Pharmacologic Modulation of STAT1 and Wnt Signaling in Chronic Lymphocytic Leukemia

BACKGROUND: The STAT and Wnt signaling pathways play critical roles in early lymphocyte development and function, with considerable cross-talk between them.  STAT1 has been reported to be constitutively phosphorylated at SER727, and often at TYR701, in CLL patients.  STAT1 activation can regulate the function of the IRF8 transcription factor. Polymorphisms of the IRF8 gene have been associated with both familial and sporadic CLL, and can influence Wnt signaling. Hence, both STAT1 and Wnt are potential pharmacologic targets for CLL therapy, particularly in patients that are resistant to, or intolerant of BTK and PI3K inhibitors. We previously reported that the electrophilic drug dimethylfumarate (DMF, Tecfidera, Fumaderm) could inhibit Wnt signaling in CLL.  Here we have investigated its effects on constitutive and inducible STAT1 signaling, and on IRF8 expression, in primary CLL cells.

METHODS: Primary leukemia cells from patients with CLL were cultured with DMF at pharmacologically relevant doses (3 uM to 30 uM) for 4 hours prior to analysis of STAT1 phosphorylation and IRF8 expression. We assessed the effect of DMF with and without pre-stimulation by lipopolysaccharide (LPS), Wnt3a, or a TLR7 agonist. To further evaluate the effect on CLL cells in a microenvironment that stimulates these pathways, we utilized a murine CLL xenograft model. We implanted primary leukemic cells from patients with CLL into the peritoneum of Rag2 deficient immune-compromised mice. Groups of mice were then treated with DMF at a dose of 10 mg/kg by oral gavage.  CLL cells were retrieved 4 to 5 hours later by peritoneal lavage and analyzed.

RESULTS: We confirmed that both SER727 and TYR701 are phosphorylated in the majority of primary CLL cells from both VH mutated (high risk) and unmutated (low risk) clones, but not in normal peripheral blood mononuclear cells (PBMC).  A 4 hours exposure of CLL cells to DMF inhibited TYR701 phosphorylation, but had no effect on SER727 phosphorylation nor on total STAT1 levels. We also confirmed that IRF8 levels are 6-8 fold elevated in CLL cells, compared to normal PBMC. IRF8 expression was inhibited by DMF treatment in 7 out of 11 CLL patients.  Similar effects were observed in vivo.  Wnt or LPS stimulation increased IRF8 levels in normal PBMC, but did not alter the already elevated IRF8 levels in CLL cells.

CONCLUSIONS: Activation of the STAT1/IRF8 pathway, like the Wnt pathway, is highly characteristic of CLL.  DMF treatment of CLL cells can interrupt these signaling cascades, interfering with leukemia cell survival.