STAT3 Activates the Pentraxin 3 Gene in Chronic Lymphocytic Leukemia Cells.

Pentraxin-related protein 3 (PTX3), commonly produced by myeloid and endothelial cells, is a humoral pattern recognition protein of the innate immune system. Because PTX3 plasma levels of patients with chronic lymphocytic leukemia (CLL) are high and most circulating cells in patients with CLL are CLL cells, we reasoned that CLL cells produce PTX3. Western immunoblotting revealed that low-density cells from seven of seven patients with CLL produce high levels of PTX3, flow cytometry analysis revealed that the PTX3-producing cells are B lymphocytes coexpressing CD19 and CD5, and confocal microscopy showed that PTX3 is present in the cytoplasm of CLL cells. Because STAT3 is constitutively activated in CLL cells, and because we identified putative STAT3 binding sites within the PTX3 gene promoter, we postulated that phosphorylated STAT3 triggers transcriptional activation of PTX3. Immunoprecipitation analysis of CLL cells' chromatin fragments showed that STAT3 Abs precipitated PTX3 DNA. STAT3 knockdown induced a marked reduction in PTX3 expression, indicating a STAT3-induced transcriptional activation of the PTX3 gene in CLL cells. Using an EMSA, we established and used a dual-reporter luciferase assay to confirm that STAT3 binds the PTX3 gene promoter. Downregulation of PTX3 enhanced apoptosis of CLL cells, suggesting that inhibition of PTX3 might benefit patients with CLL.

STAT3-Induced Wnt5a Provides Chronic Lymphocytic Leukemia Cells with Survival Advantage.

The wingless and integration site growth factor-5a (Wnt5a) is a ligand of the receptor tyrosine kinase-like orphan receptor-1 (ROR1). Because both Wnt5a and ROR1 are expressed in circulating chronic lymphocytic leukemia (CLL) cells, and because in other cell types, STAT3, which is constitutively activated in CLL, induces Wnt5a signaling, we wondered whether STAT3 induces the expression of Wnt5a in CLL cells. Sequence analysis detected four putative STAT3 binding sites in close proximity to the Wnt5a gene promoter's start codon. Chromatin immunoprecipitation and EMSA revealed that STAT3 binds to the Wnt5a gene promoter, and a luciferase assay showed that STAT3 activates the Wnt5a gene. Additionally, transfection of peripheral blood CLL cells with STAT3 short hairpin RNA downregulated Wnt5a mRNA and protein levels, suggesting that STAT3 binds to the Wnt5a gene promoter and induces the expression of Wnt5a in CLL cells. Flow cytometry and confocal microscopy determined that both Wnt5a and its receptor ROR1 are coexpressed on the surface of CLL cells, and Western immunoblotting showed an inverse correlation between Wnt5a and ROR1 protein levels, implying that, regardless of CLL cells' ROR1 levels, blocking the interaction between Wnt5a and ROR1 might be beneficial to patients with CLL. Indeed, transfection of CLL cells with Wnt5a small interfering RNA reduced Wnt5a mRNA and protein levels and significantly increased the spontaneous apoptotic rate of CLL cells. Taken together, our data unravel an autonomous STAT3-driven prosurvival circuit that provides circulating CLL cells with a microenvironment-independent survival advantage.

The CXCR4-STAT3-IL-10 Pathway Controls the Immunoregulatory Function of Chronic Lymphocytic Leukemia and Is Modulated by Lenalidomide.

Chronic lymphocytic leukemia (CLL) cells possess regulatory functions comparable to those of normal B10 cells, a regulatory B cell subset that suppresses effector T-cell function through STAT3-mediated IL-10 production. However, the mechanisms governing IL-10 production by CLL cells are not fully understood. Here, we show that the CXC chemokine ligand 12 (CXCL12)-CXCR4-STAT3 axis regulates IL-10 production by CLL cells and their ability to suppress T-cell effector function through an IL-10 mediated mechanism. Knockdown of STAT3 significantly impaired the ability of CLL cells to produce IL-10. Furthermore, experiments to assess the role of lenalidomide, an immunomodulatory agent with direct antitumor effect as well as pleiotropic activity on the immune system, showed that this agent prevents a CXCL12-induced increase in p-S727-STAT3 and the IL-10 response by CLL cells. Lenalidomide also suppressed IL-10-induced Y705-STAT3 phosphorylation in healthy T cells, thus reversing CLL-induced T-cell dysfunction. We conclude that the capacity of CLL cells to produce IL-10 is mediated by the CXCL12-CXCR4-STAT3 pathway and likely contributes to immunodeficiency in patients. Lenalidomide appears to be able to reverse CLL-induced immunosuppression through including abrogation of the CXCL12-CXCR4-S727-STAT3-mediated IL-10 response by CLL cells and prevention of IL-10-induced phosphorylation of Y705-STAT3 in T cells.

High fluorescence in situ hybridization percentage of deletion 11q in patients with chronic lymphocytic leukemia is an independent predictor of adverse outcome.

We have analyzed patients with previously untreated chronic lymphocytic leukemia with del11q fluorescence in situ hybridization (FISH) abnormality (n = 196) in this study. Detection of the 11q22.3 used a multicolor FISH technique. Patients with del11q fell into two major FISH subsets-sole del11q (n = 64) and del11q with del13q (n = 132). FISH subsets were compared using the median del11q FISH% (>58%, high vs. ≤58%, low). Overall survival (OS) and time to first treatment (TTFT) were estimated using Kaplan-Meier plots (log rank). Multivariate analysis was performed to assess the association between FISH% of del11q and outcomes. Patients with sole del11q were similar to del11q with del13q in terms of TTFT and OS. Patients with high FISH% of del11q had significantly shorter OS and TTFT as compared with patients with low FISH%, particularly in sole del11q; this negative impact of high FISH% of del11q on OS and TTFT was diminished with coexistent del13q. In multivariate analysis, high FISH% of del11q was a significant predictor for shorter OS and TTFT. A comparison of these del11q subsets with a separate cohort of (n = 673) previously untreated patients with sole del13q showed that the high FISH% del11q cohort had a significantly shorter TTFT and OS. In addition, bulky disease by physical examination or computed tomography imaging was infrequent at presentation in patients with del11q. High FISH% of del11q can reliably discriminate higher risk patients with chronic lymphocytic leukemia. Presence of coexistent del13q should be accounted for while prognosticating patients with del11q.