TCL1 Transgenic Mice Research Articles

Effect of Antioxidant N-Acetylcysteine Treatment on the Phenotype of the TCL-1 Transgenic Mouse Model of B-CLL through Reactive Oxygen Species (ROS) Modulation

AbstractAbstract 3581Thioredoxin-interacting protein (TXNIP) binds and inactivates thioredoxin (TRX) leading to increased reactive oxygen species (ROS) production. It has been previously shown that patients with CLL have high levels of ROS compared to normal individuals and that TRX has a protective function in CLL. In an initial pilot study of 17 patients (Koshy et al., AACR Annual Meeting, 2010) we found that TXNIP levels (avg. 0.82 ± 0.18, range 0.45–1.15) and ROS (avg. 7967 ± 2515, range 2340–12340) were positively correlated (ρ = 0.92). Although not statistically significant, TXNIP expression was higher in patients under observation than those receiving treatment (0.89 ± 0.18 vs. 0.76 ± 0.15).For further insights into the role of TXNIP in CLL, we chose the TCL-1 transgenic mouse model of B-CLL. These animals develop a CD5+ B-cell leukemia and express therapeutic targets and display sensitivity to therapeutic agents relevant in the treatment of CLL. Mice develop a clonal leukemia characterized by splenomegaly and increased lymphocyte counts in peripheral blood at a median age of 10 months.To first determine if TXNIP was altered in the progression of CLL in the TCL-1 mice, we isolated peripheral blood (PB) and bone marrow (BM) from mice of varying ages; 3 months (disease free), 7 months (pre-leukemic phase) and 11 months (end-stage disease). As shown previously, end-stage animals developed severe splenomegaly (1.32±0.82g vs. 0.16±0.08; p<0.05) compared to disease-free. These animals also demonstrated a 1.4 fold increase in TXNIP RNA in BM (0.62±0.08 vs. 0.87±0.12, p<0.05) and 1.5 fold in PB cells (0.81±0.06 vs. 1.18±0.20; p<0.05) associated with a 3.2 fold increase in ROS in BM (15212±1522 vs. 46812±11563, p=0.07)and 3.9 fold increase in PB (11150±560 vs. 39230±8671; p=0.7) compared to animals with no evidence of disease.Although ROS levels are regulated by several cellular redox mechanisms, we have shown in a breast cancer model, that TXNIP upregulation play a major role in ROS control through its interaction with TRX. We chose to alter the ROS levels by antioxidant therapy on animals with end-stage disease. The reason for this choice was to assess a faster way to interfere with the final product of TXNIP upregulation, rather than the protein itself. We wanted to prove that by reducing ROS we would have changed the course of the disease. For this purpose, we administered the antioxidant, N-acetylcysteine (NAC), in the drinking water and measured the changes in both ROS and TXNIP. Mice of approximately 10 months of age were given fresh NAC water every other day. After a treatment period of 4 weeks animals were sacrificed. Peripheral blood and splenocytes were stained for ROS and B220 expression. Bone marrow cells were stained for ROS, B220 and bone marrow lineage markers and evaluated by flow cytometry.Upon gross examination of animals, spleen weight was found to be reduced 2-fold in NAC treated animals compared to that of untreated (0.69±0.40 g vs. 1.3±0.77, p<0.05) although still higher than that expected of a healthy animal of the same age (approximately 0.25g). We found that ROS was reduced in the spleen 1.5 fold (59±29 vs. 81±41; p<0.05). B220 expression was increased 1.8 fold (1155±611 vs. 836±416; p<0.05) in spleen cells. In peripheral blood, we found that NAC reduced ROS levels 1.7 -fold (51±19 vs. 29±14; p<0.05) and B220 was decreased by 1.5-fold (857±213 vs. 575±105). Total bone marrow ROS levels were decreased 2- fold by NAC treatment (79±31 vs. 38±13; p<0.01). In the bone marrow, low levels of ROS expression have been associated with primitive hematopoietic stem cells. Indeed, in our studies we found the bone marrow could be separated into a ROShi and ROS lo population and that NAC treatment increased the percentage of ROSlo cells. The ratio of ROShi/ROSlo was decreased by 1.5 fold (1.7±0.4 vs. 1.1±0.2; p<0.05) This ROSlo population encompassed the Lin−, c-kit+, Sca-1+ cells. However while NAC treatment induced changes in ROS levels, as expected, TXNIP RNA levels were not found to differ between NAC-treated and untreated animals in any of the cell types tested.In conclusion, both TXNIP and ROS levels were increased with progression of the disease. Antioxidant treatment of animals with end-stage disease resulted in decreased ROS in all cell types tested, associated with phenotype changes of the disease. This finding sets the ground for further work in identifying therapeutic tools that interfere with TXNIP-ROS axis. Disclosures:Turturro:Celgene: Speakers Bureau; Genentech: Speakers Bureau; Spectrum Pharmaceuticals: Speakers Bureau.

Abstract 401: CD5+CD23+ leukemic cell populations in Tcl1 transgenic mice show significantly increased proliferation and Akt phosphorylation

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is the most common human leukemia. Deregulation of the T-cell leukemia/lymphoma 1 (TCL1) oncogene in mouse B-cells causes a CD5 positive leukemia similar to aggressive human B-CLLs. We recently reported that TCL1 expression in B-CLL is regulated by miR29 and miR181. To determine whether treatment with microRNAs can inhibit B-CLL in mice by targeting TCL1 we generated TCL1 transgenic mice using a construct containing 3’ and 5’ UTRs of TCL1 under B-cell specific Eμ promoter (Eμ-TCL1fl). At the age of 12-18 months these mice showed B-CLL like disease. Immunophenotyping revealed accumulation of CD5+CD23+B220+ population in spleens and lymph nodes. Our results show that CD5+CD23+ B-cell populations from Eμ-TCL1fl mice actively proliferate and display significantly increased levels of phospho-Akt. Eμ-TCL1fl mice showed immunological abnormalities similar to human B-CLL, including hypoimmunoglobulinemia, abnormal levels of cytokines, and impaired immune response. These findings revealed biochemical and immunological similarities between Tcl1 driven B-CLL in mice and human B-CLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 401.

The PIM1 Oncogene Accelerates TCL1 Driven Lymphomagenesis in a Double-Transgenic Murine Model.

Abstract 2968Poster Board II-944Previously, we identified and validated PIM1 as a differentially expressed gene in mantle cell lymphoma (MCL) patient samples. Further, we have shown PIM1 to be a significant prognostic biomarker in MCL. PIM1 is a serine/threonine kinase that is transcriptionally regulated by cytokines, mitogens, and numerous growth factors. PIM1 cooperates with other oncogenes in tumorigenesis and has been implicated in the development of leukemias, lymphomas, late progression events, and most recently in prostate cancer. PIM1 is overexpressed in aggressive lymphomas, such as the blastoid variant of MCL, and the ABC-subtype of diffuse large B-cell lymphomas (DLBCL). Here, we tested the in vivo cooperation of PIM1 with TCL1 in murine lymphomagenesis by producing double transgenic murine strains. PIM1 transgenic mice overexpress murine PIM1 under the control of the immunoglobulin enhancer Eμ. TCL1 transgenic mice (pEμ-B29-TCL1) fail to down-regulate TCL1 expression in mature B and T cells and provide a unique model for mature B-cell malignancies. We hypothesized that PIM1 would either accelerate TCL1-driven lymphomagenesis, result in the development of immature lymphomas, or both. Lymphoid malignancies were examined by immunohistochemistry and classified by a hematopathologist according to “Mouse Models of Human Blood Cancers” (Li et al., 2008). A Kaplan-Meier plot demonstrated statistically significant acceleration of lymphomagenesis in the PIM1/TCL1 transgenic mice when compared with the single transgenic strains. The median lymphoma-free survival for TCL1 single transgenic mice, PIM1 single transgenic mice, or PIM1/TCL double transgenic mice were 10.0 months, 16.0 months, and 8.5 months, respectively. The results were statistically significant: TCL1 vs. PIM1/TCL1 (p=0.0008), PIM1 vs. PIM1/TCL1 (p<0.0001). PIM1 transgenic mice developed rare [1 of 31 (3.2%)] and early (< 7 months of age) T-cell lymphoblastic lymphomas and more commonly developed late (>12 months of age) B-cell lymphomas. The most common lymphomas in PIM1 single transgenic were low-grade B-cell lymphomas [12 of 31 (38.7%)], mainly follicular lymphomas. A minority of PIM1 single transgenic mice developed aggressive lymphomas [6 of 31 (19.4%)], including DLBCL and Burkitt's lymphoma. In contrast, the majority of TCL1 transgenic mice developed aggressive B-cell lymphomas [21 of 36 (58.3%)], mainly DLBCL, lymphohistiocytic subtype. The majority of PIM1/TCL1 double transgenic mice also developed aggressive B-cell lymphomas [20 of 34 (58.8%)], mainly DLBCL, lymphohistiocytic subtype. The low-grade lymphomas that developed in PIM1/TCL1 mice included 5 cases of lymphoplasmacytic lymphoma (LPL); one of these cases had transformed in addition to a DLBCL. Further, endogenous expression of PIM kinase family members was investigated in a human lymphoma cell line bank (n=40) by quantitative real-time PCR. PIM1, PIM2, and PIM3 were found to be overexpressed in cell lines derived from human lymphoid malignancies of multiple histologies. In summary, aberrant PIM1 overexpression in TCL1 transgenic mice accelerated the development of mature, aggressive B-cell lymphomas. The classification of lymphomas in PIM1/TCL1 mice revealed similar histologies as in TCL1 single transgenic mice, mainly DLBCL. Single transgenic PIM1 mice developed low-grade B-cell lymphomas after prolonged observation time. The expression of all 3 PIM kinase family members in human lymphomas implies that pan-PIM kinase inhibitors should be developed as a potential mechanism of drug resistance to more restricted PIM inhibitors could be compensatory overexpression of the non-targeted Pim family members. A clinical trial with a pan-PIM inhibitor is currently ongoing. Disclosures:No relevant conflicts of interest to declare.

The Syk Inhibitor R788 (FosD) Inhibits Tumor Growth in the TCL1 Transgenic Mouse Model of CLL by Blocking Antigen-Dependent BCR Signaling.

Abstract 887CLL B-cells depend on various signals from the microenvironment for survival and proliferation. Among these, antigenic stimuli that are propagated through the B-cell receptor (BCR) are considered particularly important for the development and progression of CLL, suggesting that the BCR signaling pathway could be an important target for therapeutic intervention. We have previously characterized some of the critical components of the signaling pathway downstream of the BCR in CLL B cells and identified the protein tyrosine kinase Syk as a promising therapeutic target. In a recent study we showed that CLL B-cells frequently have increased basal/constitutive Syk activity and are moderately sensitive to the cytotoxic effect of the selective Syk inhibitor R406 [Gobessi et al, Leukemia 2009]. More importantly, the survival signal induced by sustained BCR engagement was completely abolished by R406, suggesting that this compound may exert an even greater effect in vivo by inhibiting antigen-dependent Syk activation. We have now tested this possibility in the Eμ-TCL1 transgenic mouse model of CLL. Aged Eμ-TCL1 mice develop CD5+ B-cell leukemias that, similar to aggressive human CLL, show features of an antigen-driven process, including expression of stereotyped BCRs and reactivity with common autoantigens and microbial agents [Yan et al, Proc Natl Acad Sci USA 2006]. For our experiments we used a TCL1 leukemia (TCL1-002) that does not grow in vitro, but can be propagated in syngeneic recipients in vivo. TCL1-002 cells express an unmutated stereotyped BCR encoded by the VH12/VK4 combination, which reacts with phosphatidylcholine, an autoantigen exposed on the surface of senescent erythrocytes.In vitro experiments showed that R406 is not cytotoxic for TCL1-002 cells, although it completely inhibited both the basal and BCR-induced activation of signaling pathways downstream of Syk. The absence of a direct cytotoxic effect provided a unique opportunity to investigate whether inhibition of BCR signaling will affect leukemia growth in vivo. For this purpose, 1×107 TCL1-002 cells were injected intraperitoneally in 18 syngeneic mouse recipients. Three days later treatment was started in 8 mice with R788, which is the water-soluble prodrug of R406, at a daily dose of 80mg/kg during 18 consecutive days. Because of the rapid clearance of the drug (serum half-life <2 hours) R788 was administered in 3 divided doses at 4 hour intervals. Two weeks after the end of treatment leukemia developed in all mice from the control group (median WBC counts 131×106/ml, range 12-300×106/ml), whereas all R788-treated mice showed normal WBC numbers (median 6×106/ml, range 3-8×106/ml, P<0.001). Three weeks later all mice in the control group had died (median survival 46 days), whereas all mice in the R788 group were still alive and only two of them had detectable leukemic cells. R788 also showed some efficacy in the treatment of mice with overt TCL1-002 leukemias (WBC >50×106/ml). Whereas all mice from the control group (n=9) died between 6 and 18 days from the beginning of therapy, 4 out of 9 mice from the R788 group survived for more than 33 days. The mechanism of R788 activity was primarily related to inhibition of leukemic cell proliferation, as evidenced by a substantial decrease in the percentage of Ki67-positive cells after 7 days of treatment (30% before, 5% after therapy, P<0.001).To investigate whether R788 will also be effective against other TCL1 tumors we treated five TCL1 mice with preleukemic mono- or oligoclonal B-cell expansions during a four week period. R788 reduced the percentage of CD5+/B220+ cells in 2 cases, whereas in 2 other cases the percentage increased. Interestingly, the pattern of clonal Ig gene rearrangements changed during therapy, suggesting that only certain TCL1 clones are sensitive to R788 treatment. In summary, this study shows that R788 can effectively inhibit the growth of certain TCL1 tumors and provides the first in vivo experimental evidence suggesting that inhibition of antigen-dependent BCR signaling could be an effective therapeutic approach in CLL. Disclosures:No relevant conflicts of interest to declare.

High TCL1 levels are a marker of B-cell receptor pathway responsiveness and adverse outcome in chronic lymphocytic leukemia

AbstractAlthough activation of the B-cell receptor (BCR) signaling pathway is implicated in the pathogenesis of chronic lymphocytic leukemia (CLL), its clinical impact and the molecular correlates of such response are not clearly defined. T-cell leukemia 1 (TCL1), the AKT modulator and proto-oncogene, is differentially expressed in CLL and linked to its pathogenesis based on CD5+ B-cell expansions arising in TCL1-transgenic mice. We studied here the association of TCL1 levels and its intracellular dynamics with the in vitro responses to BCR stimulation in 70 CLL cases. The growth kinetics after BCR engagement correlated strongly with the degree and timing of induced AKT phospho-activation. This signaling intensity was best predicted by TCL1 levels and the kinetics of TCL1-AKT corecruitment to BCR membrane activation complexes, which further included the kinases LYN, SYK, ZAP70, and PKC. High TCL1 levels were also strongly associated with aggressive disease features, such as advanced clinical stage, higher white blood cell counts, and shorter lymphocyte doubling time. Higher TCL1 levels independently predicted an inferior clinical outcome (ie, shorter progression-free survival, P < .001), regardless of therapy regimen, especially for ZAP70+ tumors. We propose TCL1 as a marker of the BCR-responsive CLL subset identifying poor prognostic cases where targeting BCR-associated kinases may be therapeutically useful.

Epigenetic Silencing of Stk39 in B-Cell Lymphoma Inhibits Apoptosis from Genotoxic Stress

B-cell lymphomas, the most frequent human immune system malignancies, often contain dysregulated TCL1 oncogene expression. TCL1 transgenic (TCL1-tg) mice develop a spectrum of B-cell malignancies, supporting an oncogenic role for TCL1 in B cells. Our prior global survey of DNA methylation patterns in TCL1-tg B-cell lymphomas identified many lymphoma-specific candidate hypermethylated genes, including Stk39. The Stk39 encoded protein, sterile 20-like-related proline-alanine-rich kinase (SPAK), regulates cell stress responses, and microarray studies identified reduced SPAK expression in metastatic prostate and treatment-resistant breast cancers, suggesting that its loss may have a role in cancer progression. Here we identified DNA hypermethylation and SPAK silencing in TCL1-tg B-cell lymphomas and SPAK silencing without DNA methylation in multiple subtypes of human B-cell lymphomas. SPAK knockdown by shRNA protected B cells from caspase-dependent apoptosis induced by DNA double-strand breaks but not apoptosis in response to osmotic or oxidative cell stressors. Caspase 3 activation by cleavage was impaired with SPAK repression in DNA damaged B cells. Interestingly, c-Jun NH(2)-terminal kinase is potentially activated by SPAK and pharmacological inhibition of c-Jun NH(2)-terminal kinase in SPAK-expressing B cells recapitulated the cell-protective phenotype of SPAK knockdown. Taken together, these data indicate that SPAK loss in B-cell lymphomas promotes increased cell survival with DNA damage and provides a potential mechanism for increased resistance to genotoxic stress in cancer.

Eμ- TCL1 mice represent a model for immunotherapeutic reversal of chronic lymphocytic leukemia-induced T-cell dysfunction

Preclinical animal models have largely ignored the immune-suppressive mechanisms that are important in human cancers. The identification and use of such models should allow better predictions of successful human responses to immunotherapy. As a model for changes induced in nonmalignant cells by cancer, we examined T-cell function in the chronic lymphocytic leukemia (CLL) Emu-TCL1 transgenic mouse model. With development of leukemia, Emu-TCL1 transgenic mice developed functional T-cell defects and alteration of gene and protein expression closely resembling changes seen in CLL human patients. Furthermore, infusion of CLL cells into young Emu-TCL1 mice induced defects comparable to those seen in mice with developed leukemia, demonstrating a causal relationship between leukemia and the T-cell defects. Altered pathways involved genes regulating actin remodeling, and T cells exhibited dysfunctional immunological synapse formation and T-cell signaling, which was reversed by the immunomodulatory drug lenalidomide. These results further demonstrate the utility of this animal model of CLL and define a versatile model to investigate both the molecular mechanisms of cancer-induced immune suppression and immunotherapeutic repair strategies.

PKCβ is essential for the development of chronic lymphocytic leukemia in the TCL1 transgenic mouse model: validation of PKCβ as a therapeutic target in chronic lymphocytic leukemia

AbstractThe development and the propagation of chronic lymphocytic leukemia (CLL) has been linked to signaling via the B-cell receptor (BCR). Protein kinase C β (PKCβ) is an essential signaling element of the BCR and was recently shown to be overexpressed in human CLL. We used the TCL1 transgenic mouse model to directly target PKCβ in the development of murine CLL. TCL1 overexpression did restore the CD5+ B-cell population that is absent in PKCβ-deficient mice. However, PKCβ-deleted TCL1 transgenic mice did not develop a CLL disease, suggesting a role of PKCβ in the establishment of the malignant clone. Moreover, targeting of PKCβ with the specific inhibitor enzastaurin led to killing of human CLL samples in vitro. We thus propose that PKCβ may be a relevant target for the treatment of CLL.

Expression of sprouty2 inhibits B-cell proliferation and is epigenetically silenced in mouse and human B-cell lymphomas

B-cell lymphoma is the most common immune system malignancy. TCL1 transgenic mice (TCL1-tg), in which TCL1 is ectopically expressed in mature lymphocytes, develop multiple B- and T-cell leukemia and lymphoma subtypes, supporting an oncogenic role for TCL1 that probably involves AKT and MAPK-ERK signaling pathway augmentation. Additional, largely unknown genetic and epigenetic alterations cooperate with TCL1 during lymphoma progression. We examined DNA methylation patterns in TCL1-tg B-cell tumors to discover tumor-associated epigenetic changes, and identified hypermethylation of sprouty2 (Spry2). Sprouty proteins are context-dependent negative or positive regulators of MAPK-ERK pathway signaling, but their role(s) in B-cell physiology or pathology are unknown. Here we show that repression of Spry2 expression in TCL1-tg mouse and human B-cell lymphomas and cell lines is associated with dense DNA hypermethylation and was reversed by inhibition of DNA methylation. Spry2 expression was induced in normal splenic B cells by CD40/B-cell receptor costimulation and regulated a negative feedback loop that repressed MAPK-ERK signaling and decreased B-cell viability. Conversely, loss of Spry2 function hyperactivated MAPK-ERK signaling and caused increased B-cell proliferation. Combined, these results implicate epigenetic silencing of Spry2 expression in B lymphoma progression and suggest it as a companion lesion to ectopic TCL1 expression in enhancing MAPK-ERK pathway signaling.

The PIM1 Oncogene Accelerates TCL1 Driven Lymphomagenesis in a Double-Transgenic Murine Model.

Previously, we identified and validated PIM-1 as a differentially expressed gene in mantle cell lymphoma (MCL) patient samples. Further, we have shown PIM-1 to be a significant prognostic biomarker in MCL. PIM-1 is an oncogenic serine/threonine kinase that is transcriptionally regulated by cytokines, mitogens, and numerous growth factors. It cooperates with other oncogenes in tumorigenesis and has been implicated in the development of leukemias, lymphomas, late progression events, and most recently in prostate cancer. PIM-1 is overexpressed in aggressive lymphomas, such as the blastoid variant of MCL, and the ABC-type of DLBCL. Here we tested the in vivo cooperation of PIM-1 with TCL1 in murine lymphomagenesis by producing double transgenic murine strains. PIM-1 transgenic mice overexpress murine PIM-1 under the control of the immunoglobulin enhancer Eμ. TCL1 transgenic mice (pEμ -B29-TCL1) fail to downregulate TCL1 expression in mature B and T cells and provide a unique model for mature B-cell malignancies, including Burkitt-like lymphoma (BLL), DLBCL, marginal zone lymphoma, and B-cell chronic lymphocytic leukemia. We hypothesized that PIM-1 would either accelerate TCL1-driven lymphomagenesis, result in the development of immature lymphomas, or both. Lymphoid malignancies were examined by immunohistochemistry and flow cytometry and classified according to the mouse models of human cancer consortium (MMHCC) ‘Bethesda’ classification scheme. Forty double transgenic mice (PIM-1/TCL1) have been generated and observed for a median follow-up of 9 months. To date, 8/40 (20 %) of the PIM-1/TCL1 mice developed lymphomas, in contrast to 9/88 (10%) PIM-1 and 11/49 (22%) TCL1 transgenic mice, with a median follow-up of 7 and 15 months, respectively. A Kaplan-Meyer plot demonstrated statistically significant acceleration of lymphomagenesis in the PIM-1/TCL1 transgenic mice when compared with single TCL1 transgenic mice (p=0.037). PIM-1 transgenic mice developed early (< 7 months of age) T-cell lymphoblastic lymphomas and late (> 20 months of age) DLBCL. TCL1 transgenic mice developed DLBCL, with single occurrences of lymphoblastic, lymphocytic and Burkitt lymphomas. PIM-1/TCL1 transgenic mice developed DLBCL, frequently with extranodal involvement (spleen, liver and lung). A single case of follicular lymphoma was seen. In addition, endogenous expression of PIM kinase family members was investigated in a human lymphoma cell line bank (n=40) by quantitative real-time PCR. PIM-1, PIM-2, and also PIM-3 were found to be overexpressed in cell lines derived from human lymphoid malignancies of multiple histologies. In summary, aberrant PIM-1 overexpression in TCL1 transgenic mice accelerated the development of mature B-cell lymphomas. To date, the classification of lymphomas in PIM-1/TCL1 mice revealed similar histologies as in TCL1 single transgenic mice, mainly DLBCL. The expression of all 3 PIM kinase family members in lymphomas implies that pan-PIM kinase inhibitors should be developed as a potential mechanism of resistance to more restricted PIM inhibitors could be compensatory overexpression of the non-targeted family members.

PKCβ Is Essential for the Development of CLL in the TCL1 Transgenic Mouse Model: Validation of PKCβ as a Therapeutic Target in CLL

The development and the propagation of chronic lymphocytic leukemia (CLL) has been linked to signalling via the B-cell receptor (BCR). Protein kinase C (PKC) beta is an essential signalling element of the BCR and was recently shown to be overexpressed in human CLL. We used the TCL1 transgenic mouse model to directly target PKC beta in the development of murine CLL. While loss of PKC beta in wild type mice leads to a complete loss of CD5+ B cells in these mice, the targeted disruption of the PKC beta gene in Tcl1 transgenic mice did not abrogate the accumulation of CD5 positive B cells in the pretumor phase in these mice. This suggests that the basic survival signal transmitted by PKC beta in B1 cells can be substituted by the augmented Akt signalling in the Tcl1 transgenic cells. Yet despite this, PKC beta deficient TCL1 transgenic mice did not develop a CLL disease, suggesting a role of PKC beta in the establishment of the malignant clone. In fact PKC beta is likely to be essential for transformation in limiting the BCR signal via a negative feedback loop, suggesting that BCR signalling strength is an important component of the transformation process. In order to provide support for the potential of PKC beta as a drug target in CLL we targeted PKC beta with the specific inhibitor Enzastaurin. This led to killing of human CLL samples in vitro and this was independent of risk factors in the samples analysed. We thus propose that PKC beta may be a relevant target for the treatment of CLL.

Loss of Id4 Accelerates CLL Progression in TCL1 Mice

The inhibitor of DNA binding protein 4 (ID4) is a member of dominant negative basic helix loop helix (bHLH) transcription factor family that has a HLH domain but lacking DNA-binding domain. Methylation of ID4 in B-cell lineage malignancies including CLL and germinal center non-Hodgkin lymphomas (GC-NHLs) has been described. To date, the impact of Id4 loss in CLL has not been demonstrated. We utilized the TCL1 transgenic mouse model of human CLL to study the importance of loss of ID4 in disease progression. We demonstrated that ID4 was silenced by methylation only at the time mice had symptomatic leukemia. We therefore sought to investigate if earlier loss of Id4 would accelerate CLL progression. We generated mice with haploid loss of Id4 in TCL1 transgenic background by breeding homozygous TCL1 transgenic mice and mice with heterozygous Id4 mutant gene. By comparing to the control littermates with heterozygous TCL1 oncogene, mice with the loss of Id4 occur to have significantly accelerated CLL disease progression with shorter overall survival (12 versus 16 months, p-value<0.0001). The pathological analysis on Id4 mutant mice demonstrated that the CLL symptoms such as the elevated white blood cell counts, enlarged spleen and lymphoid tissues. We then performed the microarray studies on 1 month old TCL1 mice with control or mutant Id4 to identify ID4-dependent transcriptional changes in primary B-cells. Among the identified targets, genes involved in cell proliferation and anti-apoptosis were then verified. Supportively, B-cells from Id4 mutant TCL1 mice have significantly (p-value<0.001) diminished apoptosis in response to dexamethasone treatment. Additionally, both significant in vitro (p-value<0.001) and in vivo (p-value=0.025) increased proliferation of Id4 mutant TCL1 B-cells after the stimulation by CpG685NO168 was demonstrated. These data provide support that Id4 acts as tumor suppressor in transformed B-lymphocytes and is silenced through the process of methylation. The Effort to identify binding partners of Id4 in CLL cells and targeting its re-expression is warranted.

T Cell Dynamics during the Pretumor and Tumor Phase in the Murine Tcl1 Transgenic Chronic Lymphocytic Leukemia Model

Chronic lymphocytic leukaemia of B cells (B-CLL) is characterised by a clonal expansion of CD5-expressing B cells. However, the absolute number of T cells in patients with B-CLL is also increased, primarily due to an enlarged CD8+ population. Despite the enhanced number of T cells, they appear to be dysfunctional as they are largely devoid of anti-tumor activity and may even support the growth and maintenance of the malignant B-CLL clone. To further investigate the contribution this T cell dysfunction has on the establishment and progression of B-CLL, we monitored the changes in the T cell compartment in Eμ-TCL1 transgenic mice of various ages, during the progression of B-CLL-like disease. The deregulated expression of TCL1 in the B cell compartment of these mice leads to a hyperplasia of CD5+ CD19+ B cells early in life that is first detectable in the peritoneal cavity and later spleen and bone marrow. Later, this develops into a clonal disease, and onset of frank leukaemia, with infiltration of the CD5+ CD19+ cells into other organs (Bichi et al, 2003). Similar to human B-CLL, leukaemic TCL1 transgenic mice have increased absolute numbers of T cells, owing mainly to an expanded CD8+ population. Analyses of the cell surface expression of CD25, CD44, and CD62L, revealed a marked increase the number of memory and effector T cells compared to naïve T cells in the CD8+ subset that is also observed in the CD4+ subset, although to a lesser degree. Importantly, a decrease in the number of naïve T cells along with a corresponding increase in T memory and effector cells has been observed in the CD4+ T cell pool from patients with unmutated B-CLL (Tinhofer et al, unpublished), and the TCL1 mouse has been shown to be a model for the unmutated form of B-CLL. The relative shift from naïve to central memory T cells occurs alongside the CD5+ CD19+ hyperplasia in the TCL1 mice. In younger mice that exhibit the CD5+ CD19+ hyperplasia only in the peritoneal cavity, the changes in the T cell compartment are also observed solely within this organ. At later stages, when the CD5+ CD19+ hyperplasia has spread to other organs, the shift to memory T cells is also detectable in all infiltrated organs. Because memory and effector T cells represent antigen-experienced cells, we analysed the complementarity determining region 3 (CDR3) of the T cell receptor (TCR) of purified CD4+ and CD8+ populations by spectratyping in order to determine the degree of clonality (mono-, oligo-, or polyclonal) of the different T cell families within the T cell pool. More clonal T cells were observed in the CD8+ T cell subset in the infiltrated organs of leukaemic mice, though we could not observe a skewing towards any particular BV gene family. Interestingly, more mono- and oligoclonal CD4+ T cells have also been observed in the peripheral blood of patients with unmutated B-CLL. Thus, in many aspects, the TCL1 mouse recapitulates many of the T cell abnormalities observed in patients with B-CLL. It should be noted that, although driven from a B-cell specific promoter, the hTCL1 transgene is also expressed in the T cells of TCL1 mice. Interestingly, we have also found that TCL1 is overexpressed in the CD4+ and CD8+ T cells of patients with unmutated B-CLL compared to mutated CLL or healthy controls. Thus the Tcl1 transgenic murine CLL model seems suitable as a model for the immune aberrations found in human unmutated CLL.

BAFF Accelerates Development of Chronic Lymphocytic Leukemia in TCL1 Transgenic Mice.

The B cell- activating factor of the tumor necrosis factor family (BAFF) is a potent regulator of normal B cells. We recently showed that BAFF supports chronic lymphocytic leukemia (CLL) B cell survival in vitro through activation of the canonical NF-kB pathway. To study the influence of BAFF on CLL development, we crossed BAFF transgenic (Tg) mice with mice that express human TCL1 under a B cell specific promoter/enhancer, and that are known to develop a lymphoproliferative disease resembling human B-CLL. BAFF/TCL1-Tg mice had a shorter mean survival than either TCL1-Tg or BAFF-Tg mice (12 mice each; BAFF/TCL1-Tg mice 9.6±3.4 months; TCL1-Tg 17.2±3.9; BAFF-Tg 17.9±3.6; B6 wildtype (wt) >19.2). To monitor for the development of CLL, mice were bled at 6-week intervals starting at 3 months of age, and blood mononuclear cells (PBMC) were analyzed via flow cytometry using fluorochrome-conjugated antibodies for murine CD5, CD3, CD45R, and human TCL1. Whereas all BAFF/TCL1-Tg mice began to develop a pathological CD5+CD3−CD45Rlo cell population at 3 months of age, such a population was not observed in TCL1-Tg mice before 6 months of age. BAFF-Tg or wt mice did not develop CD5+CD3−CD45Rlo cells over the entire observation period (26 months). CD5+CD3−CD45Rlo B cells expressed the TCL1 transgene. Over time, the CD5+CD3−CD45Rlo population increased in BAFF/TCL1-Tg mice, coming to represent >99% of the total PBMC of 9-month-old animals. To examine the capacity of these cells to propagate, 1x106 CD5+CD3−CD45Rlo B cells were transferred i.v. into either BAFF-Tg or wt mice that previously were irradiated with 600 rad. Ten days after transfer, CD5+TCL1+ cells were detected in BAFF-Tg, but not in wt recipients. Most CLL cells were located in the liver and spleen, as assessed by bioluminescent-based imaging of mice that received luciferase expressing CLL cells. Subsequent examination upon autopsy at 6 months of age, however, revealed that the majority of CLL cells populated the spleens of the recipient mice, which were massively enlarged. At this age, CLL cells also were found in wt recipient mice, although tumor burden was less than 20% of that of BAFF-Tg recipients (n=3 per group). We found that BAFF did not promote CLL cell proliferation in vitro or in vivo using assays to measure BrdU incorporation and flow cytometry to evaluate for enhanced intracellular expression of Ki67. However, BAFF induced CLL cells to express high levels of several anti-apoptotic proteins (e.g. Bcl-XL, Bcl-2, Bim, and A1/Bfl1). Also, while death-associated protein kinase 1 was repressed in CLL cells of TCL1-Tg mice, CLL cells of BAFF/TCL1-Tg mice expressed high-levels. Because of this, we examined whether treatment with BAFF-neutralizing BR3-Fc could influence the survival of CLL cells that were adoptively transferred into BAFF-Tg mice. We found that i.p. injection of 200 ug BR3-Fc into the recipient animals reduced the numbers of circulating CLL cells by nearly 20% (18.2%±5.3%; n=3) within 6 days. These data indicate that BAFF can accelerate the development of CLL cells in TCL1-Tg mice by promoting their survival. Because BAFF can similarly promote survival of human CLL cells, BAFF, and the signaling pathways it activates in neoplastic B cells, could be targeted for the development of novel therapies for this disease.

Early Fludarabine Treatment of TCL-1 Transgenic Mice Promotes Drug Resistance through a Mechanism Not Involving p53 Mutations: Implications for Patients with Chronic Lymphocytic Leukemia (CLL).

Initial therapy for Chronic Lymphocytic Leukemia (CLL) typically includes nucleoside analogs such as fludarabine. Mutations of p53 are uncommon in CLL patients at diagnosis, but are found at increased frequency in patients with prior treatment and are often associated with resistance to standard therapies including fludarabine. The causality of fludarabine treatment to p53 mutation has not been clearly defined. In previous work with the TCL-1 model of CLL, we demonstrated that mice with active leukemia do not have p53 mutations and respond to fludarabine therapy with a modest survival advantage (Johnson et al, Blood 108:1334, 2006). We therefore initiated a randomized trial of fludarabine treatment (34mg/kg daily intraperitoneal injection for five days every 28 days) versus observation (treatment with volume-matched vehicle controls on the same dosing schedule) in TCL-1 transgenic mice prior to the onset of leukemia. The goals of this study were to determine if early treatment with fludarabine would 1) delay disease onset; 2) eventually yield a fludarabine-resistant phenotype; or 3) promote development of p53 mutations in mice developing progressive leukemia. Transgenic TCL-1 mice between the ages of eight and 12 weeks were continually treated with fludarabine (n=32) or saline (n=45) until death while being screened monthly for disease progression through changes in WBC count by peripheral blood differentiation smear, lymphocyte subsets by flow cytometry, and nodal and spleen exams. Despite continued treatment with fludarabine, drug resistance as manifested by leukemia and eventual death occurred. Median time to leukemia onset was 308 days (95% CI: 224, ∞) in the fludarabine group and 338 days (95% CI: 252, ∞) in the saline group. Thus, the risk of developing leukemia was not significantly different between the two groups. Of interest, p53 mutations in exons 2–11 were not found in either group (n=10 each) at the time of death. Microarray studies are currently underway to discover other genes differentially expressed between treated and untreated mice that might contribute to fludarabine resistance and will be presented. In conclusion, our data demonstrate that early fludarabine treatment in the TCL-1 transgenic model of CLL does not prevent onset of leukemia, but instead results in disease resistant to fludarabine. The source of fludarabine resistance in this mouse model was not through acquisition of p53 mutations, but rather an alternative unidentified mechanism(s).

The Expression of the GTPase-Deficient, Hematopoietic-Specific RhoH GTPase Is Implicated in Development of Chronic Lymphocytic Leukemia (CLL).

RhoH is a hematopoietic-specific small GTPase which is constitutively active due to lack of intrinsic GTPase activity, suggesting that expression levels of RhoH regulate cellular function. Our lab has previously shown that RhoH in T-cells directly interacts with ZAP-70, a critical protein tyrosine kinase in TCR cell signaling and function (Gu et al., Nature Immunol., 2006). As in human B-CLL ZAP-70 expression has been correlated with an unmutated IgVH gene status and poor prognosis, we have investigated RhoH expression and function in B-CLL. PBMC samples from 29 B-CLL patients, all containing >90% CD19+/CD5+ human B-CLL cells, were compared to CD19+ B-cells from normal donors. Significantly higher RhoH mRNA levels were detected in CLL samples by quantitative PCR (p<0.001) with a 2–4 fold increase in the majority of samples. In addition, RhoH levels in these CLL samples were directly correlated with ZAP-70 expression (p<0.01). To further delineate the role of RhoH in the development and progression of B-CLL, we utilized a genetic mouse model of B-CLL, the Eμ-TCL1 transgenic mouse (Bichi et al. PNAS, 2002), and interbred these with Rhoh−/− mice. Starting at 2 months of age peripheral blood of these mice was analyzed every month for different blood cell lineages and in particular for the number of IGM+/CD5+/B220low cells as a readout for the progression of the CLL-like disease. In TCL1 transgenic mice with normal RhoH expression (Rhohwt; TCL1Tg) and with one Rhoh allele knocked out (Rhoh+/−; TCL1Tg) an accumulation of IgM+/CD5+ B-cells was observed which started as early as an age of 3 and 4 months, respectively, and increased dramatically thereafter. In contrast, Rhoh−/−; TCL1Tg showed no significant increase of IgM+/CD5+ B-cells until month 5 with a modest increase thereafter and values were significantly reduced in comparison to Rhohwt; TCL1Tg from month 3–5. As expected, no increase of IgM+/CD5+ B-cells was observed in the nontransgenic controls Rhoh−/+; TCL1NTg, Rhoh−/−; TCL1NTg (data not shown), and Rhohwt; TCL1NTg (Table 1).Table 1:IGM+/CD5+ cells in the peripheral blood.Age [months]Rhohwt; TCL1TgRhoh+/−;TCL1TgRhoh−/−;TCL1TgRhohwt;TCL1NTg20.12 ± 0.02 (10)0.28 ± 0.07 (8)0.15 ± 0.04 (9)0.08 ± 0.02 (9)30.33 ± 0.09 (11)0.22 ± 0.06 (8)0.07 ± 0.03 (8)*0.07 ± 0.01 (10)40.83 ± 0.38 (11)0.57 ± 0.20 (18)0.11 ± 0.05 (16)*0.08 ± 0.01 (6)53.32 ± 2.27 (6)1.35 ± 0.46 (17)0.26 ± 0.06 (15)*0.11 ± 0.03 (5)64.38 ± 2.41 (7)5.01 ± 3.01 (9)0.66 ± 0.23 (8)0.07 ± 0.02 (5)Mean ± SEM (x1000/ul); () animals analyzed; * p<0.05 in comparison to Rhohwt; TCL1NTG (data collection still in progress)When mice from each genotype were sacrificed at 6 months of age the number of IgM+/CD5+ B-cells detected in Rhoh−/− TCL1Tg vs RhohwtTCL1Tg mice was 6-fold reduced in the peritoneal cavity, 3-fold reduced in the bone marrow and a 2-fold reduced in the spleen. In summary our data strongly suggest that RhoH plays a role in the progression of the B-CLL-like disease in the transgenic Eμ-TCL1 mouse model. This will allow detailed molecular studies of the role of RhoH in pathways altered in TCL1-induced expansion of B-cell precursor populations in this model.

The Plant-Derived Agent Silvestrol Has B-Cell Selective Activity In Vitro in Chronic Lymphocytic Leukemia Patient Cells and In Vivo in the Tcl-1 Mouse Model of CLL.

Chronic Lymphocytic Leukemia (CLL) is an incurable disease with limited therapeutic options, especially for high-risk populations such as the del(17p13) patient subset. Currently available therapies for CLL, even if effective, can have significant detrimental effects on remaining T cells, leaving patients at risk of potentially lethal opportunistic infections. New agents with unique mechanisms of action, independence of key resistance pathways, and selectivity for tumor cells are crucial to make an impact on patient survival. Silvestrol, a structurally unique compound isolated from the plant genus Aglaia, exhibited potent activity against several tumor cell lines and moderate in vivo activity in the P388 mouse leukemia model (J. Org. Chem . 2004, 69:3350; ibid. 69:6156). Based on these results, we tested silvestrol against tumor cells obtained from CLL patients. The LC50 (concentration lethal to 50% of cells relative to untreated control) of silvestrol was 6.5 nM at 72 hours by MTT assay. We performed assays to determine CLL patient cell viability at 72 hours with or without drug washout at various times. In these studies, silvestrol showed up to 50% killing at 72 hours with only a four hour exposure, and reached maximum efficacy with a 24 hour exposure. Silvestrol was similarly effective against cells from CLL patients with or without del(17p13). Furthermore, there was no significant difference in silvestrol-mediated cytotoxicity between lymphoblastic cells with a ten-fold overexpression of Bcl-2 relative to control cells. In MTT assays using isolated CD3+ or CD19+ cells, and in whole blood from healthy volunteers and CLL patients, silvestrol demonstrated substantially more cytotoxicity toward B cells than T cells. We then tested silvestrol using Tcl-1 transgenic mice, which are initially normal but develop a slow-progressing B cell leukemia very similar to human CLL. Lymphocytes obtained from spleens of Tcl-1 mice with leukemia were incubated ex vivo with 80 nM silvestrol and analyzed by flow cytometry. Silvestrol produced an 88% reduction in the B cell percentage after 24 hours with no negative effect on the T cell percentage (8% increase), in contrast to 1 μM fludarabine, which affected both B cell (22% reduction) and T cell (14% reduction) subsets. Non-leukemic mice of the Tcl-1 background strain were treated with 1.0, 1.5 and 2.5 mg/kg/day silvestrol for 5 days to determine a tolerable dose. Three of five mice treated with 2.5 mg/kg/day died at the beginning of the second week of treatment. However, none of the animals treated at 1.0 or 1.5 mg/kg showed signs of toxicity or weight loss even after two full weeks of treatment and were normal at pathological examination. Tcl-1 mice with evidence of leukemia as determined by elevated leukocyte counts and enlarged spleens were then treated with silvestrol at 1.5 mg/kg/day × 5 days for two weeks. Treated mice experienced decreased overall leukocyte counts relative to vehicle controls. Furthermore, CD19+ cell numbers and percentages diminished substantially while the T cells were only mildly affected. Additional leukemic Tcl-1 mice are currently being treated and studies are underway examining the mechanism of action of silvestrol in CLL cells.

TCL1 in B-cell Tumors Retains its Normal B-cell Pattern of Regulation and is a Marker of Differentiation Stage

The high expression of the T-cell oncogene TCL1 in B-cell tumors and the emergence of B-cell lymphomas in TCL1-transgenic mice suggest a pathogenetic role for this kinase coregulator in B-cell malignancies. We compared the expression of TCL1 in B-cell tumors with their differentiation stage. As with normal B-cell subsets, uniform TCL1 expression was characteristic of tumors of pregerminal center derivation such as precursor B-cell lymphoblastic leukemia/lymphoma (85%, 47/55) and mantle cell lymphoma (84%, 49/58), and was more variable in follicular lymphoma (57%, 28/49). Large B-cell lymphoma was less frequently positive for TCL1 (36%, 18/50), especially among cases of the activated B-cell type. All types of Hodgkin lymphoma, splenic marginal zone lymphoma, and post-germinal center-derived tumors, including plasma cell myeloma and MALT lymphoma, were negative for TCL1, except for 1 case. In nearly all TCL1-expressing tumors, as with normal B cells, variations in cellular TCL1 levels were related to the proliferation and microenvironmental factors. In normal B cells, cell lines and primary B-cell tumor samples, TCL1 downmodulation occurred after prolonged cytokine treatment and/or B-cell receptor stimulation. In contrast to mature T-cell tumors where TCL1 expression is always indicative of an activating TCL1 gene translocation, TCL1 expression in B-cell tumors parallels its regulation in non-neoplastic B cells. Therefore, TCL1 expression can be used diagnostically as an indicator of the differentiation stage of a given B-cell tumor.

Global DNA methylation profiling reveals silencing of a secreted form of Epha7 in mouse and human germinal center B-cell lymphomas

Most human lymphomas originate from transformed germinal center (GC) B lymphocytes. While activating mutations and translocations of MYC, BCL2 and BCL6 promote specific GC lymphoma subtypes, other genetic and epigenetic modifications that contribute to malignant progression in the GC remain poorly defined. Recently, aberrant expression of the TCL1 proto-oncogene was identified in major GC lymphoma subtypes. TCL1 transgenic mice offer unique models of both aggressive GC and marginal zone B-cell lymphomas, further supporting a role for TCL1 in B-cell transformation. Here, restriction landmark genomic scanning was employed to discover tumor-associated epigenetic alterations in malignant GC and marginal zone B-cells in TCL1 transgenic mice. Multiple genes were identified that underwent DNA hypermethylation and decreased expression in TCL1 transgenic tumors. Further, we identified a secreted isoform of EPHA7, a member of the Eph family of receptor tyrosine kinases that are able to influence tumor invasiveness, metastasis and neovascularization. EPHA7 was hypermethylated and repressed in both mouse and human GC B-cell non-Hodgkin lymphomas, with the potential to influence tumor progression and spread. These data provide the first set of hypermethylated genes with the potential to complement TCL1-mediated GC B-cell transformation and spread.

Expression of Tcl-1 as a Potential Prognostic Factor for Treatment Outcome in B-Cell Chronic Lymphocytic Leukemia.

Chronic Lymphocytic Leukemia (CLL) is primarily characterized by the presence of leukemic B lymphocytes resistant to the process of apoptosis. Causes for this resistance include aberrant expression of multiple anti-apoptotic proteins, perhaps including Tcl-1 (1). Tcl-1 is expressed during normal development of both B and T cells and activates Akt to provide a survival signal through phosphorylation and inactivation of apoptotic pathway components. Overexpression of Tcl-1 in the B cells of transgenic mice results in a B-cell leukemia phenotype typical of human CLL, suggesting a primary role for Tcl-1 in CLL tumorigenesis. A recent report showed that Tcl-1 protein expression in CLL cells correlated with other biological parameters, but no data were available as to its clinical relevance (2). We therefore examined Tcl-1 expression in relation to clinical outcome using 51 peripheral blood mononuclear cell samples from a Phase II clinical trial testing the combination of pentostatin, cyclophosphamide, and rituximab in previously untreated CLL patients (Kay et al., ASH 2006). Tcl-1 protein expression was measured by immunoblot using GAPDH as a normalization control across lanes and lysate from a single Tcl-1 transgenic mouse spleen as a normalization control across immunoblots. All samples were run at least in duplicate and digital quantitation was performed using a ChemiDoc instrument (BioRad). Tcl-1 expression in patient cells was calculated in comparison to Tcl-1 expression in the mouse lysate, arbitrarily set to 1.0. Data were analyzed as a continuous measure as well as by specific cutoffs. Higher Tcl-1 expression was associated with failure to achieve a minimal residual disease-negative state after treatment. Specifically, Tcl-1 expression (measured as a continuous variable) was associated with residual disease measured by post-treatment CD5+/CD19+ cell percentages (continuous variable as well as dichotomized by cutpoint of 1.0% for CD5+/CD19+; p=0.03 for both comparisons). For the dichotomized Tcl-1 expression with the cutpoint of 0.275 (second tertile), this variable was marginally significantly associated with whether or not post-treatment CD5+/CD19+ was less than 1.0%. Using this same cutoff, there was also a suggestion that patients with reduced Tcl-1 were more likely to achieve a complete response. Finally, progression-free survival was shorter in patients with increased Tcl-1 expression, although these data did not reach statistical significance in this sample set.Tcl-1 < 0.275Tcl-1≥ 0.275p valueCD5/CD19≥ 1%15100.046CD5/CD19 < 1%162CR1730.064No CR1813Median PFS (mos.)36.517.10.137Statistically significant associations with other parameters including Rai stage, IgVH mutation status, ZAP-70 expression, CD38 expression, and interphase cytogenetics categories were not detected in this sample set. These data further support the clinical relevance of the Tcl-1 mouse model to human CLL, and provide preliminary evidence that Tcl-1 protein expression correlates with clinical outcome of CLL patients treated with chemoimmunotherapy. If validated in larger studies, Tcl-1 expression may serve as a novel prognostic factor.