Combined Targeting of β-Catenin and FLT-3 Is Synergistically Lethal Against Cultured and Primary AML Blast Progenitor Cells Expressing FLT3 Internal Tandem Duplication (ITD)
Authors: Saha Saikat , Fiskus Warren , Sharma Sunil , Shah Bhavin , Rogojina Anna T , Devaraj Santhana G. T. , Sun Baohua , Leveque Christopher , Zu Youli , Iyer Swaminathan Padmanabhan , Bhalla Kapil N. 
Published: October 2019

Abstract
β-catenin acts as a co-activator for the T-cell factor (TCF) 4/lymphoid enhancer factor (LEF) 1 bipartite transcription factor at the promoters of the WNT-β-catenin target genes, including cyclin D1, c-Myc and survivin. The canonical WNT-β-catenin pathway is documented to be essential for self-renewal, growth and survival of the AML stem and blast progenitor cells (BPCs), which has also been correlated with a poor prognosis in AML. In AML stem/BPCs expressing mutant FLT3-ITD, increased PI3K/AKT activity causes phosphorylation and inactivation of GSK3β, thereby preventing degradation, promoting stabilization and nuclear localization of β-catenin. Additionally, FLT3 can also directly mediate the tyrosine phosphorylation of β-catenin, thereby stabilizing and promoting the nuclear localization and binding of β-catenin to TCF4. TBL1 (transducin beta-like) is an adaptor protein, which binds to nuclear β-catenin and promotes its co-factor activity with TCF4/LEF1 in mediating transcription of the target genes, including c-Myc, cyclin D1 and survivin. Therefore, we hypothesized that targeted disruption of TBL1-β-catenin binding or depletion of TBL1 would abrogate the pro-growth and oncogenic signaling of β-catenin in AML BPCs, especially those expressing FLT3-ITD. Here, we demonstrate that treatment with 20 to 100 nM of BC2059 (β-Cat Pharmaceuticals), a small molecule, anthraquinone oxime-analog, disrupts the binding of β-catenin to TBL1 (by anti-TBL1 pull down and immunofluorescence analyses) and promotes proteasomal degradation of β-catenin, thereby attenuating the nuclear levels of β-catenin in the cultured (OCI-AML3, MOLM13 and MV4-11), as well as in primary (p) AML BPCs. Concomitantly, BC2059 treatment inhibited the mRNA and protein expression of c-Myc, cyclin D1 and survivin, while de-repressing p21 and Axin2. BC2059 also dose dependently inhibited growth and induced apoptosis of cultured and CD34+ pAML BPCs expressing FLT3-ITD (40 to 60%), but not of normal CD34+ bone marrow progenitor cells (p < 0.01). Transient knockdown of TBL1 or beta catenin (60 to 70%) by lentivirus-transduced shRNA caused loss of viability in MOLM13 cells, which was significantly enhanced by treatment with BC2059 (p < 0.01). BC2059 also induced apoptosis of MOLM13-TKIR cells that were isolated in vitro to exhibit resistance to FLT3 antagonists (approximately 50-fold). Notably, BC2059 treatment (10 mg/kg, t.i.w., by IV injection) also exerted potent in vivo anti-AML activity and significantly improved the survival of immune depleted mice engrafted with cultured and patient-derived pAML BPCs (p < 0.001). Since compared to the control OCI-AML3 cells, BC2059 demonstrated significantly greater lethality against the OCI-AML3 cells ectopically overexpressing FLT3-ITD (approximately 8-fold), we hypothesized that co-treatment with a FLT3 antagonist would further reduce the nuclear levels of β-catenin and enhance the lethal activity of FLT3-antagonist against AML BPCs expressing FLT3-ITD. Indeed, co-treatment with BC2059 (50 nM) and the FLT3-antagonist quizartinib or ponatinib (100 to 200 nM), versus each agent alone, caused more reduction in the nuclear levels and binding of β-catenin to TBL1 (by confocal immunofluorescence analysis). This was associated with greater decline in the expression of c-Myc, cyclin D1 and survivin, but increase in the levels of p21 and BIM. Compared to each agent alone, co-treatment with BC2059 and quizartinib or ponatinib also synergistically induced apoptosis of the FLT3-ITD expressing cultured (MOLM13 and MV4-11) and pAML BPCs (combination indices of < 1.0, by isobologram analyses) but not of normal CD34+ progenitor cells. Treatment with BC2059 (25 to 100 nM) also significantly increased the apoptosis observed by the shRNA mediated incomplete knockdown of TBL1 or β-catenin (approximately 70%) in MOLM13 cells (p < 0.01). Collectively, our findings support that targeted inhibition of the levels and binding of β-catenin to TBL by BC2059 and FLT3-antagonist is a promising approach to exert lethal activity against AML BPCs expressing FLT3-ITD. Further pre-clinical development of this combination therapy against FLT3-ITD expressing AML is progressing.

Disclosures
No relevant conflicts of interest to declare.