Hypomethylating Agents Correct Subcellular Localization of FOXO3a and (re)Establish Expression of Its Transcriptional Targets In AML and MDS- Cells

We previously found deregulation of DNA-damage response (DDR) signaling in MDS and AML, notably the “uncoupling” between upstream proteins (ATM) and downstream effectors (Chk-1/2) in high-risk MDS and AML (Boehrer et al., Oncogene 2009). Here, we assessed if and to which degree the hypomethylating agents azacitidine (AZA) and decitabine (DAC), widely used agents in the treatment of high-risk MDS and AML, can overcome these defects.

MDS (MOLM-13)- and AML- (HL-60, KG-1)-derived cell lines were incubated with AZA (1-2μM) and DAC (1-2μM). The drugs capacity to induce apoptosis (DiOC₆(3)/PI), cell cycle arrest (PI) and their impact on the expression level (immunoblot) and subcellular localization (immunoflourescence) of DDR-related proteins were concomitantly assessed. Functional relevance was determined by co-incubation with biochemical inhibitors of ATM (KU-55933), ATM/ATR (caffeine), Chk-1 (UCN-01), and Chk-2 (NSC-109555). Most important findings were recapitulated on MDS- and AML- patient cells (n=5) after selection for CD34-positvity by immunomagnetic beads.

As previously described, both drugs induced dose- and time-dependent apoptosis and cell cycle arrest in G2/M. Noteworthy, whereas both drugs completely abrogated expression of DNA methyltransferases 1 and 3a as early as 4h of incubation with 2μM (abrogation sustained at 24 and 48h), they differentially affected DDR-related signaling. Indeed, the increased capacity of DAC (as compared to AZA) to induce G2/M-arrest (G2/M-increase at 48h DEC: 20%, AZA: +6%) was accompanied by an earlier and more efficient activation of the checkpoint-kinases-1 and -2 (phosphorylation of Chk-1-Ser317 and Chk-2-Ser68) as well as their downstream target y-H2AX (phosphorylation on Ser139). Noteworthy, DAC (and to a lesser extent AZA) modified activation of FOXO3a (diminished phosphorylation on Ser253) and concomitantly enabled nuclear translocation of non-phosphorylated FOXO3a. In addition, nuclear translocation of FOXO3a was accompanied by upregulation of its transcriptional targets known to confer G2/M- arrest (p21, p27) and apoptosis (BH3-only protein BIM). Confirming the functional relevance of our findings, we found that inhibition of ATM (KU-55933) or ATM/ATR (caffeine) had little to no impact on AZA /DAC-induced apoptosis or inhibition of cell cycle progression, whereas inhibition of Chk-1 by UCN-01 (and to a lesser degree inhibition of Chk-2 by NSC-1095555) abrogated G2/M-arrest induced by hypomethylating agents. Finally we showed that both DAC and AZA (2μM) can induce the cytoplasmic-nuclear shift of FOXO3a in MDS- and AML-derived cell lines and to a lesser extent in patient blasts, an effect observed as early as 4h of the incubation period.

Hypomethylating agents can correct aberrant DDR-signaling in AML-and MDS blasts, and can abrogate constitutive phosphorylation of FOXO3a thereby “correcting” its aberrant cytoplasmic localization and (re-)establishing its function as a transcriptional regulator. This possible mode of action of hypomethylating agents may be important considering that aberrant phosphorylation and localization of FOXO3a is an adverse prognostic factor in AML, and that correction of its deregulation has been described as an attractive therapeutic aim.

Fenaux: CELGENE, JANSSEN CILAG, AMGEN, ROCHE, GSK, NOVARTIS, MERCK, CEPHALON: Consultancy.