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Supplementary Tables S1-S4. MMPs which are significantly over-expressed in melanoma cell lines compared to the mean expression of all cancer cell lines in the datasets with over 15 melanoma cell lines (S1); Outlier analysis of MMP16 copy number gain or over-expression using DNA and mRNA datasets containing more than 20 clinical melanoma samples (S2); MMP mRNA expression, LVI and clinicopathological data of human melanoma tumors (S3); S100B staining coverage, lymphatic and blood vessel densities of human melanoma tumors (S4)

Supplementary Data. Supplementary Materials and Methods and Supplementary Figure Legends

Supplementary Figures S1-S9. MMP14 which is over-expressed in melanoma cell lines was not associated with patient survival (S1); Vasculature and morphology of human melanoma tumors (S2); MMP16 expression in WM852 cells and its correlation with tumor weight in melanoma xenografts (S3); No significant difference in the blood and lymphatic vessel densities was observed between shScr and shMMP16 tumors (S4); MMP16-silencing does not affect mouse collagen type I in xenografts (S5); Endogenous or exogenous MMP16 regulates collagen invasion and cell-cell contacts in WM852 and Bowes melanoma cells (S6); Endogenous or recombinant MMP16 regulates LEC spheroid intravasation of WM852 and Bowes melanoma cells (S7); Silencing of L1CAM cleaved by MMP16 rescued the transmigration across BECs but not the cell junction disassembly of shMMP16 cells (S8); L1CAM immunohistochemistry of xenografts and human samples (S9).

Funding Information: The authors thank Laura Turunen, Jani Saarela, Katja Suomi, and Maria Nurmi of the High-Throughput Biomedicine Unit and the personnel of the Sequencing Laboratory at the Institute of Molecular Medicine Finland. The authors also thank Caroline Heckman, Sirpa Lepp?, and Olli Lohi for collaboration by providing the cell lines used in the study. The Biomedicum Helsinki Flow Cytometry Core Unit and the Biomedicum Virus Core are acknowledged for their services. The primary B-ALL samples were provided by the Finnish Hematology Registry and Clinical Biobank (FHRB). We thank all the patients for their generous participation. The FHRB biobank is supported by the Finnish Association of Hematology, Finnish Red Cross Blood Service, Institute for Molecular Medicine Finland, and participating hospitals in Finland. This study was supported by the Cancer Foundation Finland, the Sigrid Juselius Foundation, the Relander Foundation, state funding for university-level health research in Finland, and HiLife fellow funds from the University of Helsinki. O.D. was supported by grants from the Biomedicum Helsinki Foundation and the Finnish Medical Society. J. Koski was supported by grants from the Finnish Pediatric Cancer Foundation, the V?re Pediatric Research Foundation, the Cancer Foundation Finland, the Orion Research Foundation, and state funding for university-level health research in Finland. Funding Information: This study was supported by the Cancer Foundation Finland, the Sigrid Juselius Foundation, the Relander Foundation, state funding for university-level health research in Finland, and HiLife fellow funds from the University of Helsinki. O.D. was supported by grants from the Biomedicum Helsinki Foundation and the Finnish Medical Society. J. Koski was supported by grants from the Finnish Pediatric Cancer Foundation, the Väre Pediatric Research Foundation, the Cancer Foundation Finland, the Orion Research Foundation, and state funding for university-level health research in Finland. Funding Information: The authors thank Laura Turunen, Jani Saarela, Katja Suomi, and Maria Nurmi of the High-Throughput Biomedicine Unit and the personnel of the Sequencing Laboratory at the Institute of Molecular Medicine Finland. The authors also thank Caroline Heckman, Sirpa Leppä, and Olli Lohi for collaboration by providing the cell lines used in the study. The Biomedicum Helsinki Flow Cytometry Core Unit and the Biomedicum Virus Core are acknowledged for their services. The primary B-ALL samples were provided by the Finnish Hematology Registry and Clinical Biobank (FHRB). We thank all the patients for their generous participation. The FHRB biobank is supported by the Finnish Association of Hematology, Finnish Red Cross Blood Service, Institute for Molecular Medicine Finland, and participating hospitals in Finland. Publisher Copyright: © 2020 American Society of Hematology Chimeric antigen receptor (CAR) T-cell therapy has proven effective in relapsed and refractory B-cell malignancies, but resistance and relapses still occur. Better understanding of mechanisms influencing CAR T-cell cytotoxicity and the potential for modulation using small-molecule drugs could improve current immunotherapies. Here, we systematically investigated druggable mechanisms of CAR T-cell cytotoxicity using >500 small-molecule drugs and genome-scale CRISPR-Cas9 loss-of-function screens. We identified several tyrosine kinase inhibitors that inhibit CAR T-cell cytotoxicity by impairing T-cell signaling transcriptional activity. In contrast, the apoptotic modulator drugs SMAC mimetics sensitized B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma cells to anti-CD19 CAR T cells. CRISPR screens identified death receptor signaling through FADD and TNFRSF10B (TRAIL-R2) as a key mediator of CAR T-cell cytotoxicity and elucidated the RIPK1-dependent mechanism of sensitization by SMAC mimetics.Death receptor expression varied across genetic subtypes of B-cell malignancies, suggesting a link between mechanisms of CAR T-cell cytotoxicity and cancer genetics. These results implicate death receptor signaling as an important mediator of cancer cell sensitivity to CAR T-cell cytotoxicity, with potential for pharmacological targeting to enhance cancer immunotherapy. The screening data provide a resource of immunomodulatory properties of cancer drugs and genetic mechanisms influencing CAR T-cell cytotoxicity. Key Points: • Survey of immunomodulatory effects of >500 drugs identifies SMAC mimetics as sensitizers to CAR T-cell cytotoxicity. • Genome-scale CRISPR screen reveals essentiality of death receptor signaling for CAR T-cell cytotoxicity.

Background Adoptive T-cell therapy offers new options for cancer treatment. Clinical results suggest that T-cell persistence, depending on T-cell memory, improves efficacy. The use of interleukin (IL)-2 for in vitro T-cell expansion is not straightforward because it drives effector T-cell differentiation but does not promote the formation of T-cell memory. We have developed a cost-effective expansion protocol for chimeric antigen receptor (CAR) T cells with an early memory phenotype. Methods Lymphocytes were transduced with third-generation lentiviral vectors and expanded using CD3/CD28 microbeads. The effects of altering the IL-2 supplementation (0–300 IU/mL) and length of expansion (10–20 days) on the phenotype of the T-cell products were analyzed. Results High IL-2 levels led to a decrease in overall generation of early memory T cells by both decreasing central memory T cells and augmenting effectors. T memory stem cells (TSCM, CD95+CD45RO−CD45RA+CD27+) were present variably during T-cell expansion. However, their presence was not IL-2 dependent but was linked to expansion kinetics. CD19-CAR T cells generated in these conditions displayed in vitro antileukemic activity. In summary, production of CAR T cells without any cytokine supplementation yielded the highest proportion of early memory T cells, provided a 10-fold cell expansion and the cells were functionally potent. Discussion The number of early memory T cells in a T-cell preparation can be increased by simply reducing the amount of IL-2 and limiting the length of T-cell expansion, providing cells with potentially higher in vivo performance. These findings are significant for robust and cost-effective T-cell manufacturing., published version, peerReviewed

Indoleamine 2,3-deoxygenase 1 (IDO1) induces immune tolerance in the tumor microenvironment (TME) and is recognized as a potential therapeutic target. We studied the expression of both IDO1 and the related tryptophan 2,3-dioxygenase (TDO) in several different subtypes of cutaneous T-cell lymphoma (CTCL), and evaluated the kynurenine (KYN) pathway in the local TME and in patient sera. Specimens from the total of 90 CTCL patients, including mycosis fungoides (MF, n = 37), lymphomatoid papulosis (LyP, n = 36), primary cutaneous anaplastic large cell lymphoma (pcALCL, n = 4), subcutaneous panniculitis-like T-cell lymphoma (SPTCL n = 13), and 10 patients with inflammatory lichen ruber planus (LRP), were analyzed by immunohistochemistry (IHC), immunofluorescence (IF), quantitative PCR, and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Three CTCL cell lines also were studied. Expression of both IDO1 and TDO was upregulated in CTCL. In MF specimens and in the MF cell line MyLa2000, IDO1 expression exceeded that of TDO, whereas the opposite was true for LyP, ALCL, and corresponding Mac1/2A cell lines. The spectrum of IDO1-expressing cell types differed among CTCL subtypes and was reflected in the clinical behavior. In MF, SPTCL, and LyP, IDO1 was expressed by malignant cells and by CD33(+) myeloid-derived suppressor cells, whereas in SPTCL CD163(+) tumor-associated macrophages also expressed IDO1. Significantly elevated serum KYN/Trp ratios were found in patients with advanced stages of MF. Epacadostat, an IDO1 inhibitor, induced a clear decrease in KYN concentration in cell culture. These results show the importance of IDO1/TDO-induced immunosuppression in CTCL and emphasize its role as a new therapeutic target.

Defects in complex I due to mutations in mitochondrial DNA are associated with clinical features ranging from single organ manifestation like Leber hereditary optic neuropathy (LHON) to multiorgan disorders like mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. Specific mutations cause overlap syndromes combining several phenotypes, but the mechanisms of their biochemical effects are largely unknown. The m.3376G>A transition leading to p.E24K substitution in ND1 with LHON/MELAS phenotype was modeled here in a homologous position (NuoH-E36K) in the Escherichia coli enzyme and it almost totally abolished complex I activity. The more conservative mutation NuoH-E36Q resulted in higher apparent Km for ubiquinone and diminished inhibitor sensitivity. A NuoH homolog of the m.3865A>G transition, which has been found concomitantly in the overlap syndrome patient with the m.3376G>A, had only a minor effect. Consequences of a primary LHON-mutation m.3460G>A affecting the same extramembrane loop as the m.3376G>A substitution were also studied in the E. coli model and were found to be mild. The results indicate that the overlap syndrome-associated m.3376G>A transition in MTND1 is the pathogenic mutation and m.3865A>G transition has minor, if any, effect on presentation of the disease. The kinetic effects of the NuoH-E36Q mutation suggest its proximity to the putative ubiquinone binding domain in 49kD/PSST subunits. In all, m.3376G>A perturbs ubiquinone binding, a phenomenon found in LHON, and decreases the activity of fully assembled complex I as in MELAS.

The neuron navigator 3 (NAV3) gene on chromosome 12q21 encodes a microtubule plus end tracking protein and belongs to the navigator family of cytoskeletal regulators. Loss of heterozygosity on 12q has previously been suggested to be associated with poor prognosis in cancers of epithelial origin. In this study, we characterized copy number changes of NAV3 in 24 basal cell cancers (BCCs), eight squamous cell cancers (SCCs) and eight non-malignant inflammatory skin lesions by fluorescent in situ hybridization. To identify genes affected by NAV3, we used oligo siRNA gene silencing and gene microarrays to analyse gene expression profiles at several time points post-transfection in primary human keratinocytes. We found NAV3 copy number loss and decreased protein expression in 21% of the BCCs and 25% of the SCCs. In the nodular/superficial BCC subgroup, low-level NAV3 amplification was also observed. NAV3 aberrations were independent of the known chromosome 6 amplifications in BCC. Chromosome 12 polysomy was detected in 33% and 25% of the invasive type of BCC and SCC, respectively. Silencing of NAV3 in primary human keratinocytes revealed 22 differentially expressed genes, mostly related to inflammation. The most relevant of these were validated with qPCR or immunohistochemistry. This pilot study suggests that NAV3 is a novel cancer-associated gene that contributes to the pathogenesis of a subgroup of BCC and SCC.

Background Subcutaneous panniculitis-like T cell lymphomas represent a rare and difficult to diagnose entity of cutaneous T cell lymphomas. SPTL affects predominantly young adults and presents with multifocal subcutaneous nodules and frequently associated autoimmune features. The pathogenesis of SPTL is not completely understood. Methods The aim of this study was to unravel molecular pathways critical to the SPTL pathogenesis. Therefore, we analyzed 23 skin samples from 20 newly diagnosed SPTL patients and relevant control samples of adipose and non-malignant panniculitis tissue by using gene expression microarray, quantitative PCR, and two-colour immunohistochemistry. Results Interestingly, indoleamine 2,3-dioxygenase (IDO-1), an immunotolerance-inducing enzyme, was among the most highly overexpressed genes in all comparisons. The expression of Th1-specific cytokines, known to be associated with autoimmune inflammation (i.e. IFNG, CXCR3, CXCL9, CXCL10, CXCL11, and CCL5), were also significantly increased. Confirmed using immunohistochemistry, the morphologically malignant lymphocytes expressed CXCR3 and CXCL9. IDO-1 expression was found both in some morphologically malignant lymphocytes rimming the adipocytes and in surrounding CD11c− CD68− cells but not in CD11c+ dendritic cells in the microenvironment. The proportion of FoxP3+ cells in SPTL exceeded that in the benign panniculitis samples. Conclusions Our results indicate that the up regulation of the tolerogenic IDO-1 together with the up regulation of IFNG, CXCR3 ligands, and CCL5 are features of SPTL lesions. We anticipate that the IFNG-inducible IDO-1 expression contributes to the formation of an immunosuppressive microenvironment, favorable for the malignant T cells. This study provides a relevant molecular basis for further studies exploring novel therapeutic means for subcutaneous T cell lymphoma. Electronic supplementary material The online version of this article (doi:10.1186/s13023-014-0160-2) contains supplementary material, which is available to authorized users.

Lymphatic invasion and accumulation of continuous collagen bundles around tumor cells are associated with poor melanoma prognosis, but the underlying mechanisms and molecular determinants have remained unclear. We show here that a copy-number gain or overexpression of the membrane-type matrix metalloproteinase MMP16 (MT3-MMP) is associated with poor clinical outcome, collagen bundle assembly around tumor cell nests, and lymphatic invasion. In cultured WM852 melanoma cells derived from human melanoma metastasis, silencing of MMP16 resulted in cell-surface accumulation of the MMP16 substrate MMP14 (MT1-MMP) as well as L1CAM cell adhesion molecule, identified here as a novel MMP16 substrate. When limiting the activities of these trans-membrane protein substrates toward pericellular collagen degradation, cell junction disassembly, and blood endothelial transmigration, MMP16 supported nodular-type growth of adhesive collagen-surrounded melanoma cell nests, coincidentally steering cell collectives into lymphatic vessels. These results uncover a novel mechanism in melanoma pathogenesis, whereby restricted collagen infiltration and limited mesenchymal invasion are unexpectedly associated with the properties of the most aggressive tumors, revealing MMP16 as a putative indicator of adverse melanoma prognosis. Cancer Res; 75(10); 2083–94. ©2015 AACR.

Copy number changes or reduced expression of the Neuron navigator 3 (NAV3) gene occurs in neuroblastomas and malignancies of epithelial or lymphoid origin. To elucidate whether NAV3 has a role in the tumorigenesis of nervous system tumors in general, we studied central and peripheral nervous system tumors for NAV3 copy number changes. In search for common tumorigenic denominators, we analyzed 113 central and peripheral nervous system tumors, including glial tumors (grades I–IV gliomas), medulloblastomas, and neuroblastomas. NAV3 copy number changes were studied by fluorescence in situ hybridization and correlated to survival analyses. To identify target genes of NAV3 deletion, NAV3 was silenced by siRNA in glioblastoma cell lines and gene expression profiles were analyzed by Agilent 4×44k dual-color microarrays. Selected upregulations were confirmed by immunohistochemistry and quantitative polymerase chain reaction. We found NAV3 amplifications to dominate in neuronally differentiated tumors, whereas glial tumors showed almost equal proportions of NAV3 deletion and amplification. However, Grade IV gliomas had more frequent NAV3 deletions than grades I–III gliomas. Silencing of NAV3 in glioma cell lines led to the upregulation of receptor genes associated with gonadotropin-releasing hormone and Jak-Stat signaling pathways. Kaplan–Meier analysis of the entire clinical tumor material showed association between NAV3 amplifications and favorable prognosis, as well as NAV3 deletions and unfavorable prognosis. With Cox regression model, a hazard ratio of 0.51 was observed for NAV3 amplifications and 1.36 for NAV3 deletions. We conclude that NAV3 may be a potential new prognostic biomarker and a potential therapeutic target. © 2012 Wiley Periodicals, Inc.

The neuron navigator 3 (NAV3) gene on chromosome 12q21 encodes a microtubule plus end tracking protein and belongs to the navigator family of cytoskeletal regulators. Loss of heterozygosity on 12q has previously been suggested to be associated with poor prognosis in cancers of epithelial origin. In this study, we characterized copy number changes of NAV3 in 24 basal cell cancers (BCCs), eight squamous cell cancers (SCCs) and eight non-malignant inflammatory skin lesions by fluorescent in situ hybridization. To identify genes affected by NAV3, we used oligo siRNA gene silencing and gene microarrays to analyse gene expression profiles at several time points post-transfection in primary human keratinocytes. We found NAV3 copy number loss and decreased protein expression in 21% of the BCCs and 25% of the SCCs. In the nodular/superficial BCC subgroup, low-level NAV3 amplification was also observed. NAV3 aberrations were independent of the known chromosome 6 amplifications in BCC. Chromosome 12 polysomy was detected in 33% and 25% of the invasive type of BCC and SCC, respectively. Silencing of NAV3 in primary human keratinocytes revealed 22 differentially expressed genes, mostly related to inflammation. The most relevant of these were validated with qPCR or immunohistochemistry. This pilot study suggests that NAV3 is a novel cancer-associated gene that contributes to the pathogenesis of a subgroup of BCC and SCC.

Aims Subcutaneous panniculitis-like T cell lymphomas represent a rare and difficult to diagnose entity of cutaneous T cell lymphomas. SPTL affects predominantly young adults and presents with multifocal subcutaneous nodules and frequently associated autoimmune features. The pathogenesis of SPTL is not completely understood. The aim of this study is to unravel molecular pathways critical to the pathogenesis for the first time. Methods We analyzed 23 skin samples from 20 newly diagnosed SPTL patients and relevant control samples of adipose and non-malignant panniculitis tissue by using gene expression microarray, quantitative PCR, and two-colour immunohistochemistry. Results Interestingly, indoleamine 2,3-dioxygenase ( IDO-1 ), an immunotolerance-inducing enzyme, was among the most highly overexpressed genes in all comparisons. The expression of Th1-specific cytokines, known to be associated with autoimmune inflammation (i.e. IFNG, CXCR3, CXCL9, CXCL10, CXCL11, and CCL5 ), were also significantly increased. Confirmed with immunohistochemistry, the morphologically malignant lymphocytes expressed CXCR3 and CXCL9. IDO-1 expression was found both in some morphologically malignant lymphocytes rimming the adipocytes and in surrounding CD11c − CD68 − cells but not in CD11c + dendritic cells in the microenvironment. The proportion of FoxP3 + cells in SPTL exceeded that in the panniculitis samples. Conclusions Our results indicate that the upregulation of the tolerogenic IDO-1 together with the upregulation of IFNG, CXCR3 ligands, and CCL5 features SPTL lesions. We anticipate that the IFNG-inducible IDO-1 expression contributes to the formation of an immunosuppressive microenvironment, favourable for the malignant T cells. This study provides a relevant molecular basis for further studies exploring novel therapeutic means for subcutaneous T cell lymphoma.

MtDNA sequence variation is presumed to be neutral in effect, but associations with diseases and mtDNA haplogroups have been reported. The aim here was to evaluate the functional consequences of m.4216T>C present in haplogroup J. Furthermore, we evaluated m.3866T>C in MT-ND1, a variant detected in a child belonging to haplogroup J and with an isolated complex I deficiency. Homologous substitutions were introduced into Escherichia coli. NADH dehydrogenase domain activity of NDH-1 with either one or both mutations was markedly decreased suggesting that m.4216T>C and m.3866T>C may have an effect on the structural integrity of complex I.

Seven of the 45 subunits of mitochondrial NADH:ubiquinone oxidoreductase (complex I) are mitochondrially encoded and have been shown to harbor pathogenic mutations. We modeled the human disease-associated mutations A4136G/ND1-Y277C, T4160C/ND1-L285P and C4171A/ND1-L289M in a highly conserved region of the fourth matrix-side loop of the ND1 subunit by mutating homologous amino acids and surrounding conserved residues of the NuoH subunit of Escherichia coli NDH-1. Deamino-NADH dehydrogenase activity, decylubiquinone reduction kinetics, hexammineruthenium (HAR) reductase activity, and the proton pumping efficiency of the enzyme were assayed in cytoplasmic membrane preparations. Among the human disease-associated mutations, a statistically significant 22% decrease in enzyme activity was observed in the NuoH-L289C mutant and a 29% decrease in the double mutant NuoH-L289C/V297P compared with controls. The adjacent mutations NuoH-D295A and NuoH-R293M caused 49% and 39% decreases in enzyme activity, respectively. None of the mutations studied significantly affected the K(m) value of the enzyme for decylubiquinone or the amount of membrane-associated NDH-1 as estimated from the HAR reductase activity. In spite of the decrease in enzyme activity, all the mutant strains were able to grow on malate, which necessitates sufficient NDH-1 activity. The results show that in ND1/NuoH its fourth matrix-side loop is probably not directly involved in ubiquinone binding or proton pumping but has a role in modifying enzyme activity.

Low il-2 concentration favors generation of early memory t cells over terminal eefectors during car t-cell expansion