Shaun Willimott

CD154 induces a switch in pro-survival Bcl-2 family members in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia cells survive and proliferate in patients but rapidly die in culture. The microenvironment that sustains leukaemic cells in vivo contains both stromal cell elements and T cells. We defined changes in Bcl‐2 family protein expression on culture with CD40 ligand (CD154) expressed on mouse fibroblast L cells, and interleukin‐4 (IL‐4; CD154/IL‐4 system): conditions that support survival and proliferation. Unexpectedly, Bcl‐2 protein expression decreased whilst pro‐survival Bcl‐xL (as well as A1 and Mcl‐1) increased. However, the CD154‐L cell/IL‐4 system also increased the pro‐apoptotic proteins, Bid and Noxa, suggesting that an increased pool of pro‐survival factors and not the effects of a single protein mediate survival. Most pro‐apoptotic proteins were not induced in drug or spontaneous apoptosis, but expression of Bcl‐xS, a pro‐apoptotic BCL2L1 isoform, was associated with cell death. This was post‐transcriptionally controlled, and, therefore, alternative splicing at the Bcl‐x locus appears to have a role in the regulation of chronic lymphocytic leukaemia (CLL) cell survival. This study demonstrated a switch in pro‐survival proteins associated with the transition from quiescence to CD154‐driven proliferation. CLL therapies targeting Bcl‐2 may need to be modified to antagonize proliferation centre‐specific pro‐survival proteins.

miR-125b and miR-155 contribute to BCL2 repression and proliferation in response to CD40 ligand (CD154) in human leukemic B-cells.

Background: BCL2 repression occurs in normal and leukemic B-lymphocytes in lymph nodes. Results: miR-155 and miR-125b repress BCL2 translation and, in part, mediate the proliferative response to CD40 ligand (CD154). Conclusion: miR-155 and miR-125b contribute to proliferative responses to CD154 and one of their targets is BCL2. Significance: CD154 drives proliferation of mature B-cell malignancies and abrogating the effects of miRNA induced by CD154 may be therapeutically useful. Developmental stage-specific regulation of BCL2 occurs during B-cell maturation and has a role in normal immunity. CD40 signaling promotes proliferation and rescues B-cells from apoptosis, partly through induction of BCL2L1 and BCL2A1 and repression of BCL2. We previously showed that a stromal cell/CD40 ligand (CD154) culture system reproduced this switch in survival protein expression in primary human leukemic B-cells and we employed this model system to investigate BCL2 repression. BCL2 was post-transcriptionally regulated and the repressed BCL2 mRNA was associated with non-polysomal, but dense fractions on sucrose density gradients. Microarrays identified a set of miRNA that were induced by culture conditions and potentially able to bind to the BCL2 3′-UTR. Luciferase reporter assays demonstrated that miR-125b and miR-155 repressed BCL2 mRNA but while stromal cell contact alone was sufficient to induce strongly miR-125b this did not cause BCL2 repression. miR-155, which is the most abundant miRNA under basal conditions, specifically required CD154 for further induction above a threshold to exert its full repressive effects. Anti-miR-125b and anti-miR-155 prevented CD154-mediated repression of BCL2 and reduced CD154-mediated proliferation in the MEC1 B-cell line. We suggest that miR-155 and miR-125b, which are induced by CD154 and stromal cell signals, contribute to regulating proliferation and that BCL2 is one of their target mRNAs.

Enhancement of CD154/IL4 proliferation by the T follicular helper (Tfh) cytokine, IL21 and increased numbers of circulating cells resembling Tfh cells in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) cells encounter T‐cells and proliferate in response to T‐cell signals in the lymph node microenvironment. In this report we determined interleukin 21 (IL21) function in CLL and showed that IL21 and interleukin 4 (IL4) act co‐operatively to promote leukaemic cell proliferation without apoptosis or differentiation We further show that IL21 increased side population (SP) cells, which are associated with resistance to chemotherapy and increased self‐renewal capacity in CLL. IL21 and IL4 are the major cytokines produced by the recently described CD4+ T follicular helper (Tfh) cell subset. Determination of Tfh cells in peripheral blood showed that patients had significantly increased numbers as compared to normal subjects although no association was found between Tfh numbers and IGHV gene mutational status or clinical stage. Our data suggests that the Tfh cytokines, IL4 and IL21, contribute to driving leukaemic cell proliferation in the lymph node microenvironment, and may contribute to the specific production of cells resistant to conventional chemotherapy. We suggest that increased circulating Tfh cells is a component of T‐cell dysregulation in CLL. Our findings have implications for the therapeutic use of IL21.

Regulation of CD38 in proliferating chronic lymphocytic leukemia cells stimulated with CD154 and interleukin-4

Background and Objectives Chronic lymphocytic leukemia (CLL) cells, like normal B-cells, exist in two populations in vivo: quiescent cells in the peripheral circulation and proliferating cells in lymph nodes. The surface marker CD38 has roles in cell adhesion and signaling. Its expression correlates with poor clinical outcome and is associated with expression of the signaling intermediate ZAP-70, which is also a marker of poor prognosis. We investigated the regulation of CD38 and ZAP-70 in proliferating CLL cells. Design and Methods We cultured CLL cells on a stromal cell layer that maintains viability and also with some stromal cells expressing CD40 ligand (CD154) in order to measure changes in expression of CD38 and ZAP-70. Results We demonstrated up-regulation of CD38 expression by CD154. The degree of up-regulation did not correlate with clinical stage or mutational status. In addition in the majority of cases tested ZAP-70 expression increased in parallel with up-regulation of CD38 although discordant cases were also observed. Interpretation and Conclusions Overall we demonstrated that regulation of CD38 in CLL is dynamic and dependent on signals from CD154 and a stromal cell layer. We speculate that CD38 and ZAP-70 are expressed in lymph node leukemic cells in both good and poor prognosis patients, but, in cases with good clinical outcome, these molecules are down-regulated in the peripheral blood whereas in cases with poor prognosis their expression is maintained.

Poly(ADP‐ribose) polymerase‐1 (Parp‐1)‐deficient mice demonstrate abnormal antibody responses

Poly(ADP‐ribosylation) of acceptor proteins is an epigenetic modification involved in DNA strand break repair, recombination and transcription. Here we provide evidence for the involvement of poly(ADP‐ribose) polymerase‐1 (Parp‐1) in antibody responses. Parp‐1−/− mice had increased numbers of T cells and normal numbers of total B cells. Marginal zone B cells were mildly reduced in number, and numbers of follicular B cells were preserved. There were abnormal levels of basal immunoglobulins, with reduced levels of immunoglobulin G2a (IgG2a) and increased levels of IgA and IgG2b. Analysis of specific antibody responses showed that T cell‐independent responses were normal but T cell‐dependent responses were markedly reduced. Germinal centres were normal in size and number. In vitro purified B cells from Parp‐1−/− mice proliferated normally and showed normal IgM secretion, decreased switching to IgG2a but increased IgA secretion. Collectively our results demonstrate that Parp‐1 has essential roles in normal T cell‐dependent antibody responses and the regulation of isotype expression. We speculate that Parp‐1 forms a component of the protein complex involved in resolving the DNA double‐strand breaks that occur during class switch recombination.

Cap-Translation Inhibitor, 4EGI-1, Restores Sensitivity to ABT-737 Apoptosis through Cap-Dependent and -Independent Mechanisms in Chronic Lymphocytic Leukemia

Purpose: The lymph node microenvironment promotes resistance to chemotherapy in chronic lymphocytic leukemia (CLL), partly through induction of BCL2 family prosurvival proteins. Currently available inhibitors do not target all BCL2 family prosurvival proteins and their effectiveness is also modified by proapoptotic BCL2 homology domain 3 (BH3) only protein expression. The goal of this study was to evaluate synergy between the eIF4E/eIF4G interaction inhibitor, 4EGI-1, and the BH3 mimetic, ABT-737. Experimental Design: CLL cells were cultured in conditions to mimic the lymph node microenvironment. Protein synthesis and cap-complex formation were determined. Polysome association of mRNAs from BCL2 family survival genes was analyzed by translational profiling. The effects of 4EGI-1 and the BCL2/BCL2L1 antagonist, ABT-737, on CLL cell apoptosis were determined. Results: Protein synthesis was increased approximately 6-fold by stromal cell/CD154 culture in a phosphoinositide 3-kinase α (PI3Kα)–specific manner and was reduced by 4EGI-1. PI3K inhibitors and 4EGI-1 also reduced cap-complex formation but only 4EGI-1 consistently reduced BCL2L1 and BCL2A1 protein levels. 4EGI-1, but not PI3K inhibitors or rapamycin, induced an endoplasmic reticulum stress response including proapoptotic NOXA and the translation inhibitor phosphorylated eIF2α. 4EGI-1 and ABT-737 synergized to cause apoptosis, independent of levels of prosurvival protein expression in individual patients. Conclusions: Overall protein synthesis and cap-complex formation are induced by microenvironment stimuli in CLL. Inhibition of the cap-complex was not sufficient to repress BCL2 family prosurvival expression, but 4EGI-1 inhibited BCL2A1 and BCL2L1 while inducing NOXA through cap-dependent and -independent mechanisms. 4EGI-1 and ABT-737 synergized to produce apoptosis, and these agents may be the basis for a therapeutically useful combination. Clin Cancer Res; 19(12); 3212–23. ©2013 AACR.

Separate cell culture conditions to promote proliferation or quiescent cell survival in chronic lymphocytic leukemia

Chronic lymphocytic leukemia cells circulating in the peripheral blood are in the resting phase (G0/G1) of the cell cycle, yet, over time, leukaemic cells accumulate. Recently, proliferation rates in vivo have been shown to be substantial, with the most clinically aggressive cases having the highest proliferation rates [1]. The sites where CLL cell division takes place are proliferation centres found in lymph nodes [2]. The characteristics and proliferative capacity of leukaemic cells within the lymph nodes or bone marrow are quite different to those of cells in the peripheral blood, and simple culture system that can separately maintain quiescent (representing peripheral blood CLL cells) and proliferating CLL cells will be useful in analysing the biology of CLL and testing the effects of drugs and new biological agents. Contact with a specialized stromal cell layer (bone marrow derived stromal cells) has been demonstrated to maintain CLL cell viability [3] and prevent spontaneous apoptosis [4]. Others have shown similar effects with in vitro derived nurse-like cells [5]. Both of these cell types are demanding to produce and difficult to maintain. Proliferation of CLL cells in vitro has been produced in various systems. CD40 ligand (CD154) is a T-cell surface molecule that is essential for B-cell activation and the formation of germinal centres by normal B-cells. It has also been used extensively in CLL research [6 – 9]. Modest proliferation of CLL cells in vitro can be produced by signalling through CD40 but this is greatly enhanced by co-activation with IL-4 [7]. This effect is specific to IL-4 and the addition of IL-2 to CD40 signalling does not produce significant enhancement of proliferation [7]. CD154 is expressed on T-cells within lymph node proliferation centres, and is physiologically relevant for CLL cells [2]. The combination of soluble CD40 with IL-4 has also been used to culture CLL cells [10] and produce proliferation. Autologous primary T-cells, activated by anti-CD3, are an alternative means of providing signals to drive CLL cell proliferation [9]. In a conceptually entirely different approach, CLL cells have been driven into cell-cycle by CpG-oligonucleotide combined with IL-2 [11]. Previous work has not specifically addressed the issue of how to compare biological and drug responses of viable but quiescent and proliferating CLL cells. We first compared survival and proliferation of CLL cells cultured on mouse fibroblast L-cells (NT-L) and a specialized stromal cell layer (human bone marrow derived mesenchymal stem cells (MSC) gift of Professor Francesco Dazzi, London) [Figure 1(a)]. Both cell types were irradiated before culture with CLL cells. MSC were used because they are important for haemopoietic cells [12,13] and can specifically support the growth of B-cell lymphomas. Blood samples were obtained from CLL patients with a diagnosis confirmed using the NCI Working Group definition. Patients in this cohort were from all clinical stages and had not been treated for at least 12 weeks prior to the study. Patient age ranged from 44 – 85 years (mean, 69 years). Patients with white cell counts 450610 l were selected for this study. CLL cells were isolated from heparinized venous blood by density gradient centrifugation and

Stromal cells and CD40 ligand (CD154) alter the miRNome and induce miRNA clusters including, miR-125b/miR-99a/let-7c and miR-17-92 in chronic lymphocytic leukaemia.

Stromal cells and CD40 ligand (CD154) alter the miRNome and induce miRNA clusters including, miR-125b/miR-99a/let-7c and miR-17-92 in chronic lymphocytic leukaemia

CD40 and B-cell receptor signalling induce MAPK family members that can either induce or repress Bcl-6 expression

Bcl-6 is essential for germinal centre development and normal antibody responses, and has major roles in controlling B-cell proliferation and differentiation. Bcl-6 expression is tightly controlled, but neither the nature of all the regulatory signals nor their interactions are known. Bcl-6 expression is induced in Bcr-Abl expressing lymphoid cell lines by the tyrosine kinase inhibitor, imatinib. We show that p38 MAPK mediates induction of Bcl-6 following inhibition of Bcr-Abl by imatinib. Next we analyze p38 function in a germinal centre B-cell line, Ramos. p38 is phosphorylated under basal conditions, and studies with p38 inhibitors show that it induces Bcl-6 expression. Membrane bound CD40 ligand activates p38 but also other MAPK pathways that strongly repress Bcl-6 and the overall effect is reduction in Bcl-6 expression. Surprisingly soluble CD40 ligand induces Bcl-6 by activating p38 without activating the repressive pathways. Hence different types of CD40 signalling are associated with varying effects on Bcl-6 expression. Transcription reporter assays demonstrate p38 responsive sequences at about 4.5 kb from the transcription start site. Immunocytochemistry of tonsil sections show phosphorylated p38 in a minor population of germinal centre B-cells. We demonstrate for the first time that p38 induces Bcl-6 transcription, but increased protein expression occurs only when the strong pathways repressing Bcl-6 are not activated.

The level of BCR-ABL1 kinase activity before treatment does not identify chronic myeloid leukemia patients who fail to achieve a complete cytogenetic response on imatinib

This study investigated the in vitro inhibition of Crkl phosphorylation by imatinib in CD34+ cells from patients with chronic myeloid leukemia, and showed that it does not correlate with the cytogenetic response, possibly indicating that BCR-ABL1-independent resistance mechanisms exist. Imatinib is currently the first line therapy for newly diagnosed patients with chronic myeloid leukemia. However, 20–25% of patients do not achieve durable complete cytogenetic responses. The mechanism underlying this primary resistance is unknown, but variations in BCR-ABL1 kinase activity may play a role and can be investigated by measuring the autophosphorylation levels of BCR-ABL1 or of a surrogate target such as Crkl. In this study we used flow cytometry to investigate the in vitro inhibition of Crkl phosphorylation by imatinib in CD34+ cells in diagnostic samples from two groups of patients distinguished by their cytogenetic response. No difference in inhibition of Crkl phosphorylation was observed in the two groups. The observation that increasing the dose of imatinib in vivo did not increase the level of cytogenetic response in some non-responders suggests that in at least a proportion of patients imatinib resistance may be due to activation of BCR-ABL1-independent pathway.