Src-Homology 2 Domain-Containing Phosphatase 2 in Resected EGFR Mutation-Positive Lung Adenocarcinoma.

INTRODUCTION: EGFR mutation-positive lung adenocarcinoma (LUAD) displays impaired phosphorylation of ERK and Src-homology 2 domain-containing phosphatase 2 (SHP2) in comparison with EGFR wild-type LUADs. We hypothesize that SHP2 expression could be predictive in patients positive with resected EGFR mutation versus patients with EGFR wild-type LUAD. METHODS: We examined resected LUAD cases from Japan and Spain. mRNA expression levels of AXL, MET, CDCP1, STAT3, YAP1, and SHP2 were analyzed by quantitative reverse transcriptase polymerase chain reaction. The activity of SHP2 inhibitors plus erlotinib were tested in EGFR-mutant cell lines and analyzed by cell viability assay, Western blot, and immunofluorescence. RESULTS: A total of 50 of 100 EGFR mutation-positive LUADs relapsed, among them, patients with higher SHP2 mRNA expression revealed shorter progression-free survival, in comparison with those having low SHP2 mRNA (hazard ratio: 1.83; 95% confidence interval: 1.05-3.23; p = 0.0329). However, SHP2 was not associated with prognosis in the remaining 167 patients with wild-type EGFR. In EGFR-mutant cell lines, the combination of SHP099 or RMC-4550 (SHP2 inhibitors) with erlotinib revealed synergism via abrogation of phosphorylated AKT (S473) and ERK1/2 (T202/Y204). Although erlotinib translocates phosphorylated SHP2 (Y542) into the nucleus, either RMC-4550 alone, or in combination with erlotinib, relocates SHP2 into the cytoplasm membrane, limiting AKT and ERK1/2 activation. CONCLUSIONS: Elevated SHP2 mRNA levels are associated with recurrence in resected EGFR mutation-positive LUADs, but not in EGFR wild-type. EGFR tyrosine kinase inhibitors can enhance SHP2 activation, hindering adjuvant therapy. SHP2 inhibitors could improve the benefit of adjuvant therapy in EGFR mutation-positive LUADs.

Targeting PKCι-PAK1 signaling pathways in EGFR and KRAS mutant adenocarcinoma and lung squamous cell carcinoma.

INTRODUCTION: p21-activated kinase 1 (PAK1) stimulates growth and metastasis in non-small cell lung cancer (NSCLC). Protein kinase C iota (PKCι) is an enzyme highly expressed in NSCLC, regulating PAK1 signaling. In the present study we explored whether the PKCι-PAK1 signaling pathway approach can be an efficient target in different types of NSCLC cell and mouse models. METHODS: The effect of IPA-3 (PAK1 inhibitor) plus auranofin (PKCι inhibitor) combination was evaluated by cell viability assay, colony formation and western blotting assay, using three types of NSCLC cell lines: EGFR or KRAS mutant adenocarcinoma and squamous cell carcinoma with PAK1 amplification. In addition, for clinical availability, screening for new PAK1 inhibitors was carried out and the compound OTSSP167 was evaluated in combination with auranofin in cell and mice models. RESULTS: The combination of IPA-3 or OTSSP167 plus auranofin showed high synergism for inhibiting cell viability and colony formation in three cell lines. Mechanistic characterization revealed that this drug combination abrogated expression and activation of membrane receptors and downstream signaling proteins crucial in lung cancer: EGFR, MET, PAK1, PKCι, ERK1/2, AKT, YAP1 and mTOR. A nude mouse xenograft assay demonstrated that this drug combination strongly suppressed tumor volume compared with single drug treatment. CONCLUSIONS: Combination of IPA-3 or OTSSP167 and auranofin was highly synergistic in EGFR or KRAS mutant adenocarcinoma and squamous cell carcinoma cell lines and decreased tumor volume in mice models. It is of interest to further test the targeting of PKCι-PAK1 signaling pathways in EGFR mutant, KRAS mutant and squamous NSCLC patients.

Osimertinib and dihydroartemisinin: a novel drug combination targeting head and neck squamous cell carcinoma.

BACKGROUND: Recurrent and metastatic head and neck squamous cell carcinoma (HNSCC) has a dismal prognosis with limited progression-free survival and overall survival, even when treated with different combinations of chemotherapy, targeted therapies and immunotherapy. We explored in vitro and in vivo the effect of the epidermal growth factor receptor (EGFR) inhibitor, osimertinib, alone and in combination with dihydroartemisinin (DHA) in HNSCC. METHODS: The combination of osimertinib with DHA was tested in the FaDu and CAL27 HNSCC cell lines. Tumor cell proliferation assays were conducted in cultured cells and mouse xenografts. Western blotting analysis of related signal pathways was performed to investigate the molecular mechanisms of the inhibitory effect of DHA and the combination. Other compounds, which inhibit signal transducer and activator of transcription 3 (STAT3), Src-family kinases (SFKs), sphingosine kinase 1 (SPHK1), or the receptor tyrosine kinase (RTK) AXL were also combined with osimertinib in vitro. RESULTS: Osimertinib exerted synergistic cytotoxicity toward FaDu and CAL27 HNSCC cells when combined with DHA. DHA reversed the osimertinib-induced STAT3 and Src phosphorylation. The double combination inhibited AXL expression. The anticancer potential of osimertinib plus DHA combination was validated in vivo on FaDu and CAL27 xenografts in mice without notable side effects. CONCLUSIONS: The results illustrate that the combinatory therapy of osimertinib and DHA, as a repurposing anticancer drug, could be a novel therapeutic strategy for recurrent and/or metastatic HNSCC patients. The findings strongly indicate that a clinical trial is warranted to confirm the benefit of the combination.

A retinoic acid-dependent stroma-leukemia crosstalk promotes chronic lymphocytic leukemia progression.

In chronic lymphocytic leukemia (CLL), the non-hematopoietic stromal microenvironment plays a critical role in promoting tumor cell recruitment, activation, survival, and expansion. However, the nature of the stromal cells and molecular pathways involved remain largely unknown. Here, we demonstrate that leukemic B lymphocytes induce the activation of retinoid acid synthesis and signaling in the microenvironment. Inhibition of RA-signaling in stromal cells causes deregulation of genes associated with adhesion, tissue organization and chemokine secretion including the B-cell chemokine CXCL13. Notably, reducing retinoic acid precursors from the diet or inhibiting RA-signaling through retinoid-antagonist therapy prolong survival by preventing dissemination of leukemia cells into lymphoid tissues. Furthermore, mouse and human leukemia cells could be distinguished from normal B-cells by their increased expression of Rarγ2 and RXRα, respectively. These findings establish a role for retinoids in murine CLL pathogenesis, and provide new therapeutic strategies to target the microenvironment and to control disease progression.

mTOR intersects antibody-inducing signals from TACI in marginal zone B cells.

Mechanistic target of rapamycin (mTOR) enhances immunity in addition to orchestrating metabolism. Here we show that mTOR coordinates immunometabolic reconfiguration of marginal zone (MZ) B cells, a pre-activated lymphocyte subset that mounts antibody responses to T-cell-independent antigens through a Toll-like receptor (TLR)-amplified pathway involving transmembrane activator and CAML interactor (TACI). This receptor interacts with mTOR via the TLR adapter MyD88. The resulting mTOR activation instigates MZ B-cell proliferation, immunoglobulin G (IgG) class switching, and plasmablast differentiation through a rapamycin-sensitive pathway that integrates metabolic and antibody-inducing transcription programs, including NF-κB. Disruption of TACI-mTOR interaction by rapamycin, truncation of the MyD88-binding domain of TACI, or B-cell-conditional mTOR deficiency interrupts TACI signaling via NF-κB and cooperation with TLRs, thereby hampering IgG production to T-cell-independent antigens but not B-cell survival. Thus, mTOR drives innate-like antibody responses by linking proximal TACI signaling events with distal immunometabolic transcription programs.

Human Secretory IgM Emerges from Plasma Cells Clonally Related to Gut Memory B Cells and Targets Highly Diverse Commensals.

Secretory immunoglobulin A (SIgA) enhances host-microbiota symbiosis, whereas SIgM remains poorly understood. We found that gut IgM+ plasma cells (PCs) were more abundant in humans than mice and clonally related to a large repertoire of memory IgM+ B cells disseminated throughout the intestine but rare in systemic lymphoid organs. In addition to sharing a gut-specific gene signature with memory IgA+ B cells, memory IgM+ B cells were related to some IgA+ clonotypes and switched to IgA in response to T cell-independent or T cell-dependent signals. These signals induced abundant IgM which, together with SIgM from clonally affiliated PCs, recognized mucus-embedded commensals. Bacteria recognized by human SIgM were dually coated by SIgA and showed increased richness and diversity compared to IgA-only-coated or uncoated bacteria. Thus, SIgM may emerge from pre-existing memory rather than newly activated naive IgM+ B cells and could help SIgA to anchor highly diverse commensal communities to mucus.

Innate lymphoid cells integrate stromal and immunological signals to enhance antibody production by splenic marginal zone B cells.

Innate lymphoid cells (ILCs) regulate stromal cells, epithelial cells and cells of the immune system, but their effect on B cells remains unclear. Here we identified RORγt(+) ILCs near the marginal zone (MZ), a splenic compartment that contains innate-like B cells highly responsive to circulating T cell-independent (TI) antigens. Splenic ILCs established bidirectional crosstalk with MAdCAM-1(+) marginal reticular cells by providing tumor-necrosis factor (TNF) and lymphotoxin, and they stimulated MZ B cells via B cell-activation factor (BAFF), the ligand of the costimulatory receptor CD40 (CD40L) and the Notch ligand Delta-like 1 (DLL1). Splenic ILCs further helped MZ B cells and their plasma-cell progeny by coopting neutrophils through release of the cytokine GM-CSF. Consequently, depletion of ILCs impaired both pre- and post-immune TI antibody responses. Thus, ILCs integrate stromal and myeloid signals to orchestrate innate-like antibody production at the interface between the immune system and circulatory system.

Free radicals-mediated damage in transmitochondrial cells harboring the T14487C mutation in the ND6 gene of mtDNA.

We have studied the production of reactive oxygen species (ROS) in transmitochondrial cells, harboring homoplasmic levels of the T14487C mtDNA mutation in the ND6 gene of mitochondrial DNA (mtDNA). Previous work has shown that this mutation causes complex I deficiency. Here, we show that this mutation causes an overproduction of ROS leading to an increase in the oxidation of lipids and mtDNA without modification of antioxidant enzyme activities. We suggest that mutations in mtDNA affecting complex I activity may result in oxidative cellular damage, and reinforce the possible role of ROS-mediated mechanisms participating in some mtDNA-related disorders.