P-056: HUWE1 orchestrates DNA Repair in response to Replicative Stress in Multiple Myeloma

Abstract

Background Multiple Myeloma (MM) is an incurable B cell neoplasm characterised by heightened levels of genomic instability that contribute to both development and progression of the disease. HUWE1, an E3 ubiquitin ligase, has been implicated in the DNA damage response (DDR) and genome integrity. Past studies identified more than 5% of patients present with a HUWE1 mutation. Our lab has determined that both MM patients and cell lines with HUWE1 mutations exhibit increased levels of genomic instability manifested by heightened mutation rates (p=0.0023) and increased incidents of micronuclei formation (p<0.0001). This study aimed to elucidate HUWE1’s role in DNA replication and to determine how it influences DNA repair in MM. Methods Cells were transfected with SMARTvector Inducible Human HUWE1 shRNA or with a non-targeting control (NTC) shRNA (Dharmacon, Chicago IL, USA). Co-immunoprecipitation was carried out using the Co-IP kit (Thermo Fisher). Replicative stress was induced with 2mM Hydroxyurea (HU) and assessed using immunofluorescence. DNA repair was investigated in (2Gy) irradiated HUWE1 knockdown cells by immunofluorescence staining. Results In line with previous studies, we found that knockdown of HUWE1 in MM cell lines led to an S-phase arrest, suggesting a role for HUWE1 in DNA replication. Using proteomic profiling and co-immunoprecipitation we identified novel putative substrates of HUWE1 that are involved in DNA replication and repair. To address HUWE1’s role in the replicative stress response we treated cells with 2mM HU to elicit replication fork stalling and used replication protein A (RPA) foci counts as a measure of the response. We found that HUWE1 depleted cells exhibited significantly less foci and therefore reduced recruitment of replication proteins when treated with HU for 6hrs compared to the NTC control (p=0.0064). This reduced response to replicative stress in HUWE1 knockdown cells was coupled with significantly higher levels of DNA damage at 6hrs (p=0.00421) and this damage persisted at 24hrs after treatment (p=0.0219). To further examine HUWE1’s role in the DDR cells were stained for the double strand break (DSB) marker, 53BP1 following irradiation (IR). HUWE1 knockdown cells displayed a reduced capacity to repair DSBs with more 53BP1 foci present at 1hrs (p=0.00254), 4hrs (p=0.0469) and 24hrs (p=0.025) post-IR compared to their NTC counterparts. Conclusion Here we demonstrate that knockdown of HUWE1 results in increased replication stress and a dampened DNA repair capacity in MM cells, most likely underpinned by reduced recruitment of repair machinery. This data coupled with our previous work demonstrating a role for HUWE1 mutations as a driver for genomic instability, outlines a clear position for HUWE1 in maintaining genome integrity in MM. Further exploration of these dysregulated repair pathways in the presence of HUWE1 mutations may offer potential therapeutic targets for a subset of patients in the future. Multiple Myeloma (MM) is an incurable B cell neoplasm characterised by heightened levels of genomic instability that contribute to both development and progression of the disease. HUWE1, an E3 ubiquitin ligase, has been implicated in the DNA damage response (DDR) and genome integrity. Past studies identified more than 5% of patients present with a HUWE1 mutation. Our lab has determined that both MM patients and cell lines with HUWE1 mutations exhibit increased levels of genomic instability manifested by heightened mutation rates (p=0.0023) and increased incidents of micronuclei formation (p<0.0001). This study aimed to elucidate HUWE1’s role in DNA replication and to determine how it influences DNA repair in MM. Cells were transfected with SMARTvector Inducible Human HUWE1 shRNA or with a non-targeting control (NTC) shRNA (Dharmacon, Chicago IL, USA). Co-immunoprecipitation was carried out using the Co-IP kit (Thermo Fisher). Replicative stress was induced with 2mM Hydroxyurea (HU) and assessed using immunofluorescence. DNA repair was investigated in (2Gy) irradiated HUWE1 knockdown cells by immunofluorescence staining. In line with previous studies, we found that knockdown of HUWE1 in MM cell lines led to an S-phase arrest, suggesting a role for HUWE1 in DNA replication. Using proteomic profiling and co-immunoprecipitation we identified novel putative substrates of HUWE1 that are involved in DNA replication and repair. To address HUWE1’s role in the replicative stress response we treated cells with 2mM HU to elicit replication fork stalling and used replication protein A (RPA) foci counts as a measure of the response. We found that HUWE1 depleted cells exhibited significantly less foci and therefore reduced recruitment of replication proteins when treated with HU for 6hrs compared to the NTC control (p=0.0064). This reduced response to replicative stress in HUWE1 knockdown cells was coupled with significantly higher levels of DNA damage at 6hrs (p=0.00421) and this damage persisted at 24hrs after treatment (p=0.0219). To further examine HUWE1’s role in the DDR cells were stained for the double strand break (DSB) marker, 53BP1 following irradiation (IR). HUWE1 knockdown cells displayed a reduced capacity to repair DSBs with more 53BP1 foci present at 1hrs (p=0.00254), 4hrs (p=0.0469) and 24hrs (p=0.025) post-IR compared to their NTC counterparts. Here we demonstrate that knockdown of HUWE1 results in increased replication stress and a dampened DNA repair capacity in MM cells, most likely underpinned by reduced recruitment of repair machinery. This data coupled with our previous work demonstrating a role for HUWE1 mutations as a driver for genomic instability, outlines a clear position for HUWE1 in maintaining genome integrity in MM. Further exploration of these dysregulated repair pathways in the presence of HUWE1 mutations may offer potential therapeutic targets for a subset of patients in the future.