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.

The propensity for tumorigenesis in human induced pluripotent stem cells is related with genomic instability

The discovery of induced pluripotent stem cells (iPSCs) is a promising advancement in the field of regenerative medicine. Previous studies have indicated that the teratoma-forming propensity of iPSCs is variable; however, the relationship between tumorigenic potential and genomic instability in human iPSCs (HiPSCs) remains to be fully elucidated. Here, we evaluated the malignant potential of HiPSCs by using both colony formation assays and tumorigenicity tests. We demonstrated that HiPSCs formed tumorigenic colonies when grown in cancer cell culture medium and produced malignancies in immunodeficient mice. Furthermore, we analyzed genomic instability in HiPSCs using whole-genome copy number variation analysis and determined that the extent of genomic instability was related with both the cells′ propensity to form colonies and their potential for tumorigenesis. These findings indicate a risk for potential malignancy of HiPSCs derived from genomic instability and suggest that quality control tests, including comprehensive tumorigenicity assays and genomic integrity validation, should be rigorously executed before the clinical application of HiPSCs. In addition, HiPSCs should be generated through the use of combined factors or other approaches that decrease the likelihood of genomic instability.

Snail部分通过下调TEL2促进鼻咽癌转移

背景与目的转移是鼻咽癌(nasopharyngeal carcinoma,NPC)患者治疗失败的主要原因。我们之前报道,SERPINE1的负调节因子TEL2可以抑制鼻咽癌转移到淋巴结。方法我们进行了一系列体内和体外实验来阐明Snail和TEL2之间的调控关系,分别在3株Snail低表达的鼻咽癌细胞系(S26、6?10B、HK1)与2株Snail高表达的鼻咽癌细胞系(S18、5?8F)中检测了TEL2的表达水平。我们采用荧光素酶和染色质免疫沉淀方法分析Snail和TEL2的相互作用,采用侵袭实验检测Snail/TEL2途径在细胞迁移和鼻咽癌细胞侵袭中的作用,采用裸鼠尾静脉注射鼻咽癌细胞来检测肺转移情况。结果 Snail异位表达下调了TEL2的mRNA和蛋白水平,shRNA敲低Snail则可上调TEL2的表达水平。荧光素酶和染色质免疫沉淀实验表明Snail可与TEL2的启动子直接结合。Snail异位表达增强了NPC细胞的迁移和侵袭,并且TEL2过表达削弱了这种作用。TEL2过表达也减弱了缺氧诱导的细胞迁移和侵袭并且增加了转移性肺结节的数量。Snail过表达减少了转移性肺结节的数量。结论 TEL2是Snail的新靶点,可抑制Snail诱导的鼻咽癌的迁移、侵袭和转移。

Low expression of centrosomal protein 78(CEP78)is associated with poor prognosis of colorectal cancer patients

Background: Centrosomal protein 78(CEP78) has been characterized as a component of the centrosome required for the regulation of centrosome-related events during the cell cycle, but its role in human cancers remains unclear. This study aimed to investigate the role and the clinical value of CEP78 in colorectal cancer(CRC).Methods: Quantitative real-time polymerase chain reaction(q RT-PCR) and immunohistochemistry were performed to examine CEP78 expression in CRC tissues and adjacent noncancerous tissues. The association between CEP78 expression and clinical outcomes of CRC patients was determined. The effect of CEP78 on cell growth was examined in vitro by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT) assay, colony formation, and flow cytometry assays and in vivo using a nude mouse model.Results: The expression level of CEP78 was significantly lower in tumor tissues than in the adjacent normal tissues(P < 0.01). Low CEP78 expression was significantly associated with poor differentiation(P etastasis(P = 0.003), large tumor size(P = 0.017), lymphatic mtient= 0.034), distant metastasis(P s with low CEP78 expression h= 0.029), and advanced stage(P Meier analysis indicated that paad shorter survival than those wit= 0.011). Kaplan–h high CEP78 expression(P < 0.01). Overexpression of CEP78 in CRC cells significantly reduced cell viability and colony formation in vitro and halted tumor growth in vivo. Further study showed that CEP78 reintroduction in CRC cells resulted in G2/M phase arrest rather than cell apoptosis.Conclusions: CEP78 might function as a tumor suppressor and serve as a novel prognostic marker in CRC.

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.

MFN2 suppresses clear cell renal cell carcinoma progression by modulating mitochondria-dependent dephosphorylation of EGFR

Background:Clear cell renal cell carcinoma(ccRCC)is the most lethal renal cancer.An overwhelming increase of patients experience tumor progression and unfavorable prognosis.However,the molecular events underlying ccRCC tumorigenesis and metastasis remain unclear.Therefore,uncovering the underlying mechanisms will pave the way for developing novel therapeutic targets for ccRCC.In this study,we sought to investigate the role of mitofusin-2(MFN2)in supressing ccRCC tumorigenesis and metastasis.Methods:The expression pattern and clinical significance of MFN2 in ccRCC were analyzed by using the Cancer Genome Atlas datasets and samples from our independent ccRCC cohort.Both in vitro and in vivo experiments,including cell proliferation,xenograft mouse models and transgenic mouse model,were used to determine the role of MFN2 in regulating the malignant behaviors of ccRCC.RNA-sequencing,mass spectrum analysis,co-immunoprecipitation,bio-layer interferometry and immunofluorescence were employed to elucidate the molecular mechanisms for the tumor-supressing role of MFN2.Results:we reported a tumor-suppressing pathway in ccRCC,characterized by mitochondria-dependent inactivation of epidermal growth factor receptor(EGFR)signaling.This process was mediated by the outer mitochondrial membrane(OMM)protein MFN2.MFN2 was down-regulated in ccRCC and associated with favorable prognosis of ccRCC patients.in vivo and in vitro assays demonstrated thatMFN2 inhibited ccRCC tumor growth and metastasis by suppressing the EGFR signaling pathway.In a kidney-specific knockout mouse model,loss of MFN2 led to EGFR pathway activation and malignant lesions in kidney.Mechanistically,MFN2 preferably binded small GTPaseRab21 in its GTPloading form,which was colocalized with endocytosed EGFR in ccRCC cells.Through this EGFR-Rab21-MFN2 interaction,endocytosed EGFR was docked to mitochondria and subsequently dephosphorylated by the OMM-residing tyrosine-protein phosphatase receptor type J(PTPRJ).Conclusions:Our findings uncover an important non-canonicalmitochondriadependent pathway regulating EGFR signaling by the Rab21-MFN2-PTPRJ axis,which contributes to the development of novel therapeutic strategies for ccRCC.

KIF2C:a novel link between Wnt/β-catenin and mTORCI signaling in the pathogenesis of hepatocellular carcinoma

Hepatocellular carcinoma(HCC)is the most common primary liver malignancy and is the fourth-leading cause of cancer-related deaths worldwide.HCC is refractory to many standard cancer treatments and the prognosis is often poor,highlighting a pressing need to identify biomarkers of aggressiveness and potential targets for future treatments.Kinesin family member 2C(KIF2C)is reported to be highly expressed in several human tumors.Nevertheless,the molecular mechanisms underlying the role of KIF2C in tumor development and progression have not been investigated.In this study,we found that KIF2C expression was significantly upregulated in HCC,and that KIF2C up-regulation was associated with a poor prognosis.Utilizing both gain and loss of function assays,we showed that KIF2C promoted HCC cell proliferation,migration,invasion,and metastasis both in vitro and in vivo.Mechanistically,we identified TBC1D7 as a binding partner of KIF2C,and this interaction disrupts the formation of the TSC complex,resulting in the enhancement of mammalian target of rapamycin complexl(mTORCI)signal transduction.Additionally,we found that KIF2C is a direct target of the Wnt/β-catenin pathway,and acts as a key factor in mediating the crosstalk between Wnt/β-catenin and mTORCI signaling.Thus,the results of our study establish a link between Wnt/β-catenin and mTORCI signaling,which highlights the potential of KIF2C as a therapeutic target for the treatment of HCC.

Rab22a-NeoF1 fusion protein promotes osteosarcoma lung metastasis through its secretion into exosomes

It remains unknown for decades how some of the therapeutic fusion proteins positive in a small percentage of cancer cells account for patient outcome.Here,we report that osteosarcoma Rab22a-NeoF1 fusion protein,together with its binding partner PYK2,is sorted into exosomes by HSP90 via its KFERQ-like motif(RVLFLN^(142)).The exosomal Rab22a-NeoF1 fusion protein facilitates the pulmonary pre-metastatic niche formation by recruiting bone marrow-derived macrophages.The exosomal PYK2 activates RhoA in its negative recipient osteosarcoma cells and induces signal transducer and activator of transcription 3 activation in its recipient macrophages to increase M2 phenotype.Consequently,lung metastases of its recipient osteosarcoma cells are promoted by this exosomal Rab22a-NeoF1 fusion protein,and this event can be targeted by disrupting its interaction with PYK2 using a designed internalizing RGD peptide.

Snail promotes metastasis of nasopharyngeal carcinoma partly by down-regulating TEL2

Background:Metastasis is the major cause of treatment failure in patients with nasopharyngeal carcinoma(NPC).We previously reported that TEL2,a negative regulator of SERPINE1,could inhibit NPC metastasis to lymph nodes.Method:A series of in vivo and in vitro assays were performed to elucidate the regulation between Snail and TEL2.TEL2 expression was analyzed in three representative NPC cell lines expressing low levels of Snail(S26,6-10B,HK1)and two cell lines expressing high levels of Snail(S18,5-8F).Luciferase and chromatin immunoprecipitation assays were used to analyze the interaction between Snail and TEL2.The roles of the Snail/TEL2 pathway in cell migration and invasion of NPC cells were examined using transwell assays.Metastasis to the lungs was examined using nude mouse receiving NPC cells injection through the tail vein.Results:Ectopic Snail expression down-regulated TEL2 at the mRNA and protein levels,whereas knockdown of Snail using short hairpin RNA up-regulated TEL2.Luciferase and chromatin immunoprecipitation assays indicated that Snail binds directly to the TEL2 promoter.Ectopic Snail expression enhanced migration and invasion of NPC cells,and such effects were mitigated by TEL2 overexpression.TEL2 overexpression also attenuated hypoxia-induced cell migration and invasion,and increased the number of metastatic pulmonary nodules.Snail overexpression reduced the number of metastatic pulmonary nodules.Conclusions:TEL2 is a novel target of Snail and suppresses Snail-induced migration,invasion and metastasis in NPC.

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.

Cannabis suppresses antitumor immunity by inhibiting JAK/STAT signaling in T cells through CNR2

The combination of immune checkpoint blockade(ICB)with chemotherapy significantly improves clinical benefit of cancer treatment.Since chemotherapy is often associated with adverse events,concomitant treatment with drugs managing side effects of chemotherapy is frequently used in the combination therapy.However,whether these ancillary drugs could impede immunotherapy remains unknown.Here,we showed that∆9-tetrahydrocannabinol(THC),the key ingredient of drugs approved for the treatment of chemotherapy-caused nausea,reduced the therapeutic effect of PD-1 blockade.The endogenous cannabinoid anandamide(AEA)also impeded antitumor immunity,indicating an immunosuppressive role of the endogenous cannabinoid system(ECS).

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.