Katja Zirlik

Expansion of Bcr-Abl-positive leukemic stem cells is dependent on Hedgehog pathway activation.

Resistance of Bcr-Abl-positive leukemic stem cells (LSCs) to imatinib treatment in patients with chronic myeloid leukemia (CML) can cause relapse of disease and might be the origin for emerging drug-resistant clones. In this study, we identified Smo as a drug target in Bcr-Abl-positive LSCs. We show that Hedgehog signaling is activated in LSCs through upregulation of Smo. While Smo(-/-) does not impact long-term reconstitution of regular hematopoiesis, the development of retransplantable Bcr-Abl-positive leukemias was abolished in the absence of Smo expression. Pharmacological Smo inhibition reduced LSCs in vivo and enhanced time to relapse after end of treatment. Our results indicate that Smo inhibition might be an effective treatment strategy to reduce the LSC pool in CML.

Chronic lymphocytic leukaemia is driven by antigen-independent cell-autonomous signalling

B-cell antigen receptor (BCR) expression is an important feature of chronic lymphocytic leukaemia (CLL), one of the most prevalent B-cell neoplasias in Western countries. The presence of stereotyped and quasi-identical BCRs in different CLL patients suggests that recognition of specific antigens might drive CLL pathogenesis. Here we show that, in contrast to other B-cell neoplasias, CLL-derived BCRs induce antigen-independent cell-autonomous signalling, which is dependent on the heavy-chain complementarity-determining region (HCDR3) and an internal epitope of the BCR. Indeed, transferring the HCDR3 of a CLL-derived BCR provides autonomous signalling capacity to a non-autonomously active BCR, whereas mutations in the internal epitope abolish this capacity. Because BCR expression was required for the binding of secreted CLL-derived BCRs to target cells, and mutations in the internal epitope reduced this binding, our results indicate a new model for CLL pathogenesis, with cell-autonomous antigen-independent signalling as a crucial pathogenic mechanism.

Essential role of stromally induced hedgehog signaling in B-cell malignancies

Interaction of cancer cells with their microenvironment generated by stromal cells is essential for tumor cell survival and influences the localization of tumor growth. Here we demonstrate that hedgehog ligands secreted by bone-marrow, nodal and splenic stromal cells function as survival factors for malignant lymphoma and plasmacytoma cells derived from transgenic Eμ-Myc mice or isolated from humans with these malignancies. Hedgehog pathway inhibition in lymphomas induced apoptosis through downregulation of Bcl2, but was independent of p53 or Bmi1 expression. Blockage of hedgehog signaling in vivo inhibited expansion of mouse lymphoma cells in a syngeneic mouse model and reduced tumor mass in mice with fully developed disease. Our data indicate that stromally induced hedgehog signaling may provide an important survival signal for B- and plasma-cell malignancies in vitro and in vivo. Disruption of this interaction by hedgehog pathway inhibition could provide a new strategy in lymphoma and multiple myeloma therapy.

Spleen tyrosine kinase inhibition prevents chemokine- and integrin-mediated stromal protective effects in chronic lymphocytic leukemia

The microenvironment provides essential growth and survival signals to chronic lymphocytic leukemia (CLL) cells and contributes to their resistance to cytotoxic agents. Pharmacologic inhibition of spleen tyrosine kinase (SYK), a key mediator of B-cell receptor (BCR) signaling, induces apoptosis in primary CLL cells and prevents stroma contact-mediated cell survival. This report demonstrates a role of SYK in molecularly defined pathways that mediate the CLL-microenvironmental crosstalk independent from the BCR. Chemokine and integrin stimulation induced SYK phosphorylation, SYK-dependent Akt phosphorylation, and F-actin formation in primary CLL cells. Inhibition of SYK by 2 pharmacologic inhibitors and siRNA-knockdown abrogated downstream SYK signaling and morphologic changes induced by these stimuli. CLL cell migration toward CXCL12, the major homing attractor, and CLL cell adhesion to VCAM-1, a major integrin ligand expressed on stromal cells, were markedly reduced by SYK inhibition. In combination with fludarabine, the SYK inhibitor R406 abrogated stroma-mediated drug resistance by preventing up-regulation of the antiapoptotic factor Mcl-1 in CLL cells. SYK blockade in CLL is a promising therapeutic principle not only for its inhibition of the BCR signaling pathway, but also by inhibiting protective stroma signals in a manner entirely independent of BCR signaling.

Spleen Tyrosine Kinase Is Overexpressed and Represents a Potential Therapeutic Target in Chronic Lymphocytic Leukemia

B-cell receptor signaling contributes to apoptosis resistance in chronic lymphocytic leukemia (CLL), limiting the efficacy of current therapeutic approaches. In this study, we investigated the expression of spleen tyrosine kinase (SYK), a key component of the B-cell receptor signaling pathway, in CLL and its role in apoptosis. Gene expression profiling identified enhanced expression of SYK and downstream pathways in CLL compared with healthy B cells. Immunoblotting showed increased expression and phosphorylation of SYK, PLCgamma(2), signal transducers and activators of transcription 3, and extracellular signal regulated kinase 1/2 in CLL compared with healthy B cells, suggesting enhanced activation of these mediators in CLL. SYK inhibitors reduced phosphorylation of SYK downstream targets and induced apoptosis in primary CLL cells. With respect to prognostic factors, SYK inhibitors exerted stronger cytotoxic effects in unmutated and ZAP70(+) cases. Cytotoxic effects of SYK inhibitors also associated with SYK protein expression, potentially predicting response to therapy. Combination of fludarabine with SYK Inhibitor II or R406 increased cytotoxicity compared with fludarabine therapy alone. We observed no stroma-contact-mediated drug resistance for SYK inhibitors as described for fludarabine treatment. CD40 ligation further enhanced efficacy of SYK inhibition. Our data provide mechanistic insight into the recently observed therapeutic effects of the SYK inhibitor R406 in CLL. Combination of SYK inhibitors with fludarabine might be a novel treatment option particularly for CLL patients with poor prognosis and should be further evaluated in clinical trials.

CD40L induces inflammation and adipogenesis in adipose cells - a potential link between metabolic and cardiovascular disease

CD40L figures prominently in atherogenesis. Recent data demonstrate elevated levels of sCD40L in the serum of patients with the metabolic syndrome (MS). This study investigated the role of CD40L in pro-inflammatory gene expression and cellular differentiation in adipose tissue to obtain insight into mechanisms linking the MS with atherosclerosis. Human adipocytes and preadipocytes expressed CD40 but not CD40L. Stimulation with recombinant CD40L or membranes over-expressing CD40L induced a time- and dose-dependent expression of IL-6, MCP-1, IL-8, and PAI-1. Supernatants of CD40L-stimulated adipose cells activated endothelial cells, suggesting a systemic functional relevance of our findings. Neutralising antibodies against CD40L attenuated these effects substantially. Signalling studies revealed the involvement of mitogen-activated protein kinases and NFkB. Furthermore, stimulation with CD40L resulted in enhanced activation of C/EBPa and PPARg and promoted adipogenesis of preadipose cells in the presence and absence of standard adipogenic conditions. Finally, patients suffering from the metabolic syndrome with high levels of sCD40L also displayed high levels of IL-6, in line with the concept that CD40L may induce the expression of inflammatory cytokines in vivo in this population. Our data reveal potent metabolic functions of CD40L aside from its known pivotal pro-inflammatory role within plaques. Our data suggest that CD40L may mediate risk at the interface of metabolic and atherothrombotic disease.

Tumor Necrosis Factor Receptor–Associated Factor 1 (TRAF1) Deficiency Attenuates Atherosclerosis in Mice by Impairing Monocyte Recruitment to the Vessel Wall

Background— Members of the tumor necrosis factor superfamily, such as tumor necrosis factor-&agr;, potently promote atherogenesis in mice and humans. Tumor necrosis factor receptor–associated factors (TRAFs) are cytoplasmic adaptor proteins for this group of cytokines. Methods and Results— This study tested the hypothesis that TRAF1 modulates atherogenesis in vivo. TRAF1−/−/LDLR−/− mice that consumed a high-cholesterol diet for 18 weeks developed significantly smaller atherosclerotic lesions than LDLR−/− (LDL receptor–deficient) control animals. As the most prominent change in histological composition, plaques of TRAF1-deficient animals contained significantly fewer macrophages. Bone marrow transplantations revealed that TRAF1 deficiency in both hematopoietic and vascular resident cells contributed to the reduction in atherogenesis observed. Mechanistic studies showed that deficiency of TRAF1 in endothelial cells and monocytes reduced adhesion of inflammatory cells to the endothelium in static and dynamic assays. Impaired adhesion coincided with reduced cell spreading, actin polymerization, and CD29 expression in macrophages, as well as decreased expression of the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in endothelial cells. Small interfering RNA studies in human cells verified these findings. Furthermore, TRAF1 messenger RNA levels were significantly elevated in the blood of patients with acute coronary syndrome. Conclusions— TRAF1 deficiency attenuates atherogenesis in mice, most likely owing to impaired monocyte recruitment to the vessel wall. These data identify TRAF1 as a potential treatment target for atherosclerosis.

The Oral Spleen Tyrosine Kinase Inhibitor Fostamatinib Attenuates Inflammation and Atherogenesis in Low-Density Lipoprotein Receptor–Deficient Mice

Objective— Spleen tyrosine kinase (SYK) has come into focus as a potential therapeutic target in chronic inflammatory diseases, such as rheumatoid arthritis and asthma, as well as in B-cell lymphomas. SYK has also been involved in the signaling of immunoreceptors, cytokine receptors, and integrins. We therefore hypothesized that inhibition of SYK attenuates the inflammatory process underlying atherosclerosis and reduces plaque development. Methods and Results— Low-density lipoprotein receptor–deficient mice consuming a high-cholesterol diet supplemented with 2 doses of the orally available SYK inhibitor fostamatinib for 16 weeks showed a dose-dependent reduction in atherosclerotic lesion size by up to 59±6% compared with the respective controls. Lesions of fostamatinib-treated animals contained fewer macrophages but more smooth muscle cells and collagen—characteristics associated with more stable plaques in humans. Mechanistically, fostamatinib attenuated adhesion and migration of inflammatory cells and limited macrophage survival. Furthermore, fostamatinib normalized high-cholesterol diet –induced monocytosis and inflammatory gene expression. Conclusion— We present the novel finding that the SYK inhibitor fostamatinib attenuates atherogenesis in mice. Our data identify SYK inhibition as a potentially fruitful antiinflammatory therapeutic strategy in atherosclerosis.

Insulin-like growth factor-1 receptor (IGF1R) as a novel target in chronic lymphocytic leukemia

The receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor (IGF1R) is implicated in various tumor entities including chronic lymphocytic leukemia (CLL), but its functional significance in this disease remains poorly characterized. Here, we show that the IGF1R protein is overexpressed in various CLL subsets, suggesting a contribution to CLL pathology. Indeed, we show that IGF1R knockdown in primary human CLL cells compromised their viability. Likewise, IGF1R inhibition with 3 structurally distinct compounds induced apoptosis, even in the presence of protective stroma components. Furthermore, IGF1R inhibition effectively limited CLL development in Eμ-TCL1 transgenic mice and of primary human CLL xenografts. In agreement with its prosurvival function, IGF1R inhibition affected the phosphorylation and/or expression of multiple signaling proteins. The multikinase inhibitor sorafenib yielded similar effects on these signaling elements as IGF1R inhibitors. Indeed, IGF1R appears to be a direct sorafenib target because sorafenib decreased IGF1R expression and phosphorylation, counteracted insulin-like growth factor-1 (IGF-1) binding to CLL cells, and lowered the in vitro kinase activity of recombinant, purified IGF1R. Thus, we demonstrate that blockade of IGF1R-mediated signaling represents a novel mechanism of action for sorafenib in CLL. Importantly, IGF1R inhibitors compromise CLL viability in their microenvironment context, implicating this RTK as a promising therapeutic target.

Spleen tyrosine kinase (Syk) is a potent target for GvHD prevention at different cellular levels.

Acute graft-versus-host disease (GvHD) limits the applicability of allogeneic hematopoietic cell transplantation for the treatment of leukemia. GvHD occurs as a consequence of multiple activating events in antigen-presenting cells (APCs) and T cells (Tcs). Spleen tyrosine kinase (Syk) is an intracellular non-receptor tyrosine kinase involved in multiple signaling events of immune cells. Therefore, we hypothesized that Syk may be a promising target to inhibit GvHD, which involves activation of different immune cell populations. In vivo expansion of luciferase+ donor Tcs in mice developing GvHD was reduced by treatment with the Syk inhibitor Fostamatinib, which led to increased survival and reduced histologically confirmed GvHD severity. Importantly, in vivo and in vitro cytotoxicity against leukemia target cells and anti-murine cytomegalovirus immune responses were not impacted by Fostamatinib. In APCs Syk inhibition reduced the expression of costimulatory molecules and disrupted cytoskeletal organization with consecutive APC migratory defects in vitro and in vivo while phagocytic activity remained intact. On the basis of these immunomodulatory effects on different cell populations, we conclude that Syk targeting in alloantigen-activated Tcs and APCs with pharmacologic inhibitors, already applied successfully in anti-lymphoma therapy, has clinical potential to reduce GvHD, especially as anti-leukemia and anti-viral immunity were preserved.