Acute leukemias induced by MLL chimeric oncoproteins are among the subset of cancers distinguished by a paradoxical dependence on GSK-3 kinase activity for sustained proliferation. In this study, we demonstrate that GSK-3 maintains the MLL LSC transcriptional program by promoting the conditional association of CREB and its co-activators with MEIS1, a critical component of the MLL-subordinate HOX/MEIS transcription complex, which in turn facilitates HOX-mediated transcription. This mechanism also applies to cells transformed by a variety of homeodomain proteins, including CDX2, which is highly expressed in a majority of acute myeloid leukemias, suggesting that the spectrum of leukemias susceptible to GSK-3 inhibitory therapies may be much broader than previously anticipated.
Our conclusions are based on the following key observations: i) HOXA/MEIS leukemia cells are sensitive to GSK-3 inhibition, essentially phenocopying MLL leukemia sensitivity both in vitro and in vivo; ii) CREB along with its co-activators CBP and TORC physically associates with MEIS1, and modulates HOX/MEIS transcriptional activity on an authentic promoter; iii) CREB association with MEIS1 is regulated by phosphorylation at Ser129 of CREB, a known target of GSK-3; and iv) gene programs linked with HOX/MEIS as well as CREB are down-regulated by GSK-3 inhibition in MLL leukemia cells. Together, these observations suggest a model () whereby GSK-3 activity maintains the physical and functional association of CREB with MEIS to promote critical target gene expression responsible for HOX-mediated transformation.
Schematic model depicting role of GSK-3 in promoting HOX-mediated transcription through CREB phosphorylation
In our studies, CREB enhanced the transcriptional activity of a HOX/MEIS/PBX complex on the Hoxb1
ARE, an authentic MEIS1 target promoter, and its role in this context appears to be regulated by GSK-3 phosphorylation. CREB is a multi-functional transcriptional activator that is involved in many physiological pathways under normal and pathologic conditions. CREB activity is regulated by phosphorylation at Ser133, which is a target of various kinases depending on the specific signaling stimulus and cell type. PKA, which is activated by cAMP, is the major kinase that targets Ser133 in many processes. Ser133 phosphorylation primes CREB for phosphorylation by GSK-3 at Ser129. However, unlike Ser133 phosphorylation, which is linked with CREB activation, the physiologic consequences of Ser129 phosphorylation are not well defined, although evidence suggests that it also is linked with CREB activation (Boer et al., 2008
; Horike et al., 2008
; Tyson et al., 2002
). We found that CREB S129A mutation, similar to S133A mutation, antagonized HOX/MEIS activity and decreased colony-forming abilities of MLL or HOX/MEIS transformed cells. Consistent with our study, S129 phosphorylation of CREB by GSK-3 is required for recruitment of CBP and subsequent induction of PEPCK-C
gene expression (Horike et al., 2008
). Thus, our results support a positive role of S129 phosphorylation by GSK-3 for CREB activation of specific transcriptional programs in MLL-transformed cells.
Our studies demonstrate that MEIS1 is a molecular link that integrates the transcriptional activities of CREB and its co-activators as a higher order complex whose physical and functional integrity is dependent on GSK-3 activity. Previous studies have shown that MEIS1-mediated transcriptional activation is stimulated by PKA and dependent on CBP (Huang et al., 2005
). Further evidence in support of a MEIS-CREB nexus is provided by the recent demonstration that MEIS1 interacts with TORC (Goh et al., 2009
), a co-activator that also associates with CREB and modulates its activity (Conkright et al., 2003
; Siu and Jin, 2007
). MEIS1 interaction with TORC is also dependent on PKA signaling, which has been shown to regulate the nuclear translocation of PBX1, an obligate dimerization partner of MEIS1. Despite the prominent role of PKA in CREB signaling pathways, treatment with several PKA inhibitors was equally toxic for leukemia cells transformed by MLL and non-MLL oncogenes (data not shown), unlike the selective anti-proliferative effects of GSK-3 inhibition. This likely reflects the essential role of PKA in various physiological processes that mask any selective role promoting CREB-MEIS interactions in HOX-transformed cells. Thus, it remains to be determined whether GSK-3 may function in concert with PKA to affect a higher order association of CREB with MEIS1 mediated through TORC and CBP.
The underlying biochemical mechanism for how GSK-3 inhibition or CREB S129 phosphorylation affects MEIS association with the TORC/CREB/CBP complex is not entirely clear. Nevertheless, several conclusions can be drawn from our study and others. First, the association is likely mediated through the MEIS1 C-terminus since its deletion abrogates MEIS1 transcriptional activation and association with TORC (Goh et al., 2009
) and CREB (). Second, TORC likely bridges CREB and MEIS1 association since TORC-enhanced activation by MEIS1 depends on CREB (), and MEIS1/CREB association depends on TORC (). Furthermore, CREB S133 and S129 phosphorylation brings CBP/p300 to MEIS1 and this likely further stabilizes MEIS1 and CREB association. Loss of S133 or S129 phosphorylation, which decreases affinity of MEIS1 and CREB, may involve: i) loss of CBP binding that reduces CREB and MEIS1 association, and ii) conformational changes of the TORC/CREB complex that alter TORC/CREB binding (Heinrich et al., 2009
) (data not shown). CREB may also directly modulate MEIS1 and PBX expression (Esparza et al., 2008
) adding to the complexity of their interrelated function. However, the detailed mechanisms remain to be determined and other unappreciated factors may also be involved.
Multiple genes are down-regulated in MLL-transformed cells following GSK-3 inhibition. Our studies focused on FOS
as a prototype since it was one of the most differentially expressed genes in MLL leukemia cells. It encodes a member of the AP-1 transcription factor family, and as an early response gene induced by CREB activation, its role in cancer has been widely studied and shown to enhance the proliferation of transformed cells. In addition to increased proliferation, forced expression of FOS
in our studies rendered MLL leukemia cells more resistant to GSK-3 inhibition whereas its knockdown rendered cells more sensitive, suggesting a downstream role for FOS
in mediating the response to GSK-3 inhibitors. FOS
expression levels are likely to be regulated by HOX transcription complexes as originally suggested by enforced expression of HOXB4
, which induced FOS
expression in fibroblasts or epithelial cells, respectively (Krosl and Sauvageau, 2000
; Potter et al., 2006
). Moreover, FOS
levels are decreased following deletion of MEIS1 (Figure S4C
). In our studies, FOS
transcript levels were increased in human leukemia cells following forced expression of HOXA9 and MEIS1, but not HOXA9 alone, indicating a regulatory role for the HOX/MEIS complex. However, reduced FOS
levels alone are unlikely to account for the cell cycle arrest associated with GSK-3 inhibition since knockdown of FOS
to similar low levels achieved by GSK-3 inhibition did not result in complete cell cycle arrest. Consistent with our expression profiling studies, multiple downstream genes are likely to mediate the adverse effects of GSK-3 inhibition on MLL leukemia cells.
In conclusion, GSK-3 promotes conditional association of CREB and its co-activators with MEIS1 to facilitate HOX-mediated transcription and oncogenesis. This provides a mechanistic basis for the paradoxical dependence of MLL-associated leukemias on GSK-3 activity, which critically maintains the MLL LSC transcriptional program, and suggests a therapeutic approach to molecularly target HOX-associated transcription. In addition to MLL leukemias, accumulating evidence indicates that GSK-3 inhibition blocks proliferation or induces apoptosis in a variety of cancers including melanoma, myeloma, glioblastoma and pancreatic cancer among others (Korur et al., 2009
; Miyashita et al., 2009
; Smalley et al., 2007
; Wilson and Baldwin, 2008
; Zhou et al., 2008
). It will be of interest to determine how much of a role, if any, CREB-MEIS interactions contribute to the GSK-3 dependence of these malignancies, which are candidates for GSK-3 inhibitor therapies.