ALBERT

Description: The IgVH mutation status is an independent predictor of time to treatment and survival in CLL. However, the biological basis of this difference is unknown. Clinical use of the IgVH mutation status as a prognostic marker is limited due to the technical difficulties of assessing this feature. As such, we probed for differences in protein expression by CLL cells with unmutated (UM) vs mutated (M) IgVH using phage peptide display libraries (PDPL). Methods: PDPL consists of phages engineered to express random peptides (7-mer) on the coat protein. From approximately 2x1011 phages displaying all possible permutations of 7-mer peptide, we selected phages binding to UM cells by incubation with 106 UM cells and elution of bound phages. Then, from numerous phages initially binding to UM cells, we subtracted phages binding to M cells by multiple incubations with M cells (106). Thus, the phages that bound only to UM cells were selected. The procedure was repeated multiple times to increase selection specificity. The peptide sequences displayed by selected phages were evaluated. We identified phage targets on UM cells by immunoprecipitation (IP) of CLL protein with specific phage and by sequencing of precipitated protein. The expression of target protein in CLL cells was confirmed by immunobloting and flow cytometry. Results: Sequencing of phages from the 3rd round of selection (UM v. M) revealed several peptides, with the most predominant being FPSAHFL. IP identified the cellular target for FPSAHFL as vimentin, an intermediate filament protein involved in cell motility and activation. The level of vimentin expressed by CLL cells varied between patient samples (n=15). UM CLL cases were found to express higher levels of vimentin (the mean MFI was 1002; SD 381), than cases of M CLL (mean MFI 547; SD 109), p=0.0128. To evaluate the prognostic significance of vimentin, we assessed vimentin expression by flow cytometry in a separate subgroup of 40 CLL patients. Archived cell samples collected between 6/2000 – 6/2001 were used. The median follow up was 81 months (40–233). Vimentin expression (MFI) as a continuous variable was a risk factor for a shortened time to treatment (TTT) with a proportional hazard ratio of 4.152 (95% CI 1.33–15.220, p=0.0317). Using MFI closest to the median (and rounded to 2 decimal places) as a cutoff, we stratified patients in high (n=21) and low (n=19) vimentin expression groups. The median times to treatment (TTT) in patients with high vs low level expression of vimentin were 2.8 and 12.9 years respectively, p=0.0025 (Fig.). Conclusion: Using novel proteomic technology, we found that CLL cells with unmutated IgVH expressed higher levels of vimentin than CLL cells with mutated IgVH. A high-level of vimentin expression defined by flow cytometry appears to be associated with high-risk disease. Validation of these findings could allow for the vimentin level to be used as a surrogate marker for expression of unmutated IgVH in CLL. Figure Figure

Description: We recently reported that chronic lymphocytic leukemia (CLL) cells synthesize and release vascular endothelial growth factor (VEGF) under normoxic and hypoxic conditions. CLL B cells also express VEGF membrane receptors (VEGF-R1 and VEGF-R2), suggesting that they use VEGF as a survival factor. To assess the mechanism of apoptosis resistance related to VEGF, we determined the impact of VEGF on CLL B cells, and we studied the impact of epigallocatechin-3-gallate (EGCG), a known receptor tyrosine kinase (RTK) inhibitor, on VEGF receptor status and viability of CLL B cells. VEGF165 significantly increased apoptotic resistance of CLL B cells, and immunoblotting revealed that VEGF-R1 and VEGF-R2 are spontaneously phosphorylated on CLL B cells. EGCG significantly increased apoptosis/cell death in 8 of 10 CLL samples measured by annexin V/propidium iodide (PI) staining. The increase in annexin V/PI staining was accompanied by caspase-3 activation and poly–adenosine diphosphate ribose polymerase (PARP) cleavage at low concentrations of EGCG (3 μg/mL). Moreover, EGCG suppressed the proteins B-cell leukemia/lymphoma-2 protein (Bcl-2), X-linked inhibitor of apoptosis protein (XIAP), and myeloid cell leukemia-1 (Mcl-1) in CLL B cells. Finally, EGCG (3-25 μg/mL) suppressed VEGF-R1 and VEGF-R2 phosphorylation, albeit incompletely. Thus, these results suggest that VEGF signaling regulates survival signals in CLL cells and that interruption of this autocrine pathway results in caspase activation and subsequent leukemic cell death.

Description: The molecular mechanism of autocrine regulation of vascular endothelial growth factor (VEGF) in chronic lymphocytic leukemia (CLL) B cells is unknown. Here, we report that CLL B cells express constitutive levels of HIF-1α under normoxia. We have examined the status of the von Hippel-Lindau gene product (pVHL) that is responsible for HIF-1α degradation and found it to be at a notably low level in CLL B cells compared with normal B cells. We demonstrate that the microRNA, miR-92-1, overexpressed in CLL B cells, can target the VHL transcript to repress its expression. We found that the stabilized HIF-1α can form an active complex with the transcriptional coactivator p300 and phosphorylated-STAT3 at the VEGF promoter and recruit RNA polymerase II. This is initial evidence that pVHL, without any genetic alteration, can be regulated by microRNA and explains the aberrant autocrine VEGF secretion in CLL.

Description: Chronic lymphocytic leukemia (CLL) represents one of the most common hematological malignancies and is characterized by the expansion of a clonal B cell population. Commonly, CLL is defined as a disease of failed apoptosis of the leukemic B cells, and it is this feature that makes them refractory to common chemotherapeutic agents designed to kill CLL cells. The mechanism controlling apoptosis in CLL is complex and influenced by numerous factors, including NFkB-mediated expression of antiapoptotic proteins, such as Bcl-2 and XIAP. We recently demonstrated that glycogen synthase kinase-3 (GSK-3) b positively regulates NFkB-mediated gene transcription and cell survival in pancreatic cancer cells (Cancer Research 2005. 65:2076). Similar to pancreatic cancer, we have found that pharmacological inhibition of GSK-3 results in decreased expression of NFkB target genes Bcl-2, Mcl-1 and XIAP in CLL B cells. In addition we noted a dose dependent increase in apoptosis levels for both the MEC1 cell line and primary CLL B cell patient samples. Using CLL blood B cells, we have found that both GSK-3b and NFkB accumulate in the nucleus of CLL cells, but not normal blood B cells. In addition, we observed that inhibition of GSK-3 leads to a decrease in NFkB-mediated gene transcription, without affecting the nuclear accumulation of NFkB. However, using chromatin immunoprecipitation, we found that NFkB binding to its target gene promoters (XIAP, Bcl-2) is significantly affected in CLL B cells treated with a GSK-3 inhibitor. These results suggest that GSK-3 regulates the nuclear activity of NFkB in CLL cells by affecting the binding of p65/p50 to the promoters of a subset of its target genes. Our results establish that inhibition of GSK-3 induces apoptosis of CLL cells and suggest GSK-3b as a potential therapeutic target in the treatment of CLL.

Description: BACKGROUND: We have demonstrated the green tea extract epigallocatechin-3-gallate (EGCG) has anticancer activity in primary CLL B-cells (Lee, Blood 2004). After dissemination of our in vitro findings by the lay press, many patients with CLL and other low grade non-Hodgkin lymphomas (NHL) began using over the counter green tea extracts as an alternative treatment strategy. We recently reported a case series of 3 patients with CLL and 1 patient with follicular lymphoma who appeared to derive objective clinical benefit from such treatment (Shanafelt, Leukemia Research 2006). Based on these findings EGCG has entered clinical testing for treatment of CLL at Mayo Clinic. Here we explore the in vitro antitumor activity of EGCG against other types of non-Hodgkin lymphoma. METHODS: Five established human B-cell lymphoma cell lines (HT, DOHH2, KARPAS, Ramos, RL) and primary lymphoma cells from 7 patients with various B-cell NHL sub-types [DLBC, FL, SMZ (2), MCL, SLL(2)] were used to evaluate the in vitro sensitivity of human lymphoma cells to EGCG. Freshly isolated primary lymphoma cells harvested in suspension from lymph nodes/spleen were obtained from patients with NHL who provided written informed consent. All patients were untreated at the time of biopsy. Lymphoma cell lines and primary lymphoma cells (n=7) were cultured with increasing doses of purified EGCG (3.12–50 ug/ml) for 24–72 hrs. Viability was assessed by using annexin/PI staining by FACS analysis. RESULTS: EGCG-induced dose dependent cell death in both established human B-cell lymphoma cell lines (average LD50 at 24 hrs between 25–50 ug/mL) and primary NHL cells (average LD50 at 24 hrs between 25–50ug/mL). In contrast, EGCG had minimal effect on purified normal B-cells (n=3) at the highest doses tested (50 ug/mL). By immunoblotting, EGCG-induced death in primary cells and cell lines was associated with PARP cleavage, suggesting the agent induced apoptotic cell death. Despite this finding, EGCG had no effect on levels of MCL-1, XIAP, or Bcl-1 by either immunoblot or FACS analysis. Based on reports that EGCG induces cell death in some cancer cell types through generation of oxidative stress (Furukawa, 2003; Nakazato, 2005), we explored this mechanism in lymphoma cells. To determine whether reactive oxygen species (ROS) generation was necessary for EGCG-induced cell death, lymphoma cell lines were cultured with or without catalase (which catalyzes the conversion of hydrogen peroxide to water and oxygen) for 30 min prior to subsequent 24 hr EGCG exposure (50 and 100 mg/ml). Pre-treatment with catalase (100 U) provided dramatic protection against cell death in both primary NHL cells and NHL cell lines suggesting that EGCG-induced cell death in lymphoma cells is dependent on ROS generation (Fig. 1 shows an example for a patient with mantle cell lymphoma and a patient with splenic marginal zone lymphoma). CONCLUSION: EGCG has in vitro anti-tumor activity against a variety of B-cell NHLs. Given its known favorable toxicity profile in vivo, EGCG is an attractive agent for clinical testing in patients with indolent NHL who otherwise are currently being managed with observation. Figure Figure

Description: CLL B cells have significant opportunity to interact with multiple tissue sites that are critical to the leukemic cell’s ability to survive. The marrow stromal elements (MSE) in B-chronic lymphocytic leukemia (B-CLL) are very likely one of the most frequently involved in this important cell-cell interaction. We have utilized a novel bone marrow biopsy technique (Alvi S, Leuk Res, 2001) that consistently generates robust and long-lived marrow stromal elements from B-CLL patients in order to study CLL B cell apoptosis status in relation to angiogenic factors. The latter angiogenic parameters were studied as we have previously detected both a VEGF based autocrine pathway for CLL B cells and found an extensive neovascularization in CLL marrows. Bone biopsy material from B-CLL patients (N=25) yielded active cell growth in vitro which expresses the features of marrow stromal constituents. Importantly, these marrow constituents were able to be sustained for several months (range, 3 – 52 weeks) and also could be transferred repetitively to new culture dishes. CLL B cells were able to spontaneously release VEGF and TSP-1 as we have previously published (Kay N, Leukemia, 2002). MSE from CLL marrow (N=5) were found to spontaneously secrete basic fibroblast growth factor (bFGF) 5.2 ± 0.9 pg/ml (mean ± one SEM), vascular endothelial growth factor (VEGF) 162 ± 48 pg/ml and thrombospondin-1 (TSP-1) 90 ± 26 ng/ml into the culture medium. Significant reduction in CLL B cell spontaneous apoptosis,measured by Annexin/PI incorporation (mean ± one SEM), was seen after exposure to the CLL MSE (N=6) both after 24 hours (i.e., Non exposed CLL B cells vs. MSE co-cultured CLL B cells was 70% ± 9 vs. 39% ± 10 respectively for 24 hours, p 〈 0.01) and 72 hrs (data not shown). Exposure to MSE also enhanced the in vitro drug resistance of CLL B cells to chlorambucil (C). Thus, for CLL B cells (n=5) cultured with CLL MSE and C (1μM) for 24 hours, apoptosis levels were reduced from 58.8% ± 7.8 for B cells without MSE compared to 39.8% ± 3.8 for B cells exposed to MSE. Apoptosis resistance enhancement for CLL B cells was found to be similar with normal marrow stromal elements and a stromal cell line (HS-5). Anti-apoptotic proteins detected by immunoblot were found to increase for CLL B cells exposed to stromal elements from bone marrow biopsies and included Mcl-1, Bcl-2, Survivin and XIAP. While Mcl-1 was found to increase, there was a decrease in Bcl-2 and Survivin detected by immunoblot when CLL B cells were exposed to the HS-5 cell line implying a different mechanism for apoptosis enhancement. When CLL B cells were added to CLL MSE for 24 hours, there was an increase in VEGF released into the culture medium (283 pg ± 114 for the co-culture vs. 14 pg ± 1.7 and 162 pg ± 48.8 for CLL B cells and MSE cultured alone respectively). In contrast co-culture of CLL B cells with MSE (24 hours) resulted in less TSP-1 in the culture medium than expected (106 ng ± 28.8 for co-cultured CLL and MSE cells vs. 89.5 ng ± 27.5 and 90.9 ng ± 26.4 for CLL B cells and MSE cultured alone respectively). This change in the ratio of pro- to anti-angiogenic factors favoring VEGF strongly suggests that CLL B cell interaction with stroma can facilitate an angiogenic switch and may be linked to disease progression. In addition, we have found that bone biopsies from B-CLL patients can generate long-term marrow stromal elements that are able to reliably enhance CLL B cell apoptosis resistance.

Description: We and others previously reported that leukemic CLL cells spontaneously secrete VEGF and that VEGF can enhance the leukemic B cell apoptosis resistance via unknown mechanisms. Here, we report elevated VEGF secretion is associated with CLL B cell over-expression of hypoxia inducible factor-1 alpha (HIF-1α), a transcription factor that is a key regulator of VEGF synthesis. Immunostaining analysis confirmed CLL B cells in blood and bone marrow over-expressed both HIF-1α and VEGF. The high HIF-1α levels were not linked to proline residue (402/564) mutations in oxygen dependent degradation domains; absence of proline hydroxylation dependent enzymes (PHD1, 2, 3, and FIH); or to pVHL mutation (exon 1, 2 and 3); or aberrant transcription of HIF-1α. However, pVHL, a regulator of HIF-1α degradation, was found to be lower in CLL B cells and unable to physically interact with HIF-1α in CLL B cells. Nuclear extraction assay revealed that HIF-1α interacted with its co-factor, p300/CRP in B cells, indicating that HIF-1α was transcriptionally active. We hypothesized that reduction of pVHL expression in CLL B cells could be related to repression by micro RNA function which is known to be aberrantly expressed in CLL. We focused on miR-92-1 known to be over-expressed in CLL B cells and it has pVHL as a putative target. In a luciferase assay of transfected human megakaryocytic cell line, MEG-01, we found a direct effect for miR-92-1 on pVHL, with significant repression of luciferase activity of VHL expression by miR-92-1 compared to both control vectors and a mutated target mRNA sequences. This result indicated that wild-type miR-92-1 was able to interact directly with the 3′ UTR sequence of pVHL and subsequently repress pVHL expression. To further confirm this finding, we did transient transfections of either the CLL cell line Mec-1, primary CLL B cells or normal B cells with a sense oligo miR-92-1 (both Mec-1 and CLL B cells) or wild-type miR-92-1 plasmid (normal B cells). We found the level of flow detectable pVHL lower when compared to non-transfected cells. Because we were interested in the relationship of HIF-1α to CLL B cell survival we studied if chemotherapeutic drugs can downregulate HIF-1α in CLL B cells. To do this we studied flavopiridol a drug that has very promising activity in CLL. Our results indicated that flavopiridol does down-regulate HIF-1α levels in CLL B cells. This latter change was associated with induction of apoptosis in CLL B cells. Overall, our data show that elevated VEGF secretion is related to constitutively elevated HIF-1α levels and is an explanation for the VEGF-based autocrine pathway found in CLL B cells. We also show that elevated HIF-1α levels could be partially related to depressed pVHL with a unique mechanism for its repression: the over-expressed microRNA, miR-92-1 in CLL-B cells.

Description: Background: We have previously shown that CLL B cells secrete vascular endothelial growth factor (VEGF) and express VEGF receptors: VEGFR-1 and VEGFR-2. Secreted VEGF protects CLL B cells from spontaneous and drug induced apoptosis via increased levels of Mcl-1 and XIAP; however, the exact mechanism of this process is unknown. In solid tumors, there is increasing evidence that signaling through VEGF receptor known as neuropillin-1 (NRP-1) is critical for VEGF induced resistance to apoptosis. Hypothesis: NRP-1 is expressed by CLL B cells and is critical for VEGF mediated protection from apoptosis. Methods: To demonstrate the presence of NRP-1 on CLL B cells, we conducted flow cytometry and immunoblotting. We then evaluated the ability of NRP-1 blocking antibodies to induce apoptosis of primary CLL B cells. To do this, circulating CLL B cells were isolated by density gradient centrifugation. Patient samples with greater than 80% of CD5+ CD19+ cells in a mononuclear cell population, as assessed by flow cytometry, were cultured in AIM-V media in 24-well plates at 1.5 x 106 cells/mL. To occupy NRP-1, we added NRP-1 blocking antibodies (Calbiochem, Darmstadt, Germany) at increasing concentrations (0.5 μg/mL -10 μg/mL) to cultured CLL B cells. Cell death was assessed using an annexin and propidium iodide flow assay after 24 h of in vitro culture. CLL cells cultured without antibodies and isotype nonspecific monoclonal antibodies were used as controls. Results: CLL B cells were found to express NRP-1 but not uniformly. Most patients had flow positive evidence for NRP-1 but a distinct percentage (35%) was very low (≤5%) or negative. However, immunoblot analysis revealed moderate to low levels of NRP-1 protein with evidence of tyrosine phosphorylation in all tested CLL patients (N=9). NRP-1 blocking antibodies, but not VEGF-R1 and -R2 blocking antibodies, induced apoptosis in a dose dependent manner in primary CLL cells (n=5, FIG.) at antibody concentrations starting at 1 μg/mL (p=0.003). The effect of the NRP-1 blockade varied between patients, with a median IC50, 1.5 μg/mL (range 0.5–2 μ/mL). Importantly, concentrations of 5 μg/mL and higher induced apoptosis in more than 90% of the CLL B cells. We also found that the sensitivity of CLL B cells to NRP-1 blocking antibody, in terms of apoptosis induction, was correlated with the number of NRP-1 receptors as assessed by flow cytometry. CLL B cell clones with no detectable NRP-1 had no induction of cell death when exposed to the NRP-1 blocking antibody. Finally, immunoprecipitation and immunoblot assays indicated that NRP-1 physically interacted with VEGF-R2 on CLL B cells. This suggests that NRP-1 could be enhancing VEGF binding affinity on VEGF-R2 to further increase the ability of VEGF to generate signals that lead to apoptosis modulation. Conclusion: We have found that NRP-1 blocking antibodies induce cell death in NRP-1 positive CLL B cells. Similar results using NRP-1 blocking peptides rather than blocking antibodies have been observed in breast cancer (Br J Cancer.2005 Jan 31;92(2):328–33). Our results suggest that NRP-1 represents an attractive therapeutic target in CLL and should be explored further. Figure Figure

Description: We have found that CLL B cells spontaneously secrete vascular endothelial growth factor (VEGF) and that a VEGF autocrine pathway can induce apoptosis resistance in these cells. Recently, we also found that hypoxia-inducible factor-1 alpha (HIF-1α) is highly expressed in CLL B cells. Since this protein is a potent transcription factor for the induction of VEGF, we were interested in further definition of HIF-1α regulation and its function in CLL B cells. CLL blood B cells overexpress HIF-1α protein but not mRNA for HIF-1α compared to normal blood and splenic B cells. Immunohistochemistry (IHC) showed that circulating blood CLL B cells and a subset of CLL marrow cells uniformly express HIF. Hypoxic conditions (i.e., 1% O2) did not increase the protein levels of HIF-1α nor mRNA for HIF-1α in CLL B cells, indicating that the high HIF-1α protein level is due to post-translation modification. Blockade of signaling pathways known to increase HIF-1α levels also did not alter the high levels of HIF-1α in CLL B cells. IHC and nuclear extraction assay demonstrated that HIF-1α was predominantly located in the CLL B cell nucleus. In addition, the nuclear extract when immunoprecipitated for HIF-1α was shown to be complexed with the co-activator p300, indicating that HIF-1α is transcriptionally active. Co-immunoprecipitation assay showed that HIF-1α from CLL B cells does not associate and form a complex with von Hippel-Landau protein tumor suppressor (pVHL), indicating that the proteasome dependent degradation pathway for HIF-1α protein in CLL B cells is dysfunctional. Using immunoblot or IHC methods, we were unable to detect pVHL protein in CLL B cells; however, we were able to use immunoprecipitation of CLL B cell lysates to demonstrate there is pVHL in CLL B cells. Prolyl hydroxylases (PHD 1, 2, and 3) are negative regulators for HIF-1α via hydroxylation of amino acid prolines in the oxygen degradation domain (ODD) which permits interaction with pVHL. RT-PCR results revealed that there is a subset of CLL patients who had ≥ 50% reduction of PHD 1 and 3 mRNA levels. However using a hydroxylation specific polyclonal antibody we found that HIF-1α from CLL B cells is indeed hydroxylated. Finally, silencing of HIF-1α by RNA interference in CLL B cells was associated with a selective decrease in VEGF mRNA levels but not VEGF-R1, Mcl-1 and prolyl hydroxylases (PHD 1–3) other downstream target genes of HIF-1α. These data show that the high endogenous HIF-1α levels in CLL B cells are due to a defect in HIF-1α degradation via the proteosomal pathway. We believe that this abnormality is linked to the autocrine VEGF pathway in CLL B cells and ultimately results in increases in their apoptotic resistance. Inhibition of HIF-1α levels may be of therapeutic benefit to CLL patients.

Description: The interactions between leukemic cells and the bone marrow microenvironment play a critical role in angiogenesis, disease progression and cell adhesion mediated drug resistance (CAM-DR). VLA-4 (CD49d) is one of the most important adhesion receptors involved in these interactions. The binding of VLA-4 to fibronectin was shown to protect CLL B-cell from apoptosis (de la Fuente, 1999). The introduction of monoclonal antibodies targeting VLA-4 provides the opportunity for the development of novel treatment strategies. We examined the ability of VLA-4 blocking antibodies to induce apoptosis and overcome CAM-DR of primary B cells from CLL patients. Methods: Blood samples were obtained from consenting CLL patients. Mononuclear cells were isolated by density gradient centrifugation. Only samples with over 80% CLL B cells as assessed by flow cytometry (minimum 80% CD19+/CD5+ cells) were used. HS-5 human stromal cell line was cultured in 24 well plates (10% FCS DMEM media) and grown until they reached confluence. The confluent cells were then washed with PBS and incubated with AIM-V media 24 hours before CLL cells were co-cultured. CLL B cells were incubated in AIM-V media with and without fludarabine (1 mmol/L) for 24 hours, spun and resuspended in AIM-V. CLL B cells were then incubated with or without anti VLA-4 antibodies (BD Biosciences) at 10 μg/106 CLL cells for 2 hours and directly added into a co-culture with HS-5 cells. The CLL B cells remained in the co-culture for 24 hours, and were then collected and analyzed by flow cytometry for apoptosis/cell death by annexin/propidium iodide assay (i.e., total culture time 48 hours). Paired T-test was used to compare B cell viability results. Results: After 48 hour culture, the viability cells cultured in media alone or fludarabine for the first 24 hours were 40.6% (18.2%–73.7%) and 8.5% (0.2%–17.9%) respectively. Significant rescue from fludarabine induced cell death was seen if CLL B cells were co-cultured with HS-5 stromal cells for the second 24 hours rather than media alone (86.5% vs. 8.5%; p