Protect Chronic Lymphocytic Leukemia Cells Research Articles

Ibrutinib modifies the function of monocyte/macrophage population in chronic lymphocytic leukemia.

In lymphoid organs, nurse-like cells (NLCs) show properties of tumor-associated macrophages, playing a crucial role in chronic lymphocytic leukemia (CLL) cell survival. Ibrutinib, a potent inhibitor of Bruton's tyrosine kinase (BTK), is able to counteract pro-survival signals in CLL cells. Since the effects on CLL cells have been studied in the last years, less is known about the influence of ibrutinib on NLCs properties. We sought to determine how ibrutinib modifies NLCs functions focusing on the balance between immunosuppressive and inflammatory features. Our data show that ibrutinib targets BTK expressed by NLCs modifying their phenotype and function. Treatment with ibrutinib reduces the phagocytic ability and increases the immunosuppressive profile of NLCs exacerbating the expression of M2 markers. Accordingly, ibrutinib hampers LPS-mediated signaling, decreasing STAT1 phosphorylation, while allows IL-4-mediated STAT6 phosphorylation. In addition, NLCs treated with ibrutinib are able to protect CLL cells from drug-induced apoptosis partially through the secretion of IL-10. Results from patient samples obtained prior and after 1 month of treatment with ibrutinib show an accentuation of CD206, CD11b and Tie2 in the monocytic population in the peripheral blood. Our study provides new insights into the immunomodulatory action of ibrutinib on monocyte/macrophage population in CLL.

At High Levels, Constitutively Activated STAT3 Induces Apoptosis of Chronic Lymphocytic Leukemia Cells.

In chronic lymphocytic leukemia (CLL), the increment in PBLs is slower than the expected increment calculated from the cells' proliferation rate, suggesting that cellular proliferation and apoptosis are concurrent. Exploring this phenomenon, we found overexpression of caspase-3, higher cleaved poly (ADP-ribose) polymerase levels (p < 0.007), and a higher apoptosis rate in cells from patients with high counts compared with cells from patients with low counts. Although we previously found that STAT3 protects CLL cells from apoptosis, STAT3 levels were significantly higher in cells from patients with high counts than in cells from patients with low counts. Furthermore, overexpression of STAT3 did not protect the cells. Rather, it upregulated caspase-3 and induced apoptosis. Remarkably, putative STAT3 binding sites were identified in the caspase-3 promoter, and a luciferase assay, chromatin immunoprecipitation, and an EMSA revealed that STAT3 activated caspase-3 However, caspase-3 levels increased only when STAT3 levels were sufficiently high. Using chromatin immunoprecipitation and EMSA, we found that STAT3 binds with low affinity to the caspase-3 promoter, suggesting that at high levels, STAT3 activates proapoptotic mechanisms and induces apoptosis in CLL cells.

Microenvironmental Stromal Cells Rescue CLL Cells from Apoptosis Via Hypoxia That Can be Targeted Therapeutically

Abstract Microenvironmental support protects malignant cells from apoptosis and from the effects of therapy. We hypothesized that in chronic lymphocytic leukemia (CLL) cells, the non-malignant bystander cells induce a transcriptional signature that prevents apoptosis. We aimed to use this transcriptional signature to identify compounds that target this protective microenvironmental effect. If these compounds are effective against protected CLL cells, we assume that understanding the underlying molecular pathways will open novel therapeutic options for therapy-resistant CLL. In primary CD19+ CLL cells cultured without support after isolation from peripheral blood, apoptosis can already be detected after 8 hrs. These cells can however be rescued by coculture with either human or murine bone-marrow derived stromal cell lines HS-5 and M210B4. In order to identify clinically relevant substances that counteract this pro-survival microenvironmental support, we determined the transcriptional signature that is induced in primary CLL cells by protective feeder cells at 8 timepoints before apoptosis could be detected and compared it to the connectivity map database of transcriptomes induced by 1300 compounds (cMAP). We identified 7 compounds with a transcriptome signature that is negatively correlated to the protective signature induced in CLL cells by in-vitro coculture. Of note, 5 of these substances were more potent than fludararabine to induce apoptosis in protected primary CLL cells. In contrast, none of the 10 control compounds with non-correlated signatures had an effect after 48h of coculture. Most potent were the isoquinoline alkaloids emetine and cephaeline and the cardiac glycoside ouabain that displayed IC50s of 34nM, 190nM and 287nM, while a concentration of 5.1µM of fludarabine was required for comparable induction of apoptosis. To test the efficacy of emetine against CLL in vivo, TCL1-induced murine CLL-like tumors were transplanted into immunocompetent syngeneic mice and treated with 3 high doses of emetine (24mg/kg) after CD5+CD19+ CLL cells reached 40% of leukocytes (CD45+ cells). Treated animals displayed a significant reduction in body weight compared to untreated (26.0g vs 29.1g, p&lt;0.0001). However, treatment of leukemic mice with emetine reduced total lymphocyte counts in the peripheral blood compared to untreated mice (19.3 vs 48.1x10^3/mm^3, p&lt;0.0001) and also the proportion of CLL cells in the bone marrow (4.1% vs 13.0%, p=0.0041). Most importantly, treatment increased survival of mice transplanted with syngeneic murine CLL tumors (median not reached in the treatment arm vs 15 days for untreated mice, n=7 and n=9, p=0.0012, censored for different start of treatment, panel A). This shows that emetine effectively targets murine TCL1-driven CLL even if they are microenvironmentally protected in vivo. Next we aimed to understand the molecular mode of action of therapeutic targeting of the microenvironmental support. Comparing transcriptomes of primary CLL cells cocultured with and without murine or human stromal cell support, 6 of the 10 most deregulated pathways involved redox homeostasis. Downregulation of the Von-Hippel-Lindau (VHL) gene led to an increase in hypoxia-inducible factor HIF1a activity (panel B). In fact, hypoxic in-vitro incubation (3% O2) of primary CLL cells significantly prevented apoptosis between 24h and 72h (n=8, p&lt;0.05). Functionally we found in cell lines stably expressing redox reporter fusion proteins that emetine and ouabain impair recovery from mitochondrial but not cytosolic oxidative stress (Figure, panel B). In fact, the observed effect was stronger than for 13 other redox-active compounds. In addition we could show that emetine and ouabain overcome the anti-apoptotic microenvironmental downregulation of VHL in CLL cells by direct reduction of HIF1a protein and also HIF1a activity, shedding light on their molecular mode of action (panel B). In conclusion, we identified modulation of redox homeostasis to play the most important role in microenvironmental protection of CLL cells by bone-marrow stromal cells in-vitro. In addition we could show that this opens a new target of the malignant CLL cells that reside in the stromal niche protecting them from chemotherapy. Even though unexpected for a chronic leukemia, hypoxia-regulated pathways seem to be a novel therapeutic option for resistant CLL. Figure Figure. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Epigenetic silencing of tumor suppressor miR-3151 contributes to Chinese chronic lymphocytic leukemia by constitutive activation of MADD/ERK and PIK3R2/AKT signaling pathways.

We hypothesize that miR-3151, localized to a GWAS-identified chronic lymphocytic leukemia (CLL) risk locus (8q22.3), is a tumor suppressor miRNA silenced by promoter DNA methylation in CLL. The promoter of miR-3151 was methylated in 5/7 (71%) CLL cell lines, 30/98 (31%) diagnostic primary samples, but not normal controls. Methylation of miR-3151 correlated inversely with expression. Treatment with 5-Aza-2'-deoxycytidine led to promoter demethylation and miR-3151 re-expression. Luciferase assay confirmed MAP-kinase activating death domain (MADD) and phosphoinositide-3-kinase, regulatory subunit 2 (PIK3R2) as direct targets of miR-3151. Moreover, restoration of miR-3151 resulted in inhibition of cellular proliferation and enhanced apoptosis, repression of MADD and PIK3R2, downregulation of MEK/ERK and PI3K/AKT signaling, and repression of MCL1. Lastly, miR-3151 methylation was significantly associated with methylation of miR-203 and miR-34b/c in primary CLL samples. Therefore, this study showed that miR-3151 is a tumor suppressive miRNA frequently hypermethylated and hence silenced in CLL. miR-3151 silencing by DNA methylation protected CLL cells from apoptosis through over-expression of its direct targets MADD and PIK3R2, hence constitutive activation of MEK/ERK and PI3K/AKT signaling respectively, and consequently over-expression of MCL1.

Cross-talk between chronic lymphocytic leukemia (CLL) tumor B cells and mesenchymal stromal cells (MSCs): implications for neoplastic cell survival.

Leukemic cells from Chronic Lymphocytic Leukemia (CLL) patients interact with stromal cells of the surrounding microenvironment. Mesenchymal Stromal Cells (MSCs) represent the main population in CLL marrow stroma, which may play a key role for disease support and progression. In this study we evaluated whether MSCs influence in vitro CLL cell survival. MSCs were isolated from the bone marrow of 46 CLL patients and were characterized by flow cytometry analysis. Following co-culture of MSCs and leukemic B cells, we demonstrated that MSCs were able to improve leukemic B cell viability, this latter being differently dependent from the signals coming from MSCs. In addition, we found that the co-culture of MSCs with leukemic B cells induced an increased production of IL-8, CCL4, CCL11, and CXCL10 chemokines.As far as drug resistance is concerned, MSCs counteract the cytotoxic effect of Fludarabine/Cyclophosphamide administration in vivo, whereas they do not protect CLL cells from the apoptosis induced by the kinase inhibitors Bafetinib and Ibrutinib. The evidence that leukemic clones are conditioned by environmental stimuli suggest new putative targets for therapy in CLL patients.

Nurse-like cells mediate ibrutinib resistance in chronic lymphocytic leukemia patients

Nurse-like cells mediate ibrutinib resistance in chronic lymphocytic leukemia patients

Abstract 1023: Duration of signaling through IgM and IgD receptors differentially influences cell survival and BCL6-regulated CCL3/4 chemokine production in Chronic Lymphocytic Leukemia (CLL)

Abstract B-cell receptor (BCR) signaling is a central pathogenetic mechanism in CLL. Most CLL cells express both IgM and IgD receptors, and increased responsiveness to IgM stimulation correlates with poor disease outcome and with expression of IGHV-unmutated immunoglobulin receptors (U-CLL). In contrast, the relevance of IgD signaling remains poorly understood. The aim of the present study is to characterize functional differences between IgM and IgD signaling in CLL in terms of protection from in vitro apoptosis, BCR signaling activation and CCL3/4 chemokine secretion and to explore the mechanism through which CCL3/4 are regulated, with a focus on their transcriptional repressor BCL6. CLL cells were stimulated with anti-IgM and anti-IgD; 48-hour cell viability and BCR expression were analyzed by flow cytometry; phosphorylation of the BCR-signaling related molecules HS1 and ERK after 2, 5, 15, 30, 60 minutes of stimulation were analyzed by Western Blot and flow cytometry; BCL6 protein expression by Western Blot and CCL3/4 secretion by ELISA were tested after 3, 6, 9, 12, 24 hours of stimulation. Anti-IgM stimulation protected CLL cells from in vitro apoptosis to a greater extent than anti-IgD and U-CLL were more responsive to anti-IgM as compared to cells carrying IGHV-mutated receptors (M-CLL) (p = 0.0022; n = 13 M-CLL vs. n = 11 U-CLL). In line with this finding, U-CLL expressed higher levels of surface IgM, as compared to M-CLL (p&amp;lt;0.0001; n = 40 M-CLL vs. n = 33 U-CLL). IgM stimulation induced protracted HS1 and ERK phosphorylation up to 30 minutes following receptor engagement, as analyzed in 10 M-CLL and 10 U-CLL cases. In contrast, IgD stimulation induced short-lived phosphorylation of both proteins, which rapidly returned to baseline. U-CLL cells were more responsive than M-CLL to both anti-IgM and anti-IgD stimulation. High CCL3/4 secretion was detected after anti-IgM, in particular in U-CLL. Surprisingly low, if any, induction of both chemokines was detected after stimulation of IgD receptors. Interestingly, increased CCL3/4 production and concomitant BCL6 protein downregulation were observed after 6 and up to 12 hours following anti-IgM but not anti-IgD stimulation. In conclusion, IgM stimulation induces a long-lived signaling response, increased CLL-cell survival and CCL3/4 secretion, in particular in U-CLL; in contrast, IgD signaling is short-lived and has minimal effects on protection from apoptosis and chemokine secretion. BCL6 protein downregulation induced by anti-IgM parallels CCL3/4 production, suggesting a functional link between BCL6 expression and CCL3/4. Taken together, these evidences suggest that the differential functional outcomes of IgM and IgD signaling might be related to the duration of the signaling response and support the relevance of IgM signaling in CLL pathogenesis. Citation Format: Elisa ten Hacken, Cristina Scielzo, Susan O'Brien, William G. Wierda, Michael J. Keating, Paolo Ghia, Federico Caligaris-Cappio, Jan A. Burger. Duration of signaling through IgM and IgD receptors differentially influences cell survival and BCL6-regulated CCL3/4 chemokine production in Chronic Lymphocytic Leukemia (CLL). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1023. doi:10.1158/1538-7445.AM2015-1023

Absence of Caveloin-1 Leads to Delayed Development of Chronic Lymphocytic Leukemia in Eµ-TCL1 Mouse Model

Background: Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia in the United States. Clinical heterogeneity, a characteristic feature of CLL is a major problem in the clinical management of this currently incurable leukemia. We and others have demonstrated that the tissue microenvironment, specifically the lymph node (LN), influence the biological and clinical behavior including the clinical heterogeneity of CLL. Using gene expression profiling of CLL cells from peripheral blood (PB), bone marrow (BM) and LNs, we identified Cav-1 a member of the Tolerogenic Signature (genes associated with host immune tolerance) as one of the candidate genes which might be involved in the pathogenesis of CLL. We found that Cav-1 levels were significantly elevated (11 fold) in CLL cells from LNs compared to BM and PB. Cav-1 is the major element of caveolae, which are flask-shaped membrane invaginations. Cav-1 is involved in multiple cellular processes like the regulation and transportation of cellular cholesterol and lipids, clathrin independent endocytosis and signal transduction leading to oncogenesis or tumor suppression. We have previously shown that knock down of Cav-1 results in a significant decrease in cell migration and proliferation of primary human CLL cells in vitro. We have also demonstrated that knock down of Cav-1 prevents CLL cells from forming immune synapses. These immune synapses are important for the interaction between the CLL cells and their tumor microenvironment. These results suggest that Cav-1 protect CLL cells from undergoing apoptosis and enhances their migration in vitro.Objectives and Methodology: To understand the precise role of Cav-1 in leukemic progression in vivo, we crossed Cav-1-/- mice to Eµ-TCL1 mice, which is a well-established transgenic murine model for CLL. The offspring were observed and evaluated for the development of CLL. These mice were sacrificed at the age of 12, 24, 36 and 40+ weeks and peripheral blood, bone marrow and spleen and were examined for the presence of CD5+B220+CD19+ CLL cells using flow cytometry. Spleen, lymph nodes, liver, lungs and kidney were evaluated for the presence of CLL cells using H&E staining of histologic slides.Results: To study the role of Cav-1 in Eµ-TCL1, we isolated splenic B cells and measured the expression of Cav-1. We observed a gradual increase in the expression of Cav-1 in splenic B cells from Eµ-TCL1 mice at age of 12, 24 and 36 weeks when compared with wild type mice. This suggested that Cav-1 might be playing a role in CLL progression in Eµ-TCL1 mice. Therefore, to study the role of Cav-1 in CLL disease progression we decreased the expression of Cav-1 in vivo by breeding Eµ-TCL1 with Cav1 knockout mice. We generated Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice to study the effect of Cav-1 knock down in aggressiveness of CLL in vivo. We have shown that Cav-1 is overexpressed in CLL cells from patients with poorer clinical outcome and protects CLL cells from undergoing apoptosis. Therefore, we analyze the number of CLL cells in Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice. We observed a significant reduction in the number of B220+CD5+ CLL cells population in bone marrow and spleen of Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice when compared with Eµ-TCL1-Cav1wt/wt mice. We have previously shown that Cav-1 is important for CLL cells migration in vitro. Therefore, to study its effect in vivo we analyzed infiltration of CLL cells in spleen, lymph nodes, liver, kidney and lungs in these mice. There was no or significant decrease in tumor infiltration of CLL cells in spleen, lymph nodes, liver, lungs and kidney in Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice when compared with Eµ-TCL1-Cav1wt/wt alone. Next, we wanted to examine the effect of Cav-1 knock down on splenomegaly and hepatomegaly. We found that there was a significant decrease in splenomegaly and hepatomegaly in Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice. The spleen and liver size of Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice was significantly reduced when compared with Eµ-TCL1 mice. Together these results suggest that high expression of Cav-1 in CLL cells leads to enhance proliferation and promotes disease progression in Eµ-TCL1 mice. DisclosuresNo relevant conflicts of interest to declare.

Lymphocyte Function-Associated Antigen 3 (LFA-3): Key Factor of the Interactions Between Nurse-like-Cells and B Leukemic Cells from Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western Countries. This pathology is characterized by an accumulation of monoclonal, non-functional and mature CD5+ CD19+ leukemic B-cells (CLL cells) in lymph nodes, peripheral blood and bone marrow. Despite a high resistance to the in vivo apoptosis, CLL cells die spontaneously in vitro due to a lack in ex vivo conditions of sustaining cells and factors from their microenvironment such as stroma cells (Lagneaux L et al, Blood. 1998; 91:2387-2396), follicular dendritic cells or Nurse-Like-Cells (NLC) (Burger JA et al, Blood. 2000; 96:2655-2663).NLC are derived from CD14+ cells in contact with CLL cells in vitro (Tsukada N et al, Blood. 2002; 99:1030-1037) and were found in lymph nodes of CLL patients (Ysebaert L et al, Leuk Lymphoma. 2011; 52:1404-1406). NLC were shown to have a Tumour Associated Macrophages phenotype and gene expression profile. These cells have been first described to be essential for in vitro CLL cells survival partially through the production of soluble factors such as CXCL12 (Burger JA et al, Blood. 2000; 96:2655-2663), CCL3 and CCL4 (Burger JA et al, Blood. 2009; 113:3050-3058). Thus, other mechanisms are required for CLL cells survival. Indeed, we showed that contact of CLL cells with NLC was necessary to protect CLL cells from the in vitro apoptosis. We then investigated the mechanism of these interactions at a molecular level. We also determined their influences on the in vitro CLL cells survival and on the NLC-induced chemoresistance.We observed close and strong interactions evaluated by the measurement of trogocytosis from NLC to CLL cells. Trogocytosis is an active phenomenon with transfer of membrane fragments from one cell to another. We showed that NLC/CLL cells trogocytosis is dependant to actin polymerization and SRC phosphorylation.To find possible couples of molecules involved in this contact, we compared different transcriptomic data from NLC, monocyte, CLL cells and B lymphocytes. We highlighted potential couples of molecules and confirmed their expression on CLL cells and NLC by flow cytometry analysis. Finally, we obtained 3 couples probably implicated: Lymphocyte Function-Associated Antigen 3 (LFA-3)/CD2, Platelet/Endothelial Cell Adhesion Molecule 1 (PECAM1)/CD38 and Intercellular Adhesion Molecule 1 (ICAM-1)/LFA-1.Antibody blocking strategies revealed that LFA-3, which is up-regulated in CLL cells compared to healthy donors B lymphocytes, was necessary for the interaction between CLL cells/NLC when PECAM1, ICAM-1 and their co-partners were not essential (figure 1). Furthermore, this contact, through LFA-3, induced Akt phosphorylation but not ERK1/2 phosphorylation in CLL cells. Finally, we showed that LFA-3 and its receptor CD2 are necessary to the rescue of CLL cells by NLC (figure 2).To go further, we tested the chemoprotective effect of NLC on CLL cells. We showed that NLC slightly protect CLL cells against bendamustin but not against rituximab, dasatinib or ibrutinib. We hypothesized that the contact through LFA-3 could be involved in this chemoresistance. However, we did not observed a significant effect of the combination of bendamustin and LFA-3-blocking compared to bendamustin alone suggesting that this chemoprotection of CLL cells by NLC involved another pathway.Altogether, our results indicate that overexpression of LFA-3 by CLL cells and its critical implication in the interaction with NLC might be a new therapeutic target in CLL to disturb the interaction of CLL cells with their microenvironment. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

IL-4 Exerts Opposing Effects on Surface-IgM and CXCR4 Mediated Signalling in Chronic Lymphocytic Leukaemia

The B cell receptor (BCR) is critical for survival and proliferation of chronic lymphocytic leukemia (CLL) cells and is activated following antigen/autoantigen engagement. BCR engagement in vivo triggers a partial receptor down modulation, phosphorylation of tyrosine residues in CD79A and CD79B, signalosome formation and subsequent calcium mobilization and ERK phosphorylation (ERK-P). Interestingly CLL cells incubated in vitro recover surface(s)IgM receptor expression and subsequently their ability to signal (calcium flux and ERK-P). During this recovery of sIgM, expression of a fully glycosylated “mature” form of sIgM increases. Interestingly, sIgD is not downregulated in vivo and expression does not increase in vitro. Downregulation by antigen engagement therefore appears only to affect sIgM. IL-4 appears to be present in CLL tissue sites and has a known role in protecting CLL cells from apoptosis however its effect on BCR expression and subsequent downstream signalling has not previously been demonstrated. Therefore we investigated the effects of IL-4 on BCR signalling and on the expression of surface receptors including chemokine receptors CXCR4 and MHC-class II (MHCII).CLL cells were pretreated with and without IL-4 for 24h and sIgM and sIgD expression measured by flow cytometry. sIgM naturally recovers with time and this is significantly enhanced further by IL-4 (p=0.006, n=33), whereas sIgD does not (p=0.48, n=30). The same CLL samples pre-treated with IL-4 for 24 hours followed by treatment with anti-IgM (n=22) for 15 minutes showed enhanced calcium flux in 77% (p=0.0005) of samples compared to the control. The increase in sIgM expression correlated strongly with the ability to induce calcium mobilization in IL-4 treated samples (p=0.0007, r=0.7). The fold increase in sIgM expression following pre-treatment with IL-4 was significantly greater in U-CLL compared with M-CLL (p=0.012) and in ZAP70+ve samples (p=0.02). However, analysis of IL-4R expression did not show a significant difference between M-CLL and U-CLL samples (p=0.18, n=20). The increase in sIgM expression by IL-4 coincided with an increase in CD79b protein expression (p<0.0001, n=26). Futhermore IL-4 pretreatment significantly augmented expression of the fully glycosylated µ-chain of sIgM on the surface of the CLL cells (p=0.005), as confirmed by resistance to EndoH cleavage, indicating a role of IL-4 in IgM assembly and transport to the cytoplasmic membrane. These IL-4-specific effects were blocked with either a JAK3 or STAT6 inhibitor (Tofacitinib or Axon 1992, respectively) which specifically inhibit IL-4 signalling.We next identified whether the effects observed with IL-4 was limited to sIgM. Interestingly, in contrast to the spontaneous recovery of CXCR4 expression which occurs in vitro and parallels the recovery of sIgM expression, IL-4 significantly inhibited CXCR4 recovery in vitro (p<0.0001, n=21) and prevented migration towards CXCL12 in transwell migration assays (p=0.03, n=6). This indicates that IL-4 is not simply amplifying the natural process of recovery of these receptors but is having a differential effect. For other molecules, a significant increase in MHCII expression in IL-4 pre-treated samples (p=0.0011, n=12) was observed mimicking the changes with sIgM, whilst other negative or positive BCR regulator such as CD5 (p=0.47, n=25) and CD19 (p=0.93, n=20) respectively, remained unchanged. Indicating the effect of IL-4 on receptor expression was receptor dependent.These data suggest a role for IL-4 in augmenting sIgM expression and subsequent downstream signalling, increasing MHCII expression, whilst down modulating chemokine receptors to retain CLL cells within the lymph node. Thus IL-4 may play an important biological role when CLL cells encounter antigen/autoantigen and therefore inhibition of this pathway with JAK3 inhibitors may be a potential clinical target for the treatment of CLL. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of STAT3 Reverses the Resistance to Histone Deacetylase Inhibitors Induced By Interleukin-6 in Chronic Lymphocytic Leukemia Cells

Introduction: Interleukin-6 (IL-6) is a pleiotropic cytokine produced by a variety of cell types, including bone marrow stromal cells (BMSCs) and chronic lymphocytic leukemia (CLL) cells. Levels of IL-6 are increased in patients with CLL and correlated with adverse clinical features and short survival. In preliminary studies we observed that IL-6 induced resistance to the pan-histone deacetylase (HDAC) inhibitors vorinostat (SAHA) and panobinostat (LBH589) in CLL cells. Activation of STAT3 induced by IL-6 in CLL cells was further enhanced by low concentration of SAHA and LBH589. We hypothesized that exposure of CLL cells to HDAC inhibitors would further expand the activation of STAT3 signaling by IL-6, and thus the ability of HDAC inhibitors to kill CLL cells may be blunted due to upregulation of STAT3 downstream antiapoptotic proteins. This study was designed to illuminate the role of the STAT3 pathway on the resistance to SAHA and LBH589 in CLL cells, and to evaluate the proapoptotic activity of the combination of SAHA and LBH589 with a STAT3-selective inhibitor, WP1066.Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from samples of 32 CLL patients. The characteristics of CLL patients were shown in Table 1. The PBMCs contained more than 90% of CD19+ B lymphocytes, which were detected by flow cytometry and were referred to as primary CLL cells. BMSCs were isolated from bone marrow mononuclear cells of healthy volunteers. Cytotoxicity of drugs was assessed by carrying out triplicate assays with the Cell Counting Kit-8 (CCK-8), after a 48-hour incubation of primary CLL cells in the presence or absence of recombinant human IL-6, and half-maximal inhibitory concentration (IC50) values were calculated. Effects of drugs on apoptosis of CLL cells, in the presence or absence of IL-6 or BMSCs, were evaluated by Annexin V/7AAD assay, morphological assessment of Giemsa-stained cytospin preparations and western blot analysis of the cleavage of poly(ADP ribose) polymerase (PARP). The levels of STAT3 phosphorylation (pSTAT3) and STAT3 downstream proteins were evaluated by western blotting.Results: IL-6-induced pSTAT3 was completely abolished by 4-hour incubation with 20µM of SAHA or 100nM of LBH589 before IL-6 was added. But when CLL cells were incubated with lower concentration of SAHA or LBH589 (e.g., SAHA, 1µM; LBH589 10nM), the level of pSTAT3 induced by IL-6 was further enhanced (Fig. 1A-C). Treatment of CLL cells with 10ng/mL IL-6 for 24 hours resulted in greater than 2-fold upregulation of the antiapoptotic proteins, Mcl-1 and Bcl-xL. The upregulation of Mcl-1 and Bcl-xL induced by IL-6 was further enhanced in the presence of 1µM or 2.5µM of SAHA (Fig. 1D-E). IL-6 decreased the sensitivity of CLL cells to each of the HDAC inhibitors tested, as evidenced by an increase in the mean IC50 values. Co-incubation with 5µM of WP1066 blocked the protective action of IL-6 (Fig. 1F-G). Apoptosis induction by SAHA and LBH589 was reduced in the presence of IL-6 and this reduction could be inhibited by WP1066 (Fig. 2A-B). The antiapoptotic action of IL-6 and the ability of WP1066 to restrain cytoprotection by IL-6 were also confirmed by morphological assessment and PARP cleavage analysis (Fig. 2C-E). WP1066 decreased the pSTAT3 level and inhibited increases of Mcl-1 and Bcl-xL proteins induced by IL-6 in the presence or absence of SAHA (Fig. 1D-E). Co-culture of CLL cells with BMSCs resulted in the activation of STAT3 in CLL cells and protected CLL cells from apoptosis induction by HDAC inhibitors, and this cytoprotection was reversed by WP1066 (Fig. 2F-I).Conclusions: Our studies indicated that CLL cells were protected from HDAC inhibitor-induced apoptosis by IL-6 or co-culture with BMSCs. This protection may be mediated by activation of STAT3 signal pathway. Furthermore, WP1066 reversed the resistances of CLL cells to SAHA and LBH589 induced by IL-6 or co-culture with BMSCs. Our findings suggest that targeting the STAT3 pathway may be a novel way to improve the efficacy of HDAC inhibitor in CLL patients by overcoming antiapoptotic signaling of the microenvironment.Table 1CharacteristicCase (n)Total (n)Age (years)32≥6020<6012Sex32Male25Female7Rai stage3205I9II11III4IV3IgVH mutational status25Unmutated (≥98% homology)8Mutated (<98% homology)17ZAP7022≥20%7<20%15CD3824≥20%8<20%16FISHcytogenetics21Normal karyotype1013q-711q-217p-1Trisomy 122 [Display omitted] [Display omitted] DisclosuresLu:Novartis Pharmaceuticals Inc: LBH589 was kindly provided by Novartis Pharmaceuticals Inc. Other.

Inhibition of the Notch1 Pathway in Chronic Lymphocytic Leukemia Promotes Apoptosis and Inhibits Proliferation

In chronic lymphocytic leukemia (CLL), mutations in the NOTCH1 receptor occur in 4-10% of newly diagnosed patients and 15–20% of multiply relapsed patients. Using next-generation sequencing, our group previously reported NOTCH1 p.P2514fs mutations in 15 CLL patients (9.4%) in an initial cohort of 160 CLL patients in which NOTCH1 mutations were associated with IGHV unmutated (UM) CLL (p=0.0001). Further analysis using a three-group comparison (NOTCH1 mut, IGHV UM vs. NOTCH1 wild-type [wt] IGHV UM vs. NOTCH1 wt IGHV mut) showed that NOTCH1 mutations associated with both trisomy 12 (p=0.049) and 17p deletion (p=0.0008) and poor overall survival (HR 2.99, p=0.008).Given that targeting activating mutations has proven an effective therapeutic strategy in many cancers, we explored the therapeutic potential of a Notch1 inhibitor, PF-03084014, in CLL. Previous studies in T-cell acute lymphoblastic leukemia cells harboring NOTCH1 mutations have shown that gamma secretase inhibitors can induce apoptosis by blocking Notch1 receptor activation. When we tested the gamma secretase inhibitor (GSI) PF-03084014 in 18 CLL samples with NOTCH1 mutations, it consistently induced apoptosis of all CLLs after 48 hours in culture across all cytogenetic groups tested (13.3-47.2% death with 5 μM GSI, p<0.0001)(Figure A). The induction of apoptosis was similar (GSI vs. ibrutinib, p=ns) to that of ibrutinib (n=11,11.9-74.4% death with 5 μM ibrutinib, p<0.0001). In contrast, GSI treatment only induced apoptosis in some (n=10), but not all NOTCH1 wt CLLs (n=6) (p=0.0137). We next tested the effect of GSI PF-03084014 in the context of a stromal environment. Co culture of CLL cells with CD40L-expressing fibroblasts partially mimics the lymph node and bone marrow microenvironments, which are known sites of drug-resistance and proliferation of CLL in vivo. We tested whether the interaction with stromal cells protects CLL cells from Notch1 inhibitor-induced apoptosis, as seen with other drugs including ibrutinib. We found that co culture with CD40L-expressing 3T3s decreased GSI-induced apoptosis in NOTCH1 mutant CLLs (p=0.0006) and in the majority of the NOTCH1 wt CLLs that responded to the GSI (Figure A).Since NOTCH1 mutations have been reported to be an independent marker of aggressive disease in CLL, we tested whether CLL cells with NOTCH1 mutations were more proliferative in vitro compared to NOTCH1 wt CLL cells. We showed that CLL cells upregulate Ki67 expression in co culture with 3T3-CD40L cells and in a cohort of 10 NOTCH1 mutants and 11 NOTCH1 wt, we found the NOTCH1 mutants to be more proliferative than the NOTCH1 wt (median of 7.6% vs. 2.3%, p=0.015). To then address whether blocking of the Notch1 pathway decreases proliferation, we treated CLL cells in co culture with 3T3-CD40L cells with 5 μM GSI for 7 days. GSI treatment decreased the percentage of Ki67+ CLL cells in all but one NOTCH1 mutant (median decrease 28.3%, p=0.044) as wells as in the majority of NOTCH1 wt samples (median decrease 38.7%, p=0.037).Having established that inhibition of Notch1 can reduce proliferation and induce apoptosis in CLL cells in vitro, we were interested in determining the downstream genes that may be the effectors of this activity. We therefore compared the gene expression profiles (GEP) of NOTCH1 mut vs. NOTCH1 wt CLLs, and found upregulation of genes involved in the Notch1 pathway, in apoptosis and in chemokine signaling in the NOTCH1 mutants. Furthermore, comparing GEP of high Ki67 vs. low Ki67 expressing CLL cells revealed higher expression of a range of both upstream and downstream Notch1 pathway genes in high Ki67 expressing CLL cells.In conclusion, we show that PF-03084014 induces apoptosis and decreases proliferation in both NOTCH1 mutant and wt CLL cells. We find NOTCH1 mutant CLL cells to be more proliferative than NOTCH1 wt and show upregulation of Notch1 pathway genes in NOTCH1 mutants compared to wt CLL cells and in high Ki67 expressing compared to low Ki67 expressing CLL cells. Taken together, these results emphasize the important role of Notch1 signaling in CLL in general, perhaps particularly in proliferative compartments like lymph nodes, and demonstrate that Notch1 pathway inhibitors are worthy of therapeutic investigation in CLL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

STAT3-activated GM-CSFRα translocates to the nucleus and protects CLL cells from apoptosis.

Here, it was determined that chronic lymphocytic leukemia (CLL) cells express the α subunit, but not the β subunit, of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR/CSF2R). GM-CSFRα was detected on the surface, in the cytosol, and in the nucleus of CLL cells via confocal microscopy, cell fractionation, and GM-CSFRα antibody epitope mapping. Because STAT3 is frequently activated in CLL and the GM-CSFRα promoter harbors putative STAT3 consensus binding sites, MM1 cells were transfected with truncated forms of the GM-CSFRα promoter, then stimulated with IL6 to activate STAT3 and to identify STAT3-binding sites. Chromatin immunoprecipitation (ChIP) and an electoromobility shift assay (EMSA) confirmed STAT3 occupancy to those promoter regions in both IL6-stimulated MM1 and CLL cells. Transfection of MM1 cells with STAT3-siRNA or CLL cells with STAT3-shRNA significantly downregulated GM-CSFRα mRNA and protein levels. RNA transcripts, involved in regulating cell survival pathways, and the proteins KAP1 (TRIM28) and ISG15 coimmunoprecipitated with GM-CSFRα. GM-CSFRα-bound KAP1 enhanced the transcriptional activity of STAT3, whereas GM-CSFRα-bound ISG15 inhibited the NF-κB pathway. Nevertheless, overexpression of GM-CSFRα protected MM1 cells from dexamethasone-induced apoptosis, and GM-CSFRα knockdown induced apoptosis in CLL cells, suggesting that GM-CSFRα provides a ligand-independent survival advantage. Constitutively, activation of STAT3 induces the expression of GM-CSFRα that protects CLL cells from apoptosis, suggesting that inhibition of STAT3 or GM-CSFRα may benefit patients with CLL.

Microenvironmental Interactions in Chronic Lymphocytic Leukemia: The Master Role of CD49d

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation/expansion of a clonal population of neoplastic cells with the morphologic appearance of small mature B lymphocytes in blood, bone marrow, and lymphoid organs. A combination of genetic lesions is primarily responsible for the first step(s) of neoplastic transformation, along with microenvironmental signals, which concurrently operate by enhancing proliferation and/or inhibiting apoptosis. In this context, CD49d is known to play a pivotal role in mediating both cell-cell and cell-matrix interactions in CLL-involved tissues, eventually delivering pro-survival signals and protecting CLL cells from drug-induced damages. In the present review, we address, in detail, CD49d activities in the CLL microenvironment, CD49d functional and physical interactions with other microenvironmental receptors (including CD38 and B-cell receptor), and the relationship of CD49d expression with specific cytogenetic features in CLL.

The Nedd8-Activating Enzyme Inhibitor MLN4924 Thwarts Microenvironment-Driven NF-κB Activation and Induces Apoptosis in Chronic Lymphocytic Leukemia B Cells

Stromal-mediated signaling enhances NF-κB pathway activity in chronic lymphocytic leukemia (CLL) B cells, leading to cell survival and chemoresistance. Ubiquitination of IκBα may partially account for constitutive activation of NF-κB. MLN4924 is an investigational agent that inhibits the Nedd8-activating enzyme, thereby neutralizing Cullin-RING ubiquitin ligases and preventing degradation of their substrates. We conducted a preclinical assessment of MLN4924 in CLL. Primary CLL cells were cocultured in vitro with CD40L-expressing stroma to mimic the prosurvival conditions present in lymphoid tissue. The effect of MLN4924 on CLL cell apoptosis, NF-κB pathway activity, Bcl-2 family members, and cell cycle was assessed by flow cytometry, Western blotting, PCR, and immunocytochemistry. CD40L-expressing stroma protected CLL cells from spontaneous apoptosis and induced resistance to multiple drugs, accompanied by NF-κB activation and Bim repression. Treatment with MLN4924 induced CLL cell apoptosis and circumvented stroma-mediated resistance. This was accompanied by accumulation of phospho-IκBα, decreased nuclear translocation of p65 and p52 leading to inhibition of both the canonical and noncanonical NF-κB pathways, and reduced transcription of their target genes, notably chemokines. MLN4924 promoted induction of Bim and Noxa in the CLL cells leading to rebalancing of Bcl-2 family members toward the proapoptotic BH3-only proteins. siRNA-mediated knockdown of Bim or Noxa decreased sensitivity to MLN4924. MLN4924 enhanced the antitumor activity of the inhibitors of B-cell receptor (BCR)-associated kinases. MLN4924 disrupts NF-κB activation and induces Bim expression in CLL cells, thereby preventing stroma-mediated resistance. Our data provide rationale for further evaluation of MLN4924 in CLL.

Molecular pathways: targeting the microenvironment in chronic lymphocytic leukemia--focus on the B-cell receptor.

Interactions between malignant B lymphocytes and the tissue microenvironment play a major role in the pathogenesis of chronic lymphocytic leukemia (CLL) and other B-cell malignancies. The coexistence and coevolution of CLL cells with their tissue neighbors provided the basis for discovery of critical cellular and molecular drivers of the disease and identification of new therapeutic targets. Bone marrow stromal cells (BMSC), monocyte-derived nurselike cells (NLC), and T cells are key players in the CLL microenvironment, which activate and protect CLL cells within the tissues. CLL surface molecules, such as the B-cell antigen receptor (BCR), chemokine receptors, adhesion molecules, and TNF receptor superfamily members (e.g., CD40, BCMA, and BAFF-R) engage in cross-talk with respective tissue ligands. This cross-talk results in survival and expansion of the CLL clone, and protects CLL cells from conventional cytotoxic drugs. Inhibiting these pathways represents an alternative therapeutic strategy to more conventional chemoimmunotherapy. Here, we review central components of the CLL microenvironment, with a particular emphasis on BCR signaling, and we summarize the most relevant clinical advances with inhibitors that target the BCR-associated spleen tyrosine kinase/SYK (fostamatinib), Bruton's tyrosine kinase/BTK (ibrutinib), and PI3Kδ (idelalisib).

A role for oleoylethanolamide in chronic lymphocytic leukemia

Oleoylethanolamide (OEA) is a bioactive lipid that stimulates nuclear and G protein-coupled receptors and regulates appetite and fat metabolism. It has not previously been shown to have a role in cancer. However, a mass spectrometry-based lipidomics platform revealed the presence of high amounts of OEA in the plasma of chronic lymphocytic leukemia (CLL) patients compared with normal donors. CLL cells produced OEA and the magnitude of plasma OEA levels was related directly to the circulating leukemic cell number. OEA from CLL cells was increased by URB-597, an inhibitor of fatty acid amide hydrolase (FAAH), and decreased by inflammatory mediators that downregulate expression of N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD). These enzymes degrade and synthesize OEA, respectively. Nonphysiologic doses of OEA prevented spontaneous apoptosis of CLL cells in a receptor-independent manner that was mimicked by its free fatty acid (FFA) derivative oleate. However, OEA-containing supernatants from CLL cells induced lipolysis in adipocytes, lipid products from adipocytes protected CLL cells from cytotoxic chemotherapy, and increased levels of FFAs were found in CLL plasma that correlated with OEA. We suggest OEA is a lipolytic factor produced by CLL cells to fuel their growth with a potential role in drug resistance and cancer cachexia.

CD62L as a Therapeutic Target in Chronic Lymphocytic Leukemia

Despite advances in the treatment of chronic lymphocytic leukemia (CLL), the disease remains incurable with standard therapies and relapse is inevitable. A growing body of evidence indicates that alterations in the adhesion properties of neoplastic cells play a pivotal role in the development and progression of CLL. The expression of 71 cell surface molecules was examined on CLL peripheral blood mononuclear cells (PBMCs) over 3 weeks in culture. The most highly upregulated marker, CD62L, was examined further for expression on CD5(+)/CD19(+) CLL cells in vitro and in lymph node and bone marrow biopsies. The prosurvival role of CD62L was examined using a functional blocking antibody and therapeutic potential evaluated by comparison with current chemotherapy agents. Blocking CD62L resulted in apoptosis of CLL cells but not PBMCs from healthy donors suggesting a novel role for CD62L in CLL cell survival. The beneficial effect of coculturing CLL cells with bone marrow stromal cells or endothelial cells does not protect CLL cells from anti-CD62L-related toxicity. Moreover, combining fludarabine or mafosfamide with the anti-CD62L in vitro produced an additive effect both with and without stromal cells. This is the first reported data showing that blocking the activation and homing marker, CD62L, regulates CLL cell survival in vitro. These data also suggest that therapeutic antibodies against CD62L may provide additional clinical benefit to patients with CLL receiving current standard chemotherapy protocols.

PPARα and fatty acid oxidation mediate glucocorticoid resistance in chronic lymphocytic leukemia

AbstractHigh-dose glucocorticoids (GCs) can be a useful treatment for aggressive forms of chronic lymphocytic leukemia (CLL). However, their mechanism of action is not well understood, and resistance to GCs is inevitable. In a minimal, serum-free culture system, the synthetic GC dexamethasone (DEX) was found to decrease the metabolic activity of CLL cells, indicated by down-regulation of pyruvate kinase M2 (PKM2) expression and activity, decreased levels of pyruvate and its metabolites, and loss of mitochondrial membrane potential. This metabolic restriction was associated with decreased size and death of some of the tumor cells in the population. Concomitant plasma membrane damage increased killing of CLL cells by DEX. However, the nuclear receptor peroxisome proliferator activated receptor α (PPARα), which regulates fatty acid oxidation, was also increased by DEX, and adipocyte-derived lipids, lipoproteins, and propionic acid protected CLL cells from DEX. PPARα and fatty acid oxidation enzyme inhibitors increased DEX-mediated killing of CLL cells in vitro and clearance of CLL xenografts in vivo. These findings suggest that GCs prevent tumor cells from generating the energy needed to repair membrane damage, fatty acid oxidation is a mechanism of resistance to GC-mediated cytotoxicity, and PPARα inhibition is a strategy to improve the therapeutic efficacy of GCs.

In VitroSensitivity of CLL Cells to Fludarabine May Be Modulated by the Stimulation of Toll-like Receptors

The emerging role of Toll-like receptors (TLR) in the pathogenesis of chronic lymphocytic leukemia (CLL) led us to ask whether TLR stimulation may protect CLL cells from drug-induced apoptosis. We cultured in vitro malignant B cells freshly isolated from 44 patients with CLLs in the presence or the absence of different concentrations of fludarabine before or after 24-hour TLR stimulation with specific ligands and evaluated cell viability, apoptosis, and molecular pathways involved. Heterogeneity was observed among samples. In leukemic cells from patients bearing adverse prognostic factors, TLR stimulation caused a significant increase of protection to fludarabine treatment, whereas this did not occur in the cells from patients with good prognosis. To identify novel molecular mechanisms accounting for the dichotomy of response between the two groups of patients, we conducted an apoptosis gene expression profile on leukemic cells either unstimulated or stimulated with TLR9 ligand. Strikingly, TLR9 stimulation specifically upregulated the expression of lymphotoxin-α in cells where an increased protection to fludarabine treatment was observed. Also, the expression of miR-155-3p was significantly increased after stimulation of distinct TLR in cells where fludarabine treatment was less effective. These results suggest that at least in a proportion of patients, in vitro sensitivity to fludarabine may be modulated by the stimulation of TLR, likely mimicking microenvironmental signals occurring in vivo.