B-cell chronic lymphocytic leukemia (B-CLL) is characterized by an accumulation of neoplastic B cells due to their resistance to apoptosis and increased survival. Among various factors, the tumor microenvironment is known to play a role in the regulation of cell proliferation and survival of many cancers. However, it remains unclear how the tumor microenvironment contributes to the increased survival of B-CLL cells. Therefore, we studied the influence of bone marrow stromal cell -induced hedgehog (Hh) signaling on the survival of B-CLL cells. Our results show that a Hh signaling inhibitor, cyclopamine, inhibits bone marrow stromal cell -induced survival of B-CLL cells, suggesting a role for Hh signaling in the survival of B-CLL cells. Furthermore, gene expression profiling of primary B-CLL cells (n = 48) indicates that the expression of Hh signaling molecules, such as GLI1, GLI2, SUFU, and BCL2, is significantly increased and correlates with disease progression of B-CLL patients with clinical outcome. In addition, SUFU and GLI1 transcripts, as determined by real-time PCR, are significantly overexpressed and correlate with adverse indicators of clinical outcome in B-CLL patients, such as cytogenetics or CD38 expression. Furthermore, selective downregulation of GLI1 by antisense oligodeoxynucleotides (GLI1-ASO) results in decreased BCL2 expression and cell survival, suggesting that GLI1 may regulate BCL2 and, thereby, modulate cell survival in B-CLL. In addition, there was significantly increased apoptosis of B-CLL cells when cultured in the presence of GLI1-ASO and fludarabine. Together, these results reveal that Hh signaling is important in the pathogenesis of B-CLL and, hence, may be a potential therapeutic target.

B-cell Chronic Lymphocytic Leukemia (B-CLL) is characterized by accumulation of clonal lymphocytes resistant to apoptosis. Therefore, identification of molecules responsible for the increased resistance to apoptosis is warranted. The identified molecules could be further targeted to develop effective therapy. Emerging evidence on the mechanism of resistant to apoptosis in several cancers suggests that GLI1 transcription factor, target of hedgehog signaling, may have a potential role in the increased resistance to apoptosis in B-CLL. However such studies have not been reported for B-CLL. Therefore, we investigated the role of sonic hedgehog (Shh)-GLI signaling in the pathogenesis B-CLL using 38 different patient samples and different parameters. The results were analyzed based on good and poor clinical outcome B-CLL subgroups identified on the basis of cytogenetic abnormality and/or CD38 expression levels. Microarray and RT-PCR analyses demonstrated active Shh-GLI signaling molecules in B-CLL cells including GLI1. Our results demonstrate that GLI1 transcripts were significantly (p = 0.003) over-expressed in B-CLL cells of poor clinical outcome patient subgroups (17pdel, 11qdel, trisomy12; N = 20) compared to good clinical outcome (13qdel, normal karyotype; N = 18) as observed in microarray analysis and confirmation by real time PCR. In addition, similar observation was also made with poor prognosis (high CD38 expression) versus good prognosis (low CD38 expression) B-CLL subgroups. Furthermore, higher expression of GLI1 was associated with significantly (p = 0.02) shorter time to treatment compared to lower GLI1 expressing B-CLL patients as determined by Kaplan-Meier survival analysis using log rank test, suggesting the increased expression of GLI1 and its association with the disease progression in poor clinical outcome B-CLL patient sub-groups. Addition of exogenous Shh on the significantly (p<0.01) increased cell survival and up-regulation of GLI1 transcripts, and decreased cell survival and downregulation of GLI1 transcripts in the presence of cyclopamine, known inhibitor of signaling, as determined by MTT assay and RT-PCR in vitro, suggesting the role of GLI1 in the survival of B-CLL. Downregulation of GLI1 expression in B-CLL cells using antisense oligonucleotides resulted in the significantly (p<0.01) increased susceptibility of the B-CLL cells to fludarabine mediated cytotoxicity compare to fludarabine alone or antisense alone or B-CLL cells treated with irrelevant oligonucleotides as determined by MTT assay and Annexin V method for identifying apoptotic cells. There was a significant decrease in BCL2 expression in GLI1-downregulated B-CLL cells suggesting that GLI1 may regulates BCL2 and thereby regulate cell survival/apoptosis in B-CLL. Together, these results demonstrate a role for GLI1 transcription factor for the increased resistance to apoptosis and thereby disease progression in B-CLL, and hence may be a potential therapeutic target to further improve treatment for B-CLL.

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