Uncovering immune checkpoint heterogeneity in oral squamous cell carcinoma using single cell RNA-sequencing data highlights three subgroups of patients with distinct immune phenotypes.

OBJECTIVES: In head and neck squamous cell carcinoma (HNSCC), PD-1/PD-L1 inhibitors remain inefficient in most patients, which points to the need for better characterization of immune checkpoint (ICP) molecule expression. MATERIAL AND METHODS: We evaluated the expression of 22 ICP ligands (ICPL) in 2,176 malignant cells from 10 patients in a public single-cell RNA-sequencing dataset and in two cohorts of HNSCC patients for which gene expression data are available. RESULTS: Based on ICPL expression, malignant cells formed three distinct clusters characterized either by a strong expression of ICPL together with an immune phenotype linked to IFN-γ response (cluster 1) or by a weak ICPL expression and little response to IFN-γ (clusters 2 and 3). Malignant cells from cluster 3 showed a high PD-L1 expression associated with NRF2 signature. The relevance of 3 groups of patients, i.e "high ICPL/high IFN-γ", "low ICPL/low IFN-γ" or "low ICPL/low IFN-γ/high PD-L1" was confirmed in a cohort of 259 OSCC whole tumor samples from TCGA and in the CLB-IHN cohort including patients treated with PD1/PD-L1 inhibitors. The heterogeneous expression of ICPL among patients' malignant cells was associated with immunologically distinct microenvironments, evaluated with the "hot/cold" and the Tumor microenvironment (TME) classification. Finally, the "low ICPL/low IFN-γ/high PD-L1" group 3 displayed a poor prognosis in the TCGA cohort. CONCLUSION: Hence, the global picture of ICPL gene expression in malignant cells from HNSCC patients may contribute to the broader issue of improving immunotherapy strategies though a better stratification of patients and the design of new treatment combinations.

Cisplatin unleashes Toll-like receptor 3-mediated apoptosis through the downregulation of c-FLIP in malignant mesothelioma.

Toll-like receptor 3 (TLR3) is an immune receptor that behaves like a death receptor in tumor cells, thereby providing an original target for cancer therapy. The therapeutic potential of TLR3 targeting in malignant mesothelioma, an aggressive and incurable neoplasia of the pleura and peritoneum, has so far not been addressed. We investigated TLR3 expression and sensitivity of human mesothelioma cell lines to the synthetic dsRNA Poly(I:C), alone or in combination with cisplatin, the gold standard chemotherapy in mesothelioma. Activation of TLR3 by Poly(I:C) induced apoptosis of 4/8 TLR3-positive cell lines but not of TLR3-negative cell lines. The combined cisplatin/Poly(I:C) treatment enhanced apoptosis of 3/4 Poly(I:C)-sensitive cell lines and overcame resistance to Poly(I:C) or cisplatin alone in 2/4 cell lines. Efficacy of the combined treatment relied on cisplatin-induced downregulation of c-FLIP, the main regulator of the extrinsic apoptotic pathway, leading to an enhanced caspase-8-mediated pathway. Of note, 6/6 primary cell samples isolated from patients with peritoneal mesothelioma expressed TLR3. Patient-derived cells were sensitive to Poly(I:C) alone while the combined cisplatin/Poly(I:C) treatment induced dramatic cell death. Our findings demonstrate that TLR3 targeting in combination with cisplatin presents an innovative therapeutic strategy in mesothelioma.

Human monocyte-derived dendritic cells turn into foamy dendritic cells with IL-17A.

Interleukin 17A (IL-17A) is a proinflammatory cytokine involved in the pathogenesis of chronic inflammatory diseases. In the field of immunometabolism, we have studied the impact of IL-17A on the lipid metabolism of human in vitro-generated monocyte-derived dendritic cells (DCs). Microarrays and lipidomic analysis revealed an intense remodeling of lipid metabolism induced by IL-17A in DCs. IL-17A increased 2-12 times the amounts of phospholipids, cholesterol, triglycerides, and cholesteryl esters in DCs. Palmitic (16:0), stearic (18:0), and oleic (18:ln-9c) acid were the main fatty acid chains present in DCs. They were strongly increased in response to IL-17A while their relative proportion remained unchanged. Capture of extracellular lipids was the major mechanism of lipid droplet accumulation, visualized by electron microscopy and Oil Red O staining. Besides this foamy phenotype, IL-17A induced a mixed macrophage-DC phenotype and expression of the nuclear receptor NR1H3/liver X receptor-α, previously identified in the context of atherosclerosis as the master regulator of cholesterol homeostasis in macrophages. These IL-17A-treated DCs were as competent as untreated DCs to stimulate allogeneic naive T-cell proliferation. Following this first characterization of lipid-rich DCs, we propose to call these IL-17A-dependent cells "foamy DCs" and discuss the possible existence of foamy DCs in atherosclerosis, a metabolic and inflammatory disorder involving IL-17A.

Genetic evidence of a precisely tuned dysregulation in the hypoxia signaling pathway during oncogenesis.

The classic model of tumor suppression implies that malignant transformation requires full "two-hit" inactivation of a tumor-suppressor gene. However, more recent work in mice has led to the proposal of a "continuum" model that involves more fluid concepts such as gene dosage-sensitivity and tissue specificity. Mutations in the tumor-suppressor gene von Hippel-Lindau (VHL) are associated with a complex spectrum of conditions. Homozygotes or compound heterozygotes for the R200W germline mutation in VHL have Chuvash polycythemia, whereas heterozygous carriers are free of disease. Individuals with classic, heterozygous VHL mutations have VHL disease and are at high risk of multiple tumors (e.g., CNS hemangioblastomas, pheochromocytoma, and renal cell carcinoma). We report here an atypical family bearing two VHL gene mutations in cis (R200W and R161Q), together with phenotypic analysis, structural modeling, functional, and transcriptomic studies of these mutants in comparison with classical mutants involved in the different VHL phenotypes. We demonstrate that the complex pattern of disease manifestations observed in VHL syndrome is perfectly correlated with a gradient of VHL protein (pVHL) dysfunction in hypoxia signaling pathways. Thus, by studying naturally occurring familial mutations, our work validates in humans the "continuum" model of tumor suppression.

Transcriptional silencing of the Wnt-antagonist DKK1 by promoter methylation is associated with enhanced Wnt signaling in advanced multiple myeloma.

The Wnt/ß-catenin pathway plays a crucial role in the pathogenesis of various human cancers. In multiple myeloma (MM), aberrant auto-and/or paracrine activation of canonical Wnt signaling promotes proliferation and dissemination, while overexpression of the Wnt inhibitor Dickkopf1 (DKK1) by MM cells contributes to osteolytic bone disease by inhibiting osteoblast differentiation. Since DKK1 itself is a target of TCF/ß-catenin mediated transcription, these findings suggest that DKK1 is part of a negative feedback loop in MM and may act as a tumor suppressor. In line with this hypothesis, we show here that DKK1 expression is low or undetectable in a subset of patients with advanced MM as well as in MM cell lines. This absence of DKK1 is correlated with enhanced Wnt pathway activation, evidenced by nuclear accumulation of ß-catenin, which in turn can be antagonized by restoring DKK1 expression. Analysis of the DKK1 promoter revealed CpG island methylation in several MM cell lines as well as in MM cells from patients with advanced MM. Moreover, demethylation of the DKK1 promoter restores DKK1 expression, which results in inhibition of ß-catenin/TCF-mediated gene transcription in MM lines. Taken together, our data identify aberrant methylation of the DKK1 promoter as a cause of DKK1 silencing in advanced stage MM, which may play an important role in the progression of MM by unleashing Wnt signaling.

The HGF/MET pathway as target for the treatment of multiple myeloma and B-cell lymphomas.

Hepatocyte growth factor (HGF) and its receptor MET are essential during embryonic development and throughout postnatal life. However, aberrant MET activation, due to overexpression, mutations, or autocrine ligand production, contributes to the development and progression of a variety of human cancers, often being associated with poor clinical outcome and drug resistance. B cell malignancies arise from B cells that are clonally expanded at different stages of differentiation. Despite major therapeutic advances, most mature B cell malignancies remain incurable and biologically-oriented therapeutic strategies are urgently needed. This review addresses the role of the HGF/MET pathway during B cell development and discusses how its aberrant activation contributes to the development of B cell lymphoproliferative disorders, with particular emphasis on multiple myeloma and diffuse large B cell lymphoma. These insights, combined with the recent development of clinical-grade agents targeting the MET pathway, provide the rationale to envision the HGF/MET pathway as a new promising target for the treatment of B cell malignancies.

Growth factors in multiple myeloma: a comprehensive analysis of their expression in tumor cells and bone marrow environment using Affymetrix microarrays.

BACKGROUND: Multiple myeloma (MM) is characterized by a strong dependence of the tumor cells on their microenvironment, which produces growth factors supporting survival and proliferation of myeloma cells (MMC). In the past few years, many myeloma growth factors (MGF) have been described in the literature. However, their relative importance and the nature of the cells producing MGF remain unidentified for many of them. METHODS: We have analysed the expression of 51 MGF and 36 MGF receptors (MGFR) using Affymetrix microarrays throughout normal plasma cell differentiation, in MMC and in cells from the bone marrow (BM) microenvironment (CD14, CD3, polymorphonuclear neutrophils, stromal cells and osteoclasts). RESULTS: 4/51 MGF and 9/36 MGF-receptors genes were significantly overexpressed in plasmablasts (PPC) and BM plasma cell (BMPC) compared to B cells whereas 11 MGF and 11 MGFR genes were overexpressed in BMPC compared to PPC. 3 MGF genes (AREG, NRG3, Wnt5A) and none of the receptors were significantly overexpressed in MMC versus BMPC. Furthermore, 3/51 MGF genes were overexpressed in MMC compared to the the BM microenvironment whereas 22/51 MGF genes were overexpressed in one environment subpopulation compared to MMC. CONCLUSIONS: Two major messages arise from this analysis 1) The majority of MGF genes is expressed by the bone marrow environment. 2) Several MGF and their receptors are overexpressed throughout normal plasma cell differentiation. This study provides an extensive and comparative analysis of MGF expression in plasma cell differentiation and in MM and gives new insights in the understanding of intercellular communication signals in MM.

APRIL and TACI interact with syndecan-1 on the surface of multiple myeloma cells to form an essential survival loop.

BLyS and APRIL share two receptors - transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA) - and BLyS binds to a third receptor, BAFF-R. We previously reported that TACI gene expression is a good indicator of a BLyS-binding receptor in human multiple myeloma cell lines (HMCLs), unlike BCMA, which is expressed by all HMCLs or BAFF-R which is typically not expressed by late-stage B cells. We hypothesised a link between APRIL and TACI through syndecan-1, similar to the situation reported for FGF and FGFR. We observed very strong binding of APRIL, but not BLyS, at the surface of all syndecan-1(+) HMCLs and primary multiple myeloma cells (MMC). All syndecan-1(+) HMCLs and MMC could also bind TACI-Fc, but not BCMA-Fc or BAFF-R-Fc molecules. Binding of APRIL or TACI-Fc was abrogated by heparin or cell pretreatment with heparitinase, which cleaves heparan sulfate chains. The growth factor activity of APRIL on MMC was also inhibited by heparin. Our data identify syndecan-1 as a co-receptor for APRIL and TACI at the cell surface of MMC, promoting the activation of an APRIL/TACI pathway that induces survival and proliferation in MMC.

Induction of angiogenesis by normal and malignant plasma cells.

Abundant bone marrow angiogenesis is present in almost all myeloma patients requiring therapy and correlated to treatment response and survival. We assessed the expression of 402 angiogenesis-associated genes by Affymetrix DNA microarrays in 466 samples, including CD138-purified myeloma cells (MMCs) from 300 previously untreated patients, in vivo microcirculation by dynamic contrast-enhanced magnetic resonance imaging, and in vitro angiogenesis (AngioKit-assay). Normal bone marrow plasma cells (BMPCs) express a median of 39 proangiogenic (eg, VEGFA, ADM, IGF-1) and 28 antiangiogenic genes (eg, TIMP1, TIMP2). Supernatants of BMPCs unlike those of memory B cells induce angiogenesis in vitro. MMCs do not show a significantly higher median number of expressed proangiogenic (45) or antiangiogenic (31) genes, but 97% of MMC samples aberrantly express at least one of the angiogenic factors HGF, IL-15, ANG, APRIL, CTGF, or TGFA. Supernatants of MMCs and human myeloma cell lines induce significantly higher in vitro angiogenesis compared with BMPCs. In conclusion, BMPCs express a surplus of proangiogenic over antiangiogenic genes transmitting to the ability to induce in vitro angiogenesis. Aberrant expression of proangiogenic and down-regulation of antiangiogenic genes by MMCs further increases the angiogenic stimulus, together leading to bone marrow angiogenesis at various degrees in all myeloma patients.

Expression of genes encoding for proteins involved in heparan sulphate and chondroitin sulphate chain synthesis and modification in normal and malignant plasma cells.

Syndecan-1 is a proteoglycan that concentrates heparin-binding factors on the surface of multiple myeloma cells, and probably plays a major role in multiple myeloma biology. As heparan sulphate and chondroitin sulphate are the bioactive components of syndecan-1, we analysed the signature of genes encoding 100 proteins involved in synthesis of these chains, i.e. from precursor uptake to post-translational modifications, using Affymetrix microarrays. The expression of enzymes required for heparan sulphate and chondroitin sulphate biosynthesis was shown to increase in parallel with syndecan-1 expression, throughout the differentiation of memory B cells into plasmablasts and normal bone marrow plasma cells. Sixteen genes were significantly different between normal and malignant plasma cells, nine of these genes -EXT2, CHSY3, CSGALNACT1, HS3ST2, HS2ST1, CHST11, CSGALNACT2, HPSE, SULF2 - encode proteins involved in glycosaminoglycan chain synthesis or modifications. Kaplan-Meier analysis was performed in two independent series of patients: B4GALT7, CSGALNACT1, HS2ST1 were associated with a good prognosis whereas EXT1 was linked to a bad prognosis. This study provides an overall picture of the major genes encoding for proteins involved in heparan sulphate and chondroitin sulphate synthesis and modifications that can be implicated in normal and malignant plasma cells.