Therapeutic implications of activation of the host gene (Dleu2) promoter for miR-15a/16-1 in chronic lymphocytic leukemia.

Genetic lesions and other regulatory events lead to silencing of the 13q14 locus in a majority of chronic lymphocytic leukemia (CLL) patients. This locus encodes a pair of critical proapoptotic microRNAs, miR-15a/16-1. Decreased levels of miR-15a/16-1 are critical for the increased survival exhibited by CLL cells. Similarly, in a de novo murine model of CLL, the NZB strain, germline-encoded regulation of the syntenic region resulted in decreased miR-15a/16-1. In this paper, we have identified additional molecular mechanisms regulating miR-15a/16-1 levels and have shown that the transcription factor BSAP (B-cell-specific activator protein) directly interacts with Dleu2, the host gene containing the miR-15a/16-1 loci, and by negative regulation of the Dleu2 promoter, results in repression of miR-15a/16-1 expression. CLL patient B-cell expression levels of BSAP were increased compared with control sources of B cells. With the use of small interfering RNA-mediated repression, the levels of BSAP were decreased in vitro in the NZB-derived malignant B-1 cell line, LNC, and in ex vivo CLL patient peripheral blood mononuclear cells (PBMCs). BSAP knockdown led to an increase in the expression of miR-15a/16-1 and an increase in apoptosis, and a cell cycle arrest in both the cell line and patient PBMCs. Moreover, using Dleu2 promoter analysis by chromatin immunoprecipitation assay, we have shown that BSAP directly interacts with the Dleu2 promoter. Derepression of the Dleu2 promoter via inhibition of histone deacetylation combined with BSAP knockdown increased miR-15a/16-1 expression, and also increased malignant B-cell death. In summary, therapy targeting enhanced host gene Dleu2 transcription may augment CLL therapy.

Systemic in vivo lentiviral delivery of miR-15a/16 reduces malignancy in the NZB de novo mouse model of chronic lymphocytic leukemia.

Similar to human chronic lymphocytic leukemia (CLL), the de novo New Zealand Black (NZB) mouse model has a genetically determined age-associated increase in malignant B-1 clones and decreased expression of microRNAs miR-15a and miR-16 in B-1 cells. In the present study, lentiviral vectors were employed in vivo to restore miR-15a/16, and both the short-term single injection and long-term multiple injection effects of this delivery were observed in NZB. Control lentivirus without the mir-15a/16 sequence was used for comparison. We found that in vivo lentiviral delivery of mir-15a/16 increased miR-15a/16 expression in cells that were transduced (detected by GFP expression) and in sera when compared with control lentivirus treatment. More importantly, mice treated with the miR-expressing lentivirus had decreased disease. The lentivirus had little systemic toxicity while preferentially targeting B-1 cells. Short-term effects on B-1 cells were direct effects, and only malignant B-1 cells transduced with miR-15a/16 lentivirus had decreased viability. In contrast, long-term studies suggested both direct and indirect effects resulting from miR-15a/16 lentivirus treatment. A decrease in B-1 cells was found in both the transduced and non-transduced populations. Our data support the potential use of systemic lentiviral delivery of miR-15a/16 to ameliorate disease manifestations of CLL.

Studies in NZB IL-10 knockout mice of the requirement of IL-10 for progression of B-cell lymphoma.

NZB mice develop an age-related malignant expansion of a subset of B cells, B-1 cells, with autocrine production of IL-10. IL-10, a pleiotropic cytokine with anti-inflammatory properties, is a potent growth and survival factor for malignant B cells. To further examine the in vivo requirement for IL-10 in the development and expansion of malignant B-1 clones in NZB mice, we developed a strain of homozygous IL-10 knockout (KO) mice on an NZB background. The NZB IL-10 KO mice develop peritoneal B-1 cells with approximately the same frequency as heterozygous and wild-type littermates. In contrast, the development of malignant B-1 cells in the peripheral blood and spleen, observed in wild-type NZB, rarely occurred in the NZB IL-10 KO. Phenotypic analysis of surface marker expression in splenic B cells indicated that, in contrast to the NZB with malignant B-1 splenic lymphoma, the surface marker expression of NZB IL-10 KO splenic B cells indicated that the majority of the B cells were typical B-2 cells. In the absence of IL-10, spontaneously activated B cells and antiapoptotic gene expression were reduced and lymphoma incidence was decreased. These results indicate that IL-10 is a critical factor for the progression of this B-cell malignant disease.

Cell cycle effects of IL-10 on malignant B-1 cells.

IL-10 is overexpressed in human chronic lymphocytic leukemia (CLL), and is an autocrine growth factor involved in the development of malignant B1 clones in NZB mice, a murine model for CLL. Antisense IL-10 oligonucleotide treatment induces apoptosis and cell cycle disruption in these cells both in vitro and in vivo. In addition, NZB IL-10 knock-out mice fail to develop the B-1 clones. Dampening of IL-10 protein production via antisense IL-10 oligonucleotide treatment is correlated with decreased p27/Kip1 protein expression which results in increased cyclin D2, cyclin E and cyclin A associated kinase activity. The action of the antisense oligonucleotides is through alterations in cell cycle regulation, resulting in accelerated cell cycle progression, a G2/M block which culminates in apoptosis induction in the malignant cells. This implies that the role of IL-10 as an autocrine growth factor in malignant B-1 cells lies in its ability to inhibit apoptosis induction through the maintenance of sustainable cell cycle progression in malignant cells.

The growth-regulatory role of B-cell-specific activator protein in NZB malignant B-1 cells.

The transcription factor B-cell-specific activator protein (BSAP) plays an important role in B-cell development. We explored the involvement of BSAP in the growth regulation of malignant B-1 cells derived from the NZB murine model of human chronic lymphocytic leukemia. BSAP protein was found in normal B-2 cells, elevated in normal B-1 cells, and highest in NZB malignant B-1 cells. When these malignant B-1 cells were treated with antisense oligonucleotides for BSAP, their growth was inhibited with a G2/M phase arrest. In contrast, B cell lines that did not appear to be of B-1 origin (IgG+/B220+/BSAPlow) were unaffected by treatment with antisense BSAP oligonucleotides. Centrifugal elutriation experiments showed that BSAP mRNA was expressed at the highest levels in the G2/M phases in malignant B-1 cells. Treatment with demecolcine (Colcemid), a known mitotic blocker, resulted in a decrease in the level of BSAP gene expression in malignant B-1 cells, further demonstrating links between BSAP expression and successful G2/M transition in the cell cycle. These data suggest a correlation between BSAP and the development of B-1 malignancy, perhaps through the regulation of cell-cycle progression.

The role of B-cell-specific activator protein in the response of malignant B-1 cells to LPS.

Chronic lymphocytic leukemia (CLL) results from the uncontrolled proliferation and accumulation of B-1 cells, many of which demonstrate self-reactivity. The response of B-1 cells to mitogen after undergoing malignant transformation is still unclear. Using our established malignant B-1 cell lines derived from the NZB murine model of human CLL, we investigated the response of malignant B-1 cells to the mitogen LPS. Interestingly, these malignant B-1 cells proliferated initially, but the proliferation rate decreased after a 48-h transition. Prolonged LPS treatment induced apoptosis and pathological differentiation. We studied possible underlying molecular mechanisms and found that the level of the DNA binding protein BSAP (B-cell-specific activator protein) was upregulated by LPS at the initial activation stage, followed by an increase in the apoptotic factor caspase-3 (CPP32) at 48 h and a subsequent decrease of BSAP at 72 h. The pathological differentiation induced by LPS was partially prevented by treatment with antisense BSAP. This study indicates that malignant B-1 cells could be driven to apoptosis and pathological differentiation when activated by the mitogen LPS, and BSAP may be an important factor in regulating these responses.

Antisense IL-10 abrogates the inhibitory effects of IL-10 production by transfected tumor cells.

Interleukin-10 (IL-10) is a pleiotropic cytokine that has a variety of downregulatory effects on immunologic and inflammatory processes. Ectopic tumor expression of IL-10 inhibited tumor growth, and local administration of antisense IL-10 significantly reversed the effects of IL-10 transfection in P815 mastocytoma. Tissue inhibitors of metalloproteinase (TIMPs) have been associated with decreased tumorigenesis and reduced metastasis, and TIMPs were increased in the region surrounding P815/IL-10 tumors and reduced in antisense IL-10-treated mice. In addition, the antisense IL-10 group had the largest tumor volume and poorest survival when compared with the P815/IL-10 control or sense groups. In summary, our data suggest that, in a mouse model, antisense IL-10 has substantive effects in reducing IL-10 translation and inhibiting IL-10-mediated TIMP upregulation, and, by doing so, allows IL-10-transfected mastocytoma to grow unchecked. Thus, ectopic tumor expression of IL-10 inhibits tumor growth, and antisense IL-10 administration in vivo reverses this protective effect.

Effects of cord blood transfer on the hematopoietic recovery following sublethal irradiation in MRL lpr/lpr mice.

The potential of cord blood (CB) to serve as a rich source of stem cells and stem cell factors is receiving increasing attention. In addition, perhaps because of the early ontogeny of these cells or the lack of surface antigens, cord blood stem cells do not appear to require close identity with the recipient. In the present pilot study, we investigated the presence of a hematopoiesis enhancing effect (HEE) by assaying the ability of human cord blood cells to augment hematopoiesis across a species barrier. For these experiments, autoimmune-prone MRL-Ipr/Ipr mice were exposed to sublethal levels of irradiation and cord blood administration to study the role of factors present in human cord blood in augmenting the rate of lymphopoiesis. This strain was chosen because of the increased presence of peripheral T and B subpopulations, namely the B-1 and CD4/CD8 double negative T-cell subpopulations, which do not arise directly from bone marrow precursors, but rather accumulate with age. MRL-Ipr/Ipr mice were sublethally irradiated and reconstituted with syngeneic bone marrow (BM) cells or with human cord blood cells or peripheral blood mononuclear cells (PBMC), or were left unreconstituted. At 2 weeks post-treatment, lymphoid populations in the spleen and lymph nodes were studied as a measure of hematopoiesis. Factors present in cord blood were able to augment hematopoiesis over that which occurred endogenously. At 2 weeks postirradiation, recipients of BM cells displayed the fastest rate of peripheral lymphoid recovery, nonreconstituted mice showed the slowest lymphoid recovery, and recipients of cord blood recovered their lymphoid populations at an intermediate rate. Similarly, myelopoiesis was increased in irradiated SJL/J recipients of human cord blood. Thus, human cord blood cells appear to produce/induce factors that may act as an adjunct to increase stem-cell activity.

Regulatory role of T cells in a murine model of lymphoproliferative disease.

As NZB mice age, approximately 90% of the 12-month-old mice possess an expansion of malignant B-1 (CD5+ B cells) cells with many similarities to the human lymphoproliferative disease, chronic lymphocytic leukemia. Malignant B-1 cells derived from NZB mice produce significantly higher levels of IL-10 mRNA and protein than normal B-1 or B cells. IL-10 may act as an autocrine growth factor for the expansion of B-1 cells. In this report, the infrequent animals which survived 18 months of age or longer were studied and compared to NZB mice at 12-14 months of age. Analysis of lymphoid subpopulations in the spleen and peritoneal cavity indicated that long-lived NZB mice had an expansion of CD8+ T cells rather than the typical B-1 expansion observed in the majority of NZB animals at 12 months of age. We established a CD8+ T cell clone from long-lived NZB mice which was cytotoxic for malignant B-1 cells of NZB origin both in vivo and in vitro. Analysis of the regulatory mechanisms preventing the development of genetically programmed age-dependent CLL in the murine system may elucidate possible avenues for therapeutic intervention in CLL.

The correlation of telomerase and IL-10 with leukemia transformation in a mouse model of chronic lymphocytic leukemia (CLL).

Telomerase activity is upregulated in activated and malignant lymphocytes. We studied the correlation of telomerase and IL-10 to leukemia transformation in the NZB mouse model of human chronic lymphocytic leukemia (CLL). Telomerase levels increased from early to late leukemic stages, likewise IL-10 gene expression levels increased with the leukemic progression. The inverse relationship of telomerase and IL-10 levels to the survival of NZB mice was also established. Our data suggested that telomerase and IL-10 were involved in transformation in the murine model of CLL and the detection of telomerase activities might be of value in the prediction of CLL progression.

Apoptosis induction in fludarabine resistant malignant B-1 cells by G2-M cell cycle arrest.

A malignant B-1 cell line from a mouse model of CLL was resistant to fludarabine, but could be induced to undergo apoptosis by mitotic spindle inhibitors, colcemid or nocodazole, which blocked these cells in the G2-M phases of cell cycle prior to apoptosis induction. Bax mRNA levels increased with relative constitutive expression of bcl-2 mRNA levels. The mRNA levels of B-cell-specific-activator-protein (BSAP) decreased with colcemid treatment. This study shows that mitotic spindle inhibitors can induce apoptosis in fludarabine-resistant malignant B-1 cells by altering levels of bax/ bcl-2 ratio and BSAP which play different roles in cell cycle regulation and apoptosis induction.

Basic fibroblast growth factor (bFGF) upregulates the expression of bcl-2 in B cell chronic lymphocytic leukemia cell lines resulting in delaying apoptosis.

Basic fibroblast growth factor (bFGF) is a pleiotropic cytokine which has recently been shown to delay fludarabine-induced apoptosis in B cell chronic lymphocytic leukemia (B-CLL) cells. To investigate the potential mechanism of bFGF-mediated delay of apoptosis, two EBV-transformed B prolymphocytic cell lines (JVM-2, JVM-13), one EBV-transformed B-CLL cell line (I83CLL), and one non-EBV-transformed B-CLL cell line (WSU-CLL) were used as a model for chronic lymphoid malignancies. Viability data of cells treated with fludarabine alone or in combination with bFGF demonstrated that the addition of bFGF to the cells resulted in prolonged survival. Quantitative assessment of apoptosis-associated DNA strand breaks by in situ TdT labeling showed a protective effect of bFGF on fludarabine-treated cells. The potential effect of bFGF on bcl-2 mRNA expression was analyzed by Northern blotting. Stimulation with bFGF led to a time-dependent accumulation of bcl-2 specific mRNA in all three cell lines. Maximal levels of bcl-2 mRNA expression were detected after 8 h in JVM-2, and after 18 h in JVM-13 and I83CLL. Intracellular bcl-2 protein was also found to be increased upon bFGF stimulation in both EBV- and non-EBV-transformed cells. In addition, exposure of cells from three patients with B-CLL to bFGF showed an upregulation of bcl-2 protein after 4-8 h. Our data demonstrate that bFGF upregulates the expression of bcl-2 in these cells, suggesting that this increase in bcl-2 expression may play a role in the delay of fludarabine-induced apoptosis.

Requirement for increased IL-10 in the development of B-1 lymphoproliferative disease in a murine model of CLL.

Malignant B-1 cells derived from NZB mice, a murine model of spontaneous autoimmunity and B cell lymphoproliferative disease, produce significantly higher levels of IL-10 mRNA than normal B-1 or B cells. IL-10 may act as an autocrine growth factor for the expansion of malignant B-1 cells. In order to determine if elevated endogenous production of IL-10 was a required element for the malignant transformation of B-1 cells in NZB mice, backcross animals were studied for the linkage between elevated IL-10 expression and the presence of lymphoid malignancy. The phenotypes of aged (NZB x DBA/2)F1 x NZB animals were determined and a strong correlation was found between the elevated levels of IL-10 mRNA and the development of B-1 malignant clones. In contrast, an increased level of IL-10 message was not associated with elevated serum IgM or the presence of anemia or reticulocytosis which is mainly seen in response to autoantibody production. These results indicate that, at least in NZB, the autoimmunity and lymphoproliferation phenotypes are not linked genetically. IL-10 may enhance proliferation and the development of B-1 cell malignancy rather than antibody production by the B-1 cell subpopulation. Thus, IL-10 plays an important role in B-1 malignancies, and downregulation of IL-10 could be a likely site for intervention in B cell malignancies.

Altered CD45 expression in malignant B-1 cells.

CD45 is an important surface glycoprotein which has an intrinsic tyrosine phosphatase activity and has been implicated in cell proliferation, signaling, and differentiation and is associated with the B cell receptor during signaling. In this manuscript, the role of CD45 expression in the development of B-1 malignancies in NZB mice, which serve as a model for human diseases such as chronic lymphocytic leukemia, was investigated. B-1 cells spontaneously hyperproliferate and form a clonal hyperdiploid malignant population in aging NZB mice. Phenotypic analysis indicates that the NZB malignant B-1 cells are bright for IgM, but have reduced levels of CD45 relative to normal, nonmalignant B cells (both B-1 and B-2) and are characterized by dull or negative expression of the CD45 isoform B220/6B2 normally found on all B cells. Malignant B-1 cells demonstrated decreased RNA levels of CD45 relative to IgM expression, while nonmalignant B-2 cells showed similar levels of RNA expression for both CD45 and IgM. As CD45 exists in several isoforms and B cells express the highest molecular weight isoform (B220), malignant B-1 cells were further analyzed with respect to their isoform usage. Although, at the RNA level malignant B-1 cells showed the presence of the of the B220 form of CD45, western blot analysis of B220 protein suggested a posttranslational glycosylation defect in the CD45/B220 expression recognized by the mAb 6B2. F1 recipients of premalignant NZB B-1 cells which had been sorted for IgMhi, B220/6B2negative cells developed hyperdiploid malignant donor B-1 clones earlier than did recipients of NZB B-1 cells which were bright for B220/6B2. However, all the malignant B-1 clones of NZB origin which developed in recipients of both transfer populations were B220/6B2 negative. This indicated that abnormal expression of CD45 may be prerequisite for long-term growth and malignant transformation. Thus alterations in CD45 may result in abnormal functioning of the malignant B-1 cells which may further affect the proliferation of, or signaling within, these cells.

Altered calcium signal transduction in B-1 malignant cells.

Lymphocyte proliferation is guided by receptor-mediated signal transduction pathways that dictate the immunological response/clonality of that cell. We have previously reported that NZB-derived malignant B-1 cells, which serve as a murine model for human chronic lymphocytic leukaemia, demonstrate altered expression of surface IgM and CD45 signalling molecules, and a failure to proliferate following membrane IgM stimulation. To examine receptor-mediated cytosolic calcium (Cai) signalling in B cell leukaemia, we studied IgM-induced Cai responses in malignant B-1 cells and B cells from non-leukaemic mice. Basal Cai was slightly lower in malignant B-1 cells than in non-leukaemic cells. Anti-IgM stimulation induced a sustained increase in Cai to levels 1.3-fold greater than basal Cai in conventional B cells. In contrast, leukaemic B-1 cells demonstrated a sharp but transient rise in Cai followed by a gradual increase to levels 2.3-fold greater than basal [Ca]i Ca influx from extracellular sources contributed to the early and late Cai signal in both sets of cells. Pre-incubation (2-30 min) with anti-CD45 had no effect on basal Cai or the anti-IgM Cai signal in B cells, but reduced the Cai transient in malignant B-1 cells. Additional experiments characterized the effects of phosphorylation/dephosphorylation events on the Cai profile following anti-IgM stimulation. Protein tyrosine kinase inhibitors decreased the anti-IgM-induced Cai transient in malignant B-1 cells by 80%, but only moderately affected (40%) of the Cai response in non-leukaemic B cells. Protein tyrosine phosphatase inhibitors and protein kinase C (PKC) activators attenuated the Cai response to the same degree in normal and leukaemic B cells. These results show that Cai signalling differs widely between non-malignant B cells and malignant B-1 cells, and that tyrosine phosphorylation and CD45 modulation of IgM signalling are involved in the altered Cai responses in malignant B-1 cells.

Effect of human cord blood transfer on survival and disease activity in MRL-lpr/lpr mice.

The MRL-lpr/lpr mice have a genetic defect and by 6 months of age usually die after developing severe autoimmune disease and massively enlarged lymph nodes (Cohen, P. L., and Eisenberg, R. A., Annu. Rev. Immunol. 9, 243-269, 1991). Similarly as in some genetic diseases in humans, these mice, if given an appropriate marrow transplant from a mouse not having the defect, will have partial correction of the disease process and an extension of life (Cohen, P. L., and Eisenberg, R. A., Annu. Rev. Immunol. 9, 243-269, 1991; Lenarsky, C., Kohn, D. B., Weinberg, K. I., and Parkman, R., Hematol. Oncol. Clin. N. Am. 4, 589-603, 1990). Utilizing human umbilical cord blood as a donor source for marrow transplantation, we have been able to obtain significant correction of the MRL-lpr/lpr genetic defect and double the life span. By 11 months of age, 5 months beyond their usual life span, both the animals receiving congenic marrow (MRL-+/+) and the animals with human cord blood had mild lymphadenopathy, a decrease in double-positive T cells, and an increase in double-negative T cells. Granulomatous vasculitis could be identified in the animals receiving human cells and could not be found in the animals receiving MRL-+/+ marrow.

Growth inhibition of malignant CD5+B (B-1) cells by antisense IL-10 oligonucleotide.

Malignant B-1 cells derived from NZB mice, a murine model of chronic lymphocytic leukemia, produce significantly higher levels of IL-10 mRNA than normal B-1 or B cells. IL-10 may act as an autocrine growth factor for malignant B-1 cells. By addition of antisense oligodeoxynucleotides specific for IL-10 mRNA, we were able to dramatically inhibit the growth of leukemic B-1 cells in a time and dose dependent manner. Control cell lines which do not depend on IL-10 for growth were not affected. Antisense therapy targeted at the 5' region of the IL-10 mRNA not only resulted in inhibition of malignant B-1 cell proliferation but also inhibited IL-10 production by malignant B-1 cells. Because endogenous IL-10 gene activation is critical for B-1 cell expansion, inactivation of the endogenous IL-10 gene by IL-10 antisense rather than extracellular regulation of the IL-10 gene product should be successful in controlling the malignant growth.

The natural history of a lymphoproliferative disorder in aged NZB mice.

The molecular lesions of human familial and common B-CLL remain unknown. As an approach to this problem, aged NZB mice with a B cell lymphoproliferative disorder were chosen as a murine model. Three groups of NZB mice (2 months, 6 months and > 18 months) for a total of nineteen were studied. A complete autopsy including a CBC was performed on each mouse. Spleen cells were immunophenotyped and cell cycle analysis was performed. Spleen weight, peritoneal cell counts and absolute lymphocytes counts were all elevated in the oldest group. All mice showed evidence of extramedulary hematopoiesis and the older group showed lymphocytic infiltrates in the lacrymal glands, kidneys, liver and lungs. Two of the seven aged mice had a malignant lymphoma. One was a marginal zone lymphoma and the other a lymphocytic lymphoma. Splenic immunophenotyping showed a loss of T cells with an increase in B cells as the mice age. Cell cycle analysis revealed hyperdiploidy in all of the aged mice with a decrease in the percentage G0G1 cells. This disease appears to involve an absolute lymphocytosis of the peritoneum and the peripheral blood compartment. This is associated with splenic aneuploidy. The infiltration of the spleen by malignant cells of varying morphology is a late event. The aged NZB mouse continues to be a model for human B-CLL.