Suppression of Anti-Tumor Immunity in Chronic Lymphocytic Leukemia Via Interleukin-10 Production

Abstract The most common human leukemia is B-cell chronic lymphocytic leukemia (B-CLL), which is characterized by a progressive accumulation of abnormal B-lymphocytes in blood, bone marrow and secondary lymphoid organs. Typically disease progression is slow, but as the number of leukemic cells increases, they interfere with the production of other important blood cells, causing the patients to be in an immunosuppressive state. To study the basis of this immunoregulation, we used cells from the transgenic Eμ-Tcl1 mouse, which spontaneously develop B-CLL due to a B-cell specific expression of the oncogene, Tcl1. Previously we showed that Eμ-Tcl1 CLL cells constitutively produce an anti-inflammatory cytokine, IL-10. Here we studied the role of IL-10 in CLL cell survival in vitro and the development of CLL in vivo. We found that neutralization of IL-I0 using anti-IL-10 antibodies or blocking the IL-10 receptor (IL-10R) using anti-IL-10R antibodies did not affect the survival of CLL cells in vitro. On the other hand, adoptively transferred Eμ-Tcl1 cells grew at a slower rate in IL-10R KO mice vs. wild type (WT) mice. There was a significant reduction in CLL cell engraftment in the spleen, bone marrow, peritoneal cavity and liver of the IL-10R KO compared to WT mice. Further studies revealed that IL-10 could be playing a role in the tumor microenvironment possibly by affecting anti-tumor immunity. This was seen by a reduction in the activation of CD8+ T cells as well as a significantly lower production of IFN-γ by CD4+ T cells purified from CLL-injected WT mice compared to those purified from CLL-injected IL-10R KO mice. These studies demonstrate that CLL cells suppress host anti-tumor immunity via IL-10 production. This led us to investigate possible mechanisms by which IL-10 is produced. We found a novel role of B-cell receptor (BCR) signaling pathway in constitutive IL-10 secretion. Inhibition of Src or Syk family kinases reduces the constitutive IL-10 production by Eμ-Tcl1 cells in a dose dependent manner. In addition, we found that Eμ-Tcl1 CLL cells exhibit clonal variation in their IL-10 production in response to BCR cross-linking. Further studies are being performed to understand the mechanisms by which BCR signaling affects IL-10 production. Disclosures No relevant conflicts of interest to declare.

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Defective T cell-mediated, isotype-specific immunoglobulin regulation in B cell chronic lymphocytic leukemia

Blood ◽

10.1182/blood.v71.4.1012.bloodjournal7141012 ◽

1988 ◽

Vol 71 (4) ◽

pp. 1012-1020 ◽

Cited By ~ 1

Author(s):

JS Moore ◽

MB Prystowsky ◽

RG Hoover ◽

EC Besa ◽

PC Nowell

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Activity ◽

Lymphocytic Leukemia ◽

Specific Immunoglobulin ◽

Cell Chronic Lymphocytic Leukemia

The consistent occurrence of T cell abnormalities in patients with B cell chronic lymphocytic leukemia (B-CLL) suggest that the non- neoplastic host T cells may be involved in the pathogenesis of this B cell neoplasm. Because potential defects of immunoglobulin regulation are evident in B-CLL patients, we investigated one aspect of this by studying the T cell-mediated immunoglobulin isotype-specific immunoregulatory circuit in B-CLL. The existence of class-specific immunoglobulin regulatory mechanisms mediated by Fc receptor-bearing T cells (FcR + T) through soluble immunoglobulin binding factors (IgBFs) has been well established in many experimental systems. IgBFs can both suppress and enhance B cell activity in an isotype-specific manner. We investigated the apparently abnormal IgA regulation in a B-CLL patient (CLL249) whose B cells secrete primarily IgA in vitro. Enumeration of FcR + T cells showed a disproportionate increase in IgA FcR + T cells in the peripheral blood of this patient. Our studies showed that the neoplastic B cells were not intrinsically unresponsive to the suppressing component of IgABF produced from normal T cells, but rather the IgABF produced by the CLL249 host T cells was defective. CLL249 IgABF was unable to suppress IgA secretion by host or normal B cells and enhanced the in vitro proliferation of the host B cells. Size fractionation of both normal and CLL249 IgABF by gel-filtration high- performance liquid chromatography (HPLC) demonstrated differences in the ultraviolet-absorbing components of IgABF obtained from normal T cells v that from our patient with defective IgA regulation. Such T cell dysfunction may not be restricted to IgA regulation, since we have found similar expansion of isotype-specific FcR + T cells associated with expansion of the corresponding B cell clone in other patients with B-CLL. These data suggest that this T cell-mediated regulatory circuit could be significantly involved in the pathogenesis of B-CLL.

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ZAP-70 Is Not Phosphorylated on the Activating Tyrosine Residue (Tyr319) in Chronic Lymphocytic Leukemia and B-Cell Lymphoma Cells.

Blood ◽

10.1182/blood.v106.11.178.178 ◽

2005 ◽

Vol 106 (11) ◽

pp. 178-178

Author(s):

Stefania Gobessi ◽

Aleksandar Petlickovski ◽

Luca Laurenti ◽

Dimitar G. Efremov

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Progressive Disease ◽

Cell Receptor ◽

Kinase Domain ◽

Lymphocytic Leukemia ◽

Bcr Signaling ◽

Cell Receptor Signaling

Abstract The protein tyrosine kinase ZAP-70 is expressed at high levels in leukemic B-cells from chronic lymphocytic leukemia (CLL) patients with progressive disease and short survival. ZAP-70 is a key component of the proximal T-cell receptor signaling pathway and is highly homologous to Syk, an important B-cell receptor signaling (BCR) molecule. Recent studies indicate that ZAP-70 may participate in BCR signaling as well, but the mechanism of action is still not well understood. In T-cells, upon TCR stimulation ZAP-70 becomes phosphorylated on Tyr319 by the Src-like kinase Lck, which results in the release of the ZAP-70 kinase domain from an autoinhibited state to a fully active conformation. The Tyr319 site in ZAP-70 corresponds to the Tyr352 site in Syk, which is phosphorylated in B-cells following BCR stimulation. We therefore investigated the activation status of ZAP-70 and Syk in BCR stimulated CLL B-cells, using phosphorylation of Tyr319 and Tyr352 as markers of their activation. Analysis of 10 ZAP-70-positive CLL samples by immunoblotting with the phospho-ZAP70Tyr319/SykTyr352 antibody revealed that ZAP-70 is not phosphorylated at this site either before or after BCR stimulation, although in control experiments with Jurkat T-cells ZAP-70 became phosphorylated on Tyr319 upon TCR stimulation. Moreover, the Tyr352 site in Syk was phosphorylated following BCR stimulation in 6 of the 10 CLL B-cell samples. To further investigate the reasons for the unexpected lack of ZAP-70 activation in CLL B-cells, we produced stable transfectants of the BJAB lymphoma B-cell line that expressed ZAP-70 at levels similar to those found in CLL cases with progressive disease. In agreement with the CLL B-cell experiments, the Tyr319 site in ZAP-70 was not phosphorylated either before or after BCR stimulation. Since phosphorylation of Tyr319 is Lck-dependent in T-cells, and this kinase is expressed also in CLL B-cells, we ectopically expressed Lck in the ZAP-70-positive BJAB clones. Again, the Tyr319 site was not phosphorylated, indicating that ZAP-70 does not undergo activation of the kinase domain also in this cellular system. In contrast, BCR crosslinking in BJAB cells induced significant phosphorylation of Tyr352 in Syk, which was further enhanced in the clones that coexpressed ZAP-70. Furthermore, analysis of downstream signaling pathways following BCR stimulation showed stronger and prolonged activation of ERK and to a lesser extent Akt in the ZAP-70 positive clones, whereas no difference was observed in terms of activation of PLC-γ 2, JNK and degradation of the NF-kB inhibitor IkB. These data indicate that ZAP-70 does not undergo full activation in B-cells, but can still enhance activation of certain downstream BCR signaling pathways, possibly by affecting the activity of the related PTK Syk.

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CD160 signaling mediates PI3K-dependent survival and growth signals in chronic lymphocytic leukemia

Blood ◽

10.1182/blood-2009-08-239483 ◽

2010 ◽

Vol 115 (15) ◽

pp. 3079-3088 ◽

Cited By ~ 31

Author(s):

Feng-Ting Liu ◽

Jerome Giustiniani ◽

Timothy Farren ◽

Li Jia ◽

Armand Bensussan ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Cycle Progression ◽

Lymphocytic Leukemia ◽

Cytochrome C Release ◽

Pi3k Inhibitors ◽

Phosphoinositide 3 Kinase ◽

Dose Dependent

Abstract B-cell chronic lymphocytic leukemia (CLL) expresses CD160, a glycosylphosphatidylinositol-linked receptor found on normal natural killer (NK) and T cells, but not B cells. CD160 is a multifunctional molecule in normal lymphocytes, but its role in CLL biology is unknown. In vitro, CLL cells undergo rapid spontaneous apoptosis, which CD160 activation protected against—mean cell viability increased from 67% to 79% (P < .001). This was associated with up-regulation of Bcl-2, Bcl-xL, and Mcl-1, but not Bax. As expected from these changes in Bcl-2/Bax and Bcl-xL/Bax ratios, CD160 triggering reduced mitochondrial membrane potential collapse and cytochrome c release. CD160 stimulation also induced DNA synthesis, cell cycle progression, and proliferation. B-cell antigen receptor (BCR)–induced CLL proliferation was generally greater than with CD160, but marked variation was seen. Both BCR and CD160 signaling led to CLL secretion of interleukin-6 (IL-6) and IL-8, although CD160 induced greater increases of IL-6 (51-fold) and IL-8 (15-fold). Survival and activation signals mediated by CD160 showed dose-dependent suppression by phosphoinositide-3 kinase (PI3K) inhibitors. Thus, in vitro, CLL cells can use the CD160 pathway for survival and activation, mimicking CD160 signaling in normal NK and CD8+ T cells. Establishing the pathophysiologic relevance of these findings may reveal new therapeutic targets.

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Leukemia-associated monoclonal and oligoclonal TCR-BV use in patients with B-cell chronic lymphocytic leukemia

Blood ◽

10.1182/blood-2002-03-0746 ◽

2003 ◽

Vol 101 (3) ◽

pp. 1063-1070 ◽

Cited By ~ 50

Author(s):

Mohammad-Reza Rezvany ◽

Mahmood Jeddi-Tehrani ◽

Hans Wigzell ◽

Anders Österborg ◽

Håkan Mellstedt

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cd8 T Cells ◽

Leukemia Cell ◽

Lymphocytic Leukemia ◽

Cell Chronic Lymphocytic Leukemia

Abstract T-cell receptor–B-variable (TCR-BV) gene usage and the CDR3 size distribution pattern were analyzed by reverse transcription–polymerase chain reaction (RT-PCR) in patients with B-cell chronic lymphocytic leukemia (B-CLL) to assess the T-cell repertoire. The use of TCR-BV families in CD4 and CD8 T cells stimulated with autologous activated leukemic cells was compared with that of freshly obtained blood T cells. Overexpression of individual TCR-BV families was found in freshly isolated CD4 and CD8 T cells. Polyclonal, oligoclonal, and monoclonal TCR-CDR3 patterns were seen within such overexpressed native CD4 and CD8 TCR-BV families. In nonoverexpressed TCR-BV families, monoclonal and oligoclonal populations were noted only within the CD8 subset. After in vitro stimulation of T cells with autologous leukemic B cells, analyses of the CDR3 length patterns showed that in expanded TCR-BV populations, polyclonal patterns frequently shifted toward a monoclonal/oligoclonal profile, whereas largely monoclonal patterns in native overexpressed TCR-BV subsets remained monoclonal. Seventy-five percent of CD8 expansions found in freshly obtained CD8 T cells further expanded on in vitro stimulation with autologous leukemic B cells. This suggests a memory status of such cells. In contrast, the unusually high frequency of CD4 T-cell expansions found in freshly isolated peripheral blood cells did not correlate positively to in vitro stimulation as only 1 of 9 expansions continued to expand. Our data suggest that leukemia cell–specific memory CD4 and CD8 T cells are present in vivo of patients with CLL and that several leukemia cell–associated antigens/epitopes are recognized by the patients' immune system, indicating that whole leukemia cells might be of preference for vaccine development.

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Elevated Serum IL-12 Levels Attenuate Tumor Immunity Via the TIM-3/Gal-9 Axis and Worsen Patient Prognosis in Follicular B-Cell Non-Hodgkin Lymphoma (NHL).

Blood ◽

10.1182/blood.v114.22.2668.2668 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2668-2668

Author(s):

Zhi-Zhang Yang ◽

Steven C. Ziesmer ◽

Anne J. Novak ◽

Toshiro Niki ◽

Mitsuomi Hirashima ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Tumor Immunity ◽

Cd8 T Cells ◽

Cell Lymphoma ◽

Elevated Serum ◽

B Cell Lymphoma ◽

Serum Levels

Abstract Abstract 2668 Poster Board II-644 Interleukin-12 (IL-12) has been demonstrated to induce IFN-g production by T and NK cells and thereby contribute to anti-tumor immunity. However, the administration of IL-12 to boost anti-tumor immunity in B-cell lymphoma has shown no clinical benefit. In fact, clinical trials of IL-12 in combination with rituximab in follicular B-cell lymphoma (FL) showed a lower response rate in patients treated with the combination than in patients treated with rituximab alone (Clin Cancer Res. 2006 15; 12:6056-63). The goal of this study was therefore to determine the role of IL-12 in the antitumor response in B-cell NHL. First, we measured serum levels of IL-12 in patients with untreated FL before treatment with rituximab and normal healthy controls. We found that serum IL-12 levels were elevated in FL patients compared to healthy individuals (median: 0.50 ng/ml, n=30 vs 0.32 ng/ml, n=22; p= 0.03) and that elevated serum IL-12 levels were associated with a poor outcome in these patients when treated with rituximab alone as initial therapy. Using 0.56 ng/ml as a cutoff, patients with serum IL-12 levels of greater than 0.56 ng/ml had a significantly shorter time to progression than patients with IL-12 levels less than 0.56 ng/ml (12 months versus 40 months; p=0.001). To determine the mechanism by which IL-12 may contribute to a poor prognosis, we investigated the role of IL-12 on induction of immune tolerance. First, we found that TIM-3, a member of the T cell immunoglobulin and mucin domain-containing protein (TIM) family that functions to terminate TH1-mediated immunity and promote tolerance, was constitutively expressed on a subset of intratumoral T cells accounting for approximately 15% and 25% of the intratumoral CD4+ and CD8+ T cells, respectively. In contrast, less than 2% of T cells from peripheral blood of normal individuals expressed TIM-3. TIM-3-expressing T cells were distinct from regulatory T cells since CD25+ and Foxp3+ T cells lacked TIM-3 expression. Secondly, we found that TIM-3-expressing CD4+ cells were unable to produce cytokines such as IL-2, IFN-g or IL-17 and that TIM-3-expressing CD8+ T cells failed to produce Granzyme B, IFN-g or IL-2. We also observed that TIM-3-expressing T cells lost the capacity to proliferate in response to TCR activation. These results suggest that TIM-3 expressing CD4+ and CD8+ T cells are functionally exhausted. Thirdly, we observed that TIM-3 expression on T cells could be induced by activation and that IL-12 was the strongest stimulus to induce TIM-3 expression on CD4+ and CD8+ T cells. Finally, we found by immunohistochemistry (IHC) that Galectin-9 (Gal-9), a ligand for TIM-3, was abundantly expressed on lymphoma B cells. In vitro incubation with a stable form of Gal-9 induced apoptosis of CD4+ and CD8+ T cells in a dose dependent fashion. Gal-9-mediated apoptosis of T cells was attenuated by a TIM-3 Fc protein and isolated TIM-3+ T cells exhibited a significantly higher apoptosis rate than TIM-3− T cells in response to Gal-9. These results indicate that, in contrast to the observations in vitro or in vivo in mice, IL-12 actually plays a detrimental role in lymphoma patients. Given the findings that IL-12 strongly induces TIM-3 expression on effector T cells and that the TIM-3/Gal-9 pathway impairs the immune response, we conclude that increased serum levels of IL-12 suppress anti-tumor immunity in follicular lymphoma patients and is associated with a poor prognosis. Disclosures: Witzig: Novartis: Research Funding.

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Bruton tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765

Blood ◽

10.1182/blood-2011-01-328484 ◽

2011 ◽

Vol 117 (23) ◽

pp. 6287-6296 ◽

Cited By ~ 491

Author(s):

Sarah E. M. Herman ◽

Amber L. Gordon ◽

Erin Hertlein ◽

Asha Ramanunni ◽

Xiaoli Zhang ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Tyrosine Kinase ◽

B Cell ◽

Lymphocytic Leukemia ◽

Cell Contact ◽

Irreversible Inhibitor ◽

Bcr Signaling ◽

Bruton Tyrosine Kinase

Abstract B-cell receptor (BCR) signaling is aberrantly activated in chronic lymphocytic leukemia (CLL). Bruton tyrosine kinase (BTK) is essential to BCR signaling and in knockout mouse models its mutation has a relatively B cell–specific phenotype. Herein, we demonstrate that BTK protein and mRNA are significantly over expressed in CLL compared with normal B cells. Although BTK is not always constitutively active in CLL cells, BCR or CD40 signaling is accompanied by effective activation of this pathway. Using the irreversible BTK inhibitor PCI-32765, we demonstrate modest apoptosis in CLL cells that is greater than that observed in normal B cells. No influence of PCI-32765 on T-cell survival is observed. Treatment of CD40 or BCR activated CLL cells with PCI-32765 results in inhibition of BTK tyrosine phosphorylation and also effectively abrogates downstream survival pathways activated by this kinase including ERK1/2, PI3K, and NF-κB. In addition, PCI-32765 inhibits activation-induced proliferation of CLL cells in vitro, and effectively blocks survival signals provided externally to CLL cells from the microenvironment including soluble factors (CD40L, BAFF, IL-6, IL-4, and TNF-α), fibronectin engagement, and stromal cell contact. Based on these collective data, future efforts targeting BTK with the irreversible inhibitor PCI-32765 in clinical trials of CLL patients is warranted.

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Development of a Transgenic Mouse Model to Study the Role of ZAP-70 in the Development and Progression of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v118.21.2830.2830 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2830-2830

Author(s):

Stefania Gobessi ◽

Sara Bennardo ◽

Pablo G Longo ◽

Brendan Doe ◽

Dimitar G Efremov

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Mouse Model ◽

B Cell ◽

Lymphocytic Leukemia ◽

Bcr Signaling ◽

Leukemia Development ◽

Low Levels ◽

The Impact

Abstract Abstract 2830 The protein tyrosine kinase ZAP-70 is an important prognostic factor in chronic lymphocytic leukemia (CLL). Patients that are considered ZAP-70-positive typically express 30–100% of the levels of ZAP-70 in T-cells, whereas in the remaining patients ZAP-70 is either not expressed or is expressed at lower levels. ZAP-70-positive patients have more aggressive disease and shorter survival than patients with low or absent ZAP-70. In vitro experiments with human lymphoma cell lines and primary CLL B-cells have shown that ZAP-70 is involved in B cell receptor (BCR) signaling, indicating that overexpression of ZAP-70 could affect the capacity of the leukemic cells to respond to antigen stimulation. Despite the strong association between ZAP-70 expression and prognosis, it is still not clear whether ZAP-70 directly contributes to the aggressiveness of the disease or is just a marker of more aggressive CLL. To further address this issue, we generated transgenic (tg) mice that express different levels of ZAP-70 in B cells. In these mice expression of the murine ZAP-70 transgene is targeted to the B cell compartment by a VH or a CD19 promoter (VH-ZAP70 and CD19-ZAP70 tg mice, respectively). B cells in CD19-ZAP70 tg mice express the same levels of ZAP-70 as normal murine T cells, whereas the levels of ZAP-70 in B cells of VH-ZAP70 tg mice are approximately 10 times lower. Immunophenotyping analysis of spleen and peritoneal cavity samples from wild type, VH-ZAP70 and CD19-ZAP70 tg mice did not reveal significant differences in the percentage of follicular (FO), marginal zone (MZ) and B1 B cells, indicating that ectopic expression of ZAP-70 does not affect normal B cell development and maturation. In terms of BCR signal transduction, no abnormalities were detected in VH-ZAP70 tg mice, suggesting that low levels of ZAP-70 do not affect BCR signaling. In contrast, B cells from CD19-ZAP70 tg mice showed altered phosphorylation of several molecules downstream of the BCR, such as Syk and BLNK, whereas phosphorylation of Cbl was not affected. To investigate the impact of ZAP-70 expression on leukemia development and progression, we crossed VH-ZAP70 and CD19-ZAP70 tg mice with Eμ-TCL1 tg mice. The latter mice develop leukemias that are considered a mouse model of human CLL. These leukemias are CD5+, express unmutated IGHV genes and stereotyped polyreactive BCRs, but are always ZAP-70-negative. VH-ZAP70/Eμ-TCL1 tg mice (n=11) have been followed for over a year and did not show any differences with respect to their Eμ-TCL1 littermates (n=10). Both groups, starting from the age of 7–8 months, developed leukemias with a similar rate of progression and impact on survival, suggesting that low levels of ZAP-70 do not affect the behavior of the disease. The cohort of CD19-ZAP70/Eμ-TCL1 tg mice was more recently established. These animals are currently 4 months old and still do not show signs of leukemia development. Data from the extended follow-up of these mice will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

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Fostamatinib Inhibits BCR Signaling, and Reduces Tumor Cell Activation and Proliferation in Patients with Relapsed Refractory Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v120.21.2882.2882 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2882-2882

Author(s):

Sarah E. M. Herman ◽

Paul M. Barr ◽

Erin M. McAuley ◽

Delong Liu ◽

Jonathan W. Friedberg ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Gene Signature ◽

Lymphocytic Leukemia ◽

Gene Signatures ◽

Signature Genes ◽

Survival Pathways ◽

Bcr Signaling

Abstract Abstract 2882 B-cell receptor (BCR) signaling contributes to the pathogenesis of chronic lymphocytic leukemia (CLL). Spleen tyrosine kinase (SYK) activated directly downstream of the BCR is essential for the induction of proliferation and survival pathways. The SYK inhibitor fostamatinib disrupts BCR signaling and was the first such inhibitor to show significant clinical activity in patients with mature B-cell malignancies. Fostamatinib has been shown to both induce apoptosis in unstimulated CLL cells as well as to inhibit BCR induced anti-apoptotic signals in vitro (Gobessi et al., 2009; Quiroga et al., 2009). Similarly, using the Eμ-TCL1 transgenic mouse model, fostamatinib has been shown to inhibit the growth of malignant B-cells without significant alteration of normal B-cells (Suljagic et al., 2010). In the first phase I/II clinical trial investigating fostamatinib in relapsed B-cell non-Hodgkin's lymphoma (NHL) and CLL, clinical efficacy was observed in a variety of histologies with the highest response rate in CLL/SLL patients (Friedberg et al., 2010). Eleven CLL/SLL patients enrolled in this trial donated cellular material for correlative studies. Using these primary tumor samples, we evaluated the effects of fostamatinib on CLL cells in vivo after one cycle of treatment. We first validated the on-target effect of fostamatinib by using quantitative RT-PCR to measure expression of validated pathway specific gene signatures. Fostamatinib greatly down-regulated 12/12 evaluated BCR signature genes and significantly reduced the BCR gene signature score (computed as the average expression of the pathway specific genes; p=.002) Effective inhibition of BCR signaling was confirmed by a significant reduction in the phosphorylation of both BTK and ERK; two key BCR signaling molecules located downstream of SYK activation. Interestingly, BCR signaling was inhibited in CLL cells from all patients regardless of response to therapy. We next expanded our analysis to look at NF-κB and MYC gene signatures. We found that 11/11 representative NF-κB signature genes and 5/5 MYC signature genes were also down-regulated resulting in a significant reduction in both gene signature scores (p=.004 and p=.020, respectively). Confirming these results, we also observed a significant reduction in JUNB (p<.001) and MYC (p=.026) at the protein level. Interestingly, the reduction in NF-κB and MYC signature scores was highly correlated with the degree of reduction in BCR signaling suggesting that these pathways are linked. In addition to changes in the gene signatures we also observed a significant reduction in the cellular activation immunephenotype; CD69 and CD86 expression were significantly reduced by fostamatinib (p=.033 and p=.004, respectively). Further, we found that CD38 (an activation marker with prognostic significance) was also reduced on treatment although not to a significant extent. Finally, fostamatinib significantly reduced tumor proliferation as determined by the percentage of CLL cells expressing Ki67 (p=.005). Eight of the 11 patients in this study achieved a clinically significant response; interestingly however, the 3 non-responders demonstrated significantly brighter CD38 expression with an MFI up to 9-times the CD38 MFI in responders. A possible role of CD38 as a biomarker for response should be further explored in patients treated with BCR directed kinase inhibitors. In conclusion, fostamatinib and other inhibitors of BCR-related kinases constitute a major advance in the treatment of CLL. In vitro data with these compounds suggests that interruption of BCR signaling and survival pathways activated in the tissue microenvironments are likely responsible for the observed clinical response as only a moderate direct induction of apoptosis is seen in vitro. Here we demonstrate that inhibition of BCR-mediated signaling by fostamatinib results in a reduction in CLL proliferation and activation in vivo. Together these data provide a blueprint to further study the mechanism of action and resistance mechanisms of not only fostamatinib but also other BCR targeted therapeutics. This work was supported in part by the Intramural Research Program of the National, Heart, Lung and Blood Institute and by the University of Rochester SPORE in lymphoma P50 CA13080503, Rigel and the James P. Wilmot Foundation. Disclosures: Friedberg: Rigel: Research Funding.

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MiR-181b in Chronic Lymphocytic Leukemia B Cells Is Regulated By Cellular Interaction with CD4+ T Cells and Increases the CTL Toxicity Versus the Leukemic Clone

Blood ◽

10.1182/blood.v126.23.4134.4134 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4134-4134

Author(s):

Mirco di Marco ◽

Serena Veschi ◽

Rosa Visone ◽

Giuseppe Leone ◽

Paola Lanuti ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Cd4 T Cells ◽

Lymphocytic Leukemia ◽

Healthy Donors ◽

Leukemic Clone

Abstract Clinical progression of chronic lymphocytic leukemia (CLL) is characterized by gradual reduction of the ratio T/B cells, along with immune cell dysfunction due, at least in part, to T cell defects, such as decreased expression of CD40L and reduced signaling via the TCR CD3. This compromise the ability of T cells to respond and to eliminate leukemic cell from CLL patients. Enhanced activation of either allogenic or autologous T cells can drive the death of CLL cells in vitro and in human subjects. Changes in microRNAs expression also characterize clinical progression of CLL with a strong decrease of miR-181b/a and miR-130a associated with the more aggressive phase of the disease. The miR-181b targets anti-apoptotic proteins, such as BCL-2 and MCL1 and its expression correlates with those protein levels in CLL. In this study we demonstrate that the expression of those microRNAs in CLL-B cells, are regulated by T cells. We co-cultured allogenic pure CLL-B cells with either activated (CD2, CD3 and CD28 antibodies, used to mimic antigen-presenting cells) or not activated CD4+ T cells from healthy donors. We observed a significant increase of mir-181b/a and miR-130a expression in CLL B-cells after co-culture with activated CD4+ T cells in 8 out of 11 cases. A significant increase of these miRs was also determined in purified CLL B-cells after 4 days activation of peripheral blood mononuclear cells (PBMCs) from CLL patients, even if in minor rate. By the use of specific antibodies, co-culture with Hela CD40 expressing cells and transwell experiments, we established that this effect is a T/B contact-dependent signaling mediated through CD40L-CD40 interaction. We determine that increased expression of the 3 miRs occurs at the transcriptional level. Since the expression of miR-181b showed the most significant variation in previous experiments it was selected for further analyses. We next investigated the in vivo role of the miR-181b in highly immunodeficient mice. The CLL cell line, MEC-01, infected with either the LV-miR-181b_coGFP or the LV-CTRL_coGFP was intravenously inoculated in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were sacrificed after 4 weeks and assayed for percentage of GFP+ cells in bone marrow and spleen compartments. The miR-181b did not show any specific effect into the leukemic clone. However when the same cells were inoculated in an environment hosting mature T cells, miR-181b consistently influences the death of leukemic cells (Fig 1B), suggesting that T cells are required to potentiate the apoptotic role of this miRNA. To explain what we observed in vivo, we mixed in vitro MEC-01 infected with either the LV-miR-181b or the LV-CTRL and CD8+ T cells from healthy donors. After few hours of contact T cells showed stronger cytotoxic effect on MEC-01 carrying miR-181b as compared to the control. Mixed lymphocyte reaction CD40L-activated CLL and T cells is used to generate effector CTLs. Therefore we grew T cell with CD40L-activated MEC-01 in which the expression of miR-181b was either shut down by lentiviral vector or unchanged as control. After one week, we monitored by cytofluorimetry the CD38 surface marker on T cells since its expression has been associated with more active CTLs and, by ELISA, the release of IL-10, the inhibitor of the potent inducer of CTLs INF-g. We demonstrate that activated MEC-01 with higher expression of miR-181b leads to an increase of the cell number expressing CD38 and this was accompanied by a reduced release of IL-10 from B cells through down-regulation of c-FOS, which we show to be target of the miR-181b and to promote the transcription of the IL-10. In conclusion, our data suggest a role of the miR-181b in the immune response against CLL-B cells. We show that an efficient activation of CD4+ T cells through CD3-complex pathway and a right CD40L-CD40 interaction lead to a significant increase of the some miRNAs deregulated over the progression of chronic lymphocytic leukemia, namely miR-181b. This miRNA potentiates the cytotoxicity of T cells favoring the killing of the leukemic clone. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v124.21.2196.2196 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2196-2196

Author(s):

Ashima Shukla ◽

Christine E Cutucache ◽

Karan Rai ◽

Siddharth Rai ◽

Rene Opavsky ◽

...

Keyword(s):

Bone Marrow ◽

Chronic Lymphocytic Leukemia ◽

Lymph Nodes ◽

Lymphocytic Leukemia ◽

Clinical Heterogeneity ◽

Knock Down ◽

Immune Synapses

Abstract 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. Disclosures No relevant conflicts of interest to declare.

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