Podocytes are morphologically complex cells, the junctions of which form critical elements of the final filtration barrier. Disruption of their foot processes and slit diaphragms occur early in the development of many glomerular diseases. Here, we biochemically purified fractions enriched with slit diaphragm proteins and performed a proteomic analysis to identify new components of this important structure. Several known slit diaphragm proteins were found, such as podocin and nephrin, confirming the validity of the purification scheme. However, proteins on the apical membrane such as podocalyxin were neither enriched nor identified in our analysis. The chloride intracellular channel protein 5 (CLIC5), predominantly expressed in podocytes, was enriched in these fractions and localized in the foot process apical and basal membranes. CLIC5 colocalized and associated with the ezrin/radixin/moesin complex and with podocalyxin in podocytes in vivo. It is important to note that CLIC5−/− mice were found to have significantly decreased foot process length, widespread foot process abnormalities, and developed proteinuria. The ezrin/radixin/moesin complex and podocalyxin were significantly decreased in podocytes from CLIC5−/− mice. Thus, our study identifies CLIC5 as a new component that is enriched in and necessary for foot process integrity and podocyte function in vivo.

• The Mll PTD/wt :Flt3 ITD/wt mouse is a relevant AML model in which the miR-29b-mediated epigenetics-kinome crosstalk is targetable by bortezomib.• An original liposomal formulation of bortezomib eradicates AML and yields curative therapy for MllThe coexpression of the MLL partial tandem duplication (PTD) and the FLT3 internal tandem duplication (ITD) mutations associate with a poor outcome in cytogenetically normal acute myeloid leukemia (AML). In mice, a double knock-in (dKI) of Mll PTD/wt and Flt3 ITD/wt mutations induces spontaneous AML with an increase in DNA methyltransferases (Dnmt1, 3a, and 3b) and global DNA methylation index, thereby recapitulating its human AML counterpart. We determined that a regulator of Dnmts, miR-29b, is downregulated in bone marrow of dKI AML mice. Bortezomib exerted a dose-dependent increase in miR-29b expression in AML blasts ex vivo, followed by decreased Dnmts, reduced proliferation, and increased apoptosis. In vivo, bortezomib was not active against dKI AML, yet liposomal-encapsulated bortezomib, as a single agent, reversed downregulation of miR-29b in vivo and induced a long-term (90-day) disease-free remission in 80% of dKI AML mice that exhibited high leukemic burden at the start of therapy, yet showed no signs of relapse at autopsy. Taken together, these data support that liposomal bortezomib, as a single agent, eradicates Mll PTD/wt :Flt3 ITD/wt AML in mouse and may represent a powerful and potentially curative approach to high-risk human disease. (Blood. 2013;122(23):3778-3783)

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<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<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<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