Biglycan as a mediator of proinflammatory response and target for MDS and sAML therapy.

Myelodysplastic syndromes (MDS) and their progression to secondary acute myeloid leukemia (sAML) are associated with an altered protein expression including extracellular matrix (ECM) components thereby promoting an inflammatory environment. Since the role of the proteoglycan biglycan (BGN) as an inflammatory mediator has not yet been investigated in both diseases and might play a role in disease progression, its expression and/or function was determined in cell lines and bone marrow biopsies (BMBs) of MDS and sAML patients and subpopulations of MDS stem cells by Western blot and immunohistochemistry. The bone marrow (BM) microenvironment was analyzed by multispectral imaging, patients' survival by Cox regression. ROC curves were assessed for diagnostic value of BGN. All cell lines showed a strong BGN surface expression in contrast to only marginal expression levels in mononuclear cells and CD34+ cells from healthy donors. In the MDS-L cell line, CD34-CD33+ and CD34+CD33+ blast subpopulations exhibited a differential BGN surface detection. Increased BGN mediated inflammasome activity of CD34-CD33+TLR4+ cells was observed, which was inhibited by direct targeting of BGN or NLRP3. BGN was heterogeneously expressed in BMBs of MDS and sAML, but was not detected in control biopsies. BGN expression in BMBs positively correlated with MUM1+ and CD8+, but negatively with CD33+TLR4+ cell infiltration and was accompanied by a decreased progression-free survival of MDS patients. BGN-mediated inflammasome activation appears to be a crucial mechanism in MDS pathogenesis implicating its use as suitable biomarker and potential therapeutic target. Abbreviations: Ab, antibody; alloSCT, allogenic stem cell transplant; AML, acute myeloid leukemia; BGN, biglycan; BM, bone marrow; BMB, bone marrow biopsy; casp1, caspase 1; CTLA-4, cytotoxic T lymphocyte-associated protein 4; DAMP, danger-associated molecular pattern; ECM, extracellular matrix; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HD, healthy donor; HSPC, hematopoietic stem and progenitor cell; HSC, hematopoietic stem cell; IFN, interferon; IHC, immunohistochemistry; IL, interleukin; MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasm; MSI, multispectral imaging; NGS, next-generation sequencing; NLRP3, NLR family pyrin domain containing 3; OS, overall survival; PBMC, peripheral blood mononuclear cell; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1, PFS, progression-free survival; PRR, pattern recognition receptor; SC, stem cell; SLRP, small leucine-rich proteoglycan; TGF, transforming growth factor; TIRAP, toll/interleukin 1 receptor domain-containing adapter protein; TLR, toll-like receptor; Treg, regulatory T cell.

Induction of pulmonary HLA-G expression by SARS-CoV-2 infection.

The non-classical human leukocyte antigen (HLA)-G exerts immune-suppressive properties modulating both NK and T cell responses. While it is physiologically expressed at the maternal-fetal interface and in immune-privileged organs, HLA-G expression is found in tumors and in virus-infected cells. So far, there exists little information about the role of HLA-G and its interplay with immune cells in biopsies, surgical specimen or autopsy tissues of lung, kidney and/or heart muscle from SARS-CoV-2-infected patients compared to control tissues. Heterogeneous, but higher HLA-G protein expression levels were detected in lung alveolar epithelial cells of SARS-CoV-2-infected patients compared to lung epithelial cells from influenza-infected patients, but not in other organs or lung epithelia from non-viral-infected patients, which was not accompanied by high levels of SARS-CoV-2 nucleocapsid antigen and spike protein, but inversely correlated to the HLA-G-specific miRNA expression. High HLA-G expression levels not only in SARS-CoV-2-, but also in influenza-infected lung tissues were associated with a high frequency of tissue-infiltrating immune cells, but low numbers of CD8+ cells and an altered expression of hyperactivation and exhaustion markers in the lung epithelia combined with changes in the spatial distribution of macrophages and T cells. Thus, our data provide evidence for an involvement of HLA-G and HLA-G-specific miRNAs in immune escape and as suitable therapeutic targets for the treatment of SARS-CoV-2 infections.

Biglycan as a potential regulator of tumorgenicity and immunogenicity in K-RAS-transformed cells.

The extracellular matrix component biglycan (BGN) plays an essential role in various physiological and pathophysiological processes. A deficient BGN expression associated with reduced immunogenicity was found in HER-2/neu-overexpressing cells. To determine whether BGN is suppressed by oncogene-driven regulatory networks, the expression and function of BGN was analyzed in murine and human BGNlow/BGNhigh K-RASG12V-transformed model systems as well as in different patients' datasets of colorectal carcinoma (CRC) lesions. K-RAS-mutated CRC tissues expressed low BGN mRNA and protein levels when compared to normal colon epithelial cells, which was associated with a reduced patients' survival. Transfection of BGN in murine and human BGNlow K-RAS-expressing cells resulted in a reduced growth and migration of BGNhigh vs BGNlow K-RAS cells. In addition, increased MHC class I surface antigens as a consequence of an enhanced antigen processing machinery component expression was found upon restoration of BGN, which was confirmed by RNA-sequencing of BGNlow vs. BGNhigh K-RAS models. Furthermore, a reduced tumor formation of BGNhigh versus BGNlow K-RAS-transformed fibroblasts associated with an enhanced MHC class I expression and an increased frequency of tumor-infiltrating lymphocytes in tumor lesions was found. Our data provide for the first time an inverse link between BGN and K-RAS expression in murine and human K-RAS-overexpressing models and CRC lesions associated with altered growth properties, reduced immunogenicity and worse patients' outcome. Therefore, reversion of BGN might be a novel therapeutic option for K-RAS-associated malignancies.

Distinct Molecular Mechanisms of Altered HLA Class II Expression in Malignant Melanoma.

BACKGROUND: The human leukocyte antigen (HLA) class II molecules are constitutively expressed in some melanoma, but the underlying molecular mechanisms have not yet been characterized. METHODS: The expression of HLA class II antigen processing machinery (APM) components was determined in melanoma samples by qPCR, Western blot, flow cytometry and immunohistochemistry. Immunohistochemical and TCGA datasets were used for correlation of HLA class II expression to tumor grading, T-cell infiltration and patients' survival. RESULTS: The heterogeneous HLA class II expression in melanoma samples allowed us to characterize four distinct phenotypes. Phenotype I totally lacks constitutive HLA class II surface expression, which is inducible by interferon-gamma (IFN-γ); phenotype II expresses low basal surface HLA class II that is further upregulated by IFN-γ; phenotype III lacks constitutive and IFN-γ controlled HLA class II expression, but could be induced by epigenetic drugs; and in phenotype IV, lack of HLA class II expression is not recovered by any drug tested. High levels of HLA class II APM component expression were associated with an increased intra-tumoral CD4+ T-cell density and increased patients' survival. CONCLUSIONS: The heterogeneous basal expression of HLA class II antigens and/or APM components in melanoma cells is caused by distinct molecular mechanisms and has clinical relevance.

Differential effect on different immune subsets of neoadjuvant chemotherapy in patients with TNBC.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer (BC). Due to the absence of targets such as HER2 or hormone receptors, early TNBC is treated with surgery and chemotherapy. Since TNBC is also considered the most immunogenic type of BC with tumor infiltrating lymphocytes that are predictive for chemotherapy response and prognostic for patients' survival, many different immunotherapeutic strategies are currently explored in clinical trials for the treatment of this disease. In order to efficiently combine chemotherapy with immunotherapy, it is important to evaluate the effect of chemotherapy on immune cells in vivo. METHODS: Peripheral blood was taken from 56 patients with TNBC undergoing neoadjuvant chemotherapy with nanoparticle albumin-bound paclitaxel (Nab-Pac) followed by epirubicin and cyclophosphamide (EC) at three different time points. Multicolor flow cytometry was used to characterize the immune cell composition and functional properties along neoadjuvant chemotherapy. RESULTS: Whereas the first phase of the neoadjuvant chemotherapy did not significantly alter the patients' immune cell composition, after the second phase of chemotherapeutic administration most B cells (>90%) were lost and the frequency of natural killer (NK) cells and CD4+ T lymphocytes decreased approximately to 50%. In contrast, the frequency of CD8+ T cells were less affected. CONCLUSIONS: Despite late consequences of Nab-Pac cannot be ruled out, these data suggest that different chemotherapeutics might have distinct effects on the immune cell repertoire and that different immune cell populations exhibit a specific susceptibility to these chemotherapies with B and NK cells being more affected than T cells. This might also have an impact on the combination of chemotherapies with immunotherapies. TRIAL REGISTRATION NUMBER: NCT02685059.

Targeting the coding sequence: opposing roles in regulating classical and non-classical MHC class I molecules by miR-16 and miR-744.

BACKGROUND: To control gene expression, microRNAs (miRNAs) are of key importance and their deregulation is associated with the development and progression of various cancer types. In this context, a discordant messenger RNA/protein expression pointing to extensive post-transcriptional regulation of major histocompatibility complex (MHC) class I molecules was already shown. However, only a very limited number of miRNAs targeting these molecules have yet been identified. Despite an increasing evidence of coding sequence (CDS)-located miRNA binding sites, there exists so far, no detailed study of the interaction of miRNAs with the CDS of MHC class I molecules. METHODS: Using an MS2-tethering approach in combination with small RNA sequencing, a number of putative miRNAs binding to the CDS of human leukocyte antigen (HLA)-G were identified. These candidate miRNAs were extensively screened for their effects in the HLA-G-positive JEG3 cell line. Due to the high sequence similarity between HLA-G and classical MHC class I molecules, the impact of HLA-G candidate miRNAs on HLA class I surface expression was also analyzed. The Cancer Genome Atlas data were used to correlate candidate miRNAs and HLA class I gene expression. RESULTS: Transfection of candidate miRNAs revealed that miR-744 significantly downregulates HLA-G protein levels. In contrast, overexpression of the candidate miRNAs miR-15, miR-16, and miR-424 sharing the same seed sequence resulted in an unexpected upregulation of HLA-G. Comparable results were obtained for classical MHC class I members after transfection of miRNA mimics into HEK293T cells. Analyses of The Cancer Genome Atlas data sets for miRNA and MHC class I expression further validated the results. CONCLUSIONS: Our data expand the knowledge about MHC class I regulation and showed for the first time an miRNA-dependent control of MHC class I antigens mediated by the CDS. CDS-located miRNA binding sites could improve the general use of miRNA-based therapeutic approaches as these sites are highly independent of structural variations (e.g. mutations) in the gene body. Surprisingly, miR-16 family members promoted MHC class I expression potentially in a gene activation-like mechanism.

Tumor-induced escape mechanisms and their association with resistance to checkpoint inhibitor therapy.

Immunotherapy aims to activate the immune system to fight cancer in a very specific and targeted manner. Despite the success of different immunotherapeutic strategies, in particular antibodies directed against checkpoints as well as adoptive T-cell therapy, the response of patients is limited in different types of cancers. This attributes to escape of the tumor from immune surveillance and development of acquired resistances during therapy. In this review, the different evasion and resistance mechanisms that limit the efficacy of immunotherapies targeting tumor-associated antigens presented by major histocompatibility complex molecules on the surface of the malignant cells are summarized. Overcoming these escape mechanisms is a great challenge, but might lead to a better clinical outcome of patients and is therefore currently a major focus of research.

Heterogeneous expression and functional relevance of the ubiquitin carboxyl-terminal hydrolase L1 in melanoma.

The expression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) is deregulated in human cancer cells with tumor inhibiting or promoting functions. Due to less knowledge on the role of UCHL1 in melanoma progression, the expression pattern and function of UCHL1 as well as the deregulated signaling pathways were characterized. A large number of melanoma cell lines, tissue microarrays of melanoma lesions and control tissues were analyzed for UCHL1 expression using PCR, Western blot and/or immunohistochemistry. The analysis revealed that melanocyte cultures, 24 of 331 melanoma lesions, two of 18 short-term cultures and two of 19 melanoma cell lines tested, respectively, heterogeneously expressed UCHL1. The low frequency of UCHL1 expression in melanoma cells was due to gene silencing by promoter DNA hypermethylation. Using different transfection models an enzyme activity-dependent growth promoting function of UCHL1 via the activation of the mitogen-activated protein kinase signaling pathway was found in melanoma cells. Under oxygen stress a dose-dependent effect of UCHL1 was detected, which was mediated by a dynamic modification of the PI3K-Akt signaling. Thus, the aberrant UCHL1 expression in melanoma cells is linked to dynamic changes in growth properties and signal transduction cascades suggesting that UCHL1 provides a novel marker and/or therapeutic target at least for a subset of melanoma patients.