Journal: Blood

Article Title: Salt-inducible kinase inhibition suppresses acute myeloid leukemia progression in vivo

doi: 10.1182/blood.2019001576

Figure Lengend Snippet: SIK3 and MEF2C are selectively essential for the growth of AML and multiple myeloma cells. (A) SIK3 and MEF2C essentiality scores extracted from the DepMap database of cancer cell lines.35 Shown is a boxplot distribution of the copy number–adjusted essentiality score (CERES; a normalized metric of gene essentiality) of SIK3 and MEF2C across all 558 cell lines, 16 AML lines, 18 multiple myeloma lines, and 498 solid tumor cell lines. (B-C) Scatterplots of SIK3 and MEF2C essentiality scores in human AML cell lines in the DepMap (CERES) or from Wang et al52 (CRISPR scores). (D) Western blot analysis of MEF2C in RN2 and 3T3 whole-cell lysates. (E) Competition-based proliferation assays in which cells were infected with the indicated sgRNAs linked to GFP. Bar graphs represent the mean ± standard error of the mean (SEM; n = 3). (F) Bright-field images of methylcellulose-based colony-formation assays of normal myeloid progenitors or RN2 cells on day 7 after retroviral transduction with control or Sik3 shRNAs. (G) Quantification of the immature/blast colonies shown in panel F. Mean ± SEM (n = 4). (H) Western blot analysis of SIK3 performed on day 6 after infection of the indicated shRNAs. (I) Bioluminescence imaging of wild-type C57BL/6 mice receiving transplants of Cas9-expressing RN2 cells transduced with the indicated sgRNA. Representative images are shown on the indicated day after transplantation. (J) Quantification of bioluminescence from panel I. Values represent photons per second (p/s) of bioluminescent signal detection (mean ± SEM). The P value was calculated by unpaired Student t test (n = 5). (K) Survival curves of the mice in panel I. The P value was calculated by log-rank (Mantel-Cox) test (n = 5). (L) Bioluminescence imaging of NSG (NOD-SCID/IL2Rgammanull) mice which received transplants of Cas9-expressing MV4-11 cells transduced with the indicated sgRNA. Representative images are shown on the indicated day after transplantation. (M) Quantification of bioluminescence from panel L. Values represent photons per second (p/s) of bioluminescent signal detection (mean ± SEM). The P value was calculated by unpaired Student t test (n = 5). (N) Survival curves of the mice in panel L. The P value was calculated by log-rank (Mantel-Cox) test (n = 5). sgNeg1, sgNeg2, shREN: negative controls. sgRpa3: positive control.

Article Snippet: Plasmid constructions The lentiviral single-guide RNA (sgRNA) expression vectors (LRG2.1, Addgene_108098; LRG, Addgene_65656) and the lentiviral Cas9 (LentiV_Cas9_puro, Addgene_108100) or luciferase (Lenti-luciferase-P2A-Neo, Addgene_105621) expression vectors were described previously.

Techniques: CRISPR, Western Blot, Infection, Retroviral, Transduction, Control, Imaging, Expressing, Transplantation Assay, Positive Control

Journal: Cancer discovery

Article Title: SLC5A3-dependent myo-inositol auxotrophy in acute myeloid leukemia

doi: 10.1158/2159-8290.CD-20-1849

Figure Lengend Snippet: (A) Overview of in vivo genetic screening procedure using domain-focused sgRNA libraries. GPCR, G-protein coupled receptor; NGS, Next Gen Sequencing. The numbers represent the number of genes in each sgRNA library. Figures for mouse bone marrow, spleen and NGS were created with a licensed version of BioRender . (B-E) Examples of in vivo CRISPR screening results with sgRNA libraries targeting kinases (B), transcription factors (C), chromatin regulators (D), and membrane proteins (E). The gene beta score was calculated using MAGeCK (78, 79). The beta score describes how the gene knockout alters cell fitness: a positive beta score indicates positive selection, and a negative beta score indicates negative selection. The screening results using pooled spleen samples were compared with in vitro screening results. Core fitness genes (95) are labeled in red, negative control sgRNAs are labeled in blue. (F) Venn diagram comparing genetic dependencies identified by in vivo screening with in vitro screening. The in vivo-relevant targets were determined using spleen data with p-value <0.05, FDR <0.25, beta score <0. For in vivo- and in vitro-specific targets, the absolute value of differential beta scores were shown as pie charts. Genes with a differential beta score of 2 or greater are listed.

Article Snippet: Plasmid Construction 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 ( 42 , 75 ).

Techniques: In Vivo, Sequencing, CRISPR, Membrane, Gene Knockout, Selection, In Vitro, Labeling, Negative Control

Journal: Cancer discovery

Article Title: SLC5A3-dependent myo-inositol auxotrophy in acute myeloid leukemia

doi: 10.1158/2159-8290.CD-20-1849

Figure Lengend Snippet: (A) Analysis of Project Achilles genetic screening data, evaluating the leukemia-biased essentiality of 239 in vivo-relevant dependencies. The in vivo screening dependencies were determined by p-value <0.05, FDR <0.25, beta score <0. The average gene effect scores were calculated using Achilles Gene Effect 20Q3 data. The p values compared gene effect scores in leukemia vs. other cancers using an unpaired Student’s t-test. (B) Comparison of dependencies of top SLC candidates between leukemia and other type of cancers. The dependency of SLC5A3 is the most leukemia-biased among the candidates. Data is obtained from DepMap database (20Q3). Plotted as average gene effect score with standard deviation in leukemia cell lines vs. non-leukemia cell lines. The p values were calculated using unpaired Student’s t-test. (* p-value <0.05) (C) Summary of in vivo vs in vitro CRISPR screening results of the SLC sgRNA library.

Article Snippet: Plasmid Construction 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 ( 42 , 75 ).

Techniques: In Vivo, Comparison, Standard Deviation, In Vitro, CRISPR

Journal: Cancer discovery

Article Title: SLC5A3-dependent myo-inositol auxotrophy in acute myeloid leukemia

doi: 10.1158/2159-8290.CD-20-1849

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: Plasmid Construction 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 ( 42 , 75 ).

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

Journal: Cancer discovery

Article Title: SLC5A3-dependent myo-inositol auxotrophy in acute myeloid leukemia

doi: 10.1158/2159-8290.CD-20-1849

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 (96, 97). 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 myo-inositol supplement (−MI, blue), or with 0.05 g/L (0.28 mM) myo-inositol supplement (+MI, red). (Shown as mean with standard deviation of 3 replicates with independent infections.)

Article Snippet: Plasmid Construction 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 ( 42 , 75 ).

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

Journal: Cancer discovery

Article Title: SLC5A3-dependent myo-inositol auxotrophy in acute myeloid leukemia

doi: 10.1158/2159-8290.CD-20-1849

Figure Lengend Snippet: (A) Liquid Chromatography Mass Spectrometry (LC-MS) analysis of cellular myo-inositol and glutamine levels in MOLM-13 and MV4-11 cells transduced with indicated sgRNA. The p-value was calculated using unpaired Student’s t test (n=6). (**** p<0.0001). (B-C) Depletion of myo-inositol in the cell culture medium affects the growth of SLC5A3-dependent lines MOLM-13, MV4-11, and U937 (B), but not SLC5A3-independent lines HEL, K562, and SET-2 (C). A customized RPMI cell culture medium without myo-inositol, supplied with dialyzed FBS, was used (−MI). Additional myo-inositol supplement (1.11 mM/0.2 g/L) was added into the medium for rescue (+MI). (Shown as mean with standard deviation of 2-4 biological replicates. The p values were calculated using 2way ANOVA Sidak test to compare each line graph). (D) Competition-based proliferation assays in MOLM13 and MV4-11 cells transduced with indicated sgRNA, and treated with additional myo-inositol supplement. The base level of myo-inositol in RPMI medium is 0.035 g/L (0.19 mM). To rescue the phenotype, additional myo-inositol was added to the cell culture to make myo-inositol concentration as 0.07 g/L (0.38 mM) and 0.175 g/L (0.95 mM). Myo-inositol from FBS (100% FBS contains ~0.9 mM, determined by LC-MS, 10% FBS was added to cell culture medium) was not included. (Shown as mean with standard deviation of 3 replicates with independent infections).

Article Snippet: Plasmid Construction 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 ( 42 , 75 ).

Techniques: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transduction, Cell Culture, Standard Deviation, Concentration Assay

Journal: Cancer discovery

Article Title: SLC5A3-dependent myo-inositol auxotrophy in acute myeloid leukemia

doi: 10.1158/2159-8290.CD-20-1849

Figure Lengend Snippet: (A) Western blot shows overexpression of flag-tagged ISYNA1 in indicated AML cell lines. (B) Competition-based proliferation assays in SLC5A3-dependent AML lines expressing EV (empty vector, blue) or ISYNA1(red), following infection with indicated sgRNA linked to GFP. (Shown as mean with standard deviation of 3 replicates from independent infections). (C) LC-MS analysis in MOLM-13 cells expressing EV or ISYNA1, following infection with indicated sgRNA. The p-value was calculated using 2way ANOVA (n=5). (** p=0.0011, **** p<0.0001). (D) Overexpression of ISYNA1 in MOLM-13 cells rescues the growth defect in myo-inositol deficient medium (−MI), but has no impact on the cell growth in medium with myo-inositol supplement (+MI). (Shown as mean with standard deviation of two biological replicates, each includes 3 replicates with independent infection. The p value was calculated using 2way ANOVA Sidak test.)

Article Snippet: Plasmid Construction 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 ( 42 , 75 ).

Techniques: Western Blot, Over Expression, Expressing, Plasmid Preparation, Infection, Standard Deviation, Liquid Chromatography with Mass Spectroscopy