rker 216  (R&D Systems)

Journal: Cancer Research

Article Title: Live-Cell Invasive Phenotyping Uncovers ALK2 as a Therapeutic Target in LKB1 -Mutant Lung Cancer

doi: 10.1158/0008-5472.CAN-23-2631

Figure Lengend Snippet: BMP6 expression is uniquely upregulated in response to loss of LKB1 in invasive HBECs and patients with LUAD. A, Western blot analysis of indicated proteins in a 3D invasive spheroid panel. B, Volcano plot depicting significant DEGs (upregulated, N = 583; downregulated, N = 401) between invasive KRAS /LKB1 (KL) vs. KRAS /TP53 (KP) 3D spheroids with respect to noninvasive control (C) HBEC spheroids. C, Heatmap depicting the mean log 2 -transformed expression levels of selected differentially upregulated genes (KL > KP and KL > K > C) between isogenic 3D spheroids. D, Graph depicting fold change in BMP6 gene expression from qRT-PCR validation in isogenic HBECs. E, Confocal images of immunofluorescence for BMP6 protein expression (red) in 3D HBECs of the indicated genotypes (DAPI labels nuclei, and all cells express cytoplasmic GFP). Scale bar, 100 μm. F, Graph generated using cBioPortal depicting the mean BMP6 mRNA expression from genetic subtypes of patients with LUAD. Each circle represents an individual patient sample. Error bars, SD. G, Western blot analysis of indicated proteins in isogenic 3D spheroids. H, Graph of pathway enrichment analysis sorted by significance. ****, P < 0.0001. RESM, RNA-seq by expectation maximization; TCGA, The Cancer Genome Atlas.

Article Snippet: Neutralizing BMP6 Abs (MAB507 for human, MAB6325 for mouse) were purchased from R&D Systems.

Techniques: Expressing, Western Blot, Control, Transformation Assay, Quantitative RT-PCR, Immunofluorescence, Generated, RNA Sequencing Assay

Journal: Cancer Research

Article Title: Live-Cell Invasive Phenotyping Uncovers ALK2 as a Therapeutic Target in LKB1 -Mutant Lung Cancer

doi: 10.1158/0008-5472.CAN-23-2631

Figure Lengend Snippet: LKB1 restricts BMP6 signaling using a kinase-dependent mechanism, and suppression of BMP6 signaling and ALK2 inhibition suppresses 3D proliferation and invasion in multiple KL cell lines. A, Western blot analysis of BMP6 pathway activation in stable control pLKO.1 and shLKB1 H1299 lung cancer cells. B, Western blot of BMP6-regulated Smad signaling components in LKB1-null H157 cells that express vector control, LKB1-WT, or kinase-dead LKB1 (LKB1-K78I). C, Western blot analysis of indicated proteins in control IgG (−)- or anti-BMP6 (+)–treated KL, JK43-P, and JK43-M cells. D, Representative brightfield images (top) and quantitative graphs (bottom) of control IgG-treated or anti-BMP6–treated invasive KL, JK43-P, or JK43-M spheroids. E, Graph depicting cell viability of indicated cell lines with increasing concentrations of LDN214117 (top). Table of LDN214117 IC 50 in indicated cell lines. F, Representative images (left) and quantitative graphs (right) of A549 3D spheroids assayed for Ki67. Scale bar, 70 μm. G, Representative images (left) and quantitative graphs (right) of JK43-M 3D spheroids assayed for Ki67. Scale bar, 70 μm. H, Brightfield images of 3D spheroids of the indicated cell lines either treated with vehicle control (−) or treated with the indicated concentration of LDN214117 and embedded in the invasion matrix for 72 hours. Scale bar, 100 μm. I, Quantitation of the invasive area for LDN214117-treated KL spheroids (the graph depicts the mean of three biological replicates). J, Quantitation of the invasive area for LDN214117-treated A549 spheroids (the graph depicts the mean of three biological replicates). K, Western blot of BMP6/ALK2-regulated Smad signaling in KL HBECs and A549 (LKB1-null) cells treated with increasing concentrations of LDN214117 for 24 hours. L, Western blot analysis of BMP6/ALK2-regulated Smad signaling in JK43-P and JK43-M mouse tumor cell lines (KrasG12D/Lkb1-null) treated with increasing concentrations of LDN214117 for 24 hours. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Neutralizing BMP6 Abs (MAB507 for human, MAB6325 for mouse) were purchased from R&D Systems.

Techniques: Inhibition, Western Blot, Activation Assay, Control, Plasmid Preparation, Concentration Assay, Quantitation Assay

Journal: Cancer Research

Article Title: Live-Cell Invasive Phenotyping Uncovers ALK2 as a Therapeutic Target in LKB1 -Mutant Lung Cancer

doi: 10.1158/0008-5472.CAN-23-2631

Figure Lengend Snippet: Efficacy of targeting ALK2 in LKB1-mutant lung cancer in vivo. LKB1 restricts iron homeostasis pathways using a kinase-dependent mechanism. A, Mean tumor volume from syngeneic mice (JK-M cells) treated with vehicle (6 mice/group) and LDN214117 (7 mice/group). B, Graph depicting the mean tumor weight from vehicle- and LDN214117-treated mice. C, Brightfield images of tumors isolated from vehicle- and LDN214117-treated mice. D, The mean tumor volume from NSG mice with A549 lung tumor xenografts treated with either vehicle (7 mice/group), LDN214117 (7 mice/group), or LDN193189 (7 mice/group). E, Graph depicting the mean tumor weight from vehicle-, LDN214117-, and LDN193189-treated mice. F, Brightfield images of A549 tumors isolated from vehicle-, LDN214117-, and LDN193189-treated mice. G, Representative brightfield images of tumor sections treated with vehicle or LDN214117 and stained by the indicated Ab IHC or special stain. Scale bar, 200 μm or 2 mm (TUNEL). H, Western blot to assess BMP6 and hepcidin levels in tumors from vehicle- and LDN214117-treated mice. I, Western blot of indicated proteins from JK43-M cells treated with 1 uM of LDN214117 for 24, 48, and 72 hours. J, Western blot analysis of indicated proteins in vector, LKB1-WT, or LKB1-K78I add-back H157 lung cancer cells. K, Model depicting the mechanism of altered iron homeostasis signaling in LKB1-mutant tumor cells. **, P < 0.01; ****, P < 0.0001.

Article Snippet: Neutralizing BMP6 Abs (MAB507 for human, MAB6325 for mouse) were purchased from R&D Systems.

Techniques: Mutagenesis, In Vivo, Isolation, Staining, TUNEL Assay, Western Blot, Plasmid Preparation