The team (as of August 2013)

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From left: Dr. Marco Herling, Prof. Hinrich Abken, Dr. Alexandra Schrader, Sebastian Oberbeck

Second Funding Period

Accomplishments of the first funding period

The modulation of TCR signaling by the TCL1 oncogene has been one focus of investigation by RP5. The underlying hypothesis is a functionally cooperative effect exerted by TCR activation and TCL1, leading towards clonal escape and leukemic sustenance. To this end, RP5 studied T-PLL, an aggressive and poor-prognostic leukemic MTCL, as a paradigmatic disease for oncogene-driven and TCR-mediated perturbation of normal T-cell homeostasis. RP5 thereby capitalizes on (a) a large collection of well-characterized T-PLL samples; (b) autochthonous mouse models of TCL1-driven leukemia mimicking human T-PLL with access to early disease stages; and c) chimeric antigen receptors (CARs) as engineered TCR mimics, allowing interrogations of the importance of defined proximal signaling components. Using these, RP5 showed that the T-PLL cell is a memory-type activated T-cell that is resistant to activation-induced cell death. Nearly all T-PLL are shown to express surface TCR and/or CD28 co-receptors and to be strongly responsive to in vitro cross-linking. There was no obvious preference for any particular TCRα or β usage across primary T-PLL samples. Extensive in vitro experimentation revealed an enhancing effect of TCL1 on TCR signaling and in T-cell effector function. In vivo, TCL1 compensated for a loss in TCR signaling strength, resulting in a marked growth advantage in the context of low-level (self)-antigen mediated stimulation.


T-cell prolymphocytic leukemia (T-PLL) is the most common leukemic mature T-cell lymphoma (MTCL). It is a poor prognostic, yet understudied tumor. Its hallmark is the constitutive transcriptional activation of the T-cell leukemia 1 (TCL1) oncogene. This persistent TCL1 expression in the differentiating peripheral T-cells is considered the initiating event towards T-PLL. However, it is not established how exactly TCL1 acts leukemogenic. Based on initial observations, we proposed that protein-kinase modulation, especially in the context of T-cell receptor (TCR) activation, is a central mechanism. We addressed this by capitalizing on valuable tools established by both PIs: (a) large collections of cell-line systems of defined TCR and TCL1 status and of well-characterized T-PLL samples; (b) autochthonous mouse models of TCL1-driven leukemia mimicking human T-PLL with access to early disease stages; and (c) chimeric antigen receptors (CARs) as engineered TCR mimics allowing interrogations of the importance of defined signaling components. Through the progress in funding phase-I, also involving collaborations with RPs 2-4, we arrived at the following central concept that requires further refinements and validations: T-PLL is a paradigmatic MTCL for a TCR-driven pathogenesis with TCL1 as the central oncogene acting as an enhancer of TCR-signal input through which there is a propensity of accumulating memory-type cells utilizing low-level TCR activation towards transformation. Consequently, we will address in phase-II the detailed mechanisms of the combined action of TCR input and TCL1, the facultative or obligatory relevance of a pro-tumorigenic cooperation of TCL1/TCR signaling at certain phases of leukemic development, the T-cell differentiation stage that is most susceptible to TCL1’s influence on TCR signaling, and molecular vulnerabilities that can be deduced for interventional exploitations. In detail, we study in Aim I which TCR/coreceptor components or branches are preferentially impacted by TCL1. Aim II assesses the pro-leukemic cooperation of ‘tonic’ TCR signaling in the context of overexpressed TCL1 resolved for the exposed T-cell stage vs leukemic phase. In an opposite fashion we will ablate TCR input in Aim III and by that establish the differential necessity of TCR signaling input at early and overt leukemic stages. Finally, in Aim IV we seek to identify molecular targets defined through a specific instruction by TCL1 in the context of TCR signaling that are pharmacologically exploitable for an antileukemic potential. The proposed studies will data-feed the competition systems of RP3 and the developing in-silico models on oncogenic modulation of milieu-input triggered intracellular signaling of RP4. Ultimately, by advancing the concepts of TCL1-driven leukemogenesis, our work will also serve to better understand general aspects of perturbed T-cell homeostasis.


Aim 1: Identify which TCR signaling components appear most crucial in mediating an activating TCL1 effect

The goal is to establish a molecular concept of TCL1 as a modulator in major TCR signaling axes.

Aim 2: Assess the pro-leukemic cooperation of ‘tonic’ TCR signaling in the healthy naive T-cells context of overexpressed TCL1 resolved for exposed T-cell stage and leukemic phase

It is intented to establish a refined model of phase-relevances of leukemogenic TCR/TCL1 signaling.

Aim 3: Interrogate TCL1-mediated T-cell transformation at early and fully leukemic stages for the necessity of TCR signaling input

It is planned to establish phase-specific dependencies on functional TCR in TCL1-driven leukemia.

Aim 4: Define molecular vulnerabilities specifically instructed by TCL1 in the context of TCR signaling that are exploitable as anti-leukemic targets

We aim at identifying drug candidates that intercept in TCL1/TCR dependencies for clinical testing.

The proposed work or RP5 will refine our understanding of the pathophysiology of T-PLL, particularly the functional interaction of TCL1 with TCR signaling, both major determinants of T-PLL’s ‘phenotype’. While it provides data for detailed mechanistic concepts, Aims III / IV will also establish treatment rationales. The gathered data will advance the general understanding of lymphocyte transformation by the novel class of TCL1 oncogenes in the context of antigen-receptor signaling. The results will strongly feed our consortial concepts of oncogene-mediated perturbation of TCR-based T-cell homeostasis. Our model of TCR/TCL1 driven leukemogenesis will help in discerning other scenarios of MTCL development.

First Funding Period


T-cell prolymphocytic leukemia (T-PLL), the most common mature T-cell leukemia, is a poor prognostic tumor. From incidentally diagnosed cases we increasingly learn about the stage-wise nature of disease evolution from a smoldering precursor phase to overt aggressive growth. Constitutive transcriptional activation of T-cell leukemia 1 (TCL1), resulting from chromosomal rearrangements found in the majority of T-PLL, prevents the physiological post-thymic silencing of this oncogene. The persistent expression of TCL1 in these peripheral T-cells is considered the initiating causal event towards the development of T-PLL. However, it is not established how exactly TCL1 acts oncogenic at the molecular level in the affected T-cells. In preliminary work, we and others found that protein-kinase modulation, especially in the context of T-cell receptor (TCR) activation, is a plausible central mechanism. Consequently, we address here the lack of understanding of the combined action of TCR input and TCL1, the relevance of this potential cooperation at certain phases of leukemic development, and the T-cell differentiation stage that is most susceptible to TCL1/TCR-mediated clonal outgrowth. Generally, we take advantage of 3 central tools established by the 2 investigators: a) a large collection of T-PLL samples from our clinical trials; b) mouse models of TCL1-driven leukemia mimicking human T-PLL which provide access to early tumor stages; and c) chimeric antigen receptors (CARs) as engineered TCR mimics allowing interrogation of the importance of defined proximal signaling components. Aim I mainly characterizes primary T-PLL samples of defined TCL1 and TCR/coreceptor status with respect to their kinase constitution and effector functions in the context of TCR signaling. In Aim II we quantify the impact of TCL1 on the TCR signaling outcome to define the quality of this cooperation. There we also address the necessity of key functional domains of TCL1 and of TCR/coreceptor components. Aim III seeks to provide evidence for the concept that TCR stimulation in conjunction with TCL1 represents a truly transforming drive in vivo and studies at which stages of leukemogenesis this is most relevant. Aim IV takes a histogenetically biased look at the impact of TCL1 on the TCR response of defined T-cell subsets. Overall, the ultimate goal is to advance our understanding of TCL1-driven leukemogenesis. By that our project contributes to general concepts of oncogene-based perturbations of T-cell homeostasis investigated in CONTROL-T.


Disease model of TCL1 in T-PLL and Hypotheses:

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Aim 1: Characterize the TCR signalosome and effector functions of T-PLL cells.

Aim 2: Assess TCL1 and TCR signaling for potential overlap, mutual compensation, or synergism by interrogating TCL1’s influence on TCR activation thresholds.

Aim 3: Define the phase of leukemogenesis during which TCL1 and TCR cooperate towards neoplastic transformation.

Aim 4: Identify the T-cell maturation stage that is most susceptible to the TCL1 effect.

Preliminary Data

The laboratory of PI M. Herling has been working on diagnostic algorithms for mature T-cell leukemias as well as on the molecular biology of T-PLL and treatment optimizations in these tumors[1-13].

TCR-signaling capacity and dysregulated TCR components define MTCL subsets

Based on the centrality of TCR signaling input in normal T-cells, we proposed a preliminary concept of functional categorization of mature T-cell leukemias / lymphomas (MTCL; Figure 1. Therein, we postulate that there are entities, which at some point of their evolution or even at the stage of overt lymphoma benefit from TCR input via (self-)antigen binding. Some of them may use enhancer systems such as TCL1 in T-PLL to gain a level of signal amplification that lowers their input dependence. Alternatively, clonal escape through a growth advantage, but reduced dependence on constant exogenous TCR input can be accomplished via genetic lesions constitutively activating TCR proximal kinases, as exemplified by SYK in ITK-SYK carrying PTCL-nos. Finally, subsets such as ALCL (usually of surface TCR-null phenotype) may rely on complete functional stand-in’s (e.g. ALK tyrosine kinase) of TCR signaling. These constitutive signals would feed into common, very down-stream effector pathways.

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Figure 1: There are distinguishable categories of mature T-cell lymphomas based on differential TCR-signaling activity and involvement of enhancing or substituting oncogenes[13].

T-PLL is an orphan disease with an aggressive course and a very poor prognosis

T-PLL is a tumor of peripheral T-cells[14]. The recurrent chromosomal aberrations inv(14) or t(14;14) and less frequently t(X;14) are its molecular hallmarks. They induce constitutive expression of TCL1 family oncogenes, particularly TCL1A or MTCP1.

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Figure 2: T-PLL is associated with TCL1 dysregulation.

Although being the most frequent mature T-cell leukemia, T-PLL is still rarely encountered (incidence »0.6/million)[2-4]. The median overall survival (OS) of patients is ≈20-36 months (5-year OS of 21%)[3,4,7,11,15]. T-PLL typically presents with exponentially rising PB lymphocytes and BM infiltration accompanied by lymphadenopathy and hepatosplenomegaly[3].Suggesting a protracted multi-step transformation process, few cases can coincidentally be diagnosed in a chronic asymptomatic (‘precursor’) phase (formerly ’T-CLL’)[2,16]. T-PLL characteristically does not respond well to standard multi-agent chemotherapy[11]. The monoclonal anti-CD52 antibody alemtuzumab is, so far, the only (targeted) agent that induces high remission rates, but severe immune suppression limits its use and relapses are the rule[17,18]. Sporadic cures can be accomplished in eligible patients transplanted in first major remission by an allogeneic stem cell graft[19]. However, this is too often not an option for the mostly elderly patients (median 63 years) or precluded by poor pre-transplant conditioning. As of today, T-PLL is still an under-addressed disease. More rationale-based therapeutic designs would tremendously benefit from a better understanding of T-PLL’s biology.

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Figure 3: The need to improve the clinical management in T-PLL.

Aberrant expression of TCL1 is central in the pathogenesis of T-PLL

Juxtaposition of the TCL1A locus at 14q32.1 to T-cell receptor (TCR) α/δ enhancer sequences at 14q11 is considered the initiating event in most T-PLL. It prevents TCL1’s physiological post-thymic silencing without a major differentiation block[20]. We demonstrated TCL1A overexpression in »80% of T-PLL and that variably high levels correlate with an inferior outcome7. Non-hematopoietic adult tissues do not express TCL1. The phenotype of TCL1knock-out mice supports a role for TCL1 lymphoid development and function, but also in embryogenesis[21]. TCL1A (here mostly referred to as TCL1) is the prototypic member of a paralogue family, also including TML1 (TCL1B; 15kb upstream of TCL1A) and MTCP1 (at chr. Xq28)[22]. The T-cell oncogenic potential of TCL1A and its homologue MTCP1 was formally proven in transgenic (tg) mice (Lck-TCL1 and CD2-MTCP1, respectively) as high-fidelity models of human T-PLL[23,24]. We could show that TCL1 expression modulates TCR associated kinase activity[7], which led us to propose TCL1 as a TCR sensitizer.

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Figure 4: High-level TCL1 expression defines a TCR hyper-responsive and aggressive T-PLL subset.

CARs represent valuable tools to interrogate TCR signaling

PI H. Abken has developed a large library of CARs for human and murine T-cells with tremendous combinatorial freedom for specific purposes. His laboratory developed efficient protocols of peripheral T-cell modification, adoptive T-cell therapy in appropriate mouse models, and CAR-tg animals. Originally designed for targeted redirection of T-cells towards defined epitopes on tumor cells in adoptive immunotherapy, CARs will be used here to address TCR signaling aspects. We understand here CARs as a modular tool set of a chimeric TCR with intracellular components of the physiological TCR complex.

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Figure 5: CAR engineering allows intra-cellular signaling modulation and modelling of inter-cellular TCR-mediated regulation.

In contrast to the TCR, CARs consist of an extracellular antibody-derived domain of pre-defined specificity for binding; the intracellular signaling domain derived from the TCR initiates T-cell activation upon engagement of a specific ligand. The antigen-binding domain is derived from a single chain fragment of variable regions (scFv) antibody. Due to the antibody-derived binding domain, CARs can be generated that bind antigen of any chemical composition or conformation including classical and non-classical T-cell targets like carbohydrates, as far as an antibody exists. The CAR thereby mediates T-cell activation independently of MHC engagement. A trans-membrane (TM) domain links the scFv part to an intracellular signaling domain. The latter is preferentially derived from the CD3z chain of the TCR/CD3 complex containing 3 immunoreceptor tyrosine activation motifs (ITAMs), which become phosphorylated upon receptor cross-linking. The ITAMs thereby serve as specific adaptors for downstream signaling proteins of the TCR complex, e.g. LCK. CARs of the second generation harbor in addition to the CD3z chain a costimulatory domain, e.g. CD28; 3rd generation receptors have 2 co-stimulatory domains, e.g. CD28 and 4-1BB. Different co-stimulatory domains induce different activation profiles in CD4+ and CD8+ T-cells. Moreover, effector and central memory T cells respond differentially to certain co-stimulations revealing specific requirements of certain T-cell subsets to maintain activation and prevent AICD.