Identification of Beta-Catenin As a Novel Ubiquitin-Regulated Target of Combined Flβ3/Syk- and Proteasome Inhibitors for Interruption of LSC Signaling in Poor-Risk AML

Enhanced blast clearance/remission followed for relapsed/refractory AML patients, in whose blasts were annotated by mutant Flt3 in company with additional TET2 mutation, when a combination of Sorafenib/Vorinostat was supplemented by Bortezomib (Sayar, et al. Oncotarget, 2018). Pharmacodynamic analysis of day0/4 marrow blasts demonstrated early inhibition of HOXA9/10, and/or MEIS1, which occurred upon re-regulation of Wnt pathway participants whose activity had been affected by mTET2. We hypothesized Wnt effectors which drive HOXA overexpression enlist β-Catenin-dependent transactivation of HOXA (Bei, Eklund et al. JBC, 2012), and the role of Bortezomib extends beyond previously recognized AML-specific targets: Flt3ITD, p52NFκB. Therefore a series of primary AML blasts chosen across a spectrum of cytogenetic and molecular categories were analyzed herein to examine the hypothesis that proteasome inhibitors (PI) provide novel targeting action in combination with a new Flt3/Syk inhibitor (FSI) currently in clinical trial, TAK-659 (K Pratz, Blood(abstract) 2018) or, alternatively, when using a previously-reported compound with similar FSI selectivity, R406 (Puissant, Stegmaier, et al. Cancer Cell, 2014). Either of these agents alone or in combination with Bortezomib or Ixazomib were tested to learn respective mechanism of action, and to identify sensitive molecular phenotypes. In order to study β-Catenin as target, we incubated AML blasts in culture with the following treatments: control, FSI, PI, or PI+FSI, while using informative dosing in tandem cultures for proliferation, apoptosis, gene expression and proteomic endpoints. Because ubiquitination directs substrate proteolysis as well as subcellular localization for transcriptional regulators, we isolated within each test group both cytoplasmic and nuclear protein. Also, because activity of β-Catenin involves phosphorylation-initiated and ubiquitin-directed trafficking, we probed immunoblots for both nonphosphorylated [active] (S33,S37,T41)- and phosphorylated β-Catenin species. For Flt3/Syk inhibition by TAK-659, we used concentrations at, and below, a clinically-achievable optimal concentration of 250 nM (opcit). In all cases of Flt3mutant AML's studied, we observed synergy between Flt3/Syk inhibitor with PI, including at clinically suboptimal concentrations of individual agents. This cooperative activity involved loss of active, nonphosphorylated β-Catenin (80kD) from the nucleus, and the cytoplasmic accum