B cell chronic lymphocytic leukemia (CLL) CD38 expression is variable and may predict outcome. Inducible nitric oxide synthase (NOS2) expression regulates CLL cell apoptosis. IL-4 and IFN-γ regulate B cell CD38 expression and NOS2 expression. We compared IL-4 and IFN-γ serum levels between CLL patients and normal individuals, and determined whether serum IL-4 and IFN-γ levels correlated with CLL cell CD38 expression and NOS enzyme activity. IL-4 levels, but not IFN-γ levels, differed between normal individuals and CLL patients. Furthermore, there was an association of IL-4 levels, but not IFN-γ levels, with CD38 and NOS2 expression in CLL patients.

Cobalamins (Cbl) serve as important co-factors for methionine synthase and methylmalonyl-coA mutase. They also can block the activities of nitric oxide (NO), as the cobalt binds NO and quenches its bioactivity. NO plays very important roles in normal physiology and in various pathologic processes. NO is synthesized by NO synthases (NOS), enzymes that convert L-arginine to L-citrulline and NO. We postulated that various corrin derivatives (e.g., cobalamins or cobinamides) would inhibit the enzymatic activity of NO synthase (NOS) by directly interacting with NOS. We used purified, recombinant neuronal NOS (NOS1) and inducible NOS (NOS2) in in vitro assays measuring conversion of 14-C-L-arginine to 14-C-L-citrulline. Results showed that hydroxocobalamin (OH-Cbl), cobinamide (Cbi), and dicyanocobinamide (CN2Cbi) were potent NOS1 and NOS2 inhibitors, while cyanocobalamin had much less activity. In general, NOS1 was inhibited more actively than was NOS2, but both were inhibited over 80% by the agents. Methylcobalamin and adenosylcobalamin had very little ability to inhibit NOS activity, but light illumination of these two compounds (a process that liberates the methyl and adenosyl groups from cobalamin, generating OH-Cbl) “activated” them so that they had inhibitory activity comparable to native OH-Cbl

One of the complexities of chronic lymphocytic leukemia (CLL) is that some patients die early from the disease, while others identically staged by conventional means may live more than two decades. This has led to a search for prognostic indicators that correlate better with eventual survival. Candidate markers include Rai stage, Binet stage, lymphocyte doubling time (DT), immunoglobulin heavy chain V region (IgVH) mutation status, and leukemia cell CD38 and Zap-70 expression. To determine which of these markers correlate best with survival in univariate and multivariate analyses, we examined these parameters in 150 CLL patients from a VA medical center and a university medical center. CLL cells were purified from patients not receiving active treatment with WBC >20,000/uL by deleting T cells, monocytes, neutrophils, and erythrocytes using antibodies against CD2, CD3, CD16, CD36, CD56, and glycophorin A, and centrifugation over ficoll-Hypaque (CLL purity > 97%). In univariate analyses, patients with advanced stage on presentation had shorter survival than those with low stage [14.0 yr for high modified Rai stage vs. 25.3 yr for low stage (p = 0.013), and 13.8 yr, 13.4, and 25.3 yr for Binet stages C, B, and A, respectively (p = 0.033)]. Patients with a lymphocyte DT >3 yr survived twice as long (22.6 yr) as those with a DT <3 yr (11.3 yr) (p = 0.004). There was a strong relationship of IgVH mutation status with survival (median survival 22.6 yr for mutated, 9.4 yr for unmutated; p = 0.0006). Likewise, cellular CD38 expression significantly correlated with survival (CD38 neg 25.3 yr, CD38 pos 13.4 yr, p = 0.008). Zap-70 expression determined by quantitative immunoblot [Zap-70/actin ratio (anti-Zap-70 antibody from Upstate; clone 2F3.2)] was not significantly related to survival (Zap-70 pos 20.8 yr, Zap-70 neg 22.6 yr). Other parameters found not to be related to eventual survival were WBC on presentation, maximum WBC reached before treatment, and time from diagnosis to initial treatment. IgVH mutation status was significantly related to lymphocyte DT, CD38 expression, and Zap-70 expression. In multivariate analyses using the proportional hazards model, IgVH mutation status (p = 0.001), cellular CD38 expression (p = 0.025), and Binet stage (p = 0.026) were significantly associated with survival (with p = 0.0002 overall for these three parameters in the model), but Zap-70 had no significant relationship to survival in the model. Thus, in contrast to other reports, cellular Zap-70 expression does not correlate with various parameters of disease severity (including survival) in our cohort of CLL patients. In summary, our univariate analyses show significant correlations of advanced clinical stage, short lymphocyte doubling time, high CD38 expression, and unmutated IgVH with short survival. However, multivariate analyses show that only IgVH gene mutation status, clinical stage at presentation, and cellular CD38 expression correlate significantly with survival.

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., Leukemia 17:442, 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.

Somatic mutations of immunoglobulin genes characterize mature memory B cells, and intraclonal B-cell diversification is typically associated with expansion of B-cell clones with greater affinity for antigen (antigen drive). Evidence for a role of antigen in progression of intraclonal chronic lymphocytic leukemia (CLL) cell diversification in patients with mutated immunoglobulin genes has not been previously presented. We performed a single-cell analysis of immunoglobulin heavy and light chains in 6 patients with somatically mutated CLL-cell immunoglobulin genes and identified 2 patients with multiple related (oligoclonal) subgroups of CLL cells. We constructed genealogic trees of these oligoclonal CLL-cell subgroups and assessed the effects of immunoglobulin somatic mutations on the ratios of replacement and silent amino acid changes in the framework and antigen-binding regions (CDRs) of the immunoglobulin heavy and light chains from each oligoclonal CLL-cell population. In one subject, the amino acid changes were consistent with an antigen-driven progression of clonally related CLL-cell populations. In the other subject, intraclonal diversification was associated with immunoglobulin amino acid changes that would have likely lessened antigen affinity. Taken together, these studies support the hypothesis that in some CLL cases intraclonal diversification is dependent on antigen interactions with immunoglobulin receptors.

Chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived non-dividing CD5 + B cells. Nitric oxide (NO) is an important regulator of apoptosis, and the viability of cultured B-CLL cells may be dependent on the autocrine production of nitric oxide by inducible nitric oxide synthase (NOS2). We performed this study to determine whether cytokine factors that prevent spontaneous in vitro apoptosis of B-CLL cells induce B-CLL cell NOS2 enzyme activity. B-CLL cells expressed NOS enzyme activity and NOS2 protein and mRNA. IL-4 and IFN-γ increased B-CLL cell NOS2 enzyme activity and protein expression during in vitro culture. IFN-γ, but not IL-4, increased NOS2 mRNA expression in cultured B-CLL cells suggesting that IL-4-mediated changes of NOS2 protein expression occurred at the post-transcriptional level. We were unable to detect increased concentrations of nitrite or nitrate (NO x ) as surrogate markers of NO production in B-CLL cell cultures treated with IL-4 or IFN-γ. IL-4 and IFN-γ diminished NOS inhibitor-induced B-CLL cell death. In summary, we found that B-CLL cells expressed NOS2 and that IL-4 and IFN-γ increased B-CLL NOS2 expression. Cytokine-mediated expression of NOS2 by B-CLL cells may promote their survival, and therapeutic strategies that target NOS2 or quench NO may be beneficial in patients with B-CLL.