Epidermal growth factor receptor (EGFR) mutations identify patients with lung cancer who derive benefit from kinase inhibitors. However, most patients eventually develop resistance, primarily due to the T790M second‐site mutation. Irreversible inhibitors (e.g., osimertinib/AZD9291) inhibit T790M‐EGFR, but several mechanisms, including a third‐site mutation, C797S, confer renewed resistance. We previously reported that a triple mixture of monoclonal antibodies, 3×mAbs, simultaneously targeting EGFR, HER2, and HER3, inhibits T790M‐expressing tumors. We now report that 3×mAbs, including a triplet containing cetuximab and trastuzumab, inhibits C797S‐expressing tumors. Unlike osimertinib, which induces apoptosis, 3×mAbs promotes degradation of the three receptors and induces cellular senescence. Consistent with distinct mechanisms, treatments combining 3×mAbs plus sub‐inhibitory doses of osimertinib synergistically and persistently eliminated tumors. Thus, oligoclonal antibodies, either alone or in combination with kinase inhibitors, might preempt repeated cycles of treatment and rapid emergence of resistance.

Because of emergence of resistance to osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), no targeted treatments are available for patients with lung cancer who lose sensitivity due to new mutations or bypass mechanisms. We examined in animals and an alternative therapeutic approach making use of antibodies. An osimertinib-sensitive animal model of lung cancer, which rapidly develops drug resistance, has been employed. To overcome compensatory hyperactivation of ERK, which we previously reported, an anti-EGFR antibody (cetuximab) was combined with other antibodies, as well as with a subtherapeutic dose of osimertinib, and cancer cell apoptosis was assayed. Our animal studies identified a combination of three clinically approved drugs, cetuximab, trastuzumab (an anti-HER2 mAb), and osimertinib (low dose), as an effective and long-lasting treatment that is able to prevent onset of resistance to osimertinib. A continuous schedule of concurrent treatment was sufficient for effective tumor inhibition and for prevention of relapses. Studies employing cultured cells and analyses of tumor extracts indicated that the combination of two mAbs and a subtherapeutic TKI dose sorted EGFR and HER2 for degradation; cooperatively enhanced apoptosis; inhibited activation of ERK; and reduced abundance of several bypass proteins, namely MET, AXL, and HER3. Our assays and animal studies identified an effective combination of clinically approved drugs that might overcome resistance to irreversible TKIs in clinical settings. The results we present attribute the long-lasting effect of the drug combination to simultaneous blockade of several well-characterized mechanisms of drug resistance. .

Mutations mimicking growth factor-induced proliferation and motility characterize aggressive subtypes of mammary tumors. To unravel currently unknown players in these processes, we performed phosphoproteomic analysis on untransformed mammary epithelial cells (MCF10A) that were stimulated in culture with epidermal growth factor (EGF). We identified ladinin-1 (LAD1), a largely uncharacterized protein to date, as a phosphorylation-regulated mediator of the EGF-to-ERK pathway. Further experiments revealed that LAD1 mediated the proliferation and migration of mammary cells. LAD1 was transcriptionally induced, phosphorylated, and partly colocalized with actin stress fibers in response to EGF. Yeast two-hybrid, proximity ligation, and coimmunoprecipitation assays revealed that LAD1 bound to actin-cross-linking proteins called filamins. Cosedimentation analyses indicated that LAD1 played a role in actin dynamics, probably in collaboration with the scaffold protein 14-3-3σ (also called SFN). Depletion of LAD1 decreased the expression of transcripts associated with cell survival and inhibited the growth of mammary xenografts in an animal model. Furthermore, LAD1 predicts poor patient prognosis and is highly expressed in aggressive subtypes of breast cancer characterized as integrative clusters 5 and 10, which partly correspond to triple-negative and HER2-positive tumors. Thus, these findings reveal a cytoskeletal component that is critically involved in cell migration and the acquisition of oncogenic attributes in human mammary tumors.

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Anti-cancer therapies have been limited by 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 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, while 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.

Highlights d Unlike promoter-mediated PD-L1 induction by IFN-g, EGFR rapidly stabilizes PD-L1 mRNA d Once induced, PD-L1 enhances metastasis in vivo and chemotaxis toward EGF d PD-L1 physically binds with and enhances activation of phospholipase C-g1 by EGFR d PLC-g1 binds a PD-L1's cytoplasmic segment implicated in protection from cytotoxicity

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Each group of the 56 receptor tyrosine kinases (RTK) binds with one or more soluble growth factors and coordinates a vast array of cellular functions. These outcomes are tightly regulated by inducible post-translational events, such as tyrosine phosphorylation, ubiquitination, ectodomain shedding, and regulated intramembrane proteolysis. Because of the delicate balance required for appropriate RTK function, cells may become pathogenic upon dysregulation of RTKs themselves or their post-translational covalent modifications. For example, reduced ectodomain shedding and decreased ubiquitination of the cytoplasmic region, both of which enhance growth factor signals, characterize malignant cells. Whereas receptor phosphorylation and ubiquitination are reversible, proteolytic cleavage events are irreversible, and either modification might alter the subcellular localization of RTKs. Herein, we focus on ectodomain shedding by metalloproteinases (including ADAM family proteases), cleavage within the membrane or cytoplasmic regions of RTKs (by gamma-secretases and caspases, respectively), and complete receptor proteolysis in lysosomes and proteasomes. Roles of irreversible modifications in RTK signaling, pathogenesis, and pharmacology are highlighted.