Extracellular vesicles released by CD40/IL-4-stimulated CLL cells confer altered functional properties to CD4+ T cells.

The complex interplay between cancer cells, stromal cells, and immune cells in the tumor microenvironment (TME) regulates tumorigenesis and provides emerging targets for immunotherapies. Crosstalk between CD4(+) T cells and proliferating chronic lymphocytic leukemia (CLL) tumor B cells occurs within lymphoid tissue pseudofollicles, and investigating these interactions is essential to understand both disease pathogenesis and the effects of immunotherapy. Tumor-derived extracellular vesicle (EV) shedding is emerging as an important mode of intercellular communication in the TME. In order to characterize tumor EVs released in response to T-cell-derived TME signals, we performed microRNA (miRNA [miR]) profiling of EVs released from CLL cells stimulated with CD40 and interleukin-4 (IL-4). Our results reveal an enrichment of specific cellular miRNAs including miR-363 within EVs derived from CD40/IL-4-stimulated CLL cells compared with parental cell miRNA content and control EVs from unstimulated CLL cells. We demonstrate that autologous patient CD4(+) T cells internalize CLL-EVs containing miR-363 that targets the immunomodulatory molecule CD69. We further reveal that autologous CD4(+) T cells that are exposed to EVs from CD40/IL-4-stimulated CLL cells exhibit enhanced migration, immunological synapse signaling, and interactions with tumor cells. Knockdown of miR-363 in CLL cells prior to CD40/IL-4 stimulation prevented the ability of CLL-EVs to induce increased synapse signaling and confer altered functional properties to CD4(+) T cells. Taken together, these data reveal a novel role for CLL-EVs in modifying T-cell function that highlights unanticipated complexity of intercellular communication that may have implications for bidirectional CD4(+) T-cell:tumor interactions within the TME.

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.

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.

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

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.

Apoptosis induces Bcl-XS and cleaved Bcl-XL in chronic lymphocytic leukaemia.

The Bcl-X gene has both pro-survival, Bcl-XL, and pro-apoptotic, Bcl-XS, gene products, which are produced by alternative splicing. The function of these proteins has previously been characterised in cell lines, often by transfecting expression constructs, and primary cell systems capable of dynamically regulating Bcl-XL and Bcl-XS have not been described. Such a system is potentially important to allow testing of agents that promote apoptosis by increasing the amount of Bcl-XS at the expense of Bcl-XL. In this report we characterise Bcl-X gene products in primary human leukaemic B-cells in culture conditions associated with survival and apoptosis. We found that Bcl-XS was induced in spontaneous and drug-induced apoptosis and that apoptosis induced in cells cultured on mouse fibroblasts expressing CD40 ligand with IL-4 (CD154/IL-4), a condition mimicking the tissue microenvironment, additionally produced expression of cleavage products of Bcl-XL. Both Bcl-XS and Bcl-XL were produced in a caspase dependent manner. We tested emetine, an agent previously reported to increase Bcl-XS but found that it did not have this effect in primary human B-cells. Therefore, there are two mechanisms-cleavage of Bcl-XL and production of Bcl-XS-by which Bcl-X gene products could enhance apoptosis in CLL but neither appeared to have a primary role in inducing leukaemic cell death.

Post-transcriptional and post-translational regulation of Bcl2.

Bcl2 is an important pro-survival protein that has an essential function in normal immunity and whose constitutive expression leads to the development of lymphomas. Although transcriptional control of Bcl2 has been reported, increasing evidence suggests an important component of Bcl2 regulation is post-transcriptional. Phosphorylation of Bcl2 has been shown to enhance activity to allow response to extracellular growth-factor-mediated signals. Bcl2 mRNA contains regulatory elements in both its 5'- and 3'-UTRs (untranslated regions). An IRES (internal ribosome entry sequence) in the 5'-UTR permits continued translation in the presence of cellular stresses that reduce cap-dependent translation. The 3'-UTR of Bcl2 mRNA is 5.2 kb in length and contains multiple predicted miRNA (microRNA) and RNA-BP (RNA-binding protein)-binding sites. miR-15a and miR-16-1 have been found to inhibit Bcl2 expression in B-cells, whereas the RNA-BP nucleolin has been shown to increase Bcl2 expression by binding to the 3'-UTR and enhancing mRNA stability. Both decreased expression of miR-15a and miR-16-1 and increased nucleolin have been shown to be associated with increased Bcl2 expression and resistance to apoptosis in the common human disease, chronic lymphocytic leukaemia. miRNA-based therapeutic approaches to treat cancer are emerging. Bcl2 is highly regulated by miRNAs and is therefore an excellent candidate for such approaches.

An in vitro based investigation of the cytotoxic effect of water extracts of the Chinese herbal remedy LD on cancer cells.

BACKGROUND: Long Dan Xie Gan Wan (LD), a Chinese herbal remedy formulation, is traditionally used to treat a range of conditions, including gall bladder diseases, hepatitis, hyperthyroidism, migraines but it is not used for the management or treatment of cancer. However some of its herbal constituents, specifically Radix bupleuri, Radix scutellariae and Rhizoma alismatis have been shown to inhibit the growth of cancer cells. Thus, the aim of the study was to investigate the impact of LD on cancer cells in vitro. METHODS: HL60 and HT29 cancer cell lines were exposed to water extracts of LD (1:10, 1:50, 1:100 and/or 1:1000 prepared from a 3 mg/30 ml stock) and for both cell lines growth, apoptotic induction, alterations in cell cycle characteristics and genotoxicity were investigated. The specificity of the action of LD on these cancer cell lines was also investigated by determining its effect on human peripheral blood lymphocytes. Preliminary chemical analysis was carried out to identify cytotoxic constituents of LD using HPLC and LCMS. RESULTS: LD was significantly cytotoxic to, and induced apoptosis in, both cell lines. Apoptotic induction appeared to be cell cycle independent at all concentrations of LD used (1:10, 1:50 and 1:100) for the HL60 cell lines and at 1:10 for the HT29 cell line. At 1:50 and 1:100 apoptotic induction by LD appeared to be cell cycle dependent. LD caused significant genotoxic damage to both cell lines compared to their respective controls. The specificity study showed that LD exerted a moderate cytotoxic action against non-proliferating and proliferating blood lymphocytes but not apoptosis. Chemical analysis showed that a number of fractions were found to exert a significant growth inhibitory effect. However, the molecular weights of compounds within these fractions did not correspond to those from the herbal constituents of LD. CONCLUSION: It is possible that LD may have some chemotherapeutic potential. However, further studies are required to determine its cytotoxic constituents.

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.

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.

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.

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.

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.

Apoptotic effect of Oldenlandia diffusa on the leukaemic cell line HL60 and human lymphocytes.

Oldenlandia diffusa is traditionally prescribed in the treatment of a number of cancers and studies suggest that it exerts a cytotoxic action specific to cancer cells. To further investigate this suggested action, the effect(s) of Oldenlandia diffusa on leukaemic cells (HL60) and stimulated and unstimulated human blood lymphocytes (PBLs) was investigated. For the HL60s, cell growth, apoptotic induction, alterations in cell cycle characteristics and genotoxicity were investigated. For the PBLs, apoptotic induction and alterations in cell cycle characteristics were investigated. Preliminary chemical analysis to identify the cytotoxic constituents of Oldenlandia diffusa was also carried out. Results showed that Oldenlandia diffusa significantly inhibited the growth of the HL60s and induced apoptosis in a cell cycle-independent fashion, possibly through the induction of genotoxic damage. Oldenlandia diffusa did not induce apoptosis in the PBLs however progression through the cell cycle was not evident (in stimulated PBLs) suggesting some degree of cytotoxicity. Preliminary chemical analysis indicated that a number of compounds appear to be responsible for the cytotoxic action of Oldenlandia diffusa. These results indicate that HL60s are more sensitive to Oldenlandia diffusa than stimulated PBLs and thus support a cytotoxic action for Oldenlandia diffusa that has some degree of specificity.

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-x(L) (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-x(S), 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.