ABSTRACT
The clinical success of B-cell receptor (BCR) signaling pathway inhibitors in chronic lymphocytic leukemia (CLL) is attributed to inhibition of adhesion in and migration towards the lymph node. Proliferation of CLL cells is restricted to this protective niche, but the underlying mechanism(s) is/are not known. Treatment with BCR pathway inhibitors results in rapid reductions of total clone size, while CLL cell survival is not affected, which points towards inhibition of proliferation. In vitro, BCR stimulation does not induce proliferation of CLL, but triggering via Toll-like receptor, tumor necrosis factor or cytokine receptors does. Here, we investigated the effects of clinically applied inhibitors that target BCR signaling, in the context of proliferation triggered either via CD40L/IL-21 or after CpG stimulation. CD40L/IL-21-induced proliferation could be inhibited by idelalisib and ibrutinib. We demonstrate this was due to blockade of CD40L-induced ERK-signaling. Targeting JAKs, but not SYK, blocked CD40L/IL-21-induced proliferation. In contrast, PI3K, BTK as well as SYK inhibition prevented CpG-induced proliferation. Knockdown experiments showed that CD40L/IL-21 did not co-opt upstream BCR components such as CD79A, in contrast to CpG-induced proliferation. Our data indicate that currently applied BTK/PI3K inhibitors target antigen-independent proliferation in CLL, and suggest that targeting of JAK and/or SYK might be clinically useful.
Subject(s)
Antigens, Neoplasm/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Phosphotransferases/antagonists & inhibitors , Biomarkers , CD40 Antigens/immunology , CD40 Antigens/metabolism , Cell Line, Tumor , Cell Proliferation , Humans , Interleukins/metabolism , Janus Kinases/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , NF-kappa B/metabolism , Phosphotransferases/metabolism , Protein Kinase Inhibitors/pharmacology , Receptors, Antigen, B-Cell/metabolism , STAT Transcription Factors/metabolism , Signal Transduction/drug effects , Tumor Cells, CulturedABSTRACT
Protective interactions with bystander cells in micro-environmental niches, such as lymph nodes (LNs), contribute to survival and therapy resistance of chronic lymphocytic leukemia (CLL) cells. This is caused by a shift in expression of B-cell lymphoma 2 (BCL-2) family members. Pro-survival proteins B-cell lymphoma-extra large (BCL-XL), BCL-2-related protein A1 (BFL-1) and myeloid leukemia cell differentiation protein 1 (MCL-1) are upregulated by LN-residing T cells through CD40L interaction, presumably via nuclear factor (NF)-κB signaling. Macrophages (Mφs) also reside in the LN, and are assumed to provide important supportive functions for CLL cells. However, if and how Mφs are able to induce survival is incompletely known. We first established that Mφs induced survival because of an exclusive upregulation of MCL-1. Next, we investigated the mechanism underlying MCL-1 induction by Mφs in comparison with CD40L. Genome-wide expression profiling of in vitro Mφ- and CD40L-stimulated CLL cells indicated activation of the phosphoinositide 3-kinase (PI3K)-V-Akt murine thymoma viral oncogene homolog (AKT)-mammalian target of rapamycin (mTOR) pathway, which was confirmed in ex vivo CLL LN material. Inhibition of PI3K-AKT-mTOR signaling abrogated MCL-1 upregulation and survival by Mφs, as well as CD40 stimulation. MCL-1 can be regulated at multiple levels, and we established that AKT leads to increased MCL-1 translation, but does not affect MCL-1 transcription or protein stabilization. Furthermore, among Mφ-secreted factors that could activate AKT, we found that induction of MCL-1 and survival critically depended on C-C motif chemokine receptor-1 (CCR1). In conclusion, this study indicates that two distinct micro-environmental factors, CD40L and Mφs, signal via CCR1 to induce AKT activation resulting in translational stabilization of MCL-1, and hence can contribute to CLL cell survival.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Macrophages/pathology , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Receptors, CCR1/metabolism , Animals , Apoptosis/physiology , CD40 Ligand/genetics , CD40 Ligand/metabolism , Cell Line, Tumor , Cell Survival/physiology , Down-Regulation , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Macrophages/metabolism , Mice , Myeloid Cell Leukemia Sequence 1 Protein/genetics , NIH 3T3 Cells , Receptors, CCR1/genetics , Signal Transduction , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , TransfectionABSTRACT
Resistance to chemotherapy-induced apoptosis in CLL is associated with overexpression of antiapoptotic proteins induced by signals from the microenvironment. In vitro, dasatinib effectively inhibits expression of anti-apoptotic regulators and restores fludarabine sensitivity in activated CLL. The aim of this study was to evaluate efficacy of one cycle of dasatinib monotherapy (100mg/day, days 1-28) followed by combination of dasatinib with fludarabine (40mg/m²/day, days 1-3 every 28 day) for a total of 6 cycles in fludarabine-refractory CLL. The primary endpoint was overall response rate according to the IWCLL'08 criteria. 20 patients were enrolled: 18 completed at least one cycle of treatment of which 67% finished at least 2 cycles of combination treatment. 3 of these 18 patients reached a formal PR (16.7%). Majority of patients obtained some reduction in lymph node (LN) size. Most frequent toxicity was related to myelosuppression. NF-κB RNA expression levels of circulating CLL cells decreased whereas the levels of pro-apoptotic NOXA increased during treatment. In conclusion, dasatinib/fludarabine combination has modest clinical efficacy in fludarabine-refractory patients.
