Journal: bioRxiv
Article Title: Group comparison based on genetic information reveals lineage-specific therapeutic vulnerabilities in acute myeloid leukemia
doi: 10.1101/2023.05.18.541265
Figure Lengend Snippet: PCYT1A is essential for the growth of monocytic AML. ( a ) Human MLL-AF9 cells, mouse MLL-AF9 cells, and cSAM cells were transduced with Cas9 together with non-targeting (NT) sgRNA or PCYT1A/Pcyt1a-targeting sgRNAs (sgPCYT1A/Pcyt1a) co-expressing tRFP657. Changes in the frequency of tRFP657 + cells (sgRNA-transduced cells) in cell cultures are shown. Results are normalized to the frequency of tRFP657 + cells at day 4, set to 1. These MLL-AF9 expressing AML cells and MLL-WT monocytic AML cells are not registered in DepMap dataset. ( b ) FPKM values obtained via RNA-seq analysis. The data were retrieved from GSE135008 (MLL-AF9: n=3, cSAM: n=3) . ( c, d ) OCIAML3 and U937 cells were transduced with Cas9 together with non-targeting (NT) sgRNA or PCYT1A-targeting sgRNAs (sgPCYT1A) co-expressing tRFP657. PCYT1A depletion by each sgRNA was confirmed. ( c ) Changes in the frequency of tRFP657 + cells (sgRNA-transduced cells) in cell cultures (triplicates) are shown. ( d ) Results are normalized to the frequency of tRFP657 + cells at day 4, set to 1. Data are shown as mean ± s.d. ns not significant, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, two-way ANOVA multiple comparisons. ( e-f ) mRNA expression levels of PCYT1A and PCYT1B in 162 AML patients’ samples. The data were retrieved from cBioPortal and TCGA. M0 – M7 indicates the FAB classification. Data are shown as mean ± s.e.m. ** P <0.01, *** P <0.001, **** P <0.0001, Brown-Forsythe and Welch ANOVA tests.
Article Snippet: Annealed oligonucleotides were cloned into the pLKO5.sgRNA.EFS.tRFP657 vector, pLKO5.sgRNA.EFS.GFP vector or lentiGuide-Puro vector to generate single-guide (sg)RNA expression vector. pLKO5.sgRNA.EFS.tRFP657 (Addgene plasmid # 57824 ; http://n2t.net/addgene:57824 ; RRID:Addgene_57824) and pLKO5.sgRNA.EFS.GFP (Addgene plasmid # 57822 ; http://n2t.net/addgene:57822 ; RRID:Addgene_57822) were gifts from Benjamin Ebert. lentiGuide-Puro was a gift from Feng Zhang (Addgene plasmid # 52963 ; http://n2t.net/addgene:52963 ; RRID:Addgene_52963).
Techniques: Transduction, Expressing, RNA Sequencing Assay