Inhibition of Wnt Signaling By Dimethyl Fumarate Results in in Vitro and in Vivo Clearance of Chronic Lymphocytic Leukemia Cells and Has Additive Activity with Ibrutinib

Background: Small molecules that inhibit B cell survival pathways are effective treatments for patients with chronic lymphocytic leukemia (CLL). However, such therapies are not curative, and resistance can develop in some patients. Combination therapies with agents that inhibit several CLL survival pathways may allow for more complete responses, and help prevent treatment resistance. Previous data has shown that the pro-survival Wnt pathway is highly active in CLL and is a negative prognostic factor, and therefore is an attractive target for novel therapies to combine with agents like ibrutinib.

Dimethyl fumarate (DMF) is an orally bioavailable fumaric acid ester with immunomodulatory properties, including inhibition of the NF-kB signaling cascade. DMF has been evaluated as a systemic treatment of psoriasis as well as multiple sclerosis. Our group previously observed anti-CLL effects of DMF, mediated in part through oxidative stress. Herein we describe a novel mechanism of action of DMF and ibrutinib, mediated by inhibition of the Wnt signaling pathway.

Methods: Effects of DMF and ibrutinib on Wnt signaling were determined using a cell-based LEF/TCF beta-lactamase reporter gene FRET assay. In vitro activity was assessed in primary CLL from patients with indolent and aggressive disease. In vivo activity was evaluated in Rag2^-/- gamma chain^-/- immunodeficient (RG-KO) mice, which were engrafted with human CLL cells by intraperitoneal injection. DMF and/or ibrutinib were administrated to mice by oral gavage, at clinically used doses and schedules.

Results: Both DMF and ibrutinib have an alpha-beta unsaturated ketone that can react with essential free cysteines in the Wnt-driven LEF1 transcription factor. This effect was confirmed by a cell-based reporter gene assay in which DMF inhibited LEF/TCF dependent gene expression at low μM levels. Ibrutinib also inhibited Wnt signaling activity in the same assay. In short term cultures, DMF was cytotoxic to primary CLL cells from patients with both indolent and aggressive disease, at low uM concentrations. The combination of DMF and ibrutinib resulted in a higher degree of CLL cell clearance than achieved by either agent alone (p < 0.05 after multiple comparison adjustments, Dunnet’s method).

To evaluate the effect in a preclinical CLL xenograft animal model, we administered DMF by oral lavage to RG-KO mice engrafted with human CLL cells. Doses ranging from 3 to 30 mg/kg BID for 7 days resulted in dose dependent clearance of CLL cells compared to vehicle controls, without observable toxicity to the recipient animals. Moreover, the combination of DMF and ibrutinib resulted in a higher degree of CLL cell clearance than achieved by either agent alone. Preliminary FACS analyses revealed that DMF selectively targets CLL subpopulations of cells with aggressive characteristics, as assessed by CD38 expression. Further molecular analyses of predictive or correlative biomarkers are ongoing.

Conclusions: DMF inhibits Wnt signaling, and has single agent activity as a treatment for CLL. The combination of DMF and ibrutinib is more effective than either agent alone, particularly in aggressive disease, and is well tolerated. Clinical trials of DMF in CLL are warranted, and are planned.

This work is supported by a Leukemia and Lymphoma Society Specialized Center of Research Grant (7005-14) and by the CLL Research Consortium (5P01CA081534-14).