Abstract 2576: BMI1 and RING1A are involved in H2A ubiquitination at sites of platinum-induced damage
Authors: Sriramkumar Shruthi , O'Hagan Heather M. 
Published: August 2019

Ovarian cancer (OC) is a lethal gynecological malignancy with a 5-year survival rate of 46%. Surgical debulking followed by platinum and taxane based chemotherapy is the standard of care for OC patients. However, the major obstacle in the use of these agents for treatment of OC patients is development of chemoresistance. An epigenetic mechanism commonly associated with development of platinum resistance is promoter DNA methylation and associated gene silencing. Transcriptional silencing of tumor suppressor genes like MutL homolog 1 (MLH1) by this mechanism has been well established in development of platinum resistance. However, the mechanism of initiation of this aberrant promoter DNA methylation is not known. Platinum agents are DNA damaging agents which crosslink guanines and form intra-strand or inter-strand adducts. We hypothesize that platinum induced DNA damage or repair of the damage results in recruitment of proteins involved in transcriptional repression to sites of damage. Recruitment of repressive proteins results in transient repression of transcription in the vicinity of damage to promote repair. However, similar to our findings with enzyme induced double strand breaks, repair of damage can occasionally result in retention of repressive proteins at sites of damage causing persistent transcriptional repression and gene silencing. Such persistent repression of key loci potentially contributes to the development of platinum resistance. We demonstrate that treatment of platinum sensitive OC cells with the IC50 dose of cisplatin for 8 hours resulted in ubiquitination of H2A/H2AX by western blot analysis. We hypothesize that this ubiquitination occurs on K119 of H2A/H2AX. H2A/H2AX ubiquitination at K119 mediated by Polycomb repressive complex 1 (PRC1) has been associated with transcriptional repression and gene silencing during development and differentiation and also during DNA repair. We observe a reduction in platinum induced H2AX ubiquitination on knockdown of PRC1 complex member RING1A using western blot analysis. Using immunofluorescence, we observe that BMI1, another PRC1 complex member, localizes to sites of platinum induced DNA damage. Platinum induced lesions are repaired by different repair pathways including – Nucleotide excision repair pathway (NER), Fanconi Anemia repair pathway (FA) and Homologous recombination repair (HRR). We demonstrate that knockdown of proteins in global genome NER and HRR pathways results in a decrease in platinum induced ubiquitination. Through on-going studies we seek to further elucidate the mechanism of recruitment of BMI1, RING1A to sites of platinum induced DNA damage and their role in long-term gene silencing. Understanding this mechanism will enable us to design inhibitors to prevent the occurrence of platinum resistant tumors in OC patients.

Citation Format: Shruthi Sriramkumar, Heather M. O'Hagan. BMI1 and RING1A are involved in H2A ubiquitination at sites of platinum-induced damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2576.