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No relevant conflicts of interest to declare.

In chronic lymphocytic leukemia (CLL) cells, signal transducer and activator of transcription 3 (STAT3) is constitutively phosphorylated on serine 727 residues, and phosphoserine STAT3 induces the transcription and proliferation of anti-apoptosis genes including c-Myc, BCL2, Bcl-XL,and Mcl-1(Hazan-Halevy, 2010). Therefore, we hypothesized that the CLL cells of patients with high peripheral blood absolute lymphocyte counts (ALCs) have a high level of STAT3 protein expression and a low rate of spontaneous apoptosis. Using Western blotting and flow cytometry, we quantitated STAT3 levels and apoptosis rates in CLL cells from 64 patients with high (N=32) and low (N=32) ALCs. As expected, the levels of STAT3 expression were significantly higher in cells from patients with high ALCs (145,000 ± 49,738/µL) than in cells from patients with low ALCs (12,800 ± 4,654/µL). However, contrary to our hypothesis, Annexin V/PI staining revealed that the levels of procaspase-3, its activated (cleaved) form caspase-3, and cleaved PARP as well as the apoptosis rates of cells from patients with high ALCs were significantly higher than those in cells from patients with low ALCs. These findings suggest that cells from patients with high ALCs are prone to spontaneous apoptosis. An RNA microarray analysis revealed that the levels of apoptotic pathway genes were upregulated in cells from patients with high ALC (P = 0.002), and Reverse-transcriptaction PCR of Caspase-3, Calpain 9, MAPK 8, KRAS, PLCγ-2, and PKCvalidated the array data. Intrigued by these findings, we sought to determine whether high levels of intracellular STAT3 induce apoptosis. Indeed, overexpression of STAT3 in interleukin-6–stimulated MM1 cells upregulated caspase-3and caspase-3 protein levels and induced apoptosis. Similarly, in CLL cells levels of caspase3 and procaspase3 remained stable across a wide range of STAT3 levels, but when STAT3 reached a threshold, level of caspase3 and procaspase3 markedly increased.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

MicroRNA (miR) deregulation is a hallmark of chronic lymphocytic leukemia (CLL). However, little is known about the mechanisms of miR gene transcription. In CLL, signal transducer and activator of transcription 3 (STAT3) is constitutively phosphorylated on serine 727 residues, and phosphoserine STAT3 activates protein-coding genes known to be induced by phosphotyrosine STAT3. Therefore, we sought to determine whether phosphoserine STAT3 also activates miR genes. Because an analysis of publically available data, revealed that STAT3 binds to the promoter of nearly 25% of all human miR genes, we transfected unstimulated CLL cells with STAT3 small hairpin (sh)-RNA. Using an RNA microarray, we found that STAT3-shRNA downregulated the levels of 63 miR genes and upregulated the levels of 9 miR genes. Using qRT-PCR, we confirmed the microarray results in 5 of the 6 differentially expressed miR genes. Together, these data suggested that STAT3 affects miR gene levels. An analysis of chromatin immunoprecipitation followed by sequencing (ChIP-seq) data revealed STAT3-miR promoter binding sites in only 60% of the STAT3-regulated miR genes. This suggested to us that mechanisms unrelated to miR promoter binding are involved in the STAT3-mediated regulation of miR expression. Indeed, computer simulation of complementary binding of STAT3-targeted microRNA genes to STAT3 mRNA provided indirect evidence that STAT3 mRNA acts as a “sponge”, soaking up miRs and altering their level and function. Several miRs that are downregulated by STAT3-shRNA, including miR-15, miR-16, miR-21, and miR-155, play crucial roles in CLL pathobiology. In particular, miR-155 is involved in tumorigenesis, and its overexpression induces lymphoma in mice; also, CLL has a high expression level of miR-155. Therefore, we sought to confirm that STAT3 directly regulates the transcription of miR-155 in CLL cells. Electrophoretic mobility shift assay analysis revealed that STAT3 bound to the miR-155 promoter in CLL cells, and ChIP analysis revealed that STAT3 bound the 700–709 bp but not the 615–624 bp in the miR-155 promoter. We then co-transfected MM1 cells with truncated miR-155 promoter constructs plus STAT3 small interfering (si)-RNA and found that the STAT3-siRNA reduced the luciferase activity of the construct harboring the 700–709 bp miR-155 promoter region. Taken together, our data suggest that STAT3 directly and indirectly deregulates miR gene levels in CLL.

Abstract 3910

For several decades CLL has been defined as a chronic leukemia characterized by a passive accumulation of small neoplastic lymphocytes that do not proliferate and do not die. This definition has been revised in recent years as it was shown that CLL cells do proliferate mostly in proliferation centers. Because the increase in peripheral blood (PB) CLL cell count was lower than their proliferation rate, it was intuitively assumed that proliferation of CLL cells is accompanied by spontaneous apoptosis, and as proliferation rate increases so does the apoptosis rate. Because STAT3 is constitutively activated in CLL cells and provides CLL cells with survival advantage (Hazan-Halevy I. et al. Blood 115:2852, 2010), we wondered whether decreased levels of intracellular STAT3 would correlate with increased apoptosis of CLL cell.Assessment of apoptosis rate by flow cytometry using propidium iodide (PI) and annexin V staining demonstrated that a significant fraction of freshly obtained PB CLL cells undergo spontaneous apoptosis in samples of 4 out of 4 patients with CLL. Spontaneous apoptosis was detected in 23% of CLL cells from a patient with a white blood cell count (WBC) of 16,000 (*10⋀6/L), in 20% of CLL cells from a patient with a WBC of 32,800, in 41 % of CLL cells from a patient with a WBC of 55,600, and in 65% of CLL cells from patient with a WBC of 101,000 (*10⋀6/L), suggesting that spontaneous apoptosis rates correlate with the number of circulating CLL cells. Because apoptotic cells are removed by the reticuloendothelial system, early apoptosis of CLL cells was assessed by quantification of cleaved PARP levels in CLL cells from 36 patients using an enzyme linked immunosorbent assay (ELISA). WBC in half of those patients ranged from 5,000 to 17,000 (*10⋀6/L) (median: 14,500) and in the other half from 151,000 to 680,000 (*10⋀6/L) (median: 237,000). The median level of cleaved PARP was three times higher in cells from patients with a high lymphocyte count than in cells from patients with a low lymphocyte count (P = 0.007), confirming our hypothesis that as disease burden increases so does CLL cell apoptosis rate. Because STAT3 plays a key role in CLL cell survival we sought to determine whether CLL cell apoptosis rates correlate with intracellular STAT3 levels. We quantified STAT3 levels in PB CLL cells from 185 CLL patients using an ELISA. Our data revealed a linear correlation between the number of CLL cells and intracellular STAT3 levels. The higher the lymphocyte counts, the lower were STAT3 levels (rp = 0.28, P<0.0001). Unlike WBC, STAT3 levels did not correlated with disease stage, cytogenetic abnormality, β2M levels, IgH mutation status, ZAP-70, or CD38. However, STAT3 levels were significantly higher in previously treated patients (No. = 24; P = 0.01), suggesting that previous exposure to chemotherapy activated compensatory survival pathways.Spontaneous apoptosis occurs in PB CLL cells of all patients regardless of disease stage or cytogenetic abnormality. High PB lymphocyte count linearly correlates with increased spontaneous apoptosis rates and decreased intracellular STAT3 levels.No relevant conflicts of interest to declare.