Cooperativity between Flt3/Syk and Proteasome Inhibitors in Flt3mutant/Wildtype AML, By Acting on the Oxphos and Wnt Endpoints of Syk and p62SQSTM1 Pathways Prohibits Protective Autophagy; While Downregulation of NRF2, NQO1, Jun, and b-Catenin Bypasses Interfering RAS or WT1 Co-Mutations
Authors: Rangaraju Sravanti , Marino Silvia , Roodman G. David , Cripe Larry D. , Ramdas Baskar , Palam Lakshmi Reddy , Sargent Katie J , Sayar Hamid , Konig Heiko , Kapur Reuben , Boswell H. Scott 
Published: November 2019

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 =/<250 nM, significantly broadened the ED30-66 concentrations for these PIs when acting on AML blasts annotated for the presence of mutations Flt3, DNMT3A, IDH1/2, NPM1, where inhibitory activity has been reported for all other Flt3 inhibitors (JW Tyner et al, 2018). We identified that activity involved reduction by the combination of PI with TAK-659, of p62SQSTM1, the scaffolding adaptor protein linked to both sequestration/disposal of ubiquitinated and damaged proteins, as well as to mitochondrial remodeling for OXPHOS. P62, jun, NRF2, NQO1, and b-catenin were reduced by the combination, to levels that equaled or exceeded effects by TAK-659 alone, with heightened impact on blast cell extinction by visible apoptosis and thymidine uptake. However, the combination of PI Ixazomib/Bortezomib with TAK-659 demonstrated unexpected heightened activity also for Flt3mutant and Flt3WT AML blasts with RAS or WT1 mutations, resulting in significant shifts in the ED concentrations for blasts in response to PI alone or the combination with TAK-659 at the clinically relevant dose of 250nM. In addition, blasts annotated for RAS mutation, without or with WT1 mutation (which abrogates expression of the nuclear chaperone for b-catenin, TBL1X) had heightened nuclear content for RAC1, which can participate in b-catenin chaperoning. TAK/PI combinations reduced nuclear RAC content as a measure of the antileukemic cooperativity, while also diminishing levels of nuclear b-catenin and its activating (PAK-dependent) phosphorylation on S675. The combination was also active for AML blasts with MLL-PTD, without or with additional RAS mutation. Further, genetic epistasis experiments were performed with siRNA transduced into MV-411 cells to establish the functional relationship of the individual effectors. P62 knockdown had a dominant effect on reducing NRF2 and NQO1 content, as well reducing nuclear/active expressions of c-jun, and b-catenin, thus establishing its importance in both OXPHOS and Wnt/b-catenin pathways. On the other hand, siRNA knockdown of Syk supported its role as a signaling substation for b-catenin abundance, also dependent on c-jun. Whether the consistent ability for TAK-659 to inhibit p62 expression in primary blasts depends upon it's inhibition of Syk vs. Flt3 is under further investigation. However, both NRF2 and c-jun are known to transactivate p62 gene, and p62 product can serve as scaffolding for JNK1. Indeed, an OXPHOS phenotype is dependent upon jun as well as NRF2, and jun also affects b-catenin en route to HOXA9/10, where overlapping expression of HOXA's and OXPHOS species is described. On the other hand, RAS mutations have been found to occupy an AML signaling space that is OXPHOS independent (I Baccelli, et al. Cancer Cell, 2019). In summary, we have identified common intracellular effectors for OXPHOS as well as Wnt/b-catenin-> 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.