Journal: bioRxiv

Article Title: In vivo genetic screen identifies a SLC5A3-dependent myo-inositol auxotrophy in acute myeloid leukemia

doi: 10.1101/2020.12.22.424018

Figure Lengend Snippet: (A) Summary of in vitro validation experiments of individual sgRNAs (linked to GFP) in competition-based proliferation assays in AML cell lines. Plotted is the log2 transformed fold change of GFP positive cell percentage at day 18 vs. day 3 post the infection of sgRNA lentivirus. Shown as mean with standard deviation of 2~7 biological replicates. (B) Procedure of in vivo validation of SLC5A3 dependency in human AML. MOLM-13 cells expressing Cas9 and Luciferase were transduced with sgROSA or sgSLC5A3-2 (linked to GFP/bsr), and then selected with Blasticidin. The SLC5A3 knock-out cells (sgSLC5A3) and control cells (sgROSA) were then transplanted into NSG mice via tail vein injection. (C) Bioluminescence imaging of NSG mice transplanted with MOLM-13 cells transduced with the indicated sgRNA. The images were taken on day 13, 16, and 19 post-transplantation. (D) Quantification of bioluminescence from (C). Values represent total flux as photons per second (Mean ± Standard Deviation). The p -value was calculated using unpaired Student’s t test (n=5). (E) Survival curves of mice shown in (C). The p -value was calculated using Log-rank (Mantel-Cox) test (n=5).

Article Snippet: The lentiviral Cas9 vector (LentiV_Cas9_puro/Addgene #108100), the lentiviral sgRNA vectors (LRG2.1-GFP/Addgene #108098, LRG2.1-mCherry/Addgene #108099, the lentiviral luciferase vector (LentiV_Neo_Luc/Addgene #105621), and the lentiviral cDNA expression vector (LentiV_Neo/Addgene #108101) have been described in previous studies ( , ).

Techniques: In Vitro, Biomarker Discovery, Transformation Assay, Infection, Standard Deviation, In Vivo, Expressing, Luciferase, Transduction, Knock-Out, Control, Injection, Imaging, Transplantation Assay

Journal: bioRxiv

Article Title: In vivo genetic screen identifies a SLC5A3-dependent myo-inositol auxotrophy in acute myeloid leukemia

doi: 10.1101/2020.12.22.424018

Figure Lengend Snippet: (A) Summary of two major sources of cellular myo-inositol: transported from extracellular environment by SLC5A3; de novo biosynthesis from glucose. The rate limiting de-novo myo-inositol biosynthesis enzyme is inositol-3-phosphate synthase 1, encoded by the ISYNA1 gene. (B) SLC5A3 dependency in AML (and K562) cell lines correlates with ISYNA1 expression. Data from DepMap database (Achilles 20Q3). (C) Expression of ISYNA1 in AML (and K562) cell lines. The ISYNA1 gene mRNA expression data was obtained from Cancer Cell Line Encyclopedia (CCLE) database. The ISYNA1 protein expression in AML lines and K562 (CML) was detected by Western blot, shown as one of two replicates. (D) A double knock-out construct that co-targets two genes by two sgRNAs driven by human U6 (hU6) or bovine U6 (bU6) promoter, respectively. The sgRNAs are linked to GFP. (E) Competition-based proliferation assays in Cas9 expressing SLC5A3-independent cell lines, following infection with indicated co-targeting sgRNA constructs. Double knock-out both ISYNA1 and SLC5A3 leads to growth phenotype (red). (Shown as mean with standard deviation of 3 replicates of independent infections.). (F) Western blot shows the depletion of ISYNA1 protein in HEL and K562 cells following infection with indicated sgRNA. (G) Competition-based proliferation assays in Cas9 expressing HEL and K562 cells following infection with indicated sgRNA. The experiment was performed in myo-inositol deficient RPMI medium without myoinositol supplement (-MI, blue), or with 0.2 g/L (1.11 mM) myo-inositol supplement (+MI, red). (Shown as mean with standard deviation of 3 replicates with independent infections.)

Article Snippet: The lentiviral Cas9 vector (LentiV_Cas9_puro/Addgene #108100), the lentiviral sgRNA vectors (LRG2.1-GFP/Addgene #108098, LRG2.1-mCherry/Addgene #108099, the lentiviral luciferase vector (LentiV_Neo_Luc/Addgene #105621), and the lentiviral cDNA expression vector (LentiV_Neo/Addgene #108101) have been described in previous studies ( , ).

Techniques: Expressing, Western Blot, Knock-Out, Construct, Infection, Standard Deviation