Overexpression and Phosphorylation of eIF4E Is Required for Beta-Catenin Activation in Blast Crisis Chronic Myelogenous Leukemia.
Authors: Lim Sharon , Saw T. Yih , Chuah Charles , Ong S. Tiong 
Published: October 2019

Abstract 41

Despite the success of tyrosine kinase inhibitors (TKI) in chronic phase (CP) CML, blast crisis (BC) CML remains difficult to control, with most patients eventually developing either primary or secondary TKI-resistance (TKI-R). In addition to TKI-R, myeloid BC GMPs acquire the ability to self-renew, a feature that is mediated by b-catenin activation, and which may contribute to disease persistence. The mechanisms underlying b-catenin activation remain obscure, including its dependence on Bcr-Abl activity. Long-term control of BC CML will require overcoming the mechanisms mediating TKI-R, as well as reversing b-catenin-mediated self-renewal. In this study, we investigated the role of the mRNA translation machinery in mediating b-catenin-mediated self-renewal, since our prior work had implicated aberrant mRNA translation in TKI-R and BC pathophysiology (Ly et al. Cancer Research 2003; Prabhu et al. Oncogene, 2007; Zhang et al. MCB, 2008). First, we showed that primary CD34+ BC cells overexpressed eIF4E, a rate-limiting translation initiation factor, compared to CP cells. We also found that both eIF4E overexpression and phosphorylation at serine 209 (S209) occurred in a Bcr-Abl-independent manner. Next, by using a combination of genetic and biochemical approaches, we showed that forced overexpression of eIF4E was sufficient to activate b-catenin in the K562 BC cell line, as measured by a b-catenin reporter (a 2-fold increase in TOP/FOP ratio) and by an increase in nuclear b-catenin (a 2.5-fold increase in mean fluorescence intensity using immunofluorescence). Importantly, overexpression of an eIF4E S209A mutant did not show increased b-catenin activity, whereas overexpression of a phospho-mimetic S209D mutant did, indicating b-catenin activation was dependent on S209 phosphorylation. Next, we found that siRNA knock-down or small molecule inhibition (CGP57380) of the Mnk 1/2 kinases (which are responsible for in vivo phosphorylation of eIF4E) prevented the increased b-catenin activity induced by eIF4E overexpression. Also, Mnk1/2 inactivation had no effect on b-catenin activity in the S209D mutants. Because recent work has implicated GSK3b inactivation as a contributor to b-catenin activity in BC GMPs (Abrahamsson et al. PNAS, 2009), we repeated these experiments in the presence of the GSK3b inhibitor, BIO. Here, we found that both eIF4E overexpression and BIO treatment cooperated to further augment b-catenin activity (a 3 to 4-fold increase in the TOP/FOP ratio, and 3-fold increase in nuclear b-catenin), and that this was an effect that required eIF4E phosphorylation. Based on these data, we performed biochemical and functional analyses of primary CP and BC CML cells, as well as normal cord blood (CB) progenitors. As reported by others, we found increased nuclear b-catenin in CD34+CD38+ BC cells compared CP or CB cells. Inhibition of Mnk 1/2 was also able to prevent accumulation of nuclear b-catenin in CD34+CD38+ BC cells, as was a known b-catenin inhibitor (CGP049090), but not imatinib. Functionally, Mnk kinase inhibition by CGP57380 (2.5-10 microM) also significantly decreased the ability of BC CFCs to serially replate (>8 weeks) in methylcellulose (by >90%) compared to CB CFCs (25-50%). Finally, since recent work has demonstrated that activation of the serine/threonine phosphatase, PP2A, can impair b-catenin activity and self-renewal in CML stem cells (Neviani et al, ASH Meeting Abstracts, 2008), we tested the ability of the PP2A activator, FTY720, to inhibit b-catenin activity in the context of eIF4E overexpression. We found that while FTY720 was able to inhibit b-catenin activity in cells overexpressing wild-type eIF4E, it could not do so in the presence of the S209D mutants. In summary, our results indicate that: (i) increased expression and phosphorylation of eIF4E at S209 is sufficient to activate b-catenin in CML cells; (ii) GSK3b inactivation and eIF4E phosphorylation cooperate to augment b-catenin activity; (iii) the serial-replating ability of BC CML progenitors is dependent on eIF4E phosphorylation; (iv) dephosphorylation of eIF4E at S209 is important in enabling the PP2A activator, FTY720, to inhibit b-catenin activity. Taken together, our data show that targeting eIF4E function, by modulating the activity of the kinases or phosphatases which regulate S209 phosphorylation, is a novel approach to overcome b-catenin-mediated self-renewal in BC CML cells, but not normal progenitors.

Disclosures:

No relevant conflicts of interest to declare.