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
Authors: Rangaraju Sravanti , Marino Silvia , Petrusca Daniela Nicoleta , Cripe Larry D , Sargent Katie J , Roodman G. David , Sayar Hamid , Ramdas Baskar , Palam Lakshmi Reddy , Shanmugam Rajasubramaniam , Rogozea Adriana , Konig Heiko , Kapur Reuben , Boswell H. Scott 
Published: October 2019

Abstract
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 accumulation of large ubiquitin-laden species of phospho-β-Catenin, ranging from 100-175kD, under conditions where apoptotic cell death occurred. A cytoplasmically-localized gatekeeper for stopping canonical Wnt pathway activation, Axin1, was not limiting for the loss of nuclear β-Catenin by the combination: FSI plus PI, but was modestly upregulated by PI alone. However, nuclear-localized FOXM1, a known nuclear chaperone for β-Catenin, was frequently significantly reduced by PI or the combination of FSI/PI. Both Flt3ITD (cytoplasmic) and p52NFκB (nuclear) were lost in additive fashion by combination treatments. At the transcriptional level (real-time RT-PCR), c-jun (a transactivator for FOXM1 and CTNNB1), Syk, as well as FOXM1, HOXA and MEIS1 were additively repressed by combination FSI plus PI in select AML blasts. Certain AML molecular phenotypes demonstrated heightened sensitivity to the combination of TAK-659 plus PIs. Within blasts bearing hierarchically-related mutant effectors: IDH2, TET2, or WT1-all recognized to dampen Wnt-inhibitory pathways, we observed that the combination of TAK-659 with PI was cooperatively active in both Flt3mutant and Flt3wild-type AMLs, while accompanied by an inhibitory effect on β-Catenin. In summary, proteasome inhibition with Ixazomib or Bortezomib cooperated with Flt3/Syk inhibition to cause apoptotic cell death in both Flt3mutant and wild-type AML's with Wnt pathway DNA/chromatin modifying enzyme mutations, a process which appeared to depend upon loss of nuclear-active β-Catenin and FOXM1, and accompanied by accumulation of large, ubiquitin-laden cytoplasmic β-Catenin species. Thus, proteasome inhibition in combination with inhibition of Flt3/Syk holds a central role in targeted therapy of certain poor-risk de novo AMLs, by interdiction of a leukemic stem cell (LSC) signature and causing apoptosis of AML blasts.

Disclosures
Roodman: Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.