Abstract Novel analytic tools are needed to elucidate the molecular basis of leukemia-relevant gene mutations in the post-genome era. We generated isogenic leukemia cell clones in which the FLT3 gene was disrupted in a single allele using TALENs. Isogenic clones with mono-allelic disrupted FLT3 were compared to an isogenic wild-type control clone and parental leukemia cells for transcriptional expression, downstream FLT3 signaling and proliferation capacity. The global gene expression profiles of mutant K562 clones and corresponding wild-type controls were compared using RNA- seq . The transcriptional levels and the ligand-dependent autophosphorylation of FLT3 were decreased in the mutant clones. TALENs-mediated FLT3 haplo-insufficiency impaired cell proliferation and colony formation in vitro . These inhibitory effects were maintained in vivo , improving the survival of NOD/SCID mice transplanted with mutant K562 clones. Cluster analysis revealed that the gene expression pattern of isogenic clones was determined by the FLT3 mutant status rather than the deviation among individual isogenic clones. Differentially expressed genes between the mutant and wild-type clones revealed an activation of nonsense-mediated decay pathway in mutant K562 clones as well as an inhibited FLT3 signaling. Our data support that this genome-editing approach is a robust and generally applicable platform to explore the molecular bases of gene mutations.
B cell lymphoma can co-occur with multiple myeloma (MM), and the prognosis in this case is usually poor. We propose the combination of CD19-chimeric antigen receptor (CAR) T cells and BCMA-CAR T cells for the treatment of such patients to obtain a superior prognosis.
Liquid biopsy is a promising technique for tumor genetic profiling and evaluating disease progression. Recent advances in circulating tumor DNA (ctDNA) research highlight its utility in solid tumors and hematological diseases. Extramedullary multiple myeloma (EMM) is an advanced type of multiple myeloma, defined by the presence of clonal plasma cells outside of the bone marrow. The potential of ctDNA to reveal the holistic genomic landscape of EMM is of great interest. Moreover, the mutational concordance between ctDNA and extramedullary plasmacytoma biopsies requires further investigation. In this study, using time-matched extramedullary plasmacytoma biopsies, bone marrow aspirates, and plasma samples from eight EMM patients, we conducted deep sequencing targeting the coding regions of 22 multiple myeloma-related genes. Sequencing results were further verified using droplet digital PCR. Gene mutations in mitogen-activated protein kinase pathways were identified in all 8 tissue biopsies and 7/8 (87.5%) time-matched plasma samples. The ctDNA to tissue biopsy concordance and bone marrow aspirate to tissue biopsy concordance were 0.873 (P = 8.66e−7) and 0.621 (P = 0.109), respectively. In conclusion, our results suggest that ctDNA can be a reliable surrogate for extramedullary plasmacytoma biopsy to characterize the genomic landscape and track disease progression, particularly when extramedullary plasmacytomas are inaccessible.Funding Statement: This work was supported in part by the National Science Foundation for Young Scientists of China (No. 81700160), National Natural Science Foundation of China (General Program, No. 81873452), National Natural Science Foundation of China (Key Program, No. 81630006).Declaration of Interests: The authors stated: "No relevant conflicts of interest to declare."Ethics Approval Statement: This study was approved by Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The approval file number is TJ-IRB20180813.
PAX5 encodes a transcription factor essential for B-cell differentiation, and PAX5 haploinsufficiency is involved in tumorigenesis. There were few studies on how PAX5 haploinsufficiency regulated genes expression to promote tumorigenesis. In this study, we constructed the cell model of PAX5 haploinsufficiency using gene editing technology in Raji cells, detected differentially expressed genes in PAX5 haploinsufficiency Raji cells, and used protein-protein interaction networks and cluster analysis to comprehensively investigate the cellular pathways involved in PAX5 haploinsufficiency. The clusters of gene transcription, inflammatory and immune response, and cancer pathways were identified as three important pathways associated with PAX5 haploinsufficiency in Raji cells. These changes hinted that the mechanism of PAX5 haploinsufficiency promoting tumorigenesis may be related to genomic instability, immune tolerance, and tumor pathways.
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