Daniela Asslaber

MicroRNA-34a expression correlates with MDM2 SNP309 polymorphism and treatment-free survival in chronic lymphocytic leukemia

In chronic lymphocytic leukemia (B-CLL), aberrations along the p53 axis lead to decreased overall survival and therapy resistance. Recent studies identified microRNA-34a (miR-34a) as a major downstream target of p53. We monitored the expression of miR-34a during disease development in a murine B-CLL model. miR-34a was up-regulated more than 20-fold during the leukemic but not during the preleukemic phase. In the human system, B-CLL cells also had 4.6-fold higher miR-34a expression compared with B cells of healthy controls. In B-CLL cells of patients with p53 aberrations, miR-34a expression was consistently low. The broad distribution of miR-34a levels in p53 wild-type patients prompted us to study the correlation between single nucleotide polymorphism 309 (SNP309) in the intronic promoter of MDM2 and miR-34a expression. B-CLL cells of patients with the SNP309 GG genotype had significantly lower miR-34a expression levels compared with patients with the TT genotype (P = .002). Low miR-34a levels were able to predict shorter time to treatment (P = .003) and were associated with an abbreviated lymphocyte doubling time. Further, overexpression of miR-34a in primary B-CLL cells induced apoptosis. These findings suggest miR-34a as a possible therapeutic avenue and a sensitive indicator of the activity of the p53 axis in B-CLL.

Development of CLL in the TCL1 transgenic mouse model is associated with severe skewing of the T-cell compartment homologous to human CLL

Chronic lymphocytic leukemia (CLL) cells require complex microenvironmental and immunologic interactions to survive and proliferate. Such interactions might be best recreated in animal models; however, this needs extensive verification. We therefore investigated the composition of the T-cell compartment in the Eμ-TCL1 transgenic mouse, currently the most widely used murine model for CLL. Immunophenotyping and transplant approaches were used to define T-cell subsets at various stages of CLL. Analogous to human CLL, we observed a skewing of T-cell subsets from naive to antigen-experienced memory T cells that was more pronounced in lymph nodes than in blood. Transplantation of CLL into non-transgenic recipients was feasible without immunosuppression in a pure C57BL/6 background and resulted in the prominent skewing of the T cells of the recipient mice. Both in spontaneously developed CLL and in the transplantation setting, a loss in T-cell receptor diversity was observed, with a relevant number of clonal T-cell populations arising. This suggests that antigen-dependent differentiation toward the T memory pool is initiated by murine CLL cells. In summary, we validate the TCL1 transgenic mouse model for analysis of T-cell phenotypes and suggest a CLL-dependent antigen-driven skewing of T cells in these mice.

Tiam1/Rac1 signals contribute to the proliferation and chemoresistance, but not motility, of chronic lymphocytic leukemia cells

Signals from the tumor microenvironment promote the migration, survival, and proliferation of chronic lymphocytic leukemia (CLL) cells. Rho GTPases control various signaling pathways downstream of microenvironmental cues. Here, we analyze the function of Rac1 in the motility and proliferation of CLL cells. We found decreased transcription of the Rac guanine nucleotide exchange factors Tiam1 and Vav1 in unstimulated peripheral blood CLL cells with almost complete loss of Tiam1 but increased transcription of the potential Rac antagonist RhoH. Consistently, stimulation of CLL cells with the chemokine CXCL12 induced RhoA but not Rac1 activation, whereas chemokine-induced CLL cell motility was Rac1-independent. Coculture of CLL cells with activated T cells induced their activation and subsequent proliferation. Here, Tiam1 expression was induced in the malignant cells in line with increased Ki-67 and c-Myc expression. Rac1 or Tiam1 knockdown using siRNA or treatment with the Tiam1/Rac inhibitor NSC-23766 attenuated c-Myc transcription. Furthermore, treatment of CLL cells with NSC-23766 reduced their proliferation. Rac inhibition also antagonized the chemoresistance of activated CLL cells toward fludarabine. Collectively, our data suggest a dynamic regulation of Rac1 function in the CLL microenvironment. Rac inhibition could be of clinical use by selectively interfering with CLL cell proliferation and chemoresistance.

CD40-Mediated Activation of Chronic Lymphocytic Leukemia Cells Promotes Their CD44-Dependent Adhesion to Hyaluronan and Restricts CCL21-Induced Motility

Microenvironmental interactions are crucial for the survival and proliferation of chronic lymphocytic leukemia (CLL) cells. CD4+ T cells that express CD40 ligand (CD40L), along with other accessory immune and stromal cells within CLL lymph nodes, provide signals needed for activation and outgrowth of the tumor clone. Furthermore, correct positioning of CLL cells within lymphoid subcompartments is essential for the transmission of these supportive signals. Thereby, interstitial cell migration and adhesion events, influenced by activational stimuli, determine CLL cell localization. CD44 has been implicated in cell activation, migration, and tissue retention via binding to its extracellular matrix ligand hyaluronan (HA). In this study, we investigated the role of CD44-HA interactions for CLL positioning and interaction with supportive microenvironments in peripheral lymph nodes, focusing on its regulation via CD40L-dependent, T-cell-mediated activation of CLL cells. We found that hyaluronan triggered a robust CCL21-induced motility of resting CLL cells. However, CD40L stimulation promoted the firm, CD44-mediated adhesion of CLL cells to hyaluronan, antagonizing their motile behavior. N-linked glycosylations of CD44, particularly associated with the variant isoform CD44v6 after CD40L activation, seemed to facilitate hyaluronan recognition by CD44. We propose that the CD40L-CD40 signaling axis provides a stop signal to motile CLL cells within lymph node compartments by inducing high avidity CD44-HA adhesion. This might retain CLL cells close to T-cell stimuli and facilitate essential interactions with hyaluronan-bearing stromal cells, collectively promoting CLL cell proliferation and survival.

Mimicking the microenvironment in chronic lymphocytic leukaemia – where does the journey go?

Emerging evidence suggests that the growth and survival of chronic lymphocytic leukaemia (CLL) cells requires the support and stimulation by T cells and stromal cells in the lymphoid microenvironment (Herishanu et al, 2011). Substantial proliferation rates have been observed in vivo (Messmer et al, 2005), and particularly, CLL cells adjacent to activated CD40 Ligand (CD40LG)-expressing CD4 T cells in the nodes are prone to proliferate (Patten et al, 2008). Nevertheless, experimental in vitro studies of proliferating CLL cells are still impaired by the difficulties in mimicking this complex microenvironment. Moreover, preclinical testing of potential new CLL therapies has mainly been restricted to cytotoxicity assays of CLL cells, partly taking the protective effect of stromal or nurse-like cells into account (Burger et al, 2009). However, physiologically relevant models should not only provide pro-survival support of the G0-arrested peripheral blood CLL cells but also stimulate their proliferation. Hence, several CD40LG-based assays have been suggested (Hallaert et al, 2008; Plander et al, 2009). The growing number of available co-culture systems raises difficulties in comparing the data achieved with these highly variable approaches. More back-to-back characterizations of resulting phenotypes, including the extent of proliferation and/or survival support, would help to select the appropriate in vitro system and also to rate and evaluate data that have been gained in a certain system. Hamilton et al (2012) characterized three different co-culture systems mimicking the tumour microenvironment. They co-cultured CLL cells with fibroblasts that overexpressed either CD40LG or CD31, or with endothelial cells. While all approaches resulted in an activated CLL phenotype with increased expression of the early activation marker CD69, the ectoenzyme CD38, and the adhesion molecules CD44 and CD49d, only CD40LGor CD31-transfected fibroblasts induced relevant CLL cell proliferation, coinciding with increased ZAP-70 expression. These data demonstrated that in CLL, multiple cell types are capable of providing prosurvival signals with remarkably similar resulting phenotypes. However, this contact-dependent activation does not necessarily translate into induction of proliferation. In order to add to the study of Hamilton et al (2012) and as a result of the high relevance of CLL-T cell interactions in vivo (Patten et al, 2008; Bagnara et al, 2011; Hofbauer et al, 2011), we have compared co-culture systems based on activated (CD40LG) T cells or CD40LG-transfected fibroblasts mimicking activated T cells. First, anti-CD3/CD28 microbeads were used to activate autologous T cells in peripheral blood mononuclear cells (PBMCs) from CLL patients. The co-culture system also contained a murine fibroblast layer. Using this system, we detected CLL cell cycle induction after 3 d (Fig 1A, left) and actual cell division after 5 d (Fig 1A, right). The presence of the fibroblast layer resulted in increased viability and CD69 expression of CLL cells but did not induce proliferation by itself (Fig 1B). Moreover, co-culture systems containing fibroblasts in addition to activated T cells yielded higher proliferation rates (Fig 1C), probably by lowering the activation threshold or as a result of sustained CLL cell viability. Notably, increased surface expression of the co-stimulatory molecules CD80 and CD86 on CLL cells coincided with their proliferation (Fig 1C). Thus, we propose CD80 and CD86 rather than the often-suggested CD69 as suitable markers for induction of CLL cell proliferation. Hypothesizing that an increased CD4 T:CLL cell ratio could further increase CLL cell proliferation, we next isolated autologous CD4 T cells from CLL samples, activated them using microbeads, and re-combined them with the corresponding purified CLL cells in a 1:3 ratio on a fibroblast layer, resulting in striking cell division rates of about 25% (Fig S1A). Using this high T:CLL ratio, a relevant amount of CLL cells even proliferated without the need of prior T cell activation. However, as the ratio of autologous T:CLL cells is highly variable among the patient samples, this approach is not easily applicable for routine measurements. Therefore, we also performed proliferation assays using isolated allogeneic CD4 T lymphocytes from a healthy donor, investigating the effect of escalating T:CLL ratios. This allogeneic setup revealed comparable proliferation rates to the autologous setting without evidence of a clear dose-effect (Fig S1B). We next tested the potential of CD40LG-stimulation to mimic activated T cells and thereby induce CLL proliferation. CLL cells co-cultured in direct contact with CD40LG-overexpressing fibroblasts developed a CD80 CD86 phenotype and efficiently proliferated in a CD40-CD40LG-dependent manner (Fig 2A), consistent with the findings of Hamilton et al (2012). However, when CLL cells were separated from the fibroblast layer by a transwell insert, thus preventing direct cell-cell contact, proliferation was abrogated. Moreover, soluble crosslinked CD40LG, additionally added to CLL cells in the upper part of the transwell, could significantly but not completely, restore their activation and proliferation Correspondence

Targeting proliferation of chronic lymphocytic leukemia (CLL) cells through KCa3.1 blockade

Although the dependence of Ca2+ signaling and mitosis on K+ channel activity in lymphocytes has been thoroughly examined,1 the therapeutic significance of these findings for malignant hematological diseases is largely unexplored. Out of approximately 80 different K+ channel genes in humans, T and B cells express the voltage-dependent K+ channel, Kv1.3, and the Ca2+-activated K+ channel, KCa3.1. Expression levels of K+ channels vary with lymphocyte maturation and activation state.1, 2 Accordingly, selective blockade of the predominant K+ channel type allows lymphocyte subset specific inhibition of proliferation.1, 2 Given the importance of controlling Ca2+-influx, there is growing interest in selective K+ channel blockers to suppress cell proliferation in autoimmune diseases and cancer.3, 4, 5

CD1d expression on chronic lymphocytic leukemia B cells affects disease progression and induces T cell skewing in CD8 positive and CD4CD8 double negative T cells

Chronic lymphocytic leukemia develops within a complex network driven by genetic mutations and microenvironmental interactions. Among the latter a complex interplay with the immune system is established by the clone. Next to a proposed recruitment of support from T and myeloid cells, potential anti-CLL immune reactions need to be subverted. By using TCL1 mice as a CLL model, we show that TCR-Vβ7+ NK1.1+ T cells are overrepresented in this disease model and constitute a main subset of peripheral CD3+ cells with biased TCR usage, showing that these cells account for a major part for T cell skewing in TCL1 mice. Moreover, we show that overrepresentation is dependent on CD1d expression in TCL1 mice, implicating that these cells belong to a NKT-like cell fraction which are restricted to antigen presented by the MHC-like surface marker CD1d. Accordingly, we observed a high fraction of CD161+ cells within overrepresented T cells in CLL patients and we found downregulation of CD1d on the surface of CLL cells, both in TCL1 mice and patients. Finally, we show that in TCL1 mice, CD1d deficiency resulted in shortened overall survival. Our results point to an interaction between CLL and CD161+ T cells that may represent a novel therapeutic target for immune modulation.

B cell receptor usage correlates with the sensitivity to CD40 stimulation and the occurrence of CD4+ T cell clonality in chronic lymphocytic leukemia

The mutation status of the B-cell receptor (BCR) is a strong prognostic factor in chronic lymphocytic leukemia (CLL), dividing patients into BCR mutated and unmutated CLL (CLL-Mut, CLL-UM), the latter predicting for worse prognosis.[1][1]–[3][2] Furthermore, the occurrence of highly similar BCRs

ILK Induction in Lymphoid Organs by a TNFα–NF-κB–Regulated Pathway Promotes the Development of Chronic Lymphocytic Leukemia

The proliferation of chronic lymphocytic leukemia (CLL) cells requires communication with the lymphoid organ microenvironment. Integrin-linked kinase (ILK) is a multifunctional intracellular adaptor protein that transmits extracellular signals to regulate malignant cell motility, metastasis, and cell-cycle progression, but is poorly characterized in hematologic malignancies. In this study, we investigated the role of ILK in the context of CLL and observed high ILK expression in patient samples, particularly in tumor cells harboring prognostic high-risk markers such as unmutated IGHV genes, high Zap70, or CD38 expression, or a signature of recent proliferation. We also found increased numbers of Ki67 (MKI67)-positive cells in regions of enhanced ILK expression in lymph nodes from CLL patients. Using coculture conditions mimicking the proliferative lymph node microenvironment, we detected a parallel induction of ILK and cyclin D1 (CCND1) expression in CLL cells that was dependent on the activation of NF-κB signaling by soluble TNFα. The newly synthesized ILK protein colocalized to centrosomal structures and was required for correct centrosome clustering and mitotic spindle organization. Furthermore, we established a mouse model of CLL in which B-cell-specific genetic ablation of ILK resulted in decelerated leukemia development due to reduced organ infiltration and proliferation of CLL cells. Collectively, our findings describe a TNFα-NF-κB-mediated mechanism by which ILK expression is induced in the lymph node microenvironment and propose that ILK promotes leukemogenesis by enabling CLL cells to cope with centrosomal defects acquired during malignant transformation. Cancer Res; 76(8); 2186-96. ©2016 AACR.

CD4+ T cells, but not non-classical monocytes, are dispensable for the development of chronic lymphocytic leukemia in the TCL1-tg murine model

CD4+ T cells, but not non-classical monocytes, are dispensable for the development of chronic lymphocytic leukemia in the TCL1-tg murine model