Super-enhancer-based identification of a BATF3/IL-2R-module reveals vulnerabilities in anaplastic large cell lymphoma

Anaplastic large cell lymphoma (ALCL), an aggressive CD30-positive T-cell lymphoma, comprises systemic anaplastic lymphoma kinase (ALK)-positive, and ALK-negative, primary cutaneous and breast implant-associated ALCL. Prognosis of some ALCL subgroups is still unsatisfactory, and already in second line effective treatment options are lacking. To identify genes defining ALCL cell state and dependencies, we here characterize super-enhancer regions by genome-wide H3K27ac ChIP-seq. In addition to known ALCL key regulators, the AP-1-member BATF3 and IL-2 receptor (IL2R)-components are among the top hits. Specific and high-level IL2R expression in ALCL correlates with BATF3 expression. Confirming a regulatory link, IL-2R-expression decreases following BATF3 knockout, and BATF3 is recruited to IL2R regulatory regions. Functionally, IL-2, IL-15 and Neo-2/15, a hyper-stable IL-2/IL-15 mimic, accelerate ALCL growth and activate STAT1, STAT5 and ERK1/2. In line, strong IL-2Rα-expression in ALCL patients is linked to more aggressive clinical presentation. Finally, an IL-2Rα-targeting antibody-drug conjugate efficiently kills ALCL cells in vitro and in vivo. Our results highlight the importance of the BATF3/IL-2R-module for ALCL biology and identify IL-2Rα-targeting as a promising treatment strategy for ALCL

Oncogenic role of miR-155 in anaplastic large cell lymphoma lacking the t(2;5) translocation.

Anaplastic large cell lymphoma (ALCL) is a rare, aggressive, non-Hodgkin's lymphoma that is characterized by CD30 expression and disease onset in young patients. About half of ALCL patients bear the t(2;5)(p23;q35) translocation, which results in the formation of the nucleophosmin-anaplastic lymphoma tyrosine kinase (NPM-ALK) fusion protein (ALCL ALK(+)). However, little is known about the molecular features and tumour drivers in ALK-negative ALCL (ALCL ALK(-)), which is characterized by a worse prognosis. We found that ALCL ALK(-), in contrast to ALCL ALK(+), lymphomas display high miR-155 expression. Consistent with this, we observed an inverse correlation between miR-155 promoter methylation and miR-155 expression in ALCL. However, no direct effect of the ALK kinase on miR-155 levels was observed. Ago2 immunoprecipitation revealed miR-155 as the most abundant miRNA, and enrichment of target mRNAs C/EBPβ and SOCS1. To investigate its function, we over-expressed miR-155 in ALCL ALK(+) cell lines and demonstrated reduced levels of C/EBPβ and SOCS1. In murine engraftment models of ALCL ALK(-), we showed that anti-miR-155 mimics are able to reduce tumour growth. This goes hand-in-hand with increased levels of cleaved caspase-3 and high SOCS1 in these tumours, which leads to suppression of STAT3 signalling. Moreover, miR-155 induces IL-22 expression and suppresses the C/EBPβ target IL-8. These data suggest that miR-155 can act as a tumour driver in ALCL ALK(-) and blocking miR-155 could be therapeutically relevant. Original miRNA array data are to be found in the supplementary material (Table S1).This work was supported by the SCRI-LIMCR GmbH, the “Jubiläumsfond der Österreichischen Nationalbank” (grant-no. 14856 to O.M.), R.G. was supported by grant SFB P021 from the Austrian Science Funds (FWF), L.K. was supported by grant FWF, P26011, R.M. was supported by FWF grants SFB F28 and SFB F47. S.D.T. is a Senior Lecturer supported with funding from Leukemia and Lymphoma Research.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1002/path.453

The first trimester human trophoblast cell line ACH-3P: A novel tool to study autocrine/paracrine regulatory loops of human trophoblast subpopulations – TNF-α stimulates MMP15 expression

<p>Abstract</p> <p>Background</p> <p>The trophoblast compartment of the placenta comprises various subpopulations with distinct functions. They interact among each other by secreted signals thus forming autocrine or paracrine regulatory loops. We established a first trimester trophoblast cell line (ACH-3P) by fusion of primary human first trimester trophoblasts (week 12 of gestation) with a human choriocarcinoma cell line (AC1-1).</p> <p>Results</p> <p>Expression of trophoblast markers (cytokeratin-7, integrins, matrix metalloproteinases), invasion abilities and transcriptome of ACH-3P closely resembled primary trophoblasts. Morphology, cytogenetics and doubling time was similar to the parental AC1-1 cells. The different subpopulations of trophoblasts e.g., villous and extravillous trophoblasts also exist in ACH-3P cells and can be immuno-separated by HLA-G surface expression. HLA-G positive ACH-3P display pseudopodia and a stronger expression of extravillous trophoblast markers. Higher expression of insulin-like growth factor II receptor and human chorionic gonadotropin represents the basis for the known autocrine stimulation of extravillous trophoblasts.</p> <p>Conclusion</p> <p>We conclude that ACH-3P represent a tool to investigate interaction of syngeneic trophoblast subpopulations. These cells are particularly suited for studies into autocrine and paracrine regulation of various aspects of trophoblast function. As an example a novel effect of TNF-α on matrix metalloproteinase 15 in HLA-G positive ACH-3P and explants was found.</p

STAT3 regulated ARF expression suppresses prostate cancer metastasis.

Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19(ARF) as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14(ARF) expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.Lukas Kenner and Jan Pencik are supported by FWF, P26011 and the Genome Research-Austria project “Inflammobiota” grants. Helmut Dolznig is supported by the Herzfelder Family Foundation and the Niederösterr. Forschungs-und Bildungsges.m.b.H (nfb). Richard Moriggl is supported by grant SFB-F2807 and SFB-F4707 from the Austrian Science Fund (FWF), Ali Moazzami is supported by Infrastructure for biosciences-Strategic fund, SciLifeLab and Formas, Zoran Culig is supported by FWF, P24428, Athena Chalaris and Stefan Rose-John are supported by the Deutsche Forschungsgemeinschaft (Grant SFB 877, Project A1and the Cluster of Excellence --“Inflammation at Interfaces”). Work of the Aberger lab was supported by the Austrian Science Fund FWF (Projects P25629 and W1213), the European FP7 Marie-Curie Initial Training Network HEALING and the priority program Biosciences and Health of the Paris-Lodron University of Salzburg. Valeria Poli is supported by the Italian Association for Cancer Research (AIRC, No IG13009). Richard Kennedy and Steven Walker are supported by the McClay Foundation and the Movember Centre of Excellence (PC-UK and Movember). Gerda Egger is supported by FWF, P27616. Tim Malcolm and Suzanne Turner are supported by Leukaemia and Lymphoma Research.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms873

Super-enhancer-based identification of a BATF3/IL-2R-module reveals vulnerabilities in anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL), an aggressive CD30-positive T-cell lymphoma, comprises systemic anaplastic lymphoma kinase (ALK)-positive, and ALK-negative, primary cutaneous and breast implant-associated ALCL. Prognosis of some ALCL subgroups is still unsatisfactory, and already in second line effective treatment options are lacking. To identify genes defining ALCL cell state and dependencies, we here characterize super-enhancer regions by genome-wide H3K27ac ChIP-seq. In addition to known ALCL key regulators, the AP-1-member BATF3 and IL-2 receptor (IL2R)-components are among the top hits. Specific and high-level IL2R expression in ALCL correlates with BATF3 expression. Confirming a regulatory link, IL-2R-expression decreases following BATF3 knockout, and BATF3 is recruited to IL2R regulatory regions. Functionally, IL-2, IL-15 and Neo-2/15, a hyper-stable IL-2/IL-15 mimic, accelerate ALCL growth and activate STAT1, STAT5 and ERK1/2. In line, strong IL-2Rα-expression in ALCL patients is linked to more aggressive clinical presentation. Finally, an IL-2Rα-targeting antibody-drug conjugate efficiently kills ALCL cells in vitro and in vivo. Our results highlight the importance of the BATF3/IL-2R-module for ALCL biology and identify IL-2Rα-targeting as a promising treatment strategy for ALCL

Correction: Dependency on the TYK2/STAT1/MCL1 axis in anaplastic large cell lymphoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

TYK2 is a dependency in anaplastic large cell lymphoma

Hintergrund: Mutationen in Mitgliedern der Janus-Kinase-Familie JAK1, JAK2, JAK3 und TYK2, treten häufig bei hämatologischen Malignomen auf. Dies führte zur Entwicklung von mittlerweile durch die FDA zugelassene Pan-JAK-Inhibitoren Tofacitinib (2012) und Ruxolitinib (2014) die zur Behandlung von Polycythaemia Vera oder Rheumatoider Arthritis eingesetzt werden. Die Tyrosin-kinase 2 (TYK2) ist ein wichtiger Akteur des Immunsystems und ist an IFN / , IL-6, IL-10, IL-12, IL-13, IL-22 und IL-23 Signalwegen beteiligt. In den letzten 5 Jahren wurden TYK2 Mutationen oder Fusionen im Zusammenhang mit Krebs beschrieben, einschließlich solider Tumoren wie peripheren Nervenhüllentumoren sowie Tumoren des blutbildenden Systems wie die T-Zell Akute Lymphoblas-tische Leukämie (T-ALL), Akute Myeloische Leukämie (AML) und das Anaplastische Großzellige Lymphom (ALCL).^ ^ALCL ist ein aggressives CD30-positives Non-Hodgkin-T-Zell-Lymphom, das vor allem Kinder und junge Erwachsene betrifft. Verschiedene Subtypen von ALCL, die von der aktualisierten WHO Klassifikation in 2016 beschrieben wurden, zeigen unterschiedliche klinische Verhaltensweisen und molekulare Merkmale einschließlich ALK-Translokationen in etwa 50% der Patienten (ALCL, ALK+). Therapiemöglichkeiten für ALCL umfassen Chemotherapie und CD30-Antikörper. Darüber hinaus sind ALK-Inhibitoren eine weitere Option für ALCL, ALK + Patienten. Patienten ohne ALK-Fusionen fehlt jedoch diese Möglichkeit, was die Entwicklung alternativer the-rapeutischer Ansätze dringend notwendig macht. Interessanterweise wurden TYK2-Fusionsproteine, einschließlich PABPC4-TYK2, NFkB2-TYK2 und NPM1-TYK2, in ALCL identifi-ziert. Die beiden letzteren führten zu konstitutionell aktivierten TYK2- und STAT3-Signalen in HEK293T-Zellen und NFkB1-TYK2 führte zu einer verstärkten Koloniebildung in 3T3-Zellen.^ Eine genaue Rolle von TYK2 bei der Lymphomagenese und Tumorprogression wurde bisher noch nicht geklärt. Diese Studie untersucht die TYK2-Funktion in ALCL und zeigt erstmals die TYK2-Abhängigkeit in einem In-vivo-Modell von Krebs. Darüber hinaus erwiesen sich selektive TYK2-Inhibitoren hochwirksam und betonen somit die TYK2-Kinase als potentielles Therapieziel bei ALCL. Ergebnisse: TYK2 war in primärem Tumorgewebe von ALCL Patienten stark exprimiert was wir auch in Analysen von RNAseq Daten von 23 ALCL Patienten sehen konnten. TYK2 Gen-Knockout in der N-terminalen FERM- und C-terminalen Kinase-Domäne führte zum Zelltod in humanen ALCL-Zellen. Die Behandlung mit selektiven TYK2-Inhibitoren führte zu einer massiven Apoptose-Induktion in ALCL-Zellen, die die therapeutische Relevanz von TYK2 bestätigten. Der Verlust von TYK2 in T-Zellen führte zu signifikant erhöhten Überlebensraten von einem transgenen NPM-ALK Mausmodell.^ Wir konnten sehen, dass der Verlust von TYK2 zu einer reduzierten Phosphorylierung von STAT1 und STAT3 führte. Um deren relative Bedeutung zu beurteilen, haben wir entweder STAT1 oder STAT3 unter Verwendung von Lentiviral-transduzierten shRNAs herunterreguliert. Inte-ressanterweise konnte STAT1, aber nicht STAT3 Suppression den Effekt des TYK2 Verlustes imi-tieren. Darüber hinaus identifizierten wir IL-10 und IL-22 als nachgeschaltete Effektoren von TYK2, die den durch TYK2-Verlust verursachten Wachstumsphänotyp retten können. Schließlich wurde das BCL-2-ähnliche Apoptose-hemmende Protein MCL-1 in ALCL hoch exprimiert und durch TYK2 reguliert. Methoden: Die in vivo-Relevanz von TYK2 wurde durch TYK2-Knockout (CD4-NPM-ALKLCKTYK2) unter Verwendung des Lck Cre-Systems in einem CD4-NPM-ALK ALCL-Mausmodell untersucht.^ In humanen ALCL, ALK+ und ALCL, ALK- Zelllinien wurde TYK2 mittels CRISPR / CAS9 Methode in der N-terminalen FERM und in der C-terminalen Kinase-Domäne ausgeschaltet. TYK2-, STAT1- und STAT3-Knockdown wurden in humanen ALCL-Zelllinien unter Verwendung von validierten shRNAs durchgeführt. XTT-Assays und Immunoblot-Analyse von TYK2 Downstream Targets wurden nach TYK2 CRISPR / Cas9-Knock-Out oder Small-Molecule-Inhibition (Bayer-18, Symansis, TYK2 # 1, Genentech) durchgeführt. Schlussfolgerung: ALCL ist TYK2 abhängig und die Behandlung mit selektiven TYK2 Inhibitoren führte zum Absterben der ALCL Zellen. Die Entwicklung von oral verträglichen TYK2 Inhibitoren eröffnet neue therapeutische Möglichkeiten, die besonders für Patienten ohne ALK-Translokation, die bisher keine gezielte Therapie bekommen, hilfreich sein können.Background: Mutations in members of the Janus kinase family including JAK1, JAK2, JAK3 and TYK2 occur frequently in hematological malignancies. This prompted the development of now FDA approved pan-JAK inhibitors Tofacitinib (2012) and Ruxolitinib (2014) for treatment of polycythemia vera or rheumatoid arthritis. Tyrosine kinase 2 (TYK2) plays an important role in the immune system and is involved in IFN/, IL-6, IL-10, IL-12, IL-13, IL-22 and IL-23 signaling pathways. In the last 5 years, TYK2 mutations or fusions were described in the context of cancer including solid tumors such as malignant peripheral nerve sheath tumors, as well as hematopoietic cancers such as T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML) and anaplastic large cell lymphoma (ALCL). ALCL is an aggressive, CD30 positive non-Hodgkin T-cell lymphoma that primarily affects children and young adults.^ ^Different subtypes of ALCL described by the updated WHO classification in 2016, involve ALK-kinase fusions in around 50% of the patients (ALCL, ALK+) but also prognosis-relevant translocations like DUSP22 and TP63 in ALCL, ALK-. ALCL treatment options include chemo-therapy and armed-CD30 antibodies. Moreover ALK inhibition is a further option for ALCL, ALK+ patients. However, patients without ALK fusions lack this opportunity making alternative therapeutic approaches highly warranted. Interestingly, TYK2 fusion proteins including PABPC4-TYK2, NFkB2-TYK2 and NPM1-TYK2 were identified in ALCL. The latter two led to constitutively active TYK2 and STAT3 signaling in HEK293T cells and NFkB1-TYK2 led to enhanced colony formation in 3T3 cells. However, a precise role of TYK2 in lymphomagenesis and tumor progression remains to be shown. In this study, we investigate TYK2 function in ALCL and demonstrate for the first time TYK2 dependency in an in vivo model of cancer.^ Moreover, selective TYK2 inhibitors are highly effective, emphasizing the TYK2 kinase as a potential therapy target in ALCL. Results: We found enhanced TYK2 expression in primary ALCL patient derived tumor tissue and RNAseq data of 23 ALCL cases revealed TYK2 to be highly expressed in ALCL patients. Gene knockout of TYK2 in the N-terminal FERM and C-terminal kinase domain both led to cell death in human ALCL cells. Treatment with selective TYK2 inhibitors led to apoptosis induction in ALCL cells corroborating the therapeutic relevance of TYK2 inhibition. Conditional loss of TYK2 in T-cells of a transgenic CD4-NPM-ALK driven lymphoma mouse model resulted in significantly increased mean-survival time. We found that loss of TYK2 led to reduced phosphorylation of STAT1 and STAT3. To assess their respective importance, we down-regulated either STAT1 or STAT3 using lentiviral transduced shRNAs. Interestingly, STAT1 but not STAT3 knockout could mimic the effect of TYK2 depletion.^ Moreover, we identified IL-10 and IL-22 as downstream effectors of TYK2 that can rescue the growth phenotype caused by TYK2 loss. Finally, the BCL-2 like survival protein MCL-1 was identified be highly expressed in ALCL and regulated by TYK2. Methods: CRISPR/Cas9 mediated genomic TYK2 deletion was performed in the N-terminal FERM and C-terminal kinase domain in ALCL, ALK+ and ALCL, ALK- cell lines. TYK2, STAT1 and STAT3 knockdown were performed in human ALCL cell lines using validated shRNAs. XTT assays and immunoblot analysis of downstream targets was performed after TYK2 CRISPR/Cas9 knock-out or small molecule inhibition (Bayer-18, Symansis; TYK2#1, Genentech). T-cell specific TYK2 knockout (CD4-NPM-ALKLCKTYK2) using the Lck Cre system in a CD4 promoter driven NPM-ALK ALCL mouse model was performed. Conclusion: TYK2 depletion is lethal in ALCL cell lines and prolongs overall survival in a transgenic mouse model.^ Selective TYK2 inhibitors are currently under development opening a new avenue of potential ALCL therapeutics in the future.submitted by Mag. art. Nicole Prutsch, MScZusammenfassung in deutscher Sprache2017, Dissertatio

Oncogenic role of miR155 in anaplastic large cell lymphoma lacking the t(2;5) translocation

Anaplastic large cell lymphoma (ALCL) is a rare, aggressive, nonHodgkin's lymphoma that is characterized by CD30 expression and disease onset in young patients. About half of ALCL patients bear the t(2;5)(p23;q35) translocation, which results in the formation of the nucleophosminanaplastic lymphoma tyrosine kinase (NPMALK) fusion protein (ALCL ALK+). However, little is known about the molecular features and tumour drivers in ALKnegative ALCL (ALCL ALK), which is characterized by a worse prognosis. We found that ALCL ALK, in contrast to ALCL ALK+, lymphomas display high miR155 expression. Consistent with this, we observed an inverse correlation between miR155 promoter methylation and miR155 expression in ALCL. However, no direct effect of the ALK kinase on miR155 levels was observed. Ago2 immunoprecipitation revealed miR155 as the most abundant miRNA, and enrichment of target mRNAs C/EBP and SOCS1. To investigate its function, we overexpressed miR155 in ALCL ALK+ cell lines and demonstrated reduced levels of C/EBP and SOCS1. In murine engraftment models of ALCL ALK, we showed that antimiR155 mimics are able to reduce tumour growth. This goes handinhand with increased levels of cleaved caspase3 and high SOCS1 in these tumours, which leads to suppression of STAT3 signalling. Moreover, miR155 induces IL22 expression and suppresses the C/EBP target IL8. These data suggest that miR155 can act as a tumour driver in ALCL ALK and blocking miR155 could be therapeutically relevant. Original miRNA array data are to be found in the supplementary material (Table S1). © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.(VLID)484329

STAT3 couples activated tyrosine kinase signaling to the oncogenic core transcriptional regulatory circuitry of anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL) is an aggressive, CD30+ T cell lymphoma of children and adults. ALK fusion transcripts or mutations in the JAK-STAT pathway are observed in most ALCL tumors, but the mechanisms underlying tumorigenesis are not fully understood. Here, we show that dysregulated STAT3 in ALCL cooccupies enhancers with master transcription factors BATF3, IRF4, and IKZF1 to form a core regulatory circuit that establishes and maintains the malignant cell state in ALCL. Critical downstream targets of this network in ALCL cells include the protooncogene MYC, which requires active STAT3 to facilitate high levels of MYC transcription. The core autoregulatory transcriptional circuitry activity is reinforced by MYC binding to the enhancer regions associated with STAT3 and each of the core regulatory transcription factors. Thus, activation of STAT3 provides the crucial link between aberrant tyrosine kinase signaling and the core transcriptional machinery that drives tumorigenesis and creates therapeutic vulnerabilities in ALCL.This work was supported by NIH grants R35CA210064 (A.T.L.), R35CA210030 (K.S.) and K08CA245251 (A.D.D), and the St. Jude Children’s Research Hospital Collaborative Research Consortium on 3D Regulatory Nuclear Landscape of Pediatric Cancer Cells (A.T.L., K.S., B.J.A.). N.P. was supported by a grant from the Lymphoma Research Foundation. A.D.D. and B.J.A. are supported by the American Lebanese Syrian Associated Charities (ALSAC). N.V.D. was supported by the Julia’s Legacy of Hope St. Baldrick’s Foundation Fellowship. SDT is supported by the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU and SDT, JDM and LH by the Cancer Research UK Cambridge Centre [CTRQQR-2021\100012]. M.W.Z. was supported by grants from Alex’s Lemonade Stand Foundation, Charles A. King Trust, and Claudia Adams Barr Foundation