Aberrant Expression of and Cell Death Induction by Engagement of the MHC-II Chaperone CD74 in Anaplastic Large Cell Lymphoma (ALCL)

SIMPLE SUMMARY: Anaplastic large cell lymphoma (ALCL) is a lymphoid malignancy considered to be derived from T cells. Currently, two types of systemic ALCL are distinguished: anaplastic lymphoma kinase (ALK)-positive and ALK-negative ALCL. Although ALK(+) and ALK(−) ALCL differ at the genomic and molecular levels, various key biological and molecular features are highly similar between both entities. We have developed the concept that both ALCL entities share a common principle of pathogenesis. In support of this concept, we here describe a common deregulation of CD74, which is usually not expressed in T cells, in ALCL. Ligation of CD74 induces cell death of ALCL cells in various conditions, and an anti-CD74-directed antibody-drug conjugate efficiently kills ALCL cell lines. Furthermore, we reveal expression of the proto-oncogene and known CD74 interaction partner MET in a fraction of ALCL cases. These data give insights into ALCL pathogenesis and might help to develop new treatment strategies for ALCL. ABSTRACT: In 50–60% of cases, systemic anaplastic large cell lymphoma (ALCL) is characterized by the t(2;5)(p23;q35) or one of its variants, considered to be causative for anaplastic lymphoma kinase (ALK)-positive (ALK(+)) ALCL. Key pathogenic events in ALK-negative (ALK(−)) ALCL are less well defined. We have previously shown that deregulation of oncogenic genes surrounding the chromosomal breakpoints on 2p and 5q is a unifying feature of both ALK(+) and ALK(−) ALCL and predisposes for occurrence of t(2;5). Here, we report that the invariant chain of the MHC-II complex CD74 or li, which is encoded on 5q32, can act as signaling molecule, and whose expression in lymphoid cells is usually restricted to B cells, is aberrantly expressed in T cell-derived ALCL. Accordingly, ALCL shows an altered DNA methylation pattern of the CD74 locus compared to benign T cells. Functionally, CD74 ligation induces cell death of ALCL cells. Furthermore, CD74 engagement enhances the cytotoxic effects of conventional chemotherapeutics in ALCL cell lines, as well as the action of the ALK-inhibitor crizotinib in ALK(+) ALCL or of CD95 death-receptor signaling in ALK(−) ALCL. Additionally, a subset of ALCL cases expresses the proto-oncogene MET, which can form signaling complexes together with CD74. Finally, we demonstrate that the CD74-targeting antibody-drug conjugate STRO-001 efficiently and specifically kills CD74-positive ALCL cell lines in vitro. Taken together, these findings enabled us to demonstrate aberrant CD74-expression in ALCL cells, which might serve as tool for the development of new treatment strategies for this lymphoma entity

Genomic loss of the putative tumor suppressor gene E2A in human lymphoma

The transcription factor E2A is essential for lymphocyte development. In this study, we describe a recurrent E2A gene deletion in at least 70% of patients with Sézary syndrome (SS), a subtype of T cell lymphoma. Loss of E2A results in enhanced proliferation and cell cycle progression via derepression of the protooncogene MYC and the cell cycle regulator CDK6. Furthermore, by examining the gene expression profile of SS cells after restoration of E2A expression, we identify several E2A-regulated genes that interfere with oncogenic signaling pathways, including the Ras pathway. Several of these genes are down-regulated or lost in primary SS tumor cells. These data demonstrate a tumor suppressor function of E2A in human lymphoid cells and could help to develop new treatment strategies for human lymphomas with altered E2A activity

Funktionelle Charakterisierung linien-fremder Signalwege für Wachstum, Überleben und Reprogrammierung lymphatischer Zellen

Cytokine steuern die Kommunikation von verschiedenen Zelltypen untereinander und regulieren deren Überleben, Differenzierung und Wachstum. Kommt es zu einer Deregulation der Expression von Cytokinen oder deren Rezeptoren, kann es zu autoimmunen oder malignen Erkrankungen kommen. Ein besonderes Beispiel der aberranten Cytokinexpression ist das klassische Hodgkin Lymphom. Die malignen Hodgkin/Reed-Sternberg (HRS) Zellen des Hodgkin Lymphoms stammen ursprünglich aus Keimzentrums B-Zellen ab, haben aber ihren B-Zell Phänotyp verloren. Des Weiteren exprimieren sie eine Vielzahl von verschiedenen Cytokinen und Cytokinrezeptoren, die ursprünglich nicht in einem Genexpressionsprogramm von B-Zellen vorkommen. In dieser Arbeit wurden zwei dieser Cytokin-Rezeptorsysteme (IL-21/IL-21R und CSF-1/CSF1R) hinsichtlich ihrer Funktionen für die HRS Zellen des Hodgkin Lymphoms charakterisiert. Die Expression des T-Zell assoziierten Cytokins IL-21 konnte in dieser Arbeit erstmals in HRS Zellen nachgewiesen werden. Für die Expression des myeloiden CSF1R zeigen Ergebnisse dieser Arbeit eine neuartige Regulation durch ein Long Terminal Repeat (LTR) Element, welche zu einem bis dahin unbekannten mRNA Transkript des Protoonkogens CSF1R in den HRS Zellen führt. Sowohl für IL-21 als auch für CSF1R konnte in der Doktorarbeit die Expression und Funktionalität des jeweilig korrespondierenden Rezeptors (IL-21R) bzw. Cytokins (CSF-1) nachgewiesen werden. Die Bedeutung dieser B-Zell fremden Gene für die HRS Zellen lag hauptsächlich in der Stimulation von Wachstum und Überleben und der Induktion von wichtigen Signalwegen (z.B. STAT3). Die Ergebnisse der Dissertation können als Ausgangspunkt für neue Strategien in der Diagnostik und der spezifischeren Therapie von Hodgkin Lymphom Patienten dienen. Der außergewöhnliche Mechanismus der Genregulation des CSF1R Gens über ein endogenes LTR Element kann in anderen Tumorentitäten ebenfalls ein Grund für die Aktivierung von Onkogenen sein.Cytokines in the human body are responsible for cell-cell communication and regulate survival, differentiation and proliferation of different cell types. Deregulation of expression levels of cytokines might contribute to autoimmune diseases or tumor growth. One of the most prominent examples of aberrant cytokine expression is the classical Hodgkin Lymphoma. The malign Hodgkin/Reed-Sternberg (HRS) cells of classical Hodgkin Lymphoma are derived from germinal centre B cells, however they lost their B cell-specific phenotype. Moreover they express a huge variety of cytokines and cytokine receptors, normally not expressed in B cells. Two of these cytokine-receptor systems (IL-21/IL-21R and CSF-1/CSF1R) and their expression and function in HRS cells are subject of this dissertation. The expression of the T cell-associated cytokine IL-21 has been shown for the first time in HRS cells. The results for the myeloid-specific proto-oncogene CSF1R identified a unique, so far unknown mRNA transcript, expressed due to activation of a long terminal repeat (LTR) element. For both, IL-21 and CSF1R, the expression and functionality of the corresponding receptor (IL-21R) or cytokine (CSF-1), respectively, was demonstrated in this dissertation. Protection from apoptosis, proliferation and stimulation of several pathways are the main functional consequences of auto- and paracrine stimulation of HRS cells with either IL-21 or CSF-1. These results might lead to new diagnostic and more specific treatment strategies for Hodgkin Lymphoma patients. Regarding the unusual expression of CSF1R via LTR activation this mechanism might also be the reason for oncogene activation in several other tumor entities

Gene deregulation and spatial genome reorganization near breakpoints prior to formation of translocations in anaplastic large cell lymphoma

Although the identification and characterization of translocations have rapidly increased, little is known about the mechanisms of how translocations occur in vivo. We used anaplastic large cell lymphoma (ALCL) with and without the characteristic t(2;5)(p23;q35) translocation to study the mechanisms of formation of translocations and of ALCL transformation. We report deregulation of several genes located near the ALCL translocation breakpoint, regardless of whether the tumor contains the t(2;5). The affected genes include the oncogenic transcription factor Fra2 (located on 2p23), the HLH protein Id2 (2p25), and the oncogenic tyrosine kinase CSF1-receptor (5q33.1). Their up-regulation promotes cell survival and repression of T cell-specific gene expression programs that are characteristic for ALCL. The deregulated genes are in spatial proximity within the nuclear space of t(2;5)-negative ALCL cells, facilitating their translocation on induction of double-strand breaks. These data suggest that deregulation of breakpoint-proximal genes occurs before the formation of translocations, and that aberrant transcriptional activity of genomic regions is linked to their propensity to undergo chromosomal translocations. Also, our data demonstrate that deregulation of breakpoint-proximal genes has a key role in ALCL

Derepression of an endogenous long terminal repeat activates the CSF1R proto-oncogene in human lymphoma

Mammalian genomes contain many repetitive elements, including long terminal repeats (LTRs), which have long been suspected to have a role in tumorigenesis. Here we present evidence that aberrant LTR activation contributes to lineage-inappropriate gene expression in transformed human cells and that such gene expression is central for tumor cell survival. We show that B cell-derived Hodgkin's lymphoma cells depend on the activity of the non-B, myeloid-specific proto-oncogene colony-stimulating factor 1 receptor (CSF1R). In these cells, CSF1R transcription initiates at an aberrantly activated endogenous LTR of the MaLR family (THE1B). Derepression of the THE1 subfamily of MaLR LTRs is widespread in the genome of Hodgkin's lymphoma cells and is associated with impaired epigenetic control due to loss of expression of the corepressor CBFA2T3. Furthermore, we detect LTR-driven CSF1R transcripts in anaplastic large cell lymphoma, in which CSF1R is known to be expressed aberrantly. We conclude that LTR derepression is involved in the pathogenesis of human lymphomas, a finding that might have diagnostic, prognostic and therapeutic implications

Mapping of transcription factor motifs in active chromatin identifies IRF5 as key regulator in classical Hodgkin lymphoma

Deregulated transcription factor (TF) activities are commonly observed in hematopoietic malignancies. Understanding tumorigenesis therefore requires determining the function and hierarchical role of individual TFs. To identify TFs central to lymphomagenesis, we identified lymphoma type-specific accessible chromatin by global mapping of DNaseI hypersensitive sites and analyzed enriched TF-binding motifs in these regions. Applying this unbiased approach to classical Hodgkin lymphoma (HL), a common B-cell-derived lymphoma with a complex pattern of deregulated TFs, we discovered interferon regulatory factor (IRF) sites among the top enriched motifs. High-level expression of the proinflammatory TF IRF5 was specific to HL cells and crucial for their survival. Furthermore, IRF5 initiated a regulatory cascade in human non-Hodgkin B-cell lines and primary murine B cells by inducing the TF AP-1 and cooperating with NF-{kappa}B to activate essential characteristic features of HL. Our strategy efficiently identified a lymphoma type-specific key regulator and uncovered a tumor promoting role of IRF5

Structural insights into the mechanism of GTPase activation in the GIMAP family

SummaryGTPases of immunity-associated proteins (GIMAPs) are regulators of lymphocyte survival and homeostasis. We previously determined the structural basis of GTP-dependent GIMAP2 scaffold formation on lipid droplets. To understand how its GTP hydrolysis is activated, we screened for other GIMAPs on lipid droplets and identified GIMAP7. In contrast to GIMAP2, GIMAP7 displayed dimerization-stimulated GTP hydrolysis. The crystal structure of GTP-bound GIMAP7 showed a homodimer that assembled via the G domains, with the helical extensions protruding in opposite directions. We identified a catalytic arginine that is supplied to the opposing monomer to stimulate GTP hydrolysis. GIMAP7 also stimulated GTP hydrolysis by GIMAP2 via an analogous mechanism. Finally, we found GIMAP2 and GIMAP7 expression differentially regulated in several human T cell lymphoma lines. Our findings suggest that GTPase activity in the GIMAP family is controlled by homo- and heterodimerization. This may have implications for the differential roles of some GIMAPs in lymphocyte survival