ALBERT

Description: 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.

Description: Multiple myeloma (MM) is incurable and 80% of MM patients develop MM bone disease (MMBD). MMBD lesions do not heal due to the persistent suppression of bone formation, which markedly increases mortality and contributes to MM drug resistance. Current treatments for MMBD, such as bisphosphonates and denosumab, target bone destruction but do not result in new bone formation. Although proteasome inhibitors (PIs) have greatly improved survival of MM patients, they only have transient bone anabolic effects. Further, development of drug resistance to PIs remains a major clinical problem. We previously showed that the ZZ domain of p62 (sequestosome-1),plays an important role in both MM growth and suppression of osteoblast (OB) differentiation in the MM microenvironment, by regulating multiple signaling pathways and acting as a cargo-receptor for autophagy. Recently, our collaborators showed that the p62-ZZ is a high-affinity N-recognin of the N-end rule pathway (NERP). p62-ZZ also serves as the molecular switch for necroptotic versus apoptotic cell death pathways. We previously reported that MM cells or TNFα prevent OB differentiation by inducing persistent epigenetic repression of the Runx2-P1 promoter in MM patient bone marrow stromal cells (BMSCs), via the transcriptional repressor Gfi1. We found that blocking p62-ZZ by saturating it with a novel synthetic p62-ZZ/NERP competing ligand, XRK3F2, prevented and reversed MM-induced Gfi1 occupancy at the Runx2-P1 promoter, allowing BMSCs to increase OB marker gene expression and to mineralize. These results suggest that targeting the p62-ZZ/N-end rule pathway would enhance the bone anabolic effects of PIs. To test this hypothesis, we first exposed normal OBs to different doses of bortezomib (Btz) or XRK3F2 or their combination. The combination significantly increased OB differentiation markers Runx2 (60%), Osterix (20%) and ATF4 (60%), and induced mineral deposition compared with either drug alone. The combination also blocked TNFα up-regulation of Gfi1 and suppression of OB differentiation. Interestingly, none of the concentrations tested decreased OB viability. Studies with MM patient-BMSCs showed that XRK3F2 reversed suppression of OB differentiation induced by MM cells, allowing them to mineralize. Importantly, our preliminary in vivo data showed that administration of XRK3F2 to mice with established MM induced dramatic cortical bone formation in MM-containing bones but had no effect on tumor burden. We and others previously showed that MM cells subjected to sustained proteasomal inhibition, rely on p62-mediated autophagic degradation to reduce the proteotoxic load caused by excessive immunoglobulin synthesis. We recently found that targeting the p62-ZZ domain in human MM cells, increases Btz-induced MM cell death, independently of their p53 status. The combination also significantly reduced cell viability in Btz resistant cells although no caspase 3 activation was observed, suggesting a caspase-3 independent cell death. To determine the mechanism(s) responsible for MM cell death induced by the combination, we pretreated MM cell lines and primary CD138+ MM cells with Z-DEVD (20μM), bafilomycin (Baf, 40nM), or necrostatin1 (NEC1, 50μM). The anti-MM effects of XRK3F2 or Btz+XRK3F2 were fully blocked by NEC1, an inhibitor of necroptosis, but not by inhibitors of caspase-dependent apoptosis (Z-DEVD) or autophagy (Baf), supporting that p62-ZZ regulates necroptosis in MM cells. RNAseq analysis of the additive effect of Btz+XRK3F2 on gene expression showed a total of 583 differentially regulated genes, including 374 significantly down-regulated and 209 significantly upregulated. GO term analysis of up-regulated DEGs identified an enrichment in the endoplasmic reticulum (ER) stress and ER unfolded protein response, and regulation of transcription in response to stress and autophagy. In summary, our results demonstrate that targeting the p62-ZZ/N-end rule pathway in combination with PIs in MM significantly reduces MM cell viability by activating multiple death pathway and overcomes PI-resistance of MM cells. In addition, targeting the p62-ZZ in OBs potentiates the bone anabolic action of PIs and reverses the persistent OB suppression induced by MM cells to allow bone formation. Thus, p62-ZZ plays a critical role in MM and bone cells and identifies p62-ZZ as an important molecular target for the treatment of MMBD. Disclosures Xie: Oxis Biotech: Consultancy; ID4Pharma: Other: Founder. Roodman:Amgen: Membership on an entity's Board of Directors or advisory committees.

Description: Recent attempts at single agent targeted therapy of AMLs described by mutation of Flt3 or nuclear epigenetic effectors, has led to the conclusion that combination targeted approaches will be required (CM McMahon et al. Cancer Discov 2019). The simplest combination therapy would involve inhibitors directed at mutant drivers at each level (receptor, nuclear). However, the number of those inhibitors is limited. A broader strategy would target common endpoints for converging pathways such as Wnt/beta-catenin activation elicited by mutation of IDH1/2, TET2, DNMT3A. We found marked cytoplasmic accumulation of ubiquitinated protein (especially inactive b-catenin excluded from the nucleus) by treatment with proteasome inhibitor(PI) to be an efficient, dose-dependent inducer of endoplasmic reticulum (ER) stress apoptosis in mutant Flt3/Wnt effector AML's, requiring concentrations =/〉100nM Ixazomib, or =/〉20nM Bortezomib, when used alone on cultured blasts. Indeed, a compensatory pathway to protective autophagic escape from PI in poor-risk AML is linked to the levels of NRF2, a major transcriptional activator of NADPH quinone reductase1 (NQO1) that buffers oxidative stress. However, the Flt3/Syk inhibitor (FSI) in clinical trial, TAK-659, at =/ HOXA in AML, as compared to the RAS and WT1 pathways, and have established a combination therapy (TAK-659 plus PI) that affects the inhibitory effectors elicited by these co-mutational states, which are responsible for negating activity for most Flt3 selective targeted agents, so as to allow antileukemic response. Disclosures Roodman: Amgen: Membership on an entity's Board of Directors or advisory committees. Konig:Agios: Consultancy; Amgen: Honoraria.