ALBERT

Description: Survival of CLL cells requires sustained activation of the anti-apoptotic PI-3-kinase/Akt pathway, and many therapies for CLL cause leukemia cell death by triggering apoptosis. Blood lipoprotein particles are either pro- or anti-apoptotic. High density lipoprotein (HDL) particles are anti-apoptotic through sphingosine-1-phosphate receptor 3 (S1P3)-mediated activation of the PI-3-K/Akt pathway. We have noted that apoE4-very low density lipoprotein (VLDL) (but not apoE2- or apoE3-VLDL) particles increase apoptosis of endothelial cells by recruiting the phosphoinositol phosphatase SHIP-2 to the plasma membrane, thereby directly inhibiting the anti-apoptotic activity of HDL (DeKroon R, et al., Circ Res99:829–836, 2006). Since apoE4-VLDL increases apoptosis of certain cells, and since increased leukemia cell apoptosis favors longer survival in CLL, we hypothesized that APOE4 genotype would beneficially influence the clinical course of CLL. Of the 193 CLL patients (50 women and 133 men) studied, 29% had an APOE4 genotype. In the entire group, survival of men and women was not statistically different. However, women (but not men) with an APOE4 genotype had markedly longer survival than non-APOE4 patients (median 〉25 yr vs. 14.0 yr; p = 0.02) (Figure). Figure Figure Men and women had the same time-to-treatment (treatment-free survival) irrespective of APOE genotype. VLDL is metabolized to LDL by the actions of LPL. Patients had shorter survival and time-to-treatment if their CLL cell lipoprotein lipase (LPL) mRNA levels were high (p = 0.002). In analyzing APOE genotype and LPL levels in the same patients, we demonstrated that APOE4 was a more important determinant of survival than was the LPL level (p = 0.0007). The beneficial effect of APOE4 in CLL survival is likely mediated through allele-specific influences of APOE4 on serum lipoproteins increasing leukemia cell apoptosis. The frequency of the APOE alleles in the CLL patient population was not significantly different than that of a control population (16 and 13%, respectively). APOE genotype therefore does not appear to affect susceptibility to CLL, but influences the clinical course of disease, particularly after therapy is initiated. In contrast, APOE genotype does influence susceptibility to other diseases, most notably Alzheimer’s disease in which APOE4 markedly increases risk. The beneficial impact of APOE4 in CLL and its deleterious impact on Alzheimer’s disease expression may relate to a common mechanism of APOE4 in enhancing apoptotic cell death. APOE genotyping of patients with CLL may provide important clinical prognostic information, particularly in women. Most importantly, the allele-specific influence of APOE on disease progression may provide important new insights into the mechanisms of disease and response to therapy, and lead to new agents for treatment.

Description: Abstract 1785 Background: Chronic lymphocytic leukemia (CLL), the most common hematopoietic malignancies in adults, remains incurable, and new approaches to treatment are needed. Previous research has defined certain molecular characteristics of CLL (e.g., cytogenetic abnormalities, dysregulated signaling pathways, and alterations of apoptotic proteins) that improve our understanding of this disease and provide prognostic information. In addition to these molecular signatures, it is now clear from multiple settings that changes in cellular metabolism can impact the survival and promote the growth and proliferation of malignant cells. Unlike many cancers, however, CLL cells can have a very low rate of proliferation. Because targeting cancer metabolism may attack a fundamental feature of cancer biology, we sought to characterize the metabolic program of CLL in hopes of establishing new prognostic markers and identifying novel treatment targets. We evaluated the glycolytic and lipid metabolism profiles of B-CLL cells from low- and high-risk CLL patients. Methods: We collected blood from CLL patients seen at the Duke Center for CLL and enrolled in IRB-approved protocols at the Duke University and Durham VA Medical Centers. CLL lymphocytes were isolated using negative selection yielding greater than 95% purity of CLL lymphocytes. Standard prognostic testing was done as we have described before. Glut1 expression was detected by immunoblot. Glycolytic rate and lipid oxidation rate was measured by using 3H-labled glucose or palmitic acid respectively as described in the literature. Cell viability was measured by propidium iodide exclusion assay. Results: CLL cells exhibited higher expression of the major glucose transporter Glut1 compared to naïve B cells purified from healthy donors (n=20 for CLL and n=3 for normal). CLL cells from a subset of high-risk patients (unmutated IGVH; total n=6) had higher glycolytic rate compared to those from low-risk (mutated IGVH; total n=11) patients. In contrast, no significant change in lipid oxidation was observed between high-risk (IGVH unmutated; n=2) to low-risk (IGVH mutated; n=4) patients. CLL cells were also sensitive to glycolysis inhibitor 2-deoxyglucose (2-DOG), with 2-DOG (5uM) mediating cytotoxicity in vitro. Conclusion: We establish for the first time that purified, malignant CLL cells overexpress the glucose transporter Glut1, and that the glycolysis inhibitor 2-DOG is cytotoxic for CLL cells in vitro. In addition, our results demonstrate that cells from high-risk CLL patients have enhanced glucose metabolism relative to leukemia cells from low-risk patients. Therefore, glucose metabolism in these malignant B cells may represent a novel prognostic marker and a novel therapeutic target in this incurable disease. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Chronic lymphocytic leukemia (CLL) is an incurable cancer of mature B-lymphocytes that infil­trate the bone marrow, secondary lymphoid organs, and blood. Much progress has been made during the past decade in understanding the pathogenesis and prognosis in CLL. Patients with unmutated immunoglobulin gene heavy chain variable region (IGHV) genes have inferior clinical outcomes when compared to patients with mutated IGHV. While there is robust data regarding clinical outcomes in patients with single IGHV rear­range­ments in CLL, there is limited data regarding double IGHV rearrangements. Thus, we investigated the preva­lence of double IGHV rearrangements in our institutional cohort to determine molecular features and the prog­nos­tic significance of CLL cases with double IGHV rearrangements. Methods: Patients with CLL receiving care at Duke University and Durham Veterans Affairs (VA) Medical Centers were enrolled into a longitudinal cohort study between 1999 and 2014. IGHV mutation analysis was performed on DNA from all study patients. Patients were followed clinically for disease progression, need for treatment, and overall survival. The primary end points were time to treatment (TTT) or overall survival (OS). Survival curves were estimated using Kaplan-Meier method. Statistical differences were tested using propor­tional hazards tests for a clinical significance of p 〈 0.05. CLL cell isolation and IGHV mutational analysis were done as we have previously described (BLOOD 109:1559-1567, 2007). Statistical analysis was performed using SAS enterprise guide 5.1 and JMP Pro 11. Results: A total of 489 CLL patients were studied. Of these, 420 had one IGVH gene rearrangement (single IGHV; 86%). In 69 patients (14%), we amplified two IGHV rearrangements, indicating likely biallelic IGHV. The median TTT for the single IGHV group was 5.4 years, and the median OS for the single IGHV group was 15.3 years. For the double IGHV group, the mean TTT was 6.5 years (p = 0.44), and the median OS was 15.8 years (p = 0.73). Patients with single mutated IGHV (m) had better survival (median OS = 20.1 years) than those with single unmutated IGHV (u) (median OS = 11.3 yrs; p

Description: B-cell chronic lymphocytic leukemia (CLL), an incurable leukemia, is characterized by defective apoptosis. We found that the SET oncoprotein, a potent inhibitor of the protein phosphatase 2A (PP2A) tumor suppressor, is overexpressed in primary CLL cells and B-cell non-Hodgkin lymphoma (NHL) cell line cells. In CLL, increased levels of SET correlated significantly with disease severity (shorter time to treatment and overall survival). We developed SET antagonist peptides that bound SET, increased cellular PP2A activity, decreased Mcl-1 expression, and displayed selective cytotoxicity for CLL and NHL cells in vitro. In addition, shRNA for SET was cytotoxic for NHL cells in vitro. The SET antagonist peptide COG449 inhibited growth of NHL tumor xenografts in mice. These data demonstrate that SET is a new treatment target in B-cell malignancies and that SET antagonists represent novel agents for treatment of CLL and NHL.

Description: Abstract 4576 Background: Chronic lymphocytic leukemia (CLL) has a highly variable clinical course. Some patients require treatment early while others can be monitored without therapy. CD38 expression has been shown in multiple cohorts to have prognostic significance. An elevated percentage of CD38 positive CLL lymphocytes at the time of diagnosis is correlated with a more rapid need for therapy and a shorter overall survival. The extent to which CD38 varies during the course of CLL, including after therapy, has only been evaluated in a limited fashion. Methods: From a cohort of over 500 CLL patients at the Duke University and Durham VA Medical Centers, we selected 136 patients in whom we had measured CD38 expression by flow cytometry on two or more occasions. We determined the first, maximum, minimum, and range (maximum – minimum) CD38 values. We compared these values to other molecular prognostic markers using Wilcoxon tests and assessed the prognostic significance of these values using Cox proportional hazard models and Kaplan-Meier analyses. Results: Of the 136 patients, 70% were male and 88% Caucasian, with a median age of 60. The majority had low clinical stage at diagnosis—either Rai stage 0 (68%) or 1 (19%). Molecular prognostic markers were also generally favorable. Eighty-two (67%) patients had mutated IGHV status, 69 (51%) were ZAP70 negative, and 76 (63%) had either 13q deletion or normal cytogenetics, determined by fluorescent in situ hybridization. CD38 expression was measured a median of 5.5 times (2 – 19). The median time between the first and last CD38 measurements was 1206 days (81 – 4109). The median values were 6% (0.6 – 99) for maximum CD38, 1.5% (0 to 84.5) for minimum CD38, and 4.9% (0.2 to 95.3) for CD38 range. Maximum, minimum, and CD38 range were significantly lower in patients with mutated compared to unmutated IGHV status (p 〈 0.005 for all parameters, Wilcoxon rank sum test). Elevated maximum and CD38 range were significantly associated with a more rapid time to therapy (TTT) and shorter overall survival (OS) in a univariate Cox proportional hazards model (p 〈 0.03 for all, Wald test). In a multivariate Cox proportional hazards model including first CD38 and maximum CD38 values, only maximum CD38 remained statistically significant. We found that patients with high CD38 variation (CD38 range greater than the median) had significantly shorter TTT and OS than patients with low CD38 variation (p = 0.002 for both, log rank test). Using receiver operator characteristic analyses, we determined that the best cut-off for dichotomizing the first CD38 according to TTT and OS in the entire Duke/Durham VA CLL cohort was 11%. Using this cut-off, 15 patients (11%) converted from CD38 negative to CD38 positive. Using the standard 30% cut-off, 14 patients (10%) converted from CD38 negative to CD38 positive. Patients with a first CD38 measurement less than 11% and subsequent measurements above 11% had a favorable OS, similar to patients with low CD38 for all measurements (p = 0.002, log rank test). However, patients with a first CD38 measurement less than 30% who had subsequent measurements above 30% had an inferior OS, similar to patients with high CD38 for all measurements (p = 0.006, log rank test). Lastly, among 24 patients with CD38 measurements before and after first therapy, the percentage of CD38 positive cells increased in 19 patients (79%), with a median value of 3.2% before to 6.9% after therapy (p = 0.005, Wilcoxon signed rank test). Conclusions: CD38 values vary as patients transition across the disease trajectory. This variation appears to have prognostic significance, with high variation associated with faster time to first therapy and shorter overall survival. Additionally, in our cohort, a patient's maximum CD38 value had more prognostic significance than a single initial measurement. Thus, longitudinally measuring CD38 throughout the clinical course of CLL could aid in the management of CLL patients, refining the initial prognostic assessment, and improving patient counseling and decision making. Disclosures: No relevant conflicts of interest to declare.

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: Introduction Although chronic lymphocytic leukemia (CLL) is a generally indolent malignancy, there is a spectrum of disease aggressiveness. Clinical and molecular prognostic markers are helpful for the clinician and for the patient, in terms of disease management and life planning. Additional prognostic markers can help with further risk stratification, especially for CLL patients with “low-risk” disease. The prognostic value of absolute monocyte count (AMC) has been evaluated in various malignancies, including CLL where elevated AMC at diagnosis was shown to be associated with rapid time to first therapy (TTT), and in one series, inferior overall survival (OS). The mechanism by which elevated AMC is associated with worse treatment free survival is not known. However, CD14, which is secreted by monocytes, improves in vitro CLL cell survival, and is found at high levels in the serum of CLL patients. We hypothesized that elevated AMC at the time of CLL diagnosis is associated with inferior survival and that elevated serum CD14 is associated with high AMC and worse survival. Methods CLL patients followed at the Duke University and Durham VA Medical Centers and enrolled in an IRB approved protocol to collect clinical data and blood samples were evaluated. We selected patients for whom AMC was measured between three months prior to diagnosis to three months after diagnosis. We evaluated the correlation between AMC and TTT and OS, with AMC as a continuous and as a dichotomized variable. We also assessed the prognostic capability of AMC in relation to other clinical and molecular prognostic markers, such as Rai stage, race, interphase cytogenetics by FISH, CD38 and ZAP70 expression, and IGHV mutation status. We measured serum CD14 levels using an ELISA assay, and evaluated the correlation between CD14 levels and clinical outcomes or AMC. Cox proportional hazard models were used to evaluate time to event outcomes, Wilcoxon rank sum test and Kruskal-Wallis rank sum test were used to compare AMC to other prognostic markers, and Pearson’s correlation test was used to compare continuous variables. Results From a cohort of over 600 CLL patients, we selected 222 patients with AMC measured ± three months from the date of diagnosis. AMC ranged from 0 to 7.63 cells/mL. With a median follow up of 5.2 years (range 0.1 – 18.2), 102 patients (46%) had been treated, and 59 patients (27%) died. This was not significantly different from the entire cohort. Higher AMC was significantly correlated with shorter TTT (p = 0.002, hazard ratio 1.37, 95% CI 1.12 – 1.68) and inferior OS (p = 0.017, hazard ratio 1.39, 95% CI 1.06 – 1.83). There was no significant difference in AMC in patients stratified by Rai stage, race, interphase cytogenetics, CD38 or ZAP70 expression, or IGHV mutation status. When combined with molecular prognostic markers (IGHV mutation status, CD38 and ZAP70 expression, and interphase cytogenetics) in multivariate models, AMC retained significant prognostic power for TTT and OS. The serum soluble CD14 levels were measured in CLL patients from this cohort, with a mean CD14 level of 2.3 ug/mL. The prognostic significance of serum CD14 and correlation with AMC will be presented. Conclusions Absolute monocyte count at the time of CLL diagnosis is associated with inferior clinical outcomes – both TTT and OS. These results confirm and extend other reports evaluating the prognostic significance of circulating monocytes in CLL. Our evaluation of serum CD14, a monocyte-derived secreted protein that promotes CLL cell viability, in concert with AMC may provide a possible explanation for the associations identified in this cohort of patients. As an easily measured clinical marker, AMC can be readily used and/or combined with other prognostic markers to improve risk stratification and patient counseling at the time of diagnosis. Disclosures: No relevant conflicts of interest to declare.

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: The viability of CLL cells may be dependent on the autocrine production of nitric oxide because nitric oxide synthase (NOS) inhibitors induce CLL cell apoptosis and CLL cells express inducible NOS (NOS2). Our previous study indicated that the non-specific NOS inhibitor NMMA induced CLL cell apoptosis but only at high concentrations (〉 1 mM) (Levesque et al., Leukemia17:442, 2003). Therefore, we performed the current study to identify NOS inhibitors that induce CLL cell apoptosis at lower concentrations and to understand factors that promote NOS inhibitor-induced CLL cell toxicity. We isolated and enriched CLL cells from the blood of CLL patients and cultured the CLL cells in media containing various concentrations of 21 different NOS inhibitors. We determined CLL cell viability following culture with each NOS inhibitor. We found that NOS inhibitors with specificity for neuronal NOS (NOS1) induced CLL cell death at concentrations lower than non-specific NOS inhibitors and lower than inducible NOS (NOS2) specific inhibitors. There was a weak correlation (r2 = 0.29, p = 0.1608) of the NOS1 (but not NOS2) half-maximal inhibitory concentration (IC50) of each NOS inhibitor for purified recombinant NOS and its ability to induce CLL cell death. We confirmed the specificity of the NOS inhibitors by inhibition of purified recombinant NOS1 and NOS2 enzyme activity, and we confirmed that NOS1 specific inhibitors induced CLL cell death by apoptosis. Because there was only a weak correlation of the NOS1 IC50 with NOS inhibitor induced CLL cell death, we considered whether other factors such as the Kd and hydrophobicity of each compound correlated with CLL cell death. We found that there was a direct correlation between the NOS1 (but not NOS2) dissociation constant (Kd) of NOS inhibitors and CLL cell death (r2 = 0.77, p = 0.0041) and a direct correlation of the partitioning coefficient (a measure of hydrophobicity) of each NOS inhibitor and its ability to induce CLL cell death (r2 = 0.68, p 〈 0.0001). Therefore, NOS inhibitors that bound tightly to NOS1 and were hydrophobic induced CLL cell death at lower concentrations. There was variable expression of CLL cell NOS1 mRNA (6 of 28 samples positive) and we were unable to demonstrate CLL cell expression of NOS1 protein by immunoblotting. This suggests that if NOS1 is present in CLL cells, it exists at very low levels. Taken together, we believe that low level NO production promotes CLL cell viability and that inhibition of CLL NOS induces CLL cell apoptosis. Importantly, our studies provide direction for the rational design and selection of NOS inhibitors that may be useful as CLL therapeutics.