GLI1 Inhibitor GANT61 Deregulates stat3 Phosphorylation In Chronic Lymphocytic Leukemia Cells

Hedgehog(Hh) family has come to be recognized as key fundamental mediators of many carcinomas, but conflicting results exist about its role in chronic lymphocytic leukemia (CLL). Here we examined the effect of GLI inhibitor GANT61 to investigate the role of the Hh-signaling pathway in CLL.

We conduct real-time PCR for Hedgehog family members on isolated mononuclear cells from peripheral blood (n=35) and bone marrow cells(n=6) to evaluate the presence of the Hh-signaling pathway in CLL. There's no significant difference between peripheral blood and bone marrow cells in levels of Hh members. Profiling of cognate Hh pathway members revealed reduced expression of three key Hh signaling effectors, Patched, Smoothened (SMOH) and GLI, in peripheral blood mononuclear cells (PBMC), whereas transcription levels of other investigated members(SHH, IHH, DHH, GLI2, GLI3 etc.) resembled normal B-lymphocyte levels. However, we found a great heterogeneity for the expression levels of the Hh family with a subset of about 25% of CLL PBMC samples showing high transcript levels ( 1.5-fold than the median) for GLI1 and SMO. There is a direct positive correlation between GLI1 expression and SMO expression. We performed western-blot in CLL PBMC samples and found a positive correlation between phosphorylation of stat3 and GLI1 (figure 1). We examined the activity of GANT61 on viability of cell lines and primary CLL cells (N=3) in vitro by CCK8. GANT61 reduced the cell viability to 65% ± 14% after 24 hours of culture at concentration of 20uM (mean +/¨C SD, P < 0.05). We found that the capacity of GANT61 to inhibit CLL cell viability was associated with stat3 phosphorylation, which is time and dose dependent (figure 2).

These results suggest that in CLL Hh pathway is closely related to stat3 pathway. Moreover, these studies reveal a potential mechanism for the anti-leukemia activity of GANT61 which might inhibit viability of CLL cells by deregulating stat3 phosphorylation.

Figure 1

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stat3 phosphorylation status in CLL patients. Three CLL patients with high level of GLI1 (lane 1¨C3) and 3 with low level (lane 4¨C6) were tested. Lane1 Lane2 Lane3 Lane4 Lane5 Lane6

Figure 1

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stat3 phosphorylation status in CLL patients. Three CLL patients with high level of GLI1 (lane 1¨C3) and 3 with low level (lane 4¨C6) were tested. Lane1 Lane2 Lane3 Lane4 Lane5 Lane6

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Figure 2

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Western blot was performed after culture with GANT61. Lane 1:20um-24hour control. Lanes 2 : 20um-24hour Lane 3:20um-48hour control. Lanes 4 : 20um-48hour Lane 1:20um-24hour control.  Lanes 2 : 20um-24hour  Lane 3:20um-48hour control  Lanes 4 : 20um-48hour

Figure 2

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Western blot was performed after culture with GANT61. Lane 1:20um-24hour control. Lanes 2 : 20um-24hour Lane 3:20um-48hour control. Lanes 4 : 20um-48hour Lane 1:20um-24hour control.  Lanes 2 : 20um-24hour  Lane 3:20um-48hour control  Lanes 4 : 20um-48hour

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No relevant conflicts of interest to declare.