ALBERT

Description: Abstract 1737 Poster Board I-763 We previously reported the efficacy and B-cell selectivity of the natural product silvestrol in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), using both primary cells and B-cell lines. We also showed that silvestrol inhibits translation, resulting in rapid depletion of the short half-life protein Mcl-1 followed by mitochondrial damage and apoptosis. Cencic et al. reported that silvestrol directly blocks translation initiation by aberrantly promoting interaction of eIF4A with capped mRNA (PLoS One 2009; 4(4):e5223). However, the loss of Mcl-1 in breast and prostate cancer cell lines is delayed relative to what we observe in B-leukemias (48 hr vs. 4-6 hr in CLL and ALL cells). Additionally, silvestrol does not reduce Mcl-1 expression in normal T-cells to the same extent that it does in B-cells, potentially explaining in part the relative resistance of T-cells to this agent. We therefore investigated cell-type differences, as well as the importance of Mcl-1, in silvestrol-mediated cytotoxicity. We incubated the ALL cell line 697 with gradually increasing concentrations of silvestrol to generate a cell line (697-R) with resistance to 30 nM silvestrol (IC50 of parental 697 〈 5 nM). No differences between 697-R and the parental line were detected upon detailed immunophenotyping. However, cytogenetic analysis revealed a balanced 7q;9p translocation in 697-R not present in the parental 697 cell line that may be related to the emergence of a resistant clone. We also detected no difference in expression of multi-drug resistance proteins MDR-1 and MRP, which can contribute to resistance to complex amphipathic molecules such as silvestrol. In contrast, we found that baseline Mcl-1 protein expression is strikingly increased in 697-R cells relative to the parental line, although these cells still show similar percent-wise reduction in Mcl-1 upon re-exposure to 80 nM silvestrol. To investigate whether this resistance to silvestrol is reversible, 697-R cells were maintained without silvestrol for 6 weeks (∼18 passages). During this time, viability remained near 99%. Cells were then treated with 30 nM silvestrol. Viability was 94% at 48 hr post-treatment and returned to 99% within a week, while parental 697 cells with the same treatment were completely dead. Baseline Mcl-1 levels remained elevated in 697-R even with prolonged silvestrol-free incubation. These results indicate that the resistance phenotype is not rapidly reversible, as is seen with transient upregulation of multi-drug resistance or stress-response proteins. Additionally, silvestrol moderately induces the transcription of several pro-apoptotic Bcl-2 family members and results in elevated levels of these proteins despite its translation inhibitory activity. Interestingly, no such activity is detected in silvestrol-treated normal T-cells. Together, these results support the hypothesis that in B-cells, silvestrol induces cell death by altering the balance of pro- and anti-apoptotic factors, and that increased Mcl-1 protein can force the balance back toward survival. This work further underscores the importance of Mcl-1 in silvestrol-mediated cytotoxicity. We are now investigating the mechanism of Mcl-1 upregulation in 697-R cells to identify a factor or pathway that can be targeted therapeutically to circumvent resistance. Silvestrol is currently undergoing preclinical pharmacology and toxicology investigation by the U.S. National Cancer Institute Drug Development Group at the Stage IIA level to facilitate its progression to Phase I clinical testing. Disclosures No relevant conflicts of interest to declare.

Description: Therapeutic options for advanced B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) are limited. Available treatments can also deplete T lymphocytes, leaving patients at risk of life-threatening infections. In the National Cancer Institute cell line screen, the structurally unique natural product silvestrol produces an unusual pattern of cytotoxicity that suggests activity in leukemia and selectivity for B cells. We investigated silvestrol efficacy using primary human B-leukemia cells, established B-leukemia cell lines, and animal models. In CLL cells, silvestrol LC50 (concentration lethal to 50%) is 6.9 nM at 72 hours. At this concentration, there is no difference in sensitivity of cells from patients with or without the del(17p13.1) abnormality. In isolated cells and whole blood, silvestrol is more cytotoxic toward B cells than T cells. Silvestrol causes early reduction in Mcl-1 expression due to translational inhibition with subsequent mitochondrial damage, as evidenced by reactive oxygen species generation and membrane depolarization. In vivo, silvestrol causes significant B-cell reduction in Eμ-Tcl-1 transgenic mice and significantly extends survival of 697 xenograft severe combined immunodeficient (SCID) mice without discernible toxicity. These data indicate silvestrol has efficacy against B cells in vitro and in vivo and identify translational inhibition as a potential therapeutic target in B-cell leukemias.

Description: Chronic Lymphocytic Leukemia (CLL) is an incurable disease with limited therapeutic options. The development of drug resistance through multiple pathways, especially in advanced disease, further restricts these options. Thus, new agents with unique mechanisms of action are crucial to make an impact on patient survival. Silvestrol, a rocaglate derivative with an unusual dioxanyloxy unit, was isolated from Aglaia species using bioassay-guided fractionation. Silvestrol exhibited potent in vitro cytotoxic activity against several tumor cell lines. Silvestrol was further evaluated in vivo in the hollow fiber test and in the murine P-388 leukemia model, in which it demonstrated promising anti-tumor activity with no significant weight loss up to 2.5 mg/kg (3.7 μM, assuming equal distribution) (1). Based on these results, we tested silvestrol against tumor cells obtained from CLL patients. Silvestrol exhibited significant antitumor activity with an estimated LC50 (concentration lethal to 50% of cells relative to untreated control) of 10 nM at 72 hours by MTT assay. In contrast, at this same timepoint using normal human peripheral blood mononuclear cells, an LC50 for silvestrol could not be defined even up to 4.0 μM. Under identical conditions, silvestrol was 50 to 100 fold more potent than the active metabolite of fludarabine, commonly used in the treatment of CLL. To determine the minimum exposure time required for silvestrol to have an effect, cells were incubated for various times in 80 nM silvestrol, then washed and resuspended in media with or without drug and incubated for a total of 72 hours. With only a four hour exposure, an average of 56% cytotoxicity was observed relative to untreated cells, and with a 24 hour exposure, results between samples in which the drug was removed and those incubated continuously were indistinguishable. T cell depletion and concomitant immunodeficiency is a serious risk with therapies currently available for CLL. We therefore tested the relative effects of silvestrol on B and T cells. By MTT assay with selected cells and in whole blood incubations followed by flow cytometry, using blood from both CLL patients and healthy volunteers, silvestrol demonstrated significantly more cytotoxicity toward B cells than T cells. Although some variability was observed between patient samples, silvestrol had activity against all samples tested and there was no detectable difference in average potency against cells from patients with a 17p13 deletion (chromosomal site of p53) relative to those without this risk factor. 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. Together, these data demonstrate that silvestrol has efficacy against CLL cells in vitro and in whole blood, has highly unusual B-cell specificity, and is independent of key CLL resistance mechanisms. Our data strongly support further investigation of silvestrol as an antitumor agent in CLL. Studies are underway to determine the precise mechanism of action of this compound in CLL cells.

Description: Epstein-Barr virus (EBV) is an oncogenic herpes virus associated with the development of a range of malignant B-cell lymphoproliferative diseases that are associated with immune suppression, aggressive clinical courses, and poor outcomes. Current treatment options typically lead to further immune suppression, increasing the risk of EBV reactivation and other potentially lethal opportunistic infections. These complications make it essential to identify novel therapeutic approaches targeting mechanisms of immune evasion while promoting tumor immune surveillance. Immune evasion of tumors is supported by aberrant ligand and cytokine expression within the microenvironment, and tumor-associated macrophages (TAM) play an important role in inhibiting anti-tumor immune responses. In particular, TAMs are known to suppress T cell activation, and increased TAM density and ratio to cytotoxic T lymphocytes (CTL) in the tumor microenvironment are negative prognostic indicators. Here we demonstrate the use of an in vitro co-culture (CoCx) system in which EBV-transformed lymphoblastoid cell lines (LCL) are cultured with autologous peripheral blood mononuclear cells (PBMC). Media from these CoCx collected after 48 hrs., showed elevated levels of the monocyte/macrophage associated cytokines and chemokines MCP, IL-8, IP-10, MIG, GRO and MMP-9, which were absent in media from LCL or PBMC cultures. Furthermore, LCL+PBMC CoCx showed a notable outgrowth of a CD14+ monocyte/macrophage population. To analyze the direct effect of these secreted factors, conditioned media was collected from cultures of PBMC, LCL or CoCx and added to purified autologous monocytes. MTS assays at 7 days showed pronounced proliferation of monocytes in CoCx conditioned media relative to monocytes in PBMC or LCL conditioned media. Microscopy indicated that monocytes cultured with CoCx conditioned media formed non-adherent, proliferating colonies, whereas monocytes incubated in unconditioned, PBMC conditioned, or LCL conditioned media remained adherent. Depletion of CD8+ lymphocytes (but not CD4+, CD19+, CD56+ or CD14+ subsets) from CoCx led to a decrease in proliferation of monocytes, indicating a role of CTL in this phenomenon. This monocyte-derived cell population retained monocyte markers CD14 and CD11b and showed pronounced increases in both canonical M1 and M2 macrophage markers (HLA-DR and CD163, respectively), consistent with a TAM phenotype. Furthermore, the T cell inhibitory molecule PD-L1 was prominently expressed on these cells. This phenotype was confirmed to be the same as for the CD14+ cells that expanded from PBMC cultured with LCL. The functional activity of these macrophages on T cells was assessed by incubating autologous T cells alone or with macrophages derived from CoCx conditioned media for 24 hrs. Quantitative flow cytometric analysis showed a moderate decrease in CD4+ T helper cells and CD8+ CTL in the CoCx, compared to T cells cultured alone. However, if T cells were first activated (anti-CD3/CD28), the total numbers of viable CD3+ cells in the CoCx dropped 10-fold compared with activated T cells cultured alone. We previously reported that the eIF4A-specific translation initiation inhibitor silvestrol elicits marked anti-tumor activity against EBV-driven lymphoma. This occurs both by direct cytotoxicity to EBV-transformed B cells and by facilitating the activation, proliferation and cytotoxic activity of anti-tumor immune subsets including EBV-specific CTL. Here, the addition of 10nM silvestrol into CoCx blocked the release of monocyte/macrophage-associated cytokines, and silvestrol-treated CoCx conditioned medium did not drive proliferation or colony expansion of TAM-like macrophages. This finding indicates that selective targeting of the eIF4F translation initiation complex, which includes eIF4A, may block TAM formation in the setting of EBV-driven lymphoma. In summary, we have identified an immune evasion mechanism by which exposure of normal PBMC to EBV+ lymphoma cells results in expansion of a TAM-like macrophage population with potent cytotoxic activity against T cells. Low-dose silvestrol treatment abrogates the outgrowth of this TAM-like population and allows expansion of EBV-specific CTL. This finding provides an entirely new approach to modulate the immune response in this challenging group of EBV-related diseases. Disclosures No relevant conflicts of interest to declare.

Description: Natural killer (NK) cells are known to play a pivotal role in regulating immune defense against tumors and viruses. However, very few studies describe the effects of natural products or their derivatives on NK cell function. We previously showed that the natural product lignan phyllanthusmin C enhances human NK cell IFN-gamma production. In this study, we successfully synthesized dozens of phyllanthusmin derivatives and screened their activities to enhance IFN-gamma production by NK cells. We found that three of these derivatives possessed capacities to induce IFN-gamma production by NK cells. Among them, derivative No. 17 had the highest efficacy and was significantly superior (P 〈 0.05) to that of the original phyllanthusmin C. These three phyllanthusmin derivatives had no adverse effect on human NK cell survival or proliferation. All derivatives alone or in combination with IL-12 or IL-15 induced human NK cell interferon (IFN)-gamma in comparison with the corresponding vehicle control or cytokine alone (P 〈 0.05). These derivatives stimulated IFN-gamma production in both CD56bright and CD56dim human NK cell subsets (P 〈 0.01). Mechanistically, immunoblotting assays and chromatin immunoprecipitation (ChIP) analyses combined with promoter-reporter luciferase assays revealed that these phyllanthusmin derivatives induced the phosphorylation of NF-κB and STAT3, resulting in their increased binding on theIFN G promoter, which was dependent on the Toll-like receptor (TLR) 1 and the TLR3 signaling pathways, respectively. STAT3 knockdown with lentivirus shRNA and inhibition of NF-kB signaling with a specific inhibitor (TPCK) significantly attenuated IFN-gamma production induced by these phyllanthusmin derivatives in human NK cells (P 〈 0.05). Blockade of TLR1 or TLR3 with their corresponding neutralizing antibodies nearly abolished activation of NF-κB or STAT3 as well as IFN-γ induction by the phyllanthusmin derivatives (P 〈 0.05). In conclusion, we have successfully synthesized and screened phyllanthusmin derivatives and identified three compounds that induce IFN-gamma production in human NK cells, one of which had optimal potential. The induction of human NK cell IFN-gamma production by phyllanthusmin derivatives is mediated by TLR1-NF-κB and TLR3-STAT3 signaling pathways, rendering their potential for use as agents for cancer prevention or treatment. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3498 During cell cycle progression, D class cyclins activate cyclin dependent kinases (CDK) 4 and 6 to phosphorylate and inactivate Rb, allowing E2F-1 mediated transcription of additional cell cycle genes including cyclin E to drive S phase entry. This critical pathway is nearly universally dysregulated in cancer, providing tumor cells a strong growth advantage and escape from normal mitotic control. Substantial research is being directed toward targeting this pathway in many cancer types, with some preliminary successes being achieved with pharmacologic inhibitors of CDK4/6. However the development of alternative strategies to block this pathway could potentially provide broad therapeutic benefit. A prime example of a tumor with a disrupted cyclin D axis is Mantle Cell Lymphoma (MCL), in which the t(11;14) translocation places CCND1, the gene for cyclin D1, under the control of an immunoglobulin promoter. This results in sustained cyclin D1 expression in tumor cells and concomitant Rb inactivation, S phase entry and cell division. MCL is a relatively uncommon subset of Non-Hodgkin Lymphoma, but accounts for a disproportionate number of deaths. Treatments are limited and relapse is nearly universal; thus, new treatment strategies are essential for this disease. Silvestrol is a structurally unique, plant-derived cyclopenta[b]benzofuran with potent in vitro and in vivo anti-tumor activity in several model systems including B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). Silvestrol inhibits the initiation step of translation by preventing assembly of eIF4A and capped mRNA into the eIF4F complex, leading to selective loss of short half-life proteins such as Mcl-1 and cyclin D1. We therefore hypothesized that silvestrol, through the depletion of cyclin D1, would demonstrate efficacy in MCL. Silvestrol showed low nanomolar IC50 values in the JeKo-1 (13 nM), Mino (17 nM) and SP-53 (43 nM) MCL cell lines at 48 hr (MTS assay; cell death confirmed by propidium iodide flow cytometry). This potency was similar in primary MCL tumor cells. Longer exposure times substantially improved the cytotoxicity of silvestrol assessed at 48 hr (approximately 50% effect achieved with a 16 hr exposure vs. 80% effect with a 24 hr exposure), suggesting that the cellular impacts of this agent increase with exposure time. Cyclins D1 and D3 were dramatically reduced in MCL cell lines with just 10 nM silvestrol at 16 hr (cyclin D2 was undetectable in these cells), with subsequent loss of Rb phosphorylation as well as cyclin E mRNA and protein, culminating in G1 cell cycle arrest. Similar to what we previously showed in CLL and ALL cells, silvestrol treatment under these conditions also caused loss of Mcl-1 protein with concurrent mitochondrial depolarization, although the exact mechanism of silvestrol-mediated cytotoxicity in these cells is still under investigation. In an aggressive xenograft mouse model of MCL, silvestrol produced a highly significant improvement in survival [median survival of vehicle vs. silvestrol treated mice (1.5 mg/kg every 48 hr) = 27 vs. 38 days; P

Description: Abstract 2616 Despite the progress made in the diagnosis, classification, prognostication and treatment of AML, the majority of patients still fail to achieve long-term survival and die of their disease. Several cytogenetic and molecular markers including gene mutations and aberrant expression have been shown to impact the outcome of AML patients and some of these are also potential therapeutic targets. Receptor tyrosine kinases (RTKs) regulate cell survival, proliferation and differentiation in normal hematopoiesis. Gain-of-function mutations of genes encoding RTKs, such as internal tandem duplication of FLT3 (FLT3-ITD) and mutations of KIT, result in constitutive tyrosine kinase activity and have been associated with poor outcome in distinct cytogenetic (i.e., normal karyotype and core binding factor, respectively) subsets of AML. In addition, overexpression of FLT3 and wild type (wt) KIT was also found to negatively impact outcome. Tyrosine kinase (TK) inhibitors (Is) (e.g., imatinib, dasatinib or PKC412) have been shown to suppress the aberrant activity of these mutants, but their activities as single agents have been limited by their relatively elevated IC50 values for myeloid blasts and early onset of resistance. Indeed, even in combination with chemotherapy, TKIs have not yet been shown to impact favorably on AML patient outcome. This supports the need for development of novel strategies that effectively interfere with aberrant TK activity in leukemia blasts. Silvestrol, a cyclopenta[b]benzofuran derivative isolated from Aglaia foveolata, a plant that grows in the tropical rain forests of Southeast Asia, has been shown to exhibit potent inhibitory activity against a panel of human cancer cell lines and shows in vitro and in vivo efficacy in B-cell malignancies. The mechanism of action has been thought to involve inhibition of translation initiation, which in turn results in selective depletion of actively translated, short half-lives proteins. We hypothesized that silvestrol-mediated inhibition of the translation of mutated and/or overexpressed RTKs results in a significant antileukemic activity in AML cells that rely on the aberrant TK activity of these proteins. Silvestrol showed antiproliferative and proapoptotic activities in representative AML cell lines harboring activated TK proteins: FLT3-ITD-positive MV4-11, FLT3-overexpressing THP-1 KIT-mutated Kasumi-1 and also BCR-ABL-positive K562, an erythroblastic cell line notoriously resistant to chemotherapy. Importantly, silvestrol treatment reduced the expression levels of FLT3 in MV4-11 (92 %) and THP-1 (84 %), KIT in Kasumi-1 (46 %), and BCR-ABL in K562 (64 %), compared to the respective vehicle-treated controls. The silvestrol IC50 at 48 hr was less than 10nM for all cell lines (MTS assay). Silvestrol also showed antiproliferative activity in FLT3-ITD-positive AML patient blasts (IC50 24 nM at 48 hr). To evaluate in vivo activity, NOD-SCID Gamma (NSG) mice were engrafted with FLT3-ITD-positive MV4-11 cells. Twenty-one days post-engraftment, mice were treated intraperitoneally with vehicle or silvestrol (1.5 mg/kg every 48 hr for 3 weeks). After 3 doses (day 6 of treatment), spleens were obtained from three mice from each group. Spleens from silvestrol treated mice were 60% decreased in size (P=0.016) and showed 80% FLT3 protein downregulation (P=0.002) compared to vehicle-treated controls. Silvestrol treated leukemic mice (n=10) survived significantly longer than vehicle treated control (n=6) (median survival: 55.5 days vs. 29 days from engraftment day respectively; P

Description: Abstract 104 Epstein-Barr virus (EBV) is an oncogenic human herpes virus that infects more than 90% of people worldwide and is associated with a broad spectrum of malignant lymphoproliferative disorders (EBV-LPD), and nasopharyngeal and gastric carcinomas. Chemotherapy often leads to prolonged immune suppression, development of opportunistic infections, including EBV reactivation, and increased risk of relapsed disease. The poor prognosis of EBV+ diseases makes it essential to identify novel agents that can deliver direct anti-tumor activity while preserving innate and EBV-specific adaptive immune surveillance. Silvestrol is a cyclopenta[b]benzofuran derived from the plant genus Aglaia and has been shown to possess potent anti-tumor activity against hematologic and solid tumors. Silvestrol interferes with the translation of mRNA with complex 5' untranslated regions often found on pro-survival oncoproteins. Silvestrol exhibits anti-tumor activity against malignant B-cell lines while causing minimal toxicity to peripheral blood mononuclear cells (PBMC) and resting T cells. To examine the potential selective anti-tumor activity and functionally address the effects of silvestrol on adaptive and innate immune function, we utilized in vitro and in vivo EBV lymphomagenesis models. Fully transformed EBV+ Lymphoblastoid lines (LCL) were derived from EBV-LPD tumors of severe combined immune deficient (SCID) mice engrafted with PBMC from EBV-positive donors (hu-PBL-SCID). EBV-LCLs were plated in the presence of silvestrol (2 – 50nM) and proliferation (MTS assay) and apoptosis (Annexin V/PI) was evaluated (24, 72, 120hr). While silvestrol showed potent anti-proliferative activity at these concentrations, we observed minimal cell death (IC50 = 10nM). The anti-proliferative activity of silvestrol was associated with loss of LMP-1 expression, an EBV oncogene essential for B-cell transformation. Examination of LMP1 induced pathways showed decrease in pAkt levels and an increase in NFkB/p65 levels (total and phospho). To examine the functional consequences of silvestrol on immune surveillance, we utilized a co-culture system where EBV+ LCLs are plated in the presence of autologous PBMCs. Autologous LCL/PBMC co-cultures were plated with silvestrol or DMSO vehicle control and allowed to incubate for 10 days. When EBV+ LCLs are irradiated, expansion of memory EBV-specific CD3+/CD8+ cytotoxic T cells (CTLs), capable of cytotoxicity and IFNg production, is observed. The addition of silvestrol (2–10nM) to co-cultures did not hinder CTL expansion or IFNg production. When unirradiated LCLs were plated in co-cultures, CTL expansion was seen, however, EBV+ LCLs became the dominant population in control conditions by day 10 of culture. Addition of silvestrol to unirradiated co-cultures (2–10nM) led to marked expansion of memory CD3/CD8+ CTLs as well as CD56br NK cells. A dose dependent ablation of viable EBV-LCLs was observed in unirradiated co-cultures supporting the notion that silvestrol allowed for the expansion of both innate and adaptive immune effectors that were capable promoting anti-tumor activity. Immune effector populations that expanded in the presence of silvestrol showed preservation of direct cytotoxicity (adaptive immunity) and antibody dependent cell-mediated cytotoxicity assays (ADCC) against EBV+ LCLs (innate immunity) that was comparable to untreated effector populations. We next examined the efficacy of silvestrol in vivo using the Hu-PBL SCID model depleted of murine NK cells. On d14, mice were randomized to receive treatment with either silvestrol (1.5mg/kg) or vehicle control by IP injection every 48hr. Human cell engraftment was measured by quantifying human IgG in blood. While silvestrol treatment did not affect cell engraftment, all treated mice showed improved survival when compared to mice treated with vehicle control (100% silvestrol vs. 22% control alive at day 140, p ≤0.001). Control mice showed decreased body weight (85% decrease) and splenomegally (spleen wt 421.6 mg vs 72.9 mg for control vs silvestrol) on necropsy at day 140, p≤.0004). There were no toxicities observed in silvestrol treated mice. Low dose silvestrol promotes direct anti-proliferative activity of EBV-transformed LCLs while sparing antigen specific adaptive and innate immune effector function that is capable of delivering potent anti-tumor activity in vitro and in vivo. Disclosures: No relevant conflicts of interest to declare.

Description: 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.

Description: Although successful pregnancy is not an uncommon outcome in patients with homozygous beta thalassemia over the last 20 years, most of these have been reported in case reports and small series, while many publications do not appear to distinguish between pregnancies in thalassemia intermedia and those in thalassemia major. For example, of the total of 150 pregnancies in women with thalassemia reported in the literature from 1984 to 2004, 74 pregnancies appear clearly to be documented in patients with thalassemia major, while in the other 76, a reportedly late onset of transfusions suggests a milder phenotype of thalassemia intermedia. We examined the records of all thalassemia patients managed at the Toronto General Hospital, Canada (n = 82 patients with thalassemia major; 23 patients with thalassemia intermedia; 7 patients with hemoglobin E thalassemia = 112 patients over 18 years, of whom 54 are female) to determine the incidence and complications of pregnancy in our population. A surprisingly high number (17) and proportion (31%) of all female thalassemia patients over age 18 years have attempted to become pregnant. A total of 14 (82% of those who attempted pregnancy) of patients (9 thalassemia major; 4 thalassemia intermedia; 1 Hemoglobin E thalassemia) successfully conceived 26 times. The maternal age at first attempt to become pregnant was 29.6 ± 4.9 (range 18–37) years; average age at delivery was 27.2 ± 4.1 (range 19–33) years. Of the 26 conceptions, 20 were carried to term; 19 live births resulted. Six abortions and one fetal death occurred. Deferoxamine was avoided in all patients during pregnancy; control of body iron was followed using hepatic iron concentration. Pre-pregnancy hepatic iron concentration, obtained 2.8 ± 1.8 (range 1–7) years prior to conception, was 9.3 ± 7.9 (range 0.9–31) mg/gram dry weight; post partum hepatic iron concentration, obtained 3 ± 3.2 (range 0.2–10) years following parturition, was 17.4 ± 9.6 (range 1.25–32.5) mg/gram dry weight. There were one reported significant worsening in cardiac function during pregnancy; diabetes (present in one woman prior to pregnancy) developed in another woman during pregnancy. Other complications occurring or worsening during pregnancy included immune thrombocytopenic purpura and post- transfusional thrombocytopenia. In 13 of the 58 men in this population, 13 (8 with thalassemia major; 4 with thalassemia intermedia and one with Hemoglobin E thalassemia) fathered 20 children at 33.5 ± 4.8 (range 24–44) years. These data report the largest North American series of pregnancy in thalassemics and their partners and while raising issues of the safety of pregnancy in thalassemic women offer encouragement to those patients managed with adequate control of body iron burden.