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How the microenvironment wires the natural history of chronic lymphocytic leukemia

Seminars in Cancer Biology, pages 43 - 48

Abstract

The investigation on the mechanisms that govern the development and progression of cancer is constantly swaying between “seed” and “soil”. Chronic lymphocytic leukemia (CLL) makes no exception. Its natural history, including response to treatment and drug resistance, is determined both by causal and influential genes and by the relationships that leukemic cells entertain with their supportive microenvironments. Therefore dissecting the role of microenvironment may provide new strategies of diagnosis and treatment. CLL, though phenotypically homogeneous, is clinically heterogeneous and despite major therapeutic advances remains incurable. Conceivably the host of new non-genotoxic drugs that operate at the forefront between tumor cells and their milieu will modify the present therapeutic perspective by re-shaping the tumor cell/microenvironment cross talk.

Keywords: Chronic lymphocytic leukemia, Microenviroment, Mouse models, Cytoskeleton.

1. CLL key events occur in tissues

CLL key events occur mainly in peripheral lymphoid organs and in the bone marrow (BM) where conducive microenvironments are established and maintained through a dynamic, interactive co-evolution of tumor and normal bystander cells [1] ( Fig. 1 ). CLL microenvironments share the general properties of cancer microenvironment: new vessels provide nutrients, growth factors are produced locally and leukemic cells are protected from immune elimination. The main actors of CLL cell/microenvironment co-evolution are yet incompletely defined populations of stromal, endothelial and immune cells. Pseudofollicular proliferation centers (PC) scattered in infiltrated tissues are the source of most cellular generation in CLL, a highly dynamic process that spawns a daily birth rate of around 1–2% [2] and [3]. The progeny that escapes apoptosis accumulate in tissues and may then flow into the peripheral blood (PB). Circulating clonal cells may re-enter the tissues to start a new other rounds of proliferation.

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Fig. 1 Schematic model of CLL development, trafficking and homing. Yet undetected genetic alterations possibly occur already at the stage of HSC and predispose B cells to the CLL onset. Circulating monoclonal CD5+ B cells are attracted by different stimuli, interact with the cellular elements of the microenvironment and transmigrate into the lymph nodes and bone marrow. T-dependent or T-independent immune response, persistent microenvironmental interactions and accumulating genetic aberrations ultimately contribute to the progression of MBL into overt CLL. Figure was produced using Servier Medical Art: www.servier.com .

Leukemic cells infiltrating different tissues are exposed to different microenvironmental conditions. One example is the hypoxic gradient in the BM, another example is the striking heterogeneity of bystander cells in peripheral lymphoid organs with the compartmentalization in B and T cell areas. These differences influence the emergence and evolution of tissue-related intraclonal heterogeneity and dictate the importance of investigating which rules govern the traffic of leukemic cells and their homing to specific tissue microenvironmental niches.

2. CLL conducive microenvironments

The complex cross talk between CLL cells and their microenvironments are largely dependent upon a functional leukemic B-cell receptor (BCR) that allows antigen (Ag) interaction [4] . A debate is ongoing on the nature of the stimulating Ags, whether they are exogenous Ags provided by pathogenic bacteria [5] or fungi [6] or self Ags presented by apoptotic cells [7] . An intriguing possibility is that surface monoclonal immunoglobulins (Ig) themselves may somehow autonomously act as triggering Ags [8] . Irrespective of the nature and source of Ag stimulation, the outcome is a stimulation of the BCR-triggered pathway [9] . The evident therapeutic relevance of this observation is reinforced by the in vivo finding that CLL cells in infiltrated tissues have signs of BCR-induced activation [10] . In vitro CLL cells from different patients differ significantly in their capacity to signal through the BCR: some (most expressing unmutated IgVH genes) carry more competent BCRs and others (usually showing mutated IgVH genes) appear to be unresponsive [11] .

Differences in signal transduction may be ascribed to the nature of the Ag and/or to the receptor affinity. It is reasonable to postulate that in responsive cases an on going antigenic stimulation might promote CLL survival and possibly also growth, while in unresponsive cases a continuous antigenic binding might lead to receptor desensitization and cell anergy [12] and [13]. It is however unknown where the stimulatory Ags are located and why the proliferation occurs essentially in areas that take the form of PC. This leads also to consider that in every patient all leukemic cells carry the same monoclonal BCR, hence have the same potential reactivity, while only a very limited proportion enter the cell cycle. The implication is that either the relevant Ags are only intermittently present or that Ag stimulation is important in triggering the initial clonal expansion but less so in maintaining the malignancy. These possibilities would be especially true in case of foreign Ag, difficult to explain if Ags are presented by apoptotic cells, even more unlikely if surface monoclonal Ig provide an autonomous signal. An alternatively hypothesis is that Ag stimulation might continuously tickle individual cells and lead them to the decisional crossroad between apoptosis and proliferation, the outcome of such decision being influenced by the microenvironment organization that leads to the formation of PC. This hypothesis would be easily understandable if BCR stimulation is triggered by leukemic monoclonal surface Ig themselves. Admittedly a reductionist hypothesis is more easily experimentally testable, still it is more than conceivable that different Ags may be operating and explains the complexity and heterogeneity of disease evolution in different patients.

Within this general context several other potential abnormalities have to be taken into account. As an example a critical aspect of CLL clonal expansion is the incapacity of leukemic cells to differentiate into Ab-producing cells able to somehow neutralize the stimulating Ag. The implication is that the triggering Ag perpetuates an unabated reaction. Another potential abnormality is an alteration of the signal transduction system that following BCR stimulation leads to the cytoskeleton modification that are needed for cell proliferation and trafficking [14] and [15].

Several other key molecules act at the forefront between CLL B cells and their microenvironments including CD40 [16] , Toll-like receptors (TLR) [17] , BAFF and April [18] receptors. The individual pathogenetic weight of each molecule is unclear as it is unknown to what extent they cooperate with the BCR stimulation in different patients. Evidence is increasing that membrane-associated as well as endosomal TLRs have a role in CLL development and progression [19] and [20]. It has been reported that TLR signaling pathways in the lymph node microenvironment could contribute to NF-κB activation, expression of costimulatory molecules and regulation of survival of CLL cells [21] . Furthermore different subgroups of CLL cases (with different BCR molecular features) have distinct expression profiles of TLR signaling molecules [22] . Also, at least in a proportion of patients, in vitro CLL cell sensitivity to fludarabine may be modulated by the stimulation of TLR, likely mimicking microenvironmental signals occurring in vivo[23] and [24]. These differences coupled with the fact that in a mouse model the lack of the inhibitory receptor TIR8 (Toll IL-1R 8) has been shown to trigger progression of CLL [24] suggest that specific modalities of BCR/TLR cooperation and/or cross-regulation may impact on the behavior of the malignant clone.

Taken together these observations indicate that a molecular blueprint of microenvironment influence will have to be promoted and applied to individual cases to help developing a personalized treatment approach.

3. From hematopoietic stem cells (HSCs) to monoclonal B cell lymphocytosis (MBL): old sins have long shadows

An elegant study has suggested that newborn NSG mice transplanted with HSC purified from CLL patients show the propensity to develop monoclonal B-cell populations, though not a full-blown CLL [25] . CLL-HSC gave rise to significantly higher numbers of human pro-B cells expressing CD5 as compared to healthy donors HSC. In addition, the mature B cell progeny showed mono- or oligoclonal IGH rearrangements, though VDJ recombinations were always independent from the recombinations originally expressed by the patient's CLL clone. HSC from CLL patients appear to be skewed toward B-cell lineage thereby suggesting the existence of yet undetected intrinsic abnormalities. These findings are well in keeping with the well known familial tendency of CLL [26] hinting at a genetic susceptibility. Whether and how this putative abnormality relates to the gene variants associated with B-cell function described by the genome-wide association studies (GWAS) [26] still has to be clarified.

CLL is virtually always preceded by MBL [27] . Tiny monoclonal B-cell populations phenotypically CLL-like can be detected in the PB of about 3.5% of healthy individuals, have the same gender and age predisposition as CLL (reaching a frequency greater than 10% in people > 60 years) and have a significantly higher incidence in the relatives of CLL patients. It has been shown that MBL may progress into CLL with a frequency of 1–2%/year. It thus appears logical to hypothesize that MBL might be a precursor state for CLL, reminiscent of the relationship between monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma. That notwithstanding, as with MGUS, we do not have a clue as to whether individual subjects with MBL will/will not progress into fully fledged CLL nor when this event will occur. To this end it has to be noted that MBL may be further divided into two different general subgroups, high and low-count MBL. High count MBL are associated with lymphocytosis, have a concentration of clonal B cells greater than ∼1500/μL and represent a continuum with CLL [28] and [29]. Low count MBL are only detected during screening studies of healthy individuals using highly sensitive technical procedures and have generally less than ∼50 clonal B cells per μL. The prevalence of low-count MBL is at least 100-fold greater than that of CLL and the propensity to progress is negligible if any. That notwithstanding low-count MBL have the cytogenetic hallmarks of CLL including 13q deletions, trisomy 12 and 17p deletion.

As all cancers CLL is condemned to becoming and never to being. The still unresolved question is how far back can we trace the oncogenic process and which role has the microenvironment along this trail.

4. Animal models

Animal models represent the best tool to recreate in vivo the whole complexity of microenvironmental interactions that allow CLL cells to survive and proliferate. Besides the naturally occurring model of CLL which is the New Zealand Black (NZB) strain [30] , the first transgenic model of the disease was the Eμ-TCL1 transgenic mouse expressing the TCL1 (T cell leukemia/lymphoma 1) oncogene under the control of a VH promoter-IgH-enhancer targeting the transgene expression in B cells [31] . These mice develop a CD5+ CLL-like disease around 8–18 months of age with IgV-region rearrangements replicating those typical of the aggressive form of human CLL [31] and [32]. To reproduce the most frequent genetic aberration of human CLL, the deletion of chromosomal region 13q14, Klein et al. [33] developed a transgenic mouse model lacking the minimal deleted region (MDR). The MDR region includes the long non-coding RNA deleted in leukemia (DLEU)-2 and the first exon of the DLEU-1 gene (that contains mir15 and mir16 whose deletion was considered to be the main driving lesion). This mouse model develops a full spectrum of lymphoproliferative disorders ranging from Monoclonal B-cell lymphocytosis (MBL), to CLL, to more aggressive CD5 diffuse large B-cell lymphomas (DLBCL), somehow recapitulating the natural history of CLL that may evolve from MBL to CLL to Richter Syndrome. The Eμ-TCL1 transgenic mouse is the most widely used model of CLL and it has been extensively used to exploit in the last years pharmacologic, genetic and biologic studies. As an example this model has been used to dissect in vivo the leukemogenic role of several molecules such as frizzled-6 (Fzd6) and PKCβ, in B cell leukemogenesis [34] and [35]. It has been also used to investigate the influence of factors, such as CD257, on leukemic cell-turnover during disease development. Indeed Tcl-1/BAFF double transgenic mice, having high level expression of Tcl-1 and systemic high levels of CD257, develop more aggressive features of leukemia confirming the significant role of microenvironmental factors on disease progression [36] .

Furthermore the Eμ-TCL1 transgenic mouse extensively helped to study the role of microenvironmental signals and accessory cells (i.e. T cells and macrophages) involved in CLL pathogenesis. Gorgun et al. examined changes in gene-expression profile, protein expression, and function of T cells (CD4 and CD8) from Eμ-TCL1 transgenic mice demonstrating, as already observed in patients, that during CLL development a T-cell impairment is correlated to leukemia [37] . It has also been demonstrated a skewing of T-cell subsets from naïve to antigen-experienced memory T cells [38] , a phenomenon previously observed in human CLL and associated with stage and disease progression [39] .

The importance of macrophages activated through CD40 and TLR has also been observed [40] and [41]. The role of the monocyte/macrophage lineage in the pathogenesis of CLL has been later more extensively analyzed in vivo by Reinart et al.: in the TCL1 model the genetic inactivation of the macrophage migration inhibitory factor (MIF) delays the development of leukemia by reducing the survival of CLL cells and the number of macrophages in both spleen and BM [41] . These results indicate that the interaction of leukemic cells with elements of the monocyte/macrophage lineage may significantly support the development of CLL [41] .

The role of normal by-stander cells present within the CLL microenvironment has been extensively elucidated also in CLL xenotransplantation studies. Different xenograft models confirmed the relevant role of autologous T cells [42], [43], and [44]. B–T cell interactions, either directly or indirectly, are critical for the growth of CLL cells, though it has still to be fully clarified how many of these interactions are advantageous for the leukemic cells or are conversely detrimental. Possibly the different outcome is based on the stage of the disease as well as on the site of the interaction, notable being the differences between tissues. A recent model revealed that antigen presenting cells (APCs – CD14+ or CD19+ cells) are another important piece of the puzzle as they were found to elicit in vivo an increased CLL cell survival and proliferation of CLL cells probably facilitating the T-cell influence on CLL cells [44] .

In summary both transgenic and xenograft models of CLL gave important insights on the role of the microenvironment in the pathogenesis of leukemia and represent an important tool for investigating novel therapeutic strategies targeting the microenvironment.

5. The issue of cell trafficking and homing

Many of the physiologic mechanisms of tissue-specific trafficking and homing are reproduced in CLL infiltrated tissues and entail a very complex multifactorial system of chemokine-mediated migratory signals ( Fig. 1 ). These signals are modulated by cell–cell contacts between CLL cells and microenvironmental elements (especially stromal cells) mediated by integrins, selectins, metalloprotease and cytoskeletal proteins [3], [45], [46], and [47]. Though the precise mechanisms are currently unknown, it is likely that lymphocyte trafficking and homing to specialized microenvironments depend on one side from the sequential engagement of adhesion molecules and chemokine receptors activation and on the other side from leukemic cell behavior.

Chemokines such as CCL3, CCL4, CCL22 and IL-8 are secreted by CLL cells and can recruit T cells and other accessory cells to promote survival. BM and lymph node stromal cells secrete CXCL12, CXCL13, CXCL9,10,11, and CCL19 and 21 which bind CXCR3, CXCR4 and CXCR5 receptors different expressed by CLL B cells [48] . The ligand/receptor interactions have been shown in vitro to direct leukemia-cell chemotaxis, suggesting the possibility that the same mechanism may also occur in vivo in tissue microenviroments. Recent studies [49] show that the majority of CLL cells express reduced levels of the two major lymphocyte integrins, LFA-1 and VLA-4 and underlie the critical role of these receptors by showing that CLL cells with reduced LFA-1 and VLA-4 have a reduced capacity to adhere and transmigrate through multiple vascular endothelial beds and poorly home to lymphoid organs other than the spleen. A consistent divergence has been shown [50] in the signaling mechanics that control integrin activation in CLL patients as compared to normal B cells. While LFA-1 was found to be modulated by RhoA in all CLL patients, Rac1 and CDC42 had a consistent variability among different cases. Along this line it has been observed [51] that the chemokine-induced transendothelial migration (TEM) is defective in CLL due to defective Rap1 and alphaLbeta2 integrin activation.

Cell movement and migration involve complex molecular mechanisms brought about by cytoskeletal reorganization. A time-honored observation is that CLL cells have a high fragility and undergo morphologic deformities in the preparation of blood films, which suggests that the so-called “smudge” cells may be accounted for a cytoskeletal alteration. This possibility is also supported by numerous CLL cell features including impaired cell motility, diminished capping by multivalent ligands, increased shedding of membrane proteins and enhanced susceptibility to microtubule-disrupting drugs [52] and [53]. Many studies have highlighted new F-actin-regulatory molecules controlling various aspects of leukocyte biology. Much attention has been drawn onto proteins that regulate actin dynamics through their effects on the Arp2/3 complex (i.e. WASP and VAV family) [54] . Furthermore it has been recently observed that the expression of RhoH (a GTPases family member) correlates with the expression of ZAP70 in CLL and a potential role in the progression of the disease has been suggested [55] .

ZAP-70+ CLL B cells have been shown to respond in vitro more readily than ZAP-70− CLL and normal B cells to chemokine-driven migration by modulating CCR7 expression and increasing responsiveness to its ligands CCL19 and CCL21 [47] . In addition, ZAP-70+ CLL cells exhibit sustained ERK activation following stimulation with CXCL12 (SDF-1) as compared to ZAP-70− cells.

In this scenario we identified HS1 protein (hematopoietic lineage cell-specific protein 1) as a central regulator of cytoskeleton remodeling that controls lymphocyte trafficking and homing and significantly influences the tissue invasion and infiltration in CLL [56] . HS1 has been shown to be an important actin regulator at the T-cell immunological synapse and also to influence numerous functions of NK cells and DCs including lysis of target cells, cell adhesion, chemotaxis and actin assembly at the lytic synapse [57] .

Taken together, these findings suggest the potential importance of interfering with CLL trafficking and homing by targeting cytoskeletal organization. HS1 is a promising regulator candidate. We recently demonstrated that targeting LYN/HS1 axis in vitro with dasatinib leads to the concomitant reduction of cytoskeletal activity, BCR signaling and cell survival in the selected subset of patients whose cells have activated LYN/HS1. Moreover in a transplantable mouse model based on the Eμ-TCL1 transgenic mouse, the pharmacological inhibition LYN/HS1 signaling interferes with CLL progression and lymphoid organ infiltration [58] .

Major unresolved questions are (i) through which mechanisms resting CLL cells flow into the PB, why they tend to accumulate and be sheltered in peripheral lymphoid tissues in small lymphocytic lymphoma (SLL) and (ii) how and why proliferating cells cluster in specific niches most evident in lymph nodes (pseudo follicles) while resting malignant B cells that have survived the therapeutic hit tend to lurk in the BM.

6. Concluding remarks: where are we heading with microenvironment-targeting drugs

The concept that active microenvironments co-evolving with the leukemic clone have a key role in the natural history of CLL ( Fig. 1 ) acquires a clinical relevance considering the growing number of new non-genotoxic drugs that affect B-cell signaling pathways or different elements of the tumor microenvironment [59] and [60].

Clinical benefits have been observed in patients treated with (i) the second-generation immune-modulating agent lenalidomide that influences the patient's immune system; (ii) various inhibitors of the cascade of BCR downstream effector kinases, such as fostamatinib disodium (Syk), dasatinib (Lyn kinase), GS1101 (phosphatidylinositol 3-kinase delta), and ibrutinib (PCI-32765; Bruton's tyrosine kinase inhibitor [BTK]) as well as with (iii) the mammalian target of rapamycin (mTOR) inhibitors. A variety of doses, schedules and combinations are now tested in numerous clinical trials. The results have generated great excitement because of their clinical efficacy and safety and are also leading to a number of general considerations. First, it is likely that the present list of drugs will be enriched by new categories of agents that e.g. interfere with TLR-signaling or with cytoskeleton activities [58] or may reverse malignant B-cell anergy [13] . Next it is conceivable that, in order to achieve the optimal result (and select the optimal drug), individual molecular prognostic blueprints will have to be developed [58] and patients will have to be stratified accordingly. Finally, it is well known that some kinases targeted by the new agents (examples being ibrutinib and dasatinib) are not strictly B-cell specific, rather they are also present in other cell [61] . Targeting these other cell populations may also influence the agent activity.

In this dynamic perspective the key role of active microenvironments leads finally to ask whether we have to modify our treatment policy with a treatment anticipation. According to the IWCLL guidelines [62] treating patients who do not have an active/progressive disease is not warranted especially with alkylating agents as their use in early stage diseases does not prolong survival and may even be associated with an increased frequency of fatal epithelial cancers. It may be argued that this policy has to be reconsidered and that the possibility of pre emptive therapy with microenvironment-influencing non-genotoxic drugs has to be taken into account.

Conflict of interest

The authors declare no competing financial interests.

Acknowledgements

This work was supported by: Associazione Italiana per la Ricerca sul Cancro AIRC (Investigator Grant and Special Program Molecular Clinical Oncology–5 per mille #9965), “CLLGRF – U.S./European Alliance for the Therapy of CLL” and PRIN – Ministero Istruzione, Università e Ricerca (MIUR), Roma.

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Footnotes

a San Raffaele Scientific Institute, Division of Molecular Oncology, Unit of Lymphoid Malignancies, Milano, Italy

b Università Vita-Salute San Raffaele, Milano, Italy

lowast Corresponding author at: Università Vita-Salute San Raffaele, San Raffaele Scientific Institute, Division of Molecular Oncology, Unit of Lymphoid Malignancies, Via Olgettina 58, 20132 Milano, Italy. Tel.: +39 02 2643 2471; fax: +39 02 2643 4723.