IntroductionB-cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal CD5 ϩ B cells in blood, secondary lymphoid tissues, and the bone marrow. 1 Most of the circulating leukemia cells are arrested in the G 0 /G 1 phase of the cell cycle; therefore, the primary defect may be one of resistance to apoptosis rather than accelerated cell division. 2 However, in vitro CLL cells undergo spontaneous apoptosis, suggesting that such ex vivo conditions lack factors necessary for leukemia-cell survival and that the resistance to apoptosis is not intrinsic to the leukemia B cell. In vitro apoptosis of CLL cells can be prevented by coculture with different accessory cells that are part of the CLL microenvironment, such as monocyte-derived nurselike cells (NLCs), [3][4][5][6] mesenchymal marrow stromal cells (MSCs), 3,7,8 or follicular dendritic cells, 9 which provide survival signals to CLL cells. NLCs differentiate from monocytes into large, round, adherent cells that attract CLL cells and protect them from undergoing spontaneous or drug-induced cell death in a contact-dependent fashion. 3,4,10 Because these cells share features in common with thymic nurse cells that nurture developing thymocytes, 11 we designated these cells "nurselike cells." 3 NLCs can be found in the spleen and secondary lymphoid tissue of patients with CLL 4 and thus represent a model for the microenvironment in secondary lymphatic tissues. High levels of CD68 make NLCs comparable with CD68 ϩ lymphoma-associated macrophages in follicular lymphoma. 12 Although still controversial, some studies demonstrated that a high CD68 ϩ cell content in the microenvironment is associated with an aggressive clinical course and poor outcome in follicular lymphoma, [12][13][14] suggesting that cell-cell interactions between the lymphoma cells and accessory cells of monocyte/ macrophage lineage play a role in supporting neoplastic B-cell growth and drug resistance in lymphatic tissues. In addition, T cells are an integral part of the microenvironment in CLL. In CLL pseudofollicles (PFs), CLL cells are interspersed with T cells in proliferation clusters. 15,16 PFs are a hallmark finding in CLL histopathology and are considered the proliferative compartment of this disease. [17][18][19] In PFs, T cells are in intimate contact with CLL cells and express activation markers, such as CD40 ligand (CD154). 15,20 Moreover, contact with activated CD4 ϩ T cells induces Survivin 15 and CD38 expression 16 in CLL cells in PFs, suggesting that T cells promote CLL cell activation and proliferation in these areas. 16,17 However, the factors promoting colocalization of T cells with CLL cells are largely unknown. 16 Several molecules involved in cross talk between CLL cells and their microenvironment have recently been identified based on in vitro work and correlative studies on CLL tissue specimen. We characterized CXCL12 (SDF-1), a chemokine constitutively secreted by MSCs 21 and NLCs, 3 as a chemotactic and antiapoptotic factor for CLL cells, acting th...

IntroductionWith the establishment of more effective treatments for patients with chronic lymphocytic leukemia (CLL) over the past decade, complete remissions are no longer the exception. 1 Despite these major improvements in CLL treatment, we still consider CLL an incurable disease, because patients generally relapse from minimal residual disease (MRD). 2 There is growing evidence suggesting that CLL cells are protected from conventional drugs in tissue microenvironments, such as the bone marrow and secondary lymphoid organs, with facilitation of residual disease that is drug resistant and ultimately paving the way to clonal evolution and relapses. The complex cellular and molecular contexts in the tissues, collectively referred to as the CLL microenvironment, provide signals for the expansion of the CLL clone and for primary drug resistance. This is largely dependent on direct contact between the malignant B cells and stromal cells, 3 and therefore has been designated as cell adhesion-mediated drug resistance. 4 Disrupting cross talk between leukemia cells and their milieu is an attractive novel but yet incompletely tested strategy for treating CLL. Appropriately, there is growing interest in understanding the biology of CLL-stroma cross talk to find ways to eliminate residual CLL cells that are "hiding" in stromal niches within the marrow and the lymphatic tissues.Importantly, once CLL cells are removed from the in vivo microenvironment and placed in suspension cultures without supportive stroma, they undergo spontaneous apoptosis, highlighting the importance of external signals from accessory cells. 5 Previous studies have shown that CLL cell cocultures with different adherent cell types, collectively referred to as stromal cells, induce leukemia cell survival, migration, and drug resistance. These stromal cells include mesenchymal marrow stromal cells (MSCs), 3,6,7 CD68 ϩ nurselike cells derived from monocytes, 7-10 and follicular dendritic cells. 11 Immunohistochemistry showed that in situ, ␣SMA ϩ mesenchymal stromal cells, 12 the in vivo counterpart of MSCs, are a dominant stromal cell population in the CLL microenvironment, which is in contrast to other B-cell lymphomas, particularly high-grade lymphomas, which harbor larger numbers of CD68 ϩ hemangiogenic cells. 12 MSCs regulate normal hematopoiesis by providing attachment sites and secreted or surface-bound growth factors that constitute the marrow microenvironment. 13 During B-cell development in the marrow, programmed cell death regulates B-cell homeostasis by diverting a large fraction of immature B cells into an apoptotic death pathway to eliminate functionless or potentially harmful cells. 14,15 Critical factors for the survival of selected B cells are interactions with MSCs in the marrow microenvironment, [16][17][18] expression of surface immunoglobulin molecules, and expression of apoptosis-regulatory proteins, such as Bcl-2. 19 In patients with CLL, the marrow invariably is infiltrated with CLL B cells, and the For personal use only. on May 7,...

Antigenic stimulation through the B-cell antigen receptor (BCR) is considered to promote the expansion of chronic lymphocytic leukemia (CLL) B cells. The spleen tyrosine kinase (Syk), a key component of BCR signaling, can be blocked by R406, a small-molecule Syk inhibitor, that displayed activity in CLL patients in a first clinical trial. In this study, we investigated the effects of BCR stimulation and R406 on CLL cell survival and migration. The prosurvival effects promoted by anti-IgM stimulation and nurselike cells were abrogated by R406. BCR triggering up-regulated adhesion molecules, and increased CLL cell migration toward the chemokines CXCL12 and CXCL13. BCR activation also enhanced CLL cell migration beneath marrow stromal cells. These responses were blocked by R406, which furthermore abrogated BCR-dependent secretion of T-cell chemokines (CCL3 and CCL4) by CLL cells. Finally, R406 inhibited constitutive and BCR-induced activation of Syk, extracellular signal-regulated kinases, and AKT, and blocked BCR-induced calcium mobilization. These findings suggest that BCR activation favors CLL cell homing, retention, and survival in tissue microenvironments. R406 effectively blocks these BCR-dependent responses in CLL cells, providing an explanation for the activity of R406 in patients with CLL.

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