BRD7 Suppresses Chemosensitivity By Inhibiting USP1-mediated Deubiquitination Of CHK1

CHK1(Checkpoint Kinase 1)plays a critical role in DNA damage caused by a variety of endogenous and exogenous agents in cellular response.Defending the integrity of the genome is the DDR,an extensive network of pathways which detects and signals DNA damage for subsequent processing.CHK1 as a key effector in DDR,is a multifunctional Ser/Thr kinase protein highly conserved through evolution and represents a crucial component in all cell cycle checkpoints.The function of CHK1 is blocking cell cycle and allowing cells time to repair their damaged DNA before moving to the next stage of the cell cycle when the cells are challenged by DNA damaging agents.The two major DNA damage-induced checkpoint pathways are ATM/CHK2 and ATR/CHK1.ATM/CHK2 activates the p53-associated G1/S-phase checkpoint,while ATR/CHK1 triggers the intra-S-and G2/M-phase checkpoints.However,most cancer cells have dysregulated G1 checkpoints,due to p53 mutations,making them dependent on S and G2 checkpoints,which are activated by ATR/CHK1 signaling.Thus,CHK1 plays a crucial function in the whole DDR signaling.Moreover,as a protective mechanism of cellular response,ATR/CHK1 also have multiple additional roles in protecting cells from DNA damage.That’s also why the current studies demonstrate CHK1 is overexpressed in many cancer types and CHK1 overexpression or high activity significantly increases chemo-radiotherapy resistance of tumor cells in clinic.Those functions of CHK1 make it ideal therapeutic targets.CHK1 inhibitors have been developed and are currently used in clinical studies.However,these inhibitors have been terminated in clinical trials because of their cellular or tissue toxicity,indicating in order to improve the clinical therapeutic effect of tumor treatment and reduce side effects,in addition to the development of a new generation of CHK1 inhibitors with reduced toxicity in the future,it will be more necessary to explore the mechanisms that regulate CHK1.BRD7 is a member of the family of bromodomain-containing proteins(BCPs),and involved in forming PBAF-specific SWI/SNF chromatin remodeling complex which plays a role in regulating transcription.Recently,accumulating studies have shown that BRD7 is involved in tumorgenesis and progression as a tumor suppressor gene and is deficient in various kinds of tumors.BRD7 has been reported to play a tumor-suppress function through regulating multiple signaling pathways,as well as interact with many key proteins and plays significant roles in transcriptional regulation and cell cycle control,involving in cellular growth,mobility and apoptosis,thus relating with tumorgenesis.However,present studies on BRD7 have been more focused on its role in tumorgenesis and progression,while less on the mechanism and function of BRD7 involving in cell cycle control when tumor cells response to the later but equally important cancer therapy.In our study,we found that BRD7 could bind to CHK1 and negatively regulate CHK1 at both protein and m RNA levels.Further,by half-life assays,we found silencing of BRD7 slowed down the degradation rate and extended half-life of CHK1 protein,suggesting that BRD7 may affect the stability of CHK1 by regulating its degradation.Because that 80-90% of proteins in cells are degraded by UPS,combined with our research background,by gain-of-function and loss-of-function experiments,we confirmed that BRD7 could promote CHK1 ubiquitination.To further determine the mechanism how BRD7 controled CHK1 ubiquitination and degradation,we detected currently reported three E3 ligases and three deubiquitinases of CHK1 respectively and found only USP1 presented corresponding change after BRD7 silencing,which suggesting USP1 may be the bridge in BRD7 regulating CHK1.And our hypothesis was verified by protein stability and ubiquitination rescue assays which showed that silencing of USP1 reversed the effects of BRD7 on CHK1 stability and polyubiquitination.Physiologically,BRD7 deficiency in tumor cells significantly increased expression and activity of CHK1.And the results of IC50,flow cytometry and WB all showed high expression and activity of CHK1 significantly sensitized tumor cells to CHK1 inhibitors,accelerated tumor cells to apoptosis,thus enabling a lower dose of CHK1 inhibitors to achieve a better tumor killing effect.Taken together,our research found BRD7 is a novel negative regulator of CHK1 in tumor cells.And BRD7 affected the stability of CHK1 by negatively regulating the deubiquitination of CHK1 mediated by USP1.Moreover,the accumulation of CHK1 caused by BRD7 deficiency can sensitize tumor cells to CHK1 inhibitors and promote the apoptosis of tumor cells.Controlling the level of BRD7 in tumor cells could affect the expression,activity and degradation of CHK1,thereby enhancing the effect of genotoxic agents on killing tumor cells.In the future,BRD7 may be used as a potential new gene target and drug target to play an important role in improving the efficacy of CHK1 targeted chemotherapy drugs and clinical tumor intervention.