Interaction between tumor cell TNFR2 and monocyte membrane-bound TNF-α triggers tumorigenic inflammation in neuroblastoma.

BACKGROUND: Tumor progression and resistance to therapy in children with neuroblastoma (NB), a common childhood cancer, are often associated with infiltration of monocytes and macrophages that produce inflammatory cytokines. However, the mechanism by which tumor-supportive inflammation is initiated and propagated remains unknown. Here, we describe a novel protumorigenic circuit between NB cells and monocytes that is triggered and sustained by tumor necrosis factor alpha (TNF-α). METHODS: We used NB knockouts (KOs) of TNF-α and TNFRSF1A mRNA (TNFR1)/TNFRSF1B mRNA (TNFR2) and TNF-α protease inbitor (TAPI), a drug that modulates TNF-α isoform expression, to assess the role of each component in monocyte-associated protumorigenic inflammation. Additionally, we employed NB-monocyte cocultures and treated these with clinical-grade etanercept, an Fc-TNFR2 fusion protein, to neutralize signaling by both membrane-bound (m) and soluble (s)TNF-α isoforms. Further, we treated NOD/SCID/IL2Rγ(null) mice carrying subcutaneous NB/human monocyte xenografts with etanercept and evaluated the impact on tumor growth and angiogenesis. Gene set enrichment analysis (GSEA) was used to determine whether TNF-α signaling correlates with clinical outcomes in patients with NB. RESULTS: We found that NB expression of TNFR2 and monocyte membrane-bound tumor necrosis factor alpha is required for monocyte activation and interleukin (IL)-6 production, while NB TNFR1 and monocyte soluble TNF-α are required for NB nuclear factor kappa B subunit 1 (NF-κB) activation. Treatment of NB-monocyte cocultures with clinical-grade etanercept completely abrogated release of IL-6, granulocyte colony-stimulating factor (G-CSF), IL-1α, and IL-1ß and eliminated monocyte-induced enhancement of NB cell proliferation in vitro. Furthermore, etanercept treatment inhibited tumor growth, ablated tumor angiogenesis, and suppressed oncogenic signaling in mice with subcutaneous NB/human monocyte xenografts. Finally, GSEA revealed significant enrichment for TNF-α signaling in patients with NB that relapsed. CONCLUSIONS: We have described a novel mechanism of tumor-promoting inflammation in NB that is strongly associated with patient outcome and could be targeted with therapy.

Comparative Analysis and Isoform-Specific Therapeutic Vulnerabilities of KRAS Mutations in Non-Small Cell Lung Cancer.

PURPOSE: Activating missense mutations of KRAS are the most frequent oncogenic driver events in lung adenocarcinoma (LUAD). However, KRAS isoforms are highly heterogeneous, and data on the potential isoform-dependent therapeutic vulnerabilities are still lacking. EXPERIMENTAL DESIGN: We developed an isogenic cell-based platform to compare the oncogenic properties and specific therapeutic actionability of KRAS-mutant isoforms. In parallel, we analyzed clinicopathologic and genomic data from 3,560 patients with non-small cell lung cancer (NSCLC) to survey allele-specific features associated with oncogenic KRAS mutations. RESULTS: In isogenic cell lines expressing different mutant KRAS isoforms, we identified isoform-specific biochemical, biological, and oncogenic properties both in vitro and in vivo. These exclusive features correlated with different therapeutic responses to MEK inhibitors, with KRAS G12C and Q61H mutants being more sensitive compared with other isoforms. In vivo, combined KRAS G12C and MEK inhibition was more effective than either drug alone. Among patients with NSCLCs that underwent comprehensive tumor genomic profiling, STK11 and ATM mutations were significantly enriched among tumors harboring KRAS G12C, G12A, and G12V mutations. KEAP1 mutation was significantly enriched among KRAS G12C and KRAS G13X LUADs. KRAS G13X-mutated tumors had the highest frequency of concurrent STK11 and KEAP1 mutations. Transcriptomic profiling revealed unique patterns of gene expression in each KRAS isoform, compared with KRAS wild-type tumors. CONCLUSIONS: This study demonstrates that KRAS isoforms are highly heterogeneous in terms of concurrent genomic alterations and gene-expression profiles, and that stratification based on KRAS alleles should be considered in the design of future clinical trials.

Combination of Type I and Type II MET Tyrosine Kinase Inhibitors as Therapeutic Approach to Prevent Resistance.

MET-targeted therapies are clinically effective in MET-amplified and MET exon 14 deletion mutant (METex14) non-small cell lung cancers (NSCLCs), but their efficacy is limited by the development of drug resistance. Structurally distinct MET tyrosine kinase inhibitors (TKIs) (type I/II) have been developed or are under clinical evaluation, which may overcome MET-mediated drug resistance mechanisms. In this study, we assess secondary MET mutations likely to emerge in response to treatment with single-agent or combinations of type I/type II MET TKIs using TPR-MET transformed Ba/F3 cell mutagenesis assays. We found that these inhibitors gave rise to distinct secondary MET mutant profiles. However, a combination of type I/II TKI inhibitors (capmatinib and merestinib) yielded no resistant clones in vitro The combination of capmatinib/merestinib was evaluated in vivo and led to a significant reduction in tumor outgrowth compared with either MET inhibitor alone. Our findings demonstrate in vitro and in vivo that a simultaneous treatment with a type I and type II MET TKI may be a clinically viable approach to delay and/or diminish the emergence of on target MET-mediated drug-resistance mutations.

Oncogenic switch and single-agent MET inhibitor sensitivity in a subset of EGFR-mutant lung cancer.

The clinical efficacy of epidermal growth factor receptor (EGFR)­targeted therapy in EGFR-mutant non­small cell lung cancer is limited by the development of drug resistance. One mechanism of EGFR inhibitor resistance occurs through amplification of the human growth factor receptor (MET) proto-oncogene, which bypasses EGFR to reactivate downstream signaling. Tumors exhibiting concurrent EGFR mutation and MET amplification are historically thought to be codependent on the activation of both oncogenes. Hence, patients whose tumors harbor both alterations are commonly treated with a combination of EGFR and MET tyrosine kinase inhibitors (TKIs). Here, we identify and characterize six patient-derived models of EGFR-mutant, MET-amplified lung cancer that have switched oncogene dependence to rely exclusively on MET activation for survival. We demonstrate in this MET-driven subset of EGFR TKI-refractory cancers that canonical EGFR downstream signaling was governed by MET, even in the presence of sustained mutant EGFR expression and activation. In these models, combined EGFR and MET inhibition did not result in greater efficacy in vitro or in vivo compared to single-agent MET inhibition. We further identified a reduced EGFR:MET mRNA expression stoichiometry as associated with MET oncogene dependence and single-agent MET TKI sensitivity. Tumors from 10 of 11 EGFR inhibitor­resistant EGFR-mutant, MET-amplified patients also exhibited a reduced EGFR:MET mRNA ratio. Our findings reveal that a subset of EGFR-mutant, MET-amplified lung cancers develop dependence on MET activation alone, suggesting that such patients could be treated with a single-agent MET TKI rather than the current standard-of-care EGFR and MET inhibitor combination regimens.