L858R emerges as a potential biomarker predicting response of lung cancer models to anti-EGFR antibodies: Comparison of osimertinib vs. cetuximab.

EGFR-specific tyrosine kinase inhibitors (TKIs), especially osimertinib, have changed lung cancer therapy, but secondary mutations confer drug resistance. Because other EGFR mutations promote dimerization-independent active conformations but L858R strictly depends on receptor dimerization, we herein evaluate the therapeutic potential of dimerization-inhibitory monoclonal antibodies (mAbs), including cetuximab. This mAb reduces viability of cells expressing L858R-EGFR and blocks the FOXM1-aurora survival pathway, but other mutants show no responses. Unlike TKI-treated patient-derived xenografts, which relapse post osimertinib treatment, cetuximab completely prevents relapses of L858R+ tumors. We report that osimertinib's inferiority associates with induction of mutagenic reactive oxygen species, whereas cetuximab's superiority is due to downregulation of adaptive survival pathways (e.g., HER2) and avoidance of mutation-prone mechanisms that engage AXL, RAD18, and the proliferating cell nuclear antigen. These results identify L858R as a predictive biomarker, which may pave the way for relapse-free mAb monotherapy relevant to a large fraction of patients with lung cancer.

AXL and Error-Prone DNA Replication Confer Drug Resistance and Offer Strategies to Treat EGFR-Mutant Lung Cancer.

Anticancer therapies have been limited by the emergence of mutations and other adaptations. In bacteria, antibiotics activate the SOS response, which mobilizes error-prone factors that allow for continuous replication at the cost of mutagenesis. We investigated whether the treatment of lung cancer with EGFR inhibitors (EGFRi) similarly engages hypermutators. In cycling drug-tolerant persister (DTP) cells and in EGFRi-treated patients presenting residual disease, we observed upregulation of GAS6, whereas ablation of GAS6's receptor, AXL, eradicated resistance. Reciprocally, AXL overexpression enhanced DTP survival and accelerated the emergence of T790M, an EGFR mutation typical to resistant cells. Mechanistically, AXL induces low-fidelity DNA polymerases and activates their organizer, RAD18, by promoting neddylation. Metabolomics uncovered another hypermutator, AXL-driven activation of MYC, and increased purine synthesis that is unbalanced by pyrimidines. Aligning anti-AXL combination treatments with the transition from DTPs to resistant cells cured patient-derived xenografts. Hence, similar to bacteria, tumors tolerate therapy by engaging pharmacologically targetable endogenous mutators. SIGNIFICANCE: EGFR-mutant lung cancers treated with kinase inhibitors often evolve resistance due to secondary mutations. We report that in similarity to the bacterial SOS response stimulated by antibiotics, endogenous mutators are activated in drug-treated cells, and this heralds tolerance. Blocking the process prevented resistance in xenograft models, which offers new treatment strategies. This article is highlighted in the In This Issue feature, p. 2483.

Nucleoporin-93 reveals a common feature of aggressive breast cancers: robust nucleocytoplasmic transport of transcription factors.

By establishing multi-omics pipelines, we uncover overexpression and gene copy-number alterations of nucleoporin-93 (NUP93), a nuclear pore component, in aggressive human mammary tumors. NUP93 overexpression enhances transendothelial migration and matrix invasion in vitro, along with tumor growth and metastasis in animal models. These findings are supported by analyses of two sets of naturally occurring mutations: rare oncogenic mutations and inactivating familial nephrotic syndrome mutations. Mechanistically, NUP93 binds with importins, boosts nuclear transport of importins' cargoes, such as ß-catenin, and activates MYC. Likewise, NUP93 overexpression enhances the ultimate nuclear transport step shared by additional signaling pathways, including TGF-ß/SMAD and EGF/ERK. The emerging addiction to nuclear transport exposes vulnerabilities of NUP93-overexpressing tumors. Congruently, myristoylated peptides corresponding to the nuclear translocation signals of SMAD and ERK can inhibit tumor growth and metastasis. Our study sheds light on an emerging hallmark of advanced tumors, which derive benefit from robust nucleocytoplasmic transport.

TSHZ2 is an EGF-regulated tumor suppressor that binds to the cytokinesis regulator PRC1 and inhibits metastasis.

Unlike early transcriptional responses to mitogens, later events are less well-characterized. Here, we identified delayed down-regulated genes (DDGs) in mammary cells after prolonged treatment with epidermal growth factor (EGF). The expression of these DDGs was low in mammary tumors and correlated with prognosis. The proteins encoded by several DDGs directly bind to and inactivate oncoproteins and might therefore act as tumor suppressors. The transcription factor teashirt zinc finger homeobox 2 (TSHZ2) is encoded by a DDG, and we found that overexpression of TSHZ2 inhibited tumor growth and metastasis and accelerated mammary gland development in mice. Although the gene TSHZ2 localizes to a locus (20q13.2) that is frequently amplified in breast cancer, we found that hypermethylation of its promoter correlated with down-regulation of TSHZ2 expression in patients. Yeast two-hybrid screens and protein-fragment complementation assays in mammalian cells indicated that TSHZ2 nucleated a multiprotein complex containing PRC1/Ase1, cyclin B1, and additional proteins that regulate cytokinesis. TSHZ2 increased the inhibitory phosphorylation of PRC1, a key driver of mitosis, mediated by cyclin-dependent kinases. Furthermore, similar to the tumor suppressive transcription factor p53, TSHZ2 inhibited transcription from the PRC1 promoter. By recognizing DDGs as a distinct group in the transcriptional response to EGF, our findings uncover a group of tumor suppressors and reveal a role for TSHZ2 in cell cycle regulation.

Host-Dependent Phenotypic Resistance to EGFR Tyrosine Kinase Inhibitors.

Lung cancers driven by mutant forms of EGFR invariably develop resistance to kinase inhibitors, often due to secondary mutations. Here we describe an unconventional mechanism of resistance to dacomitinib, a newly approved covalent EGFR kinase inhibitor, and uncover a previously unknown step of resistance acquisition. Dacomitinib-resistant (DR) derivatives of lung cancer cells were established by means of gradually increasing dacomitinib concentrations. These DR cells acquired no secondary mutations in the kinase or other domains of EGFR. Along with resistance to other EGFR inhibitors, DR cells acquired features characteristic to epithelial-mesenchymal transition, including an expanded population of aldehyde dehydrogenase-positive cells and upregulation of AXL, a receptor previously implicated in drug resistance. Unexpectedly, when implanted in animals, DR cells reverted to a dacomitinib-sensitive state. Nevertheless, cell lines derived from regressing tumors displayed renewed resistance when cultured in vitro. Three-dimensional and cocultures along with additional analyses indicated lack of involvement of hypoxia, fibroblasts, and immune cells in phenotype reversal, implying that other host-dependent mechanisms might nullify nonmutational modes of resistance. Thus, similar to the phenotypic resistance of bacteria treated with antibiotics, the reversible resisters described here likely evolve from drug-tolerant persisters and give rise to the irreversible, secondary mutation-driven nonreversible resister state. SIGNIFICANCE: This study reports that stepwise acquisition of kinase inhibitor resistance in lung cancers driven by mutant EGFR comprises a nonmutational, reversible resister state. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/14/3862/F1.large.jpg.

PD-L1 recruits phospholipase C and enhances tumorigenicity of lung tumors harboring mutant forms of EGFR.

Cancer immunotherapy focuses on inhibitors of checkpoint proteins, such as programmed death ligand 1 (PD-L1). Unlike RAS-mutated lung cancers, EGFR mutant tumors have a generally low response to immunotherapy. Because treatment outcomes vary by EGFR allele, intrinsic and microenvironmental factors may be involved. Among all non-immunological signaling pathways surveyed in patients' datasets, EGFR signaling is best associated with high PD-L1. Correspondingly, active EGFRs stabilize PD-L1 transcripts and depletion of PD-L1 severely inhibits EGFR-driven tumorigenicity and metastasis in mice. The underlying mechanisms involve the recruitment of phospholipase C-γ1 (PLC-γ1) to a cytoplasmic motif of PD-L1, which enhances PLC-γ1 activation by EGFR. Once stimulated, PLC-γ1 activates calcium flux, Rho GTPases, and protein kinase C, collectively promoting an aggressive phenotype. Anti-PD-L1 antibodies can inhibit these intrinsic functions of PD-L1. Our results portray PD-L1 as a molecular amplifier of EGFR signaling and improve the understanding of the resistance of EGFR+ tumors to immunotherapy.

ETS Proteins Bind with Glucocorticoid Receptors: Relevance for Treatment of Ewing Sarcoma.

The glucocorticoid receptor (GR) acts as a ubiquitous cortisol-dependent transcription factor (TF). To identify co-factors, we used protein-fragment complementation assays and found that GR recognizes FLI1 and additional ETS family proteins, TFs relaying proliferation and/or migration signals. Following steroid-dependent translocation of FLI1 and GR to the nucleus, the FLI1-specific domain (FLS) binds with GR and strongly enhances GR's transcriptional activity. This interaction has functional consequences in Ewing sarcoma (ES), childhood and adolescence bone malignancies driven by fusions between EWSR1 and FLI1. In vitro, GR knockdown inhibited the migration and proliferation of ES cells, and in animal models, antagonizing GR (or lowering cortisol) retarded both tumor growth and metastasis from bone to lung. Taken together, our findings offer mechanistic rationale for repurposing GR-targeting drugs for the treatment of patients with ES.

Synthesis, characterization, and evaluation of Cd[L-proline]2, a novel histone deacetylase inhibitor that induces epigenetic modification of histone deacetylase isoforms in A549 cells.

Histone deacetylases (HDACs) play an important role in the epigenetic regulation of gene expression through their effects on the compact chromatin structure. In clinical studies, several classes of histone deacetylase inhibitors (HDACi) have demonstrated potent anticancer activities with metal complexes. Hence, we synthesized cadmium-proline complexes using both the D- and L-isomers of proline and evaluated their biological activities by observing the efficiency of their inhibition of HDAC activity, ability to reduce the expression of HDAC isoforms in A549 cells and effect on apoptosis. The synthesized compounds were characterized by UV, IR, NMR spectroscopy and elemental analysis. In-vitro cell toxicity was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and the 50% inhibitory concentration (IC50; 2 µM) was obtained at 12 h. The morphological study at nuclear levels was performed by acridine orange/ethidium bromide (AO/EB) and Hoechst staining, and the results showed an association with cell cycle arrest at the G2/M phase. Both cadmium-proline complexes intensely inhibited HDAC activity at 2 µM concentration. Interestingly, Cd[L-proline]2 was found to be a potent inhibitor for all HDAC isoforms, whereas Cd[D-proline]2 inhibited only HDAC1 and 2. HDACi are novel chemotherapeutic drugs that induce hyperacetylation of histones H3 and H4, counteracting the aberrant repression of genes, such as insulin-like growth factor-binding protein 3 (IGFBP-3), p53, and p21. ERK/MAPK signaling pathway resulted in the downregulation of the expression of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), contributing to the inhibition of metastasis in A549 cells. Apoptosis induction was accompanied by the activation of death receptors and their ligands which recruit initiator caspase 8, decrease in mitochondrial membrane potential (ΔΨm), as well as increased Bax/Bcl2 ratio, followed by activation of caspases 9 and 3. Our finding suggests that Cd[L-proline]2 complex accelerates epigenetic rearrangement by HDAC inhibition, which may be the key mechanism for its anticancer activity.