Abstract 3249: Targeting the miR29b/Sp1/Dnmt pathway for curative therapy in Mll-PTD/Flt3-ITD acute myeloid leukemia.
Authors: Bernot Kelsie M. , Nemer John S. , Santhanam Ramasamy , Liu Shujun , Marcucci Gabriel G. , Zorko Nicholas A. , Whitman Susan P. , Yan Pearlly , Frankhouser David , Bundschuh Ralf , Zhang Mengzi , Siebenaler Ronald F. , Ahmed Elshafa H. , McConnell Kathleen K. , Munoz Maura , Brook Daniel L. , Dorrance Adrienne M. , Dickerson Katy E. , Zhang Xiaoli , Zhang Jianying , Jarjoura David , Lee Robert , Blum William , Caligiuri Michael A. , Marcucci Guido 
Published: August 2014

Abstract
The partial tandem duplication (PTD) of the MLL gene is a molecular defect in human myelodysplastic syndrome and acute myeloid leukemia (AML) and associates with poor patient outcomes, especially when other molecular prognostic markers are co-present such as the internal tandem duplication (ITD) of FLT3, i.e., FLT3-ITD. In human AML, MLL-PTD alters the epigenome via aberrant H3K4 methylation activity but is also associated with increased DNA methylation through an unknown mechanism (Whitman, et al, Blood 2008). Using a double knock-in mouse model of AML with Mll-PTD and Flt3-ITD, we analyzed the DNA methylome using a methyl binding protein (MBD2) pull down followed by next generation sequencing. Leukemic mouse bone marrow (BM, n=8) exhibited a 1.7 fold increase in global DNA methylation compared to non-leukemic controls (n=9, P=0.017). Consistent with this finding, DNA methyltransferases (Dmnt) 1, 3a, and 3b were overexpressed (1.5 fold P=0.03, 2.3 fold P=0.01, and 5.3 fold P=0.03, respectively). We previously showed that the non-coding microRNA 29b (miR-29b) expression directly downmodulates DNMT3a and 3b, but indirectly inhibits DNMT1 via miR-29b-driven SP1 suppression. In the murine Mll-PTD/Flt3-ITD AML, the precursor to the mature miR-29b (pri-miR-29b-2) was downregulated in leukemic BM (0.7-fold, P&lt;0.001), whereas Sp1 mRNA was upregulated compared to wildtype (1.6-fold P=0.01). Bortezomib is a proteasome inhibitor that also downregulates Dnmt's (Liu, et al, Blood, 2008) by increasing the expression of miR-29b (Liu, et al, Cancer Cell, 2010). In vitro, bortezomib induced a dose-dependent reduction in proliferation of Mll-PTD/Flt3-ITD leukemic blasts (MTS assay, EC50 23.83nM) and apoptosis. Prior to cell death in bortezomib-treated cells, expression of pri-miR-29b-2 increased by 1.7 fold over vehicle treated cells (P=0.01) while Dnmt3b mRNA (P=0.001) and protein levels decreased to half of the vehicle treated level. Targeting of this pathway in vivo using liposomal-bortezomib (1mg/kg IV twice per week for 2 weeks followed by 2mg/kg IV twice per week for 2 weeks) significantly increased survival of AML transplanted mice, compared with vehicle or free-bortezomib with 80% alive 90 days post-transplant (P&lt;0.001). At day 90, treated mice had normal spleen weights (mean 86mg) and absence of leukemic blasts in BM, liver, or spleen compared to vehicle-treated mice that showed significant blast infiltration (mean spleen weight 396mg, P&lt;0.001). Taken together, these data support the miR-29b/SP1/DNMT pathway of epigenetic modulation as active in Mll-PTD/Flt3-ITD AML, and indicate bortezomib as a valuable treatment option to be explored in patients with MLL-PTD/FLT3-ITD-associated AML.
Citation Format: Kelsie M. Bernot, John S. Nemer, Ramasamy Santhanam, Shujun Liu, Gabriel G. Marcucci, Nicholas A. Zorko, Susan P. Whitman, Pearlly Yan, David Frankhouser, Ralf Bundschuh, Mengzi Zhang, Ronald F. Siebenaler, Elshafa H. Ahmed, Kathleen K. McConnell, Maura Munoz, Daniel L. Brook, Adrienne M. Dorrance, Katy E. Dickerson, Xiaoli Zhang, Jianying Zhang, David Jarjoura, Robert Lee, William Blum, Michael A. Caligiuri, Guido Marcucci. Targeting the miR29b/Sp1/Dnmt pathway for curative therapy in Mll-PTD/Flt3-ITD acute myeloid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3249. doi:10.1158/1538-7445.AM2013-3249