Differential Effects Of Triggering Via Toll Like Receptors In Stable Or Aggressive Chronic Lymphocytic Leukemia

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in the peripheral blood, secondary lymphoid tissues and bone marrow of functionally defective clonal B lymphocytes with prolonged survival in vivo. Despite therapeutic achievements have been accomplished in the management of this disease, however CLL remains incurable partially because of the resistance to apoptosis of CLL B-cells and of the altered immune system of CLL patients. CLL is hetereogenous, but its evolution is mostly slow, probably linked to some level of immune control of the leukemic cells. Indeed, clinical observations have been reported of spontaneous remissions associated with the intense immunological activity following a viral infection. Clinical responses have also been observed after treatment by immunomodulating cytokines and long-term survival is described, without disease, after allogeneic stem cells transplantation. All these data suggest that immunotherapy could be useful in the treatment of CLL, possibly as an adjuvant therapy after classical immunochemotherapy schedules. Toll like receptors (TLR) are proteins of the innate immune system belonging to the family of Pattern Recognition receptors (PRRs). Recognition of their ligands by the TLR present on neutrophils, macrophages, dendritic cells and B-cells are important in the initiation of adaptive immune responses. TLR-7 and 9 are expressed by CLL B-cells (Grandjenette, Hematologica, 2007). Previous studies have shown that stimulation of TLR-9 by CpG ODN (oligodinucleotides) induces an activation of CLL B-cells while triggering TLR-7 increases in vitro apoptosis of these cells. Here we show that these effects of TLR-9 and TLR-7 stimulation differ depending on the clinical form (stable or aggressive) of the disease and on the mutational status of CLL B-cells. In vitro stimulation with three doses of Imiquimod R837 or ODN CpG M362 was carried out for three days on cells from 40 patients (22 stable, 18 aggressive, mutational status known for 35, 18 IgVH mutated, 17 unmutated). Flow cytometry was used to measure apoptosis, proliferation after carboxyfluorescein succinimidyl ester (CFSE) labeling and modulation of surface differentiation antigens. Signaling pathways after incubation were further studied by antibody arrays and western blot. Spontaneous apoptosis occurring in vitro was demonstrated to involve the mitochondrial pathway. CLL B-cells were also confirmed to proliferate strongly and produce large amounts of IL-6 and IL-8 upon triggering by phorbol myrsistate (positive control), this compound almost completely aborting in vitro apoptosis. Cells from patients with a stable disease were significantly more prone to rapid apoptosis after TLR-7 triggering with Imiquimod, compared to cells from patients with an aggressive disease which displayed only spontaneous apoptosis. This rapid apoptosis in stable patients involved the p38 MAP-Kinase pathway. It was concomitant to an important production of IL-8 and IL-6. Conversely, CpG ODN conferred a protection against apoptosis to CLL B-cells from patients with an aggressive disease. This was accompanied by the activation of numerous anti-apoptotic proteins in the cells. CpG ODN also significantly increased CLL B-cells proliferation, concomitantly to the phosphorylation of Erk and Akt proteins. ODN finally increased the expression of CD20 and CD19 on the cells’surface. The same differences in reactivity were observed comparing mutated (∼stable) and unmutated (∼aggressive) cases. These data indicate that CLL B-cells from patients with a stable or aggressive (mutated/unmutated) disease answer differently when triggered through their surface TLRs. This might have an incidence on the behavior of these cells in vivo, in answer to stimulations by microbial compounds naturally binding these structures. These properties could also be used to develop adjuvant immunotherapies by loading CpG ODN-activated CLL B-cells with autologous apoptotic fragments issued from stimulation of part of the same cells with Imiquimod. Disclosures: No relevant conflicts of interest to declare.

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Reversible anergy of sIgM-mediated signaling in the two subsets of CLL defined by VH-gene mutational status

Blood ◽

10.1182/blood-2006-11-056648 ◽

2007 ◽

Vol 109 (10) ◽

pp. 4424-4431 ◽

Cited By ~ 139

Author(s):

C. Ian Mockridge ◽

Kathleen N. Potter ◽

Isla Wheatley ◽

Louise A. Neville ◽

Graham Packham ◽

...

Keyword(s):

B Cells ◽

Direct Evidence ◽

Lymphocytic Leukemia ◽

Downstream Signaling ◽

Specific Effects ◽

Mutational Status ◽

Differential Signaling ◽

Vh Gene

Abstract The 2 subsets of chronic lymphocytic leukemia (CLL), of worse or better prognosis, likely derive from pre-GC unmutated B cells, or post-GC mutated B cells, respectively. Different clinical behavior could relate to the ability of tumor cells to respond to surface (sIg)–mediated signals. Unmutated cases (U-CLL) have an increased ability to phosphorylate p72Syk in response to sIgM ligation compared to mutated cases (M-CLL). We now confirm and further investigate this differential signaling in a large cohort by [Ca2+]i mobilization. Cases responding to sIgM ligation express higher levels of CD38, ZAP-70, and sIgM. However, CD38 does not influence signaling in vitro or associate with response in bimodal CD38-expressing cases. Similarly, ZAP-70 expression is not required for response in either U-CLL or M-CLL. Strikingly, partially or completely anergized sIgM responses from each subset can recover both sIgM expression and signal capacity spontaneously in vitro or following capping/endocytosis. This provides direct evidence for engagement of putative antigen in vivo. Signaling via sIgD differs markedly being almost universally positive in both U-CLL and M-CLL, with no association with CD38 or ZAP-70 expression. Downstream signaling pathways, therefore, appear intact in CLL, locating anergy to sIgM, mainly in M-CLL. Integration of differential isotype-specific effects mediated by (auto)antigen may determine tumor behavior.

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Receptors for tumor necrosis factor on neoplastic B cells from chronic lymphocytic leukemia are expressed in vitro but not in vivo

Blood ◽

10.1182/blood.v76.8.1607.1607 ◽

1990 ◽

Vol 76 (8) ◽

pp. 1607-1613 ◽

Cited By ~ 18

Author(s):

W Digel ◽

W Schoniger ◽

M Stefanic ◽

H Janssen ◽

C Buck ◽

...

Keyword(s):

B Cells ◽

Tumor Necrosis ◽

Lymphocytic Leukemia ◽

Receptor Expression ◽

Tnf Alpha ◽

Tnf Receptor ◽

Tnf Receptors ◽

Necrosis Factor

Abstract Recombinant tumor necrosis factor-alpha (TNF-alpha) is a cytokine that induces proliferation of neoplastic B cells from patients with chronic lymphocytic leukemia (CLL). To gain insight into the mechanisms involved in regulating TNF responsiveness, we have examined TNF receptor expression on neoplastic B-CLL cells. We have demonstrated that freshly isolated neoplastic B cells from patients with CLL did not express TNF receptors. After 1 day of incubation in culture medium, TNF receptors were detectable in the range of 540 to 1,500/cell. Kinetic experiments revealed that receptor expression was half-maximal after 3 hours of culturing and required de novo protein synthesis. The Scatchard plots of TNF-alpha binding indicated a single set of high- affinity TNF receptors with a dissociation constant of 70 pmol/L. TNF receptor expression in vitro was found in all examined cases. All cytokines tested, with the exception of IL-2, did not influence the expression of TNF receptors. The TNF receptor expression is enhanced in B-CLL cells cultured in the presence of interleukin-2 when compared with the receptor expression of cells cultured in medium alone. Our data suggest that neoplastic B-CLL cells in patients with stable disease do not express TNF receptors in vivo and that an unknown mechanism suppressing TNF receptor expression in vivo may play a role in growth regulation of neoplastic B cells.

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Monoclonal antibody to the interferon-inducible protein Leu-13 triggers aggregation and inhibits proliferation of leukemic B cells

Blood ◽

10.1182/blood.v76.12.2583.2583 ◽

1990 ◽

Vol 76 (12) ◽

pp. 2583-2593 ◽

Cited By ~ 64

Author(s):

SS Evans ◽

DB Lee ◽

T Han ◽

TB Tomasi ◽

RL Evans

Keyword(s):

B Cells ◽

Dna Synthesis ◽

Lymphocytic Leukemia ◽

Intercellular Adhesion Molecule ◽

Intercellular Adhesion Molecule 1 ◽

Adhesive Properties ◽

Prolymphocytic Leukemia ◽

Ifn Alpha

Abstract Interferon (IFN)-alpha inhibits DNA synthesis stimulated by low molecular weight B-cell growth factor (BCGF) in hairy cells in vitro, suggesting that the therapeutic efficacy of IFN-alpha in hairy cell leukemia (HCL) involves growth inhibition of malignant B cells. Evidence that the 16-Kd cell surface protein Leu-13 mediates an antiproliferative signal in T lymphocytes and is IFN-inducible in endothelial cells prompted us to examine the expression and functional role of this molecule in leukemic B cells. Leu-13 density, determined by flow cytometry, was upregulated in vitro and in vivo by IFN-alpha on malignant B cells from patients with HCL, chronic lymphocytic leukemia, and prolymphocytic leukemia. Monoclonal anti-Leu-13 triggered homotypic aggregation of leukemic B cells via an adhesion pathway that was not inhibited by antibodies to leukocyte function associated antigen-1 (LFA- 1) or intercellular adhesion molecule-1 (ICAM-1). Moreover, anti-Leu-13 potentiated the inhibitory effects of IFN-alpha on BCGF-stimulated DNA synthesis, assessed by [3H]-thymidine and [3H]-deoxyadenosine incorporation into DNA. These results indicate that Leu-13 is part of a novel IFN-inducible signaling pathway which may modify the growth and adhesive properties of leukemic B cells under physiologic or therapeutic conditions.

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Chronic Lymphocytic Leukemia B Cells Are Resistant to the Apoptotic Effects of Transforming Growth Factor-β

Blood ◽

10.1182/blood.v89.3.941 ◽

1997 ◽

Vol 89 (3) ◽

pp. 941-947 ◽

Cited By ~ 64

Author(s):

Raymond S. Douglas ◽

Renold J. Capocasale ◽

Roberta J. Lamb ◽

Peter C. Nowell ◽

Jonni S. Moore

Keyword(s):

B Cells ◽

Growth Factor ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Lymphocytic Leukemia ◽

Western World ◽

Spontaneous Apoptosis

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia of the western world and is characterized by a slowly progressing accumulation of clonal CD5+ B cells. Our laboratory has investigated the role of transforming growth factor-β (TGF-β) and interleukin-4 (IL-4) in the pathogenesis of B-cell expansion in CLL. In vitro addition of TGF-β did not increase spontaneous apoptosis of B cells from most CLL patients, as determined using the TUNEL method, compared with a twofold increase observed in cultures of normal B cells. There was similar expression of TGF-β type II receptors on both CLL B cells and normal B cells. In contrast to apoptosis, CLL B-cell proliferation was variably inhibited with addition of TGF-β. In vitro addition of IL-4, previously reported to promote CLL B-cell survival, dramatically reduced spontaneous apoptosis of CLL B cells compared with normal B cells. CLL B-cell expression of IL-4 receptors was increased compared to normal B cells. Thus, our results show aberrant apoptotic responses of CLL B cells to TGF-β and IL-4, perhaps contributing to the relative expansion of the neoplastic clone.

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Growth- and differentiation-associated expression of bcl-2 in B-chronic lymphocytic leukemia cells

Blood ◽

10.1182/blood.v79.11.2981.2981 ◽

1992 ◽

Vol 79 (11) ◽

pp. 2981-2989 ◽

Cited By ~ 150

Author(s):

M Schena ◽

LG Larsson ◽

D Gottardi ◽

G Gaidano ◽

M Carlsson ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Messenger Rna ◽

Phorbol Esters ◽

Lymphocytic Leukemia ◽

Mantle Zone ◽

Cell Stage ◽

High Level

Abstract The bcl-2 gene is translocated into the Ig loci in about 80% of human follicular lymphomas and in 10% of B-type chronic lymphocytic leukemias (B-CLL), resulting in a high level of expression. We have compared the expression of bcl-2 transcripts and protein in B-CLL cells in their normal equivalent CD5+ B cells and in normal B-cell populations representative of different in vivo and in vitro stages of activation and proliferation. We report here that bcl-2 was expressed in 11 of 11 cases of CD5+ B-CLL clones, contrasting with the absent expression in normal CD5+ B cells. Activation of 173 and 183 B-CLL cells by phorbol esters (12-O-tetradecanoylphorbol-13-acetate [TPA]) to IgM secretion without concomitant DNA synthesis resulted in a rapid but transient downregulation of bcl-2 expression. In contrast, the reduction of bcl-2 at both the messenger RNA and protein levels was sustained after mitogenic stimulation, suggesting that bcl-2 expression and proliferation are inversely related in these cells. This notion was further supported by immunocytochemical analysis showing that bcl-2 was primarily expressed in small resting lymphocytes and in cells differentiating to the plasma cell stage, but less expressed in Ki67- positive proliferating B blasts. Moreover, it was also supported by the low level of bcl-2 in exponentially growing Epstein-Barr virus-carrying lymphoblastoid and B-CLL cell lines. The regulation of bcl-2 expression in B-CLL resembled that of normal tonsillar follicular B cells, in which a high level of expression was found in resting mantle zone B cells but not in the proliferating germinal center B cells. Based on these findings and the role of bcl-2 in maintaining B-cell memory, we propose that the phenotype of B-CLL cells corresponds to a mantle zone memory-type B cell.

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Interleukin-13 inhibits interleukin-2-induced proliferation and protects chronic lymphocytic leukemia B cells from in vitro apoptosis

Blood ◽

10.1182/blood.v87.3.1022.bloodjournal8731022 ◽

1996 ◽

Vol 87 (3) ◽

pp. 1022-1029 ◽

Cited By ~ 56

Author(s):

N Chaouchi ◽

C Wallon ◽

C Goujard ◽

G Tertian ◽

A Rudent ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Interleukin 2 ◽

Intermediate Stage ◽

Lymphocytic Leukemia ◽

Spontaneous Apoptosis ◽

Interleukin 13 ◽

B Cell Maturation ◽

Cd23 Expression

Human interleukin-13 (IL-13) acts at different stages of the normal B- cell maturation pathway with a spectrum of biologic activities overlapping those of IL-4. B chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of slow-dividing and long-lived monoclonal B cells, arrested at the intermediate stage of their differentiation. In vitro, B-CLL cells exhibit a spontaneous apoptosis regulated by different cytokines. In this report, we show that IL-13 (10 to 200 ng/mL) acts directly on monoclonal B-CLL cells from 12 patients. (1) IL-13 enhances CD23 expression and induces soluble CD23 secretion by B-CLL cells but does not exhibit a growth factor activity. (2) IL-13 inhibits IL-2 responsiveness of B-CLL cells, activated either with IL-2 alone or through crosslinking of lgs or ligation of CD40 antigen. (3) IL-13 protects B-CLL cells from in vitro spontaneous apoptosis. The effects of IL-13 on neoplasic B cells were slightly less than those of IL-4 and occurred independently of the presence of IL-4. The present observations show that IL-13 may exhibit a negative regulatory effect on neoplasic B cells in contrast with that observed in normal B cells, and suggest that IL-13 could be an important factor in the pathogenesis of CLL by preventing the death of monoclonal B cells. Moreover, B-CLL may be an interesting model to study the regulation of the expression of IL-13 receptor and/or signal transduction pathways.

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Chronic Lymphocytic Leukemia B Cells Can Undergo Somatic Hypermutation and Intraclonal Immunoglobulin VHDJH Gene Diversification

Journal of Experimental Medicine ◽

10.1084/jem.20011693 ◽

2002 ◽

Vol 196 (5) ◽

pp. 629-639 ◽

Cited By ~ 74

Author(s):

Carmela Gurrieri ◽

Peter McGuire ◽

Hong Zan ◽

Xiao-Jie Yan ◽

Andrea Cerutti ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Lymphocyte ◽

Somatic Hypermutation ◽

Point Mutations ◽

Single Strand Conformation Polymorphism ◽

Lymphocytic Leukemia ◽

Gene Diversification

Chronic lymphocytic leukemia (CLL) arises from the clonal expansion of a CD5+ B lymphocyte that is thought not to undergo intraclonal diversification. Using VHDJH cDNA single strand conformation polymorphism analyses, we detected intraclonal mobility variants in 11 of 18 CLL cases. cDNA sequence analyses indicated that these variants represented unique point-mutations (1–35/patient). In nine cases, these mutations were unique to individual submembers of the CLL clone, although in two cases they occurred in a large percentage of the clonal submembers and genealogical trees could be identified. The diversification process responsible for these changes led to single nucleotide changes that favored transitions over transversions, but did not target A nucleotides and did not have the replacement/silent nucleotide change characteristics of antigen-selected B cells. Intraclonal diversification did not correlate with the original mutational load of an individual CLL case in that diversification was as frequent in CLL cells with little or no somatic mutations as in those with considerable mutations. Finally, CLL B cells that did not exhibit intraclonal diversification in vivo could be induced to mutate their VHDJH genes in vitro after stimulation. These data indicate that a somatic mutation mechanism remains functional in CLL cells and could play a role in the evolution of the clone.

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IL-21 Plus CpG ODN Induces Granzyme B-Dependent Induction of Apoptosis in CD5-Positive B Cells Including B-CLL Cells.

Blood ◽

10.1182/blood.v108.11.2823.2823 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2823-2823

Author(s):

Sue E. Blackwell ◽

Bernd Jahrsdoerfer ◽

James E. Wooldridge ◽

Jian Huang ◽

Melinda W. Andreski ◽

...

Keyword(s):

B Cells ◽

Cord Blood ◽

Granzyme B ◽

Lymphocytic Leukemia ◽

Cpg Odn ◽

T And B Cells ◽

B1 Cells ◽

Lymphocyte Populations

Abstract Interleukin 21 (IL-21), a recently discovered cytokine with structural homology to IL-2, IL-4 and IL-15, has pleiotropic effects on lymphocyte populations including NK, T and B cells and is currently undergoing early clinical evaluation. We explored the effect of the combination of IL-21 and immunostimulatory CpG ODN on B chronic lymphocytic leukemia (B-CLL), and other CD5-positive B cells. IL-21 plus CpG ODN were synergistic in their ability to induce apoptosis of the B-CLL cells, and also induced production and secretion of granzyme B from the B-CLL cells. B-CLL cells treated with IL-21 and CpG ODN were capable of inducing apoptosis of untreated autologous B-CLL cells. This bystander killing was inhibited by anti-granzyme B antibodies. The effect was observed in all cases of CD5-positive B-CLL, but not in CD5-negative B-CLL samples. IL-21 plus CpG ODN also induced granzyme B production and apoptosis of benign CD5-positive B1 cells obtained from umbilical cord blood. In contrast, the number of CD5-negative B2 cells increased in the same samples during in vitro culture, resulting in a decreased ratio of CD5-positive to CD5-negative cord blood B cells (Fig. 1). Our results indicate the combination of IL-21 and CpG ODN is able to induce apoptosis of both benign and malignant CD5-positive B cells. Given the suspected role of B1 cells in autoimmune diseases, our findings could have important implications for the understanding of their pathogenetic mechanisms. These results might also open new avenues for the development of novel therapies for both autoimmune dieseases and CD5-positive B-CLL. Figure 1. IL- 21 and CpG ODN therapy selectively eliminates CD5 positive B cells in cord blood. Figure 1. IL- 21 and CpG ODN therapy selectively eliminates CD5 positive B cells in cord blood.

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Spontaneous in Vitro Death of CLL Cells Can Be Prevented by Mitochondrial Stabilization Via CD160 Signaling.

Blood ◽

10.1182/blood.v114.22.4372.4372 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4372-4372

Author(s):

Feng-Ting Liu ◽

Li Jia ◽

Timothy Farren ◽

Jerome Giustiniani ◽

Armand Bensussan ◽

...

Keyword(s):

Mitochondrial Membrane ◽

Conflicts Of Interest ◽

Membrane Integrity ◽

Lymphocytic Leukemia ◽

Cellular Growth ◽

Spontaneous Apoptosis ◽

Cytochrome C Release ◽

Phosphorylated Akt

Abstract Abstract 4372 B-cell chronic lymphocytic leukemia (CLL) is an incurable disease, which is at least partly attributable to the majority of cells being in the G0/G1 phase of the cell cycle and expressing high levels of anti-apoptotic Bcl-2 family proteins. Despite their prolonged survival in vivo, CLL cells rapidly undergo spontaneous apoptosis in vitro, suggesting that survival signals in vivo have been lost in in vitro culture conditions. CD160, a glycosylphosphatidylinositol-linked surface antigen, was found to be expressed by CLL cells. In normal NK and T-cells, CD160 mediates cellular growth and activation, but its role in CLL is unclear. Using monoclonal antibodies to CD160 (CL1-R2 or BY55 - non cross blocking) led to increased expression of Bcl-2, Bcl-xL and Mcl-1 anti-apoptotic proteins and protected CLL from spontaneous apoptosis in vitro - mean cell viability increased from 66.8 to 79.4% (n = 17, p = 0.02). These CD160-mediated events were also accompanied by decreased cytochrome C release and prevention of mitochondrial membrane potential collapse, indicating stabilization of both inner and outer mitochondrial membrane integrity. PI3K/AKT signalling is a well known survival pathway in cancer cells and in normal lymphocytes CD160 has been shown to act via PI3K/AKT. Activation of CD160 in CLL led to phosphorylated AKT, while inhibition of PI3K by wortmannin completely blocked AKT phosphorylation and CD160-mediated protection from apoptosis. In summary, the activation of CD160 protected CLL cells from spontaneous cell death in vitro via a PI3-kinase/AKT pathway. This improved survival was also associated with increased Bcl-2, Bcl-xL and Mcl-1 expression and preservation of mitochondrial function. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of Wnt Signaling By Dimethyl Fumarate Results in in Vitro and in Vivo Clearance of Chronic Lymphocytic Leukemia Cells and Has Additive Activity with Ibrutinib

Blood ◽

10.1182/blood.v124.21.4683.4683 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4683-4683 ◽

Cited By ~ 1

Author(s):

Christina C.N. Wu ◽

Fitzgerald Lao ◽

Hongying Li ◽

Laura Rassenti ◽

Thomas J. Kipps ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Wnt Signaling ◽

Dimethyl Fumarate ◽

Lymphocytic Leukemia ◽

Aggressive Disease ◽

Cell Clearance ◽

Survival Pathways ◽

A Cell

Abstract Background: Small molecules that inhibit B cell survival pathways are effective treatments for patients with chronic lymphocytic leukemia (CLL). However, such therapies are not curative, and resistance can develop in some patients. Combination therapies with agents that inhibit several CLL survival pathways may allow for more complete responses, and help prevent treatment resistance. Previous data has shown that the pro-survival Wnt pathway is highly active in CLL and is a negative prognostic factor, and therefore is an attractive target for novel therapies to combine with agents like ibrutinib. Dimethyl fumarate (DMF) is an orally bioavailable fumaric acid ester with immunomodulatory properties, including inhibition of the NF-kB signaling cascade. DMF has been evaluated as a systemic treatment of psoriasis as well as multiple sclerosis. Our group previously observed anti-CLL effects of DMF, mediated in part through oxidative stress. Herein we describe a novel mechanism of action of DMF and ibrutinib, mediated by inhibition of the Wnt signaling pathway. Methods: Effects of DMF and ibrutinib on Wnt signaling were determined using a cell-based LEF/TCF beta-lactamase reporter gene FRET assay. In vitro activity was assessed in primary CLL from patients with indolent and aggressive disease. In vivo activity was evaluated in Rag2-/- gamma chain-/- immunodeficient (RG-KO) mice, which were engrafted with human CLL cells by intraperitoneal injection. DMF and/or ibrutinib were administrated to mice by oral gavage, at clinically used doses and schedules. Results: Both DMF and ibrutinib have an alpha-beta unsaturated ketone that can react with essential free cysteines in the Wnt-driven LEF1 transcription factor. This effect was confirmed by a cell-based reporter gene assay in which DMF inhibited LEF/TCF dependent gene expression at low μM levels. Ibrutinib also inhibited Wnt signaling activity in the same assay. In short term cultures, DMF was cytotoxic to primary CLL cells from patients with both indolent and aggressive disease, at low uM concentrations. The combination of DMF and ibrutinib resulted in a higher degree of CLL cell clearance than achieved by either agent alone (p < 0.05 after multiple comparison adjustments, Dunnet’s method). To evaluate the effect in a preclinical CLL xenograft animal model, we administered DMF by oral lavage to RG-KO mice engrafted with human CLL cells. Doses ranging from 3 to 30 mg/kg BID for 7 days resulted in dose dependent clearance of CLL cells compared to vehicle controls, without observable toxicity to the recipient animals. Moreover, the combination of DMF and ibrutinib resulted in a higher degree of CLL cell clearance than achieved by either agent alone. Preliminary FACS analyses revealed that DMF selectively targets CLL subpopulations of cells with aggressive characteristics, as assessed by CD38 expression. Further molecular analyses of predictive or correlative biomarkers are ongoing. Conclusions: DMF inhibits Wnt signaling, and has single agent activity as a treatment for CLL. The combination of DMF and ibrutinib is more effective than either agent alone, particularly in aggressive disease, and is well tolerated. Clinical trials of DMF in CLL are warranted, and are planned. This work is supported by a Leukemia and Lymphoma Society Specialized Center of Research Grant (7005-14) and by the CLL Research Consortium (5P01CA081534-14). Disclosures No relevant conflicts of interest to declare.

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