Wolfgang W. Müller

Up‐regulation of c‐FLIPshort and reduction of activation‐induced cell death in CD28‐co‐stimulated human T cells

Efficient activation of antigen‐specific T cells requires co‐stimulatory signals provided e.g. by CD28. Re‐exposure to antigen and CD28 co‐stimulation reduces activation‐induced cell death (AICD) and increases the number of T cells performing effector functions. AICD is mediated predominantly by CD95 (APO‐1/Fas) and its cognate ligand (CD95L). In an in vitro model system, using human peripheral activated T cells, we demonstrate here that co‐stimulation prevents CD95L expression. Moreover, we show that co‐stimulation reduces the activity of the CD95 death‐inducing signaling complex and procaspase‐8 activation. In parallel, co‐stimulation strongly increases expression of the short form of the FLICE‐inhibitory protein c‐FLIPshort and of Bcl‐xL. These data provide important new insight into the molecular mechanisms of apoptosis resistance in co‐stimulated T cells.

TCR-Mediated Up-Regulation of c-FLIP short Correlates with Resistance Toward CD95-Mediated Apoptosis by Blocking Death-Inducing Signaling Complex Activity

To investigate apoptosis resistance upon restimulation in human peripheral blood T lymphocytes, we used the following in vitro model. This model represents the main features of T cell reactivity: freshly isolated PHA-activated T cells cultured in IL-2 for a prolonged period of time develop a CD95 (APO-1/Fas) apoptosis-sensitive phenotype. These T cells represent activation-induced cell death-sensitive T cells during the down phase of an immune response. A fraction of apoptosis-sensitive activated T cells becomes apoptosis resistant upon TCR/CD3 restimulation. CD95 apoptosis sensitivity requires formation of a functional receptor associated death-inducing signaling complex (DISC), i.e., a protein complex of CD95 receptors, the adaptor Fas-associated death domain protein (FADD)/MORT1 and caspase-8 (FADD-like IL-1β-converting enzyme (FLICE), MACH, Mch5). We identified activation of procaspase-8 at the DISC as the main target for the protective activity of TCR/CD3 restimulation. We found that procaspase-8 cleavage is reduced in T cells after TCR/CD3 restimulation. In addition, we detected up-regulation of c-FLIPS (the short splice variant of the cellular FLICE inhibitory protein) and strongly enhanced recruitment of c-FLIPS into the DISC. These data suggest that the recruitment of c-FLIPS into the DISC results in reduced DISC and caspase-8 activity.

The natural anticancer compounds rocaglamides inhibit the Raf-MEK-ERK pathway by targeting prohibitin 1 and 2.

Rocaglamides are potent natural anticancer products that inhibit proliferation of various cancer cells at nanomolar concentrations. We have recently shown that these compounds prevent tumor growth and sensitize resistant cancer cells to apoptosis by blocking the MEK-ERK-eIF4 pathway. However, their direct molecular target(s) remain(s) unknown. In this study, using an affinity chromatography approach we discovered that prohibitin (PHB) 1 and 2 are the direct targets of rocaglamides. Binding of rocaglamides to PHB prevents interaction between PHB and CRaf and, thereby, inhibits CRaf activation and subsequently CRaf-MEK-ERK signaling. Moreover, knockdown of PHB mimicked the effects of rocaglamides on the CRaf-MEK-ERK pathway and cell cycle progression. Thus, our finding suggests that rocaglamides are a new type of anticancer agent and that they may serve as a small-molecular tool for studying PHB-mediated cellular processes.

A PP4 Holoenzyme Balances Physiological and Oncogenic Nuclear Factor-Kappa B Signaling in T Lymphocytes

Signal transduction to nuclear factor-kappa B (NF-κB) involves multiple kinases and phosphorylated target proteins, but little is known about signal termination by dephosphorylation. By RNAi screening, we have identified protein phosphatase 4 regulatory subunit 1 (PP4R1) as a negative regulator of NF-κB activity in T lymphocytes. PP4R1 formed part of a distinct PP4 holoenzyme and bridged the inhibitor of NF-κB kinase (IKK) complex and the phosphatase PP4c, thereby directing PP4c activity to dephosphorylate and inactivate the IKK complex. PP4R1 expression was triggered upon activation and proliferation of primary human T lymphocytes and deficiency for PP4R1 caused sustained and increased IKK activity, T cell hyperactivation, and aberrant NF-κB signaling in NF-κB-addicted T cell lymphomas. Collectively, our results unravel PP4R1 as a previously unknown activation-associated negative regulator of IKK activity in lymphocytes whose downregulation promotes oncogenic NF-κB signaling in a subgroup of T cell lymphomas.

Sustained JNK signaling by proteolytically processed HPK1 mediates IL-3 independent survival during monocytic differentiation.

We studied monocytic differentiation of primary mouse progenitor cells to understand molecular mechanisms of differentiation. We found a tightly controlled non-apoptotic activation of caspase-3 that correlated with differentiation. Although caspase activity was already detected during monocytic differentiation, a caspase-3 target has not been identified yet. We show that hematopoietic progenitor kinase 1 (HPK1) is processed towards its N- and C-terminal fragments during monocytic differentiation. While HPK1 is an immunoreceptor-proximal kinase in T and B cells, its role in myeloid cells is elusive. Here, we show that the N-terminal cleavage product, HPK1-N, comprising the kinase domain, confers progenitor cell survival independent of the growth factor IL-3. Furthermore, HPK1-N causes differentiation of progenitor cells towards the monocytic lineage. In contrast to full-length kinase, HPK1-N is constitutively active causing sustained JNK activation, Bad phosphorylation and survival. Blocking of caspase activity during differentiation of primary mouse progenitor cells leads to reduced HPK1-N levels, suppressed JNK activity and attenuated monocytic differentiation. Our work explains growth factor-independent survival during monocytic differentiation by caspase-mediated processing of HPK1 towards HPK1-N.

Rocaglamide sensitizes leukemic T cells to activation-induced cell death by differential regulation of CD95L and c-FLIP expression

Drugs with tumor selectivity may have an important benefit in chemotherapies. We have previously shown that Rocaglamide(s), derived from the medicinal plant Aglaia, kills various leukemic cells through the mitochondrial apoptosis pathway with only minor toxicities to normal lymphocytes. Here, we show further that Rocaglamide preferentially promotes activation-induced cell death in malignant T cells by differential regulation of c-FLIP and CD95L expression. Rocaglamide enhances and also prolongs activation-induced JNK activation in malignant T cells leading to downregulation of c-FLIP but upregulation of CD95L expression. We also show that malignant T cells express a significantly higher amount of Bid – the molecular linker that bridges the receptor-mediated to the mitochondria-mediated apoptosis pathway. Conversely, a substantially lower amount of c-FLIP in response to T-cell stimulation compared to normal T cells is observed. This difference may provide a therapeutic window for cancer treatment. The effect of Rocaglamide on sensitization of activation-induced cell death in malignant T cells was further demonstrated in vivo in a mouse model. Our study demonstrates that Rocaglamide may be a potential anticancer drug that simultaneously targets both c-FLIP and CD95L expressions in tumor cells. This study may also provide a new clue to design a more efficient chemotherapy by using a combination of stimuli that engage the receptor-mediated and the mitochondria-mediated death pathway.

Targeting CDK9 by wogonin and related natural flavones potentiates the anti-cancer efficacy of the Bcl-2 family inhibitor ABT-263

Tumor initiation, progression and resistance to therapies are tightly associated with over‐expression of anti‐apoptotic proteins Bcl‐2, Bcl‐xL, Bcl‐w and Mcl‐1. ABT‐263 (Navitoclax), an orally bio‐available small‐molecule mimetic of the Bcl‐2 homology domain 3, inhibits Bcl‐2, Bcl‐xL, and Bcl‐w and has shown anti‐cancer effects mainly on lymphomas and lymphocytic leukemia. Despite promising results obtained from the clinical trials, the use of ABT‐263 in patients is dose‐limited due to causing thrombocytopenia via inhibition of Bcl‐xL in platelets. ABT‐199 specifically inhibits Bcl‐2; however, its use is limited to tumors over‐expressing only Bcl‐2. Besides, many tumors resist treatment due to high levels of Mcl‐1 expression or develop resistance via up‐regulation of Mcl‐1 during long‐term exposure. These obstacles highlight the demand to improve the ABT‐263‐based therapy. In this study, we show that anti‐cancer flavones, e.g., wogonin, baicalein, apigenin, chrysin and luteolin enhance ABT‐263‐induced apoptosis in different cancer cell lines and in primary AML and ALL cells by down‐regulation of Mcl‐1 expression. Importantly, wogonin does not enhance the toxicity of ABT‐263 to proliferating normal T cells and thrombocytes. Wogonin also potentiates the lethality of ABT‐263 in cancer cells which have acquired resistance to ABT‐263. Furthermore, we show that combination of wogonin with ABT‐263 promotes in vivo tumor regression in a human T‐cell leukemia xenograft mouse model. Our study demonstrates that wogonin (and related flavones) reduce the effective dose of ABT‐263 thereby possibly decreasing the risk of adverse side effects.

Targeting the IL‐4/IL‐13 signaling pathway sensitizes Hodgkin lymphoma cells to chemotherapeutic drugs

Hodgkin/Reed‐Sternberg lymphoma (HL) is a clonal B‐cell‐related malignancy. Although many patients with HL can be cured by the current regimen of high‐dose multi‐agent chemotherapy, the treatment causes high risks of later pathologies including secondary malignancies. This fact highlights the demand to develop rational treatment for HL. Survival and growth of HL cells are largely dependent on their microenvironment. In this study, using the HL cell lines L1236 and KM‐H2 as model systems, we investigated the role of IL‐4/IL‐13 signaling in regulation of drug sensitivity and resistance in HL. We show that specific blocking of IL‐4 and IL‐13‐mediated STAT6 activation by either an IL‐4‐binding fusion protein APG598 or an IL‐4R antagonist APG201 (R121D/Y124D) renders HL cells more prone to apoptotic killing by chemotherapeutic drugs such as Mitomycin C, 5‐Fluorouracil, Etopside, Doxorubicin and Paclitaxel. This effect is due to inhibition of STAT6‐mediated elevation of expression of the anti‐apoptotic Bcl‐2 family protein Bcl‐xL. Employing ChIP analysis in combination with APG201 or STAT6‐specific siRNA we identified a defined STAT6‐binding site in the Bcl‐xL promoter region from −1967 to −1957 of the transcription start site. Our data demonstrate that the IL‐4/IL‐13‐STAT6‐Bcl‐xL axis may be an important target for HL treatment. This study also suggests that combination of classical chemotherapeutic drugs with the IL‐4/IL‐13 antagonists may enhance efficacy and reduce risks of toxicity from high dose of drugs in HL treatment.