Frequency and Genomic Aspects of Intrinsic Resistance to Vismodegib in Locally Advanced Basal Cell Carcinoma.

PURPOSE: Vismodegib is approved for the treatment of locally advanced basal cell carcinoma (laBCC), but some cases demonstrate intrinsic resistance (IR) to the drug. We sought to assess the frequency of IR to vismodegib in laBCC and its underlying genomic mechanisms. EXPERIMENTAL DESIGN: Response to vismodegib was evaluated in a cohort of 148 laBCC patients. Comprehensive genomic and transcriptomic profiling was performed in a subset of five intrinsically resistant BCC (IR-BCC). RESULTS: We identified that IR-BCC represents 6.1% of laBCC in the studied cohort. Prior treatment with chemotherapy was associated with IR. Genetic events that were previously associated with acquired resistance (AR) in BCC or medulloblastoma were observed in three out of five IR-BCC. However, IR-BCCs were distinct by highly rearranged polyploid genomes. Functional analyses identified hyperactivation of the HIPPO-YAP and WNT pathways at RNA and protein levels in IR-BCC. In vitro assay on the BCC cell line further confirmed that YAP1 overexpression increases the cell proliferation rate. CONCLUSIONS: IR to vismodegib is a rare event in laBCC. IR-BCCs frequently harbor resistance mutations in the Hh pathway, but also are characterized by hyperactivation of the HIPPO-YAP and WNT pathways.

Mature tertiary lymphoid structures predict immune checkpoint inhibitor efficacy in solid tumors independently of PD-L1 expression.

Only a minority of patients derive long-term clinical benefit from anti-PD1/PD-L1 monoclonal antibodies. The presence of tertiary lymphoid structures (TLS) has been associated with improved survival in several tumor types. Here, using a large-scale retrospective analysis of three independent cohorts of cancer patients treated with anti-PD1/PD-L1 antibodies, we showed that the presence of mature TLS was associated with improved objective response rate, progression-free survival, and overall survival independently of PD-L1 expression status and CD8+ T-cell density. These results pave the way for using TLS detection to select patients who are more likely to benefit from immune checkpoint blockade.

IDO Targeting in Sarcoma: Biological and Clinical Implications.

Sarcomas are heterogeneous malignant mesenchymal neoplasms with limited sensitivity to immunotherapy. We recently demonstrated an increase in Kynurenine Pathway (KP) activity in the plasma of sarcoma patients treated with pembrolizumab. While the KP has already been described to favor immune escape through the degradation of L-Tryptophan and production of metabolites including L-Kynurenine, Indoleamine 2,3 dioxygenase (IDO1), a first rate-limiting enzyme of the KP, still represents an attractive therapeutic target, and its blockade had not yet been investigated in sarcomas. Using immunohistochemistry, IDO1 and CD8, expression profiles were addressed within 203 cases of human sarcomas. At a preclinical level, we investigated the modulation of the KP upon PDL1 blockade in a syngeneic model of sarcoma through mRNA quantification of key KP enzymes within the tumor. Furthermore, in order to evaluate the possible anti-tumor effect of IDO blockade in combination with PDL1 blockade, an innovative IDO inhibitor (GDC-0919) was used. Its effect was first assessed on Kynurenine to Tryptophan ratio at plasmatic level and also within the tumor. Following GDC-0919 treatment, alone or in combination with anti-PDL1 antibody, tumor growth, immune cell infiltration, and gene expression profiling were measured. IDO1 expression was observed in 39.1% of human sarcoma cases and was significantly higher in tumors with high CD8 infiltration. In the pre-clinical setting, blockade of PDL1 led to a strong anti-tumor effect and was associated with an intratumoral inflammatory cytokines signature driven by Ifng but also with a modulation of the KP enzymes including Ido1 and Ido2. IDO1 inhibition using GDC-0919 resulted in (i) a significant decrease of plasmatic Kynurenine to Tryptophan ratio and in (ii) a decrease of tumoral Kynurenine. However, GDC-0919 used alone or combined with anti-PDL1, did not show anti-tumoral activity and did not affect the tumor immune cell infiltrate. In order to elucidate the mechanism(s) underlying the lack of effect of GDC-0919, we analyzed the gene expression profile of intratumoral biopsies. Interestingly, we have found that GDC-0919 induced a downregulation of the expression of pvr and granzymes, and an upregulation of inhba and Dtx4 suggesting a potential role of the IDO pathway in the control of NK function.

Tumor uptake and associated greater efficacy of anti-Her2 immunoliposome does not rely on Her2 expression status: study of a docetaxel-trastuzumab immunoliposome on Her2+ breast cancer model (SKBR3).

Nanoparticles have been used for decades in breast cancer. More recently, anti-human epidermal receptor 2 (Her2) immunoliposomes are of rising interest. However, recent studies have questioned the actual relevance of using anti-Her2 antibodies to improve liposome distribution and efficacy. Using standard thin-film method and maleimide linker, we have synthesized a 140-nm docetaxel-trastuzumab immunoliposome. This nanoparticle was then tested on a canonical Her2-overexpressing breast cancer model (i.e., SKBR3), using 3D spheroids and xenografted mice. Its efficacy was compared with free docetaxel + trastuzumab, liposomal docetaxel + free trastuzumab and to reference antibody-drug conjugate trastuzumab-emtansine (T-DM1). Immunoliposomes resulted in better efficacy as compared with all other treatments, both in vitro and in vivo. To explain such an improvement, immunoliposome biodistribution was investigated using live imaging in xenografted mice. Surprisingly, no difference in tumor uptake was found between anti-Her2 immunoliposomes and standard docetaxel liposomes (i.e., 1.9 ± 1.2 vs. 1.7 ± 0.5% at the end of treatment and 1.4 ± 0.6 vs. 1.6 ± 0.4% at the end of the study, respectively, P > 0.05). We hypothesized that passive targeting (i.e., enhanced permeation and retention effect) contributed more to tumor distribution than active targeting and that the observed differences in efficacy could come from a better internalization of immunoliposomes into Her2+ cells as compared with standard liposomes, and not from a higher specificity towards tumor tissue.

A Tridimensional Model for NK Cell-Mediated ADCC of Follicular Lymphoma.

Follicular lymphoma (FL) is the second most frequent subtype of B non-Hodgkin's lymphomas (NHL) for which the treatment is based on the use of anti-CD20 mAbs. NK cells play a crucial role in their mechanism of action and the number of these cells mediating antibody-dependent cell cycotoxicity (ADCC) in the peripheral blood of FL patients predict the outcome. However, their presence in FL biopsies, their activation and their role have been poorly investigated. Moreover, in vitro studies have not deciphered the exact signaling cascades triggered by NK cells in presence of anti-CD20 mAbs on both effector and target cells in a relevant FL model. We performed in silico analyses and ex vivo functional assays to determine the presence and the activation status of NK cells in FL biopsies. We modelized ADCC phenomenon by developing a co-culture model composed by 3D-cultured FL cells and NK cells. Thus, we investigated the biological effect of anti-CD20 mAbs by fluorescent microscopy and the phosphorylation status of survival pathways by cell bar coding phosphoflow in target cells. In parallel, we measured the status of activation of downstream FcγRIIIa signaling pathways in effector cells and their activation (CD69, perforin, granzyme B, IFNγ) by flow cytometry. We determined by in vivo experiments the effects of anti-CD20 mAbs in presence of NK cells in SCID-Beige engrafted FL mice. Here, we show that functional NK cells infiltrate FL biopsies, and that their presence tends to correlate with the survival of FL patients. Using our 3D co-culture model, we show that rituximab and GA101 are able to promote degranulation, CD69 expression, IFNγ production and activate FcγRIIIa signaling cascade in NK cells, and inhibit survival pathways and induce apoptosis in FL cells. The effect of GA101 seems to be more pronounced as observed in vivo in a xenograft FL model. This study strongly supports the role of NK cells in FL and highlights the application of the 3D co-culture model for in vitro validation.

Endometrial Tumor Microenvironment Alters Human NK Cell Recruitment, and Resident NK Cell Phenotype and Function.

Endometrial Cancer is the most common cancer in the female genital tract in developed countries, and with its increasing incidence due to risk factors such as aging and obesity tends to become a public health issue. However, its immune environment has been less characterized than in other tumors such as breast cancers. NK cells are cytotoxic innate lymphoid cells that are considered as a major anti-tumoral effector cell type which function is drastically altered in tumors which participates to tumor progression. Here we characterize tumor NK cells both phenotypically and functionally in the tumor microenvironment of endometrial cancer. For that, we gathered endometrial tumors, tumor adjacent healthy tissue, blood from matching patients and healthy donor blood to perform comparative analysis of NK cells. First we found that NK cells were impoverished in the tumor infiltrate. We then compared the phenotype of NK cells in the tumor and found that tumor resident CD103+ NK cells exhibited more co-inhibitory molecules such as Tigit, and TIM-3 compared to recruited CD103- NK cells and that the expression of these molecules increased with the severity of the disease. We showed that both chemokines (CXCL12, IP-10, and CCL27) and cytokines profiles (IL-1ß and IL-6) were altered in the tumor microenvironment and might reduce NK cell function and recruitment to the tumor site. This led to hypothesize that the tumor microenvironment reduces resident NK cells cytotoxicity which we confirmed by measuring cytotoxic effector production and degranulation. Taken together, our results show that the tumor microenvironment reshapes NK cell phenotype and function to promote tumor progression.

Resistance to lysosomotropic drugs used to treat kidney and breast cancers involves autophagy and inflammation and converges in inducing CXCL5.

Lysosomotropic agents such as sunitinib, lapatinib, and chloroquine belong to a drug family that is being used more frequently to treat advanced cancers. Sunitinib is standard care for metastatic renal cell carcinomas (mRCC) and lapatinib is used for trastuzumab/pertuzumab-refractory cancers. However, patients ineluctably relapse with a delay varying from a few months to a few years. To improve reactivity prior to relapse it is essential to identify the mechanisms leading to such variability. We showed previously that sunitinib became sequestered in lysosomes because of its basic pKa. Methods: Modifications to gene expression in response to sunitinib and in sunitinib resistant cells were analyzed by transcriptomic and proteomic analysis. ROS production was evaluated by FACS. Nuclear Factor kappa B (NFkB)-dependent transcriptional regulation of inflammatory gene expression was evaluated with a reporter gene. Correlation of CXCL5 with survival was analyzed with an online available data base (TCGA) and using a cohort of patients enrolled in the SUVEGIL clinical trial (NCT00943839). Results: We now show that sunitinib sequestration in lysosomes induced an incomplete autophagic process leading to activation of the NFkB inflammatory pathway. We defined a subset of inflammatory cytokines that were up-regulated by the drug either after an acute or chronic stimulus. One of the most up-regulated genes in sunitinib-resistant cells was the CXCL5 cytokine. CXCL5 was also induced in RCC by chloroquine and in a model of HER2 positive breast cancer cell lines after acute or chronic treatment with lapatinib. CXCL5 correlated to shorter survival in RCC and to the most aggressive forms of breast cancers. The levels of CXCL5 present in the plasma of patients treated with sunitinib were predictive of the efficacy of sunitinib but not of the VEGF-directed antibody bevacizumab. Conclusion: This translational study identified CXCL5 as a biomarker of efficacy of lysosomotropic drugs, a potential asset for personalized medicine.

Role of Calcium Signaling in GA101-Induced Cell Death in Malignant Human B Cells.

GA101/obinutuzumab is a novel type II anti-CD20 monoclonal antibody (mAb), which is more effective than rituximab (RTX) in preclinical and clinical studies when used in combination with chemotherapy. Ca2+ signaling was shown to play a role in RTX-induced cell death. This report concerns the effect of GA101 on Ca2+ signaling and its involvement in the direct cell death induced by GA101. We reveal that GA101 triggered an intracellular Ca2+ increase by mobilizing intracellular Ca2+ stores and activating Orai1-dependent Ca2+ influx in non-Hodgkin lymphoma cell lines and primary B-Cell Chronic Lymphocytic Leukemia (B-CLL) cells. According to the cell type, Ca2+ was mobilized from two distinct intracellular compartments. In Raji, BL2, and B-CLL cells, GA101 induced a Ca2+ release from lysosomes, leading to the subsequent lysosomal membrane permeabilization and cell death. Inhibition of this calcium signaling reduced GA101-induced cell death in these cells. In SU-DHL-4 cells, GA101 mobilized Ca2+ from the endoplasmic reticulum (ER). Inhibition of ER replenishment, by blocking Orai1-dependent Ca2+ influx, led to an ER stress and unfolded protein response (UPR) which sensitized these cells to GA101-induced cell death. These results revealed the central role of Ca2+ signaling in GA101's action mechanism, which may contribute to designing new rational drug combinations improving its clinical efficacy.

Boosting γδ T cell-mediated antibody-dependent cellular cytotoxicity by PD-1 blockade in follicular lymphoma.

Follicular lymphoma (FL) is a common non Hodgkin's lymphoma subtype in which immune escape mechanisms are implicated in resistance to chemo-immunotherapy. Although molecular studies point to qualitative and quantitative deregulation of immune checkpoints, in depth cellular analysis of FL immune escape is lacking. Here, by functional assays and in silico analyses we show that a subset of FL patients displays a 'high' immune escape phenotype. These FL cases are characterized by abundant infiltration of PD1+ CD16+ TCRVγ9Vδ2 γδ T lymphocytes. In a 3D co-culture assay (MALC), γδ T cells mediate both direct and indirect (ADCC in the presence of anti-CD20 mAbs) cytolytic activity against FL cell aggregates. Importantly, PD-1, which is expressed by most FL-infiltrating γδ T lymphocytes with ADCC capacity, impairs these functions. In conclusion, we identify a PD1-regulated γδ T cell cytolytic immune component in FL. Our data provide a treatment rational by PD-1 blockade aimed at boosting γδ T cell anti-tumor functions in FL.

From 3D spheroids to tumor bearing mice: efficacy and distribution studies of trastuzumab-docetaxel immunoliposome in breast cancer.

PURPOSE: Nanoparticles are of rising interest in cancer research, but in vitro canonical cell monolayer models are not suitable to evaluate their efficacy when prototyping candidates. Here, we developed three-dimensional (3D) spheroid models to test the efficacy of trastuzumab-docetaxel immunoliposomes in breast cancer prior to further testing them in vivo. MATERIALS AND METHODS: Immunoliposomes were synthesized using the standard thin film method and maleimide linker. Two human breast cancer cell lines varying in Her2 expression were tested: Her2+ cells derived from metastatic site: mammary breast MDA-MB-453 and triple-negative MDA-MB-231 cells. 3D spheroids were developed and tested with fluorescence detection to evaluate viability. In vivo efficacy and biodistribution studies were performed on xenograft bearing nude mice using fluorescent and bioluminescent imaging. RESULTS: In vitro, antiproliferative efficacy was dependent upon cell type, size of the spheroids, and treatment scheduling, resulting in subsequent changes between tested conditions and in vivo results. Immunoliposomes performed better than free docetaxel + free trastuzumab and ado-trastuzumab emtansine (T-DM1). On MDA-MB-453 and MDA-MB-231 cell growth was reduced by 76% and 25%, when compared to free docetaxel + free trastuzumab and by 85% and 70% when compared to T-DM1, respectively. In vivo studies showed tumor accumulation ranging from 3% up to 15% of the total administered dose in MDA-MB-453 and MDA-MB-231 bearing mice. When compared to free docetaxel + free trastuzumab, tumor growth was reduced by 89% (MDA-MB-453) and 25% (MDA-MB-231) and reduced by 66% (MDA-MB-453) and 29% (MDA-MB-231) when compared to T-DM1, an observation in line with data collected from 3D spheroids experiments. CONCLUSION: We demonstrated the predictivity of 3D in vitro models when developing and testing nanoparticles in experimental oncology. In vitro and in vivo data showed efficient drug delivery with higher efficacy and prolonged survival with immunoliposomes when compared to current anti-Her2 breast cancer strategies.

Macrophages of distinct origins contribute to tumor development in the lung.

Tissue-resident macrophages can self-maintain without contribution of adult hematopoiesis. Herein we show that tissue-resident interstitial macrophages (Res-TAMs) in mouse lungs contribute to the pool of tumor-associated macrophages (TAMs) together with CCR2-dependent recruited macrophages (MoD-TAMs). Res-TAMs largely correlated with tumor cell growth in vivo, while MoD-TAMs accumulation was associated with enhanced tumor spreading. Both cell subsets were depleted after chemotherapy, but MoD-TAMs rapidly recovered and performed phagocytosis-mediated tumor clearance. Interestingly, anti-VEGF treatment combined with chemotherapy inhibited both Res and Mod-TAM reconstitution without affecting monocyte infiltration and improved its efficacy. Our results reveal that the developmental origin of TAMs dictates their relative distribution, function, and response to cancer therapies in lung tumors.

Docetaxel-trastuzumab stealth immunoliposome: development and in vitro proof of concept studies in breast cancer.

BACKGROUND: Trastuzumab plus docetaxel is a mainstay to treat HER2-positive breast cancers. However, developing nanoparticles could help to improve the efficacy/toxicity balance of this doublet by improving drug trafficking and delivery to tumors. This project aimed to develop an immunoliposome in breast cancer, combining docetaxel encapsulated in a stealth liposome engrafted with trastuzumab, and comparing its performances on human breast cancer cell lines with standard combination of docetaxel plus trastuzumab. METHODS: Several strategies to engraft trastuzumab to pegylated liposomes were tested. Immunoliposomes made of natural (antibody nanoconjugate-1 [ANC-1]) and synthetic lipids (ANC-2) were synthesized using standard thin film method and compared in size, morphology, docetaxel encapsulation, trastuzumab engraftment rates and stability. Antiproliferative activity was tested on human breast cancer models ranging from almost negative (MDA-MB-231), positive (MDA-MB-453) to overexpressing (SKBR3) HER2. Finally, cell uptake of ANC-1 was studied by electronic microscopy. RESULTS: ANC-1 showed a greater docetaxel encapsulation rate (73%±6% vs 53%±4%) and longer stability (up to 1 week) as compared with ANC-2. Both ANC presented particle size ≤150 nm and showed similar or higher in vitro antiproliferative activities than standard treatment, ANC-1 performing better than ANC-2. The IC50s for docetaxel combined to free trastuzumab were 8.7±4, 2±0.7 and 6±2 nM with MDA-MB-231, MDA-MB-453 and SKBR3, respectively. The IC50s for ANC-1 were 2.5±1, 1.8±0.6 and 3.4±0.8 nM and for ANC-2 were 1.8±0.3 nM, 2.8±0.8 nM and 6.8±1.8 nM with MDA-MB-231, MDA-MB-453 and SKBR3, respectively. Cellular uptake appeared to depend on HER2 expression, the higher the expression, the higher the uptake. CONCLUSION: In vitro results suggest that higher antiproliferative efficacy and efficient drug delivery can be achieved in breast cancer models using nanoparticles.

Mitochondrial reactive oxygen species regulate the induction of CD8+ T cells by plasmacytoid dendritic cells.

Cross-presentation allows exogenous antigen presentation in association with major histocompatibility complex class I molecules, a process crucial for the priming of CD8+ T-cell responses against viruses and tumors. By contrast to conventional dendritic cells (cDC), which cross-present antigens in the steady state, plasmacytoid dendritic cells (pDC) acquire this ability only after stimulation by Toll-like receptor (TLR) ligands. The intracellular pathways accounting for this functional difference are still unknown. Here we show that the induction of cross-presentation by pDCs is regulated by mitochondria through a reactive oxygen species (ROS)-dependent mechanism, involving pH alkalization and antigen protection. The reduction of mitochondrial ROS production dramatically decreases the cross-presentation capacity of pDCs, leading to a strong reduction of their capacity to trigger CD8+ T-cell responses. Our results demonstrate the importance of mitochondrial metabolism in pDC biology, particularly for the induction of adaptive immune responses.

Esophageal cancer cells resistant to T-DM1 display alterations in cell adhesion and the prostaglandin pathway.

Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate that specifically targets HER2 thanks to its antibody component trastuzumab. In spite of responses to this novel agent, acquired resistance to treatment remains a major obstacle. Prolonged in vitro exposure of the gastroesophageal junction cancer cell line OE-19 to T-DM1, in the absence or presence of ciclosporin A resulted in the selection of two resistant cell lines to T-DM1. T-DM1-resistant cells presented an increased expression of adhesion genes, altered spreading and higher sensitivity to anoikis than parental cells. A resistant cell line showed decreased adhesion strength, increased migration speed and increased sensitivity to RhoA inhibition. Genes involved in the prostaglandin pathway were deregulated in resistant models. Addition of prostaglandin E2 to T-DM1 partially restored its cytotoxic activity in resistant models. This work demonstrates that T-DM1-resistance may be associated with alterations of cell adhesion and the prostaglandin pathway, which might constitute novel therapeutic targets.

Combinations of Bevacizumab and Erlotinib Show Activity in Colorectal Cancer Independent of RAS Status.

Purpose: There is extensive cross-talk between VEGF- and EGFR-pathway signaling in colorectal cancer. However, combinations of VEGF- and EGFR-targeted monoclonal antibodies (mAb) show disappointing activity, in particular for patients with mutant RAS Previous results show that tyrosine kinase inhibitors (TKI) can be active in colorectal cancer models resistant to mAbs. This prompted us to examine whether the activity of bevacizumab can be increased by combination with erlotinib.Experimental Design: The antitumor activity of bevacizumab, erlotinib, and their combination was determined in colorectal cancer models with different RAS status and bevacizumab sensitivity. EGFR/VEGF pathway activation was characterized by immunohistochemistry, Western blot, and ELISA assays. The influence of cetuximab and erlotinib on EGF-mediated migration and the EGFR-EGF ligand feedback loop was established in colorectal cancer cell lines with different RAS status.Results: The addition of erlotinib increased bevacizumab activity in all models independent of RAS status. Bevacizumab exposure was accompanied by marked EGFR activation in tumor cells as well as in tumor-associated endothelial cells (TECs) and resulted in strong accumulation of intracellular EGFR, which could be attenuated by erlotinib. In cellular models, erlotinib was able to attenuate EGF-mediated functions in all cell lines independent of RAS status while cetuximab only showed activity in RAS wild-type cells.Conclusions: These results should provide a molecular framework to better understand the increased activity of the bevacizumab-erlotinib combination, compared with bevacizumab alone, in the GERCOR DREAM phase III clinical trial. Differential activity of mAbs and TKIs targeting the same signaling pathway is likely applicable for other tumor types. Clin Cancer Res; 24(11); 2548-58. ©2018 AACR.

Shifting the Balance of Activating and Inhibitory Natural Killer Receptor Ligands on BRAFV600E Melanoma Lines with Vemurafenib.

Over 60% of human melanoma tumors bear a mutation in the BRAF gene. The most frequent mutation is a substitution at codon 600 (V600E), leading to a constitutively active BRAF and overactivation of the MAPK pathway. Patients harboring mutated BRAF respond to kinase inhibitors such as vemurafenib. However, these responses are transient, and relapses are frequent. Melanoma cells are efficiently lysed by activated natural killer (NK) cells. Melanoma cells express several stress-induced ligands that are recognized by activating NK-cell receptors. We have investigated the effect of vemurafenib on the immunogenicity of seven BRAF-mutated melanoma cells to NK cells and on their growth and sensitivity to NK-cell-mediated lysis. We showed that vemurafenib treatment modulated expression of ligands for two activating NK receptors, increasing expression of B7-H6, a ligand for NKp30, and decreasing expression of MICA and ULBP2, ligands for NKG2D. Vemurafenib also increased expression of HLA class I and HLA-E molecules, likely leading to higher engagement of inhibitory receptors (KIRs and NKG2A, respectively), and decreased lysis of vemurafenib-treated melanoma cell lines by cytokine-activated NK cells. Finally, we showed that whereas batimastat (a broad-spectrum matrix metalloprotease inhibitor) increased cell surface ULBP2 by reducing its shedding, vemurafenib lowered soluble ULBP2, indicating that BRAF signal inhibition diminished expression of both cell-surface and soluble forms of NKG2D ligands. Vemurafenib, inhibiting BRAF signaling, shifted the balance of activatory and inhibitory NK ligands on melanoma cells and displayed immunoregulatory effects on NK-cell functional activities. Cancer Immunol Res; 5(7); 582-93. ©2017 AACR.

Sensitization of EGFR Wild-Type Non-Small Cell Lung Cancer Cells to EGFR-Tyrosine Kinase Inhibitor Erlotinib.

The benefit of EGFR-TKI in non-small cell lung cancer has been demonstrated in mutant EGFR tumors as first-line treatment but the benefit in wild-type EGFR tumors is marginal as well as restricted to maintenance therapy in pretreated patients. This work aimed at questioning the effects of cisplatin initial treatment on the EGFR pathway in non-small cell lung cancer and the functional consequences in vitro and in in vivo animal models of patient-derived xenografts (PDX). We establish here that cisplatin pretreatment specifically sensitizes wild-type EGFR-expressing cells to erlotinib, contrary to what happens in mutant EGFR cells and with a blocking EGFR antibody, both in vitro and in vivo The sensitization entails the activation of the kinase Src upstream of EGFR, thereafter transactivating EGFR through a ligand-independent activation. We propose a combination of markers that enable to discriminate between the tumors sensitized to erlotinib or not in PDX models, which should be worth testing in patients. These markers might be useful for the selection of patients who would benefit from erlotinib as a maintenance therapy. Mol Cancer Ther; 16(8); 1634-44. ©2017 AACR.

IL-26 Confers Proinflammatory Properties to Extracellular DNA.

In physiological conditions, self-DNA released by dying cells is not detected by intracellular DNA sensors. In chronic inflammatory disorders, unabated inflammation has been associated with a break in innate immune tolerance to self-DNA. However, extracellular DNA has to complex with DNA-binding molecules to gain access to intracellular DNA sensors. IL-26 is a member of the IL-10 cytokine family, overexpressed in numerous chronic inflammatory diseases, in which biological activity remains unclear. We demonstrate in this study that IL-26 binds to genomic DNA, mitochondrial DNA, and neutrophil extracellular traps, and shuttles them in the cytosol of human myeloid cells. As a consequence, IL-26 allows extracellular DNA to trigger proinflammatory cytokine secretion by monocytes, in a STING- and inflammasome-dependent manner. Supporting these biological properties, IL-10-based modeling predicts two DNA-binding domains, two amphipathic helices, and an in-plane membrane anchor in IL-26, which are structural features of cationic amphipathic cell-penetrating peptides. In line with these properties, patients with active autoantibody-associated vasculitis, a chronic relapsing autoimmune inflammatory disease associated with extensive cell death, exhibit high levels of both circulating IL-26 and IL-26-DNA complexes. Moreover, in patients with crescentic glomerulonephritis, IL-26 is expressed by renal arterial smooth muscle cells and deposits in necrotizing lesions. Accordingly, human primary smooth cells secrete IL-26 in response to proinflammatory cytokines. In conclusion, IL-26 is a unique cationic protein more similar to a soluble pattern recognition receptor than to conventional cytokines. IL-26 expressed in inflammatory lesions confers proinflammatory properties to DNA released by dying cells, setting up a positive amplification loop between extensive cell death and unabated inflammation.

B Cell Homeostasis and Functional Properties Are Altered in an Hypochlorous Acid-Induced Murine Model of Systemic Sclerosis.

INTRODUCTION: During systemic sclerosis (SSc), peripheral B cells display alterations in subset homeostasis and functional properties and are a promising therapeutic target. However, there is only few data regarding whether these anomalies are accurately reproduced in animal models of SSc. OBJECTIVE: In this work, we assessed the B cell homeostasis modifications in an experimental model of SSc [hypochlorous acid (HOCl)-induced mouse], both at a phenotypic and functional level, during the course of the disease. METHODS: Balb/c mice underwent daily intradermal injections of HOCl (or phosphate-buffered saline) and were then sacrificed at day 21 (early inflammatory stage) or day 42 (late fibrotic stage). For phenotypic studies, the distribution of the main spleen cell subsets (B cells, T CD4 and CD8 cells, NK cells, macrophages) and splenic B cell subsets (immature, mature naïve, germinal center, antibody-secreting, memory, B1) was assessed by flow cytometry. For functional studies, splenic B cells were immediately MACS-sorted. Production of interleukin (IL)-6, CCL3, IL-10, and transforming growth factor (TGF)-ß was assessed ex vivo by RT-PCR and after 48 h of culture by ELISA. Regulatory B cell (Breg) counts were quantified by flow cytometry. RESULTS: Phenotypic analyses showed an early expansion of transitional B cells, followed by a late expansion of the mature naive subset and decrease in plasmablasts and memory B cells. These anomalies are similar to those encountered in SSc patients. Functional analyses revealed a B-cell overproduction of pro-inflammatory cytokines (IL-6 and CCL3) and an impairment of their anti-inflammatory capacities (decreased production of IL-10 and TGF-ß, reduced levels of Bregs) at the early inflammatory stage; and an overproduction of pro-fibrotic cytokines (TGF-ß and IL-6) at the late fibrotic stage. These results approximate the anomalies observed in human SSc. CONCLUSION: This work reports the existence of anomalies in B cell homeostasis and functional properties in an animal model of SSc that approximate those displayed by SSc patients. These anomalies vary over the course of the disease, which pleads for their participation in inflammatory and fibrotic events. This makes the HOCl mouse a relevant experimental model for the study of B cells, and therefore, B-cell-targeted therapies in SSc.

Unexpected cross-reactivity of anti-cathepsin B antibodies leads to uncertainties regarding the mechanism of action of anti-CD20 monoclonal antibody GA101.

GA101, also known as obinutuzumab or Gazyva (Gazyvaro), is a glycoengineered type II humanized antibody that targets the CD20 antigen expressed at the surface of B-cells. This novel anti-CD20 antibody is currently assessed in clinical trials with promising results as a single agent or as part of therapeutic combinations for the treatment of B-cell malignancies. Detailed understanding of the mechanisms of GA101-induced cell death is needed to get insight into possible resistance mechanisms occurring in patients. Although multiple in vitro and in vivo mechanisms have been suggested to describe the effects of GA101 on B-cells, currently available data are ambiguous. The aim of our study was to clarify the cellular mechanisms involved in GA101-induced cell death in vitro, and more particularly the respective roles played by lysosomal and mitochondrial membrane permeabilization. Our results confirm previous reports suggesting that GA101 triggers homotypic adhesion and caspase-independent cell death, two processes that are dependent on actin remodeling and involve the production of reactive oxygen species. With respect to lysosomal membrane permeabilization (LMP), our data suggest that lack of specificity of available antibodies directed against cathepsin B may have confounded previously published results, possibly challenging current LMP-driven model of GA101 action mode.