ALBERT

Description: Abstract 2358 Poster Board II-335 Background: Chronic Lymphocytic Leukemia (CLL) is a common incurable hematologic malignancy. When therapy is required, maximizing durable responses is often at the risk of increasing toxicity. Thus, developing novel therapeutic agents that have minimal overlapping toxicity with currently used chemotherapy would be advantageous. To this end, we investigated LMP-420, a boronic acid containing purine nucleoside analogue, that potently inhibits tumor necrosis factor alpha (TNF) transcription in stimulated peripheral blood mononuclear cells (PBMCs) without affecting cell viability. Since TNF has been implicated in promoting CLL cell viability and can be produced by CLL cells themselves, we hypothesized that LMP-420 would be cytotoxic for CLL lymphocytes, either alone or in combination with fludarabine. Methods: To test the activity of LMP-420, we negatively selected circulating CLL cells from blood collected from patients using the RosetteSep B-cell enrichment cocktail (StemCell Technologies) and a Ficoll-Hypaque gradient. This method yields greater than 95% purity of malignant lymphocytes, determined by immunophenotyping CD5+CD19+ cells. Prognostic markers such as IgVH mutation status, CD38 and ZAP70 expression, and interphase cytogenetics were determined as previously described. We assessed the fractional toxicity and 50% effective dose (ED50) of LMP-420, fludarabine, or the combination, with the MTS colorimetric cytotoxicity assay, in which CLL cells were incubated for three days in Hybridoma media + 10% fetal bovine serum with serial dilutions of drug alone or in combination. Apoptosis was measured via annexin V flow cytometry methods and caspase 3/7 activity assays. We determined the effect of LMP-420 compared to fludarabine on the normal hematopoietic system by testing serial dilutions of both agents in killing normal PBMCs and in suppressing erythroid and myeloid colony formation. Results: The median ED50 of LMP-420 for CLL cells was 423 nM (range 0.01 to 2224 nM, n = 21). Two patients had high-risk cytogenetics (17p or 11q deletions), and their ED50 values for LMP-420 were 691 and 90 nM, respectively. The cytotoxic effect of fludarabine was potentiated on average 80 or 261 fold with the addition of LMP-420 at concentrations of 62 or 250 nM, respectively (ranges 1.14 – 947 and 1.19 – 2754). This agent killed malignant lymphocytes by apoptotic mechanisms in a dose-responsive fashion, as demonstrated by both Annexin V staining and caspase 3/7 activity assays. While LMP-420 has potent anti-CLL activity, it has minimal effects on normal hematologic cells. For example, fludarabine suppresses erythroid and myeloid colony formation by greater than 50% at a concentration of 1 uM, while this level of inhibition is seen for LMP-420 at a concentration of 90 uM. The average cytotoxic ED50 of LMP-420 on normal PBMCs using the MTS assay was greater than 90 uM, whereas for fludarabine, it was 5.3 uM. This finding was confirmed with apoptosis assays. Conclusions: The results of these experiments demonstrate that LMP-420, a novel inhibitor of TNF expression, has cytotoxic activity against CLL cells, including those with high-risk features. LMP-420 appears to increase the cytotoxic effect of the chemotherapy agent fludarabine, while imparting minimal increase in hematologic toxicity. Thus, LMP-420 is promising new therapeutic agent in CLL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2850 Background: Upon encountering cognate antigen, naïve B cells may undergo germinal center reaction. This results in DNA modification including somatic hypermutations (HSM) of the immunoglobulin heavy chain variable region gene (IGHV) and immunoglobulin class switch recombination (CSR). HSM and CSR are unlinked, but share initiating and mechanistic factors. Prior evidence indicates CLL B cells are antigen experienced; however, half of all CLL patients have an unmutated IGHV and follow more aggressive clinical courses. The biologic importance of the CLL immunoglobulin isotype and CSR in B cell receptor (BCR)-mediated signaling is not well understood. We hypothesized the immunoglobulin isotype and expression density of surface immunoglobulin would correlate with IGHV mutation status and other clinical parameters, and predict BCR responsiveness in vitro. Methods: 195 samples from our CLL specimen repository with detailed clinical and biologic characterization were evaluated. Surface expression of IgD, IgM and IgG was determined using multi-parameter flow cytometry and samples were classified as either IgG+ or IgM+IgD+ co-expressing. An ROC analysis was performed to identify the most discriminate value for dichotomization of IgM surface expression as a function of IGHV mutation status. IgMhiIgD+, and IgMlowIgD+ patients were then analyzed for associations with clinical and biologic parameters. To investigate the biological mechanisms of potential associations, BCR-mediated signaling after isotype specific crosslinking in vitro was measured by quantification of downstream phospho-proteins (SYK, AKT, ERK, MEK1, NFκB) by flow cytometry (phospho-flow). Samples were crosslinked with IgM (or IgG if IgG+), IgD, and isotype control and incubated for 0, 15, 30, 60, and 120 minutes. The extent of phosphorylation at each timepoint was expressed as mean fluorointensity (MFI) of the examined proteins. Crosslinked samples were also analyzed using oligonucleotide microarray gene analysis (GEP) to assess differential transcription. Results: A correlation was identified between the percentage of CLL cells expressing surface IgM and IGHV mutation status (r2=0.33); higher surface expression of IgM correlated with unmutated IGHV. The most discriminate value of IgM expression for prediction of IGHV mutation was 47%, with an area under ROC curve of 0.72. Of the 195 patient samples analyzed, 91 (47%) were IgMhiIgD+; 85 (43%) were IgMlowIgD+; and 19 (10%) were IgG+. IgMhiIgD+ samples showed increased expression of CD38 (p

Description: B cell chronic lymphocytic leukemia (CLL), the most common subtype of leukemia in the United States of America and in Europe, is treatable but incurable. New drugs are needed for its management. Phosphodiesterase inhibitors (PDEi) block catabolism of cyclic nucleotides resulting in accumulation of cellular cAMP and cGMP. These PDEi also decrease production of inflammatory cytokines such as tumor necrosis factor. Furthermore, they inhibit expression of inducible nitric oxide synthase (NOS2) mRNA and NO production. Researchers have previously noted that nonspecific PDE inhibitors such as theophylline as well as a relatively specific PDE4 inhibitor (rolipram) cause CLL cell death in vitro, while relatively sparing normal peripheral blood mononuclear cells (PBMC). The purpose of this study was to evaluate the effectiveness of CD160130 in the killing of freshly isolated CLL cells in vitro. CD160130 is a novel, orally available heterocyclic pyrimido-indole derivative that is relatively PDE4-specific. Patients were from the V.A. and Duke University Medical Centers, and normal controls were from the community. Control normal PBMC were isolated by ficoll-Hypaque centrifugation, and CLL cells were purified using negative selection with antibodies. We determined cytotoxicity using the MTS colorimetric assay. Samples from 10 CLL patients (6 male and 4 female) and 10 normal controls were examined. Nine of 10 patients were stage 0 at presentation, and 1 was stage 2. They had been followed 6.1 yr (median; range 0.7–19.1 yr). One of 10 was CD38 positive, and 6 of 10 were Zap-70 positive. Of nine analyzed, one had unmutated IgVH gene, and 9 were mutated. Six patients had not been treated, and 4 had been treated with chlorambucil, fludarabine, and/or rituximab. Four had normal cytogenetics by FISH analysis; one had trisomy 12; three had 13q14 del; and one had 17p del. Of seven determined, two had elevated CLL cell lipoprotein lipase mRNA elevated. CD160130 induced cell death in a dose-dependent fashion in all patients’ samples, with a mean cytotoxicity of 96% at 12.5 uM. The mean ED50 for killing was 233 nM in media with FBS and 314 nM in serum-free medium, while that for PBMC was higher at 7500 nM with FBS and 5210 nM with serum-free medium. The agent was 32.2 fold more potent for killing of CLL cells compared to PBMC in medium with FBS and 16.6 fold more potent for CLL cells in serum-free medium. In summary, the PDE4 inhibitor CD160130 potently kills freshly isolated CLL cells in vitro in the presence or absence of serum. The killing is relatively selective for CLL cells compared to normal PBMC. In vivo trials of CD160130 in patients with CLL should help determine the toxicity and efficacy in patients.

Description: Abstract 1842 Perifosine (Keryx Biopharmaceuticals) is an oral alkylphospholipid that has been shown to have clinical activity in multiple myeloma and Waldenstrom's macroglobulinemia. Pre-clinical data suggest that its activity is due to inhibition of the Akt signal transduction pathway. The Akt pathway is known to be important for viability in CLL, another B-cell malignancy. Therefore, we investigated the in vitro activity of perifosine in freshly isolated primary CLL cells. CLL patients at the Duke University and Durham VA Medical Centers were enrolled in an IRB-approved research protocol for blood sample collection. CLL cells were negatively selected using the RosetteSep B-cell enrichment cocktail (StemCell Technologies) and a ficoll-Hypaque gradient. This method yields greater than 95% purity of malignant lymphocytes, determined by flow cytometry. Prognostic markers such as IgVH mutation status, CD38 and ZAP70 expression, and interphase cytogenetics were determined. We found the 50% effective dose (ED50) of perifosine for inducing cytotoxicity in CLL cells after a three-day incubation using the MTS colorimetric assay to be 510 nM (n = 29, range 120 – 1540 nM). CLL cells were obtained from patients with generally poor prognostic markers: 52% CD38+, 93% ZAP70+, 78% IgVH unmutated, 42% 17p deletion, 8% 11q deletion, 27% trisomy 12, 12% normal, and 12% 13q deletion as a sole abnormality. There were no statistical differences in ED50 between cells obtained from patients in high or low risk prognostic groups. Perifosine induced apoptosis in a dose- and time-dependent manner, measured by Annexin V and PI staining (n = 4). Based upon these pre-clinical results, we initiated a phase II study of perifosine (50 mg orally, twice daily) in relapsed or refractory CLL/small lymphocytic lymphoma (SLL) (NCT00873457). The primary objective of this study is to assess the response rate at 3 and 6 months of perifosine treatment in patients with relapsed or refractory CLL/SLL. Secondary objectives are to monitor toxicity, evaluate overall survival, progression-free survival and response duration, and perform laboratory correlates. Early interim results of this study are presented. Since trial initiation in September 2009, 13 patients have been enrolled. Nine patients had Rai stage III/IV disease at the time of therapy, and 4 patients were fludarabine-refractory. Patients had extensive prior treatment (median 4, range 1 – 11). Many patients had high-risk prognostic features: 9/11 IgVH unmutated, 10/13 CD38+, and 11/13 ZAP70+. Evaluation of interphase and metaphase cytogenetics demonstrated 4 patients with 17p deletion, two with 11q deletion, 2 with trisomy 12, 4 normal, and 4 with other complex cytogenetic anomalies. Of 12 patients who began therapy, 5 patients withdrew from the study prior to 3 months, 6 patients received at least 3 months of therapy, and 1 patient completed 6 months of therapy. Of the patients who received at least 3 months of therapy, there were 5 patients with stable disease, and one patient with partial response, using iwCLL response criteria. Grade 3/4 toxicities included anemia (n=2), fatigue (n=2), dehydration (n=1), febrile neutropenia (n=1), hyperbilirubinemia (n=1), hyponatremia (n=1), cough (n=1), and dyspnea (n=1). One patient required a dose reduction to 50 mg daily and two patients required dose delays due to toxicities. In conclusion, perifosine has potent in vitro activity against primary CLL cells. Preliminary results of this ongoing phase II study of oral perifosine in relapsed or refractory CLL/SLL demonstrate mostly disease stabilization in a group of very high-risk patients and an acceptable toxicity profile. Completion of this clinical study is necessary to determine if perifosine monotherapy has a potential role in the treatment of CLL/SLL. Disclosures: Sportelli: Keryx Biopharmaceutical: Employment, Equity Ownership. Weinberg:Keryx Biopharmaceuticals: Research Funding.

Description: Cancer patients with relapsed or refractory disease often require repeated sequential therapies. This approach may induce resistance to conventional chemotherapy and may drive selection for cancer cells that rely on pro-survival signals. Such changes in the molecular constitution of the cancer at the time of each treatment have implications in the drive to personalize cancer therapy. We investigated this phenomenon in Chronic Lymphocytic Leukemia (CLL), a common incurable leukemia that often requires multiple therapeutic regimens over time. Using stored purified CLL cells and serum from a cohort of patients followed at the Duke University and Durham VA Medical Centers, we identified twenty pairs of samples collected from patients prior to and after therapy, and eight pairs of samples collected from patients where no therapy was administered. There were no significant differences in time between paired sample collection or prognostic factors such as Rai stage, cytogenetic aberrations, or IgVH mutational, CD38 or ZAP70 status between these two groups of patients. In the group of sample pairs collected before therapy and upon progression, there was a lower white blood cell count in the second sample (p = 0.04, Wilcoxon signed rank), but no significant change in percentage of cells expressing CD38 or ZAP70 by flow cytometry. The therapies given to patients included alkylating agents alone (14/20), R-CHOP (1/20), Fludarabine-containing regimens (4/20), and single agent-Rituximab (1/20). We profiled gene expression of malignant lymphocytes using Affymetrix U133 Plus 2.0 GeneChips and measured serum levels of circulating cytokines and cytokine receptors from these paired samples in order to identify consistent changes that occurred with therapy. Using supervised analyses of the genomic data, we identified 207 gene probes that were differentially expressed in the twenty pairs of samples where treatment was given. Importantly, these gene probes were not altered in the pairs of samples where no therapy was administered. We next analyzed genomic pathways using gene ontology, Gene Set Enrichment Analysis, and genomic signatures of oncogenic deregulation. We found that after therapy, there is upregulation of genes involved in cellular and nucleic acid metabolism, cell interaction, and signal transduction, with the phosphoinositol 3-kinase and beta-catenin pathways specifically affected. In addition, upregulation of the myc pathway prior to therapy was associated with a shorter duration of response to therapy. Upon studying serum cytokine and cytokine receptor levels in these patients, we found significantly different levels of EGF, EGFR, G-CSF, and RAGE before therapy compared to those on progression of disease. Higher levels of pre-treatment serum cytokines such as GM-CSF and IL-6 were associated with shorter durations of response to therapy. The results of these experiments demonstrate that there are consistent intra- and extra-cellular signals in CLL that are altered after heterogeneous therapies. These signals could be responsible for maintaining leukemic cells despite therapy, and thus are potential targets for future therapies, specifically in the relapsed and refractory patient.

Description: Background and Significance : Even though we have treatments for CLL, it remains an incurable leuke mia. We need new and better treatments for this disease. The Akt kinase is usually constitutively acti vated (phosphorylated) in CLL, and it acts to maintain CLL cell viability. Chromosome deletion at 11q22–23 is frequently seen in CLL, and this cytogenetic abnormality portends a bad prognosis. The PPP2R1B gene that encodes the Ab constant regulatory subunit of the tumor suppressor protein phos phatase 2a (PP2a) is within the deleted segment in CLL patients with deleted 11q22–23. The resulting underexpression noted in those with deleted 11q22–23 leads to decreased PP2a activity in CLL cells. PP2a is important in deactivation of Akt, the mitogen activated protein kinases (MAPK) p38, JNK, ERK, and nuclear factor kB (through IkK). We have developed apolipoprotein E-mimetic therapeutic peptides that potently decrease phosphorylation of Akt, decrease TNF and nitric oxide (NO) and NO synthase (NOS) expression, and display anti-inflammatory activity in vitro and in vivo. NOS is overexpressed in CLL, and its product NO inhibits CLL cell apoptosis. Methods : Patients were from the V.A. and Duke University Medical Centers, and normal controls were from the community. Control normal PBMC were isolated by ficoll-Hypaque centrifugation, and CD19+ CLL or normal B cells were purified using negative selection with antibodies. We determined cytotoxicity using the MTS colorimetric assay. The apoE-mimetic peptides were prepared by chemical synthesis. Results : The apoE-mimetic COG compounds are peptides of 10 to 34 amino acids derived from the ligand-binding region of the apolipoprotein E holoprotein. Samples from 6 early stage CLL patients and 11 normal individuals were examined. Five of 6 patients were Rai stage 0 at presentation, and one was stage 1. They had been followed 5.6 yr (median; range 1.9–10.6 yr). Five of 6 were CD38 negative, and 2 of 6 were Zap-70 positive. Of 5analyzed, all had mutated IgVH gene. Five of 6 patients had not been treated. Of 6 peptides examined, all displayed some cytotoxicity for CLL cells in vitro. Peptide COG 112 was the most potent, while the control peptide with an inverted sequence (COG 056) had very little or no activity (Table). CLL PBMC Agent ED50 (nM) ED50 range (n) ED50 (nM) ED50 range (n) COG 112 (active) 215 64 to 351 (6) 5,150 1,100 to 〉12,500 (6) COG 056 (control) 13,546 2771 to 20,418 (6) 〉25,000 20,470 to 〉25,500 (6) COG 112 induced cell death in a dose-dependent fashion in all patients’ samples.The ED50 of COG 112 for normal B cells was very high (〉 24,400 nM). COG 112 was approximatel 24 to 116 fold more potent for killing of CLL cells compared to normal PBMC or purified B cells. In vivo studies in normal mice using COG 112 revealed no toxicity even with doses of 100 mg/kg. Conclusions : ApoE mimetic peptides kill CLL cells in vitro with high efficacy and potency (ED50s in the low nanomolar range). The cytotoxicity is very specific for CLL cells compared to normal PBMC and B cells (24 to 116 fold more potent for CLL cells). Preliminary studies show that the peptide is nontoxic in vivo in normal mice. In vivo trials with apoE peptides in patients with CLL should help determine the toxicity and efficacy in patients.

Description: Chronic Lymphocytic Leukemia (CLL) is notable for variation in aggressiveness of disease. Many patients with low-risk disease at diagnosis can be followed expectantly, while others rapidly require therapy. While a number of factors aid in determining prognosis, they have no role in predicting response to chemotherapy. We collected clinical data from a cohort of 220 patients with CLL followed at Duke University and the Durham VA Medical Centers. We assessed factors such as stage, leukocyte doubling time, IgVH mutational status, CD38 and ZAP-70 expression, cytogenetics, and time to treatment. We isolated RNA from purified CD5+, CD19+ leukemia cell samples from low and intermediate risk CLL patients, assessed the RNA expression with Affymetrix U133 Plus 2.0 GeneChips, and analyzed differential gene expression using previously developed methods of Bayesian binary regression. In a group of seventy-nine CLL patients who either eventually required (n = 45) or did not (n = 34) require therapy, CD38 status, ZAP70 expression, and cytogenetics were not statistically different (p 〉 0.5) while IgVH mutational status and leukocyte doubling time were different (p 〈 0.003). Of the patients treated, 69% were treated with chlorambucil, 51% with fludarabine, 31% with cyclophosphamide, and 49% with rituximab. Using genomic expression data, we developed a 100 gene expression signature that correlated with the need for eventual therapy. The accuracy of this signature was 94% using the leave-one-out cross validation method. Further validation using published data is currently being conducted, and data will be presented. Genes associated with the need for treatment include cell cycle regulators (such as E2F2 and CDK6), inhibitors of apoptosis (such as BAG1), and apolipoprotein B. In addition, using techniques described previously (Potti A et al, Nature Medicine, 2006, 12(11):1294), gene expression data coupled with either in vitro drug sensitivity in the NCI-60 panel of cell lines or in vivo drug response data were used to generate genomic models predictive of sensitivity to drugs commonly used in CLL including fludarabine, cyclophosphamide, and rituximab with accuracies on leave-one-out cross validation of 93%, 96%, and 79% respectively. These predictive models are currently being applied to gene expression data from leukemia samples of CLL patients to predict response to therapy (data to be presented). Thus, a genomic approach can be used to determine which CLL patients will require treatment. Importantly, models of chemosensitivity may predict response to therapy, and thus may ultimately help target the appropriate treatment for each patient.

Description: Abstract 802 Background and Significance: Even though we have treatments for CLL, it remains an incurable leukemia. We need new and better treatments for this disease. The Akt kinase is usually constitutively activated (phosphorylated) in CLL, and it acts to maintain CLL cell viability. The tumor suppressor protein phosphatase 2A is important in deactivation of Akt, the mitogen activated protein kinases (MAPK) p38, JNK, ERK, and NFkB (through IkK). We developed apoE-mimetic peptides that potently decrease phosphorylation of Akt and MAPKs, decrease TNF and nitric oxide synthase expression, and display anti-inflammatory activity in vitro and in vivo by antagonism of SET, a potent physiological inhibitor of PP2A. Others have reported that PP2A activity is reduced and that SET is overexpressed in cells of chronic myelocytic leukemia patients. Increased SET expression with consequent decreased PP2A activity leads to dysregulated kinase signaling. We show here that SET is also overexpressed in CLL cells, and that SET antagonist apoE-mimetic peptides kill CLL cells. Methods: Patients were from the Duke University and V.A. Medical Centers, and normal controls were from the community. Control normal PBMC were isolated by ficoll-Hypaque centrifugation, and CD19+ CLL or normal B cells were purified using negative selection with antibodies. We determined cytotoxicity using the MTS colorimetric assay. ApoE-mimetic peptides were prepared by chemical synthesis. Western blotting was used with anti-SET and anti-beta-actin antibodies. Apoptosis assays were performed with the annexin-V:propidium iodide staining method. Results: Samples from 17 CLL patients and 5 normal volunteers were examined by Western blotting. SET protein levels were 6.2-fold higher in CLL cells than in normal B cells. The apoE-mimetic COG compounds are peptides of 17 to 34 amino acids derived from the ligand-binding region of the apolipoprotein E holoprotein, and they act by binding to SET and preventing the inhibition of PP2A. This results in a net increase in PP2A activity in CLL cells. We have examined 11 COG peptides. Each of the 11 peptides displayed some cytotoxicity for CLL cells in vitro, irrespective of the patients' stages and other good or bad prognostic findings. 12 of 17 CLL patients were Rai stage 0 at presentation, and 5 were stage 1 or 2. They had been followed 4.2 yr (median; range 1.0 – 24.1 yr). 2 of 16 were CD38 positive, and 6 of 15 were Zap-70 positive. Of 15 analyzed, 6 had unmutated IgVH gene. 11 of 17 patients had not been treated. Peptide COG449 was the most potent, while a control peptide with an inverted apoE sequence had no activity. COG449 induced cell death in a dose-dependent fashion in all patients' samples, with a mean ED50 of 80 nM. The ED50 of COG449 for normal B cells was very high (〉 10,000 nM). Annexin-V staining indicated that apoptosis was induced at concentrations in good agreement with the ED50 for cytotoxicity of the compounds tested. In vivo studies in normal mice using COG449 show no toxicity even at doses of 100 mg/kg when delivered by subcutaneous injection. Conclusions: We demonstrated SET overexpression in CLL cells and that apoE-mimetic peptides bind SET to de-inhibit PP2A. This results in apoptosis and death of CLL cells in vitro with high efficacy and potency (low nanomolar ED50s). CLL cells are killed preferentially compared to normal PBMC and B cells. Preliminary studies show that the peptide is nontoxic in normal mice. Trials in CLL patients will help determine the efficacy in vivo. Disclosures: Christensen: Cognosci Inc.: Employment. Oddo:Cognosci Inc.: Employment. Vitek:Cognosci Inc.: Employment, Equity Ownership.