Franziska Haderk

CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR-17-5p, and miR-20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk.

Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts

Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected α-smooth actin-positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts.

Obstacles and opportunities in the functional analysis of extracellular vesicle RNA – an ISEV position paper

ABSTRACT The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge – of the nature of EV(-RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data.

Tumor-derived exosomes modulate PD-L1 expression in monocytes.

Transfer of exosomal RNA from leukemic cells to monocytes induces immunosuppression. Messaging with RNAs Understanding interactions between tumor cells and immune cells is essential for tailoring immunocentric therapies to tumors. Here, Haderk et al. have identified a key role for tumor-derived exosomes in modulating immune responses to chronic lymphocytic leukemia (CLL). They report that CLL-derived exosomal RNAs promote monocytes in CLL patients to adopt an immunosuppressive phenotype, including promoting expression of PD-L1. They identify noncoding RNA hY4 as a key functional component of CLL-derived exosomes and show that hY4 promotes exosome-dependent skewing of monocytes in a TLR7-dependent manner. Using mouse models, they found that inhibition of TLR7 delayed progression of CLL, opening up the possibility that the TLR7 pathway could be therapeutically targeted in CLL. In chronic lymphocytic leukemia (CLL), monocytes and macrophages are skewed toward protumorigenic phenotypes, including the release of tumor-supportive cytokines and the expression of immunosuppressive molecules such as programmed cell death 1 ligand 1 (PD-L1). To understand the mechanism driving protumorigenic skewing in CLL, we evaluated the role of tumor cell–derived exosomes in the cross-talk with monocytes. We carried out RNA sequencing and proteome analyses of CLL-derived exosomes and identified noncoding Y RNA hY4 as a highly abundant RNA species that is enriched in exosomes from plasma of CLL patients compared with healthy donor samples. Transfer of CLL-derived exosomes or hY4 alone to monocytes resulted in key CLL-associated phenotypes, including the release of cytokines, such as C-C motif chemokine ligand 2 (CCL2), CCL4, and interleukin-6, and the expression of PD-L1. These responses were abolished in Toll-like receptor 7 (TLR7)–deficient monocytes, suggesting exosomal hY4 as a driver of TLR7 signaling. Pharmacologic inhibition of endosomal TLR signaling resulted in a substantially reduced activation of monocytes in vitro and attenuated CLL development in vivo. Our results indicate that exosome-mediated transfer of noncoding RNAs to monocytes contributes to cancer-related inflammation and concurrent immune escape via PD-L1 expression.

Extracellular vesicles in chronic lymphocytic leukemia.

Abstract Extracellular vesicles (EVs) are membrane-enclosed nanoparticles 30 to 1000 nm in size and represent a novel mechanism of cell communication. By transferring RNA and protein from their cell of origin, they can reprogram target cells and thus are involved in changes within the cellular microenvironment – a key player in CLL pathogenesis. In the current study, we were able to isolate EVs of 20 to 300 nm from blood plasma of CLL patients as well as from supernatant of primary CLL cells in culture. Further, proteomic profiling by Coomassie staining of SDS-PAGE gels and by mass spectrometry revealed an EV-specific protein profile. These findings suggest that EVs represent an important mean of CLL cells to interact with other cells, which might contribute to the establishment of a pro-survival microenvironment for CLL cells.

Efficacy of SHP2 phosphatase inhibition in cancers with nucleotide-cycling oncogenic RAS, RAS-GTP dependent oncogenic BRAF and NF1 loss

Oncogenic alterations in the RAS-RAF-MEK-ERK pathway, including mutant forms of KRAS, BRAF, and loss of the tumor suppressor and RAS GTPase-activating protein (GAP) NF1, drive the growth of a wide spectrum of human cancers. While BRAF and MEK inhibitors are effective in many patients with oncogenic BRAF V600E, there are no effective targeted therapies for individuals with cancers driven by other pathway alterations, including oncogenic KRAS, non-V600E BRAF, and NF1 loss. Here, we show that targeting the PTPN11/SHP2 phosphatase with a novel small molecule allosteric inhibitor is effective against cancers bearing nucleotide-cycling oncogenic RAS (e.g. KRAS G12C), RAS-GTP dependent oncogenic BRAF (e.g. class 3 BRAF mutants), or NF1 loss in multiple preclinical models in vitro and in vivo. SHP2 inhibition suppressed the levels of RAS-GTP and phosphorylated ERK in these models and induced growth inhibition. Expression of a constitutively active mutant of the RAS guanine nucleotide exchange factor (GEF) SOS1 rescued cells from the effects of SHP2 inhibition, suggesting that SHP2 blockade decreases oncogenic RAS-RAF-MEK-ERK signaling by disrupting SOS1-mediated RAS-GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS activation and downstream signaling in cancers with nucleotide-cycling oncogenic RAS, RAS-GTP dependent oncogenic BRAF, and NF1 loss. SHP2 inhibition thus represents a rational, biomarker-driven therapeutic strategy to be tested in patients with cancers of diverse origins bearing these oncogenic drivers and for which current treatments are largely ineffective.

RAS nucleotide cycling underlies the SHP2 phosphatase dependence of mutant BRAF-, NF1- and RAS-driven cancers

Oncogenic alterations in the RAS/RAF/MEK/ERK pathway drive the growth of a wide spectrum of cancers. While BRAF and MEK inhibitors are efficacious against BRAFV600E-driven cancers, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including non-V600E oncogenic BRAF, RAS GTPase-activating protein (GAP) NF1 (neurofibromin 1) loss and oncogenic KRAS. Here, we show that targeting the SHP2 phosphatase (encoded by PTPN11) with RMC-4550, a small-molecule allosteric inhibitor, is effective in human cancer models bearing RAS–GTP-dependent oncogenic BRAF (for example, class 3 BRAF mutants), NF1 loss or nucleotide-cycling oncogenic RAS (for example, KRASG12C). SHP2 inhibitor treatment decreases oncogenic RAS/RAF/MEK/ERK signalling and cancer growth by disrupting SOS1-mediated RAS–GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS/MAPK pathway activation in cancers with RAS–GTP-dependent oncogenic BRAF, NF1 loss and nucleotide-cycling oncogenic KRAS. SHP2 inhibition is a promising molecular therapeutic strategy for patients with cancers bearing these oncogenic drivers.Nichols et al. identify an SHP2 inhibitor that disrupts SOS1-mediated RAS–GTP loading with demonstrated efficacy in various types of tumour driven by mutant BRAF, NF1 or RAS.

Non-Canonical Thinking for Targeting ALK-Fusion Onco-Proteins in Lung Cancer

Anaplastic lymphoma kinase (ALK) gene rearrangements have been identified in lung cancer at 3–7% frequency, thus representing an important subset of genetic lesions that drive oncogenesis in this disease. Despite the availability of multiple FDA-approved small molecule inhibitors targeting ALK fusion proteins, drug resistance to ALK kinase inhibitors is a common problem in clinic. Thus, there is an unmet need to deepen the current understanding of genomic characteristics of ALK rearrangements and to develop novel therapeutic strategies that can overcome ALK inhibitor resistance. In this review, we present the genomic landscape of ALK fusions in the context of co-occurring mutations with other cancer-related genes, pointing to the central role of genetic epistasis (gene-gene interactions) in ALK-driven advanced-stage lung cancer. We discuss the possibility of targeting druggable domains within ALK fusion partners in addition to available strategies inhibiting the ALK kinase domain directly. Finally, we examine the potential of targeting ALK fusion-specific neoantigens in combination with other treatments, a strategy that could open a new avenue for the improved treatment of ALK positive lung cancer patients.

Abstract A30: Chronic lymphocytic leukemia-derived extracellular vesicles mediate NFkB signaling and pro-inflammatory cytokine release in monocytes

The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Extracellular vesicles (EV) released by CLL cells are taken up by non-malignant cells in the microenvironment and mediate major disease-related changes in recipient cells. In the current study, we characterized CLL EVs, especially focusing on vesicle-incorporated RNA transcripts, and defined the role of CLL EVs in changing the myeloid tumor microenvironment. EVs were isolated from blood plasma of CLL patients and healthy donors as well as from supernatant of the CLL cell line MEC-1 by a serial centrifugation protocol. Characterization of EVs by electron microscopy, Nanoparticle Tracking Analysis (NTA) and Western blotting revealed vesicles 30 to 350 nm in size, which are positive for various EV marker proteins such as Rab5a and Hsp70. Quantification of blood plasma-derived EVs indicated an enrichment of B-cell derived EVs in plasma of CLL patients compared to healthy donors, although absolute EV counts were not altered in CLL. Focusing on RNA analysis, an enrichment of small RNAs in EVs was observed. Subsequent small RNA sequencing revealed a unique microRNA signature of MEC-1 EVs, with CLL-relevant miRNAs such as miR-21, miR-155 and miR-146a being the most abundant miRNAs. Moreover, full length and 59end fragment forms of Y RNAs, another class of small non-coding RNAs, were enriched in MEC-1 EVs and CLL plasma EVs. Further evaluating the functional relevance of CLL EVs within the tumor microenvironment, a rapid uptake of CLL cell-derived EVs by human monocytes and macrophages was observed. Uptake of CLL EVs in monocytes induced NFkB signaling and the release of multiple pro-inflammatory cytokines such as CCL2, CCL3, IL-6 and IL-8, which are also upregulated in plasma of CLL patients. In conclusion, tumor-derived EVs harbor a distinct set of non-coding RNAs. The uptake of EVs in recipient cells and the concomitant transfer of incorporated RNAs mediate substantial phenotypic changes in target cells. In the current study, this is exemplified for monocytes, which present several disease-relevant alterations upon EV uptake, including cellular activation and secretion of pro-inflammatory cytokines. Citation Format: Franziska Haderk, Laura Llao Cid, Etienne Moussay, Jerome Paggetti, Karolin Willmund, Jana Seiler, Sven Diederichs, Maria Goebel, Jan Duerig, Thorsten Zenz, Stephan Stilgenbauer, Marc Zapatka, Peter Lichter, Martina Seiffert. Chronic lymphocytic leukemia-derived extracellular vesicles mediate NFkB signaling and pro-inflammatory cytokine release in monocytes. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A30.