Systematic screening for potential therapeutic targets in osteosarcoma through a kinome-wide CRISPR-Cas9 library

Objective:Osteosarcoma is the most common primary malignant bone tumor.However,the survival of patients with osteosarcoma has remained unchanged during the past 30 years,owing to a lack of efficient therapeutic targets.Methods:We constructed a kinome-targeting CRISPR-Cas9 library containing 507 kinases and 100 nontargeting controls and screened the potential kinase targets in osteosarcoma.The CRISPR screening sequencing data were analyzed with the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout(MAGeCK)Python package.The functional data were applied in the 143B cell line through lenti-CRISPR-mediated gene knockout.The clinical significance of kinases in the survival of patients with osteosarcoma was analyzed in the R2:Genomics Analysis and Visualization Platform.Results:We identified 53 potential kinase targets in osteosarcoma.Among these targets,we analyzed 3 kinases,TRRAP,PKMYT1,and TP53RK,to validate their oncogenic functions in osteosarcoma.PKMYT1 and TP53RK showed higher expression in osteosarcoma than in normal bone tissue,whereas TRRAP showed no significant difference.High expression of all 3 kinases was associated with relatively poor prognosis in patients with osteosarcoma.Conclusions:Our results not only offer potential therapeutic kinase targets in osteosarcoma but also provide a paradigm for functional genetic screening by using a CRISPR-Cas9 library,including target design,library construction,screening workflow,data analysis,and functional validation.This method may also be useful in potentially accelerating drug discovery for other cancer types.

Protein stability regulators screeningassay (Pro-SRSA)： protein degradation meets theCRISPR-Cas9 library

The regulation of protein stability is a fundamental issue for biophysical processes,but there has not previously been a convenient and unbiased method of identifying regulators of protein stability.However,as reported in the article entitled "A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25 A," recently published in Cell Discovery,our team developed a protein stability regulators screening assay(Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9(CRISPR-Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability.Based on our findings,we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability.

Human KIAA1018/FAN1 nuclease is a new mitotic substrate of APC/C^(Cdh1)

A recently identified protein, FAN1 (FANCD2-associated nuclease 1, previously known as KIAA1018), is a novel nuclease associated with monoubiquitinated FANCD2 that is required for cellular resistance against DNA interstrand crosslinking (ICL) agents. The mechanisms of FAN1 regulation have not yet been explored. Here, we provide evidence that FAN1 is degraded during mitotic exit, suggesting that FAN1 may be a mitotic substrate of the anaphase-promoting cyclosome complex (APC/C). Indeed, Cdh1, but not Cdc20, was capable of regulating the protein level of FAN1 through the KEN box and the D-box. Moreover, the up-and down-regulation of FAN1 affected the progression to mitotic exit. Collectively, these data suggest that FAN1 may be a new mitotic substrate of APC/C Cdh1 that plays a key role during mitotic exit.

CRISPR/Cas9 screening identifies a kinetochore-microtubule dependent mechanism for Aurora-A inhibitor resistance in breast cancer

Background:Overexpression of Aurora-A(AURKA)is a feature of breast cancer and associates with adverse prognosis.The selective Aurora-A inhibitor alisertib(MLN8237)has recently demonstrated promising antitumor responses as a single agent in various cancer types but its phase III clinical trial was reported as a failure since MLN8237 did not show an apparent effect in prolonging the survival of patients.Thus,identification of potential targets that could enhance the activity of MLN8237 would provide a rationale for drug combination to achieve better therapeutic outcome.Methods:Here,we conducted a systematic synthetic lethality CRISPR/Cas9 screening of 507 kinases using MLN8237 in breast cancer cells and identified a number of targetable kinases that displayed synthetic lethality interactions with MLN8237.Then,we performed competitive growth assays,colony formation assays,cell viability assays,apoptosis assays,and xenograft murine model to evaluate the synergistic therapeutic effects of Haspin(GSG2)depletion or inhibition with MLN8237.For mechanistic studies,immunofluorescence was used to detect the state of microtubules and the localization of Aurora-B and mitotic centromere-associated kinesin(MCAK).Results:Among the hits,we observed that Haspin depletion or inhibition marginally inhibited breast cancer cell growth but could substantially enhance the killing effects of MLN8237.Mechanistic studies showed that co-treatment with Aurora-A and Haspin inhibitors abolished the recruitment of Aurora-B and mitotic centromere-associated kinesin(MCAK)to centromeres which were associated with excessive microtubule depolymerization,kinetochore-microtubule(KT-MT)attachment failure,and severe mitotic catastrophe.We further showed that the combination of MLN8237 and the Haspin inhibitor CHR-6494 synergistically reduced breast cancer cell viability and significantly inhibited both in vitro and in vivo tumor growth.Conclusions:These findings establish Haspin as a synthetic lethal target and demonstrate CHR-6494 as a potential combinational drug for promoting the therapeutic effects of MLN8237 on breast cancer.

SUMOylation stabilizes hSSB1 and enhances the recruitment of NBS1 to DNA damage sites

Human single-stranded DNA-binding protein 1(hSSB1)is required for the efficient recruitment of the MRN complex to DNA doublestrand breaks and is essential for the maintenance of genome integrity.However,the mechanism by which hSSB1 recruits NBS1 remains elusive.Here,we determined that hSSB1 undergoes SUMOylation at both K79 and K94 under normal conditions and that this modification is dramatically enhanced in response to DNA damage.SUMOylation of hSSB1,which is specifically fine-tuned by PIAS2α,and SENP2,not only stabilizes the protein but also enhances the recruitment of NBS1 to DNA damage sites.Cells with defective hSSB1 SUMOylation are sensitive to ionizing radiation,and global inhibition of SUMOylation by either knocking out UBC9 or adding SUMOylation inhibitors significantly enhances the sensitivity of cancer cells to etoposide.Our findings reveal that SUMOylation,as a novel posttranslational modification of hSSB1,is critical for the functions of this protein,indicating that the use of SUMOylation inhibitors(e.g.,2-D08 and ML-792)may be a new strategy that would benefit cancer patients being treated with chemo-or radiotherapy.

RMI2 plays crucial roles in growth and metastasis of lung cancer

Dear Editor,Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer death in the world,but its therapeutic targets are still being explored.Genome instability as a key hallmark of cancer not only contributes to cancer initiation and progression,1 but also creates vulnerabilities that are relatively specific to cancer cells,which may be potential therapeutic targets for cancer patients.During DNA Double-Strand Breaks(DSBs)repair,BTR(BLM-Topo IIIα-RMI1/RMI2)complex promotes the dissolution of double Holliday junctions to form non-crossover products and is often considered as a tumor suppressor.2 However,the function of each individual component of this BTR complex in cancer remains largely unknown.