD) was determined by spectrophotometer at 600nm. The population survival rate was determined by the average ratio between the OD of the treated and untreated cells.
Results :
The two UM cell lines Mel202 and 92.1 did not survive BAP1 knock down. B16F10 cells survived and multiple different clones of homozygous and heterozygous BAP1 KO cells were generated. BAP1 deletion and loss of expression were confirmed by sequencing and Western blot, respectively. Two B16F10 BAP1 KO clones in addition to the wild type were used for assessment. We observed increased cell death at different doses of radiation in BAP1 KO cell lines compared to BAP1 WT cells. Moreover, loss of BAP1 minimally increased the sensitivity of B16F10 cell line towards rucaparib.
Conclusions :
Our results suggest that combination therapy of PARP inhibitor with IR could help to reduce the IR dosage in BAP1 mutant melanoma cells. Further studies on naturally occurring BAP1 mutant and wild type UM cell lines are ongoing.
This is a 2021 ARVO Annual Meeting abstract.