Systematic screening reveals synergistic interactions that overcome MAPK inhibitor resistance in cancer cells

Objective:Effective adjuvant therapeutic strategies are urgently needed to overcome MAPK inhibitor(MAPKi)resistance,which is one of the most common forms of resistance that has emerged in many types of cancers.Here,we aimed to systematically identify the genetic interactions underlying MAPKi resistance,and to further investigate the mechanisms that produce the genetic interactions that generate synergistic MAPKi resistance.Methods:We conducted a comprehensive pair-wise sgRNA-based high-throughput screening assay to identify synergistic interactions that sensitized cancer cells to MAPKi,and validated 3 genetic combinations through competitive growth,cell viability,and spheroid formation assays.We next conducted Kaplan-Meier survival analysis based on The Cancer Genome Atlas database and conducted immunohistochemistry to determine the clinical relevance of these synergistic combinations.We also investigated the MAPKi resistance mechanisms of these validated synergistic combinations by using co-immunoprecipitation,Western blot,qRTPCR,and immunofluorescence assays.Results:We constructed a systematic interaction network of MAPKi resistance and identified 3 novel synergistic combinations that effectively targeted MAPKi resistance(ITGB3+IGF1R,ITGB3+JNK,and HDGF+LGR5).We next analyzed their clinical relevance and the mechanisms by which they sensitized cancer cells to MAPKi exposure.Specifically,we discovered a novel protein complex,HDGF-LGR5,that adaptively responded to MAPKi to enhance cancer cell stemness,which was up-or downregulated by the inhibitors of ITGB3+JNK or ITGB3+IGF1R.Conclusions:Pair-wise sgRNA library screening provided systematic insights into elucidating MAPKi resistance in cancer cells.ITGB3-+IGF1R-targeting drugs(cilengitide+linsitinib)could be used as an effective therapy for suppressing the adaptive formation of the HDGF-LGR5 protein complex,which enhanced cancer stemness during MAPKi stress.

EMT transcription factors activated circuits:A novel tool to study EMT dynamics and its therapeutic implications

The epithelial mesenchymal transition(EMT)plays significant roles in the progression of cancer and fibrotic disease.Moreover,this process is reversible,resulting in mesenchymal epithelial transition(MET),which plays an important role in cancer metastasis.There is a lack of methods to trace and target EMT cells using synthetic biology circuits,which makes it difficult to study the cell fate or develop targeted treatments.In this study,we introduced responsive EMT sensing circuits,which sense the EMT using specific promoters that respond to transcription factors typical of EMT activation(EMT-TFs).The transcriptional strength of EMT-sensing promoters decreased more than 13-fold in response to the overexpression of the EMT-TF.Then,the NOT gate circuits were built by placing the tetR transcription repressor under the control of EMT sensing promoters and expressed an output signal using the constitutive CMV promoter modified with tetO sites This circuit is named EMT sensing and responding circuits.When the EMT transcription factors was present,we observed a 5.8-fold signal increase in the system.Then,we successfully distinguished mesenchymal breast cancer cells from epithelial cancer cells and repressed the proliferation of EMT tumor cells using our circuits.The EMT sensing and responding circuits are promising tools for the identification of EMT cells,which is crucial for EMT-related disease therapy and investigating the mechanisms underlying the reversible EMT process.

3D bioactive cell-free-scaffolds for in-vitro/in-vivo capture and directed osteoinduction of stem cells for bone tissue regeneration

Hydrophilic bone morphogenetic protein 2(BMP2)is easily degraded and difficult to load onto hydrophobic carrier materials,which limits the application of polyester materials in bone tissue engineering.Based on soybean-lecithin as an adjuvant biosurfactant,we designed a novel cell-free-scaffold of polymer of poly(ε-caprolactone)and poly(lactide-co-glycolide)-co-polyetherimide with abundant entrapped and continuously released BMP2 for in vivo stem cell-capture and in situ osteogenic induction,avoiding the use of exogenous cells.The optimized bioactive osteo-polyester scaffold(BOPSC),i.e.SBMP-10SC,had a high BMP2 entrapment efficiency of 95.35%.Due to its higher porosity of 83.42%,higher water uptake ratio of 850%,and sustained BMP2 release with polymer degradation,BOPSCs were demonstrated to support excellent in vitro capture,proliferation,migration and osteogenic differentiation of mouse adipose derived mesenchymal stem cells(mADSCs),and performed much better than traditional BMP-10SCs with unmodified BMP2 and single polyester scaffolds(10SCs).Furthermore,in vivo capture and migration of stem cells and differentiation into osteoblasts was observed in mice implanted with BOPSCs without exogenous cells,which enabled allogeneic bone formation with a high bone mineral density and ratios of new bone volume to existing tissue volume after 6 months.The BOPSC is an advanced 3D cell-free platform with sustained BMP2 supply for in situ stem cell capture and osteoinduction in bone tissue engineering with potential for clinical translation.

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

Human pluripotent stem cells （hPSCs） are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR.ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline （Dox） inducible dCasg-VP64-p65-Rta （dCas9-VPR） transcription activator and a reverse Tet transactivator （rtTA） expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG pro- moter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naive pluripotsnt gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass （ICM） of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

Hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p inhibitors can reduce the cytotoxicity of Ebola virus glycoprotein in vitro

Ebola virus （EBOV） causes a highly lethal hemorrhagic fever syndrome in humans and has been associated with mortality rates of up to 91% in Zaire, the most lethal strain. Though the viral envelope glycoprotein （GP） mediates widespread inflammation and cellular damage, these changes have mainly focused on alterations at the protein level, the role of microRNAs （miRNAs） in the molecular pathogenesis underlying this lethal disease is not fully understood. Here, we report that the miRNAs hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p were induced in human umbilical vein endothelial cells （HUVECs） following expression of EBOV GP. Among the proteins encoded by predicted targets of these miRNAs, the adhesion-related molecules tissue factor pathway inhibitor （TFPI）, dystroglycan! （DAG1） and the caspase 8 and FADD-like apoptosis regulator （CFLAR） were significantly downregulated in EBOV GP-expressing HUVECs. Moreover, inhibition of hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p, or overexpression of TFPI, DAG1 and CFLAR rescued the cell viability that was induced by EBOV GP. Our results provide a novel molecular basis for EBOV pathogenesis and may contribute to the development of strategies to protect against future EBOV pandemics.

Construction of a CRISPR-based paired-sgRNA library for chromosomal deletion of long non-coding RNAs

Background:Derived from an adaptive bacterial immune system,the clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated 9(Cas9)system has shown great potential in high-throughput functional genomic screening,especially for protein-coding genes.However,it is still challenging to apply the similar strategy to study non-coding genomic elements such as long non-coding RNAs(lncRNAs)or clusters of microRNAs,because short insertions or deletions may not be sufficient to generate loss-of-function phenotypes.Methods:Here,we presented a systematic strategy for designing a CRISPR-based paired-sgRNA library for highthroughput screening in non-coding regions.Due to the abundance of lncRNAs and their diverse regulatory roles in vivo,we repurposed microarray datasets to select 600 highly expressed lncRNAs in non-small-cell lung cancer and designed two schemes for lncRNA deletion with^20 paired-sgRNAs for each lncRNA.Through Golden-Gate assembly,we generated a pooled CRISPR-based library with a total of 12,878 sgRNA pairs.Results:Over 80%of paired-sgRNAs were recovered from final pooled library with a relarively even distribution.Cleavage efficiency of sgRNA pairs was validated through experiments of transient transfection and viral infection.Moreover,randomly selected paired-sgRNAs showed that efficient deletion of genomic DNA could be achieved with a deletion size within the range of 500 to 3000 bp.Conclusions:In summary,we have demonstrated a strategy to design and construct a pooled paired-sgRNA library to generate genomic deletion in the lncRNA regions,validated their deletion efficiency and explored the relationship of deletion efficiency with respect to deletion size.This method would be also suitable for investigation of other uncharacterized non-coding genomic regions in mammalian cells in an efficient and cost-efTective manner.

Synthetic gene circuits moving into the clinic

On November 28th 2020,SyngenTech,a China-based biotechnology company,announced that the company’s leading oncolytic virotherapeutic product,SynOVl.l,was approved for clinical trial by the U.S.Food and Drug Administration.After synthetic bacterial or cell therapy[1,2],this is the first introduction of a viral therapy engineered using synthetic gene circuits into the clinic.Together with the clinical development of variable living therapeutics,we anticipate that synthetic gene circuits will receive increasing attention as a revolutionary technology to improve disease-treatment efficacy.

Explore the dominant factor in prime editing via a view of DNA processing

Prime editing is a revolutionary gene-editing method that is capable of introducing insertions,deletions and base substitutions into the genome.However,the editing efficiency of Prime Editor(PE)is limited by the DNA repair process.Here,we show that overexpression of the flap structure-specific endonuclease 1(FEN1)and the DNA ligase 1(LIG1)increases the efficiency of prime editing,which is similar to the dominant negative mutL homolog 1(MLH1dn).In addition,MLH1 is still the dominant factor over FEN1 and LIG1 in prime editing.Our results help to further understand the relationship of proteins involved in prime editing and envisage future directions for the development of PE.

A personal journey on cracking the genomic codes

In February 2001,the IHGSC(International Human Genome Sequencing Consortium)[1]and Celera Genomics[2]each reported draft sequences providing a first overall view of the human genome.On the occasion of the 20th anniversary celebration,I would like to provide some of my personal account on how the HGP(Human Genome Project)has impacted my academic research interests and to offer some candid advice for the new comers.1.

Modulating the aggregation of amyloid proteins by macrocycles

The aggregation of amyloid proteins has been suggested to be the main cause of multiple human disorders;for example,amyloidβaggregates in Alzheimer’s disease andα-synuclein aggregates in Parkinson’s disease.In the search for therapeutic medicines,many molecules have been discovered and developed to modulate the aggregation of amyloid proteins.This century has witnessed the flourishing growth of supramolecular chemistry,and some biocompatible macrocycles have been proven to inhibit the aggregation of some amyloid proteins via host-guest interactions and could thus be used for the prevention or treatment of related diseases.Here,we review the application of macrocycles in modulating the aggregation of amyloid proteins.