CRISPR-Cas9 Gene Editing Technology Explores The Resistance Mechanism Of Homoharringtonine (HHT) In Acute Myeloid Leukemi

BackgroundAcute myeloid leukemia(AML)is a group of highly heterogeneous malignant clonal proliferative disorders due to blocking in hematopoietic stem cell differentiation and childhood AML accounts for approximately 10-15%of childhood acute leukemias.Over the past 30 years,there has been a marked improvement in the outcome of childhood AML thanks to enhanced chemotherapy regimens and hematopoietic stem cell transplantation[1].As the molecular mechanisms of AML pathogenesis continue to be studied in depth,clinical trials of small molecule targeted drugs or immunotherapy have been conducted for AML patients who are drug-resistant or intolerant to conventional chemotherapy,and some of these drugs have been approved for marketing in China and abroad[2].Even so,nearly 20-30%of pediatric AML patients relapsed in early or late periods and the 5-year event-free survival(EFS)significantly increased(88.0±6.5%vs.60.2±9.6%)[3].The GeCKO(Genome-Scale CRISPR-Cas9 Knockout)method developed by Zhang’s team enables high-throughput genome-wide screening of drug resistance genes in human cells infected with human genome-wide sg library virus[4].Further exploration of the underlying mechanisms of HHT drug resistance in AML and the search for alternative treatment options that may reverse drug resistance is important for the precise targeting of relapsed/refractory AML(R/R AML)and may improve prognosis in those patients.ObjectivesThe utilization of CRISPR-Cas9 gene editing technology and high-throughput sequencing to screen for candidate genes that may cause HHT drug resistance in AML and explore the mechanisms of HHT resistance in AML.Methods(1)Lentivirus production and viral infection of cells:lenti-X 293T cells were transfected using PEI with target plasmids and packaging plasmids.The virus is concentrated and used to infect cells.(2)CRISPR-Cas9 library screening monoclonal Cas9-expressing Molm13 cells were constructed using pKLV2-EF1a-Cas9Bsd-W(Addgene#68343).Loss-of-Function of the targeted gene was performed using the human genomic sgRNA library(Addgene#67989),which targeted the 18010 genes with 90709 sgRNA.Cas9/molm13 cells were used for viral transduction at a low multiplicity of infection(MOI)of 0.3 and treated with HHT or DMSO for 4 weeks.Cells were collected every 2-3 days and genomic DNA was extracted and isolated at the end of the resistance screen.The HHT resistance screen was repeated three times.The CRISPR-Screen library was prepared by a two-step PCR.(3)Individual genes were inactivated by cloning sgRNA into pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W(Addgene#67974)and according to the manufacturer’s suggestions.(4)The drug sensitivity assay was performed using Cell Counting Kit-8.The competition culture assay was performed to validate the competitive advantage of the cells.(5)Apoptosis assay,cell cycle assay,qPCR,and western blotting were performed for Ex vivo functional screen.Results(1)Monoclonal Cas9-expressing Molm13 cell lines were constructed and then transfected with pKLV2-U6gRNA5(gGFP)-PGKBFP2AGFP-W(Addgene#67980)viruses to verify the successful expression of Cas9 protein.(2)HHT resistance screening was performed three times using Cas9/molm13 cells infected with human genome-wide sg library virus,and MAGeCK analysis found the top 10 candidate genes that may be associated with HHT resistance in AML.(3)In vitro phenotypic validation:Three candidate resistance genes with the greatest log fold change(1gf)including TP53,PELO,and PPRC1 were selected and knockout in Cas9/molm13 cells,which had a competitive proliferation advantage and higher half maximal inhibitory concentration(IC50).(4)Loss-of-function of the TP53 gene induces HHT resistance through impaired regulation of cell apoptosis and cell cycle in Molm13 cells.Conclusion(1)The high-throughput screening of leukemia drug resistance genes by GeCKO is highly efficient,and several candidate genes associated with HHT drug resistance in AML were obtained.(2)Loss-of-function of the TP53 gene is related to HHT drug resistance in AML and loss-of-function of the TP53 gene mediates resistance to HHT through regulation of apoptosis and the cell cycle.ObjectiveTo investigate the clinical features,early treatment response,and prognostic correlation analysis of childhood acute T lymphoblastic leukemia(T-ALL)with CDKN2A/B deletion.MethodsA total of 63 pediatric T-ALL patients enrolled in the CCCG-ALL 2015 regimen at our center were retrospectively analyzed and their clinical outcomes were tracked.ResultsNearly 46.0%(29/63)of the 63 pediatric acute T-lymphocytic leukemia patients carried CDNK2A/B gene deletion.Pediatric T-ALL had higher initial white blood cell counts when carrying CDKN2A/B deletion(174.09 vs.57.1×109/L,P=0.024),and there was no significant difference between gender,age at diagnosis,or initial cerebrospinal fluid(CNS)status,and concurrence of gene mutations(P>0.05).CDKN2A/B gene deletion did not increase the risk of poor early treatment response in pediatric T-ALL,including poor prednisone response(PPR)(41.4%vs.47.1%,P=0.651),bone marrow MRD positive on day 19 of induction remission therapy(44.8%vs.58.8%,P=0.268),bone marrow MRD positive on day 46 of induction remission therapy(20.7%vs.14.7%,P=0.268).There was no significant effect of CDKN2A/B gene deletion on 3-year overall survival(90.6±5.2%vs.89.7±5.7%)and 3-year event-free survival(72.9±8.8%vs.81.4±7.6%)of pediatric T-ALL.ConclusionCDKN2A/B gene deletion detected by FISH is a recurrent cytogenetic abnormality in childhood T-ALL,which had a higher initial white blood cell count.CDKN2A/B deletion did not serve as a poor prognostic factor in pediatric T-ALL.