Publications by authors named "Anton G Henssen"

27 Publications

  • Page 1 of 1

CD28 Co-Stimulus Achieves Superior CAR T Cell Effector Function against Solid Tumors Than 4-1BB Co-Stimulus.

Cancers (Basel) 2021 Mar 2;13(5). Epub 2021 Mar 2.

Department of Pediatric Oncology and Hematology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 13353 Berlin, Germany.

Spacer or co-stimulatory components in chimeric antigen receptor (CAR) design influence CAR T cell effector function. Few preclinical mouse models optimally support CAR candidate pre-selection for clinical development. Here we use a model in which murine CAR T cells can be exploited with human tumor xenografts. This mouse-in-mouse approach avoids limitations caused by species-specific factors crucial for CAR T cell survival, trafficking and function. We compared trafficking, expansion and tumor control for T cells expressing different CAR construct designs targeting two antigens (L1CAM or HER2), structurally identical except for spacer (long or short) or co-stimulatory (4-1BB or CD28) domains to be evaluated. Using monoclonal, murine-derived L1CAM-specific CAR T cells in Rag-/- mice harboring established xenografted tumors from a human neuroblastoma cell line revealed a clear superiority in CAR T cell trafficking using CD28 co-stimulation. L1CAM-targeting short spacer-CD28/ζ CAR T cells expanded the most at the tumor site and induced initial tumor regression. Treating patient-derived neuroblastoma xenografts with human L1CAM-targeting CAR T cells confirmed the superiority of CD28 co-stimulus. CD28 superiority was also demonstrated with HER2-specific CAR T cells (targeting ovarian carcinoma xenografts). Our findings encourage incorporating CD28 signaling into CAR design for adoptive T cell treatment of solid tumors.
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http://dx.doi.org/10.3390/cancers13051050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7958604PMC
March 2021

Combined inhibition of Aurora-A and ATR kinase results in regression of -amplified neuroblastoma.

Nat Cancer 2021 Mar 11;2(3):312-326. Epub 2021 Feb 11.

Theodor Boveri Institute, Department of Biochemistry and Molecular Biology, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.

Amplification of is the driving oncogene in a subset of high-risk neuroblastoma. The MYCN protein and the Aurora-A kinase form a complex during S phase that stabilizes MYCN. Here we show that MYCN activates Aurora-A on chromatin, which phosphorylates histone H3 at serine 10 in S phase, promotes the deposition of histone H3.3 and suppresses R-loop formation. Inhibition of Aurora-A induces transcription-replication conflicts and activates the Ataxia telangiectasia and Rad3 related (ATR) kinase, which limits double-strand break accumulation upon Aurora-A inhibition. Combined inhibition of Aurora-A and ATR induces rampant tumor-specific apoptosis and tumor regression in mouse models of neuroblastoma, leading to permanent eradication in a subset of mice. The therapeutic efficacy is due to both tumor cell-intrinsic and immune cell-mediated mechanisms. We propose that targeting the ability of Aurora-A to resolve transcription-replication conflicts is an effective therapy for -driven neuroblastoma (141 words).
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http://dx.doi.org/10.1038/s43018-020-00171-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610389PMC
March 2021

Enhancer hijacking determines extrachromosomal circular MYCN amplicon architecture in neuroblastoma.

Nat Commun 2020 11 16;11(1):5823. Epub 2020 Nov 16.

Berlin Institute of Health, Anna-Louisa-Karsch-Str. 2, 10178, Berlin, Germany.

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA (ecDNA). The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyze the MYCN amplicon structure using short-read and Nanopore sequencing and its chromatin landscape using ChIP-seq, ATAC-seq and Hi-C. This reveals two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplifies a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons is characterized by high structural complexity, lacks key local enhancers, and instead contains distal chromosomal fragments harboring CRC-driven enhancers. Thus, ectopic enhancer hijacking can compensate for the loss of local gene regulatory elements and explains a large component of the structural diversity observed in MYCN amplification.
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http://dx.doi.org/10.1038/s41467-020-19452-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7669906PMC
November 2020

Extrachromosomal DNA is associated with oncogene amplification and poor outcome across multiple cancers.

Nat Genet 2020 09 17;52(9):891-897. Epub 2020 Aug 17.

The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA.

Extrachromosomal DNA (ecDNA) amplification promotes intratumoral genetic heterogeneity and accelerated tumor evolution; however, its frequency and clinical impact are unclear. Using computational analysis of whole-genome sequencing data from 3,212 cancer patients, we show that ecDNA amplification frequently occurs in most cancer types but not in blood or normal tissue. Oncogenes were highly enriched on amplified ecDNA, and the most common recurrent oncogene amplifications arose on ecDNA. EcDNA amplifications resulted in higher levels of oncogene transcription compared to copy number-matched linear DNA, coupled with enhanced chromatin accessibility, and more frequently resulted in transcript fusions. Patients whose cancers carried ecDNA had significantly shorter survival, even when controlled for tissue type, than patients whose cancers were not driven by ecDNA-based oncogene amplification. The results presented here demonstrate that ecDNA-based oncogene amplification is common in cancer, is different from chromosomal amplification and drives poor outcome for patients across many cancer types.
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http://dx.doi.org/10.1038/s41588-020-0678-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484012PMC
September 2020

Partner independent fusion gene detection by multiplexed CRISPR-Cas9 enrichment and long read nanopore sequencing.

Nat Commun 2020 06 5;11(1):2861. Epub 2020 Jun 5.

Department of Genetics, Center for Molecular Medicine, University Medical Center Utrecht and Utrecht University, Utrecht, Netherlands.

Fusion genes are hallmarks of various cancer types and important determinants for diagnosis, prognosis and treatment. Fusion gene partner choice and breakpoint-position promiscuity restricts diagnostic detection, even for known and recurrent configurations. Here, we develop FUDGE (FUsion Detection from Gene Enrichment) to accurately and impartially identify fusions. FUDGE couples target-selected and strand-specific CRISPR-Cas9 activity for fusion gene driver enrichment - without prior knowledge of fusion partner or breakpoint-location - to long read nanopore sequencing with the bioinformatics pipeline NanoFG. FUDGE has flexible target-loci choices and enables multiplexed enrichment for simultaneous analysis of several genes in multiple samples in one sequencing run. We observe on-average 665 fold breakpoint-site enrichment and identify nucleotide resolution fusion breakpoints within 2 days. The assay identifies cancer cell line and tumor sample fusions irrespective of partner gene or breakpoint-position. FUDGE is a rapid and versatile fusion detection assay for diagnostic pan-cancer fusion detection.
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http://dx.doi.org/10.1038/s41467-020-16641-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275081PMC
June 2020

Oncogenic hijacking of a developmental transcription factor evokes vulnerability toward oxidative stress in Ewing sarcoma.

Nat Commun 2020 05 15;11(1):2423. Epub 2020 May 15.

Max-Eder Research Group for Pediatric Sarcoma Biology, Institute of Pathology, Faculty of Medicine, LMU Munich, Munich, Germany.

Ewing sarcoma (EwS) is an aggressive childhood cancer likely originating from mesenchymal stem cells or osteo-chondrogenic progenitors. It is characterized by fusion oncoproteins involving EWSR1 and variable members of the ETS-family of transcription factors (in 85% FLI1). EWSR1-FLI1 can induce target genes by using GGAA-microsatellites as enhancers.Here, we show that EWSR1-FLI1 hijacks the developmental transcription factor SOX6 - a physiological driver of proliferation of osteo-chondrogenic progenitors - by binding to an intronic GGAA-microsatellite, which promotes EwS growth in vitro and in vivo. Through integration of transcriptome-profiling, published drug-screening data, and functional in vitro and in vivo experiments including 3D and PDX models, we discover that constitutively high SOX6 expression promotes elevated levels of oxidative stress that create a therapeutic vulnerability toward the oxidative stress-inducing drug Elesclomol.Collectively, our results exemplify how aberrant activation of a developmental transcription factor by a dominant oncogene can promote malignancy, but provide opportunities for targeted therapy.
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http://dx.doi.org/10.1038/s41467-020-16244-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228971PMC
May 2020

Central memory phenotype drives success of checkpoint inhibition in combination with CAR T cells.

Mol Carcinog 2020 07 25;59(7):724-735. Epub 2020 Apr 25.

Department of Pediatric Oncology and Hematology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt - Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

The immunosuppressive microenvironment in solid tumors is thought to form a barrier to the entry and efficacy of cell-based therapies such as chimeric antigen receptor (CAR) T cells. Combining CAR T cell therapy with checkpoint inhibitors has been demonstrated to oppose immune escape mechanisms in solid tumors and augment antitumor efficacy. We evaluated PD-1/PD-L1 signaling capacity and the impact of an inhibitor of this checkpoint axis in an in vitro system for cancer cell challenge, the coculture of L1CAM-specific CAR T cells with neuroblastoma cell lines. Fluorescence-activated cell sorting-based analyses and luciferase reporter assays were used to assess PD-1/PD-L1 expression on CAR T and tumor cells as well as CAR T cell ability to kill neuroblastoma cells. Coculturing neuroblastoma cell lines with L1CAM-CAR T cells upregulated PD-L1 expression on neuroblastoma cells, confirming adaptive immune resistance. Exposure to neuroblastoma cells also upregulated the expression of the PD-1/PD-L1 axis in CAR T cells. The checkpoint inhibitor, nivolumab, enhanced L1CAM-CAR T cell-directed killing. However, nivolumab-enhanced L1CAM-CAR T cell killing did not strictly correlate with PD-L1 expression on neuroblastoma cells. In fact, checkpoint inhibitor success relied on strong PD-1/PD-L1 axis expression in the CAR T cells, which in turn depended on costimulatory domains within the CAR construct, and more importantly, on the subset of T cells selected for CAR T cell generation. Thus, T cell subset selection for CAR T cell generation and CAR T cell prescreening for PD-1/PD-L1 expression could help determine when combination therapy with checkpoint inhibitors could improve treatment efficacy.
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http://dx.doi.org/10.1002/mc.23202DOI Listing
July 2020

Tumor-Derived Extracellular Vesicles Impair CD171-Specific CD4 CAR T Cell Efficacy.

Front Immunol 2020 31;11:531. Epub 2020 Mar 31.

Department of Pediatric Oncology and Hematology, Berlin Institute of Health, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany.

Chimeric antigen receptor (CAR) T cell efficacy against solid tumors is currently limited by several immune escape mechanisms, which may include tumor-derived extracellular vesicles. Advanced neuroblastoma is an aggressive childhood tumor without curative treatment options for most relapsed patients today. We here evaluated the role of tumor-derived extracellular vesicles on the efficacy of CAR T cells targeting the neuroblastoma-specific antigen, CD171. For this purpose, CAR T cell activation, cytokine production, exhaustion, and tumor cell-directed cytotoxicity upon co-culture was evaluated. Tumor-derived extracellular vesicles isolated from SH-SY5Y neuroblastoma cells neither affected CAR T cell activation nor expression of inhibitory markers. Importantly, exposure of CD4 CD171-specific CAR T cells to tumor-derived extracellular vesicles significantly impaired tumor cytotoxicity of CAR T cells. This effect was independent of neurotrophic receptor tyrosine kinases 1 or 2 (NTRK1, NTRK2) expression, which is known to impact immune responses against neuroblastoma. Our results demonstrate for the first time the impact of tumor-derived extracellular vesicles and non-cell-mediated tumor-suppressive effects on CD4 CAR T cell efficacy in a preclinical setting. We conclude that these factors should be considered for any CAR T cell-based therapy to make CAR T cell therapy successful against solid tumors.
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http://dx.doi.org/10.3389/fimmu.2020.00531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7137471PMC
March 2021

High Specificity of BCL11B and GLG1 for EWSR1-FLI1 and EWSR1-ERG Positive Ewing Sarcoma.

Cancers (Basel) 2020 Mar 10;12(3). Epub 2020 Mar 10.

Max-Eder Research Group for Pediatric Sarcoma Biology, Institute of Pathology, Faculty of Medicine, LMU Munich, 80337 Munich, Germany.

Ewing sarcoma (EwS) is an aggressive cancer displaying an undifferentiated small-round-cell histomorphology that can be easily confused with a broad spectrum of differential diagnoses. Using comparative transcriptomics and immunohistochemistry (IHC), we previously identified BCL11B and GLG1 as potential specific auxiliary IHC markers for -positive EwS. Herein, we aimed at validating the specificity of both markers in a far larger and independent cohort of EwS (including -positive cases) and differential diagnoses. Furthermore, we evaluated their intra-tumoral expression heterogeneity. Thus, we stained tissue microarrays from 133 molecularly confirmed EwS cases and 320 samples from morphological mimics, as well as a series of patient-derived xenograft (PDX) models for BCL11B, GLG1, and CD99, and systematically assessed the immunoreactivity and optimal cut-offs for each marker. These analyses demonstrated that high BCL11B and/or GLG1 immunoreactivity in CD99-positive cases had a specificity of 97.5% and an accuracy of 87.4% for diagnosing EwS solely by IHC, and that the markers were expressed by -positive EwS. Only little intra-tumoral heterogeneity in immunoreactivity was observed for differential diagnoses. These results indicate that BCL11B and GLG1 may help as specific auxiliary IHC markers in diagnosing EwS in conjunction with CD99, especially if confirmatory molecular diagnostics are not available.
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http://dx.doi.org/10.3390/cancers12030644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139395PMC
March 2020

Current and Future Treatment Strategies for Rhabdomyosarcoma.

Front Oncol 2019 20;9:1458. Epub 2019 Dec 20.

Department of Pediatric Oncology/Hematology, Charité-Universitätsmedizin Berlin, Berlin, Germany.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, and can be subcategorized histologically and/or based on PAX-FOXO1 fusion gene status. Over the last four decades, there have been no significant improvements in clinical outcomes for advanced and metastatic RMS patients, underscoring a need for new treatment options for these groups. Despite significant advancements in our understanding of the genomic landscape and underlying biological mechanisms governing RMS that have informed the identification of novel therapeutic targets, development of these therapies in clinical trials has lagged far behind. In this review, we summarize the current frontline multi-modality therapy for RMS according to pediatric protocols, highlight emerging targeted therapies and immunotherapies identified by preclinical studies, and discuss early clinical trial data and the implications they hold for future clinical development.
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http://dx.doi.org/10.3389/fonc.2019.01458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933601PMC
December 2019

Small-Molecule Dual PLK1 and BRD4 Inhibitors are Active Against Preclinical Models of Pediatric Solid Tumors.

Transl Oncol 2020 Feb 21;13(2):221-232. Epub 2019 Dec 21.

Department of Pediatric Oncology and Hematology, Charité-Universitätsmedizin Berlin, Berlin, Germany; Experimental and Clinical Research Center (ECRC) of the Charité and the Max-Delbrück-Center for Molecular Medicine (MDC) in the Helmholtz Association, Berlin, Germany; Deutsches Konsortium für Translationale Krebsforschung, Berlin, Germany; Berlin Institute of Health, Berlin, Germany. Electronic address:

Simultaneous inhibition of multiple molecular targets is an established strategy to improve the continuance of clinical response to therapy. Here, we screened 49 molecules with dual nanomolar inhibitory activity against BRD4 and PLK1, best classified as dual kinase-bromodomain inhibitors, in pediatric tumor cell lines for their antitumor activity. We identified two candidate dual kinase-bromodomain inhibitors with strong and tumor-specific activity against neuroblastoma, medulloblastoma, and rhabdomyosarcoma tumor cells. Dual PLK1 and BRD4 inhibitor treatment suppressed proliferation and induced apoptosis in pediatric tumor cell lines at low nanomolar concentrations. This was associated with reduced MYCN-driven gene expression as assessed by RNA sequencing. Treatment of patient-derived xenografts with dual inhibitor UMB103 led to significant tumor regression. We demonstrate that concurrent inhibition of two central regulators of MYC protein family of protooncogenes, BRD4, and PLK1, with single small molecules has strong and specific antitumor effects in preclinical pediatric cancer models.
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http://dx.doi.org/10.1016/j.tranon.2019.09.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6931204PMC
February 2020

Extrachromosomal circular DNA drives oncogenic genome remodeling in neuroblastoma.

Nat Genet 2020 01 16;52(1):29-34. Epub 2019 Dec 16.

Department of Pediatric Oncology/Hematology, Charité-Universitätsmedizin Berlin, Berlin, Germany.

Extrachromosomal circularization of DNA is an important genomic feature in cancer. However, the structure, composition and genome-wide frequency of extrachromosomal circular DNA have not yet been profiled extensively. Here, we combine genomic and transcriptomic approaches to describe the landscape of extrachromosomal circular DNA in neuroblastoma, a tumor arising in childhood from primitive cells of the sympathetic nervous system. Our analysis identifies and characterizes a wide catalog of somatically acquired and undescribed extrachromosomal circular DNAs. Moreover, we find that extrachromosomal circular DNAs are an unanticipated major source of somatic rearrangements, contributing to oncogenic remodeling through chimeric circularization and reintegration of circular DNA into the linear genome. Cancer-causing lesions can emerge out of circle-derived rearrangements and are associated with adverse clinical outcome. It is highly probable that circle-derived rearrangements represent an ongoing mutagenic process. Thus, extrachromosomal circular DNAs represent a multihit mutagenic process, with important functional and clinical implications for the origins of genomic remodeling in cancer.
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http://dx.doi.org/10.1038/s41588-019-0547-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7008131PMC
January 2020

Prohibitin is a prognostic marker and therapeutic target to block chemotherapy resistance in Wilms' tumor.

JCI Insight 2019 08 8;4(15). Epub 2019 Aug 8.

Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

Wilms' tumor is the most common type of childhood kidney cancer. To improve risk stratification and identify novel therapeutic targets for patients with Wilms' tumor, we used high-resolution mass spectrometry proteomics to identify urine tumor markers associated with Wilms' tumor relapse. We determined the urine proteomes at diagnosis of 49 patients with Wilms' tumor, non-Wilms' tumor renal tumors, and age-matched controls, leading to the quantitation of 6520 urine proteins. Supervised analysis revealed specific urine markers of renal rhabdoid tumors, kidney clear cell sarcomas, renal cell carcinomas as well as those detected in patients with cured and relapsed Wilms' tumor. In particular, urine prohibitin was significantly elevated at diagnosis in patients with relapsed as compared with cured Wilms' tumor. In a validation cohort of 139 patients, a specific urine prohibitin ELISA demonstrated that prohibitin concentrations greater than 998 ng/mL at diagnosis were significantly associated with ultimate Wilms' tumor relapse. Immunohistochemical analysis revealed that prohibitin was highly expressed in primary Wilms' tumor specimens and associated with disease stage. Using functional genetic experiments, we found that prohibitin was required for the growth and survival of Wilms' tumor cells. Overexpression of prohibitin was sufficient to block intrinsic mitochondrial apoptosis and to cause resistance to diverse chemotherapy drugs, at least in part by dysregulating factors that control apoptotic cytochrome c release from mitochondrial cristae. Thus, urine prohibitin may improve therapy stratification, noninvasive monitoring of treatment response, and early disease detection. In addition, therapeutic targeting of chemotherapy resistance induced by prohibitin dysregulation may offer improved therapies for patients with Wilms' and other relapsed or refractory tumors.
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http://dx.doi.org/10.1172/jci.insight.127098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693841PMC
August 2019

Reflection of neuroblastoma intratumor heterogeneity in the new OHC-NB1 disease model.

Int J Cancer 2020 02 1;146(4):1031-1041. Epub 2019 Aug 1.

Department of Pediatric Hematology and Oncology, Charité - Universitätsmedizin Berlin, Berlin, Germany.

Accurate modeling of intratumor heterogeneity presents a bottleneck against drug testing. Flexibility in a preclinical platform is also desirable to support assessment of different endpoints. We established the model system, OHC-NB1, from a bone marrow metastasis from a patient diagnosed with MYCN-amplified neuroblastoma and performed whole-exome sequencing on the source metastasis and the different models and passages during model development (monolayer cell line, 3D spheroid culture and subcutaneous xenograft tumors propagated in mice). OHC-NB1 harbors a MYCN amplification in double minutes, 1p deletion, 17q gain and diploid karyotype, which persisted in all models. A total of 80-540 single-nucleotide variants (SNVs) was detected in each sample, and comparisons between the source metastasis and models identified 34 of 80 somatic SNVs to be propagated in the models. Clonal reconstruction using the combined copy number and SNV data revealed marked clonal heterogeneity in the originating metastasis, with four clones being reflected in the model systems. The set of OHC-NB1 models represents 43% of somatic SNVs and 23% of the cellularity in the originating metastasis with varying clonal compositions, indicating that heterogeneity is partially preserved in our model system.
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http://dx.doi.org/10.1002/ijc.32572DOI Listing
February 2020

Prohibitin promotes de-differentiation and is a potential therapeutic target in neuroblastoma.

JCI Insight 2019 04 18;5. Epub 2019 Apr 18.

Department of Pediatric Oncology and Hematology, Charité-Universitätsmedizin Berlin, Berlin, Germany.

Gain of the long arm of chromosome 17 (17q) is a cytogenetic hallmark of high-risk neuroblastoma, yet its contribution to neuroblastoma pathogenesis remains incompletely understood. Combining whole-genome and RNA sequencing of neuroblastomas, we identified the prohibitin (PHB) gene as highly expressed in tumors with 17q gain. High PHB expression correlated with poor prognosis and was associated with loss of gene expression programs promoting neuronal development and differentiation. PHB depletion induced differentiation and apoptosis and slowed cell cycle progression of neuroblastoma cells, at least in part through impaired ERK1/2 activation. Conversely, ectopic expression of PHB was sufficient to increase proliferation of neuroblastoma cells and was associated with suppression of markers associated with neuronal differentiation and favorable neuroblastoma outcome. Thus, PHB is a 17q oncogene in neuroblastoma that promotes tumor cell proliferation, and de-differentiation.
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http://dx.doi.org/10.1172/jci.insight.127130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542629PMC
April 2019

Synergistic activity of BET inhibitor MK-8628 and PLK inhibitor Volasertib in preclinical models of medulloblastoma.

Cancer Lett 2019 03 4;445:24-33. Epub 2019 Jan 4.

Department of Pediatric Oncology/Hematology, Charité-Universitätsmedizin Berlin, Germany; German Consortium for Translational Cancer Research (DKTK), Berlin, Germany; Berlin Institute of Health (BIH), Berlin, Germany. Electronic address:

Medulloblastoma is the most prevalent central nervous system tumor in children. Targeted treatment approaches for patients with high-risk medulloblastoma are needed as current treatment regimens are not curative in many cases and cause significant therapy-related morbidity. Medulloblastoma harboring MYC amplification have the most aggressive clinical course and worst outcome. Targeting the BET protein BRD4 has significant anti-tumor effects in preclinical models of MYC-amplified medulloblastoma, however, in most cases these are not curative. We here assessed the therapeutic efficacy of the orally bioavailable BRD4 inhibitor, MK-8628, in preclinical models of medulloblastoma. MK-8628 showed therapeutic efficacy against in vitro and in vivo models of MYC-amplified medulloblastoma by inducing apoptotic cell death and cell cycle arrest. Gene expression analysis of cells treated with MK-8628 showed that anti-tumor effects were accompanied by significant repression of MYC transcription as well as disruption of MYC-regulated transcriptional programs. Additionally, we found that targeting of MYC protein stability through pharmacological PLK1 inhibition showed synergistic anti-medulloblastoma effects when combined with MK-8628 treatment. Thus, MK-8628 is effective against preclinical high-risk medulloblastoma models and its effects can be enhanced through simultaneous targeting of PLK1.
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http://dx.doi.org/10.1016/j.canlet.2018.12.012DOI Listing
March 2019

Emerging functions of DNA transposases and oncogenic mutators in childhood cancer development.

JCI Insight 2018 10 18;3(20). Epub 2018 Oct 18.

Departments of Pediatrics, Pharmacology, and Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York, USA.

Our understanding of the molecular pathogenesis of childhood cancers has advanced substantially, but their fundamental causes remain poorly understood. Recently, multiple mechanisms of DNA damage and repair have been associated with mutations observed in human cancers. Here, we review the physiologic functions and oncogenic activities of transposable genetic elements. In particular, we focus on the recent studies implicating DNA transposases RAG1/2 and PGBD5 as oncogenic mutators that promote genomic rearrangements in childhood leukemias and solid tumors. We outline future studies that will be needed to define the contributions of transposons to mutational processes that become dysregulated in cancer cells. In addition, we discuss translational approaches, including synthetic lethal strategies, for identifying and developing improved clinical therapies to target oncogenic transposons and transposases.
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http://dx.doi.org/10.1172/jci.insight.123172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237467PMC
October 2018

Therapeutic targeting of PGBD5-induced DNA repair dependency in pediatric solid tumors.

Sci Transl Med 2017 Nov;9(414)

Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

Despite intense efforts, the cure rates of childhood and adult solid tumors are not satisfactory. Resistance to intensive chemotherapy is common, and targets for molecular therapies are largely undefined. We have found that the majority of childhood solid tumors, including rhabdoid tumors, neuroblastoma, medulloblastoma, and Ewing sarcoma, express an active DNA transposase, , that can promote site-specific genomic rearrangements in human cells. Using functional genetic approaches, we discovered that mouse and human cells deficient in nonhomologous end joining (NHEJ) DNA repair cannot tolerate the expression of PGBD5. In a chemical screen of DNA damage signaling inhibitors, we identified AZD6738 as a specific sensitizer of PGBD5-dependent DNA damage and apoptosis. We found that expression of PGBD5, but not its nuclease activity-deficient mutant, was sufficient to induce sensitivity to AZD6738. Depletion of endogenous PGBD5 conferred resistance to AZD6738 in human tumor cells. PGBD5-expressing tumor cells accumulated unrepaired DNA damage in response to AZD6738 treatment and underwent apoptosis in both dividing and G-phase cells in the absence of immediate DNA replication stress. Accordingly, AZD6738 exhibited nanomolar potency against most neuroblastoma, medulloblastoma, Ewing sarcoma, and rhabdoid tumor cells tested while sparing nontransformed human and mouse embryonic fibroblasts in vitro. Finally, treatment with AZD6738 induced apoptosis and regression of human neuroblastoma and medulloblastoma tumors engrafted in immunodeficient mice in vivo. This effect was potentiated by combined treatment with cisplatin, including substantial antitumor activity against patient-derived primary neuroblastoma xenografts. These findings delineate a therapeutically actionable synthetic dependency induced in PGBD5-expressing solid tumors.
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http://dx.doi.org/10.1126/scitranslmed.aam9078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5683417PMC
November 2017

PGBD5 promotes site-specific oncogenic mutations in human tumors.

Nat Genet 2017 Jul 15;49(7):1005-1014. Epub 2017 May 15.

Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

Genomic rearrangements are a hallmark of human cancers. Here, we identify the piggyBac transposable element derived 5 (PGBD5) gene as encoding an active DNA transposase expressed in the majority of childhood solid tumors, including lethal rhabdoid tumors. Using assembly-based whole-genome DNA sequencing, we found previously undefined genomic rearrangements in human rhabdoid tumors. These rearrangements involved PGBD5-specific signal (PSS) sequences at their breakpoints and recurrently inactivated tumor-suppressor genes. PGBD5 was physically associated with genomic PSS sequences that were also sufficient to mediate PGBD5-induced DNA rearrangements in rhabdoid tumor cells. Ectopic expression of PGBD5 in primary immortalized human cells was sufficient to promote cell transformation in vivo. This activity required specific catalytic residues in the PGBD5 transposase domain as well as end-joining DNA repair and induced structural rearrangements with PSS breakpoints. These results define PGBD5 as an oncogenic mutator and provide a plausible mechanism for site-specific DNA rearrangements in childhood and adult solid tumors.
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http://dx.doi.org/10.1038/ng.3866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5489359PMC
July 2017

Targeting tachykinin receptors in neuroblastoma.

Oncotarget 2017 Jan;8(1):430-443

Department of Pediatric Oncology/Hematology, Charité- Universitätsmedizin Berlin, Germany.

Neuroblastoma is the most common extracranial tumor in children. Despite aggressive multimodal treatment, high-risk neuroblastoma remains a clinical challenge with survival rates below 50%. Adding targeted drugs to first-line therapy regimens is a promising approach to improve survival in these patients. TACR1 activation by substance P has been reported to be mitogenic in cancer cell lines. Tachykinin receptor (TACR1) antagonists are approved for clinical use as an antiemetic remedy since 2003. Tachykinin receptor inhibition has recently been shown to effectively reduce growth of several tumor types. Here, we report that neuroblastoma cell lines express TACR1, and that targeting TACR1 activity significantly reduced cell viability and induced apoptosis in neuroblastoma cell lines. Gene expression profiling revealed that TACR1 inhibition repressed E2F2 and induced TP53 signaling. Treating mice harboring established neuroblastoma xenograft tumors with Aprepitant also significantly reduced tumor burden. Thus, we provide evidence that the targeted inhibition of tachykinin receptor signaling shows therapeutic efficacy in preclinical models for high-risk neuroblastoma.
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http://dx.doi.org/10.18632/oncotarget.13440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352132PMC
January 2017

Forward genetic screen of human transposase genomic rearrangements.

BMC Genomics 2016 Aug 4;17:548. Epub 2016 Aug 4.

Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Background: Numerous human genes encode potentially active DNA transposases or recombinases, but our understanding of their functions remains limited due to shortage of methods to profile their activities on endogenous genomic substrates.

Results: To enable functional analysis of human transposase-derived genes, we combined forward chemical genetic hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) screening with massively parallel paired-end DNA sequencing and structural variant genome assembly and analysis. Here, we report the HPRT1 mutational spectrum induced by the human transposase PGBD5, including PGBD5-specific signal sequences (PSS) that serve as potential genomic rearrangement substrates.

Conclusions: The discovered PSS motifs and high-throughput forward chemical genomic screening approach should prove useful for the elucidation of endogenous genome remodeling activities of PGBD5 and other domesticated human DNA transposases and recombinases.
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http://dx.doi.org/10.1186/s12864-016-2877-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4973553PMC
August 2016

Genomic DNA transposition induced by human PGBD5.

Elife 2015 Sep 25;4. Epub 2015 Sep 25.

Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States.

Transposons are mobile genetic elements that are found in nearly all organisms, including humans. Mobilization of DNA transposons by transposase enzymes can cause genomic rearrangements, but our knowledge of human genes derived from transposases is limited. In this study, we find that the protein encoded by human PGBD5, the most evolutionarily conserved transposable element-derived gene in vertebrates, can induce stereotypical cut-and-paste DNA transposition in human cells. Genomic integration activity of PGBD5 requires distinct aspartic acid residues in its transposase domain, and specific DNA sequences containing inverted terminal repeats with similarity to piggyBac transposons. DNA transposition catalyzed by PGBD5 in human cells occurs genome-wide, with precise transposon excision and preference for insertion at TTAA sites. The apparent conservation of DNA transposition activity by PGBD5 suggests that genomic remodeling contributes to its biological function.
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http://dx.doi.org/10.7554/eLife.10565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4625184PMC
September 2015

Mutational dynamics between primary and relapse neuroblastomas.

Nat Genet 2015 Aug 29;47(8):872-7. Epub 2015 Jun 29.

1] Pediatric Oncology and Hematology, University Children's Hospital Essen, University of Duisburg-Essen, Essen, Germany. [2] Pediatric Oncology and Hematology, Charité University Medicine, Berlin, Germany. [3] German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), Heidelberg, Germany.

Neuroblastoma is a malignancy of the developing sympathetic nervous system that is often lethal when relapse occurs. We here used whole-exome sequencing, mRNA expression profiling, array CGH and DNA methylation analysis to characterize 16 paired samples at diagnosis and relapse from individuals with neuroblastoma. The mutational burden significantly increased in relapsing tumors, accompanied by altered mutational signatures and reduced subclonal heterogeneity. Global allele frequencies at relapse indicated clonal mutation selection during disease progression. Promoter methylation patterns were consistent over disease course and were patient specific. Recurrent alterations at relapse included mutations in the putative CHD5 neuroblastoma tumor suppressor, chromosome 9p losses, DOCK8 mutations, inactivating mutations in PTPN14 and a relapse-specific activity pattern for the PTPN14 target YAP. Recurrent new mutations in HRAS, KRAS and genes mediating cell-cell interaction in 13 of 16 relapse tumors indicate disturbances in signaling pathways mediating mesenchymal transition. Our data shed light on genetic alteration frequency, identity and evolution in neuroblastoma.
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http://dx.doi.org/10.1038/ng.3349DOI Listing
August 2015