Molecular Pathways: Targeting the Microenvironment in Chronic ...

Published OnlineFirst December 9, 2013; DOI: 10.1158/1078-0432.CCR-13-0226

Clinical Cancer Research

Molecular Pathways

Molecular Pathways: Targeting the Microenvironment in Chronic Lymphocytic Leukemia—Focus on the B-Cell Receptor Elisa ten Hacken and Jan A. Burger

Abstract Interactions between malignant B lymphocytes and the tissue microenvironment play a major role in the pathogenesis of chronic lymphocytic leukemia (CLL) and other B-cell malignancies. The coexistence and coevolution of CLL cells with their tissue neighbors provided the basis for discovery of critical cellular and molecular drivers of the disease and identification of new therapeutic targets. Bone marrow stromal cells (BMSC), monocyte-derived nurselike cells (NLC), and T cells are key players in the CLL microenvironment, which activate and protect CLL cells within the tissues. CLL surface molecules, such as the B-cell antigen receptor (BCR), chemokine receptors, adhesion molecules, and TNF receptor superfamily members (e.g., CD40, BCMA, and BAFF-R) engage in cross-talk with respective tissue ligands. This cross-talk results in survival and expansion of the CLL clone, and protects CLL cells from conventional cytotoxic drugs. Inhibiting these pathways represents an alternative therapeutic strategy to more conventional chemoimmunotherapy. Here, we review central components of the CLL microenvironment, with a particular emphasis on BCR signaling, and we summarize the most relevant clinical advances with inhibitors that target the BCR-associated spleen tyrosine kinase/SYK (fostamatinib), Bruton’s tyrosine kinase/BTK (ibrutinib), and PI3Kd (idelalisib). Clin Cancer Res; 20(3); 548–56. 2013 AACR.

Disclosure of Potential Conflicts of Interest J. Burger has received commercial research grants from Pharmacyclics, Gilead, and Noxxon, and is a consultant/advisory board member for Noxxon and Pharmacyclics. No potential conflicts of interest were disclosed by the other author.

CME Staff Planners' Disclosures The members of the planning committee have no real or apparent conflict of interest to disclose.

Learning Objectives Upon completion of this activity, the participant should have an overview of the cellular and molecular players in the chronic lymphocytic leukemia (CLL) microenvironment. The participant should also have a better understanding of the central role of B-cell receptor (BCR) signaling in CLL pathogenesis, and how novel therapies targeting BCR signaling disrupt the CLL-microenvironment cross-talk.

Acknowledgment of Financial or Other Support This activity does not receive commercial support.

Background Anatomy of the CLL microenvironment Chronic lymphocytic leukemia (CLL) cells recirculate between peripheral blood and tissue compartments (i.e.,

Authors' Affiliation: Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, Texas Corresponding Author: Jan A. Burger, Department of Leukemia, The University of Texas MD Anderson Cancer Center, 1400 Holcombe Boulevard, Unit 428, PO Box 301402, Houston, TX 77230-1402. Phone: 713-5631487; Fax: 713-794-4297; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-13-0226 2013 American Association for Cancer Research.

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the bone marrow and secondary lymphatic organs), in which they proliferate in distinct tissue areas termed "proliferation centers" or "pseudofollicles." Proliferation in these pseudofollicles accounts for a daily birth rate of approximately 1% to 2% of the entire clone, as determined by deuterated water labeling (1). Studying the cross-talk between CLL and tissue stromal cells led to the discovery of key mechanisms involved in CLL homing, proliferation, and survival, providing a rationale for therapeutic targeting of the CLL—stroma cross-talk (Fig. 1 and Table 1). Tissue stromal cells, such as stromal cells and monocytederived nurselike cells (NLC) are critical elements of the tissue microenvironment in CLL, and the latter share lineage and function with lymphoma-associated macrophages

Clin Cancer Res; 20(3) February 1, 2014

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B-Cell Receptor Targeting in CLL

A BMSC

PKC-βll NF-κB VCAM-1 FN

CXCL12

VLA-4

CXCR4 G Tissue homing survival proliferation

CLL cell

G

PI3Kδ BCMA TACI BAFF-R Figure 1. The CLL microenvironment. A, in the CLL microenvironment, CLL cells interact with bone marrow stromal cells (BMSC) and NLCs through adhesion molecules and chemokine receptors, expressed on CLL cells. These interactions, in addition to B-cell receptor engagement, promote CLL survival, þ proliferation, and homing to tissues. B, CD4 T cells are recruited into the tissue microenvironment by CLL cell–derived chemokines, including CCL3 and CCL4, to support CLL cell survival and proliferation. Inhibitory receptors expressed by CLL cells induce defective immune synapse formation between CLL and T cells. Cytotoxic granule þ secretion by CD8 T cells also is defective, and production of soluble factors by CLL cells suppresses NK cell cytotoxicity, favoring immune evasion of CLL cells.

CD38

Syk

CXCR4, CXCR5

BTK

BCR CXCL12 CXCL13

CD31 BAFF APRIL

NLC B

CD4+ T cells

CCL3/4 CCL17/22 Inhibitory receptors

CD40L CD40

Survival proliferation

CLL Cell

Defective synapse formation

Defective cytotoxicity

NKp30

Inhibitory receptors Soluble BAG6

Defective synapse formation

CD107a Cytotoxic granules

NK cells

CD8+ T cells © 2013 American Association for Cancer Research

www.aacrjournals.org



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ten Hacken and Burger

Table 1. Cellular components of the CLL microenvironment Cell type

Function in normal immune responses

Function in CLL microenvironment

BMSC

Formation of hematopoietic stem-cell niches and regulation of hematopoiesis; constitutive secretion of CXCL12 and various other hematopoietic cytokines

Tissue retention of CLL cells (2); CLL cell drug resistance (2) and protection from apoptosis (2); and PKC-bII activation in BMSCs induced by CLL cells (11)

NLC

Not applicable

Tissue retention of CLL cells (3); CLL cell drug resistance (3) and protection from apoptosis (3); CLL cell attraction by CXCL12 and CXCL13 production (3); BAFF and APRIL expression (12); activation of BCR and NF-kB pathway genes in CLL cells (8); disease progression in vivo (13, 14); and interaction with CD38-expressing cells via CD31 (24)

CD4þ T cells/Th cells

Adaptive immune responses; antigen recognition; T-antigen-presenting cell (APC) immune synapse formation; APC activation via CD40/CD40L interaction

CLL engraftment in vivo (15); defective immune synapse formation upon contact with CLL cells (16, 17); CD40Lþ CD4þ T cells induce CLL cells to produce CCL17 and CCL22 (30, 31); and increased expression of inhibitory receptors (e.g., PD, ref. 17)

CD4þCD25high/Treg

Inhibition of proliferation and cytokine release by CD4þ T cells; inhibition of antitumor immunity

High proportion of CD4þCD25high Treg may contribute to CLL immune evasion (15)

CD8þ T cells/cytotoxic T cells

Adaptive immune responses; antigen recognition when presented on MHC class I molecules by target cells (e.g., cancer cells, virally infected cells); apoptosis induction of target cells

Defective immune synapse formation upon contact with CLL cells (16, 17); exhausted phenotype and impaired cytotoxicity (18)

NK cells

Innate immune responses; antigen recognition when presented on MHC class I molecules by target cells (e.g., cancer cells, virally infected cells); apoptosis induction of target cells

Reduced cytotoxicity (19); low expression of NKp30 activating receptor (20)

described in other B-cell malignancies. Mesenchymal stromal cells, such as bone marrow stromal cells (BMSC), provide "feeder" layers for hematopoietic progenitor cells and are part of hematopoietic stem-cell niches in the normal bone marrow. They protect CLL cells from spontaneous and drug-induced apoptosis in a contact-dependent fashion (2, 3). These interactions not only take place in the marrow, mesenchymal stromal cells also are commonly found in secondary lymphatic tissues in patients with CLL (4), in which they can provide survival and migration signals to CLL cells. In contrast, NLCs spontaneously develop in vitro from monocytes in CLL peripheral blood mononuclear cell cultures (5), in which they form feeder layers and maintain CLL cell viability by activating numerous surface receptors and signaling pathways. NLCs can be found in lymphoid organs from patients with CLL (6, 7), and gene expression profiles (GEP) of CLL cells after coculture with NLCs (8) are highly similar to those of CLL cells isolated from CLL lymph nodes (9), suggesting that NLCs are a highly relevant model system for studying the microenvironment.

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Stromal cells constitutively secrete chemokines, which organize CLL cell trafficking and tissue homing (10), and provide additional signals that support survival and growth and protect CLL cells from drug-induced apoptosis. Not only CLL cells benefit from bone marrow stroma contact, the stromal cells, in turn, also become activated by the CLL cells, with induction of protein kinase C (PKC)-bII expression and subsequent NF-kB pathway activation (11). In vivo, NLCs are found in lymphoid organs from patients with CLL (6) and activate prosurvival signaling pathways in CLL cells (8). NLCs attract and protect leukemic CLL cells through secretion of chemokines (CXCL12, ref. 5; CXCL13, ref. 7), support their survival via expression of the TNF family members BAFF and APRIL (12), and promote CLL disease progression, as demonstrated in recent CLL animal models (13, 14). The similarity between NLC-induced GEPs in CLL cells (8) and GEP of CLL cells isolated from lymphatic tissues (9) suggests that NLCs are a valid and probably the best model system to imitate the in vivo microenvironment.





T and NK cells CLL cells influence T-cell and natural killer (NK)–cell composition and function in the microenvironment to escape from immune-mediated cytotoxicity (see Table 1; ref. 15). Contact between T cells (either Th CD4þ or cytotoxic CD8þ lymphocytes) and CLL cells prevents appropriate immune synapse formation by inducing changes in cytoskeletal gene transcription (16) and defective actin polymerization and T-cell motility (17), which can be restored by revlimide (16, 17). Increased expression of inhibitory receptors [e.g., Programmed-death-receptor 1 (PD-1)] on T cells from patients with CLL and high numbers of CD4þCD25high T regulatory cells (Treg) in the CLL microenvironment may contribute to increased evasion from antitumor immunity (15). CD8þ cytotoxic T cells have an exhausted phenotype and fail to establish cytotoxic immune synapses, with impaired granzyme B packaging and impaired degranulation (18). NK cell–mediated antitumor activity is also limited, as NK cells from patients with CLL express low levels of the activating receptor NKp30 (19, 20) and show reduced cytotoxicity in response to soluble BAG6 ligand produced by CLL cells (19). Taken together, it is evident that CLL cells actively shape an immunoprotected environment, in which the leukemia cells can escape immune-mediated destruction. CLL tissue homing: role of chemokines and adhesion molecules CLL cells traffic between peripheral blood and tissues is organized by chemokines that are secreted by tissue stromal cells, attracting CLL cells via corresponding chemokine receptors. CXCR4 is the receptor for the chemokine CXCL12 (previously called stromal cell–derived factor-1), which is constitutively secreted by BMSCs. CXCL12 regulates CXCR4 surface expression through receptor endocytosis (21), resulting in low CXCR4 surface expression in the presence of high CXCL12 tissue levels. CXCR4 activation causes actin polymerization, CLL cell chemotaxis, transendothelial migration, and tissue homing (21). High CXCR4 expression on CLL cells is associated with a higher degree of lymphoid organ infiltration (22). Furthermore, CLL cells expressing high levels of the intracellular adaptor ZAP-70 (23), CD38 (24), or VLA-4 integrins (25) display higher migratory potential toward CXCL12. VLA-4 integrins cooperate with chemokine receptors and CD38 (26) in CLL cell adhesion to stromal cells (27). In addition, high CD38 (28) and VLA-4 (29) expression are also predictors of an inferior clinical outcome, supporting the notion that disease aggressiveness is linked to tissue homing and adhesion of CLL cells. Another layer of complexity is added by chemokines secreted by CLL cells in response to activation. B-cell antigen receptor (BCR) triggering and NLC-contact induces secretion of CCL3 and CCL4 (8), and costimulation through CD40 ligand engagement by CD40þ T cells induces CCL17 (30) and CCL22 (30, 31). Increased CCL3 plasma levels correlate with poor outcome and are an independent prognostic marker in CLL (32), demonstrating the relevance of CLL-produced chemokines. CCL17 and CCL22 induce


transendothelial migration of activated T cells (30), but the function of these chemokines in CLL pathogenesis remains ill defined. The function of CCL3 and CCL4 is better defined; these chemokines attract CD4þ T cells and monocytes to activated B cells (33), contributing to normal B or CLL cells support at tissue sites (34). B-cell receptor signaling in CLL There is accumulating evidence to support that BCR signaling plays a key role in CLL pathogenesis (35–37). The BCR (Fig. 2) is composed of the antigen-specific surface membrane immunoglobulin (smIg), and Ig-a/Ig-b heterodimers (CD79A and CD79B). Antigen binding to the smIg induces phosphorylation of immunoreceptor tyrosinebased activation motifs (ITAM) in the cytoplasmatic tails of CD79A and CD79B by LYN kinase. This, in turn, activates SYK, BTK, and phosphoinositide 3-d (PI3Kd) kinases and downstream pathways, including calcium mobilization, activation of phospholipase C g2, PKCb, NF-kB signaling, mitogen-activated protein kinases, and nuclear transcription. The precise mechanism triggering BCR activation, for example, the importance of ligand (antigen)-dependent and -independent (autonomous) signaling remains controversial, but indirect and some more direct evidence points toward the central role of the BCR. First, the prognostic importance of the mutational status of immunoglobulin heavy chain variable (IGHV) genes indicates that CLL BCRs encounter antigens, which ultimately promote a certain degree of somatic hypermutations, which in turn influences the clinical behavior of the disease. Second, the expression of quasi-identical ("stereotyped") BCRs among different patients with CLL suggest that a set of common antigens contributed to the stereotypy of the BCR in individual patients. Third, GEP studies demonstrated that BCR signaling is the key regulatory pathway activated in CLL cells in lymph nodes, the sites of CLL cell proliferation (9). Na€ve B cells develop in the bone marrow and are characterized by the presence of a functional surface immunoglobulin of the M isotype (sIgM). Further maturation takes place in secondary lymphoid organs, in which na€ve B cells undergo further maturation, including expression of immunoglobulins of the D isotype (sIgD; ref. 38). As CLL cells show features of mature B cells, most of them express both sIgM and sIgD isotypes (39, 40). Previous studies suggested a dominant role of sIgM signaling (39, 40) and differential responsiveness to IgM stimulation was demonstrated for CLL cases carrying unmutated IGHV genes (U-CLL) versus mutated IGHV (M-CLL). U-CLL cases typically are more responsive to BCR triggering, with activation of robust intracellular signaling, whereas cells from patients with M-CLL generally are less responsive to BCR cross-linking (39). Prolonged extracellular signal–regulated (ERK) kinase activation after sIgM triggering supports expression of the proto-oncogene MYC (40), suggesting that IgM signaling promotes cell-cycle entry and CLL cell growth. The role of sIgD signaling is less defined. The majority of CLL clones seem to respond to IgD triggering (39, 40), and anti-IgD responsiveness was described to impact prognosis (41).



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BCR

P

P

P

SHIP P

SHP1/2

PI3Kδ

P LYN

PTPN22

P

P

LYN P

P P F-Actin

HS1 P

SYK P

BLNK P P BTK

Fostamatinib Ibrutinib

CD19

P

CD79B

P

CD79A

P

CD79A

P

CD79B

CD5

CD22

FcγRlllb P

P

PLCγ2 Idelalisib Figure 2. Schematic representation of the BCR signaling pathway and small molecule–kinase inhibitors used to interfere with BCR signaling.

Ca2+ PKC

ERK

NF-κB

Cytosol Nucleus Transcription factors

© 2013 American Association for Cancer Research

Given that both IgM and IgD BCRs have the same antigen specificity, we currently assume that both, sIgM- and sIgDderived signals govern overall BCR pathway activation. The caveat with interpreting these in vitro data, however, remains that they are based on somewhat artificial modes of BCR activation, relaying on generally high concentrations of soluble or immobilized BCR ligands used for CLL cell stimulation, which likely differs from in vivo BCR ligation. In addition, the BCR signaling is modulated by receptor endocytosis, signaling of positive (e.g., CD19) and negative (e.g., CD5 and CD22) regulatory coreceptors, intracellular kinases (e.g., SYK, BTK, and PI3K), and phosphatases (e.g., FcbRIIIb, SHIP, SHP-1, and PTPN22; ref. 42), which finetune the functional outcome of BCR signaling (Fig. 2; ref. 35). With regard to potential antigens responsible for BCR stimulation in CLL, several studies suggested that liganddependent BCR signaling is a main mechanism. Generally, BCRs from U-CLL cells have low-affinity binding to a broader range of self-antigens, whereas affinity-matured BCRs from M-CLL cases have high-affinity binding to restricted, more specific antigens (43–45). Recently, defined epitopes within the BCR third complementarity-determining region of the heavy chain have been described as targets of BCR (self-) recognition in CLL, representing an alternative form of autoantigen (46, 47). This form of autonomous BCR signaling likely contributes to CLL growth and survival, in concert with extrinsic

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BCR ligands. In contrast with ABC subtype diffuse large Bcell lymphoma, CLL cells have no activating mutations within BCR signaling components. The variability and complexity of BCR signaling in CLL are mirrored by variable levels of activation of BCR downstream signaling molecules, including LYN (48), the cytoskeletal protein HS1 (48, 49), and ERK kinase (50).

Clinical–Translational Advances The current standard of care for younger patients with CLL is chemoimmunotherapy, and one of the most commonly used regimens is FCR (fludarabine, cyclophosphamide, and rituximab). The inferior outcomes in subsets of patients, especially those with high-risk cytogenetics (i.e., del17p) and poor tolerability of chemoimmunotherapy in patients older than 70 (which is the majority of patients with CLL) has promoted the introduction of additional agents, including ofatumumab, alemtuzumab, or bendamustine, and prompted the development of alternative therapeutics that target interactions between CLL cells and the microenvironment, including lenalidomide, inhibitors of BCR signaling, and chemokine receptors. Targeting BCR-associated kinases BTK, PI3Kd, and SYK BTK, PI3Kd, and SYK kinases are rapidly activated following BCR triggering, but also involved in other pathways,





such as chemokine and integrin signaling, they promote signal transduction to downstream partners, and regulate actin polymerization, cell–cell and cell-matrix adhesion, and chemokine secretion (CCL3 and CCL4). BTK, PI3Kd, and SYK knockout mouse models show deficiencies in Bcell development and function, supporting the development of specific inhibitors that target these kinases (45). Several orally bioavailable inhibitors are currently tested in clinical trials in patients with CLL, generating excitement because of promising responses and benign side effect profiles (36, 37, 45, 51). BTK is a nonreceptor tyrosine kinase of the Tec family, and is rapidly activated by both LYN and SYK kinases upon BCR engagement, resulting in the activation of NF-kB signaling, B-cell proliferation, and differentiation. In addition, BTK is also involved in regulation of migration and adhesion via CXCR4/CXCR5 and integrin signaling (52). Ibrutinib, previously called PCI-32765, is an irreversible BTK inhibitor that blocks BTK kinase phosphorylation and activity. Ibrutinib alone induces only modest CLL cell apoptosis in vitro, but it is capable of overcoming prosurvival signals derived from NLC-contact, CD40 ligation, BAFF, fibronectin, interleukin (IL)-6, IL-4, TNF-a (53), and BCR stimulation (54). Ibrutinib inhibits CLL cell proliferation (54), chemotaxis toward CXCL12 and CXCL13 (54, 55), integrin-mediated adhesion (55), and CLL cells release of CCL3 and CCL4, in vitro and in patients with CLL receiving therapy with ibrutinib (54). Ibrutinib also reduces tumor burden in mouse models of human CLL (54, 56). The results of a phase Ib-2 multicenter study conducted on 85 patients have recently been published and show high rates of durable remissions in patients with relapsed or refractory CLL, including patients with high-risk genetic lesions (51). Early lymphocytosis and organomegaly reduction was observed, followed by lymphocyte count normalization, which occurred more frequently and more rapidly in patients carrying unmutated IGHV genes. The estimated progression-free survival rate was 75% at 26 months, with an overall survival rate of 83%. Ibrutinib is currently explored in combination with either chemotherapy or monoclonal antibodies to shorten lymphocytosis and to increase complete remission rates (57). Whether higher response rates translate into longer progression-free survival will be critical for further development of combination strategies. PI3Ks are divided into three classes (I through III) and class I is further composed by four different isoforms (a, b, g, and d). PI3Ks regulate several cellular functions, including survival, migration, and cell growth after BCR, chemokine receptor and integrin signaling activation. The predominant form expressed by hematopoietic cells is PI3Kd, which plays a critical role in B-cell homeostasis and function. Idelalisib, previously called GS-1101 or CAL-101, is a reversible, highly selective PI3Kd inhibitor (58). Similar to BTK inhibition, PI3Kd inhibition is not very effective in causing high levels of apoptosis in unstimulated CLL cells (59), but it effectively thwarts survival signals from microenvironmental triggers, such as NLC-contact (60), CD40


ligation, TNF-a, fibronection and BCR stimulation (59), and reduction of CLL cell chemotaxis toward CXCL12 and CXCL13 (60). In addition, CCL3/4 release by CLL cells was reduced by idelalisib in vitro and in patients receiving idelalisib therapy (60). Similar to patients receiving ibrutinib therapy (51, 61), patients receiving idelalisib show early lymphocytosis after treatment start, due to CLL cell redistribution from tissues into the blood. Reduced CLL cell adhesion to stromal cells may promote their mobilization to the peripheral blood early after treatment start; once in the peripheral circulation, reduced responsiveness to CXCL12 and CXCL13 may interfere with CLL cell tissue homing. Subsequently, the lack of prosurvival signals in the peripheral blood ultimately sensitizes CLL cells to apoptosis. SYK belongs to the SYK/ZAP70 family of nonreceptor kinases, and activates signaling pathways downstream of the BCR, chemokine, and integrin receptor, suggesting that SYK participates in tissue homing and retention of activated B cells. Fostamatinib (FosD, R788) is an orally available inhibitor of SYK and rapidly converts in vivo into the bioactive form called R406 (62). R406 inhibits CLL cell migration, chemokine secretion, and BCR signaling (63) and inhibits tumor growth in a CLL mouse model (64). On the clinical side, partial responses in a number of relapsed patients with CLL were reported in phase I/II study (65), but further development of this drug focused on rheumatoid arthritis (66). Additional SYK-specific inhibitors are under development and preclinical testing, showing effective inhibition of CLL cell survival and response to microenvironmental cues (67). Targeting the CXCR4–CXCL12 axis The CXCR4–CXCL12 axis is an attractive therapeutic target, based on the importance of CXCR4 signaling for tissue homing, adhesion, and survival of CLL cells (21, 22). Several classes of CXCR4 inhibitors have been developed, including small molecule CXCR4 antagonists (Plerixafor; T140 analogs), small molecule CXCL12 antagonists (NOXA12), and antibodies to CXCR4 (MDX-1338/BMS 93656). CXCR4 antagonists inhibit CXCL12-induced signaling, chemotaxis, and stromal cell–mediated drug resistance (68). One of the early concerns in clinical development of these inhibitors was their capacity to mobilize normal hematopoietic stem cells (69), which may result in increased toxicity if cytotoxic drugs are used in combination. However, no major hematotoxicity of plerixafor in combination with chemotherapy was observed in patients with relapsed acute myelogenous leukemia (70). In a recent phase I clinical trial in relapsed patients with CLL, patients were treated with rituximab in combination with plerixafor, and a plerixafor dose-dependent mobilization of CLL cells from the tissues into the blood was reported (71). Given the robust and sustained mobilizing effects of BCR kinase inhibitors (BTK, PI3Kd, and SYK inhibitors), it is unclear in which setting the more specific CXCR4 or CXCL12 antagonists may have advantages.



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Conclusions and Perspective A plethora of cellular and molecular players in the CLL microenvironment promote the survival and evolution of CLL leukemic cells. Over the past few years, the clinical success of kinase inhibitors targeting BCR-associated kinases put the spotlight on the CLL microenvironment. The rapid translational development of these agents, along with basic discoveries, identified BCR signaling as a central pathomechanism, and BCR-related novel biomarkers that predict disease progression and response to therapy with these novel agents. The BTK inhibitor ibrutinib and the PI3Kd inhibitor idelalisib induce highly encouraging responses in CLL and will change the landscape of CLL therapy, and already have changed our view of CLL disease biology. The redistribution of CLL cells from tissue sanctuaries into the peripheral blood represents a peculiar clinical activity of these new drugs and provides a rationale for combination treatment with B cell–directed antibodies that generally have limited activ-

ity in tissue sites. That notwithstanding, as some patients may become refractory to treatment and eventually relapse after initial response to these novel agents, a more complete understanding of the in vivo mechanisms of action of these drugs and of the relationships between CLL cells and the immune microenvironment is needed, to identify additional survival pathways responsible for drug resistance and treatment failure. Authors' Contributions Conception and design: J.A. Burger Writing, review, and/or revision of the manuscript: E. ten Hacken, J.A. Burger

Grant Support This work was supported by a Cancer Prevention and Research Institute of Texas (CPRIT) grant (to J.A. Burger) and a Leukemia and Lymphoma Society Scholar Award in Clinical Research (to J.A. Burger). Received August 29, 2013; revised November 14, 2013; accepted November 15, 2013; published OnlineFirst December 9, 2013.

References 1.

Messmer BT, Messmer D, Allen SL, Kolitz JE, Kudalkar P, Cesar D, et al. In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic leukemia B cells. J Clin Invest 2005;115:755–64. 2. Lagneaux L, Delforge A, Bron D, De Bruyn C, Stryckmans P. Chronic lymphocytic leukemic B cells but not normal B cells are rescued from apoptosis by contact with normal bone marrow stromal cells. Blood 1998;91:2387–96. 3. Kurtova AV, Balakrishnan K, Chen R, Ding W, Schnabl S, Quiroga MP, et al. Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance. Blood 2009;114:4441–50. 4. Ruan J, Hyjek E, Kermani P, Christos PJ, Hooper AT, Coleman M, et al. Magnitude of stromal hemangiogenesis correlates with histologic subtype of non-Hodgkin's lymphoma. Clin Cancer Res 2006;12: 5622–31. 5. Burger JA, Tsukada N, Burger M, Zvaifler NJ, Dell'Aquila M, Kipps TJ. Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor1. Blood 2000;96:2655–63. 6. Tsukada N, Burger JA, Zvaifler NJ, Kipps TJ. Distinctive features of "nurselike" cells that differentiate in the context of chronic lymphocytic leukemia. Blood 2002;99:1030–7. 7. Burkle A, Niedermeier M, Schmitt-Graff A, Wierda WG, Keating MJ, Burger JA. Overexpression of the CXCR5 chemokine receptor, and its ligand, CXCL13 in B-cell chronic lymphocytic leukemia. Blood 2007; 110:3316–25. 8. Burger JA, Quiroga MP, Hartmann E, Burkle A, Wierda WG, Keating MJ, et al. High-level expression of the T-cell chemokines CCL3 and CCL4 by chronic lymphocytic leukemia B cells in nurselike cell cocultures and after BCR stimulation. Blood 2009;113:3050–8. 9. Herishanu Y, Perez-Galan P, Liu D, Biancotto A, Pittaluga S, Vire B, et al. The lymph node microenvironment promotes B-cell receptor signaling, NF-kappaB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood 2011;117:563–74. 10. Burger JA, Ghia P, Rosenwald A, Caligaris-Cappio F. The microenvironment in mature B-cell malignancies: a target for new treatment strategies. Blood 2009;114:3367–75. 11. Lutzny G, Kocher T, Schmidt-Supprian M, Rudelius M, Klein-Hitpass L, Finch AJ, et al. Protein kinase c-beta-dependent activation of NFkappaB in stromal cells is indispensable for the survival of chronic lymphocytic leukemia B cells in vivo. Cancer Cell 2013;23:77–92.

554


12. Nishio M, Endo T, Tsukada N, Ohata J, Kitada S, Reed JC, et al. Nurselike cells express BAFF and APRIL, which can promote survival of chronic lymphocytic leukemia cells via a paracrine pathway distinct from that of SDF-1alpha. Blood 2005;106:1012–20. 13. Troeger A, Johnson AJ, Wood J, Blum WG, Andritsos LA, Byrd JC, et al. RhoH is critical for cell-microenvironment interactions in chronic lymphocytic leukemia in mice and humans. Blood 2012; 119:4708–18. 14. Reinart N, Nguyen PH, Boucas J, Rosen N, Kvasnicka HM, Heukamp L, et al. Delayed development of chronic lymphocytic leukemia in the absence of macrophage migration inhibitory factor. Blood 2013; 121:812–21. 15. Riches JC, Gribben JG. Understanding the immunodeficiency in chronic lymphocytic leukemia: potential clinical implications. Hematol Oncol Clin North Am 2013;27:207–35. 16. Ramsay AG, Johnson AJ, Lee AM, Gorgun G, Le Dieu R, Blum W, et al. Chronic lymphocytic leukemia T cells show impaired immunological synapse formation that can be reversed with an immunomodulating drug. J Clin Invest 2008;118:2427–37. 17. Ramsay AG, Clear AJ, Fatah R, Gribben JG. Multiple inhibitory ligands induce impaired T-cell immunologic synapse function in chronic lymphocytic leukemia that can be blocked with lenalidomide: establishing a reversible immune evasion mechanism in human cancer. Blood 2012;120:1412–21. 18. Riches JC, Davies JK, McClanahan F, Fatah R, Iqbal S, Agrawal S, et al. T cells from CLL patients exhibit features of T-cell exhaustion but retain capacity for cytokine production. Blood 2013;121:1612–21. 19. Reiners KS, Topolar D, Henke A, Simhadri VR, Kessler J, Sauer M, et al. Soluble ligands for NK cell receptors promote evasion of chronic lymphocytic leukemia cells from NK cell anti-tumor activity. Blood 2013;121:3658–65. 20. Veuillen C, Aurran-Schleinitz T, Castellano R, Rey J, Mallet F, Orlanducci F, et al. Primary B-CLL resistance to NK cell cytotoxicity can be overcome in vitro and in vivo by priming NK cells and monoclonal antibody therapy. J Clin Immunol 2012;32:632–46. 21. Burger JA, Burger M, Kipps TJ. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. Blood 1999;94:3658–67. 22. Calissano C, Damle RN, Hayes G, Murphy EJ, Hellerstein MK, Moreno C, et al. In vivo intraclonal and interclonal kinetic heterogeneity in B-cell chronic lymphocytic leukemia. Blood 2009;114:4832–42.





23. Richardson SJ, Matthews C, Catherwood MA, Alexander HD, Carey BS, Farrugia J, et al. ZAP-70 expression is associated with enhanced ability to respond to migratory and survival signals in B-cell chronic lymphocytic leukemia (B-CLL). Blood 2006;107:3584–92. 24. Deaglio S, Vaisitti T, Aydin S, Bergui L, D'Arena G, Bonello L, et al. CD38 and ZAP-70 are functionally linked and mark CLL cells with high migratory potential. Blood 2007;110:4012–21. 25. Brachtl G, Sahakyan K, Denk U, Girbl T, Alinger B, Hofbauer SW, et al. Differential bone marrow homing capacity of VLA-4 and CD38 high expressing chronic lymphocytic leukemia cells. PLoS ONE 2011;6: e23758. 26. Zucchetto A, Vaisitti T, Benedetti D, Tissino E, Bertagnolo V, Rossi D, et al. The CD49d/CD29 complex is physically and functionally associated with CD38 in B-cell chronic lymphocytic leukemia cells. Leukemia 2012;26:1301–12. 27. Burger JA, Zvaifler NJ, Tsukada N, Firestein GS, Kipps TJ. Fibroblastlike synoviocytes support B-cell pseudoemperipolesis via a stromal cell-derived factor-1- and CD106 (VCAM-1)-dependent mechanism. J Clin Invest 2001;107:305–15. 28. Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999;94:1840–7. 29. Gattei V, Bulian P, Del Principe MI, Zucchetto A, Maurillo L, Buccisano F, et al. Relevance of CD49d protein expression as overall survival and progressive disease prognosticator in chronic lymphocytic leukemia. Blood 2008;111:865–73. 30. Ghia P, Strola G, Granziero L, Geuna M, Guida G, Sallusto F, et al. Chronic lymphocytic leukemia B cells are endowed with the capacity to attract CD4þ, CD40Lþ T cells by producing CCL22. Eur J Immunol 2002;32:1403–13. 31. Scielzo C, Apollonio B, Scarfo L, Janus A, Muzio M, Ten Hacken E, et al. The functional in vitro response to CD40 ligation reflects a different clinical outcome in patients with chronic lymphocytic leukemia. Leukemia 2011;25:1760–7. 32. Sivina M, Hartmann E, Kipps TJ, Rassenti L, Krupnik D, Lerner S, et al. CCL3 (MIP-1{alpha}) plasma levels and the risk for disease progression in chronic lymphocytic leukemia (CLL). Blood 2011; 117:1662–9. 33. Krzysiek R, Lefevre EA, Zou W, Foussat A, Bernard J, Portier A, et al. Antigen receptor engagement selectively induces macrophage inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta chemokine production in human B cells. J Immunol 1999;162:4455–63. 34. Bystry RS, Aluvihare V, Welch KA, Kallikourdis M, Betz AG. B cells and professional APCs recruit regulatory T cells via CCL4. Nature Immunol 2001;2:1126–32. 35. Stevenson FK, Krysov S, Davies AJ, Steele AJ, Packham G. B-cell receptor signaling in chronic lymphocytic leukemia. Blood 2011;118: 4313–20. 36. Woyach JA, Johnson AJ, Byrd JC. The B-cell receptor signaling pathway as a therapeutic target in CLL. Blood 2012;120:1175–84. 37. Burger JA, Chiorazzi N. B cell receptor signaling in chronic lymphocytic leukemia. Trends Immunol 2013;34:592–601. 38. Shapiro-Shelef M, Calame K. Regulation of plasma-cell development. Nat Rev Immunol 2005;5:230–42. 39. Lanham S, Hamblin T, Oscier D, Ibbotson R, Stevenson F, Packham G. Differential signaling via surface IgM is associated with VH gene mutational status and CD38 expression in chronic lymphocytic leukemia. Blood 2003;101:1087–93. 40. Krysov S, Dias S, Paterson A, Mockridge CI, Potter KN, Smith KA, et al. Surface IgM stimulation induces MEK1/2-dependent MYC expression in chronic lymphocytic leukemia cells. Blood 2012;119:170–9. 41. Morabito F, Cutrona G, Gentile M, Fabbi M, Matis S, Colombo M, et al. Prognostic relevance of in vitro response to cell stimulation via surface IgD in binet stage a CLL. Br J Haematol 2010;149:160–3. 42. Negro R, Gobessi S, Longo PG, He Y, Zhang ZY, Laurenti L, et al. Overexpression of the autoimmunity-associated phosphatase PTPN22 promotes survival of antigen-stimulated CLL cells by selectively activating AKT. Blood 2012;119:6278–87. 43. Young RM, Staudt LM. Targeting pathological B cell receptor signalling in lymphoid malignancies. Nat Rev Drug Discov 2013;12:229–43.


44. Hoogeboom R, van Kessel KP, Hochstenbach F, Wormhoudt TA, Reinten RJ, Wagner K, et al. A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi. J Exp Med 2013;210:59–70. 45. Burger JA, Montserrat E. Coming full circle: 70 years of chronic lymphocytic leukemia cell redistribution, from glucocorticoids to inhibitors of B-cell receptor signaling. Blood 2013;121:1501–9. 46. Duhren-von Minden M, Ubelhart R, Schneider D, Wossning T, Bach MP, Buchner M, et al. Chronic lymphocytic leukaemia is driven by antigen-independent cell-autonomous signalling. Nature 2012;489: 309–12. 47. Binder M, Muller F, Frick M, Wehr C, Simon F, Leistler B, et al. CLL Bcell receptors can recognize themselves: alternative epitopes and structural clues for autostimulatory mechanisms in CLL. Blood 2013; 121:239–41. 48. ten Hacken E, Scielzo C, Bertilaccio MT, Scarfo L, Apollonio B, Barbaglio F, et al. Targeting the LYN/HS1 signaling axis in chronic lymphocytic leukemia. Blood 2013;121:2264–73. 49. Scielzo C, Ghia P, Conti A, Bachi A, Guida G, Geuna M, et al. HS1 protein is differentially expressed in chronic lymphocytic leukemia patient subsets with good or poor prognoses. J Clin Invest 2005;115: 1644–50. 50. Apollonio B, Scielzo C, Bertilaccio MT, Ten Hacken E, Scarfo L, Ranghetti P, et al. Targeting B-cell anergy in chronic lymphocytic leukemia. Blood 2013;121:3879–88, S1–8. 51. Byrd JC, Furman RR, Coutre SE, Flinn IW, Burger JA, Blum KA, et al. Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. N Engl J Med 2013;369:32–42. 52. de Gorter DJ, Beuling EA, Kersseboom R, Middendorp S, van Gils JM, Hendriks RW, et al. Bruton's tyrosine kinase and phospholipase Cgamma2 mediate chemokine-controlled B cell migration and homing. Immunity 2007;26:93–104. 53. Herman SE, Gordon AL, Hertlein E, Ramanunni A, Zhang X, Jaglowski S, et al. Bruton's tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765. Blood 2011;117:6287–96. 54. Ponader S, Chen SS, Buggy JJ, Balakrishnan K, Gandhi V, Wierda WG, et al. The Bruton tyrosine kinase inhibitor PCI-32765 thwarts chronic lymphocytic leukemia cell survival and tissue homing in vitro and in vivo. Blood 2012;119:1182–9. 55. de Rooij MF, Kuil A, Geest CR, Eldering E, Chang BY, Buggy JJ, et al. The clinically active BTK inhibitor PCI-32765 targets B-cell receptorand chemokine-controlled adhesion and migration in chronic lymphocytic leukemia. Blood 2012;119:2590–4. 56. Herman SE, Sun X, McAuley EM, Hsieh MM, Pittaluga S, Raffeld M, et al. Modeling tumor-host interactions of chronic lymphocytic leukemia in xenografted mice to study tumor biology and evaluate targeted therapy. Leukemia 2013;27:2311–21. 57. Jain N, O'Brien S. Ibrutinib (PCI-32765) in chronic lymphocytic leukemia. Hematol Oncol Clin North Am 2013;27:851–60. 58. Lannutti BJ, Meadows SA, Herman SE, Kashishian A, Steiner B, Johnson AJ, et al. CAL-101, a p110{delta} selective phosphatidylinositol-3-kinase inhibitor for the treatment of B-cell malignancies, inhibits PI3K signaling and cellular viability. Blood 2011;117: 591–4. 59. Herman SE, Gordon AL, Wagner AJ, Heerema NA, Zhao W, Flynn JM, et al. Phosphatidylinositol 3-kinase-d inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals. Blood 2010; 116:2078–88. 60. Hoellenriegel J, Meadows SA, Sivina M, Wierda WG, Kantarjian H, Keating MJ, et al. The phosphoinositide 30 -kinase delta inhibitor, CAL101, inhibits B-cell receptor signaling, and chemokine networks in chronic lymphocytic leukemia. Blood 2011;118:3603–12. 61. Advani RH, Buggy JJ, Sharman JP, Smith SM, Boyd TE, Grant B, et al. Bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) has significant activity in patients with relapsed/refractory B-cell malignancies. J Clin Oncol 2013;31:88–94. 62. Braselmann S, Taylor V, Zhao H, Wang S, Sylvain C, Baluom M, et al. R406, an orally available spleen tyrosine kinase inhibitor blocks fc



555



63.

64.

65.

66.

556

receptor signaling and reduces immune complex-mediated inflammation. J Pharmacol Exp Ther 2006;319:998–1008. Quiroga MP, Balakrishnan K, Kurtova AV, Sivina M, Keating MJ, Wierda WG, et al. B cell antigen receptor signaling enhances chronic lymphocytic leukemia cell migration and survival: specific targeting with a novel Syk inhibitor, R406. Blood 2009;114: 1029–37. Suljagic M, Longo PG, Bennardo S, Perlas E, Leone G, Laurenti L, et al. The Syk inhibitor fostamatinib disodium (R788) inhibits tumor growth in the Emu- TCL1 transgenic mouse model of CLL by blocking antigen-dependent B-cell receptor signaling. Blood 2010; 116:4894–905. Friedberg JW, Sharman J, Sweetenham J, Johnston PB, Vose JM, Lacasce A, et al. Inhibition of Syk with fostamatinib disodium has significant clinical activity in non-Hodgkin lymphoma and chronic lymphocytic leukemia. Blood 2010;115:2578–85. Weinblatt ME, Kavanaugh A, Genovese MC, Musser TK, Grossbard EB, Magilavy DB. An oral spleen tyrosine kinase (Syk) inhibitor for rheumatoid arthritis. New England J Med 2010;363:1303–12.


67. Hoellenriegel J, Coffey GP, Sinha U, Pandey A, Sivina M, Ferrajoli A, et al. Selective, novel spleen tyrosine kinase (Syk) inhibitors suppress chronic lymphocytic leukemia B-cell activation and migration. Leukemia 2012;26:1576–83. 68. Burger JA, Peled A. CXCR4 antagonists: targeting the microenvironment in leukemia and other cancers. Leukemia 2009;23:43–52. 69. Hendrix CW, Flexner C, MacFarland RT, Giandomenico C, Fuchs EJ, Redpath E, et al. Pharmacokinetics and safety of AMD-3100, a novel antagonist of the CXCR-4 chemokine receptor, in human volunteers. Antimicrob Agents Chemother 2000;44:1667–73. 70. Uy GL, Rettig MP, Motabi IH, McFarland K, Trinkaus KM, Hladnik LM, et al. A phase 1/2 study of chemosensitization with the CXCR4 antagonist plerixafor in relapsed or refractory acute myeloid leukemia. Blood 2012;119:3917–24. 71. Andritsos L, Byrd JC, Jones JA, Hewes B, Kipps TJ, Hsu FJ, et al. Preliminary results from a phase I dose escalation study to determine the maximum tolerated dose of plerixafor in combination with rituximab in patients with relapsed chronic lymphocytic leukemia. Blood 2010;116:1017a.




Molecular Pathways: Targeting the Microenvironment in Chronic Lymphocytic Leukemia−−Focus on the B-Cell Receptor Elisa ten Hacken and Jan A. Burger Clin Cancer Res 2014;20:548-556. Published OnlineFirst December 9, 2013.

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