Martina Seiffert

Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts

Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected α-smooth actin-positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts.

Soluble CD14 is a novel monocyte-derived survival factor for chronic lymphocytic leukemia cells, which is induced by CLL cells in vitro and present at abnormally high levels in vivo.

Accumulation of leukemic cells in patients with chronic lymphocytic leukemia (CLL) is due to prolonged cell survival rather than increased proliferation. Survival of CLL cells depends on microenvironmental factors. Even though long-lived in vivo, CLL cells rapidly die by spontaneous apoptosis in vitro unless cocultured with stromal cells or their conditioned medium. In the present study, we show that survival of CLL cells is maintained in high cell density cultures, where the main prosurvival activity is delivered by monocytes. Cytokine array and enzyme-linked immunosorbent assay studies revealed increased expression of soluble CD14 by monocytes in the presence of CLL cells. The addition of recombinant soluble CD14 to primary CLL cells resulted in significantly increased cell survival rates, which were associated with higher activity nuclear factor κB. Quantification of serum levels of soluble CD14 revealed abnormally high levels of this protein in CLL patients, indicating a potential role of soluble CD14 in vivo. In summary, the presented data show that monocytes help in the survival of CLL cells by secreting soluble CD14, which induces nuclear factor κB activation in these cells, and that CLL cells actively shape their microenvironment by inducing CD14 secretion in accessory monocytes.

Inflammatory cytokines and signaling pathways are associated with survival of primary chronic lymphocytic leukemia cells in vitro: a dominant role of CCL2

Background Chronic lymphocytic leukemia cells show prolonged survival in vivo, but rapidly die by spontaneous apoptosis in vitro, unless they are co-cultured with stromal cells or non-malignant leukocytes. The objective of this study was to characterize the survival-inducing cross-talk of chronic lymphocytic leukemia cells with their microenvironment to identify novel therapeutic targets. Design and Methods We analyzed and compared microarray-based expression profiles of chronic lymphocytic leukemia cells before and after three different survival-inducing culture conditions: (i) stromal cell co-culture, (ii) stromal cell conditioned medium and (iii) high cell density cultures of unsorted peripheral blood mononuclear cells. Cytokine antibody arrays were applied to study the composition of soluble factors present in these cultures. Results The different survival-supportive culture conditions induced distinct gene expression changes, the majority of which were common to all three conditions. Pathway analyses identified – in addition to known signaling networks in chronic lymphocytic leukemia – novel pathways, of which Toll-like receptor signaling, nuclear respiratory factor-2 (NRF2)-mediated oxidative stress response, and signaling via triggering receptor expressed on myeloid cells-1 (TREM1) were the most relevant. A high proportion of up-regulated genes were inflammatory cytokines, of which chemokine (C-C motif) ligand 2 (CCL2) was shown to be induced in monocytes by the presence of chronic lymphocytic leukemia cells in vitro. In addition, increased serum levels of this chemokine were detected in patients with chronic lymphocytic leukemia. Conclusions Our data provide several lines of evidence that an inflammatory microenvironment is induced in survival-supportive cultures of chronic lymphocytic leukemia cells which might be directly or indirectly involved in the prolonged survival of the malignant cells.

Efficient nucleofection of primary human B cells and B-CLL cells induces apoptosis, which depends on the microenvironment and on the structure of transfected nucleic acids

Accumulation of neoplastic cells in B-cell chronic lymphocytic leukemia (B-CLL) is thought to be due to intrinsic defects in the apoptotic machinery of the leukemic cells or to an altered, survival-stimulating microenvironment in vivo. Despite their long survival in vivo, B-CLL cells undergo rapid spontaneous apoptosis ex vivo. To maintain survival in vitro, we established a coculture system using the human bone marrow-derived stromal cell line HS-5. The microenvironment in these cocultures lead to B-CLL cell survival for at least several months and therefore provided a tool for valid in vitro analysis, mimicking the in vivo situation. Although primary B lymphocytes are notoriously resistant to most gene transfer techniques, we achieved high transfection efficiency and cell viability in this coculture system by using a nucleofection-based strategy. Surprisingly, the introduction of circular plasmid DNA into B cells and B-CLL cells induced rapid apoptosis, which was independent of the type of transgene used, but dependent on the DNA concentration. However, transfection of these cells with mRNA was highly efficient and resulted in sustained cell viability and potent transgene expression. The described procedure represents a new approach to study gene function in primary B cells and B-CLL cells.

Depletion of CLL-associated patrolling monocytes and macrophages controls disease development and repairs immune dysfunction in vivo.

Chronic lymphocytic leukemia (CLL) is characterized by apoptosis resistance and a dysfunctional immune system. Previous reports suggested a potential role of myeloid cells in mediating these defects. However, the composition and function of CLL-associated myeloid cells have not been thoroughly investigated in vivo. Using the Eμ-TCL1 mouse model, we observed severe skewing of myeloid cell populations with CLL development. Monocytes and M2-like macrophages infiltrated the peritoneal cavity of leukemic mice. Monocytes also accumulated in the spleen in a CCR2-dependent manner, and were severely skewed toward Ly6Clow patrolling or nonclassical phenotype. In addition, the percentage of MHC-IIhi dendritic cells and macrophages significantly dropped in the spleen. Gene expression profiling of CLL-associated monocytes revealed aberrantly high PD-L1 expression and secretion of multiple inflammatory and immunosuppressive cytokines like interleukin-10, tumor necrosis factor-α and CXCL9. In vivo myeloid cell depletion using liposomal Clodronate resulted in a significant control of CLL development accompanied by a pronounced repair of innate immune cell phenotypes and a partial resolution of systemic inflammation. In addition, CLL-associated skewing of T cells toward antigen-experienced phenotypes was repaired. The presented data suggest that targeting nonmalignant myeloid cells might serve as a novel immunotherapeutical strategy for CLL.

Lenalidomide reduces survival of chronic lymphocytic leukemia cells in primary cocultures by altering the myeloid microenvironment

Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental stimuli for their survival, provided for example by monocyte-derived nurse-like cells (NLCs). The immunomodulatory drug lenalidomide shows therapeutic effects in subgroups of CLL patients, and is believed to act via the microenvironment. To investigate the effects of lenalidomide on the survival support of NLCs, cocultures of monocytes and CLL cells were treated for 14 days with lenalidomide, which resulted in significantly decreased viability of CLL cells. Among the changes induced by this drug, we observed reduced expression of HLA-DR in NLCs as well as increased secretion of interleukin-10 (IL-10), indicating an altered inflammatory milieu in the cocultures. The increase in IL-10 levels lead to an induction of STAT1 phosphorylation in CLL cells and to enhanced cell-surface expression of intercellular adhesion molecule 1 and altered expression of cytoskeletal and migration-related genes. Chemotaxis assays with lenalidomide-treated CLL cells revealed an impaired migration capability. Our data show that lenalidomide reduces the survival support of NLCs for CLL cells in vitro, suggesting that this drug affects the myeloid microenvironment in CLL in vivo. Furthermore, lenalidomide acts on the migratory potential of CLL cells, which may affect circulation and homing of CLL cells in vivo.

Chaetoglobosin A preferentially induces apoptosis in chronic lymphocytic leukemia cells by targeting the cytoskeleton

Chronic lymphocytic leukemia (CLL) is an incurable malignancy of mature B cells. One of the major challenges in treatment of CLL is the achievement of a complete remission to prevent relapse of disease originating from cells within lymphoid tissues and subsequent chemoresistance. In search for novel drugs that target CLL cells in protective microenvironments, we performed a fungal extract screen using cocultures of primary CLL cells with bone marrow-derived stromal cells. A secondary metabolite produced by Penicillium aquamarinium was identified as Chaetoglobosin A (ChA), a member of the cytochalasan family that showed preferential induction of apoptosis in CLL cells, even under culture conditions that mimic lymphoid tissues. In vitro testing of 89 CLL cases revealed effective targeting of CLL cells by ChA, independent of bad prognosis characteristics, like 17p deletion or TP53 mutation. To provide insight into its mechanism of action, we showed that ChA targets filamentous actin in CLL cells and thereby induces cell-cycle arrest and inhibits membrane ruffling and cell migration. Our data further revealed that ChA prevents CLL cell activation and sensitizes them for treatment with PI3K and BTK inhibitors, suggesting this compound as a novel potential drug for CLL.

Tumor-derived exosomes modulate PD-L1 expression in monocytes.

Transfer of exosomal RNA from leukemic cells to monocytes induces immunosuppression. Messaging with RNAs Understanding interactions between tumor cells and immune cells is essential for tailoring immunocentric therapies to tumors. Here, Haderk et al. have identified a key role for tumor-derived exosomes in modulating immune responses to chronic lymphocytic leukemia (CLL). They report that CLL-derived exosomal RNAs promote monocytes in CLL patients to adopt an immunosuppressive phenotype, including promoting expression of PD-L1. They identify noncoding RNA hY4 as a key functional component of CLL-derived exosomes and show that hY4 promotes exosome-dependent skewing of monocytes in a TLR7-dependent manner. Using mouse models, they found that inhibition of TLR7 delayed progression of CLL, opening up the possibility that the TLR7 pathway could be therapeutically targeted in CLL. In chronic lymphocytic leukemia (CLL), monocytes and macrophages are skewed toward protumorigenic phenotypes, including the release of tumor-supportive cytokines and the expression of immunosuppressive molecules such as programmed cell death 1 ligand 1 (PD-L1). To understand the mechanism driving protumorigenic skewing in CLL, we evaluated the role of tumor cell–derived exosomes in the cross-talk with monocytes. We carried out RNA sequencing and proteome analyses of CLL-derived exosomes and identified noncoding Y RNA hY4 as a highly abundant RNA species that is enriched in exosomes from plasma of CLL patients compared with healthy donor samples. Transfer of CLL-derived exosomes or hY4 alone to monocytes resulted in key CLL-associated phenotypes, including the release of cytokines, such as C-C motif chemokine ligand 2 (CCL2), CCL4, and interleukin-6, and the expression of PD-L1. These responses were abolished in Toll-like receptor 7 (TLR7)–deficient monocytes, suggesting exosomal hY4 as a driver of TLR7 signaling. Pharmacologic inhibition of endosomal TLR signaling resulted in a substantially reduced activation of monocytes in vitro and attenuated CLL development in vivo. Our results indicate that exosome-mediated transfer of noncoding RNAs to monocytes contributes to cancer-related inflammation and concurrent immune escape via PD-L1 expression.

BRAF inhibition in hairy cell leukemia with low-dose vemurafenib.

The activating mutation of the BRAF serine/threonine protein kinase (BRAF V600E) is the key driver mutation in hairy cell leukemia (HCL), suggesting opportunities for therapeutic targeting. We analyzed the course of 21 HCL patients treated with vemurafenib outside of trials with individual dosing regimens (240-1920 mg/d; median treatment duration, 90 days). Vemurafenib treatment improved blood counts in all patients, with platelets, neutrophils, and hemoglobin recovering within 28, 43, and 55 days (median), respectively. Complete remission was achieved in 40% (6/15 of evaluable patients) and median event-free survival was 17 months. Response rate and kinetics of response were independent of vemurafenib dosing. Retreatment with vemurafenib led to similar response patterns (n = 6). Pharmacodynamic analysis of BRAF V600E downstream targets showed that vemurafenib (480 mg/d) completely abrogated extracellular signal-regulated kinase phosphorylation of hairy cells in vivo. Typical side effects also occurred at low dosing regimens. We observed the development of acute myeloid lymphoma (AML) subtype M6 in 1 patient, and the course suggested disease acceleration triggered by vemurafenib. The phosphatidylinositol 3-kinase hotspot mutation (E545K) was identified in the AML clone, providing a potential novel mechanism for paradoxical BRAF activation. These data provide proof of dependence of HCL on active BRAF signaling. We provide evidence that antitumor and side effects are observed with 480 mg vemurafenib, suggesting that dosing regimens in BRAF-driven cancers could warrant reassessment in trials with implications for cost of cancer care.

Exploiting biological diversity and genomic aberrations in chronic lymphocytic leukemia

There is remarkable heterogeneity in the clinical course and biological characteristics of patient subgroups with chronic lymphocytic leukemia (CLL). Mutations of key tumor suppressors (ATM, miR-15a/16-1 and TP53) have been identified in CLL, and these aberrations are important “drivers” of the disease and some of its clinical characteristics. While some mutations are associated with poor outcome [particularly del(17p) and TP53 mutation], others are linked to a favorable clinical course [e.g. del(13q) as sole aberration]. In addition to genetic aberrations, antigen drive and microenvironmental interactions contribute to the pathogenesis of CLL. How the genetic aberrations impact on the process of antigen drive or microenvironmental interactions is currently unclear. Our improved understanding of the biology and clinical course of specific genetic subgroups is beginning to be translated into more specific and targeted treatment approaches. As a result, genetic subgroups are treated in distinct protocols. This review summarizes the contribution of the microenvironment and the most important genetic aberrations in CLL and how our improved knowledge of the biology of CLL may translate into improved treatment results.