Sanne H. Tonino

Dichotomy in NF-kappaB signaling and chemoresistance in immunoglobulin variable heavy-chain-mutated versus unmutated CLL cells upon CD40/TLR9 triggering.

Chronic lymphocytic leukemia (CLL) cells circulating in peripheral blood (PB) differ from the leukemic fraction in lymph nodes (LNs) with respect to cell division and drug sensitivity. CD40 stimulation of PB CLL cells in vitro results in chemoresistance and provides a partial model for the LN microenvironment. The TLR9 ligand CpG induces proliferation in immunoglobulin variable heavy-chain-unmutated CLL, but apoptosis in immunoglobulin variable heavy-chain-mutated CLL. To juxtapose proliferative with antiapoptotic signals, we investigated the effects of CpG in the context of CD40 ligation in mutated versus unmutated CLL cells in this study. Prolonged CD40 ligation induced classical, followed by alternative nuclear factor-κB (NF-κB), activity in both subgroups, correlating with enhanced Bfl-1 and Bcl-XL levels, respectively. A dichotomy in NF-κB signaling occurred on combined CD40/TLR9 triggering. This induced declining p52 and Bcl-XL levels, and reversed chemoresistance only in mutated cells, whereas unmutated cells proliferated, maintained p52 and Bcl-XL and remained chemoresistant. The pivotal contribution of Bcl-XL to chemoresistance was shown by the BH3 mimetic ABT-737 and RNA interference. Finally, in ex vivo LN samples, p52, p65 and Bcl-XL levels were highly expressed, corroborating the in vitro findings. Thus, a distinction in NF-κB activation and drug susceptibility in mutated versus unmutated (LN-like) CLL cells was uncovered, which was causally linked to Bcl-XL levels.

ROS-mediated upregulation of Noxa overcomes chemoresistance in chronic lymphocytic leukemia

In recent years considerable progress has been made in treatment strategies for chronic lymphocytic leukemia (CLL). However, the disease remains incurable because of the development of chemoresistance. Strategies to overcome resistance mechanisms are therefore highly needed. At least two mechanisms contribute to the development of resistance to drugs; acquired mutations resulting in a dysfunctional p53 response and shifts in the balance between apoptosis-regulating proteins. Platinum-based compounds have been successfully applied in relapsed lymphoma and recently also in high-risk CLL. In this study we investigated the efficacy and mechanism of action of cisplatinum (CDDP) in chemorefractory CLL. Independent of p53-functional status, CDDP acted synergistically with fludarabine (F-ara-A). The response involved generation of reactive oxygen species (ROS), which led to specific upregulation of the proapoptotic BH3-only protein Noxa. Induction of Noxa resulted in cell death by apoptosis as inhibition of caspase activation completely abrogated cell death. Furthermore, drug-resistance upon CD40-ligand stimulation, a model for the protective stimuli provided in lymph nodes, could also be overcome by CDDP/F-ara-A. ROS accumulation resulted in Noxa upregulation mainly at the transcriptional level and this was, at least in part, mediated by the mitogen-activated protein kinase p38. Finally, Noxa RNA-interference markedly decreased sensitivity to CDDP/F-ara-A, supporting a key role for Noxa as mediator between ROS signaling and apoptosis induction. Our data indicate that interference in the cellular redox balance can be exploited to overcome chemoresistance in CLL.

How does lenalidomide target the chronic lymphocytic leukemia microenvironment

Immunotherapy has emerged as a viable clinical strategy to harness endogenous antitumor T-cell immunity. Lenalidomide is an oral immunomodulatory drug that repairs antitumor T-cell function and is showing efficacy in ongoing chronic lymphocytic leukemia (CLL) and lymphoma clinical trials. This article focuses on advances in our understanding of its mechanism of action in the tumor microenvironment and provides a clinical update in CLL. Challenges associated with this drug and its potential use in the targeted drug treatment era are discussed.

CMV-specific CD8+ T-cell function is not impaired in chronic lymphocytic leukemia

In chronic lymphocytic leukemia (CLL), CD8(+) T cells exhibit features of exhaustion and impaired functionality. Yet, reactivations of latent viruses such as cytomegalovirus (CMV) are uncommon in untreated CLL, suggesting that antiviral responses are uncompromised. We analyzed phenotypical and functional characteristics of CMV-specific CD8(+) T cells in CLL patients in comparison with age-matched healthy controls (HCs). Despite increased expression of the inhibitory receptors PD1, CD160, and CD244 on total CD8(+) T cells in CLL, expression levels of these markers were decreased on CMV-tetramer(+)CD8(+) T cells. Second, cytokine production upon stimulation with both phorbol 12-myristate 13-acetate/ionomycin and CMV-peptide-loaded antigen-presenting cells was intact in CMV-tetramer(+)CD8(+) T cells. Third, CMV-tetramer(+)CD8(+) T cells of CLL patients and HCs were equally effective in killing CMV-peptide-loaded target cells. Finally, quantitative imaging flow cytometry revealed that the proportion of CD8(+) T cells forming immunologic synapses with CMV-peptide-loaded B cells was intact. In conclusion, despite evidence for global T-cell dysfunction in CLL, we show here that CLL-derived CMV-specific CD8(+) T cells display lower expression of exhaustion markers and are functionally intact. These data indicate that the changes in the T-cell compartment in CLL may be more heterogeneous than presently assumed.

Expansion of effector T cells associated with decreased PD-1 expression in patients with indolent B cell lymphomas and chronic lymphocytic leukemia

Abstract In patients with chronic lymphocytic leukemia (CLL), numbers of CD8 + CD45RA +/− CD27− effector T cells are expanded. We investigated whether this expansion is also present in other B cell malignancies and the possible mechanism underlying these changes. Whereas an increase in total CD4+and CD8+ T cell numbers was found only in CLL, numbers of CD4+ and CD8+ effector T cells were significantly increased in both CLL and indolent lymphoma, but not aggressive lymphoma and myeloma. Interestingly, PD-1 expression was decreased on effector T cells and inversely correlated with effector T cell numbers, suggesting a functional role for PD-1 in regulating T cell homeostasis. In vitro experiments revealed impaired up-regulation of PD-1 upon T cell activation in the presence of malignant but also healthy B cells. Our data suggest that in CLL and indolent lymphoma, the malignant B cells affect PD-1 expression on effector T cells, resulting in an expansion of these subsets.

R-DHAP is effective in fludarabine-refractory chronic lymphocytic leukemia

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No convincing evidence for a role of CD31-CD38 interactions in the pathogenesis of chronic lymphocytic leukemia.

Although CD38, a marker of poor prognosis in chronic lymphocytic leukemia (CLL), is known primarily as an ecto-enzyme, it has also been ascribed a receptor function. Interaction with its proposed ligand CD31 expressed on nurse-like cells would result in proliferative and survival-signals. Yet, in CLL, both homotypic and heterotypic CD31-CD38 interactions are expected to be rather ubiquitous. We analyzed whether CD38-CD31 interactions result in proliferative and antiapoptotic signals. We found a high expression of CD31 on CLL, irrespective of CD38 expression. Coculture of CD38(high) CLL with endothelial cells or CD31 transfected fibroblasts, with or without blocking CD31 or CD38 antibodies, did not result in increased survival or proliferation. Analysis of gene expression of most known regulators of apoptosis revealed no influence of coculture with CD31-expressing feeder cells. In conclusion, our data do not support an important contribution of CD38 triggering by CD31 to the proliferative and antiapoptotic state of the leukemic clone.

Chronic lymphocytic leukemia specific T-cell subset alterations are clone-size dependent and not present in monoclonal B lymphocytosis

Abstract In patients with chronic lymphocytic leukemia (CLL), effector and memory CD4 + and CD8 + T cells are expanded. We investigated whether these CLL specific T-cell expansions also occur in monoclonal B lymphocytosis (MBL), the pre-leukemic state of CLL, which is currently distinguished from CLL by an arbitrarily chosen cut-off value of CD19 of 5.0 × 109/L. Whereas an increase in effector and memory CD4 + and CD8 + T cells was found in CLL, these expansions could not be found in MBL. Although a significant correlation was found between absolute B cell count (CD19) and T cell numbers, correlation coefficients were rather low. Therefore, we analyzed whether an optimal threshold for CD19 number could be defined which best related to an expansion of T cells. The B-cell threshold that best predicted expansion of CD3 +, CD4 + and CD8 + T cells, respectively, was 10 × 109/L. Our study indicates that a higher cut-off value than the current 5.0 × 109/L relates better to the biological impact of CLL.

Standards for the treatment of relapsed chronic lymphocytic leukemia: a case-based study.

In recent years, considerable advances have been made in first-line treatment strategies for chronic lymphocytic leukemia (CLL). Combination of conventional chemotherapy with immunotherapeutic agents is currently considered the most active strategy, with improved progression-free survival and overall survival. However, patients are not cured and invariably experience relapsing disease requiring treatment. In contrast to the advances made in first-line treatment strategies, much less progress has been made for patients with relapsed and especially refractory CLL. The activity of most chemotherapeutic drugs used in CLL rely on intact p53 function, and repeated cycles of therapy might eventually result in drug resistance because of acquired cytogenetic alterations, mainly affecting genes involved in the p53 response. As a consequence, most commonly used treatment regimens are ineffective in patients with refractory disease. A number of promising alternative treatment approaches are currently under investigation. In this review, the approach to patients with relapsed and refractory CLL and current promising experimental treatment options for these distinct clinical patient categories are discussed.

Induction of TAp73 by platinum-based compounds to overcome drug resistance in p53 dysfunctional chronic lymphocytic leukemia

In chronic lymphocytic leukemia (CLL), strategies to overcome drug resistance due to p53 dysfunction are highly needed. Platinum-based compounds such as cisplatinum (CDDP) are active in fludarabine-refractory CLL through a largely unknown mechanism. We analyzed the mechanism of action of CDDP in the context of p53 dysfunctionality. In vitro treatment with CDDP did not induce death in quiescent CLL cells, but did induce apoptosis in CD40-ligand (and CpG) stimulated and proliferating cells, irrespective of p53 function. In the p53 dysfunctional prolymphocytic cell-line MEC1, CDDP treatment resulted in apoptosis, cell cycle arrest and ABL1-dependent expression of TAp73, CDKN1A, PUMA and BID. TAp73 RNA-interference decreased sensitivity to CDDP. Finally, both in vitro stimulated CLL cells and lymph node (LN) derived CLL cells showed increased TAp73 expression in comparison with quiescent peripheral blood derived cells. Activity of CDDP may therefore be mediated by TAp73, especially in the context of activation such as occurs in the LN microenvironment.