108P Wpływ transferu genów glikozylotransferazy na immunogeniczność czerniaka złośliwehgo u myszy

Glikozylotransferazy są swoistymi enzymami, biorącymi udział w glikozylacji białek wydzielniczych i powierzchniowych komórki. Ich niedobór lub brak wpływa na strukturę powierzchniowych antygenów i immunogenność komórki. Udoskonalenie metod transferu genów i uzyskiwania ich właściwej ekspresji umożliwiło wprowadzenie genów glikozylotransferaz do komórki.Komórki nowotworowe, pomimo ekspresji na swej powierzchni antygenów rozpoznawanych jako obce, nie ulegają eliminacji. Uważa się, że obniżona immunogenność transfoemowanych komórek wpływa na rozwój choroby nowotworowej.Cel pracyOkreślenie kinetyki wzrostu guza i czasu przeżycia myszy C57CL6/C3H, którym podskórnie podano komórki mysiego czerniaka (B78-H1) modyfikowane poprzez wprowadzenie genu (β1,4 galctosylotransferazy.Materiał i metodyKomórki słabo immunogennej linii mysiego czerniaka (B78-H1) transdukowano β1,4 galctosylotransferazą przy użyciu wektora retrowirusowego podwójnej kopii (DCCMV- β1.4GT). Poziom ekspresji mRNA dla 1.4 galaktozylotransferazy oceniano, wykorzystując swoiste 1) znakowane sondy cDNA (Northern Blott) oraz 2) primery (RT-PCR).Wyjściowe (B78-H1) i modyfikowane genetycznie (B78-1,4GT) komórki mysiego czerniaka wstrzykiwano s.c. 8 tygodniowym myszom C57BL6/C3H (5×105komórek). Analizowano dynamikę wzrostu guza, zdolność formowania przerzutów oraz czas przeżycia zwierząt.WynikiWszystkie zwierzęta, którym podano wyjściowe komórki B78-H1 (grupa kontrolna) rozwinęły guz po 3 tygodniach. U myszy, którym zaszczepiono komórki B78-1.4GT guzy pojawiły się 2 tygodnie później. Analiza średniego czasu przeżycia zwierząt wykazała, że zwierzęta zaszczepione komórkami B78-1.4GT przeżywały dłużej w porównaniu z grupą kontrolną

Effect of irradiation on interleukin 6 and soluble interleukin 6 receptor modified melanoma genetic vaccine

We have designed phase I/II human melanoma gene therapy clinical protocol. The aim of the study was to actively immunize HLA-A1 and/or HLA-A2-positive patients with melanoma with an admixture of irradiated autologous tumor cells and allogeneic melanoma cells genetically engineered to secrete IL-6 and sIL-6R in order to elicit or enhance specific and nonspecific anti-melanoma immune responses to autologous tumor cells to eradicate distant melanoma lesions. Irradiation of autologous and allogeneic tumor cells is a key step in preparation of cellular vaccine because of two major reasons, (i) it inhibits cell proliferation which is crucial in the case of autologous cells which may form a tumor; (ii) it increases melanoma vaccine immunogenicity. The aim of the study was to estimate the optimal dose of ionizing radiation which will provide sterilization of both autologous and allogeneic melanoma cells and will ensure cytokine secretion.Human melanoma cells (Mich-1) were transduced with IL-6 and sIL-6R cDNA using double copy bicistronic retroviral vector. Parental and transduced cells were seeded at in six-well tissue culture plates and were irradiated with 10, 50, 100 and 200 Gy. Secretion of both recombinant proteins into culture was analyzed before and 24, 48, 72, 96 h and 6, 7, 10 and 12 days following irradiation. At the same time adherent cells were enumerated, evaluated for viability and proliferation. At 24, 48, 72 and 96 h postirradiation specific IL-6 and sIL-6R mRNA levels were analyzed.Irradiation of gene modified cells inhibited their proliferation in the dose dependant manner. Dose of 50 Gy sufficiently affected cell proliferation, however, for safety reasons we decided to use the dose of 100 Gy for vaccine preparation. Irradiation did not inhibit secretion of IL-6 and sIL-6R. In contrary, on a per cell basis it significantly increased their secretion which lasted 12 days postirradiation. Interestingly, we did not observe dose or time dependent differences in specific mRNA cellular levels suggesting that increased secretion of both proteins is regulated not on the transcriptional but rather on the posttranscriptional level. Taking all these facts into account we concluded that irradiation of tumor cells may provide an effective and safe approach for gene-modified vaccine preparation

Effect of irradiation on interleukin 6 and soluble interleukin 6 receptor modified melanoma genetic vaccine

We have designed phase I/II human melanoma gene therapy clinical protocol. The aim of the study was to actively immunize HLA-A1 and/or HLA-A2-positive patients with melanoma with an admixture of irradiated autologous tumor cells and allogeneic melanoma cells genetically engineered to secrete IL-6 and sIL-6R in order to elicit or enhance specific and nonspecific antimelanoma immune responses to autologous tumor cells to eradicate distant melanoma lesions. Irradiation of autologous and allogeneic tumor cells is a key step in preparation of cellular vaccine because of two major reasons, (i) it inhibits cell proliferation which is crucial in the case of autologous cells which may form a tumor; (ii) it increases melanoma vaccine immunogenicity. The aim of the study was to estimate the optimal dose of ionizing radiation which will provide sterilization of both autologous and allogeneic melanoma cells and will ensure cytokine secretion.Human melanoma cells (Mich-1) were transduced with IL-6 and sIL-6R cDNA using double copy bicistronic retroviral vector. Parental and transduced cells were seeded at in six-well tissue culture plates and were irradiated with 10, 50, 100 and 200 Gy. Secretion of both recombinant proteins into culture was analyzed before and 24, 48,72,96 h and 6, 7, 10 and 12 days following irradiation. At the same time adherent cells were enumerated, evaluated’ for viability and proliferation. At 24, 48, 72 and 96 h postirradiation specific IL-6 and sIL-6R mRNA levels were analyzed.Irradiation of gene modified cells inhibited their proliferation in the dose dependant manner. Dose of 50 Gy sufficiently affected cell proliferation, however, for safety reasons we decided to use the dose of 100 Gy for vaccine preparation. Irradiation did not inhibit secretion of IL-6 and sIL-6R. In contrary, on a per cell basis it significantly increased their secretion which lasted 12 days postirradiation. Interestingly, we did not observe dose or time dependent differences in specific mRNA cellular levels suggesting that increased secretion of both proteins is regulated not on the transcriptional but rather on the posttranscriptional level. Taking all these facts into account we concluded that irradiation of tumor cells may provide an effective and safe approach for gene-modified vaccine preparation

The immunopeptidome landscape associated with T cell infiltration, inflammation and immune editing in lung cancer.

One key barrier to improving efficacy of personalized cancer immunotherapies that are dependent on the tumor antigenic landscape remains patient stratification. Although patients with CD3 <sup>+</sup> CD8 <sup>+</sup> T cell-inflamed tumors typically show better response to immune checkpoint inhibitors, it is still unknown whether the immunopeptidome repertoire presented in highly inflamed and noninflamed tumors is substantially different. We surveyed 61 tumor regions and adjacent nonmalignant lung tissues from 8 patients with lung cancer and performed deep antigen discovery combining immunopeptidomics, genomics, bulk and spatial transcriptomics, and explored the heterogeneous expression and presentation of tumor (neo)antigens. In the present study, we associated diverse immune cell populations with the immunopeptidome and found a relatively higher frequency of predicted neoantigens located within HLA-I presentation hotspots in CD3 <sup>+</sup> CD8 <sup>+</sup> T cell-excluded tumors. We associated such neoantigens with immune recognition, supporting their involvement in immune editing. This could have implications for the choice of combination therapies tailored to the patient's mutanome and immune microenvironment

Poly(ADP-ribose) polymerase family member 14 (PARP14) is a novel effector of the JNK2-dependent pro-survival signal in multiple myeloma

Copyright @ 2013 Macmillan Publishers Limited. This is the author's accepted manuscript. The final published article is available from the link below.Regulation of cell survival is a key part of the pathogenesis of multiple myeloma (MM). Jun N-terminal kinase (JNK) signaling has been implicated in MM pathogenesis, but its function is unclear. To elucidate the role of JNK in MM, we evaluated the specific functions of the two major JNK proteins, JNK1 and JNK2. We show here that JNK2 is constitutively activated in a panel of MM cell lines and primary tumors. Using loss-of-function studies, we demonstrate that JNK2 is required for the survival of myeloma cells and constitutively suppresses JNK1-mediated apoptosis by affecting expression of poly(ADP-ribose) polymerase (PARP)14, a key regulator of B-cell survival. Strikingly, we found that PARP14 is highly expressed in myeloma plasma cells and associated with disease progression and poor survival. Overexpression of PARP14 completely rescued myeloma cells from apoptosis induced by JNK2 knockdown, indicating that PARP14 is critically involved in JNK2-dependent survival. Mechanistically, PARP14 was found to promote the survival of myeloma cells by binding and inhibiting JNK1. Moreover, inhibition of PARP14 enhances the sensitization of MM cells to anti-myeloma agents. Our findings reveal a novel regulatory pathway in myeloma cells through which JNK2 signals cell survival via PARP14, and identify PARP14 as a potential therapeutic target in myeloma.Kay Kendall Leukemia Fund, NIH, Cancer Research UK, Italian Association for Cancer Research and the Foundation for Liver Research

Modeling recursive RNA interference.

An important application of the RNA interference (RNAi) pathway is its use as a small RNA-based regulatory system commonly exploited to suppress expression of target genes to test their function in vivo. In several published experiments, RNAi has been used to inactivate components of the RNAi pathway itself, a procedure termed recursive RNAi in this report. The theoretical basis of recursive RNAi is unclear since the procedure could potentially be self-defeating, and in practice the effectiveness of recursive RNAi in published experiments is highly variable. A mathematical model for recursive RNAi was developed and used to investigate the range of conditions under which the procedure should be effective. The model predicts that the effectiveness of recursive RNAi is strongly dependent on the efficacy of RNAi at knocking down target gene expression. This efficacy is known to vary highly between different cell types, and comparison of the model predictions to published experimental data suggests that variation in RNAi efficacy may be the main cause of discrepancies between published recursive RNAi experiments in different organisms. The model suggests potential ways to optimize the effectiveness of recursive RNAi both for screening of RNAi components as well as for improved temporal control of gene expression in switch off-switch on experiments

Specific Marking of hESCs-Derived Hematopoietic Lineage by WAS-Promoter Driven Lentiviral Vectors

Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in hESCs-derived hematopoietic cells. We first demonstrated that endogenous WAS gene was not expressed in undifferentiated hESCs but was evident in hemogenic progenitors (CD45−CD31+CD34+) and hematopoietic cells (CD45+). Accordingly, WAS-promoter driven LVs were unable to express the eGFP transgene in undifferentiated hESCs. eGFP+ cells only appeared after embryoid body (EB) hematopoietic differentiation. The phenotypic analysis of the eGFP+ cells showed marking of different subpopulations at different days of differentiation. At days 10–15, AWE LVs tag hemogenic and hematopoietic progenitors cells (CD45−CD31+CD34dim and CD45+CD31+CD34dim) emerging from hESCs and at day 22 its expression became restricted to mature hematopoietic cells (CD45+CD33+). Surprisingly, at day 10 of differentiation, the AWE vector also marked CD45−CD31low/−CD34− cells, a population that disappeared at later stages of differentiation. We showed that the eGFP+CD45−CD31+ population generate 5 times more CD45+ cells than the eGFP−CD45−CD31+ indicating that the AWE vector was identifying a subpopulation inside the CD45−CD31+ cells with higher hemogenic capacity. We also showed generation of CD45+ cells from the eGFP+CD45−CD31low/−CD34− population but not from the eGFP−CD45−CD31low/−CD34− cells. This is, to our knowledge, the first report of a gene transfer vector which specifically labels hemogenic progenitors and hematopoietic cells emerging from hESCs. We propose the use of WAS-promoter driven LVs as a novel tool to studying human hematopoietic development

A Distinct DNA Methylation Shift in a Subset of Glioma CpG Island Methylator Phenotypes during Tumor Recurrence

Glioma diagnosis is based on histomorphology and grading; however, such classification does not have predictive clinical outcome after glioblastomas have developed. To date, no bona fide biomarkers that significantly translate into a survival benefit to glioblastoma patients have been identified. We previously reported that the IDH mutant G-CIMP-high subtype would be a predecessor to the G-CIMP-low subtype. Here, we performed a comprehensive DNA methylation longitudinal analysis of diffuse gliomas from 77 patients (200 tumors) to enlighten the epigenome-based malignant transformation of initially lower-grade gliomas. Intra-subtype heterogeneity among G-CIMP-high primary tumors allowed us to identify predictive biomarkers for assessing the risk of malignant recurrence at early stages of disease. G-CIMP-low recurrence appeared in 9.5% of all gliomas, and these resembled IDH-wild-type primary glioblastoma. G-CIMP-low recurrence can be characterized by distinct epigenetic changes at candidate functional tissue enhancers with AP-1/SOX binding elements, mesenchymal stem cell-like epigenomic phenotype, and genomic instability. Molecular abnormalities of longitudinal G-CIMP offer possibilities to defy glioblastoma progression

An Integrated TCGA Pan-Cancer Clinical Data Resource to Drive High-Quality Survival Outcome Analytics

For a decade, The Cancer Genome Atlas (TCGA) program collected clinicopathologic annotation data along with multi-platform molecular profiles of more than 11,000 human tumors across 33 different cancer types. TCGA clinical data contain key features representing the democratized nature of the data collection process. To ensure proper use of this large clinical dataset associated with genomic features, we developed a standardized dataset named the TCGA Pan-Cancer Clinical Data Resource (TCGA-CDR), which includes four major clinical outcome endpoints. In addition to detailing major challenges and statistical limitations encountered during the effort of integrating the acquired clinical data, we present a summary that includes endpoint usage recommendations for each cancer type. These TCGA-CDR findings appear to be consistent with cancer genomics studies independent of the TCGA effort and provide opportunities for investigating cancer biology using clinical correlates at an unprecedented scale. Analysis of clinicopathologic annotations for over 11,000 cancer patients in the TCGA program leads to the generation of TCGA Clinical Data Resource, which provides recommendations of clinical outcome endpoint usage for 33 cancer types