Live-cell invasive phenotyping uncovers the ALK2/BMP6 iron homeostasis pathway as a therapeutic vulnerability in LKB1-mutant lung cancer

Abstract

The acquisition of invasive properties is a prerequisite for tumor progression and metastasis. Molecular subtypes of KRAS-driven lung cancer exhibit distinct modes of invasion that likely contribute to unique growth properties and therapeutic susceptibilities. Despite this, pre-clinical discovery strategies designed to exploit invasive phenotypes are lacking. To address this, we designed an experimental system to screen for targetable signaling pathways linked to active early invasion phenotypes in the two most prominent molecular subtypes, TP53 and LKB1, of KRAS-driven lung adenocarcinoma (LUAD). By combining live-cell imaging of human bronchial epithelial cells in a 3D invasion matrix with RNA transcriptome profiling, we identified the LKB1-specific upregulation of bone morphogenetic protein 6 (BMP6). Examination of early-stage lung cancer patients confirmed upregulation of BMP6 in LKB1-mutant lung tumors. At the molecular level, we find that the canonical iron regulatory hormone Hepcidin is induced via BMP6 signaling upon LKB1 loss, where intact LKB1 kinase activity is necessary to maintain signaling homeostasis. Furthermore, pre-clinical studies in a novel Kras/Lkb1-mutant syngeneic mouse model show that potent growth suppression was achieved by inhibiting the ALK2/BMP6 signaling axis with single agents that are currently in clinical trials. We show that alterations in the iron homeostasis pathway are accompanied by simultaneous upregulation of ferroptosis protection proteins. Thus, LKB1 is sufficient to regulate both the 'gas' and 'breaks' to finely tune iron-regulated tumor progression.

Figure 1.. Generation and characterization of in vitro transformation phenotypes and 3D invasive phenotyping.

A) Schematic depicting knockdown and overexpression strategy to generate isogenic subsets of HBEC cells for invasive phenotyping . Scale bar = 200μm B) Western blot to verify tumor suppressor, oncogene status and EMT markers in isogenic HBECs. C) Graph depicting quantitative analysis of cell cycle distribution from each isogenic HBEC genotype. D) Graph depicting mean cell proliferation rate from isogenic HBECs over the course of 72hrs. Error bar represents ± SEM of data obtained from quadruplicate samples. E) Schematic representation of 3D invasive phenotyping assay. F) Bright-field and corresponding fluorescent images of live-cell 3D phenotype of isogenic HBECs 4 days post-embedding. Scale bar represents 200 μm. G) Representative still images of focal point time-lapse microscopy (36hr post-embedding) show protruding cell clusters at the leading branching edge, followed by budding formation or ductal elongation (red line). Scale bar = 16 μm.

Figure 2.. BMP6 expression is uniquely upregulated in response to loss of LKB1 in invasive HBECs and lung adenocarcinoma patients.

Volcano plot depicting significantly differentially expressed genes (upregulated, N = 583 and downregulated, N = 401) genes between invasive KRAS/LKB1 (KL) vs. KRAS/TP53 (KP) 3D spheroids with respect to non-invasive control (Control) HBEC spheroids. B) Scatterplot of differentially expressed genes (absolute fold change of >= 2 or more and FDR <= 0.05) showing K vs. C against KL vs. K shown by significance in either or both comparisons. C) Heatmap depicting mean log2 transformed expression levels of selected differentially upregulated genes (KL>KP and KL>K>C) between isogenic 3D spheroids. D) Graph generated using cBioPortal depicting mean BMP6 mRNA expression from genetic subtypes of lung adenocarcinoma (LUAD) patients. Each circle represents an individual patient sample. Error bars represent standard deviation. E) Graph depicting fold change in BMP6 gene expression among isogenic HBECs.

Figure 3.. LKB1 negatively regulates the BMP6-induced iron homeostasis pathway through hepcidin and pharmacological inhibition of ALK2 inhibits invasion of KL cells in vitro.

A) Confocal images of immunofluorescence for BMP6 protein expression (red) in 3D HBECs 3D of the indicated genotypes (DAPI labels nuclei and all cells express cytoplasmic GFP). Western blot of the indicated proteins detected from 2D and 3D lysate and media from HBECs of the indicated genotypes. C) Western blot of BMP6-regulated Smad signaling components in LKB1-null H157 cells that express vector control, wild-type LKB1 (LKB1-WT) or kinase dead LKB1 (LKB1-K78I). D) Western blot of iron homeostasis and ferroptosis protective genes in H157 cells expressing vector control, LKB1-WT or LKB1-K78I. E) Western blot analysis of BMP6/ALK2-regulated Smad signaling in KL HBECs and A549 (LKB1-null) cells treated with increasing concentrations of LDN214117 for 24hrs. F) Brightfield images of 3D spheroids of the indicated genotypes treated with the indicated concentration of LDN214117 and embedded in invasion matrix for 72hrs. G) Quantitation of invasive area in for LDN214117-treated spheroids. (graph depicts mean of 3 biological replicates).

Figure 4.. Kras^G12D/Lkb1^fl/fl mouse tumor cells lines respond to AKL2 inhibition by downregulating BMP6-regulated iron homeostasis and ferroptosis protective proteins.

A) Schematic of mouse KL tumor cell line isolation. B) Western blot to assess indicated proteins in NIH3T3 (mouse fibroblast cell line), JK43-P and JK43-M tumor cells. C) Western blot of indicated Smad signaling proteins from JK43-P and JK43-M cells treated with increasing concentration of LDN214117. D) Western blot of indicated iron homeostasis and ferroprotective proteins from JK43-M cells treated with 1uM of LDN214117 for 24, 48 and 72hrs.

Figure 5.. In vivo efficacy of LDN214117 targeting Alk2 in syngeneic mouse model.

A) Schematic depicting pre-clinical trial workflow. B) Brightfield images of tumors isolated from vehicle and LDN214117 treated mice. C) Mean tumor volume from mice treated with vehicle ( 6 mice/group) and LDN214117 (7 mice/group). D) Graph depicting mean tumor weight from vehicle and LDN214117 treated mice. E) Western blot to assess BMP6 and hepcidin expression level in tumors from vehicle and LDN214117-treated mice. F) Representative images of immunohistochemistry for BMP6 and Hepcidin on tumor sections from mice treated with vehicle or LDN214117. F) Model depicting mechanism of altered iron homeostasis in LKB1-mutant tumor cells.