The Roles Of The Deubiquitylating Enzyme USP15 In Homologous Recombination Repair And Breast Cancer Cell Responseto Chemotherapy

The DNA damage response(DDR)pathway is predominant for genome stability maintenance,with its deficiency always leading to immunodeficiency disorders and cancer.Among different types of DNA lesions,DNA double strand breaks(DSBs)are recognized as the most severe one.In mammary cells,there are two prominent repair pathways that repair DSBs: homologous recombination repair(HR)and non-homologous end-joining(NHEJ).Unlike NHEJ,which ligates damage ends directly and is error-prone,HR is error free.HR requires the recruitment of BRCA1-BARD1 and its downstream effectors to DSBs,allows to resect damage ends and employs homologous sequence in the sister chromatid as a template to effectively repair DSBs.Thus,HR is important in lowering deleterious mutations and inhibiting tumorigenesis.Breast cancer type 1 susceptibility protein(BRCA1)plays a pivotal role in HR.The functions of BRCA1 in HR rely on its C-terminal BRCT domain and N-terminal RING domain,where most of the breast cancer associated mutations occur.Through its BRCT domain,BRCA1 forms A,B,C complexes with ABRAXAS,BACH1 and Ct IP,respectively.In response to DNA damage,BRCA1 is recruited to DSBs via the BRCA1-A complex.However,the BRCA1-A complex is not to execute HR but rather to suppress excess DNA end resection,whereas BRCA1-B,-C complexes promote HR through regulating cell cycle arrest and DNA end resection.Through its N-terminal RING domain,BRCA1 binds with BARD1 to form a RING heterodimer core complex,which further stabilize these two proteins.The recruitment of BARD1 to DSBs depends on: 1)PARP1 or poly-PAR mediated early-stage recruitment.2)heterochromatin protein HP1γ mediated BRCA1-BARD1 retention at DSBs.However,the functions and mechanisms underlying the assembly and coordination of BRCA1-BARD1 related complexes in HR remain largely unknown.Because individual tumors often have unique defects in the DNA damage response(DDR)pathway,insights into the basic mechanisms by which cells repair different DNA lesions could also guide individual therapy.A successful example is the use of PARP inhibitors in cancer patients with BRCA1 mutations.However,there is also clinical evidence for the utility of PARP inhibitors in cancers in the absence of BRCA mutations,presumably resulting from other molecular deficiencies in DNA repair.Thus,identifying novel components regulating HR may largely expand the clinical benefit from PARP inhibitors.Here,we report that the deubiquitylating enzyme USP15 affects cancer cells response to PARP inhibitors by regulating HR.Our results implied that USP15 can be used as a potential biomarker for PARP inhibitor treatment in cancers.Our project includes the following 5 sections:1.USP15 regulates HR.1)Knockout of USP15 sensitized cancer cells to multiple DNA damaging reagents,including PARP inhibitors.Using integrated reporter assay for HR and NHEJ,we observed a significantly compromised HR in USP15 knockout cells.2)Through examining the accumulation of several DDR factors at DNA lesions induced by UV laser micro-irradiation in USP15 knockout cells,we conclude USP15 mainly affected DNA end resection by regulating BRCA1-BARD1 retention at DSBs.3)Coimmunoprecipitation assay and GST pull-down assay confirmed USP15 binds BARD1 through its BRCT domain.HR reporter assay further indicated the interaction between USP15 and BARD1 is essential for HR.2.USP15 regulates HR through deubiquitinating BARD1 BRCT domain.1)By examining HR efficiency,we concluded USP15 regulates HR through its deubiquitylating enzyme activity.2)Using in vivo and in vitro deubiquitylation assay,we found that USP15 removed K63 linked ubiquitin chains from BRCT domain of BARD1.3)USP15 facilitated BARD binding with heterochromatin protein HP1γ following DNA damage.Moreover,we purified deubiquitinated BARD-BRCT domain by USP15,and examined its interaction with HP1γ in vitro.We found that USP15 deubiquitinated BARD1 BRCT domain and promoted BARD1-HP1γ interaction.4)Double factor knockdown assay further confirmed that USP15 functions through BARD1-HP1γ axis.3.USP15 is phosphorylated by ATM and recruited to DSBs.1)In response to DNA damage,USP15 is phosphorylated at Ser678 by ATM,this result is further verified through a phospho-specific antibody against Ser678.2)Phosphorylated USP15 is recruited to DSBs.Mechanistically,1)USP15 interacted with MDC1,and this interaction was dependent on USP15 Ser678 phosphorylation.2)Immunofluorescence analysis confirmed USP15 colocalized with MDC1 upon DNA damage and depletion of MDC1 impaired USP15 recruitment to DSBs.3)GST pull-down analysis confirmed that MDC1 FHA domain bound phosphorylated USP15.Immunofluorescence analysis also indicated USP15 recruitment was dependent on MDC1 FHA domain.4.Usp15 knockout mice show genome instability.1)We generated Usp15 knockout mice using CRISPR/CAS9 technology and confirmed the null of Usp15 gene by Western blot and RT-PCR.2)Usp15-/-mice were more susceptible to ionizing radiation.3)By analyzing MEF cells derived from Usp15-/-mice,we found loss of Usp15 led to more spontaneous DNA damage in mice,as well asmore aberrant chromatin structures.4)Loss of Usp15 in mice impaired HR signaling and increased sensitivity toward DNA damaging reagents.5.Research on USP15 as a potential synergistic lethal biomarker for PARP inhibitors.1)We identified two cancer associated mutations of USP15 by analyzing TCGA database.2)cancer-associated USP15 mutations,with decreased USP15-BARD1 interaction,increases PARP inhibitor sensitivity in cancer cells.3)c Biopoartal database indicated that USP15 is deep deleted in 16.67% of pancreatic cancer patients.Immunoblot analysis also confirmed the expression level of USP15 was low in multiple pancreatic cancer cell lines.By overexpressing or knockdown USP15 in these pancreatic cancer cell lines,we found the expression of USP15 also regulate the response of the pancreatic cancer cells to PARP inhibitors.Overall,our research identified one novel player in regulating HR and mediating cancer cells response to PARP inhibitors.We found that:1)USP15 regulates HR,has a little effect on NHEJ.USP15 affects DNA end resection in HR.2)In response to DNA damage,USP15 is recruited to DSBs by MDC1,which requires the FHA domain of MDC1 and phosphorylated Ser678 of USP15 by ATM.3)USP15 deubiquitinates BARD1 BRCT domain,and promotes BARD1-HP1γ interaction,resulting in BRCA1/BARD1 retention at DSBs.4)USP15 knockout mice exhibit genomic instability in vivo.5)Cancer-associated USP15 mutations,with decreased USP15-BARD1 interaction,increase PARP inhibitors sensitivity in cancer cells.6)USP15 is deeply deleted in multiply pancreatic cancer cell lines.USP15 affects pancreatic cancer cells response to PARP inhibitors.In summary,we reveal a previously unappreciated role of USP15 in cellular response to genotoxic stress and inform novel treatments of breast cancers and pancreatic cancers,especially those with the BRCAness characteristics.