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KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) is highly immunosuppressive and resistant to targeted and immunotherapies. Among the different PDAC subtypes, basal-like mesenchymal PDAC, which is driven by allelic imbalance, increased gene dosage and subsequent high expression levels of oncogenic KRAS, shows the most aggressive phenotype and strongest therapy resistance. In the present study, we performed a systematic high-throughput combination drug screen and identified a synergistic interaction between the MEK inhibitor trametinib and the multi-kinase inhibitor nintedanib, which targets KRAS-directed oncogenic signaling in mesenchymal PDAC. This combination treatment induces cell-cycle arrest and cell death, and initiates a context-dependent remodeling of the immunosuppressive cancer cell secretome. Using a combination of single-cell RNA-sequencing, CRISPR screens and immunophenotyping, we show that this combination therapy promotes intratumor infiltration of cytotoxic and effector T cells, which sensitizes mesenchymal PDAC to PD-L1 immune checkpoint inhibition. Overall, our results open new avenues to target this aggressive and therapy-refractory mesenchymal PDAC subtype.

T cell receptor (TCR)-based immunotherapies have shown great potential for the safe and efficacious treatment of patients suffering from various solid malignancies. However, novel strategies are needed to overcome the immunosuppressive tumor microenvironment (TME) and to enhance the efficacy of TCR-T cell (TCR-T) therapies in solid tumors. NY-ESO-1 is a cancer/testis antigen that is highly expressed in various solid tumor indications while its expression in healthy tissues is restricted to male germ cells. Our HLA-A2-restricted, NY-ESO-1-specific TCR used here was selected from a non-tolerized T cell repertoire of a healthy donor using Medigene’s proprietary Allo-HLA TCR priming technology and high-throughput TCR isolation and characterization processes. To prevent inhibition of TCR-T cell function via the PD1-PDL1 axis within the TME, the NY-ESO-1-TCR was co-expressed with a chimeric PD1-41BB co-stimulatory switch receptor, consisting of the extracellular domain of PD1 and the intracellular domain of the 41BB co-stimulatory receptor. Thereby, an inhibitory signal from the PD1-PD-L1 axis is turned into a co-stimulatory one, which results in a dual mechanistic benefit, and superior TCR-T cells with respect to tumor cell killing, cell proliferation and poly-cytokine secretion. Co-expression of PD1-41BB on NY-ESO-1 TCR-T cells led to a 3-fold increase in IFN-γ release and a higher proliferation index (~1.5 fold) (after antigen encounter. Under conditions of repeated antigen exposure in vitro using HLA-A2/NY-ESO-1/PDL1-positive tumor spheroids, TCR-T cells co-expressing PD1-41BB showed enhanced functional activities compared to TCR-T cells carrying the TCR alone. In vitro single-cell secretome analyses demonstrated up to a 5-fold increase in polyfunctional T cells and a higher polyfunctional strength index (PSI) for NY-ESO-1 TCR-T cells expressing PD1-41BB compared to “naked” NY-ESO-1 TCR-T cells lacking PD1-41BB. Typical Th1 cytokines/proteins like IFN-γ, TNF-α and Granzyme B mainly contributed to the superior PSI of NY-ESO-1-TCR-T cells co-expressing PD1-41BB. Importantly, our data indicate that transgenic PD1-41BB has the capacity to provide a co-stimulatory signal despite the presence of endogenous PD1 which is upregulated in stimulated “naked“ NY-ESO-1 TCR-T cells. In summary, we find that co-expression of a chimeric PD1-41BB co-stimulatory switch receptor significantly enhances in vitro NY-ESO-1 TCR-T cell proliferation, functionality and anti-tumor activity. Analyses of single-cell T cell cytokine polyfunctionality helped us to unravel the mode of action of NY-ESO-1 TCR-T cells co-expressing PD1-41BB and point to their complex display of effector, stimulatory and chemoattractant cytokines as a potential driver for superior recognition of tumor cells. Citation Format: Andrea Coluccio, Stefanie Tippmer, Petra Prinz, Maja Buerdek, Kathrin Mutze, Barbara Loesch, Kathrin Davari, Giulia Longinotti, Dolores J. Schendel. T cells co-expressing a highly potent NY-ESO-1-specific TCR and a chimeric PD1-41BB co-stimulatory switch receptor show a favorable polyfunctional profile for the treatment of solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4057.

The laboratory mouse ranks among the most important experimental systems for biomedical research and molecular reference maps of such models are essential informational tools. Here, we present a quantitative draft of the mouse proteome and phosphoproteome constructed from 41 healthy tissues and several lines of analyses exemplify which insights can be gleaned from the data. For instance, tissue- and cell-type resolved profiles provide protein evidence for the expression of 17,000 genes, thousands of isoforms and 50,000 phosphorylation sites in vivo. Proteogenomic comparison of mouse, human and Arabidopsis reveal common and distinct mechanisms of gene expression regulation and, despite many similarities, numerous differentially abundant orthologs that likely serve species-specific functions. We leverage the mouse proteome by integrating phenotypic drug (n > 400) and radiation response data with the proteomes of 66 pancreatic ductal adenocarcinoma (PDAC) cell lines to reveal molecular markers for sensitivity and resistance. This unique atlas complements other molecular resources for the mouse and can be explored online via ProteomicsDB and PACiFIC.

Most modern CPU architectures are based on the von Neumann principle, where memory and processing units are separate entities. Although processing unit performance has improved over the years, memory capacity has not followed the same trend, creating a performance gap between them. This problem is known as the "memory wall" and severely limits the performance of a microprocessor. One of the most promising solutions is the "logic-in-memory" approach. It consists of merging memory and logic units, enabling data to be processed directly inside the memory itself. Here we propose an RISC-V framework that supports logic-in-memory operations. We substitute data memory with a circuit capable of storing data and of performing in-memory computation. The framework is based on a standard memory interface, so different logic-in-memory architectures can be inserted inside the microprocessor, based both on CMOS and emerging technologies. The main advantage of this framework is the possibility of comparing the performance of different logic-in-memory solutions on code execution. We demonstrate the effectiveness of the framework using a CMOS volatile memory and a memory based on a new emerging technology, racetrack logic. The results demonstrate an improvement in algorithm execution speed and a reduction in energy consumption.

Aberrant activation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR and Ras/mitogen-activated protein kinase (MAPK) pathways is a hallmark of hepatocarcinogenesis. In a subset of hepatocellular carcinomas (HCCs), PI3K/AKT/mTOR signaling dysregulation depends on phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations, while RAS/MAPK activation is partly attributed to promoter methylation of the tumor suppressor Ras association domain-containing protein 1 (RASSF1A). To evaluate a possible cocarcinogenic effect of PIK3CA activation and RASSF1A knockout, plasmids expressing oncogenic forms of PIK3CA (E545K or H1047R mutants) were delivered to the liver of RASSF1A knockout and wild-type mice by hydrodynamic tail vein injection combined with sleeping beauty-mediated somatic integration. Transfection of either PIK3CA E545K or H1047R mutants sufficed to induce HCCs in mice irrespective of RASSF1A mutational background. The related tumors displayed a lipogenic phenotype with upregulation of fatty acid synthase and stearoyl-CoA desaturase-1 (SCD1). Galectin-1, which was commonly upregulated in preneoplastic lesions and tumors, emerged as a regulator of SCD1. Co-inhibitory treatment with PIK3CA inhibitors and the galectin-1 inhibitor OTX008 resulted in synergistic cytotoxicity in human HCC cell lines, suggesting novel therapeutic venues.

Background Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy marked by poor prognosis and profound drug resistance characterized in more than 90% of cases by KRAS mutations. To recapitulate central aspects of PDAC, we employed genetically engineered mouse models presenting KrasG12D pancreas specific expression. Through a high-throughput combination drug screen with trametinib as backbone we identified a high synergism with the multikinase inhibitor nintedanib, preferentially in mesenchymal PDAC, a subtype of this disease characterized by poor prognosis and therapeutic resistance. This combinatorial treatment, that led to the induction of apoptosis in vitro and disease regression in vivo, was accompanied by a strong tumor infiltration of CD8 positive T cells. Materials and Methods To characterize the treatment-induced adaptive immune cell infiltration in vivo, we performed orthotopic transplantations of KRAS-driven murine PDAC cell lines presenting mesenchymal and epithelial morphology. The derived control and nintedanib + trametinib treated PDAC tumors were analyzed by multi-color immunofluorescence stainings. We compared the findings to high parameter flow cytometry results. Results Confocal microscopy of the immunofluorescence stainings revealed an overall increase of tumor-infiltrating lymphocytes (TIL) in the tumors upon combinatorial treatment with substantial differences in quantity and spatial distribution. Tumors derived from a PDAC cell line of epithelial morphology were characterized by few TIL mainly located at the invasive margins of the tumors, while tumors derived from a mesenchymal PDAC cell line showed a strong increase of TIL even in the center of the tumor mass. Furthermore, an increased ratio of CD8 positive cytotoxic T cells to CD4 positive helper T cells as well as a decrease of Foxp3 and CD4 positive regulatory T cells could be observed for tumors derived from the mesenchymal PDAC cell line under combinatorial treatment. To investigate if the observed recruitment of T cells was indispensable for treatment efficacy of the combinatorial therapy, we orthotopically transplanted the mesenchymal PDAC cell line in immunodeficient CD3-Knockout (CD3ko) mice and applied an analogous combinatorial treatment scheme. In the CD3ko mice, the combinatorial treatment did not lead to an increased survival or tumor regression as observed in immunocompetent mice. However, flow cytometry and immunofluorescence stainings revealed an increase of B cells upon nintedanib + trametinib treatment. Conclusions Our findings indicate a reduced efficacy of the combinatorial treatment in T cell deficient mice, underlining the importance of T cells in treatment-induced anti-tumor responses and enlarging the understanding of the role of TIL in PDAC. Disclosure Information J. Heetmeyer: None. C. Falcomata: None. S. Barthel: None. C. Schneeweis: None. A. Coluccio: None. C. Veltkamp: None. G. Schneider: None. D. Saur: None.

International audience; During cell division, maintenance of chromatin features from the parental genome requires their proper establishment on its newly synthetized copy. The loss of epigenetic marks within heterochromatin, typically enriched in repetitive elements, endangers genome stability and permits chromosomal rearrangements via recombination. However, how histone modifications associated with heterochromatin are maintained across mitosis remains poorly understood. KAP1 is known to act as a scaffold for a repressor complex that mediates local heterochromatin formation, and was previously demonstrated to play an important role during DNA repair. Accordingly, we investigated a putative role for this protein in the replication of heterochromatic regions. We first found that KAP1 associates with several DNA replication factors including PCNA, MCM3 and MCM6. We then observed that these interactions are promoted by KAP1 phosphorylation on serine 473 during S phase. Finally, we could demonstrate that KAP1 forms a complex with PCNA and the histone-lysine methyltransferase Suv39h1 to reinstate heterochromatin after DNA replication.

ObjectivePancreatic ductal adenocarcinoma (PDAC) still carries a dismal prognosis with an overall 5-year survival rate of 9%. Conventional combination chemotherapies are a clear advance in the treatment of PDAC; however, subtypes of the disease exist, which exhibit extensive resistance to such therapies. Genomic MYC amplifications represent a distinct subset of PDAC with an aggressive tumour biology. It is clear that hyperactivation of MYC generates dependencies that can be exploited therapeutically. The aim of the study was to find and to target MYC-associated dependencies.DesignWe analysed human PDAC gene expression datasets. Results were corroborated by the analysis of the small ubiquitin-like modifier (SUMO) pathway in a large PDAC cohort using immunohistochemistry. A SUMO inhibitor was used and characterised using human and murine two-dimensional, organoid and in vivo models of PDAC.ResultsWe observed that MYC is connected to the SUMOylation machinery in PDAC. Components of the SUMO pathway characterise a PDAC subtype with a dismal prognosis and we provide evidence that hyperactivation of MYC is connected to an increased sensitivity to pharmacological SUMO inhibition.ConclusionSUMO inhibitor-based therapies should be further developed for an aggressive PDAC subtype.

Krüppel‐associated box (KRAB)‐containing zinc finger proteins (KZFPs) are encoded in the hundreds by the genomes of higher vertebrates, and many act with the heterochromatin‐inducing KAP1 as repressors of transposable elements (TEs) during early embryogenesis. Yet, their widespread expression in adult tissues and enrichment at other genetic loci indicate additional roles. Here, we characterized the protein interactome of 101 of the ~350 human KZFPs. Consistent with their targeting of TEs, most KZFPs conserved up to placental mammals essentially recruit KAP1 and associated effectors. In contrast, a subset of more ancient KZFPs rather interacts with factors related to functions such as genome architecture or RNA processing. Nevertheless, KZFPs from coelacanth, our most distant KZFP‐encoding relative, bind the cognate KAP1. These results support a hypothetical model whereby KZFPs first emerged as TE‐controlling repressors, were continuously renewed by turnover of their hosts’ TE loads, and occasionally produced derivatives that escaped this evolutionary flushing by development and exaptation of novel functions.

KRAB-containing zinc finger proteins (KRAB-ZFPs) are early embryonic controllers of transposable elements (TEs), which they repress with their cofactor KAP1 through histone and DNA methylation, a process thought to result in irreversible silencing. Using a target-centered functional screen, we matched murine TEs with their cognate KRAB-ZFP. We found the paralogs ZFP932 and Gm15446 to bind overlapping but distinguishable subsets of ERVK (endogenous retrovirus K), to repress these elements in embryonic stem cells, and to regulate secondarily the expression of neighboring genes. Most importantly, we uncovered that these KRAB-ZFPs and KAP1 control TEs in adult tissues, in cell culture and in vivo, where they partner up to modulate cellular genes. Therefore, TEs and KRAB-ZFPs establish transcriptional networks that regulate not only development but probably many physiological events. Given the high degree of species-specificity of TEs and KRAB-ZFPs, these results have important implications for understanding the biology of higher vertebrates, including humans.

Despite recent improvements in treatment regimen such as combination chemotherapies, pancreatic ductal adenocarcinoma still carries a dismal prognosis with an overall survival rate of 9%. The oncogenic transcription factor MYC is amplified in about 12% of all PDAC, which is connected to a worse survival. It is clear that hyperactivation of MYC generates dependencies that can be exploited therapeutically. Therefore, the aim of the study was to find and to target MYC-associated dependencies. Human PDAC gene expression data sets were analyzed. Moreover, analysis of the SUMO pathway in large cohort of PDAC was performed by IHC. Furthermore, a novel, pharmacological SUMO inhibitor was used and characterized by using human and murine 2D-, organoid-, and in vivo-models of PDAC. In addition, this connection was also verified by the genetically modified human and murine models. Large-scale gene expression datasets of human PDACs analysis revealed a connection of MYC to the SUMOylation machinery in PDAC. Moreover, results were also corroborated by the hyperactivation of SUMO pathway in a large patient cohort which characterized a PDAC subtype with a dismal prognosis. We furthermore corroborated these results by using the novel SUMO inhibitor ML093 by in vitro studies that hyperactivation of MYC is connected to an increased sensitivity to pharmacological SUMO inhibition. In conclusion, SUMO inhibitor-based therapies should be further developed for an aggressive PDAC subtype. Citation Format: Zonera Hassan, Alex Biederstädt, Christian Schneeweis, Markus Schick, Nilsson Jonas, Mathias Wirth, Andrea Coluccio, Felix Orben, Katja Steiger, Jolanta Slawska, Steve Langston, Dieter Saur, Roland Rad, Maximilian Reichert, Guenter Schneider, Ulrich Keller. Interaction of MYC and SUMOylation machinery in an aggressive pancreatic cancer subtype [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1793.

Mobile elements are important evolutionary forces that challenge genomic integrity. Long interspersed element-1 (L1, also known as LINE-1) is the only autonomous transposon still active in the human genome. It displays an unusual pattern of evolution, with, at any given time, a single active L1 lineage amplifying to thousands of copies before getting replaced by a new lineage, likely under pressure of host restriction factors, which act notably by silencing L1 expression during early embryogenesis. Here, we demonstrate that in human embryonic stem (hES) cells, KAP1 (KRAB [Krüppel-associated box domain]-associated protein 1), the master cofactor of KRAB-containing zinc finger proteins (KRAB-ZFPs) previously implicated in the restriction of endogenous retroviruses, represses a discrete subset of L1 lineages predicted to have entered the ancestral genome between 26.8 million and 7.6 million years ago. In mice, we documented a similar chronologically conditioned pattern, albeit with a much contracted time scale. We could further identify an L1-binding KRAB-ZFP, suggesting that this rapidly evolving protein family is more globally responsible for L1 recognition. KAP1 knockdown in hES cells induced the expression of KAP1-bound L1 elements, but their younger, human-specific counterparts (L1Hs) were unaffected. Instead, they were stimulated by depleting DNA methyltransferases, consistent with recent evidence demonstrating that the PIWI–piRNA (PIWI-interacting RNA) pathway regulates L1Hs in hES cells. Altogether, these data indicate that the early embryonic control of L1 is an evolutionarily dynamic process and support a model in which newly emerged lineages are first suppressed by DNA methylation-inducing small RNA-based mechanisms before KAP1-recruiting protein repressors are selected.

et al., This work was supported by grants from the European Commission (FP7, PERSIST), the European Research Council (ERC-2010-AdG, GT-SKIN), and the Italian Ministry of University and Research (FIRB 2008). F.L. is supported by grants PI11/0125 from Spanish ISCIII and grant P2010BMD-2359 from Comunidad de Madrid. M.D.R. is supported by grants SAF2010-16976 from MICINN and P2010BMD-2420 from Comunidad de Madrid.

In animal embryos, transcription is mostly silent for several cell divisions, until the release of the first major wave of embryonic transcripts through so-called zygotic genome activation (ZGA). Maternally provided ZGA-triggering factors have been identified in Drosophila melanogaster and Danio rerio, but their mammalian homologs are still undefined. Here, we provide evidence that the DUX family of transcription factors is essential to this process in mice and potentially in humans. First, human DUX4 and mouse Dux are both expressed before ZGA in their respective species. Second, both orthologous proteins bind the promoters of ZGA-associated genes and activate their transcription. Third, Dux knockout in mouse embryonic stem cells (mESCs) prevents the cells from cycling through a 2-cell-like state. Finally, zygotic depletion of Dux leads to impaired early embryonic development and defective ZGA. We conclude that DUX-family proteins are key inducers of zygotic genome activation in placental mammals.