DLL3 regulates Notch signaling in small cell lung cancer.

Tumor heterogeneity plays a critical role in tumor development and response to treatment. In small-cell lung cancer (SCLC), intratumoral heterogeneity is driven in part by the Notch signaling pathway, which reprograms neuroendocrine cancer cells to a less/non-neuroendocrine state. Here we investigated the atypical Notch ligand DLL3 as a biomarker of the neuroendocrine state and a regulator of cell-cell interactions in SCLC. We first built a mathematical model to predict the impact of DLL3 expression on SCLC cell populations. We next tested this model using a single-chain variable fragment (scFv) to track DLL3 expression in vivo and a new mouse model of SCLC with inducible expression of DLL3 in SCLC tumors. We found that high levels of DLL3 promote the expansion of a SCLC cell population with lower expression levels of both neuroendocrine and non-neuroendocrine markers. This work may influence how DLL3-targeting therapies are used in SCLC patients.

Engineering a potent receptor superagonist or antagonist from a novel IL-6 family cytokine ligand.

Interleukin-6 (IL-6) family cytokines signal through multimeric receptor complexes, providing unique opportunities to create novel ligand-based therapeutics. The cardiotrophin-like cytokine factor 1 (CLCF1) ligand has been shown to play a role in cancer, osteoporosis, and atherosclerosis. Once bound to ciliary neurotrophic factor receptor (CNTFR), CLCF1 mediates interactions to coreceptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). By increasing CNTFR-mediated binding to these coreceptors we generated a receptor superagonist which surpassed the potency of natural CNTFR ligands in neuronal signaling. Through additional mutations, we generated a receptor antagonist with increased binding to CNTFR but lack of binding to the coreceptors that inhibited tumor progression in murine xenograft models of nonsmall cell lung cancer. These studies further validate the CLCF1-CNTFR signaling axis as a therapeutic target and highlight an approach of engineering cytokine activity through a small number of mutations.

The MEK5-ERK5 Kinase Axis Controls Lipid Metabolism in Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is an aggressive form of lung cancer with dismal survival rates. While kinases often play key roles driving tumorigenesis, there are strikingly few kinases known to promote the development of SCLC. Here, we investigated the contribution of the MAPK module MEK5-ERK5 to SCLC growth. MEK5 and ERK5 were required for optimal survival and expansion of SCLC cell lines in vitro and in vivo. Transcriptomics analyses identified a role for the MEK5-ERK5 axis in the metabolism of SCLC cells, including lipid metabolism. In-depth lipidomics analyses showed that loss of MEK5/ERK5 perturbs several lipid metabolism pathways, including the mevalonate pathway that controls cholesterol synthesis. Notably, depletion of MEK5/ERK5 sensitized SCLC cells to pharmacologic inhibition of the mevalonate pathway by statins. These data identify a new MEK5-ERK5-lipid metabolism axis that promotes the growth of SCLC. SIGNIFICANCE: This study is the first to investigate MEK5 and ERK5 in SCLC, linking the activity of these two kinases to the control of cell survival and lipid metabolism.

Antitumor activity of an engineered decoy receptor targeting CLCF1-CNTFR signaling in lung adenocarcinoma.

Proinflammatory cytokines in the tumor microenvironment can promote tumor growth, yet their value as therapeutic targets remains underexploited. We validated the functional significance of the cardiotrophin-like cytokine factor 1 (CLCF1)-ciliary neurotrophic factor receptor (CNTFR) signaling axis in lung adenocarcinoma (LUAD) and generated a high-affinity soluble receptor (eCNTFR-Fc) that sequesters CLCF1, thereby inhibiting its oncogenic effects. eCNTFR-Fc inhibits tumor growth in multiple xenograft models and in an autochthonous, highly aggressive genetically engineered mouse model of LUAD, driven by activation of oncogenic Kras and loss of Trp53. Abrogation of CLCF1 through eCNTFR-Fc appears most effective in tumors driven by oncogenic KRAS. We observed a correlation between the effectiveness of eCNTFR-Fc and the presence of KRAS mutations that retain the intrinsic capacity to hydrolyze guanosine triphosphate, suggesting that the mechanism of action may be related to altered guanosine triphosphate loading. Overall, we nominate blockade of CLCF1-CNTFR signaling as a novel therapeutic opportunity for LUAD and potentially for other tumor types in which CLCF1 is present in the tumor microenvironment.

A chemically cross-linked knottin dimer binds integrins with picomolar affinity and inhibits tumor cell migration and proliferation.

Molecules that target and inhibit αvß3, αvß5, and α5ß1 integrins have generated great interest because of the role of these receptors in mediating angiogenesis and metastasis. Attempts to increase the binding affinity and hence the efficacy of integrin inhibitors by dimerization have been marginally effective. In the present work, we achieved this goal by using oxime-based chemical conjugation to synthesize dimers of integrin-binding cystine knot (knottin) miniproteins with low-picomolar binding affinity to tumor cells. A non-natural amino acid containing an aminooxy side chain was introduced at different locations within a knottin monomer and reacted with dialdehyde-containing cross-linkers of different lengths to create knottin dimers with varying molecular topologies. Dimers cross-linked through an aminooxy functional group located near the middle of the protein exhibited higher apparent binding affinity to integrin-expressing tumor cells compared with dimers cross-linked through an aminooxy group near the C-terminus. In contrast, the cross-linker length had no effect on the integrin binding affinity. A chemical-based dimerization strategy was critical, as knottin dimers created through genetic fusion to a bivalent antibody domain exhibited only modest improvement (less than 5-fold) in tumor cell binding relative to the knottin monomer. The best oxime-conjugated knottin dimer achieved an unprecedented 150-fold increase in apparent binding affinity over the knottin monomer. Also, this dimer bound 3650-fold stronger and inhibited tumor cell migration and proliferation compared with cilengitide, an integrin-targeting peptidomimetic that performed poorly in recent clinical trials, suggesting promise for further therapeutic development.

The role of the 3D environment in hypoxia-induced drug and apoptosis resistance.

BACKGROUND: 3D tumors express different adhesion receptors from those expressed in monolayers, leading to a distinct microenvironment. The third dimension also brings mass transport into relevance, as inadequate diffusion of oxygen produces hypoxia. This study investigates the effects of distinct 3D environments on hypoxia-associated apoptosis and drug resistance. MATERIALS AND METHODS: Under hypoxia and normoxia, U251 glioma cells and U87 astrocytoma cells were grown as spheroids on flat substrates, scaffolds seeded with dispersed cells, and spheroid-seeded scaffolds. The samples were subsequently treated with doxorubicin and resveratrol, known inducers of apoptosis. RESULTS: All 3D environments induced increased but distinct resistance to apoptosis, as evident by lower caspase-3 activity, and higher production of anti-apoptotic proteins BCL-2 and survivin. Hypoxic monolayers also exhibited higher resistance to doxorubicin and higher production of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), but lower production of BCL-2 and survivin. CONCLUSION: These findings suggest that in vitro, 3D models acquire greater apoptosis resistance via up-regulation of anti-apoptotic proteins, and that the precise mechanism depends on the individual 3D microenvironment.

Incorporation of multicellular spheroids into 3-D polymeric scaffolds provides an improved tumor model for screening anticancer drugs.

Development of cancer therapeutics requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Three-dimensional (3-D) in vitro models have therefore been investigated for drug screening. In this study, we have developed a novel in vitro model in which multicellular aggregates, or spheroids, were incorporated into 3-D porous scaffolds. Drug resistance assays showed that spheroid-seeded scaffolds have much higher drug resistance than monolayer cultures, spheroids on flat substrates, or scaffolds seeded with dispersed cells. Furthermore, spheroid-seeded scaffolds demonstrated higher lactate production leading to acidosis, and higher expression of angiogenic factors. These data suggest that the spheroid-seeded 3-D scaffolds might serve as a useful in vitro system for screening cancer therapeutics.