The biological rationale and clinical efficacy of inhibition of signaling kinases in chronic lymphocytic leukemia

Leukemia Research 37 (2013) 838–847

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Leukemia Research journal homepage: www.elsevier.com/locate/leukres

Invited review

The biological rationale and clinical efficacy of inhibition of signaling kinases in chronic lymphocytic leukemia Iris de Weerdt a , Eric Eldering b,c , Marinus H. van Oers a,c , Arnon P. Kater a,c,∗ a b c

Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands Laboratory of Experimental Medicine, Academic Medical Center, Amsterdam, The Netherlands LYMMCARE (Lymphoma and Myeloma Center Amsterdam), Academic Medical Center, The Netherlands

a r t i c l e

i n f o

Article history: Received 13 December 2012 Received in revised form 15 February 2013 Accepted 17 March 2013 Available online 15 April 2013 Keywords: Chronic lymphocytic leukemia Microenvironment Signaling BTK Syk PI-3 kinase

a b s t r a c t Chronic lymphocytic leukemia (CLL) is still incurable, with considerable resistance to the standard therapy. CLL cells receive anti-apoptotic and pro-proliferation stimuli in lymph nodes and bone marrow, mainly through B cell receptor activation and TNF-receptor family ligation. In recent years, the focus for finding new drugs has shifted to blocking signals from the microenvironment. Novel therapeutical agents interfere with these microenvironmental interactions, and include inhibitors of kinases Syk, Btk and PI3K␦. In this review we will focus on the microenvironmental interactions of CLL and the role of tyrosine kinases. Furthermore, early results from clinical trials with kinase inhibitors are discussed. © 2013 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3.

4.

5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Key factors that enhance activation of CLL cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. BCR activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ligation of TNF-receptor family members . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Nurse-like cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intracellular signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Upstream signaling events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Downstream kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Activation of transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Signaling in CLL cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kinase inhibitors in CLL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Inhibition of multiple kinases by dasatinib . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.1. Monotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.2. Combination therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Inhibitor of Btk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. Monotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. Combination therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Inhibition of Syk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1. Monotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Inhibition of PI3K␦ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.1. Monotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.2. Combination therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author at: Department of Hematology, Academic Medical Centre, Amsterdam, University of Amsterdam, The Netherlands. Tel.: +31 20 5665785; fax: +31 20 6919743. E-mail address: [email protected] (A.P. Kater). 0145-2126/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2013.03.011

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6.

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflicts of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Chronic lymphocytic leukemia (CLL) is a malignancy of mature B lymphocytes accumulating in the peripheral blood (PB), lymph nodes (LN), bone marrow (BM), spleen and liver [1]. CLL mainly affects the elderly and has a highly variable course. Standard therapy for fit patients has shifted from monotherapy with alkylating agents aiming at alleviation of symptoms to immunochemotherapy with the goal of prolonged progression free survival and improved overall survival [2]. Still, such treatments are not considered curative. In recent years it has become evident that for survival and proliferation CLL cells are highly dependent on external stimuli. Only very recently, these interactions became the focus of novel treatment options, which will likely radically change the outcome of this disease. Despite their malignant nature CLL cells retain their susceptibility to external signals in LNs and BM, largely resembling mature healthy B cells. These interactions are collectively referred to as the microenvironment. Healthy B cells become activated upon antigen ligation to the B cell receptor (BCR), resulting in proliferation and differentiation. This can be further enhanced by cytokine stimulation and co-stimulation. The various signals from the microenvironment together orchestrate the activation of B cells and likewise of CLL cells. The LNs and BM thus provide a protective niche for CLL cells, enabling progression of the disease [3]. Kinases play a key role within the signaling cascades activated upon microenvironmental interactions. Several new therapeutic strategies that are currently under investigation for CLL specifically aim to inhibit kinases. Understanding the role of signaling kinases is mandatory for designing novel trials which aim to target the microenvironment. We will first review the current vision of the dominant external stimuli present in the CLL microenvironment. We then summarize evidence for activation of key signaling cascades in CLL. Finally, we will discuss the first clinical results of kinase inhibitors in CLL. 2. Key factors that enhance activation of CLL cells A large number of factors have been studied that may contribute to the activation of CLL cells. Factors that potentially contribute include cell–cell contact, chemokines, cytokines and activation of the BCR [4,5]. While many of these factors appear to contribute in vitro, it is yet to be defined which factors are relevant for CLL cell activation in vivo. A comparative study of expression levels of apoptotic regulators in CLL cells residing in the PB versus LNs revealed overexpression of anti-apoptotic Bcl-2 family members Bcl-xL, Mcl-1 and Bfl-1 in the LNs [6]. These molecules are regulated by the transcription factor nuclear factor ␬-B (NF-␬B) [7]. In B cells TNF-receptor associated factors (TRAFs) regulate NF-␬B translocation to the nucleus [8,9]. Members of the TNF-receptor family activate TRAFs, among which are CD40, B-cell activating factor (BAFF), A Proliferation-Inducing Ligand (APRIL) and TNF-related apoptosis-inducing ligand (TRAIL). A dominant NF-␬B signature in LN-derived CLL cells was also described by Herishanu et al. [10], who performed comparative gene expression analyses to elucidate differences between PB, LNs and BM derived CLL cells [10]. The LN emerged as the pivotal site for CLL activation, while alterations in gene expression in BM were not as markedly different from the PB.

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LN-derived CLL cells were characterized by two dominant patterns: increased BCR signaling and an activated NF-␬B signature. Taken together, the dominant factors in the microenvironment appear to be BCR activation and TNF-receptor family activation, further supported by cytokines and chemokines (Fig. 1). 2.1. BCR activation In normal B cell biology, BCR activation is initiated by the encounter of antigen in the T cell-rich areas of secondary lymphoid organs. There, naive B cells get activated and become germinal center (GC) founder B cells. GC founders enter the GC to become centroblasts that proliferate and mutate their BCR. Centroblasts differentiate into centrocytes and undergo selection. Selection requires adequate recognition of antigen presented by follicular dendritic cells, as well as presentation of processed antigen to GC T cells. BCR signaling is thought to be equally important in CLL, but likely occurs in a different fashion. One possibility recently suggested by Duhren-von Minden et al. [11] is that in CLL, unlike other B cell lymphomas, BCR signaling takes place independent of antigen. The proposed underlying mechanism is the ability of the heavy chain complementarity region 3 (HCDR3) of CLL cells to bind to an intrinsic motif of the BCR. This intrinsic motif was found to be the framework region 2 (FR2) of the heavy chain variable region. On the other hand, over 30% of chronic lymphocytic leukemias can be grouped based on expression of stereotypic BCRs with characteristic HCDR3 amino acid sequences [12–14]. As HCDR3s are most decisive for the antigen specificity of immunoglobulins (Igs), this strongly suggests that specific subsets of CLL recnogize distinctive antigens. Unmutated CLL cells (U-CLL), expressing Ig heavy chain variable domains (IGHV) in germline configuration, were found to express low-affinity poly- and self-reactive BCRs [15]. In contrast, the specificity of CLL with somatically mutated IGHV (M-CLL) remains unclear. Recently, it was described that a subset of M-CLL expresses stereotypic BCRs highly specific for ␤-(1,6)glucan, which is a major antigenic determinant of yeast and molds. CLL cells expressing these stereotypic receptors became activated resulting in marked proliferation in response to ␤-(1,6)-glucan [16], establishing a group of common pathogens as functional ligands for a subset of human CLL. A synthesis of both antigenindependent and dependent BCR activation has been suggested by Chiorazzi et al., who described a model in which both autonomous and antigen-dependent cell signaling occurs [17]. Cell-autonomous signaling may enable certain cells to live longer, and lower the threshold for stimulation by other signals. Stimulation with antigens could than result in clonal expansion. 3. Ligation of TNF-receptor family members TNF receptor family members are key players in regulating immune functions. Upon activation of TNF receptors, proliferation, apoptosis and differentiation of immune cells may be modified. The ligands for TNF receptors present in the microenvironment of CLL cells include among others CD40L, BAFF and APRIL [18]. 3.1. T cells CLL cells express receptors for interactions with T cells. Most notably, CD40L on T cells can bind to CD40 on CLL cells. Using immunohistochemistry, activated T cells were detected in close

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Fig. 1. Microenvironmental interactions of CLL. BCR signaling is triggered upon antigen binding. T cells stimulate CLL cells with cytokines (e.g. TNF-␣, IL-4) and CD40-CD40L interaction. CLL secrete chemokines to attract T cells (CCL3, CCL4, CCL22). NLCs and stromal cells secrete chemokines (CXCL12, CXCL13).

proximity of CLL cells in LN biopsy specimens [19]. CD4+ T cells costimulate CLL cells through CD40–CD40L interaction, causing upregulation of prosurvival Bcl-2 proteins [20] and survivin [21]. The expression profile of CLL cells residing in LNs can be largely mimicked in vitro by prolonged CD40 stimulation [6,22]. Furthermore, T cells produce cytokines that stimulate CLL cells, such as TNF-␣, IL-10, IL-4 and IL-21. Particularly, IL-21 can induce proliferation of CLL cells, in combination with CD40 activation [23]. CLL cells attract T cells by secretion of CCL3 and CCL4. 3.2. Nurse-like cells Nurse-like cells (NLCs) are monocyte-derived cells, which develop following prolonged in vitro cultures with CLL blood samples [24]. Similar cells appear to be present in the spleen and LNs of CLL patients [25]. NLCs seem to be important players in the microenvironment, indicated by the similarity between gene expression profiles of LN-derived CLL samples and in vitro cocultures of CLL cells with NLCs [10,26]. NLCs express the TNF family members BAFF and APRIL [27]. Blocking the binding of these ligands to their cognate receptors significantly reversed pro-survival effects of CLL cells induced by coculture with NLCs [28]. In addition to expression of TNF-family ligands, both NLCs and stromal cells produce chemokines CXCL12 and CXCL13. CXCL12 is a ligand for CXCR4, expressed on CLL cells. Besides chemotaxis, CXCL12 exerts a direct prosurvival effect on CLL cells [24]. Furthermore, NLCs express CD31, which can interact with CD38 expressed by CLL cells [29]. Whether CD38 ligation results in cell survival and proliferation of CLL cells is a matter of controversy [30].

cascades of healthy B cells. To provide a better understanding of the pathogenesis of CLL, a summary of upstream signaling cascades concerning the BCR and TNFRs in healthy B cells will shortly be described. While the B cell receives numerous stimuli, including cytokines and chemokines, only signaling cascades that involve the BCR and TNFRs will be discussed here, for the sake of brevity. Upon antigen binding, BCR signaling is triggered. The BCR complex consists of immunoglobulin expressed on the surface of the B cell and the non-covalently bound heterodimer CD79␣ (Ig␣) and CD79␤ (Ig␤; see Fig. 2). Ig␣ and Ig␤ have cytoplasmic domains, which contain immunoreceptor tyrosine activating motifs (ITAMs) [31]. These ITAMs enable the BCR to transduce its signal inside the cell. Upon BCR ligation by antigen Lyn and Syk bind to the ITAMs, resulting in their phosphorylation. Consecutively, Syk and the B cell linker protein (BLNK) are involved in activating Bruton’s tyrosine kinase (Btk) and Phospholipase C␥2 (PLC␥2) [31]. PI3-Kinase (PI3K) is activated by Syk and Btk. Both Syk and Btk are important in amplification of the BCR signal [32,33]. In addition to activation of downstream pathways, Btk is also thought to play a direct role in activation of transcription factors, such as NF-␬B and NF-AT [34,35] and may act as an adapter protein [36]. Other important stimuli in the CLL microenvironment include CD40L, BAFF and APRIL. These stimuli engage CD40, BAFF-receptor, BCMA and TACI, each belonging to the same subgroup of TNF receptors. This subgroup of receptors has cytoplasmic domains with TRAF-interacting motifs. Upon receptor activation TRAFs are recruited, subsequently activating multiple pathways, including the MAPK pathway, the PI3K/Akt pathway and activation of NF␬B [9,37]. In addition, upon CD40 activation, signal transduction starts with JAK and STAT signaling [38].

4. Intracellular signaling pathways 4.2. Downstream kinases 4.1. Upstream signaling events Stimuli from the microenvironment trigger specific signaling cascades in CLL cells, which bear extensive resemblance to signaling

When PI3K is activated, PI3K phosphorylates PIP2 , creating PIP3 . PIP3 provides docking sites for 3-phosphoinositide dependent protein kinases, PDPK1 and PDPK2, which are necessary for complete

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Fig. 2. The intracellular signaling pathways of B cells following activation of receptor complexes (TNFRs, BCR and CD19/CD21/CD81; dark blue) are shown. Intertwining pathways of upstream (yellow) and downstream (red) kinases eventually lead to translocation of transcription factors (light blue). (For interpretation of the references to color in this figure caption, the reader is referred to the web version of the article.)

activation of Akt [39]. Akt subsequently activates the mTOR pathway, binds IKK and members of the Bcl-2 family [40]. The mTOR pathway is important in regulating protein synthesis and cell cycle entry. PIP3 also assists Btk and Syk in activating PLC␥2 [31]. PLC␥2 splits PIP2 into IP3 and DAG. IP3 leads to intracellular Ca2+ release. Ca2+ and DAG are involved in activation of protein kinase C (PKC). TRAF and Syk are responsible for activating Ras [31,38]. Activated Ras leads to activation of Raf-1, which triggers the MAPK pathway. The MAPK pathway may activate ERK1/2, c-Jun kinases and p38 proteins [41].

4.3. Activation of transcription factors The kinases described above activate several transcription factors, among which are Elk-1, Jun, ATF-2, NF-AT and NF-␬B. These activated transcription factors bind to promoters of cytokines and genes involved in proliferation and cell growth [41]. Ca2+ influx leads to NF-AT nuclear translocation. This transcription factor is involved in gene transcription of cytokines and their receptors [42]. NF-␬B is activated through several pathways, among which are the PI3K/Akt and PKC pathway. Two different NF-␬B pathways exist, the classical pathway and the alternative pathway, which influence expression of distinct downstream pathways [43]. NF-␬B is bound to I␬B preventing its translocation to the nucleus. The classical pathway is initiated upon phosphorylation of the IKK complex, which consists of subunits IKK␣, IKK␤ and IKK␥/NEMO [44]. Phosphorylation of the IKK complex leads to proteosomal degradation of I␬B proteins. The alternative pathway is regulated differently. Upon TNF-receptor stimulation through BAFF or CD40L the NF-␬B inducing kinase (NIK) is stabilized. Stabilization leads to NIK activation and subsequent IKK␣ phosphorylation. The NF␬B p100 precursor protein is then phosphorylated, resulting in the generation of active RelB-p52 heterodimers. Once in the nucleus, NF-␬B is involved in transcription of multiple genes regulating differentiation, proliferation and survival [44]. The latter is mostly regulated by induction of expression levels of pro-survival molecules from the Bcl-2 family [45,46].

4.4. Signaling in CLL cells CLL intracellular signaling largely resembles that of healthy B cells, but there are some important differences (see Table 1). CLL cells in the PB have increased levels of Lyn [47], Syk [48,49] Btk [50] and PI3K [51]. Akt was identified as an especially important mediator of apoptosis resistance after sustained BCR stimulation [52]. In addition, the expression level of NF-␬B in circulating CLL cells is higher than in healthy B cells [53,54]. The balance between proand anti-apoptotic members of the Bcl-2 family is altered in CLL cells in the PB. While CLL cells express high levels of anti-apoptotic Bcl-2 [55,56], pro-apoptotic Bcl-2 proteins Noxa and Bmf are also upregulated [56] consistent with the observation that PB CLL cells frequently undergo spontaneous apoptosis ex vivo [57]. Although expression levels of molecules downstream of the BCR are increased in circulating CLL cells, expression is far more pronounced in LN-derived CLL cells [10]. The same holds true for transcription factors NF-␬B and NF-AT [10]. When profiles of apoptotic regulators of LN-derived CLL cells were compared with circulating counterparts, CLL cells appeared significantly less primed for apoptosis in the LN [6]. The pro-apoptotic protein Noxa was high in peripheral blood CLL, but absent in LNs. Simultaneously, anti-apoptotic Bcl-xL, Bfl-1 and Mcl-1 were upregulated, resulting in reduced susceptibility to cell death.

Table 1 Differential BCR signaling, NF-␬B activation and apoptotic regulators in the peripheral blood and lymph nodes.

BCR signaling

NF-␬B activation Pro-apoptotic proteins Anti-apoptotic proteins

Peripheral blood

Lymph node

+ Lyn [47], Syk [48,49], Btk [50], PI3K [51] + [53,54] + Noxa, Bmf [56] + Bcl-2 [55,56]

+++ [10] +++ [10] − [6] +++ Bcl-2, Bcl-xL, Mcl-1, Survivin [6]

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Table 2 Results of clinical trials of kinase inhibitors as monotherapy. R/R: relapsed or refractory, TN: treatment naive, U-CLL: unmutated CLL, CR: complete remission, PR: partial remission, ORR: overall response rate, SD: stable disease, NR: nodal response, AE: adverse event. Multiple kinases

Btk

Syk

PI3K␦

Study design

Study drug R/R or TN Disease

Dasatinib [66] R/R CLL

Ibrutinib [90] R/R NHL/CLL

Ibrutinib [72] R/R CLL/SLL

Ibrutinib [72] TN CLL/SLL

AVL-292 [73] R/R CLL/NHL

R788 [82] R/R CLL/SLL lymphomas

CAL-101 [86] R/R CLL

Patient characteristics

Number (of which CLL) Median age Median number of prior therapy Molecular features

15 (15)

56 (16)

61 (61)

31 (31)

16 (9)

24 (11)

54 (54)

59 3

65 3

64 4

71 0

66 2

62 2.5

62 5

11q/17p: 60%

Unknown

U-CLL: 86% 17p: 37%

U-CLL: 55% 17p: 7%

U-CLL: 44% 11q/17p: 56%

Unknown

17p: 36%

Median follow-up

Unknown

Unknown

17.3 months

16.6 months

3 months

Unknown

Unknown

Response

CR PR ORR SD “NR”

0% 20% 20% Unknown 7%

13% 56% 69% 13% 6%

3% 61% 64% 5% 20%

10% 61% 71% 13% 10%

0% 0% 0% 89% Unknown

0% 55% 55% 8% Unknown

0% 33% 33% Unknown 57%

Toxicity

Three most common AEs

Neutropenia Anemia Thrombocytopenia

Pain

Diarrhea Fatigue Upper respiratory tract infection

Diarrhea Nausea Urticaria

Diarrhea Fatigue Neutropenia

Unknown

Both upstream kinases and downstream effector molecules, specifically anti-apoptotic Bcl-2 family members have recently come into focus for targeted therapy [58,59]. In the remainder of this review we will focus on the use of kinase inhibitors in CLL.

Table 2 (monotherapy) and Table 3 (combination regimens). All of these compounds are available orally and administered daily (dasatinib, ibrutinib, AVL-292, fostamatinib) or twice daily (GS-1101). 5.1. Inhibition of multiple kinases by dasatinib

5. Kinase inhibitors in CLL Inhibition of key signaling kinases aims at depriving CLL cells of microenvironmental stimuli. In vitro experiments suggest that inhibition of signaling pathways not only deprives CLL of these stimuli, but may also lead to direct apoptosis [50,60]. Currently, the broad-spectrum kinase inhibitor dasatinib as well as selective kinase inhibitors of Syk, Btk and PI3K␦ have been studied clinically in CLL. The first results with kinase inhibitors are summarized in

The reversible kinase inhibitor dasatinib was designed as a second-generation inhibitor for the treatment of chronic myeloid leukemia, as it potently inhibits the BCR-Abl kinase [61]. The experience with dasatinib in other leukemias was very positive; showing long progression-free survival (PFS) and limited toxicity [62]. Aside from inhibiting the BCR-Abl kinase, dasatinib inhibits SRC family kinases (such as Lyn) and Btk [63,64]. This launched the idea to investigate the potential of this drug in CLL [20].

Table 3 Results of clinical trials of kinase inhibitors in combination regimens. R/R: relapsed or refractory, TN: treatment naive, CR: complete remission, PR: partial remission, ORR: overall response rate, SD: stable disease, NR: nodal response, ≥50% reduction in lymph node size. R: rituximab, B: bendamustine, BR: bendamustine and rituximab. Multiple kinases

Btk

PI3K␦ Ibrutinib [77] Ofatumumab

CAL-101 [87] Bendamustine and/or rituximab

Study design

Study drug Combination drugs R/R or TN

Dasatinib [67] Fludarabine R/R

Ibrutinib [74] Bendamustine and rituximab R/R

R/R

R/R

Patient characteristics

Number Median age Median number of prior therapy Molecular features

20 68 5

30 62 2

24 66 3

51 Unknown Unknown

11q/17p: 40%

Unknown

11q/17p: 79%

Unknown

Median follow-up (months)

Unknown

4.9

6.5

R (19)

B (17)

BR (15)

CR PR ORR SD “NR”

0% 18% 18% 67% 6%

10% 80% 90% Unknown 7%

0% 100% 100% 0% 0%

0% 78% 78% 11% 6%

0% 82% 82% 6% 0%

7% 80% 87% 0% 0%

Response

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In vitro observations indicated that dasatinib effectively inhibits expression of CD40-mediated anti-apoptotic regulators and restores fludarabine sensitivity [20]. Furthermore, dasatinib has direct cytotoxic effects against CLL cells [65]. 5.1.1. Monotherapy A phase 2 study of dasatinib monotherapy included 15 patients, all of whom had previously received fludarabine [66]. The median age of the patients was 59 years. 60% of these patients had poor prognostic scores including deletion of 17p or 11q. Toxicity was generally manageable and mainly involved hematological adverse events (AEs). Most response was seen in reduction of LN size, rather than lymphocyte counts. 9 patients had a reduction of LN size, but only 3 patients matched the criteria for a partial response (PR). The patients with 11q deletions did relatively well; 2 of these patients had a PR [66]. 5.1.2. Combination therapy Based upon the in vitro observation that dasatinib effectively inhibits expression of CD40 mediated anti-apoptotic regulators and restores fludarabine sensitivity, a phase 2 trial of dasatinib and fludarabine treatment in chemo-refractory CLL patients was performed [67]. 20 fludarabine-refractory patients were included, with a median age of 69 years. 85% of the patients were in Rai stage 3 or 4 and the median number of prior therapies was 5. Treatment was initiated with monotherapy of dasatinib for all patients, with subsequent addition of fludarabine. During monotherapy with dasatinib, toxicity was modest. The toxicity during combined fludarabine and dasatinib treatment was more pronounced as expected with chemotherapy, with neutropenia and thrombocytopenia as most observed toxicities. Although only 18% of the patients achieved a PR, most patients had a reduction in LN size with a median reduction of 20%. Patients with a reduction of ≥20% had significantly improved PFS and overall survival (OS) compared to non-responding patients. 5.2. Inhibitor of Btk The best studied irreversible Btk inhibitor to date, PCI-32765 or ibrutinib, was developed in 2007. Development of additional Btk inhibitors has led to the new inhibitor AVL-292, which is currently also studied clinically [68]. In vitro treatment of CLL cells with ibrutinib led to a variable, overall modest, induction of cell death. Ibrutinib, which had little effect on the viability of healthy B cells and T cells, could to some extent reverse the protective effects of CD40L, BAFF, TNF-␣, IL-6 and IL-4 [69]. In a CLL mouse model, mice were treated with the alkylating agent busalfan and ibrutinib. Using CFSE labeling, Herman et al. found reduced proliferation of CLL cells in PB, spleen and BM. The number of CLL cells in the PB was rather similar to that of the control mice, but the spleen infiltration was substantially lower in the treated mice [50]. In mouse models and human clinical trials with kinase inhibitors, a transient lymphocytosis is seen in combination with a reduction in lymphadenopathy. This effect can be explained by the impact of ibrutinib on adhesion and migration [70]. Ibrutinib prevented anti-IgM induced adhesion, in addition to disrupting adhesion and homing of CLL cells in response to chemokines. Since this study found that ibrutinib did not affect the viability of CLL cells it suggests that ibrutinib drives CLL cells out of the LNs and BM, briefly leading to a rise in lymphocyte counts in the blood. Once in the PB, the CLL cells are no longer protected by the interactions with their microenvironment and become prone to spontaneous apoptosis or drug-induced cell death. Although evidence lacks, the same mechanism likely applies to inhibitors of Syk and PI3K␦.

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Only one full article on a Btk inhibitor clinical trial has been published to date, however several updates have been presented at hematology conferences. 5.2.1. Monotherapy In a single-agent ibrutinib trial for relapsed or refractory (R/R) non-Hodgkin lymphoma (NHL) and CLL, both a 28 days on, 7 days off and continuous dosage were evaluated. Based on Btk occupancy tests and the observation that treatment-associated lymphocytosis reversed during the 7 days off, a dose of 560 mg continuously was considered optimal. 56 patients with a median age of 65 years were enrolled, 16 of whom had CLL. Grade 3 or 4 toxicity was uncommon and there was no evidence of cumulative toxicity. Hematologic toxicity was rare, as it occurred in 12.5% (neutropenia), 7.2% (thrombocytopenia) and 7.1% (anemia) of the patients. Of the 16 CLL patients, 9 had a PR, in addition to 2 complete remissions (CR) The median PFS was 13.6 months, indicating durable responses [71]. A phase Ib/II trial with ibrutinib was undertaken for three groups of patients [72]. The first group consisted of patients with R/R CLL or small lymphocytic lymphoma (SLL), previously treated with a fludarabine derivative. 61 patients were included in the R/R group, with a median age of 64 years. 86% of the patients had U-CLL and 37% had a 17p deletion. In spite of these unfavorable prognostic factors, the overall response rate (ORR) reached 67% after a median follow-up of 17.3 months. Furthermore, 20% of the patients had LN size reduction with a remaining lymphocytosis (nodal response, NR). The second group consisted of 31 treatment naïve patients. The median age of the patients was 71 years. After a median followup of 16.6 months, an ORR of 71% was reached. 3 patients were in CR. An additional 10% of the patients had a NR. The third group consisted of 24 patients with high-risk CLL. High-risk disease was defined as deletion of 17p or a relapse within 2 years following chemoimmunotherapy. 50% of the patients had a PR after a median follow-up of 10.3 months, with an additional 29% NR. The estimated PFS after 22 months was 76% for the R/R and highrisk group combined, and 96% for the treatment naive group. Most adverse events (AEs) were ≤grade 2. The most common AEs were diarrhea, fatigue and upper respiratory tract infection. In a dose-escalating study of AVL-292 in CLL and NHL, the toxicity profile seemed similar to ibrutinib. Most toxicities were of a non-hematologic nature, including diarrhea, nausea and urticaria [73]. The most recent report showed limited activity. For all CLL patients (n = 9), the best response achieved was stable disease (SD, n = 8), despite occupancy tests showing full Btk occupancy. 5.2.2. Combination therapy Ibrutinib has also been administered in combination therapy, along with bendamustine and rituximab [74]. 30 previously treated patients were included, with a median age of 62 years. The AEs were in line with side effects related to bendamustine and rituximab. After a median follow-up of nearly five months, the ORR was 90%, including a CR of 10% [74]. O’Brien et al. compared this to an ORR of 59%, with a CR of 9% for bendamustine and rituximab alone, as reported in 2011 [75]. In another trial, 40 high-risk CLL patients received a combination of ibrutinib and rituximab [76]. High-risk disease was defined as patients with 17p deletion, R/R disease within 3 years after chemoimmunotherapy or relapsed CLL with deletion of 11q. 20 patients were evaluable at 3 months. 17 of these patients had a PR, while the other 3 had a NR. Jaglowski et al. reported on a clinical trial for relapsed or refractory CLL patients [77]. Patients received ibrutinib combined with the anti-CD20 monoclonal antibody ofatumumab. 24 patients with CLL/SLL or prolymphocytic leukemia (PLL) were included. The median age was 66 years and patients had received a median of 3 prior cycles of treatment. 79% of the patients had an unfavorable

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genomic aberration, such as a 17p or 11q deletion. All patients had a PR after a median follow-up of 6.5 months. Several phase 3 trials are currently ongoing, in addition to extension protocols, as registered on http://clinicaltrial.gov/. One trial evaluates the addition of ibrutinib to bendamustine and rituximab for R/R patients (NCT01611090). Two other trials compare single-agent ibrutinib to ofatumumab (R/R patients; NCT01578707) and chlorambucil (treatment naïve > 65 years; NCT01724346). No additional trials for AVL-292 are registered. 5.3. Inhibition of Syk Besides hematologic malignancies, inhibition of Syk is of interest in various autoimmune diseases. Most notably, Syk inhibitors are studied in rheumatoid arthritis, for which several phase III clinical trials are currently ongoing. Fostamatinib disodium (R788), a prodrug of the active compound R406, is the only compound that has been studied clinically for CLL. The specificity of fostamatinib toward Syk may be limited, but the in vitro results with more specific inhibitors such as PRT318 are similar [78]. In vitro experiments confirmed that Syk inhibitors were able to inhibit Syk and its downstream pathways [48]. Viability decreased in vitro when CLL cells were treated with fostamatinib, even when stimulated with anti-IgM [79], CD40 [49] or cocultured with NLCs [80]. In vitro treatment with fostamatinib showed synergistic activity with fludarabine. Suljagic et al. [81] performed 2 sets of experiments in the E␮-TCL1 CLL transgenic mouse model. In one experiment, treatment with fostamatinib was initiated shortly after injection of leukemic cells. The majority of the mice did not develop leukemia, although all mice in the control group did. In another experiment, mice with flaring leukemia were treated with R788. A transient lymphocytosis was seen in mice treated with R788 in the first week, after which lymphocyte counts dropped. In the control group, the rising CLL cell counts persisted. The spleens and lymph nodes of the mice from the treatment group contained significantly less CLL infiltration. 5.3.1. Monotherapy Friedberg et al. [82] performed a phase I/II clinical trial of R788 for patients with NHL or CLL. In the phase II part of the trial 11 out of 24 patients had CLL or SLL. The median age of the patients was 62 years. Diarrhea, fatigue, cytopenias and hypertension occurred in more than 20% of the patients. Lymphocytosis was seen in all CLL patients. In fact, the majority of patients experienced ≥50% increase in CLL cell counts. In the phase I trial, treatment was discontinued because of the lymphocytosis. However, this lymphocytosis is now considered to be a result of the mechanism of kinase inhibitors, rather than a sign of disease progression. 6 of 11 patients had a PR, corresponding with an ORR of 55%. 2 patients had stable disease (SD). The median PFS was 6.4 months.

cytotoxic for NK cells. Shehata et al. also found GS-1101 to increase apoptosis when CLL cells were cocultured with stromal cells from the BM, LN and spleen [85], while Hoellenriegel et al. confirmed this for NLC coculture [60]. Furthermore, GS-1101 reduces migration toward CXCL12 and CXCL13. GS-1101 treatment made CLL cells more sensitive to fludarabine, dexamethasone and bendamustine. GS-1101 appears to have a direct effect on cell survival as well as on the protective interactions with the microenvironment. 5.4.1. Monotherapy A phase I clinical trial with GS-1101 was conducted in patients with previously treated CLL [86]. 54 patients were included, with a median age of 62 years. 36% of the patients had 17p deletion. Patients had received a median of 5 prior regimens. AEs did not occur frequently and were mostly low grade. While some ≥grade 3 hematologic AEs occured, this was not clearly related to GS-1101. Serum transaminases increased in 21% of the patients early in treatment, raising concern of potential liver toxicity. The transaminase elevation appears to be reversible and patients are frequently able to restart treatment at lower doses without recurrence. GS-1101 caused a ≥50% reduction in LN size in 80% of the patients. 26% of the patients met the response criteria for a PR. Median PFS had not been reached at 11 months. 5.4.2. Combination therapy A phase 1 clinical trial combined GS-1101 with bendamustine, rituximab or both [87]. 51 patients with relapsed or refractory CLL were included. 19 patients received GS-1101 and rituximab, 17 patients GS-1101 and bendamustine and 15 patients received GS-1101, bendamustine and rituximab. Grade 3 or 4 AEs appeared to be consistent with the expectations of the single agents. These included infections and elevated liver enzymes [88]. Of the patients treated with GS-1101 and rituximab, 78% had a PR. A PR was achieved in 82% of the patients treated with GS-1101 and bendamustine, while the response rates after combination of GS-1101, bendamustine and rituximab included both PR (80%) and CR (7%). The 1-year PFS was 74%, 88% and 87% for the respective cohorts. Furthermore, a phase I/II study was conducted with GS-1101 (150 mg twice daily) and the anti-CD20 antibody ofatumumab in 28 day cycles [89]. 9 of 11 patients had a reduction in LN size in the first two months. There were no indications of significant myelosuppression. The lymphocytosis as seen with single-agent GS-1101, was reduced in both size and duration. No data on response rates are available. For GS-1101, three phase 3 trials are currently ongoing for previously treated patients. One trial compares a bendamustinerituximab regimen with and without GS-1101. Furthermore, other trials evaluate the addition of ibrutinib to monoclonal antibodies, ofatumumab (NCT01659021) or rituximab (NCT01539512). 6. Conclusion

5.4. Inhibition of PI3Kı The PI3 kinases can be subdivided into three classes. Class I contains p110␣, p110␤, p110␦ and p110␥. Both p110␣ and p110␤ are present in most human tissues and knockout mice of either isoform are non-viable. P110␦ and p110␥ are mainly present in leukocytes. While p110␥−/− mice mainly show alterations in T cells, p110␦−/− mice have reduced numbers of mature B cells and reduced proliferation of B cells. B cells depend heavily on the p110␦ isoform for signaling, making ␦-specific inhibition an attractive target [83]. The first in vitro CLL experiments with the PI3K␦ specific inhibitor GS-1101 (previously CAL-101), showed increased apoptosis of CLL cells [84]. These levels were comparable to apoptosis induced by the general PI3K inhibitors LY294002 and Wortmannin. Compared to non-␦ specific PI3K inhibitors, GS-1101 was less

Selective kinase inhibitors have recently entered the clinic, showing very promising results. Knowledge on signaling cascades and the precise role of the targeted kinases in leukemias and lymphomas is still incomplete. An important mode of action is the inhibition of microenvironmental interactions. Kinase inhibitors disrupt adhesion and migration of CLL cells in the LNs and BM, resulting in a shift toward the PB. Cells are more prone to spontaneous and drug-induced apoptosis in the PB. Besides inhibition of stimuli in the LN and BM, kinase inhibitors may have a direct cytotoxic effect. Response rates have been encouraging. Importantly, this appears to be true for patients with unfavorable prognostic markers as well, such as advanced stage disease. Notably, ibrutinib appears to produce comparable response rates for patients with

I. de Weerdt et al. / Leukemia Research 37 (2013) 838–847

17p deletion, although durability of response may be somewhat shorter [72,76]. Toxicity seems limited, manageable and mostly nonhematologic, although long-term experience with these drugs is needed. Likewise, the toxicity of kinase inhibitors in combination regimens appears to be tolerable. However, little is known about the long-term side effects, nor the probability of aggressive transformation with usage of these agents. In the ibrutinib plus ofatumumab trial, three patients with Richter’s transformation were included [77]. Two out of three patients had progressed at the time of publication of the abstract. So far, none of the studies showed complete disappearance of the malignant clone. Indeed, lymphocyte counts quickly rise when treatment with kinase inhibitors is discontinued. Thus, it is quite possible that there is a need for lifelong treatment with these agents. If so, information on long-term effects of these agents is needed even more. If this class of drugs is not capable of achieving curation as monotherapeutic agents in CLL patients, combination regimens could be beneficial. Chemotherapeutics, the less toxic monoclonal antibodies or in the more distant future perhaps the BH3-mimetics currently in clinical development [58,59] are probable candidates for combination agents. Relocation from the protective LN microenvironment toward PB potentially enables more effective eradication of the malignant cells by (immuno)chemotherapeutic regimens, reducing the risks of long-term treatment with kinase inhibitors and decreasing the probability of resistance to these agents. More data on kinase inhibitors derived from both preclinical and clinical research are eagerly awaited, to increase appreciation of these drugs. Conflicts of interest statement The authors declare no conflict of interest. Acknowledgements APK is sponsored by a fellowship from the Dutch Cancer Society. Contributions: IdW and APK wrote the manuscript. EE and MHvO reviewed the manuscript. References [1] Chiorazzi NRKFM. Chronic lymphocytic leukemia. N Engl J Med 2005:804–15. [2] Hallek M, Fischer K, Fingerle-Rowson G, Fink AM, Busch R, Mayer J, et al. Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial. Lancet 2010;9747:1164–74. [3] Burger JA, Ghia P, Rosenwald A, Caligaris-Cappio F. The microenvironment in mature B-cell malignancies: a target for new treatment strategies. Blood 2009;16:3367–75. [4] Burger JA. Nurture versus nature: the microenvironment in chronic lymphocytic leukemia. Hematol Am Soc Hematol Educ Program 2011:96–103. [5] Hayden RE, Pratt G, Roberts C, Drayson MT, Bunce CM. Treatment of chronic lymphocytic leukemia requires targeting of the protective lymph node environment with novel therapeutic approaches. Leukemia Lymphoma 2011;0:1–13. [6] Smit LA, Hallaert DY, Spijker R, de Goeij B, Jaspers A, Kater AP, et al. Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocytic leukemia cells correlates with survival capacity. Blood 2007;4:1660–8. [7] Kim HJ, Hawke N, Baldwin AS. NF-kappaB and IKK as therapeutic targets in cancer. Cell Death Differ 2006;5:738–47. [8] Endo T, Nishio M, Enzler T, Cottam HB, Fukuda T, James DF, et al. BAFF and APRIL support chronic lymphocytic leukemia B-cell survival through activation of the canonical NF-kappaB pathway. Blood 2007;2:703–10. [9] Rickert RC, Jellusova J, Miletic AV. Signaling by the tumor necrosis factor receptor superfamily in B-cell biology and disease. Immunol Rev 2011;1:115–33. [10] 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;2:563–74. [11] Duhren-von Minden M, Ubelhart R, Schneider D, Wossning T, Bach MP, Buchner M, et al. Chronic lymphocytic leukaemia is driven by antigen-independent cellautonomous signalling. Nature 2012;7415:309–12.

845

[12] Murray F, Darzentas N, Hadzidimitriou A, Tobin G, Boudjogra M, Scielzo C, et al. Stereotyped patterns of somatic hypermutation in subsets of patients with chronic lymphocytic leukemia: implications for the role of antigen selection in leukemogenesis. Blood 2008;3:1524–33. [13] Stamatopoulos K, Belessi C, Moreno C, Boudjograh M, Guida G, Smilevska T, et al. Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors: pathogenetic implications and clinical correlations. Blood 2007;1:259–70. [14] Agathangelidis A, Darzentas N, Hadzidimitriou A, Brochet X, Murray F, Yan XJ, et al. Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies. Blood 2012;19:4467–75. [15] Herve M, Xu K, Ng YS, Wardemann H, Albesiano E, Messmer BT, et al. Unmutated and mutated chronic lymphocytic leukemias derive from self-reactive B cell precursors despite expressing different antibody reactivity. J Clin Invest 2005;6:1636–43. [16] 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. [17] Chiorazzi N, Efremov DG. Chronic lymphocytic leukemia: a tale of one or two signals? Cell Res 2012. [18] Hehlgans T, Pfeffer K. The intriguing biology of the tumour necrosis factor/tumour necrosis factor receptor superfamily: players, rules and the games. Immunology 2005;1:1–20. [19] 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 CD4+CD40L+ T cells by producing CCL22. Eur J Immunol 2002;5:1403–13. [20] Hallaert DY, Jaspers A, van Noesel CJ, van Oers MH, Kater AP, Eldering E. c-Abl kinase inhibitors overcome CD40-mediated drug resistance in CLL: implications for therapeutic targeting of chemoresistant niches. Blood 2008;13:5141–9. [21] Granziero L, Ghia P, Circosta P, Gottardi D, Strola G, Geuna M, et al. Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia. Blood 2001;9:2777–83. [22] Kater AP, Evers LM, Remmerswaal EB, Jaspers A, Oosterwijk MF, van Lier RA, et al. CD40 stimulation of B-cell chronic lymphocytic leukaemia cells enhances the anti-apoptotic profile, but also Bid expression and cells remain susceptible to autologous cytotoxic T-lymphocyte attack. Br J Haematol 2004;4:404–15. [23] Eldering E, Tromp PF, Jak J, van Bochove M, Derks G, Kater I, et al. Molecular dissection of signals provided by activated cells to CLL cells indicates major role for IL-21 and CD40 in controlling proliferation and chemoresistance [abstract]. Haematologica 2012;s1:51–2. [24] 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 factor-1. Blood 2000;8:2655–63. [25] Tsukada N, Burger JA, Zvaifler NJ, Kipps TJ. Distinctive features of “nurselike” cells that differentiate in the context of chronic lymphocytic leukemia. Blood 2002;3:1030–7. [26] 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;13:3050–8. [27] 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;3:1012–20. [28] Zhang W, Kater AP, Widhopf 2nd GF, Chuang HY, Enzler T, James DF, et al. B-cell activating factor and v-Myc myelocytomatosis viral oncogene homolog (c-Myc) influence progression of chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 2010;44:18956–60. [29] Deaglio S, Vaisitti T, Bergui L, Bonello L, Horenstein AL, Tamagnone L, et al. CD38 and CD100 lead a network of surface receptors relaying positive signals for B-CLL growth and survival. Blood 2005;8:3042–50. [30] Tonino SH, Spijker R, Luijks DM, van Oers MH, Kater AP. No convincing evidence for a role of CD31–CD38 interactions in the pathogenesis of chronic lymphocytic leukemia. Blood 2008;3:840–3. [31] Kurosaki T, Hikida M. Tyrosine kinases and their substrates in B lymphocytes. Immunol Rev 2009;1:132–48. [32] Saito K, Tolias KF, Saci A, Koon HB, Humphries LA, Scharenberg A, et al. BTK regulates PtdIns-4,5-P2 synthesis: importance for calcium signaling and PI3K activity. Immunity 2003;5:669–78. [33] Rolli V, Gallwitz M, Wossning T, Flemming A, Schamel WW, Zurn C, et al. Amplification of B cell antigen receptor signaling by a Syk/ITAM positive feedback loop. Mol Cell 2002;5:1057–69. [34] Mohamed AJ, Yu L, Backesjo CM, Vargas L, Faryal R, Aints A, et al. Bruton’s tyrosine kinase (Btk): function, regulation, and transformation with special emphasis on the PH domain. Immunol Rev 2009;1:58–73. [35] Shinners NP, Carlesso G, Castro I, Hoek KL, Corn RA, Woodland RT, et al. Bruton’s tyrosine kinase mediates NF-kappa B activation and B cell survival by B cellactivating factor receptor of the TNF-R family. J Immunol 2007;6:3872–80. [36] Middendorp S, Dingjan GM, Maas A, Dahlenborg K, Hendriks RW. Function of Bruton’s tyrosine kinase during B cell development is partially independent of its catalytic activity. J Immunol 2003;11:5988–96. [37] Ha H, Han D, Choi Y. TRAF-mediated TNFR-family signaling. Curr Protoc Immunol 2009 [Unit 11 19D].

846

I. de Weerdt et al. / Leukemia Research 37 (2013) 838–847

[38] Conzelmann M, Wagner AH, Hildebrandt A, Rodionova E, Hess M, Zota A, et al. IFN-gamma activated JAK1 shifts CD40-induced cytokine profiles in human antigen-presenting cells toward high IL-12p70 and low IL-10 production. Biochem Pharmacol 2010;12:2074–86. [39] Liu P, Cheng H, Roberts TM, Zhao JJ. Targeting the phosphoinositide 3-kinase pathway in cancer. Nat Rev Drug Discov 2009;8:627–44. [40] Werner M, Hobeika E, Jumaa H. Role of PI3K in the generation and survival of B cells. Immunol Rev 2010;1:55–71. [41] Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002;5600:1911–2. [42] Muller MR, Rao A. NFAT, immunity and cancer: a transcription factor comes of age. Nat Rev Immunol 2010;9:645–56. [43] Bonizzi G, Karin M. The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol 2004;6:280–8. [44] Vallabhapurapu S, Karin M. Regulation and function of NF-kappaB transcription factors in the immune system. Annu Rev Immunol 2009:693–733. [45] Happo L, Strasser A, Cory S. BH3-only proteins in apoptosis at a glance. J Cell Sci 2012;Pt 5:1081–7. [46] Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 2008;1:47–59. [47] Contri A, Brunati AM, Trentin L, Cabrelle A, Miorin M, Cesaro L, et al. Chronic lymphocytic leukemia B cells contain anomalous Lyn tyrosine kinase, a putative contribution to defective apoptosis. J Clin Invest 2005;2:369–78. [48] Gobessi S, Laurenti L, Longo PG, Carsetti L, Berno V, Sica S, et al. Inhibition of constitutive and BCR-induced Syk activation downregulates Mcl-1 and induces apoptosis in chronic lymphocytic leukemia B cells. Leukemia 2009;4: 686–97. [49] Buchner M, Fuchs S, Prinz G, Pfeifer D, Bartholome K, Burger M, et al. Spleen tyrosine kinase is overexpressed and represents a potential therapeutic target in chronic lymphocytic leukemia. Cancer Res 2009;13:5424–32. [50] Herman SE, Gordon AL, Hertlein E, Ramanunni A, Zhang X, Jaglowski S, et al. Bruton tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765. Blood 2011;23:6287–96. [51] Ringshausen I, Schneller F, Bogner C, Hipp S, Duyster J, Peschel C, et al. Constitutively activated phosphatidylinositol-3 kinase (PI-3K) is involved in the defect of apoptosis in B-CLL: association with protein kinase Cdelta. Blood 2002;10:3741–8. [52] Longo PG, Laurenti L, Gobessi S, Sica S, Leone G, Efremov DG. The Akt/Mcl-1 pathway plays a prominent role in mediating antiapoptotic signals downstream of the B-cell receptor in chronic lymphocytic leukemia B cells. Blood 2008;2:846–55. [53] Furman RR, Asgary Z, Mascarenhas JO, Liou HC, Schattner EJ. Modulation of NF-kappa B activity and apoptosis in chronic lymphocytic leukemia B cells. J Immunol 2000;4:2200–6. [54] Lopez-Guerra M, Colomer D. NF-kappaB as a therapeutic target in chronic lymphocytic leukemia. Expert Opin Ther Targets 2010;3:275–88. [55] Hanada M, Delia D, Aiello A, Stadtmauer E. Reed JC. bcl-2 gene hypomethylation and high-level expression in B-cell chronic lymphocytic leukemia. Blood 1993;6:1820–8. [56] Mackus WJ, Kater AP, Grummels A, Evers LM, Hooijbrink B, Kramer MH, et al. Chronic lymphocytic leukemia cells display p53-dependent drug-induced Puma upregulation. Leukemia 2005;3:427–34. [57] Collins RJ, Verschuer LA, Harmon BV, Prentice RL, Pope JH, Kerr JF. Spontaneous programmed death (apoptosis) of B-chronic lymphocytic leukaemia cells following their culture in vitro. Br J Haematol 1989;3:343–50. [58] Mason KD, Khaw SL, Rayeroux KC, Chew E, Lee EF, Fairlie WD, et al. The BH3 mimetic compound, ABT-737, synergizes with a range of cytotoxic chemotherapy agents in chronic lymphocytic leukemia. Leukemia 2009;11:2034–41. [59] Del Gaizo Moore V, Brown JR, Certo M, Love TM, Novina CD, Letai A. Chronic lymphocytic leukemia requires BCL2 to sequester prodeath BIM, explaining sensitivity to BCL2 antagonist ABT-737. J Clin Invest 2007;1:112–21. [60] Hoellenriegel J, Meadows SA, Sivina M, Wierda WG, Kantarjian H, Keating MJ, et al. The phosphoinositide 3 -kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia. Blood 2011;13:3603–12. [61] Shah NP, Kasap C, Weier C, Balbas M, Nicoll JM, Bleickardt E, et al. Transient potent BCR-ABL inhibition is sufficient to commit chronic myeloid leukemia cells irreversibly to apoptosis. Cancer Cell 2008;6:485–93. [62] Kantarjian H, Shah NP, Hochhaus A, Cortes J, Shah S, Ayala M, et al. Dasatinib versus imatinib in newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2010;24:2260–70. [63] Rix U, Hantschel O, Durnberger G, Remsing Rix LL, Planyavsky M, Fernbach NV, et al. Chemical proteomic profiles of the BCR-ABL inhibitors imatinib, nilotinib, and dasatinib reveal novel kinase and nonkinase targets. Blood 2007;12:4055–63. [64] Bantscheff M, Eberhard D, Abraham Y, Bastuck S, Boesche M, Hobson S, et al. Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Nat Biotechnol 2007;9:1035–44. [65] Veldurthy A, Patz M, Hagist S, Pallasch CP, Wendtner CM, Hallek M, et al. The kinase inhibitor dasatinib induces apoptosis in chronic lymphocytic leukemia cells in vitro with preference for a subgroup of patients with unmutated IgVH genes. Blood 2008;4:1443–52. [66] Amrein PC, Attar EC, Takvorian T, Hochberg EP, Ballen KK, Leahy KM, et al. Phase II study of dasatinib in relapsed or refractory chronic lymphocytic leukemia. Clin Cancer Res 2011;9:2977–86.

[67] Kater AP, Liu SM, Beckers D, Tonino MM, Daenen SH, Doorduijn SMGH, et al. The Broad kinase inhibitor dasatinib in combination with fludarabine in patients with refractory chronic lymphocytic leukemia: a multicenter phase 2 study [abstract]. In: ASH annual meeting abstracts. 2012. p. 1798. [68] Brown JR, Sharman JP, Harb WA, Kelly KR, Schreeder MT, Sweetenham JW, et al. Phase Ib trial of AVL-292, a covalent inhibitor of Bruton’s tyrosine kinase (Btk), in chronic lymphocytic leukemia (CLL) and B-non-Hodgkin lymphoma (B-NHL). ASCO meeting abstracts 2012;15(Suppl.):8032. [69] 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;5:1182–9. [70] 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 receptor- and chemokinecontrolled adhesion and migration in chronic lymphocytic leukemia. Blood 2012;11:2590–4. [71] Byrd JC, Coutre FR, Flinn S, Burger IW, Blum JA, Sharman K, et al. The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib (PCI-32765) promotes high response rate, durable remissions, and is tolerable in treatment naive (TN) and relapsed or refractory (RR) chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) patients including patients with high-risk (HR) disease: new and updated results of 116 patients in a phase Ib/II study [abstract]. In: ASH annual meeting abstracts. 2012. p. 189. [72] Byrd JCFR, Coutre S, Flinn IW, Burger JA, Blum K, Sharman JP, et al. The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib (PCI-32765) promotes high response rate, durable remissions, and is tolerable in treatment naive (TN) and relapsed or refractory (RR) chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) patients including patients with high-risk (HR) disease: new and updated results of 116 patients in a phase Ib/II study [abstract]. In: ASH annual meeting abstracts. 2012. p. 189. [73] Mahadevan D, Brown JR, Harb W, Kelly K, Schreeder M, Sweetenham J, et al. Phase 1b trial of AVL-292, a covalent inhibitor of Bruton’s tyrosine kinase (BTK), in chronic lymphocytic leukemia (CLL) and B-Non-Hodgkin lymphoma (B-NHL) [abstract]. Haematologica 2012;s1:58–9. [74] O’Brien SM, Barrientos JC, Flinn IW, Barr PM, Burger JA, Navarro T, et al. Combination of the Bruton’s tyrosine kinase (BTK) inhibitor PCI-32765 with bendamustine (B)/rituximab (R) (BR) in patients (pts) with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL): Interim results of a phase Ib/II study. ASCO meeting abstracts, vol 15, Suppl 2012:6515. [75] Fischer K, Cramer P, Busch R, Stilgenbauer S, Bahlo J, Schweighofer CD, et al. Bendamustine combined with rituximab in patients with relapsed and/or refractory chronic lymphocytic leukemia: a multicenter phase II trial of the German Chronic Lymphocytic Leukemia Study Group. J Clin Oncol 2011;26:3559–66. [76] Burger JA, Wierda KM, Hoellenriegel WG, Ferrajoli J, Faderl A, Lerner S, et al. The Btk inhibitor ibrutinib (PCI-32765) in combination with rituximab is well tolerated and displays profound activity in high-risk chronic lymphocytic leukemia (CLL) patients [abstract]. In: ASH annual meeting abstracts. 2012. p. 187. [77] Jaglowski S, Jones J, Flynn J, Andritsos L, Maddocks KKAB, Grever M, et al. A phase Ib/II study evaluating acitivity and tolerability of BTK inhibitor PCI-32765 and ofatumumab in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and related diseases [abstract]. ASCO meeting abstracts, 15 Suppl 2012:6508. [78] 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. [79] 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 spleen tyrosine kinase inhibitor, R406. Blood 2009;5:1029–37. [80] Buchner M, Baer C, Prinz G, Dierks C, Burger M, Zenz T, et al. Spleen tyrosine kinase inhibition prevents chemokine- and integrin-mediated stromal protective effects in chronic lymphocytic leukemia. Blood 2010;22:4497–506. [81] 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;23:4894–905. [82] 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;13:2578–85. [83] Vanhaesebroeck B, Ali K, Bilancio A, Geering B, Foukas LC. Signalling by PI3K isoforms: insights from gene-targeted mice. Trends Biochem Sci 2005;4: 194–204. [84] Herman SE, Gordon AL, Wagner AJ, Heerema NA, Zhao W, Flynn JM, et al. Phosphatidylinositol 3-kinase-delta inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals. Blood 2010;12:2078–88. [85] Shehata M, Schnabl S, Demirtas D, Hilgarth M, Hubmann R, Ponath E, et al. Reconstitution of PTEN activity by CK2 inhibitors and interference with the PI3-K/Akt cascade counteract the antiapoptotic effect of human stromal cells in chronic lymphocytic leukemia. Blood 2010;14:2513–21. [86] Coutre SEBJ, Furman RR, Brown JR, Benson DM, Wagner-Johnston ND, Flinn IW, et al. Phase I study of CAL-101, an isoform-selective inhibitor of phoshpatidylinositol 3-kinase P110d, in patients with previously treated chronic lymphocytic leukemia. In: ASCO meeting abstracts. 2011. p. 6631.

I. de Weerdt et al. / Leukemia Research 37 (2013) 838–847 [87] Coutre SELJ, Furman RR, Barrientos JC, de Vos S, Flinn IW, Schreeder MT, et al. Combinations of the selective phosphatidylinositol 3-kinase-delta (PI3Kdelta) inhibitor GS-1101 (CAL-101) with rituximab and/or bendamustine are tolerable and highly active in patients with relapsed or refractory chronic lymphocytic leukemia (CLL): results from a phase 1 study [abstract]. In: ASH annual meeting abstracts. 2012. p. 191. [88] Sharman J, de Vos S, Leonard JP, Furman RR, Coutre SE, Flinn IW, et al. A phase 1 study of the selective phosphatidylinositol 3-kinase-delta (PI3K{delta}) inhibitor, CAL-101 (GS-1101), in combination with rituximab and/or bendamustine in patients with relapsed or refractory chronic lymphocytic

847

leukemia (CLL) [abstract]. In: ASH annual meeting abstracts, vol. 22. 2011. p. 1787. [89] Furman RR, Barrientos JC, Sharman JP, De Vos S, Leonard J, Coutre SE, et al. A phase I/II study of the selective phosphatidylinositol 3-kinase-delta (PI3K{delta}) inhibitor, GS-1101 (CAL-101), with ofatumumab in patients with previously treated chronic lymphocytic leukemia (CLL). In: ASCO meeting abstracts, 15 suppl. 2012. p. 6518. [90] 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;1:88–94.