Journal: EMBO Molecular Medicine

Article Title: CDK7/12/13 inhibition targets an oscillating leukemia stem cell network and synergizes with venetoclax in acute myeloid leukemia

doi: 10.15252/emmm.202114990

Figure Lengend Snippet: Cartoon illustrating at which levels GPR56 may enhance canonical Wnt signaling (turquoise dashed arrows): at the level of ligand‐receptor/co‐receptor interaction, β‐catenin, downstream of β‐catenin, indirectly via activation of Rho/SRF through Gɑ12/13. CHIR99021 is a GSK3 inhibitor that prevents degradation of β‐catenin thus enhancing Wnt signaling. Cartoon illustrating GPR56 full length (FL, left ) and the C‐terminal fragment (CTF, right ). Amino acid changes introduced in the intracellular loops are indicated by circles and letters. GAIN: GPCR autoproteolysis‐inducing domain, GPS: GPCR proteolytic site, PLL: Pentraxin/Laminin/neurexin/sex‐hormone‐binding‐globulin‐Like (Salzman et al , ). SuperTop reporter assay showing fold change of relative luminescence units (RLU) in Wnt3a conditioned culture media normalized to empty pcDNA3.1 + in the respective control (Ctrl) media after transfection of HEK293T cells with empty vector (violet), GPR56 FL (light turquoise), and GPR56 CTF (turquoise). Shown are means, SD, and individual values of four technical replicates performed in either complete media or 1:3 diluted Wnt3a conditioned media. Unpaired t ‐test, **** P < 0.0001, *** P < 0.0005, ** P < 0.005. β‐catenin protein expression in K562 cells infected with shGPR56 strong or shLuc negative control in presence and absence of Wnt3a (representative Western Blot (above) and quantification after normalization to GAPDH (below)). Shown is fold‐change compared with shLuc in control media, bars and error bars represent mean and SD of three biological replicates, unpaired t ‐test. SuperTop reporter assay showing fold‐change of RLU in presence of Wnt3a normalized to a scrambled shRNA (shSCR) in control media after transfection with shSCR, shRNAs against GPR56 with or without additional transfection with 1 ng or 2 ng of β‐catenin plasmid. Overexpression of β‐catenin fully rescues the SuperTop signal reduction caused by shRNAs against GPR56. Unpaired t ‐test, bars, and error bars represent mean and SD of four biological replicates, **** P < 0.0001, *** P < 0.0005, ** P < 0.005, * P < 0.05. SuperTop reporter assay indicating fold change in RLU normalized to control media after transfection of HEK293T cells with empty vector, GPR56‐FL, or 5 GPR56‐CTF mutants. Unpaired t ‐test, bars, and error bars represent mean and SD of four biological replicates, **** P < 0.0001, ** P < 0.005. Left : SMO‐GFP reporter RPE cell line or RPE‐1 wild‐type cells were infected with shGPR56 strong or shLuc control followed by administration of doxycycline or the SMO agonist SAG as indicated. GPR56 KD significantly reduces the baseline and doxycycline‐induced SMO‐GFP signal. Right : representative FACS histogram plots showing GFP intensity in shLuc versus shGPR56 strong RPE cells with and without doxycycline. Unpaired t ‐test, bars, and error bars represent mean and SD of three biological replicates, *** P < 0.0005, ** P < 0.005, * P < 0.05. Left : Representative immunofluorescence (IF) images showing primary cilium formation in RPE cells upon starvation after infection with shLuc (above) or shGPR56 strong (below). The non‐motile primary cilium is visualized using antibodies against γ‐tubulin, which stains the basal body, and the ciliary membrane marker ARL13B. DAPI indicates the nucleus. Images on the right show selected cells at 3× magnification. Brightness was increased in all images to enhance visibility of cilia. Right : Percentage of RPE‐1 cells with primary cilium in shLuc vs. shGPR56 strong RPE‐1 cells at different time points after the start of serum starvation. Unpaired t ‐test, bars, and error bars represent mean and SD of three biological replicate wells. **** P < 0.0001, *** P < 0.0005, ** P < 0.005, * P < 0.05.

Article Snippet: For the SuperTop Wnt reporter assay, cells were plated in 96‐well plates in 3–4 replicates and transfected with 50 ng SuperTop reporter plasmid and 2.5 ng pTK‐Renilla control (Promega) as previously described (Cruciat et al , ) together with 50 ng of plasmid DNA carrying either shRNA against GPR56 or cDNA of GPR56 FL, GPR56 CTF, or GPR56 CTF mutants (pcDNA3.1) as indicated using TurboFect transfection reagent (Thermo #R0532) following the supplier’s protocol.

Techniques: Activation Assay, Binding Assay, Reporter Assay, Control, Transfection, Plasmid Preparation, Expressing, Infection, Negative Control, Western Blot, shRNA, Over Expression, Immunofluorescence, Membrane, Marker